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Exploring novel non-Leloir β-glucosyltransferases from proteobacteria for modifying linear (β1 → 3)-linked gluco-oligosaccharide chains
Gudmundur O Hreggvidsson, Justyna M Dobruchowska, Olafur H Fridjonsson, Jon O Jonsson, Gerrit J Gerwig, Arnthor Aevarsson, Jakob K Kristjansson, Delphine Curti, Robert R Redgwell, Carl-Eric Hansen, Johannis P Kamerling, and Takoua Debeche-Boukhit
Distinct expression profiles of UDP-galactose: β-D-galactoside α1,4-galactosyltransferase and UDP-galactose: β-D-galactoside β1,4-galactosyltransferase in pigeon, ostrich and chicken
Over the years several β-glucan transferases from yeast and fungi have been reported, but enzymes with such an activity from bacteria have not been characterized so far. In this work, we describe the cloning and expression of genes encoding β-glucosyltransferase domains of glycosyl hydrolase family GH17 from three species of proteobacteria: Pseudomonas aeruginosa PAO1, P. putida KT2440 and Azotobacter vinelandii ATCC BAA-1303. The encoded enzymes of these GH17 domains turned out to have a non-Leloir trans-β-glucosylation activity, as they do not use activated nucleotide sugar as donor, but transfer a glycosyl group from a β-glucan donor to a β-glucan acceptor. More particularly, the activity of the three recombinant enzymes on linear (β1 → 3)-linked gluco-oligosaccharides (Lam-Glc4–9) and their corresponding alditols (Lam-Glc4–9-ol) was studied. Detailed structural analysis, based on thin-layer chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry, and 1D/2D 1H and 13C nuclear magnetic resonance data, revealed diverse product spectra. Depending on the enzyme used, besides (β1 → 3)-elongation activity, (β1 → 4)- or (β1 → 6)-elongation, or (β1 → 6)-branching activities were also detected.
Glycosyltransferases catalyze the transfer of a glycosyl group from a suitable carbohydrate donor to a suitable (carbohydrate) acceptor. They can be classified as Leloir or non-Leloir enzymes, depending on the type of donor used. The Leloir-type enzymes utilize nucleotide monosaccharide donors, and the non-Leloir glycosyltransferases non-nucleotide sugar donors or oligo/polysaccharides. In the latter case, they simply trans-glycosylate between oligo/polysaccharides, being mechanistically similar to retaining glycosidases (Toone et al. 1989; Wong and Whitesides 1994). The best known non-Leloir glycosyltransferases belong to the families GH13 or GH57, the amylase families, i.e. branching or elongation enzymes, that act on the (α1 → 4)- and (α1 → 6)-linked glucose units of starch or amylose.
The essential role of Leloir glycosyltransferases has been established for the synthesis of all bacterial β-d-glucans so far examined. However, in the bacterium Bradyrhizobium japonicum, the gene ndvB was shown with mutational analysis to be necessary for the formation of β-d-glucan oligosaccharides consisting predominantly of (β1 → 3)-linked glucose backbones with some (β1 → 6)-linked branches (Bhagwat and Keister 1995). Moreover, the role of ndvB in the biosynthesis of cyclic (1 → 3)-β-glucans in Pseudomonas aeruginosa was recently demonstrated (Sadovskaya et al. 2010). A preliminary bioinformatic study of this work revealed that the ndvB gene in B. japonicum appears to code for two protein domains. Besides a region coding for a Leloir glycosyltransferase of family GT2, another region codes for a domain belonging to the glycosyl hydrolase family GH17.
The members of the glycosyl hydrolase family GH17 are mostly involved in the hydrolysis of (β1 → 3) linkages in glucans as present in laminarin and lichenan, and act by a retaining mechanism. Non-Leloir β-1,3(β-1,6)-glucosyltransferase activity has been described for some of these enzymes originating from fungi (Aspergillus fumigatus; Mouyna et al. 1998; Gastebois et al. 2010) and yeasts (Candida albicans and Saccharomyces cerevisiae; Hartland et al. 1991; Goldman et al. 1995). The catalytic activity of these enzymes can be described as a two-step retaining mechanism, the first step involving the cleavage of a (β1 → 3) linkage of laminarin or related polysaccharides, and the second step the formation of a (β1 → 6) linkage to another laminarin or related polysaccharide chain.
Genes coding for a GT2 Leloir glucosyltransferase domain and a GH17 domain, homologous to the one from B. japonicum, have been identified in other bacterial genomes. However, none of them have been characterized on an enzymatic level. They have a narrow phylogenetic distribution and have been found only in the phylum of proteobacteria so far. Their function has not been determined, but they are most likely involved in the synthesis of osmoregulated periplasmic glucans (OPGs) similar to the ndvB product in B. japonicum. OPGs, being common constituents in the envelope of proteobacteria, are necessary for establishing symbiotic relationships between specific plant hosts and bacteria, such as Agrobacterium tumefaciens and B. japonicum (Bohin 2000). In other bacteria, such as P. syringae and Erwinia chrysanthemi, they mediate similar plant host interactions but rather as harmful virility factors in disease development (Mukhopadhyay et al. 1988; Talaga et al. 1994; Cogez et al. 2001). Other studies have indicated roles in antibiotic resistance and in the formation of biofilms (Mah et al. 2003; Lequette et al. 2007; Sadovskaya et al. 2010). The glucans vary in type of linkages and degree of branching, the absence or presence of various substituents and whether they are cyclic or linear. Four families of OPGs have been described on the basis of structural features of the poly-glucose backbone. Three of those, I, II and IV, have a backbone consisting predominantly of (β1 → 2)-linked linear or cyclic glucose units. Branches may be attached to the backbone by (β1 → 6) linkages, and cycles may be closed with (β1 → 6) linkages. Oligosaccharides of family III have a poly-glucose backbone predominantly composed of (β1 → 3)-linked glucose units. They can be cyclic or linear with (β1 → 6)-linked branches and can also contain (β1 → 4) linkages (Talaga et al. 1994, 2002; Bohin and Lacroix 2006). Phylogenetic distribution of a particular OPG class appears to be lineage-specific.
In this paper, we report the cloning and expression of genes encoding β-glucosyltransferase domains of family GH17 from three species of proteobacteria: glt1 from P. aeruginosa PAO1, glt3 from P. putida KT2440 and glt7 from Azotobacter vinelandii ATCC BAA-1303. The Pseudomonas and the Azotobacter gene sequences are homologous to the ndvB gene from B. japonicum, and they are a part of a larger open reading frame (ORF) that also encodes a family GH2 domain. Furthermore, we characterized transfer products obtained by incubating the recombinant enzymes in the presence of linear (β1 → 3)-linked gluco-oligosaccharides (Lam-Glc4–9) and their corresponding alditols (Lam-Glc4–9-ol). Detailed results, based on thin-layer chromatography (TLC), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization mass spectrometry (ESI-MS), and 1D/2D 1H and 13C nuclear magnetic resonance (NMR) data, are presented for the oligosaccharides(-alditols), including the enzyme-specific variety of the formed reaction products. Our study shows that the encoded enzymes of these GH17 domains have a non-Leloir trans-β-glucosylation activity, and this is the first characterization of enzymes having such activity from bacteria.
Results
Identification of ndvB homologues in bacterial genomes and analysis of primary structure
Genes homologous to the ndvB gene from B. japonicum were identified in genomes of numerous proteobacterial species by BlastP (http://blast.ncbi.nlm.nih.gov/Blast.cgi) but not to any species belonging to other phyla. The deduced gene products consisted of an N-terminal domain belonging to the glycosyl hydrolase family GH17 and a C-terminal domain belonging to the Leloir glycosyltransferase family GT2. Due to the fact that the B. japonicum ndvB product is known to be involved in the synthesis of OPGs (Bhagwat and Keister 1995) and that glucosyltransferase activity has been described for homologous GH17 enzymes from yeast and fungi (Hartland et al. 1991; Goldman et al. 1995; Mouyna et al. 1998; Gastebois et al. 2010), the observed bacterial GH17 domain-encoding sequences were proposed to be potential structural genes for a non-Leloir β-glucan synthesizing glucosyltransferase. GH17 domains from three proteobacteria species, namely P. aeruginosa, P. putida and A. vinelandii, homologous to the B. japonicum NdvB, were selected for further study. This included cloning of the corresponding gene regions, expression of the GH17 domain and analysis of activities and enzyme products.
Activity screening of the recombinant GH17 enzymes Glt7, Glt1 and Glt3 on curdlan-derived laminari-oligosaccharides
At first, the quality of a series of curdlan-derived laminari-oligosaccharides [(1 → 3)-β-d-gluco-oligosaccharides] with DP2 to DP10 (Lam-Glc2 to Lam-Glc10), to be used as substrates, was evaluated by MALDI-TOF-MS and NMR spectroscopy. The 1H NMR spectra and the 1H/13C chemical shifts of Lam-Glc2 to Lam-Glc10 at 292 K are included in the Supplementary data, Figure A and Table A, respectively.
TLC screening of the incubation of Lam-Glc6, Lam-Glc7, Lam-Glc8 and Lam-Glc9 with Glt7 for 8 h showed Pro-Glc3, Pro-Glc4, Pro-Glc5 and Pro-Glc6 as the most intense hydrolysis products, respectively. In all cases, trans-glucosylation was also observed, yielding mainly oligosaccharides in the Glc9, Glc10, Glc11 and Glc12 reference regions. These observations suggest that in the trans-glucosylation reactions, a triaose is mainly transferred. To obtain more information about the types of products formed, the oligosaccharide mixture generated from Lam-Glc6 after 2 h at 22°C was studied by MALDI-TOF-MS and 1H NMR spectroscopy. MS analysis ([M+Na]+) of the >Lam-Glc6 products in the pool indicated the presence of mainly Pro-Glc9, medium amounts of Pro-Glc8 and Pro-Glc10, and minor amounts of Pro-Glc11 and Pro-Glc12 (ratio, based on peak intensities, is 56:17:17:5:5). Although the 1H NMR spectrum of the major >Lam-Glc6 fraction, isolated by Bio-Gel P-2 size-exclusion chromatography, was very similar to that of standard Lam-Glc9 having only (β1 → 3) linkages, additional small peaks were seen at 4.22 and 4.55 ppm. When compared with the 1H NMR spectrum of gentiobiose, the signal at δ 4.22 could reflect a (β1 → 6) linkage (gentiobiose: H-6a of reducing Glc unit at δ 4.155 for α-anomer and at δ 4.209 for β-anomer; Supplementary data, Figure B) in an amount of 10%.
TLC screening of the incubation of Lam-Glc6, Lam-Glc7, Lam-Glc8 and Lam-Glc9, respectively, with Glt1 for 8 h showed Pro-Glc2, Pro-Glc3, Pro-Glc4 and Pro-Glc5 to be the most intense hydrolysis products, respectively. In all cases, trans-glucosylation was also observed, yielding mainly oligosaccharides in the Glc10, Glc11, Glc12 and Glc13 reference regions. These observations suggest that in the trans-glucosylation reaction, a tetraose is mainly transferred. In a similar way, as described for Glt7, the oligosaccharide mixture generated from Lam-Glc6 after 8 h at 22°C was subjected to MALDI-TOF-MS and 1H NMR analysis. MS analysis ([M+Na]+) of the >Lam-Glc6 products in the pool showed the occurrence of mainly Pro-Glc10, medium amounts of Pro-Glc9 and Pro-Glc11 and minor amounts of Pro-Glc8 and Pro-Glc12 (ratio, based on peak intensities, is 50:25:15:5:5). The 1H NMR spectrum of the major >Lam-Glc6 fraction, isolated by Bio-Gel P-2 size-exclusion chromatography, showed a peak pattern very similar to that of standard Lam-Glc10 having only (β1 → 3) linkages; no additional small peaks at 4.22 and 4.55 ppm were detected.
TLC screening of the incubation of Lam-Glc4, Lam-Glc5, Lam-Glc6, Lam-Glc7, Lam-Glc8 and Lam-Glc9, respectively, with Glt3 for 16 h showed a minimal release of hydrolysis products. Only for Lam-Glc5, Lam-Glc7, Lam-Glc8 and Lam-Glc9 trans-glucosylation products of higher mass could be observed, in particular, for Lam-Glc9. In a similar way, as described for Glt7, the oligosaccharide mixture generated from Lam-Glc9 after 16 h at 22°C was subjected to MALDI-TOF-MS and 1H NMR analysis. MS analysis ([M+Na]+) of the >Lam-Glc9 products in the pool indicated the presence of two major compounds Pro-Glc12 and Pro-Glc13, two medium compounds Pro-Glc11 and Pro-Glc14 and a minor compound Pro-Glc15 (ratio, based on peak intensities, is 34:28:17:16:5). The 1H NMR spectrum of the major >Lam-Glc9 fraction, isolated by Bio-Gel P-2 size-exclusion chromatography, showed a peak pattern similar to that of standard Lam-Glc10 having only (β1 → 3) linkages; no additional small peaks at 4.22 and 4.55 ppm were detected.
In view of the complexity of the 1D/2D NMR spectra and the complicated preliminary ESI-MS analysis data of the product mixtures using free laminari-oligosaccharides as substrate, next incubation studies with Glt7, Glt1 and Glt3 were carried out with their corresponding alditols, and a relevant selection of data obtained is presented below.
Activity of the Glt7 enzyme on laminaripentaitol Lam-Glc5-ol as analyzed by MALDI-TOF-MS and NMR spectroscopy
Oligosaccharide-alditol Lam-Glc5-ol was incubated with Glt7 for 24 h at 22°C (incubation for 2 h, like in the case of Lam-Glc5, did not generate enough material for detailed NMR analysis). TLC analysis of the generated oligosaccharide(-alditol) mixture gave evidence for the transfer of a triaose fragment to Lam-Glc5-ol (major band in the Glc8 region). Fractionation on Bio-Gel P-2 made it possible to isolate a series of oligosaccharide-alditol fractions of higher mass than Lam-Glc5-ol, denoted I–VIII (TLC screening), that were studied with MALDI-TOF-MS and 1D/2D NMR spectroscopy. The 2D NMR experiments comprised total correlation spectroscopy (TOCSY), rotating-frame nuclear Overhauser enhancement spectroscopy (ROESY) and 13C–1H heteronuclear single quantum coherence (HSQC) measurements and indicated that only (1 → 3) and (1 → 6) linkages were present. The various 1H and 13C assignments are compiled in Table I (for a survey of the 1D 1H NMR spectra and 1H/13C chemical shift data of the reference compounds Lam-Glc2-ol to Lam-Glc10-ol, see Supplementary data, Figure C and Table B). The rationalization behind the various assignments will be worked out in detail for fraction IV.
Table I.
1H and 13C NMR chemical shiftsa of Glc and Glc-ol residues in oligosaccharide-alditol products Pro-Glcx-ol, after incubation of Lam-Glc5-ol or Lam-Glc6-ol with the Glt7 enzyme
aThe NMR spectra have been recorded in D2O at 335 K. Chemical shifts are in ppm relative to the signal of internal acetone (δ = 2.225 for 1H and δ = 31.07 for 13C). n.d. = not determined.
bThe J1,2 coupling constants of the β-Glc residues are 8.0 Hz.
The MALDI-TOF mass spectrum of fraction I showed nearly equal peak intensities for the [M+Na]+ ions of Lam-Glc5-ol/Pro-Glc5-ol (m/z 853.6) and Pro-Glc6-ol (m/z 1015.6) (Figure 1(I)). In the 1D 1H NMR spectrum of fraction I (Figure 2(I)), three major anomeric signals are detected at positions that correspond with the occurrence of linear (β1 → 3)-linked oligosaccharide-alditols. Terminal β-d-Glcp-(1 → 3)- units are reflected by Gt H-1 at δ 4.739 (3J1,2 = 8.0 Hz), internal -(1 → 3)-β-d-Glcp-(1 → 3)- units by Gi H-1 at δ 4.785 (3J1,2 = 8.0 Hz) and -(1 → 3)-β-d-Glcp-(1 → 3)- units connected to Glc-ol O-3 by G2 H-1 at δ 4.675 (3J1,2 = 8.0 Hz). Glc-ol itself gives a G-ol H-3 signal outside the bulk signal at δ 4.06 (Supplementary data, Table B). Additionally, two minor anomeric signals of equal intensity are detected at δ 4.517 (Ga H-1) and δ 4.713 (Gb H-1), and complete assignments of these two residues followed from TOCSY experiments. Comparison of the 1H chemical shift values of the Glc residues Ga and Gb (Table I) with those given by the CASPER database showed Ga to be a terminal β-d-Glcp-(1 → 6)- unit and residue Gb an internal Glc unit in the sequence -(1 → 6)-β-d-Glcp-(1 → 3)- (Gb H-6a, δ 4.20). Based on these findings, it is concluded that Pro-Glc6-ol in fraction I consists of two structural isomers, one elongated at the “non-reducing” side of Lam-Glc5-ol with β-d-Glcp-(1 → 3)- (Glcβ3-Lam-Glc5-ol, major) and one elongated at the “non-reducing” side of Lam-Glc5-ol with β-d-Glcp-(1 → 6)- (Glcβ6-Lam-Glc5-ol, very minor) (intensity ratio of Gt H-1:Ga H-1:Gb H-1 is 10:1:1):
MALDI-TOF mass spectra of the Bio-Gel P-2 fractions I –VIII, isolated from an incubation mixture of Lam-Glc5-ol and the Glt7 enzyme. Top, calibration with Glc5-ol up to Glc12-ol. [M+Na]+ and [M+K]+ ions are seen in different ratios.
500-MHz 1D 1H NMR spectra of the Bio-Gel P-2 fractions I–VIII, isolated from an incubation mixture of Lam-Glc5-ol and the Glt7 enzyme, recorded in D2O at 335 K.
The MALDI-TOF mass spectrum of fraction II (Figure 1(II)) showed four sodiated molecular ions [M+Na]+ at m/z 853.6, 1015.6, 1177.6 and 1339.7, corresponding to Lam-Glc5-ol/Pro-Glc5-ol, Pro-Glc6-ol, Pro-Glc7-ol and Pro-Glc8-ol, respectively, with Pro-Glc6-ol as the strongly dominating one. In the 1D 1H NMR spectrum of fraction II (Figure 2(II)), three major anomeric signals (Gi, Gt and G2) belonging to linear (β1 → 3)-linked oligosaccharide-alditols are observed, together with three minor anomeric signals at δ 4.517 (Ga H-1, 3J1,2 = 8.0 Hz), δ 4.713 (Gb H-1, 3J1,2 = 8.0 Hz) and δ 4.550 (Gc H-1, 3J1,2 = 8.0 Hz). The set of 1H values of Ga and Gb are indicative for a terminal β-d-Glcp-(1 → 6)- and an internal -(1 → 6)-β-d-Glcp-(1 → 3)- unit (Table I and CASPER database), respectively. The set of Gc1H chemical shifts corresponds to the values of an internal -(1 → 3)-β-d-Glcp-(1 → 6)- unit (Table I and CASPER database). As there are no indications for branched structures and structures having a -(1 → 6)-β-d-Glcp-(1 → 6)- unit, the most likely linear (β1 → 3)-linked oligosaccharide-alditol structures are terminated by β-d-Glcp-(1 → 3)-β-d-Glcp-(1 → 3)-β-d-Glcp-(1 → 3)- (Gt-Gi-Gi), β-d-Glcp-(1 → 3)-β-d-Glcp-(1 → 6)-β-d-Glcp-(1 → 3)- (Gt-Gc-Gb) and β-d-Glcp-(1 → 6)-β-d-Glcp-(1 → 3)-β-d-Glcp-(1 → 3)- (Ga-Gb-Gi) elements.
The MALDI-TOF mass spectrum of fraction III (Figure 1(III)) showed three sodiated molecular ions [M+Na]+ at m/z 1015.6, 1177.6 and 1339.7, corresponding to Pro-Glc6-ol, Pro-Glc7-ol and Pro-Glc8-ol, respectively, with Pro-Glc8-ol yielding the major peak. In the 1D 1H NMR spectrum of fraction III (Figure 2(III)), three major anomeric signals (Gi, Gt and G2) belonging to linear (β1 → 3)-linked oligosaccharide-alditols are detected, and three minor anomeric signals at δ 4.713 (Gb H-1), δ 4.550 (Gc H-1) and δ 4.753 (Gd H-1), respectively. As deduced above, residue Gb stands for a -(1 → 6)-β-d-Glcp-(1 → 3)- unit and residue Gc for a -(1 → 3)-β-d-Glcp-(1 → 6)- unit. Comparing the set of Gd1H chemical shifts (Table I) with those present in the CASPER database identified residue Gd as a branched -(1 → 3,6)-β-d-Glcp-(1 → 3)- unit, which means that fraction III contains branched structures. It should be noted that in the 1H NMR spectrum of fraction III, the intensities of Gb H-1 and Gc H-1 have significantly increased compared with those for these signals in fraction II and that Ga H-1 [terminal β-d-Glcp-(1 → 6)- unit] has nearly disappeared. The nature of the structural isomers in Pro-Glc6-ol and Pro-Glc7-ol have already been discussed for fractions I and II. The possible structural isomers in Pro-Glc8-ol will be discussed below.
The MALDI-TOF mass spectra of fractions IV (Figure 1(IV)) and V (Figure 1(V)) showed one major sodiated molecular ion [M+Na]+ at m/z 1339.7, corresponding to Pro-Glc8-ol, and very minor peak intensities (<10%) for Pro-Glc7-ol (m/z 1177.6) and Pro-Glc9-ol (m/z 1501.7). The 1D 1H NMR spectra of both fractions are also very similar (Figure 2(IV) and (V)). In view of the MALDI-TOF mass peak patterns, it is tempting to assume that the 1H NMR spectra reflect in fact Pro-Glc8-ol, a mixture of linear and branched structural isomers.
As an illustration of the assignment of the various 1D/2D NMR spectra, of which the chemical shifts are collected in Table I, the analysis of fraction IV will be worked out in detail. The TOCSY (200 ms), ROESY (200 ms) and HSQC spectra of fraction IV are presented in Figure 3. Starting points for the interpretation of the TOCSY spectra were the anomeric signals of the various residues. Comparison of the TOCSY spectra with increasing mixing times (40–200 ms) allowed the assignments of the sequential order of the chemical shifts belonging to the same spin system. Connectivities from H-1 to H-2,3,4,5,6a,6b were traced for residues Gi, Gd, Gt, Gb, G2 and Gc but due to overlapping, some uncertainties could not be resolved on the basis of the TOCSY data alone. Additional assignments and confirmation of the assignments were obtained from ROESY cross-peaks and by correlating the 1H resonances to the corresponding 13C resonances in the HSQC spectrum.
HSQC, TOCSY (200 ms) and partial ROESY (200 ms) spectra of Bio-Gel P-2 fraction IV, isolated from an incubation mixture of Lam-Glc5-ol and the Glt7 enzyme (mainly Pro-Glc8-ol; see Figure 1(IV)), recorded in D2O at 335 K. Cross-peaks belonging to the same scalar coupling network are indicated near a dotted line starting from the corresponding diagonal peaks.
The TOCSY Gi H-1 track (δ 4.785) showed the complete scalar coupling network H-1,2,3,4,5,6a,6b, corresponding to the values found for internal -(1 → 3)-β-d-Glcp-(1 → 3)- units in the Lam-Glcx-ol reference series (Supplementary data, Table B). The substitution at position 3 of Gi is clearly supported by the downfield shift of the Gi C-3 signal (δC-3 85.5; βGlcp1Me, δC-3 76.8; ▵δ 8.7) (Bock and Thøgersen 1982; Bock et al. 1984).
The chemical shifts of the set of protons H-1,2,3,4,5,6a,6b of residue Gt (δ 4.739) are characteristic for a terminal β-d-Glcp-(1 → 3)- residue (Supplementary data, Tables A and B). The presence of terminal β-d-Glcp-(1 → 6)- residues (Ga) is excluded by the absence of Ga H-1 at δ 4.517 (compare with β-gentiobiose, Glc H-1 of non-reducing unit: δ 4.518; Supplementary data, Figure B).
The complete set of proton signals of the Gb residue, reflecting an internal -(1 → 6)-β-d-Glcp-(1 → 3)- unit, was established by combining cross-peak data from the TOCSY Gb H-1 (δ 4.713; Gb H-5,6a,6b) and Gb H-6a (δ 4.20; Gb H-2,3,4) tracks. The HSQC spectrum showed a downfield shift for Gb C-6 (δC-6 69.5; βGlcp1Me, δC-6 61.8; ▵δ 7.7), indicating the involvement of C-6 in a glycosidic linkage.
For residue Gc, the TOCSY Gc H-1 (δ 4.550) track revealed the complete spin system H-1,2,3,4,5,6a,6b, which was interpreted as belonging to an internal -(1 → 3)-β-d-Glcp-(1 → 6)- unit. The 13C data clearly reflect the 3-substitution of residue Gc (δC-3 85.6; βGlcp1Me, δC-3 76.8; ▵δ 8.8).
The complete scalar coupling network H-1,2,3,4,5,6a,6b of residue Gd followed from combining the cross-peak data from the TOCSY Gd H-1 (δ 4.753; Gd H-6a) and Gd H-6a (δ 4.21; Gd H-2,3,4,5,6b) tracks. Together with the 13C NMR data, which indicated downfield shifts for Gd C-6 (δC-6 69.5; βGlcp1Me, δC-6 61.8; ▵δ 7.7) and Gd C-3 (δC-3 85.4; βGlcp1Me, δC-3 76.8; ▵δ 8.6), residue Gd turned out to be a 3,6-di-substituted Glc residue in a -(1 → 3,6)-β-d-Glcp-(1 → 3)- sequence.
The TOCSY G2 H-1 track (δ 4.675) showed the complete spin system H-1,2,3,4,5,6a,6b. The downfield shift of G2 C-3 (δC-3 85.5; βGlcp1Me, δC-3 76.8; ▵δ 8.7) in the HSQC spectrum reflects the presence of a 3-substituted G2 residue. The 1H (Supplementary data, Figure B) and 13C NMR data of G2 are in full agreement with the -(1 → 3)-β-d-Glcp-(1 → 3)-Glc-ol sequence (δC-3 79.6; Glc-ol, δC-3 71.0; ▵δ 8.6). The 1H and 13C assignments of the -(1 → 3)-d-Glc-ol unit G-ol followed from the TOCSY and HSQC spectra in combination with the NMR data of the Lam-Glcx-ol reference compounds.
In view of the strong overlap of inter-residual cross-peaks around δ 3.80 (H-3 of G2, Gi, Gc and Gd), only a partial sequence analysis followed from the ROESY measurements. The ROESY Gc H-1 track showed inter-residual connectivities with Gd H-6a/6b (δ 4.21/3.89) and Gb H-6a/6b (δ 4.20/3.87), in agreement with Gc(1 → 6)Gd {-(1 → 3)-β-d-Glcp-(1 → 6)-[-(1 → 3)-]β-d-Glcp-(1 → 3)-} and Gc(1 → 6)Gb [-(1 → 3)-β-d-Glcp-(1 → 6)-β-d-Glcp-(1 → 3)-] sequences, respectively. ROESY cross-peaks between Gi H-1 and Gd H-3 {-(1 → 3)-β-d-Glcp-(1 → 3)-[-(1 → 6)-]β-d-Glcp-(1 → 3)-} and/or between Gt H-1 and Gd H-3 {β-d-Glcp-(1 → 3)-[-(1 → 6)-]β-d-Glcp-(1 → 3)-} provide information about the possible positions of the branching residue Gd. However, note that with respect to Gt the cross-peak at δ 3.80 will also reflect the Gt(1 → 3)Gi sequence [β-d-Glcp-(1 → 3)-β-d-Glcp-(1 → 3)-]. Furthermore, Gd can be linked to Gi as well as to G2 [-(1 → 3,6)-β-d-Glcp-(1 → 3)-β-d-Glcp-(1 → 3)-}, but is not linked to G-ol, confirming that G2 is not branched. On the Gi H-1 track, the connectivity with Gc H-3 demonstrated a Gi(1 → 3)Gc linkage [-(1 → 3)-β-d-Glcp-(1 → 3)-β-d-Glcp-(1 → 6)-]. The inter-residual ROESY cross-peak between G2 H-1 and G-ol H-3 (δ 4.06) supports the G2(1 → 3)G-ol linkage [-(1 → 3)-β-d-Glcp-(1 → 3)-d-Glc-ol].
Based on the NMR data (peak area ratios of the G2, Gc, Gd and Gb H-1 signals) and the hypothesis that the major Pro-Glc8-ol structural isomers are synthesized by a transfer of the triaose β-d-Glcp-(1 → 3)-β-d-Glcp-(1 → 3)-β-d-Glcp- to the substrate Lam-Glc5-ol, the following structural isomers are suggested to be present in fraction IV: linear (β1 → 3)-Pro-Glc8-ol:linear (β1 → 3)/(β1 → 6)-Pro-Glc8-ol:branched (β1 → 3,6)-Pro-Glc8-ol's = 30:25:45.
The MALDI-TOF mass spectra (Figure 1(VI–VIII)) and the 1H-NMR spectra (Figure 2(VI–VIII)) of fractions VI–VIII showed the oligosaccharide-alditols products Pro-Glc8-ol to Pro-Glc12-ol in different ratios. In view of the presence of the H-1 signals of the Glc residues Gb, Gc and Gd, elongation and branching are clearly indicated. As the Ga H-1 signal, reflecting terminal β-d-Glcp-(1 → 6)- units, has disappeared, predominantly elongations with (1 → 3) linkages do occur in these structures of higher degree of polymerization (DP). Interestingly, the 1D 1H NMR spectrum of fraction VIII (Pro-Glc9–12-ol) showed additional signals at δ > 4.0, namely Ge H-1 at δ 4.639 (3J1,2 = 8.0 Hz) and Gf-ol H-6a at δ 4.16 (Robijn et al. 1995). Residue Ge corresponds with a Glc residue in the sequence -(1 → 3)-β-d-Glcp-(1 → 6)-d-Glc-ol (Table I), whereas residue Gf-ol reflects a 6-substituted Glc-ol unit, indicating other forms of trans-glucosylation than seen in the lower-mass products.
Activity of the Glt7 enzyme on laminarihexaitol Lam-Glc6-ol as analyzed by MALDI-TOF-MS, NMR spectroscopy and ESI-MS
Oligosaccharide-alditol Lam-Glc6-ol was incubated with Glt7 for 24 h at 22°C (incubation for 2 h, like in the case of Lam-Glc6, did not generate enough material for detailed NMR analysis). As observed for the Lam-Glc5-ol incubation with Glt7, TLC analysis of the generated oligosaccharide(-alditol) mixture gave evidence for the transfer of a triaose fragment to Lam-Glc6-ol (major band in the Glc9 region; Pro-Glc9-ol). Fractionation on Bio-Gel P-2 made it possible to isolate a series of oligosaccharide-alditol fractions of higher mass than Lam-Glc6-ol (TLC screening) that were studied with MALDI-TOF-MS and 1D/2D NMR spectroscopy, indicating complex mixtures of Lam-Glc6-ol and Pro-Glc7–14-ol. Here, only the structural data of the fraction containing mainly Pro-Glc9-ol and Pro-Glc10-ol will be summarized.
The MALDI-TOF mass spectrum of this fraction (Figure 4A) revealed [M+Na]+ peaks at m/z 1339.7, 1501.7, 1663.7, 1825.8, 1987.8, 2149.8 and 2311.9, corresponding to Pro-Glc8-ol, Pro-Glc9-ol, Pro-Glc10-ol, Pro-Glc11-ol, Pro-Glc12-ol, Pro-Glc13-ol and Pro-Glc14-ol, respectively. Its 1D 1H NMR spectrum (Figure 4B), showing six doublets in the anomeric region between 4.8 and 4.5 ppm (residues Gi, Gd, Gt, Gb, G2 and Gc), is similar to that of fraction VII (Lam-Glc5-ol used as substrate) (Table I). The 1:4 intensity ratio of the Gb and Gc H-1 signals is in favour of more branching than chain elongation. Because of the absence of Ga residues [terminal β-d-Glcp-(1 → 6)-], all branches must be terminated with Gt [terminal β-d-Glcp-(1 → 3)-]. The ∼2:1 ratio of the Gt and G2 H-1 signals confirmed the presence of branching. Based on the NMR data (peak area ratios of the G2, Gc, Gd and Gb H-1 signals), the ratio of the linear (β1 → 3)-Pro-Glcx-ol:linear (β1 → 3)/(β1 → 6)-Pro-Glcx-ol:branched (β1 → 3,6)-Pro-Glcx-ol's is 0:25:75. Note that no indications have been found for the occurrence of a linear (β1 → 3)-linked structure. Worked out for Pro-Glc9-ol, the following structures should be taken into account:
MALDI-TOF mass spectrum ([M+Na]+) (A) and 500-MHz 1D 1H NMR spectrum recorded in D2O at 335 K (B) of the Bio-Gel P-2 fraction that contains mainly Pro-Glc9-ol, isolated from an incubation mixture of Lam-Glc6-ol and the Glt7 enzyme.
To obtain further support for these structural isomers of Pro-Glc9-ol, the use of ESI-MS was explored. At first, library ESI-MS fragmentation data were collected for a series of laminari-oligosaccharide-alditols [(1 → 3)-β-d-gluco-oligosaccharide-alditols]. To improve the abundance of cross-ring cleavages, before analysis the compounds were permethylated. As an illustration, detailed ESI-MS data will be presented for permethylated Lam-Glc9-ol.
The positive-ion mode MS1 spectrum of Lam-Glc9-ol, showing the sodium adduct [M+Na]+ ion of the nonasaccharide-alditol at m/z 1921.1, is presented in Figure 5A. The MS2 spectrum of m/z 1921.1 is depicted in Figure 5B, whereas Scheme 1 presents the fragment ions produced by cleavage at the anomeric side of the glycosidic linkages (Y refers to alditol-terminal fragment ions and B to “non-reducing side” terminal fragment ions], as well as by cross-ring cleavages (X refers to alditol-terminal fragment ions and A to “non-reducing side” terminal fragment ions) (Domon and Costello 1988; Weiskopf et al. 1997). The predominant fragment ions show 204-mass differences that are attributed to the internal methylated glucosyl units. In the absence of (1 → 4) linkages, the presence of the 2,4A series of fragments ions is in agreement with the (1 → 3) linkages present. The MS3 spectrum of the Y8 fragment ion m/z 1703.7 (Figure 5C and Scheme 2) is essentially similar to that of the MS2 spectrum of the pseudomolecular ion (Figure 5B). The Y fragment ion series is found at the same m/z values, whereas the B fragment ion series is shifted downwards by 14 mass units, due to the OH group at C-3 in the Y8 fragment ion. Higher-order MSn spectra did not add additional information (data not shown).
MS1 (A), MS2/m/z 1921 ([M+Na]+)→ (B), and MS3/m/z 1921 → m/z 1703→ (C) ESI mass spectra of permethylated Lam-Glc9-ol. For details, see Schemes 1 and 2.
Ion fragments (Y, A, B) of permethylated Lam-Glc9-ol, as seen in the MS3 spectrum of its Yt fragment ion (m/z 1921→ m/z 1703→). R = Me.
The Pro-Glc9-ol-containing fraction was permethylated and investigated by ESI-MS, demonstrating a visible range from Pro-Glc7-ol to Pro-Glc13-ol in the MS1 spectrum with Pro-Glc9-ol as the major product (Figure 6A). The sodium adduct ion [M+Na]+ at m/z 1921.3 corresponding to Pro-Glc9-ol was selected for MS2. Comparison of the MS2 spectra of permethylated Lam-Glc9-ol (Figure 5B) and permethylated Pro-Glc9-ol (Figure 6B) revealed significant differences. For instance, comparing the Y3–Y8 peak patterns in both spectra showed a more regular pattern for Lam-Glc9-ol, whereas in Pro-Glc9-ol the Y7 and Y8 fragments dominate. Moreover, in the MS2 spectrum of Pro-Glc9-ol, some additional low-intensity peaks (cross-ring cleavages) are seen that could be related with (β1 → 6) linkages (Scheme 3). Especially, the peak at m/z 737.4, interpreted as a 3,5A4 fragment, is of interest, as it is in agreement with the attachment of a Glc-Glc-Glc segment at O-6 of a next Glc unit. However, due to the complexity of the structural isomer mixture, the exact position(s) of the attachment site(s) could not be made. The MS3 spectrum of the Y8 fragment at m/z 1703.6 [Y(α/β) or Y(γ)] (Figure 6C; loss of one terminal Glc unit from permethylated Pro-Glc9-ol with the generation of an OH group) in the MS2 spectrum produced a predominant Y-type peak at m/z 1484.7, reflecting the loss of a second terminal Glc residue [loss of Y(α) or Y(β)] from permethylated Pro-Glc9-ol (Scheme 3). The subsequent loss of the two terminal Glc units upon MS3 confirmed the presence of branched structures in Pro-Glc9-ol (Scheme 3A–C). Additionally, the formed Y-type peak at m/z 1499.9 in the MS3 spectrum, produced by the loss of an internal Glc unit when starting from Y(γ), indicates the occurrence of a linear structure (Scheme 3D).
MS1 ESI mass spectrum of permethylated Pro-Glc7-13-ol (A) and MS2/m/z 1921 ([M+Na]+ of permethylated Pro-Glc9-ol)→ (B) and MS3/m/z 1921 → m/z 1703→ (C) ESI mass spectra related to Pro-Glc9-ol. For details, see Scheme 3.
Possible isomeric structures of permethylated Pro-Glc9-ol containing a (β1 → 6) linkage. In structures A, B and C, two shaded terminal Glc residues are released upon MS3 [Y(α) and Y(β) fragmentation], whereas in structure D one shaded terminal Glc residue Y(γ) followed by an internal Glc residue is released upon MS3. R = Me.
To determine the fragmentation behavior of trapped ions from the B-ion series, MS3 was performed on the tetraose B4 fragment ion at m/z 853.5 from the MS2 spectrum (data not shown). The MS3 spectrum yielded a significant increase in intensities of earlier weak ion signals, particularly the cross-ring 3,5A4 fragment with m/z 737.4, demonstrating a Glc residue 6-substituted with Glc-Glc-Glc in a linear sequence (Scheme 3D).
Taking the TLC, MALDI-TOF-MS, NMR and ESI-MS data together, the proposed structures for Pro-Glc9-ol are: a linear structure with an internal (β1 → 6) linkage, as presented in Scheme 3D, and one, two or all three 3,6-branched structural isomers with a triaosyl side chain at the 6-branch as depicted in Scheme 3A–C.
Activity of the Glt1 enzyme on laminaripentaitol Lam-Glc5-ol and laminarihexaitol Lam-Glc6-ol as analyzed by MALDI-TOF-MS and NMR spectroscopy
The oligosaccharide-alditols Lam-Glc5-ol and Lam-Glc6-ol were incubated with Glt1 for 24 h at 22°C (incubation for 8 h, like in the case of Lam-Glc5 and Lam-Glc6, did not generate enough material for detailed NMR analysis). TLC analysis of the generated oligosaccharide(-alditol) mixture from Lam-Glc5-ol gave evidence for the release of mono- up to tetrasaccharides, whereas the generated oligosaccharide(-alditol) mixture from Lam-Glc6-ol showed the release of mono- up to pentasaccharides. In contrast to the earlier findings mentioned above for Lam-Glc6–9 as substrates (mainly transfer of tetraose units), in the case of the reduced substrates a transfer of single Glc units is mainly observed. The reaction mixture generated from Lam-Glc6-ol was subjected to a more detailed analysis. Via fractionation on Bio-Gel P-2, two fractions, denoted I and II, with compounds of higher mass than the substrate (TLC analysis) were obtained, and analyzed by MALDI-TOF-MS and NMR spectroscopy.
The MALDI-TOF mass spectrum of fraction I (Figure 7A) showed four sodiated molecular ions [M+Na]+ at m/z 853.3, 1015.3, 1177.4 and 1339.4, corresponding to Pro-Glc5-ol (very minor), Lam-Glc6-ol/Pro-Glc6-ol, Pro-Glc7-ol and Pro-Glc8-ol (very minor). In the 1D 1H NMR spectrum of fraction I (Figure 8), three major β-anomeric signals (3J1,2 = 8.0 Hz) were observed at positions that agree with the presence of linear (β1 → 3)-linked oligosaccharide-alditols: Gt H-1 at δ 4.739, terminal β-d-Glcp-(1 → 3)- units; Gi H-1 at δ 4.785, internal -(1 → 3)-β-d-Glcp-(1 → 3)- units; and G2 H-1 at δ 4.675, -(1 → 3)-β-d-Glcp-(1 → 3)-d-Glc-ol (Table II). Glc-ol gives a G-ol H-3 signal outside the bulk signal at δ 4.06. In addition, two minor β-anomeric signals are present at δ 4.515 and 4.77, encoded Gk and Gl, respectively. The various 1H assignments, compiled in Table II, followed from the corresponding scalar coupling networks H-1,2,3,4,5,6a,6b, unraveled by 2D TOCSY experiments (Figure 8). Taking into account that 6-substituted Glc residues do not occur (absence of δ 4.21, H-6a's), comparison of the H-1–H-6b chemical shifts of the Glc residue Gk with those given by the CASPER database indicated a terminal Glc unit β-d-Glcp-(1 → 4)-. Following the same strategy, the Glc residue Gl matched with an internal -(1 → 4)-β-d-Glcp-(1 → 3)- unit. Taking into account that the intensity ratios of the H-1 signals of Gk and Gl and of Gt + Gk and G2 are 1:1, the presence of branched structures is not supported. As the ratio of the [M+Na]+ peak intensities of major Lam-Glc6-ol/Pro-Glc6-ol and Pro-Glc7-ol is 3:2 (Figure 7A, MALDI-TOF mass spectrum) and the ratio of the intensities of the H-1 signals of Gt and Gk is 5:1 (Figure 8, 1D 1H NMR spectrum), in Pro-Glc7-ol the elongation at the terminal Glc unit of Lam-Glc6-ol will consist for the most part of a β-d-Glcp-(1 → 3)- unit and for a minor part of a β-d-Glcp-(1 → 4)- unit, as follows:
MALDI-TOF mass spectra of Bio-Gel P-2 fraction I (A) and II (B), isolated from an incubation mixture of Lam-Glc6-ol and the Glt1 enzyme. [M+Na]+ and [M+K]+ ions are seen in different ratios.
500-MHz 1D 1H NMR and TOCSY spectrum (200 ms), recorded in D2O at 335 K, of fraction I isolated from an incubation mixture of Lam-Glc6-ol and the Glt1 enzyme. Cross-peaks belonging to the same scalar coupling network are indicated near a dotted line starting from the corresponding diagonal peaks.
Table II.
1H NMR chemical shiftsa of Glc and Glc-ol residues in oligosaccharide-alditol products Pro-Glcx-ol, after incubation of Lam-Glc6-ol with the Glt1 enzyme
aThe NMR spectra have been recorded in D2O at 335 K. Chemical shifts are in ppm relative to the signal of internal acetone (δ = 2.225 for 1H). n.d. = not determined.
bThe J1,2 coupling constants of the β-Glc residues are 8.0 Hz.
The MALDI-TOF mass spectrum of fraction II (Figure 7B) showed six sodiated molecular ions [M+Na]+ at m/z 1015.6, 1177.6, 1339.7, 1501.8, 1663.4 and 1825.6, corresponding to Lam-Glc6-ol/Pro-Glc6-ol, Pro-Glc7-ol, Pro-Glc8-ol, Pro-Glc9-ol, Pro-Glc10-ol and Pro-Glc11-ol (very minor). The anomeric region of the 1D 1H NMR spectrum of fraction II (Figure 9) is dominated by the β-anomeric signals (3J1,2 = 8.0 Hz) of Gt at δ 4.739 [terminal β-d-Glcp-(1 → 3)- units], Gi at δ 4.785 [internal -(1 → 3)-β-d-Glcp-(1 → 3)- units], and G2 at δ 4.675 [-(1 → 3)-β-d-Glcp-(1 → 3)-d-Glc-ol] units (Table II). Glc-ol gives a G-ol H-3 signal outside the bulk signal at δ 4.06. In addition, four minor β-anomeric signals (3J1,2 = 8.0 Hz) of Glc residues are present at δ 4.515 (Gk), 4.77 (Gl), 4.552 (Gm) and 4.539 (Gn), respectively, and their corresponding spin systems were unraveled by 2D TOCSY experiments (Figure 9; Table II), and assigned to specific residues by using the CASPER database. As indicated already for fraction I, the chemical shift sets of Gk and Gl support the occurrence of terminal β-d-Glcp-(1 → 4)- and internal -(1 → 4)-β-d-Glcp-(1 → 3)- units. Following the same strategy, and taking into account that 6-substituted Glc residues did not occur (absence of δ 4.21; H-6a's), the Glc residues Gm and Gn matched with internal -(1 → 3)-β-d-Glcp-(1 → 4)- and -(1 → 4)-β-d-Glcp-(1 → 4)- units. The absence of cross-peaks at δ 4.04/4.05 on assumed H-1 tracks at ∼4.71 and ∼4.52 ppm indicated the absence of 3,4-di-substituted Glc residues. Worked out for Pro-Glc8-ol, and starting from Lam-Glc6-ol, the following proposals can be formulated:
500-MHz 1D 1H NMR and TOCSY spectrum (200 ms), recorded in D2O at 335 K, of fraction II isolated from an incubation mixture of Lam-Glc6-ol and the Glt1 enzyme. Cross-peaks belonging to the same scalar coupling network are indicated near a dotted line starting from the corresponding diagonal peaks.
Activity of the Glt3 enzyme on laminarihexaitol Lam-Glc6-ol as analyzed by MALDI-TOF-MS and NMR spectroscopy
The incubation of Lam-Glc6 with the Glt3 enzyme did not give rise to trans-glucosylation products. However, when the incubation was carried out with Lam-Glc6-ol (48 h, 22°C) (incubation for 16 h, like in the case of Lam-Glc6, did not generate enough material for detailed NMR analysis), TLC analysis showed the formation of small amounts of products with a higher mass (<5%); the substrate releases mono- to pentasaccharides in low amounts. The reaction mixture was fractionated on Bio-Gel P-2, and two fractions denoted I′ and II′ containing compounds of higher mass than the substrate (TLC analysis) were obtained and analyzed by MALDI-TOF-MS and NMR spectroscopy.
The MALDI-TOF mass spectrum of fraction I′ showed two [M + Na]+ peaks at m/z 1015.3 and 1177.4, corresponding to Lam-Glc6-ol/Pro-Glc6-ol and Pro-Glc7-ol, respectively, in a peak intensity ratio of 3:1. The MALDI-TOF mass spectrum of fraction II′ revealed [M+Na]+ peaks at m/z 1177.4, 1339.7, 1501.8 and 1663.9, corresponding to Pro-Glc7-ol, Pro-Glc8-ol, Pro-Glc9-ol and Pro-Glc10-ol, respectively, in the peak intensity ratio of 2:4:2:1 (spectra not shown). The 1D 1H NMR spectra of both fractions (spectra not shown) demonstrated only the presence of H-1 signals for Gt (δ 4.739), Gi (δ 4.785) and G2 (δ 4.675), being similar to the reference Lam-Glcx-ol series. Compared with the 1H NMR spectrum of the substrate Lam-Glc6-ol, in the anomeric region only an increase for the Gi H-1 signal is seen, indicating only stepwise elongations with β-d-Glcp-(1 → 3) units at the terminal unit of the substrate (the peak intensity ratio Gt H-1:G2 H-1 remains 1:1).
Discussion
Non-Leloir GH17 glucosyltransferases active on β-glucans have been characterized in fungi and yeast only so far. Analysis of sequencing data of bacterial genomes has, however, indicated the existence of homologous trans-glucosidases in bacteria, i.e. belonging to the phylum of proteobacteria. Two such enzymes from B. japonicum and a third one from P. aeroginosa were shown, with mutational analysis, to participate in the formation of cyclic β-glucans (Bhagwat and Keister 1995; Sadovskaya et al. 2010). Many of the bacterial proteins consist of two domains, an N-terminal domain belonging to glycosyl hydrolase family GH17 and a C-terminal domain belonging to the Leloir glycosyltransferase family GT2. The hydrophobicity profile of deduced amino acid sequences of the B. japonicum ndvB encoding enzyme revealed three transmembrane helices between the domains and five transmembrane helices at the far C-terminal end. A putative hydrophobic signal peptide was also identified at the N-terminal side (data not shown). Based on these structural features and the fact that GT2 glycosyltransferases use activated sugar donors, formed in the reduced environment of the cytoplasm, we postulate that the GT2 domain resides on the cytoplasmic side of the bacterial cell membrane and the GH17 domain on the periplasmic side. The transmembrane helices, apart from the N-terminal signal peptide, are predicted to form a membrane pore through which the newly synthesized glucan chain, product of GT2, is transported. The periplasmic GH17 enzyme domain may cleave and further modify the nascent β-glucan synthesized by the GT2 domain, leading to the formation of branched and cyclic OPGs. The predicted schematic structure is shown in Figure 10. A gene with a similar structure and proposed function has been reported for a two-domain protein involved in the synthesis of (1 → 3)-α-d-glucan in Schizosaccharomyces pombe (Hochstenbach et al. 1998).
Predicted schematic structure of a two-domain protein consisting of the GT2 domain residing on the cytoplasmic side of the bacterial cell inner membrane and the GH17 domain on the periplasmic side. The eight transmembrane helices are predicted to form a membrane pore through which the newly synthesized glucan chain is transported. According to the hypothetical model, the cytoplasmic GT2 domain produces the linear β-glucan, whereas the periplasmic GH17 domain modifies the nascent β-glucan, following transport through the inner membrane.
To our knowledge, none of the bacterial GH17 domains has been characterized on the enzymatic level. The reason may be that the enzymes are difficult to isolate in active form, and production of recombinant derivatives is demanding. We succeeded in producing three bacterial GH17 β-glucosyltransferases (trans-β-glucosidases), Glt1, Glt3 and Glt7, in high amounts by cloning the encoding genes in frame with the MalE fusion protein sequence of vector pJOE3075 and expressing them in Escherichia coli; the vector contains a rhamnose promoter, and expression is positively regulated (Wegerer et al. 2008). The MalE fusion is known to enhance expression, solubility and stability of recombinant proteins (Makrides 1996).
The product formation pattern of all three enzymes, Glt1, Glt3 and Glt7, indicated that they cleave short (β1 → 3)-linked gluco-oligosaccharide substrates from the non-reducing end. Based on the results obtained with free oligosaccharides (Lam-Glcx) and their corresponding alditols (Lam-Glcx-ol), there seems to be a main cleaving site relative to that end. The enzymes release the “reducing end part”, but retain the non-reducing end part of the donor and transfer it to an acceptor molecule, another oligosaccharide. This is in accordance with a general model of reaction mechanism of glycosyltransferases. Overall, the product formation pathways are highly complex, whereby the size of the substrates and modifications at the reducing end also play a significant role. In this context, it will be shown that the situation when using 2-aminobenzamide (2AB)-labeled oligosaccharides (Lam-Glcx-2AB) as substrates is completely different.
Based on the various results obtained, it is clear that the Glt7 enzyme has both hydrolysis and transfer activities, when incubating with Lam-Glcx or Lam-Glcx-ol, starting from Lam-Glc5 and Lam-Glc5-ol on. The major cleavage site is at the third (β1 → 3) linkage from the non-reducing end, releasing the triaose β-d-Glcp-(1 → 3)-β-d-Glcp-(1 → 3)-d-Glcp [major hydrolysis product Pro-Glcx−3(-ol)] and transferring it to acceptor Lam-Glcx(-ol) [major trans-glucosylation product Pro-Glcx+3(-ol)]. The different mechanisms, whereby cleavage sites other than the third (β1 → 3) linkage from the non-reducing end has also to be taken into account, led to large ensembles of major and minor transfer products, both in terms of structural isomers and in terms of different molecular masses (e.g. incubation with Lam-Glc6-ol can give products up to Pro-Glc15-ol). The overall yield of the transfer products was approximately 10% on free laminari-oligosaccharides but much less on alditols with a maximum incubation time of 24 h. Prolonged incubation times did not produce more transfer products, but only more hydrolysis products. Compared with the Glt3 and Glt1 enzymes, the Glt7 enzyme seemed to have the most efficient trans-glucosylation activity. With respect to the regiospecificity of the transfer reactions, elongation at Gt [terminal β-d-Glcp-(1 → 3)-] of the substrate can take place via (β1 → 3) (major reaction) and (β1 → 6) linkages. There is no evidence for the presence of (β1 → 4) or (β1 → 2) linkages. The (β1 → 6) transfer activity of Glt7 led also to branching, although not at Glc residue G2, whereby a triaose β-d-Glcp-(1 → 3)-β-d-Glcp-(1 → 3)-d-Glcp can be attached. But no successive (β1 → 6) linkages are created. Furthermore, the transfer products of a high degree of polymerization have no terminal β-d-Glcp-(1 → 6)- ends (Ga). For the latter products, more than one branching site cannot be excluded. Of course, in the whole incubation setup, the use of a product as a new substrate for extensions cannot be excluded. It should be noted that the different technologies applied to elucidate the formed structures in detail were not able so far to discriminate between all structural isomers.
The results obtained with the Glt1 enzyme are different from those obtained with the Glt7 enzyme. Also the Glt1 enzyme has both hydrolysis and transfer activities, but differ between Lam-Glcx and Lam-Glcx-ol. For the free oligosaccharides Lam-Glcx (Lam-Glc6–9), the major cleavage site is at the fourth (β1 → 3) linkage from the non-reducing end, yielding Pro-Glcx−4 hydrolysis products and Pro-Glcx+4 trans-glucosylation products. However, in the case of Lam-Glcx-ol substrates, starting from Lam-Glc4-ol on, the enzyme activity generates hydrolysis and transfer products in the full range, and a single transfer of Glc units is suggested. The yields of the transfer products are low (<10%) with a maximum incubation time of 24 h. Prolonged incubation times did not produce more transfer products, but only more hydrolysis products. With respect to the regiospecificity of the transfer reactions, as determined in more detail for Lam-Glc6-ol, elongation at Gt [terminal β-d-Glcp-(1 → 3)-] of the substrate can take place via (β1 → 3) (major reaction) and (β1 → 4) linkages. In fact, the Glt1 enzyme is able to elongate the alditol substrates sequentially by β-d-Glcp-(1 → 3)- and β-d-Glcp-(1 → 4)- units. There is no evidence for the presence of (β1 → 6) or (β1 → 2) linkages. No indications of branching activity in the Glt1 enzyme have been found. Interestingly, in the higher molecular mass transfer products, successive (β1 → 4) linkages occurred in the form of β-d-Glcp-(1 → 4)-β-d-Glcp-(1 → 4)-d-Glcp elements.
In the case of the Glt3 enzyme, the hydrolysis activity generates hydrolysis products in the full range, both using Lam-Glcx and Lam-Glcx-ol as substrates. When compared with the Glt7 and Glt1 enzymes, the Glt3 enzyme is the least active one. In the series Lam-Glc4–9, Lam-Glc4 and Lam-Glc6 did not reveal trans-glucosylation products and Lam-Glc9 seemed to be the most efficient substrate with major cleavages at the third and fourth (β1 → 3) linkages. But a full range of products is seen. Testing Lam-Glc6-ol as a substrate showed no real preference for a specific (β1 → 3) linkage, and the yield of the transfer products is low (<5%) with a maximum incubation time of 48 h. Prolonged incubation times did not produce more transfer products, but only more hydrolysis products. With respect to the regiospecificity of the transfer reactions, elongation at Gt [terminal β-d-Glcp-(1 → 3)-] of the substrate takes place only via (β1 → 3) linkages in a sequential way. No evidence was obtained for the presence of (β1 → 6), (β1 → 4) or (β1 → 2) linkages.
From additional TLC studies carried out with the three enzymes, but now using fluorescently labeled substrates, i.e. 2AB-labeled Lam-Glcx (Lam-Glcx-2AB), interesting data were collected with respect to the influence of another type of modification of the reducing end site of the substrates than the reduction described above. As a typical example, incubation of the Glt1, Glt3 and Glt7 enzymes with Lam-Glc6-2AB for 16 h did not lead to the formation of longer 2AB-labeled oligosaccharides. However, when laminarin was added to the reaction mixture, the formation of longer 2AB-labeled oligosaccharides was observed, indicating the capacity of laminarin as a donor and Lam-Glc6-2AB as an acceptor. No transfer activities were seen by adding other β-glucans, such as lichenan and pustulan. Using Lam-Glc2–6-2AB as acceptors and laminarin as a donor, for the Glt3 and Glt7 enzymes, it was found that the tetraose segment of Lam-Glc5-2AB was the minimum acceptor size for efficient transfer (detailed data not shown).
As has been reported earlier, the β-1,3(β-1,6)-glucosyltransferases originating from fungi, i.e. A. fumigatus (Mouyna et al. 1998; Gastebois et al. 2010), C. albicans (Hartland et al. 1991) and Bgl2p from S. cerevisiae (Mrsa et al. 1993; Kalebina et al. 2002), showed different cleavage sites relative to the ends of linear β-glucans. Thus, the secreted β-glucan branching enzymes from C. albicans and A. fumigatus specifically cleave laminaribiose from the reducing end of a linear (1 → 3)-β-glucan and transfers the remainder to an acceptor oligosaccharide (Hartland et al. 1991; Gastebois et al. 2010). On the other hand, β-1,3-glucosyltransferase of GH family 72 from the cell wall of A. fumigatus cleaves equally well all sites that are five residues from the non-reducing end and six residues from the reducing end in a linear (1 → 3)-β-glucan, thus exhibiting clear endo-enzyme activity (Hartland et al. 1996).
In this study, we have shown that the GH17 domain-encoding genes in Pseudomonas and Azotobacter code for β-trans-glycosylating enzymes that form mainly (β1 → 3) linkages but also to a substantial extent (β1 → 6) and (β1 → 4) linkages. So far mainly OPGs of class I, which are linear glucans consisting of backbones of (β1 → 2)-linked glucose with (β1 → 6)-linked branches, have been found within these genera. However, recently the presence of (1 → 3)-β-glucans has been verified in P. aeroginosa (Sadovskaya et al. 2010). Homologous genes are widespread among proteobacteria, and the results obtained in this study suggest that a range of activities and products may be expected in the encoded enzymes.
Materials and methods
Bacterial strains, plasmids and growth conditions
E. coli BL21 C43 (F-ompT hsdSB(rB-mB-) gal dcm araB::T7RNAP-tetA; Dumon-Seignovert et al. 2004) was used as production host for recombinant β-glucosyltransferases. P. aeruginosa PAO1 (ATCC15692), P. putida KT2440 (DSMZ6125) and A. vinelandii DJ (ATCC BAA-1303) were used as sources for β-glucosyltransferase genes. The Pseudomonas and Azotobacter strains were cultivated at 30°C in an appropriate medium recommended by the Deutsche Sammlung von Mikroorganismen (DSMZ). DNA was extracted using conventional methods (Sambrook et al. 1989). Plasmid-containing E. coli strains were grown in LB medium (Sambrook et al. 1989) supplemented with ampicillin (100 µg/mL). The expression vector pJOE3075 (Wegerer et al. 2008) was used for cloning of the β-glucosyltransferase genes into E. coli.
Recombinant enzymes and DNA techniques
Recombinant DNA techniques, such as genomic and plasmid DNA preparation, cloning and agarose gel electroporation, were performed according to the conventional protocols (Sambrook et al. 1989). Restriction enzymes and ligase were purchased from New England BioLabs (Hitchin, Hertfordshire, UK) and used as recommended by the manufacturer. Pfx PCR polymerase from Invitrogen (Taastrup, Denmark) was used to amplify the genes for expression cloning. Polymerase chain reaction (PCR) and DNA restriction fragments used in the cloning were eluted from the agarose using the Gel Extraction Kit GFX™ PCR DNA and the Gel Band Purification Kit from GE Healthcare (Uppsala, Sweden).
Cloning and expression of GH17 domain gene sequences in E. coli
GH17 domain-encoding gene sequences from P. aeruginosa PAO1, P. putida KT2440 and A. vinelandii BAA 1303 designated glt1, glt3 and glt7, respectively, are available in the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html) under the accession numbers NC_002516, NC_002947 and NZ_AAAU02000003, respectively.
The domain-encoding gene sequences were amplified by PCR using a proof reading polymerase (Platinum Pfx from Invitrogen), purified chromosomal DNA as a template and the primers listed in the Supplementary data, Table C. The forward primers were targeted 30–40 bp downstream of the start codon of the ORF, thus excluding sequences encoding hydrophobic amino acids of N-terminal signal peptide sequences. The reverse primers were targeted at sequences encoding amino acids at the C-terminal boundary of the GH17 domain and a downstream transmembrane helices region, predicted according to sequence alignment and modeling (data not shown).
Several different E. coli expression vector systems were tested for each gene (data not shown). The best results were obtained by cloning the genes in frame with the MalE fusion protein sequence of vector pJOE3075 (Wegerer et al. 2008), using the primers listed in the Supplementary data, Table C for the PCR amplification. The primers included BamHI restriction sites enabling cloning into the BamHI-digested pJOE3075 expression vector, resulting in fusion of the GH17 domain gene sequences in-frame with the plasmid MalE encoding sequence. E. coli BL21 (C43) (Dumon-Seignovert et al. 2004) was transformed with the resulting vectors containing the malE-glt1, 3 and 7 fusion genes. Correct sequence and fusion was verified with sequence analysis. Expression was carried out by growing the E. coli BL21 glt clones in the LB medium with ampicillin at 37°C until the growth reached a outer diameter value (OD600) of 0.8. Then l-rhamnose was added to the culture medium for induction (end concentration 0.1%). Subsequently, the clones were cultivated overnight at 25°C. The cells were harvested by centrifugation and disrupted by sonification. Expression of recombinant proteins was analyzed by running cleared samples of supernatant and insoluble fractions on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). All three genes (glt1, glt3 and glt7) were highly expressed following induction with l-rhamnose (data not shown), and recombinant proteins with and without histidine tags (HisTag) were produced. The three enzymes with attached HisTag were purified to near homogeneity on a Nickel affinity column. The Supplementary data, Figure D, shows stained SDS polyacrylamide gels of the purified Glt1, Glt3 and Glt7 enzymes, respectively.
Protein purification and protein analysis
All purifications were run on an Akta FPLC workstation (GE Healthcare). The recombinant proteins were purified on 5 HiTrap™ chelating columns (GE Healthcare). The column preparation was done according to the protocol from the manufacturer. The binding buffer was 20 mM sodium phosphate, pH 7.6, containing 500 mM NaCl and 10 mM imidazole; the elution buffer was 20 mM sodium phosphate, pH 7.6, containing 500 mM NaCl and 500 mM imidazole. The column was washed with 1 column volume (CV) of binding buffer. Samples (15 mL) were loaded onto the column, and unbound samples were washed out with 2× CV binding buffer. Non-specifically bound proteins were eluted from the column with 20% step elution buffer. Enzymes were eluted off the column with 40% step elution buffer. Purified samples were pooled and desalted on a 5 mL HiTrap™ Desalting Column (GE Healthcare), using 20 mM Tris, pH 7.5, containing 100 mM NaCl. Protein was concentrated from small amounts of eluates by using Microcon microconcentrators (Amicon, Beverly, MA). Large-scale purifications were carried out using an XK 16/26 column, containing 50 mL of Chelating Sepharose™ Fast Flow, using the same buffer systems and gradient steps, and the proteins in the eluates were concentrated by lyophilization. Protein contents were assayed using the Bradford reagent from BioRad (Hercules, CA), according to the manufacturer's recommendations; bovine serum albumin (Sigma, St. Louis, MO) was used as a standard. The purity of the protein in column eluates was evaluated by 12% standard SDS–PAGE, using molecular mass marker proteins in the range 2–212 kDa (New England Biolabs); the gels were stained with Coomassie protein reagent solution (Sigma).
Commercially obtained oligo- and polysaccharides
Laminari-oligosaccharides of DP2–DP7 were purchased from Megazyme (Bray, Ireland) (Lam-Glc2–6) and from Seikagaku Corp. (Tokyo, Japan) (Lam-Glc7). The (1 → 3)-β-d-glucan, curdlan (from Alcaligenes faecalis), was obtained from Fluka (Buchs, Switzerland).
Preparation of curdlan-derived laminari-oligosaccharides
Laminari-oligosaccharides of DP2–DP10 (Lam-Glc2–10) were prepared by partial acid hydrolysis of curdlan. To this end, 2 g of curdlan was incubated with 200 mL of 2 M trifluoroacetic acid for 13 min at 100°C. Subsequently, the incubate was dialyzed using a 6–8 kDa cut-off dialysis tube (Spectra/Por no. 132650). Oligosaccharides of DP2–DP10 were recovered in the water external to the dialysis sac, whereas larger-sized oligosaccharides (only partially soluble) and polymeric material remained in the tube. The permeate was dried down several times from water on a rotary evaporator to eliminate trifluoroacetic acid, and final traces of acid were removed under vacuum over potassium hydroxide pellets. The oligosaccharide mixture was dissolved in 2 mL of 0.05 M ammonium bicarbonate and fractionated on a Bio-Gel P-4 column (fine, 45–90 μm, bed volume of 100 × 2.6 cm), using 0.05 M ammonium bicarbonate at a flow rate of 14 mL/h. Fractions (1.5 mL) were collected and tested for carbohydrate content by the phenol–sulfuric acid assay. Individual peaks corresponding to the oligosaccharides DP2–DP10 were pooled and re-chromatographed on the Bio-Gel P-4 column. After the first fractionation, the oligosaccharide fractions DP2–DP10 accounted for 6.4% of the original curdlan. Ultimately, recoveries were much less than this, as it was necessary to discard up to 75% of each oligosaccharide following the second fractionation (only the midpoint of each peak was taken) to raise their purity to a level which validated conclusions made from the subsequent substrate/enzyme experiments. For each compound, the MALDI-TOF mass spectra showed a major [M+Na]+ peak, indicating purity grades of >98%. Furthermore, a library of NMR data of Lam-Glc2 to Lam-Glc10, essential for the analysis of the products generated in the enzymatic incubations, was created making use of TOCSY (mixing times of 40–200 ms), 1H–13C HSQC (mixing time of 200 ms) and ROESY (mixing time of 200 ms) experiments. Because of anomeric protons/residual HOD signal overlapping, all NMR spectra were recorded at 292 K, instead of the usual 300 K, causing a downfield shift for the HOD signal.
Reduction of curdlan-derived laminari-oligosaccharides
Laminari-oligosaccharides Lam-Glc2–10 (10 mg), dissolved in 2 mL of water, were incubated with sodium borohydride (10 mg) for 2 h at room temperature. The excess of sodium borohydride was destroyed with 4 M acetic acid until pH 6, formed boric acid was removed by co-evaporation with methanol and the generated oligosaccharide-alditols Lam-Glc2–10-ol were separated from peeling products on a Bio-Gel P-2 column (46 × 1 cm). The completeness of the reduction of each sample was checked by MALDI-TOF-MS and 1D 1H NMR spectroscopy. A library of NMR data of Lam-Glc2-ol to Lam-Glc10-ol, essential for the analysis of the products generated in the enzymatic incubations, was created making use of TOCSY (mixing times of 40–200 ms), 1H–13C HSQC (mixing time of 200 ms) and ROESY (mixing time of 200 ms) experiments. Running the spectra at 335 K instead of 292 K gave sharper peaks, whereas the obtained chemical shifts can be better compared with those in the CASPER database obtained at 340 K (www.casper.organ.su.se/casper).
TLC assays for glucosyltransferase activity
TLC was used for detecting activity in clones and initial analysis of reaction products. To this end, 5–10 μL were spotted in lines on TLC sheets (Merck Kieselgel 60 F254), which were developed with a mixture of n-butanol:acetic acid:water at a ratio of 2:1:1, and bands were visualized by orcinol/sulfuric acid staining. For calibration of migration positions, in each run, a mixture of curdlan-derived laminari-oligosaccharides(-alditols) of DP2–DP10 was included.
Enzyme assays and isolation of products
Curdlan-derived laminari-oligosaccharides and laminari-oligosaccharide-alditols (100 μL; 6.25 mg/mL) were mixed with 0.5 M potassium phosphate buffer, pH 6.5 (30 μL) and incubated with the recombinant bacterial GH17 enzymes Glt7, Glt3 and Glt1 (70 μL; 1.0 mg/mL) for different times (see Results) at 22°C. The progress of product formation was monitored by TLC; at optimal incubation times, product mixtures were isolated by fractionation of the incubate on a Bio-Gel P-2 column (95 × 1 cm), eluted with 10 mM ammonium bicarbonate at a flow rate of 12 mL/h. In the high-mass elution region, fractions of 3 min were collected and analyzed by TLC. Fractions containing oligosaccharides(-alditols) of higher molecular mass (compared with that of the substrate) were further investigated by MALDI-TOF-MS, NMR spectroscopy and/or ESI-MS.
Permethylation of standard and product oligosaccharide-alditols
Oligosaccharide-alditols (∼5 mg) were dried over silica in a desiccator, then dissolved in 0.5 mL of methyl sulfoxide, using ultrasonication, for 20 min, and sodium hydroxide powder (∼5 mg) was added (Ciucanu and Kerek 1984; Weiskopf et al. 1997). After ultrasonication for 20 min, the solution was cooled on ice, and two aliquots of 100 μL methyl iodide were added with intermediate cooling and final ultrasonication for 20 min at 20°C. Then, 1 mL of 4 mM sodium thiosulfate was added, and the permethylated products were extracted using chloroform (4 × 0.5 mL). The chloroform fraction was washed four times with 0.5 mL of water and subsequently dried with anhydrous sodium sulfate. After filtration through cotton wool, the chloroform fraction was evaporated to dryness, and the residue was dissolved in methanol. Products were investigated by ESI-MS.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
MALDI-TOF-MS experiments were performed using a Voyager-DE PRO mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a nitrogen laser (337 nm, 3 ns pulse width). Positive-ion mode spectra were recorded using the reflector mode and delayed extraction (100 ns). The accelerating voltage was 20 kV with a grid voltage of 75.2%; the mirror voltage ratio was 1.12, and the acquisition mass range was 550–3000 Da. Samples were prepared by mixing on the target 1 μL of aqueous oligosaccharides(-alditols) solutions with 2 μL of aqueous 10% 2,5-dihydroxybenzoic acid as matrix solution.
Electrospray ionization mass spectrometry
Permethylated oligosaccharide-alditols were analyzed using an LCQ DecaXP ion-trap mass spectrometer equipped with an electrospray ion source (Thermo Finnigan, San Jose, CA). Samples, dissolved in methanol:water at a ratio of 1:1 (Weiskopf et al. 1997) to a concentration of approximately 180 ng/μL, were introduced via a syringe pump at a flow rate of 3 μL/min. The capillary temperature was set to 200°C. ESI mass spectra were acquired in the positive-ion mode by scanning over m/z 50–3000 with a spray voltage of 3.0 kV and varying capillary voltage of 35–50 V. Tandem mass spectra were obtained using automated MS/MS starting from the parent ion or selected fragment ions.
NMR spectroscopy
Resolution-enhanced 1D/2D 500-MHz 1H and 125-MHz 13C NMR spectra were recorded in D2O on a Bruker DRX-500 spectrometer (Bijvoet Center, Department of NMR Spectroscopy, Utrecht University, The Netherlands) at probe temperatures of 292 K (oligosaccharides) or 335 K (oligosaccharide-alditols). Prior to analysis, samples were exchanged twice in D2O (99.9 at% D, Cambridge Isotope Laboratories, Inc., Andover, MA) with intermediate lyophilization, and then dissolved in 0.6 mL of D2O. Suppression of the HOD signal was achieved by applying a water-eliminated Fourier transform pulse sequence for 1D experiments (Hård et al. 1992) and by a pre-saturation of 1 s during the relaxation delay in 2D experiments. The 2D TOCSY spectra were recorded using an MLEV-17 mixing sequence with spin-lock times of 40–200 ms. The 2D ROESY spectra were recorded using standard Bruker XWINNMR software with a mixing time of 200 ms. The carrier frequency was set at the downfield edge of the spectrum in order to minimize TOCSY transfer during spin-locking. Natural abundance 2D 13C–1H HSQC experiments were recorded without decoupling during acquisition of the 1H free induction decay. Resolution enhancement of the spectra was performed by a Lorentzian-to-Gaussian transformation for 1D spectra or by multiplication with a squared-bell function phase shifted by π/2.3 for 2D spectra, and when necessary, a fifth-order polynomial baseline correction was performed. Chemical shifts (δ) are expressed in ppm with reference to internal acetone (δ 2.225 for 1H and δ 31.07 for 13C). All NMR data were processed using in-house developed software (J.A. van Kuik, Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, The Netherlands).
Icelandic Research Fund, University of Iceland Research Fund.
Conflict of interest statement
None declared.
Abbreviations
2AB, 2-aminobenzamide; CV, column volume; DP, degree of polymerization; DSMZ, Deutsche Sammlung von Mikroorganismen; ESI-MS, electrospray ionization mass spectrometry; HSQC, heteronuclear single quantum coherence; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MLEV, composite pulse devised by M. Levitt; NMR, nuclear magnetic resonance; OPG, osmoregulated periplasmic glucan; ORF, open reading frame; PCR, polymerase chain reaction; ROESY, rotating-frame nuclear Overhauser enhancement spectroscopy; SDS, sodium dodecyl sulfate; TLC, thin-layer chromatography; TOCSY, total correlation spectroscopy.
Acknowledgement
We are grateful to Dr. Josef Altenbuchner, University of Stuttgart, for providing the pJOE expression vector.
References
Bhagwat AA, Keister DL. Site-directed mutagenesis of the β-(1→3),β-(1→6)-d-glucan synthesis locus of Bradyrhizobium japonicum. Mol Plant Microbe Interact. 1995;8(3):366–370. [Google Scholar]
Bock K, Pedersen C, Pedersen H. Carbon-13 nuclear magnetic resonance data for oligosaccharides. Adv Carbohydr Chem Biochem. 1984;42:193–225. doi:10.1016/S0065-2318(08)60125-0. [Google Scholar]
Bock K, Thøgersen H. Nuclear magnetic resonance spectroscopy in the study of mono- and oligosaccharides. Ann Rep NMR Spectrosc. 1982;13:1–57. doi:10.1016/S0066-4103(08)60307-5. [Google Scholar]
Bohin J-P. Osmoregulated periplasmic glucans in proteobacteria. FEMS Microbiol Lett. 2000;186(1):11–19. doi: 10.1111/j.1574-6968.2000.tb09075.x. [DOI] [PubMed] [Google Scholar]
Bohin J-P, Lacroix JM. Osmoregulation in the Periplasm. Washington: DC: ASM Press; 2006. pp. 325–341. [Google Scholar]
Domon B, Costello CE. A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj J. 1988;5(4):397–409. doi:10.1007/BF01049915. [Google Scholar]
Dumon-Seignovert L, Cariot G, Vuillard L. The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3) Prot Expr Purif. 2004;37(1):203–206. doi: 10.1016/j.pep.2004.04.025. doi:10.1016/j.pep.2004.04.025. [DOI] [PubMed] [Google Scholar]
Gastebois A, Mouyna I, Simenel C, Clavaud C, Coddeville B, Delepierre M, Latgé J-P, Fontaine T. Characterization of a new β(1–3)-glucan branching activity of Aspergillus fumigatus. J Biol Chem. 2010;285(4):2386–2396. doi: 10.1074/jbc.M109.077545. doi:10.1074/jbc.M109.077545. [DOI] [PMC free article] [PubMed] [Google Scholar]
Goldman RC, Sullivan PA, Zakula D, Capobianco JO. Kinetics of β-1,3 glucan interaction at the donor and acceptor sites of the fungal glucosyltransferase encoded by the BGL2 gene. Eur J Biochem. 1995;227(1–2):372–378. doi: 10.1111/j.1432-1033.1995.tb20399.x. doi:10.1111/j.1432-1033.1995.tb20399.x. [DOI] [PubMed] [Google Scholar]
Hård K, van Zadelhoff G, Moonen P, Kamerling JP, Vliegenthart JFG. The Asn-linked carbohydrate chains of human Tamm-Horsfall glycoprotein of one male: Novel sulfated and novel N-acetylgalactosamine-containing N-linked carbohydrate chains. Eur J Biochem. 1992;209(3):895–915. doi: 10.1111/j.1432-1033.1992.tb17362.x. doi:10.1111/j.1432-1033.1992.tb17362.x. [DOI] [PubMed] [Google Scholar]
Hartland RP, Emerson GW, Sullivan PA. A secreted β-glucan-branching enzyme from Candida albicans. Proc Soc R Lond. 1991;246(1316):155–160. doi: 10.1098/rspb.1991.0138. doi:10.1098/rspb.1991.0138. [DOI] [PubMed] [Google Scholar]
Hartland RP, Fontaine T, Debeaupuis J-P, Simenel C, Delepierre M, Latgé J-P. A novel β-(1–3)-glucanosyltransferase from the cell wall of Aspergillus fumigatus. J Biol Chem. 1996;271(43):26843–26849. doi: 10.1074/jbc.271.43.26843. doi:10.1074/jbc.271.43.26843. [DOI] [PubMed] [Google Scholar]
Hochstenbach F, Klis FM, van den Ende H, van Donselaar E, Peters PJ, Klausner RD. Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast. Proc Natl Acad Sci USA. 1998;95(16):9161–9166. doi: 10.1073/pnas.95.16.9161. doi:10.1073/pnas.95.16.9161. [DOI] [PMC free article] [PubMed] [Google Scholar]
Kalebina TS, Laurinavichiute DK, Packeiser AN, Morenkov OS, Ter-Avanesyan MD, Kulaev IS. Correct GPI-anchor synthesis is required for the incorporation of endoglucanase/glucanosyltransferase Bgl2p into the Saccharomyces cerevisiae cell wall. FEMS Microbiol Lett. 2002;210(1):81–85. doi: 10.1111/j.1574-6968.2002.tb11163.x. doi:10.1111/j.1574-6968.2002.tb11163.x. [DOI] [PubMed] [Google Scholar]
Lequette Y, Rollet E, Delangle A, Greenberg EP, Bohin J-P. Linear osmoregulated periplasmic glucans are encoded by opgGH locus of Pseudomonas aeruginosa. Microbiology. 2007;153(10):3255–3263. doi: 10.1099/mic.0.2007/008953-0. doi:10.1099/mic.0.2007/008953-0. [DOI] [PubMed] [Google Scholar]
Mah TF, Pitts B, Pellock B, Walker GC, Stewart PS, O'Toole GA. A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature. 2003;426(6964):306–310. doi: 10.1038/nature02122. doi:10.1038/nature02122. [DOI] [PubMed] [Google Scholar]
Makrides SC. Strategies for achieving high level expression of genes in Escherichia coli. Microbiol Rev. 1996;60(3):512–538. doi: 10.1128/mr.60.3.512-538.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
Mouyna I, Hartland RP, Fontaine T, Diaquin M, Simenel C, Delepierre M, Henrissat B, Latgé J-P. A 1,3-β-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus is a homologue of the yeast Bgl2p. Microbiology. 1998;144(11):3171–3180. doi: 10.1099/00221287-144-11-3171. doi:10.1099/00221287-144-11-3171. [DOI] [PubMed] [Google Scholar]
Mrsa V, Klebl F, Tanner W. Purification and characterization of the Saccharomyces cerevisiae BGL2 gene product, a cell wall endo-β-1,3-glucanase. J Bacteriol. 1993;175(7):2102–2106. doi: 10.1128/jb.175.7.2102-2106.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
Mukhopadhyay P, Williams J, Mills D. Molecular analysis of a pathogenicity locus in Pseudomonas syringae pv. syringae. J Bacteriol. 1988;170(12):5479–5488. doi: 10.1128/jb.170.12.5479-5488.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
Robijn GW, van den Berg DJC, Haas H, Kamerling JP, Vliegenthart JFG. Determination of the structure of the exopolysaccharide produced by Lactobacillus sake 0–1. Carbohydr Res. 1995;276(1):117–136. doi: 10.1016/0008-6215(95)00172-p. doi:10.1016/0008-6215(95)00172-P. [DOI] [PubMed] [Google Scholar]
Sadovskaya I, Vinogradov E, Li J, Hachani A, Kowalska K, Filloux A. High-level antibiotic resistance in Pseudomonas aeruginosa biofilm: The ndvB gene is involved in the production of highly glycerol-phosphorylated β-(1→3)-glucans, which bind aminoglycosides. Glycobiology. 2010;20(7):895–904. doi: 10.1093/glycob/cwq047. doi:10.1093/glycob/cwq047. [DOI] [PubMed] [Google Scholar]
Sambrook J, Fritch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. New York: Cold Spring Harbour Laboratory Press; 1989. [Google Scholar]
Wegerer A, Sun T, Altenbuchner J. Optimization of an E. coli l-rhamnose-inducible expression vector: Test of various genetic module combinations. BMC Biotechnol. 2008;8(2):1–12. doi: 10.1186/1472-6750-8-2. doi:10.1186/1472-6750-8-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
Distinct expression profiles of UDP-galactose: β-d-galactoside α1,4-galactosyltransferase and UDP-galactose:β-d-galactoside β1,4-galactosyltransferase in pigeon, ostrich and chicken
We previously identified two novel enzymes in pigeon, α1,4- and β1,4-galactosyltransferases (GalTs), which are responsible for the biosynthesis of the Galα1-4Gal and Galβ1-4Gal sequences on glycoproteins, respectively. No such glycan structures and/or enzymes have been found in mammals, suggesting that the expression of these enzymes diverged during the course of vertebrate evolution. To compare their expression profiles among avian species, we first established a method for detecting the activities of these two GalTs based on the two-dimensional high pressure liquid chromatography mapping technique, using 2-aminopyridine-derivatized asialo-biantennary N-glycans as an acceptor substrate. When we analyzed the activities of GalTs in pigeon liver extracts in the presence of uridine diphosphate (UDP)-Gal, 13 different products containing Galα1-4Galβ1-4GlcNAc, Galβ1-4Galβ1-4GlcNAc and/or Galα1-4Galβ1-4Galβ1-4GlcNAc branches were identified. The newly formed glycosidic linkages of the enzymatic products were determined by nuclear magnetic resonance and methylation analysis, as well as by galactosidase digestions. The activities of both α1,4- and β1,4-GalTs were detected in various tissues in pigeon, although their relative activities were different in each tissue. In contrast, ostrich expressed β1,4-GalT, but not α1,4-GalT, in all tissues analyzed, whereas neither α1,4- nor β1,4-GalT activity was detected in chicken. These results indicate that α1,4- and β1,4-GalTs are expressed in a species-specific manner and are distributed throughout the entire body of pigeon or ostrich when the enzymes are present.
Different glycan structures are often expressed in a species-specific fashion in animals and plants (Varki 1993; Gagneux and Varki 1999). In vertebrates, for instance, fish, amphibians and birds express some complex structures of glycans that are rarely found in mammals. However, the biological mechanism driving the gain and the loss of species-specific glycans remains to be elucidated due to the lack of sufficient information of glycan diversity (Varki et al. 2008).
Galα1-4Galβ1-4Galβ1-4GlcNAc sequences were found in N-glycans of pigeon immunoglobulin (Ig) G (Suzuki et al. 2003) and in O-glycans from salivary gland mucin of Chinese swiftlet (Wieruszeski et al. 1987). The presence of this sequence indicates that these birds are able to express both Galα1-4Gal and Galβ1-4Gal sequences, as well as Galβ1-4GlcNAc. We have previously investigated the distribution of Galα1-4Gal in egg white glycoproteins from 181 species of birds by western blot analysis using anti-P1 monoclonal antibody (mAb; Galα1-4Galβ1-4GlcNAc-specific; Suzuki et al. 2004, 2006; Suzuki and Lee 2007). We found that the Galα1-4Gal sequence was absent from the egg white glycoproteins of two large taxa of avians, i.e. Ratitae (traditionally called Palaeognathae, e.g. ostrich, rhea, emu, tinamou) and Galloanserae (e.g. chicken, duck), but present in the majority of Neoaves (e.g. pigeon, swiftlet, parrot, gull; Suzuki et al. 2009). (Nomenclatures for avian classification are based on Sibley and Ahlquist (1990). In this paper, Neoaves does not include Galloanserae.) In contrast, mammals express Galα1-4Gal on glycolipids, such as globotriosylceramide (Gb3, Galα1-4Galβ1-4Glc-Cer) and P1 antigen (Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Cer), but not on glycoproteins, except on those in the hydatid fluid caused by tapeworm (Cossey et al. 1979; Khoo et al. 1997).
Galα1-4Gal and Galβ1-4Gal sequences are biosynthesized in pigeon by the actions of uridine diphosphate (UDP)-Gal:β-galactoside α1,4-galactosyltransferase (α4GalT(Gal)) and UDP-Gal:β-galactoside β1,4-galactosyltransferase (β4GalT(Gal)), respectively. (In this paper, GalTs are conveniently abbreviated as linkageGalT(an acceptor substrate) to distinguish their acceptor substrate specificities from each other, e.g. UDP-galactose:β-d-galactoside α1,4-galactosyltransferase is designated as α4GalT(Gal).) We recently isolated cDNAs encoding these enzymes by expression cloning (Suzuki and Yamamoto 2010). The amino acid sequence of pigeon α4GalT(Gal) is similar to that of human and chicken Gb3 synthases (58.2 and 68.0% identity, respectively), which utilize lactosylceramide (Galβ1-4Glc-Cer) as the preferred acceptor substrate to produce Gb3. These enzymes belong to the glycosyltransferase family denoted GT32 in the Carbohydrate-Active Enzymes (CAZy) database (Lairson et al. 2008). However, unlike human and chicken Gb3 synthases, pigeon α4GalT(Gal) preferred to produce P1 antigen using N-acetyllactosamine (Galβ1-4GlcNAc-) as a substrate rather than Pk antigen (Galα1-4Galβ1-4Glc) using lactose as a substrate (Suzuki and Yamamoto 2010).
Another enzyme in pigeon, β4GalT(Gal), belongs to the GT92 family in the CAZy database. The members of this family are found widely in eukaryotes, including vertebrates and nonvertebrates, but not in mammals. Among birds, zebra finch, but not chicken, possess hypothetical proteins similar to pigeon β4GalT(Gal). Therefore, the gene encoding β4GalT(Gal) may have been lost in some bird lineages.
In this study, to compare the properties and expression profiles of α4GalT(Gal) and β4GalT(Gal) among various species, we first established a method to detect the activities of these enzymes in pigeon liver by high pressure liquid chromatography (HPLC). With this method, we analyzed the expression of α4GalT(Gal), β4GalT(Gal) and β4GalT(GlcNAc) in three avian species, namely pigeon, ostrich and chicken, and found that α/β4GalTs(Gal) have distinct expression patterns.
Results
Detection and structural analysis of 2-aminopyridine-derivatized glycans produced by GalTs(Gal) in pigeon liver extract
To detect specific activity of putative α/β4GalTs(Gal) in pigeon, which direct the biosynthesis of Galα/β1-4Gal on N-glycans, we utilized asialo-biantennary oligosaccharides (N-glycan A in Table I) as the acceptor substrate. When 2-aminopyridine (PA)-derivatized N-glycan A (substrate A) was incubated with 50 mg protein/mL of pigeon liver extract and UDP-Gal as described in Materials and methods, four additional peaks (i.e. I–IV) and a peak of PA-substrate A were observed on normal phase HPLC analysis with an Amide-80 column (Figure 1A, left). No product peaks were observed when the PA-substrate A was incubated with the same pigeon liver extract without UDP-Gal, suggesting that peaks I–IV are most likely produced by the catalytic activity of putative GalTs. On the basis of the elution positions on the Amide-80 column, fractions I–IV were expected to have one to four galactose residues attached to the PA-substrate A. It was confirmed by analyzing with matrix-assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI-TOF-MS; data not shown). None of the peaks eluted after fraction IV on the column were detected under the experimental conditions, suggesting that pigeon liver extract transfers no more than four galactose residues to the PA-substrate A. The products separated on the Amide-80 column were further separated on an octadecylsilica (ODS) column (Figure 1A, right). Fraction I was separated into I-1 and I-2; II into II-1, II-2 and II-3; III into III-1; and IV into IV-1. The elution times of PA-oligosaccharides applied to the ODS and Amide-80 columns were recorded in glucose units (GUs), i.e. GU(ODS) and GU(Amide), respectively (Table II). The structures of N-glycans produced by putative pigeon GalTs were determined by a two-dimensional HPLC mapping technique (Tomiya et al. 1988) as described in Supplementary data and Supplementary Figures S1–S3. The linkages of newly formed Gal-Gal sequences were confirmed to be Galα1-4Gal and Galβ1-4Gal by methylation analysis (Supplementary Figure S4) and 1H-NMR (Supplementary Figures S5 and S6). The proposed structures of the products are summarized in Table II and Supplementary Figure S3. The results indicate that the products resulting from the action of GalT(Gal) in pigeon liver extracts possessed Galα1-4Gal and/or Galα1-4Galβ1-4Gal sequences on the branches of biantennary N-glycans. This finding strongly suggested the presence of α4GalT(Gal) and β4GalT(Gal) in pigeon liver.
Table I.
Structures of PA-N-glycans for standards or substrates
HPLC profiles of PA-oligosaccharides produced by the action of avian GalTs. PA-substrate A (Table I) was incubated with pigeon (A), ostrich (B) or chicken (C) liver extracts with (+) or without (−) UDP-Gal. The products of pigeon liver extracts were separated on an Amide-80 column into fractions I, II, III and IV (A, left) and then separated on an ODS column (A, right).
Table II.
Structures of PA-N-glycans produced by α4GalT(Gal) and β4GalT(Gal) from pigeon liver extract
aRelative quantity of the PA-oligosaccharides produced by adding one to four galactose residues to the substrate was calculated based on an Amide-80 elution profile, and each PA-oligosaccharide was calculated based on ODS elution profiles. IIIa and IIIb overlap with each other on both two of the columns, so the quantity was calculated after α-galactosidase treatment.
bIc, Id and IId were produced by α-galactosidase digestion of IIIa, IIIb and IVa, respectively. IIIc and IIId were produced by partial α-galactosidase digestion of IVa. Ia', Ib', IIb' and IIc' were produced by β4-galactosidase digestion of Ia, Ib, IIb and IIc, respectively.
Analysis of GalT(Gal) activities in ostrich and chicken liver extract
We next compared the GalT(Gal) activities of pigeon with those of other avian species. Since pigeon, ostrich and chicken belong to phylogenetically distant taxa, we decided to evaluate these species. When PA-substrate A was incubated with 10 mg protein/mL of ostrich liver extract and UDP-Gal, two peaks (V and VI) were observed upon Amide-80 column chromatography (Figure 1B). No Gal residues were transferred to PA-substrate A in the absence of UDP-Gal. Fractions V and VI were expected to have one and two galactose residues, respectively, attached to the PA-substrate A, based on the elution positions on the Amide-80 column. None of the peaks eluted after fraction VI on the Amide-80 column were detected, even when the protein concentration of liver extract was increased to 50 mg/mL (data not shown), suggesting that ostrich liver extract transfers no more than two galactose residues to PA-substrate A. By comparing the elution positions on both Amide-80 and ODS columns before and after β4-galactosidase digestion (data not shown), we established that fraction V contained Ic and Id and fraction VI contained IId (Table II). The structure of fraction VI was also confirmed by methylation analysis (Supplementary Figure S4) and 1H-NMR (Supplementary Figures S5 and S6). The results indicated that the products by the action of GalT(Gal) in ostrich liver extracts possessed only the Galβ1-4Gal sequence but not the Galα1-4Gal sequence. Accordingly, the presence of β4GalT(Gal) in ostrich liver was suggested.
In contrast, when 50 mg protein/mL of chicken liver extract was used as the enzyme source, no peaks other than the peak of PA-substrate A were observed (Figure 1C). As a representative control of glycosyltransferases, we also analyzed the activity of β4GalT(GlcNAc), using asialo-agalactosyl biantennary oligosaccharides (PA-glycan B in Table I) as an acceptor substrate. Unlike GalT(Gal) activity, β4GalT(GlcNAc) activity was detected in pigeon, ostrich and chicken liver extracts, even at a concentration of 0.25 mg protein/mL (described in “Comparison of GalTs from pigeon, ostrich and chicken based on biochemical properties”), suggesting that the enzymes were extracted properly from the tissues. Therefore, the absence of products when combining chicken liver extract with PA-substrate A and UDP-Gal implies that GalT(Gal) activity is absent or undetectable in chicken liver.
Establishment of the assay method to detect GalT activities
Once we had succeeded in identifying the structures of the PA-glycans produced by the action of pigeon and ostrich α/β4GalTs(Gal), the coordinate of GU(ODS) and GU(Amide) determined for each PA-glycan (Table II, Supplementary Figure S3) was used to detect the products of α/β4GalTs(Gal) from various samples (e.g. Figure 2). The reactions presented in Figure 2 were performed with 1 mg/mL of protein at 37°C for 2 h, to obtain products that transferred only one or two galactose residues to the acceptor substrate, PA-glycan A. On the basis of the relative elution positions on both Amide-80 and ODS columns, the products of α4GalT(Gal) and β4GalT(Gal) were identified simultaneously. Moreover, since the elution positions of these products on the ODS column were clearly different from those yielded by α3GalT(Gal) (Table I), the activities of these three enzymes are easily distinguishable. By measuring the amount of products and transferred galactose residues (pmol), GalT activities (pmol/h/mg protein) were recorded, as described in the following experiments.
A typical elution pattern of PA-oligosaccharides transferred one or two galactose residues to the acceptor substrate, PA-glycan A, on Amide-80 and ODS columns. Reaction mixtures containing PA-substrate A were incubated with microsomal fractions of pigeon kidney (A) or ostrich follicle (B). The products were separated on an Amide-80 column and then separated on an ODS column. Elution profiles of products on the ODS column that transferred one galactose are presented as examples. Products of α4GalT(Gal) (Ia and Ib in Table II) and of β4GalT(Gal) (Ic and Id in Table II) were separated on the ODS column.
Comparison of GalTs from pigeon, ostrich and chicken based on biochemical properties
To compare the optimum pH of GalT activity in pigeon, ostrich and chicken, PA-substrate A (for measuring α4GalT(Gal) and β4GalT(Gal) activities) and PA-substrate B (for measuring β4GalT(GlcNAc) activity) were incubated with microsomal fractions from liver at various pHs, ranging from 5.5 to 8.0. The activities of these enzymes were found to have a pH optimum of 7.5–8.0 for α4GalT(Gal), 6.5–7.5 for β4GalT(Gal) and 5.5 for β4GalT(GlcNAc) (Figure 3). Similar pH dependencies were observed among the species, i.e. β4GalT(Gal) of pigeon and ostrich and β4GalT(GlcNAc) of pigeon, ostrich and chicken, although the relative activities in the liver extracts differed among the species.
Comparison of the activities of avian liver GalTs at various pH values. Activities of α4GalT(Gal) (solid line), β4GalT(Gal) (dashed line) and β4GalT(GlcNAc) (broken line) from pigeon (A), ostrich (B) and chicken (C) liver extracts were analyzed at pH 5.5, 6.0, 6.5, 7.0 (MES buffer, squares), 7.0, 7.5 and 8.0 (HEPES buffer, triangles). The values represent the mean ± SD of duplicate samples. Scales for α4GalT(Gal) or β4GalT(Gal) activities are indicated on the left y-axes, and those for β4GalT(GlcNAc) activities on the right y-axes of each graph. MES, 2-(N-morpholino)ethanesulfonic; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
The effect of divalent cations on α4GalT(Gal), β4GalT(Gal) and β4GalT(GlcNAc) activities was also analyzed by the same method, using various divalent cations or ethylenediaminetetraacetic acid (EDTA), and the results are summarized in Table III. All of these enzymes showed an absolute requirement for divalent cations, because no GalT activity was detected in the absence of divalent cations or in the presence of 20 mM EDTA. Mn2+ activated all three GalTs in pigeon, which was expected because most eukaryotic glycosyltransferases require this cation. As shown in Table III, the divalent cations of preference differed slightly among the three GalTs in pigeon liver. However, the cation of preference of β4GalT(Gal) in pigeon and ostrich and of β4GalT(GlcNAc) in pigeon, ostrich and chicken seemed to be conserved among these species.
aFinal concentrations of divalent cations and ethylenediaminetetraacetic acid (EDTA) were 20 mM.
bRelative activities of 100% were calculated as a percentage of the incorporation obtained with the addition of MnCl2 (set to 100%), which corresponded to 13.8 (pigeon α4GalT(Gal)), 18.4 (pigeon β4GalT(Gal)), 488 (ostrich β4GalT(Gal)), 618 (pigeon β4GalT(GlcNAc)), 271 (ostrich β4GalT(GlcNAc)) and 1930 (chicken β4GalT(GlcNAc)) pmol/h/mg protein.
cThe values represent the mean ± SD of duplicate samples. ND, not detected.
Distribution of α/β4GalT(Gal) in various tissues of pigeon, ostrich and chicken
To investigate the tissue distributions of GalTs, PA-substrate A, for α4GalT(Gal) and β4GalT(Gal) activities, and PA-substrate B, for β4GalT(GlcNAc) activity, were incubated with microsomal fractions of tissues available from birds (Figure 4). The activities of all three GalTs in pigeon varied by more than 100-fold among tissues, with the highest activity being detected in the small intestine for α4GalT(Gal), in the leukocytes for β4GalT(Gal) and in the liver for β4GalT(GlcNAc). Since microsomal fractions of brain and erythrocytes of pigeon revealed no α4GalT(Gal) activity, extracts with increased protein concentrations (30 mg/mL) were examined for these tissues. The trace amount of products synthesized by α4GalT(Gal) treatment was detected in pigeon brain extract, but no products of α4GalT(Gal) treatment were detected in the erythrocyte fraction. All α4GalT(Gal), β4GalT(Gal) and β4GalT(GlcNAc) activities in the heart, brain and erythrocytes were relatively lower than those in other tissues, probably due to the inefficient extraction of the enzymes from these other tissues, in addition to the relatively low expression levels of the enzymes. Although the activities varied among tissues, α4GalT(Gal), β4GalT(Gal) and β4GalT(GlcNAc) were widely distributed in various tissues in pigeon. The recorded activity of β4GalT(GlcNAc) in each pigeon tissue examined was almost an order of magnitude higher than that of α4GalT(Gal) and β4GalT(Gal).
Tissue distributions of α4GalT(Gal), β4GalT(Gal) and β4GalT(GlcNAc) in pigeon, ostrich and chicken. Microsomal fractions were prepared from various tissues of pigeon (A), ostrich (B) and chicken (C) as described in Materials and methods. The values represent the mean ± SD of duplicate samples. Scales for α4GalT(Gal) (solid bar) or β4GalT(Gal) (open bar) activities are indicated on the left y-axes, and those for β4GalT(GlcNAc) (hatched bar) activities on the right y-axes of each graph.
In ostrich, both β4GalT(Gal) and β4GalT(GlcNAc) activities were detected in all tissues (Figure 4), but no α4GalT(Gal) activity was detected in any tissues, as we have previously noted for the liver (Figure 3). β4GalT(Gal) and β4GalT(GlcNAc) activities were almost equivalent in ostrich tissues. The ostrich blood plasma possessed the highest β4GalT(Gal) activity, which is presumably secreted from some tissues, as often found in several other glycosyltransferases in mammals (Kaplan et al. 1983; Sarnesto et al. 1990).
In contrast, chicken β4GalT(GlcNAc) activities were detected in all tissues examined, but neither α4GalT(Gal) nor β4GalT(Gal) activities were detected in any tissues examined (Figure 4). These results indicate that both α4GalT(Gal) and β4GalT(Gal) are absent in chicken tissues, or below the limit of detection.
Antibody/lectin-blot analysis of glycoproteins containing Galα1-4Gal and/or Galβ1-4Gal in pigeon, ostrich and chicken
Since the detected activities of α4GalT(Gal) and β4GalT(Gal), as well as β4GalT(GlcNAc) in tissues, varied depending on the avian species, we also compared the presence of particular glycan structures on glycoproteins in the tissues. We used anti-P1 mAb for Galα1-4Gal, anti-(Galβ1-4Gal) mAb for Galβ1-4Gal and Erythrina cristagalli agglutinin (ECA) for Galβ1-4GlcNAc for antibody/lectin-blotting. Proteins extracted from tissues were blotted onto polyvinylidene difluoride (PVDF) membranes and visualized with Coomassie brilliant blue R-250 (CBB) staining (Figure 5). All the tissues presented in Figure 5 from pigeon, ostrich and chicken stained with ECA, confirming the presence of the Galβ1-4GlcNAc sequence, which can act as the acceptor substrate of α4GalT(Gal) and β4GalT(Gal). In pigeon, all tissues stained with anti-P1 mAb, although the intensity of staining varied depending on the tissue. The same tissue extracts from pigeon stained only faintly with anti-(Galβ1-4Gal) mAb, presumably because the Galβ1-4Gal sequence was masked with α4-galactosylation at the nonreducing termini. In ostrich, all tissues stained with anti-(Galβ1-4Gal) mAb, but not with anti-P1 mAb. These results were consistent with the presence of β4GalT(Gal) activity, and the absence of detectable α4GalT(Gal) activity in ostrich. Although the chicken tissue glycoproteins stained strongly with ECA, no glycoproteins were stained with either anti-P1 mAb or anti-(Galβ1-4Gal) mAb. These results were also consistent with the absence of detectable α4GalT(Gal) and β4GalT(Gal) activities in chicken.
Antibody/lectin staining of protein extracts from various tissues. Protein extracts (20 µg/lane) from pigeon (A), ostrich (B) and chicken (C) blotted onto PVDF membrane were visualized with CBB staining. Pigeon IgG (for CBB and anti-P1 mAb stainings) and α-galactosidase-treated pigeon IgG (for anti-(Galβ1-4Gal) mAb and ECA stainings) were used as controls.
Discussion
Research into the glycosyltransferases that regulate the expression of species-specific glycans is essential for the systematic study of glycan diversity. In this study, we isolated and quantified PA-derivatized N-glycans produced by α4GalT(Gal) and/or β4GalT(Gal) by employing the two-dimensional HPLC map technique (Tomiya et al. 1988). Since products with different structures were eluted at different positions on the columns, the activities of α4GalT(Gal) and β4GalT(Gal) were measured simultaneously. This HPLC method is more convenient than mass spectrometry (MS) analysis, which cannot easily fractionate or quantify different oligosaccharides of the same molecular masses. Moreover, using the same HPLC system, we could analyze the activities of β4GalTs(GlcNAc), which are key enzymes in the production of the Galβ1-4GlcNAc sequence. Because the genes encoding β4GalTs(GlcNAc) are highly conserved among vertebrates, including chicken (Lo et al. 1998; Amado et al. 1999), we expected that β4GalTs(GlcNAc) are also expressed in many other birds. It was indeed found that the activities of β4GalTs(GlcNAc) were commonly detected in pigeon, ostrich and chicken with similar biochemical properties (Figures 3 and 4).
Detecting the activities of α4GalT(Gal) and β4GalT(Gal) is also useful to predict the expression of their enzymatic products, i.e. Galα1-4Galβ1-4GlcNAc, Galβ1-4Galβ1-4GlcNAc and Galα1-4Galβ1-4Galβ1-4GlcNAc sequences in tissues or cells, if the presence of both donor and acceptor substrates in the same cells is also demonstrated. Although it is better to confirm the presence of these glycan structures in the tissues with specific antibodies or with structural analysis of the glycans, these strategies are not always feasible. For instance, when the glycan sequences, such as Galβ1-4Gal, are not exposed at nonreducing termini and masked by additional modifications, steric hindrance prevents the recognition of epitopes by the antibodies. Detailed structural analysis of glycans in tissues can provide more precise information of the structures, but it is time-consuming and involves tedious procedures. Thus, detection of the enzyme activities is one of the practical strategies to estimate the presence of specific glycan structures, as long as fresh tissues are available.
Modern birds are monophyletic and are divided into three large taxa, namely, Ratitae, Galloanserae and Neoaves (Sibley and Ahlquist 1990; Mindell et al. 1997; Cracraft 2001). To compare the activities of α4GalT(Gal) and β4GalT(Gal) among birds as an initial study, we selected one representative species from each taxon, i.e. pigeon from Neoaves, ostrich from Ratitae and chicken from Galloanserae. The expression of α4GalT(Gal) was found only in pigeon, which was expected, as we have previously proposed that Galα1-4Gal is absent in Ratitae and Galloanserae (Suzuki et al. 2004). In contrast, the expression of β4GalT(Gal) was found in both pigeon and ostrich, but not in chicken. The pH and cation dependencies of β4GalT(Gal) from pigeon and ostrich were quite similar, suggesting that there are cognate β4GalTs(Gal) in these species. Since pigeon and ostrich belong to distant phylogenetic taxa, the results imply that a wide range of modern birds may also express β4GalT(Gal) and Galβ1-4Gal. This possibility could be investigated by analyzing samples from many other birds, using the method established in this study.
Materials and methods
Materials
Adult female pigeons (Columba livia) and an adult female chicken (Gallus gallus) were purchased from Saitama Experimental Animals Supply Co., (Kitakatsushika, Japan). Fresh tissues from ostrich (Struthio camelus) were purchased from a local farmer in the Ibaraki area. Porcine thyroglobulin was prepared as described previously (Ui and Tarutani 1961). α-Galactosidase from green coffee beans, and N-acetyl-β-d-hexosaminidase from jack bean were purchased from Sigma (St Louis, MO). β4-Galactosidase from Streptococcus pneumoniae was purchased from Calbiochem (La Jolla, CA). Glycoamidase F (also known as N-glycosidase F or PNGase F) was from Roche Diagnostics GmbH (Penzberg, Germany). Neuraminidase from Arthrobacter ureafaciens was from Nacalai Tesque, Inc. (Kyoto, Japan). Anti-P1 mAb (mouse IgM) was from Gamma Biologicals (Houston, TX). Alkaline phosphatase (AP)–conjugated ECA was from EY Laboratories (San Mateo, CA). AP-conjugated goat anti-mouse IgG and AG1-X8 (100–200 mesh) were from Bio-Rad Laboratories (Hercules, CA). AP-conjugated goat anti-mouse IgM was from Beckman Coulter (Fullerton, CA), and AP-conjugated streptavidin was from BD Biosciences Pharmingen (San Jose, CA). The Shim-Pack CLC-ODS column (6.0 × 150 mm) was from Shimadzu (Kyoto, Japan). The TSKgel diethylaminoethyl (DEAE)-5PW column (7.5 × 75 mm) and the TSKgel Amide-80 column (4.6 × 250 mm) were from Tosoh Co. (Tokyo, Japan). Sephadex G-25 and Sephadex G-15 were from GE Healthcare UK Ltd (Buckinghamshire, UK). The carbograph cartridge (150 mg) was from Alltech Associates (Deerfield, IL). Dowex 50W-X8 (100–200 mesh) was from Muromachi Kagaku Kogyo Kaisha (Tokyo, Japan). Preparation of the anti-(Galβ1-4Gal) mAb “68” (mouse IgG1) will be described elsewhere (Suzuki et al., manuscripts in preparation).
Standard procedures and buffers
Protein concentrations were measured by the bicinchoninic acid (BCA) assay, using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL), or by the Bradford assay, using Coomassie Plus Reagent (Pierce). Bovine serum albumin was utilized as a standard. The lysis buffer contains 100 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (pH 7.5), 1% Triton X-100, 20 mM MnCl2, 1 mM phenylmethanesulfonyl fluoride (PMSF) and 1 µg/mL of leupeptin. The reaction buffer contains 100 mM HEPES (pH 7.5), 1% Triton X-100, 20 mM MnCl2, 5 mM d-galactonic acid γ-lactone and 0.2 mM deoxyfuconojirimycin.
Preparation of PA-derivatized oligosaccharides
Porcine thyroglobulin (1 g) was dissolved in 20 mL of 0.1 M sodium borate (pH 7.8) and digested with 1 mg/mL of pronase at 37°C for 48 h. After concentration and centrifugation, the supernatant was loaded on a Sephadex G-25 column (1.5 × 90 cm) in water. Fractions containing neutral sugars were monitored with the phenol/sulfuric acid method (Dubois et al. 1956), and glycopeptide fractions were pooled and lyophilized. N-Glycans were released by hydrazinolysis and re-N-acetylated. Free oligosaccharides were desalted by passage through Dowex 50W-X8 (H+ form), purified with a carbograph cartridge, as described previously (Suzuki et al. 2003), then PA-derivatized (Kondo et al. 1990). The mixture of PA-oligosaccharides was first separated by HPLC on a diethylaminoethyl cellulose column (7.5 × 75 mm) based on the sialic acid content (Nakagawa et al. 1995). The neutral oligosaccharide fractions were separated on an ODS column (6.0 × 150 mm), as described previously (Suzuki et al. 2001), and each fraction was analyzed by MALDI-TOF-MS, using 2,5-dihydroxybenzoic acid (10 mg/mL in 5 mM NaCl) as a matrix. Additional purification was performed with an amide-silica (Amide-80) column (Tomiya et al. 1988), and PA-substrates A and B (PA-glycans A and B in Table I) were obtained. Reference PA-derivatized N-glycans C–F (Table I) were prepared from human IgG, as described previously (Suzuki and Lee 2004). PA-glycans G and H were prepared as described in Results.
Preparation of tissue extracts and microsomal fractions as enzyme sources
Fresh tissues from pigeon, ostrich and chicken were kept at −80°C until used. Each tissue was homogenized at 4°C in lysis buffer with a Polytron homogenizer. The homogenate was centrifuged at 17,400 × g for 5 min at 4°C, and the supernatant filtered through a 0.45 µm membrane was used as a tissue extract for an enzyme source of α4GalT(Gal) and β4GalT(Gal).
To obtain microsomal fractions, various tissues were homogenized in nine volumes of 10 mM Tris–HCl (pH 7.4), containing 0.25 M sucrose and 1 mM EDTA. After centrifugation at 7700 × g for 30 min, each supernatant was centrifuged at 105,000 × g for 60 min. The microsomal fraction was obtained as a precipitate and dissolved with appropriate solvents, as indicated below for the following enzyme reactions.
Identification of PA-oligosaccharide structures produced by avian α4GalT(Gal) and β4GalT(Gal) using a two-dimensional mapping technique
To produce α/β-galactosylated PA-oligosaccharides, 10 or 50 mg/mL of proteins from tissue extracts was incubated in a total volume of 25 µL of reaction buffer with 20 mM UDP-Gal, 50–100 µM PA-substrate A (Table I) at 37°C for 16 h, and the reactions were terminated by heating at 100°C for 2 min. After centrifugation, the supernatant was loaded onto a Dowex 50W-X8 column (H+ form, 40 µL), and then onto an AG1-X8 column (CO2−3 form, 30 µL), and the flow-through fractions were collected.
PA-oligosaccharides in the reaction mixture were first separated by HPLC on the Amide-80 column, based on the number of added galactose residues, and then on the ODS column. The separated PA-oligosaccharides were digested with α-galactosidase from green coffee bean, β4-galactosidase from Streptococcus pneumoniae or N-acetyl-β-d-hexosaminidase from jack bean to confirm their structures. Before and after each reaction with an exoglycosidase, GUs on the ODS and Amide-80 columns were determined by the relative elution positions of PA-derivatized isomalto-oligosaccharides of polymerization degree 4–20 and plotted on the x- and y-axes, respectively, as described previously (Tomiya et al. 1988; Takahashi et al. 2001). This step was continued with other exoglycosidases, until the elution positions of the products totally matched those of the reference compound (Table I).
Assays of α/β4GalT(Gal) and β4GalT(GlcNAc)
To compare the activities of GalTs in avian tissues, the following assay was performed: the microsomal fractions from various tissues were suspended in lysis buffer, and the solution was filtered through a 0.45 µm membrane and used as an enzyme source. Blood was treated with 3.2% citric acid, and blood plasma was used as an enzyme source. The reaction mixture contained the following components in a total volume of 25 µL of reaction buffer: 20 mM UDP-Gal, 4 µM PA-substrate A or 6 µM PA-substrate B and 1 mg protein/mL of enzyme source for PA-substrate A or 0.25 mg protein/mL of enzyme source for PA-substrate B. Incubations were carried out at 37°C for 2 h, and the reaction was terminated by heating at 100°C for 2 min. If enzyme activities were not detected in microsomal fractions, the GalT assay was carried out under the same conditions, except that incubations were performed at 37°C for 4 h, using 30 mg protein/mL of tissue extract. After centrifugation, the supernatant was loaded onto a Dowex 50W-X8 column (40 µL), and then onto an AG1-X8 column (30 µL), and the flow-through fraction was collected. For the α4GalT(Gal) and the β4GalT(Gal) assays, products from PA-substrate A were separated by HPLC on the Amide-80 column and analyzed by HPLC on the ODS column. For the β4GalT(GlcNAc) assay, products from PA-substrate B were analyzed by HPLC on the Amide-80 column.
To analyze the pH dependency of the activity of GalTs, the following assay was performed: the microsomal fraction from liver was dissolved in 10 mM HEPES (pH 6.5), containing 1% Triton X-100, 20 mM MnCl2, 1 mM PMSF and 1 µg/mL of leupeptin. The homogenate was centrifuged at 17,400 × g for 5 min, and the supernatant was filtered through a 0.45 µm membrane and used as an enzyme source. The reaction mixture contained the following components in a total volume of 25 µL: 100 mM HEPES (pH 8.0, 7.5 or 7.0) or 100 mM MES (pH 7.0, 6.5, 6.0 or 5.5), 1% Triton X-100, 5 mM d-galactonic acid γ-lactone, 0.2 mM deoxyfuconojirimycin, 20 mM UDP-Gal, 4 µM PA-substrate A or 6 µM PA-substrate B and 2 mg/mL of enzyme source for PA-substrate A or 1 mg/mL of enzyme source for PA-substrate B. Incubations were carried out at 37°C for 2 h, and the reaction was terminated by heating at 100°C for 2 min. After the treatment with ion-exchange columns, the samples were analyzed by HPLC on the ODS column when PA-substrate A was used or analyzed by HPLC on the Amide-80 column when PA-substrate B was used.
For analyzing the effect of divalent cations on the activity of GalTs, the microsomal fraction from liver was dissolved in 100 mM HEPES (pH 7.5), containing 1% Triton X-100, 1 mM PMSF and 1 µg/mL of leupeptin, without 20 mM MnCl2. The GalT assay was carried out in 100 mM HEPES (pH 7.5), following the same procedures used to analyze the pH dependencies of the activity of GalTs, except that 20 mM of various divalent cation chlorides or 20 mM EDTA, were added instead of 20 mM MnCl2.
Preparation of the PA-oligosaccharide standard produced by mouse α3GalT(Gal)
cDNA encoding mouse α3GalT(Gal) (Joziasse et al. 1992) was amplified by polymerase chain reaction with two primers, 5′-CCATGTCAACAAGATCTCCATG-3′ and 5′-CAAGTGTT GCTACTTGTTTGAGG-3′, using cDNA from mouse F9 cells. The amplified fragments were inserted into the SmaI site of pBlueScriptII(SK+) (Stratagene, La Jolla, CA) to construct pBS-α3GalT. The pBS-α3GalT plasmid was digested with HindIII and NotI and the released fragment was inserted into pRc/CMV (Invitrogen, Carlsbad, CA) to construct pRc/CMV-α3GalT. pRc/CMV-α3GalT was transfected into 293T cells with Lipofectamine 2000 (Invitrogen). PA-glycan I (Table I) was produced from PA-substrate A in the transfected cell lysate, in 100 mM MES (pH 6.5), 1% Triton X-100, 20 mM MnCl2 and purified by HPLC. The GU on ODS and Amide-80 columns of PA-glycan I was recorded as shown in Table I.
Methylation analysis and gas chromatography–MS
To prepare partially methylated alditol acetates, glycans were permethylated using the NaOH/dimethyl sulfoxide slurry method, as described previously (Dell et al. 1994), followed by hydrolysis (2 M trifluoroacetic acid at 100°C for 4 h), reduction (10 mg/mL of NaBD4 in 1 M NH4OH at room temperature for 2 h) and acetylation (50% acetic anhydride and 50% pyridine at 100°C for 2 h). Gas chromatography–MS was carried out using a GCMS-QP5050 (Shimazu, Tokyo, Japan). Each sample was dissolved in chloroform before splitless injection into an HP-5MS column (30 m × 0.25 mm internal diameter, Agilent Technologies, Palo Alto, CA). The column head pressure was maintained at 56.7 kPa to give a constant flow rate of 1 mL/min using helium as the carrier gas. The initial oven temperature was held at 60°C for 1 min and increased to 90°C over 1 min and then to 290°C over 25 min.
1H-NMR. measurement
Prior to 1H-NMR spectroscopic analysis, lyophilized PA-oligosaccharides (ca. 100 nmol) were dissolved in 99.96% D2O. After repeating the exchange treatments more than twice, the lyophilized samples were redissoloved in 99.96% D2O. 1H-NMR spectroscopy was performed on a Bruker AVANCE-600 Spectrometer (1H frequency: 600.13 MHz) at 15°C and 27°C. The chemical shift of acetone was set at 2.218 ppm relative to 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt.
Electrophoresis and western blotting
Electrophoresis was performed under reducing conditions in a 12.5% sodium dodecyl sulfate–polyacrylamide gel using 20 µg of protein/lane for each tissue homogenate. The separated proteins were transferred to PVDF membranes and then detected with CBB or antibody/lectin staining. For antibody/lectin staining, the membranes were soaked in blocking solution, which consisted of Tris-buffered saline (TBS; 20 mM Tris–HCl (pH 7.5) with 150 mM NaCl) containing 3% (w/v) bovine serum albumin (for lectin/anti-(Galβ1-4Gal) mAb-staining) or 5% (w/v) skim milk (for anti-P1 mAb-staining). The blot-membranes were first incubated with anti-P1 mAb (for detection of Galα1-4Galβ1-4GlcNAc), anti-(Galβ1-4Gal) mAb or AP-labeled ECA (for detection of Galβ1-4GlcNAc). After washing with 0.1% Tween-20 in TBS, the membranes were incubated with AP-labeled secondary antibodies for antibody staining.
This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (18770081) and by the Hamaguchi Foundation for the Advancement of Biochemistry.
The authors thank Prof. Yuan C. Lee (Department of Biology, Johns Hopkins University) for his encouragement during the early days of the study, and Dr. Fumiko Matsumura (Department of Medical Genome Science, University of Tokyo) for her technical advice on NMR spectroscopy.
References
Amado M, Almeida R, Schwientek T, Clausen H. Identification and characterization of large galactosyltransferase gene families: Galactosyltransferases for all functions. Biochim Biophys Acta. 1999;1473:35–53. doi: 10.1016/s0304-4165(99)00168-3. [DOI] [PubMed] [Google Scholar]
Bouhours D, Liaigre J, Naulet J, Bovin NV, Bouhours JF. A novel pentaglycosylceramide in ostrich liver, IV4-β-Gal-nLc4Cer, with terminal Gal(β1-4)Gal, a xenoepitope recognized by human natural antibodies. Glycobiology. 2000;10:857–864. doi: 10.1093/glycob/10.9.857. doi:10.1093/glycob/10.9.857. [DOI] [PubMed] [Google Scholar]
Coppin A, Florea D, Maes E, Cogálniceanu D, Strecker G. Comparative study of carbohydrate chains released from the oviducal mucins of the two very closely related amphibian species Bombina bombina and Bombina variegata. Biochimie. 2003;85:53–64. doi: 10.1016/s0300-9084(03)00021-x. doi:10.1016/S0300-9084(03)00021-X. [DOI] [PubMed] [Google Scholar]
Coppin A, Maes E, Morelle W, Strecker G. Structural analysis of 13 neutral oligosaccharide-alditols released by reductive β-elimination from oviducal mucins of Rana temporaria. Eur J Biochem. 1999;266:94–104. doi: 10.1046/j.1432-1327.1999.00823.x. doi:10.1046/j.1432-1327.1999.00823.x. [DOI] [PubMed] [Google Scholar]
Cossey A, Dimichiel A, Dunstone J. The use of equilibrium density-gradient ultracentrifugation in the isolation and characterisation of glycoproteins with blood group P1 activity from sheep hydatid-cyst fluid. Eur J Biochem. 1979;98:53–60. doi: 10.1111/j.1432-1033.1979.tb13159.x. doi:10.1111/j.1432-1033.1979.tb13159.x. [DOI] [PubMed] [Google Scholar]
Dell A, Reason AJ, Khoo K-H, Panico M, McDowell RA, Morris HR. Mass spectrometry of carbohydrate-containing biopolymers. In: Lennarz WJ, Hart GW, editors. Methods Enzymology. San Diego (CA): Academic Press; 1994. pp. 108–132. [DOI] [PubMed] [Google Scholar]
Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem. 1956;28:350–356. doi:10.1021/ac60111a017. [Google Scholar]
Florea D, Maes E, Haddad M, Strecker G. Structural analysis of the oligosaccharide alditols released from the jelly coat of Rana dalmatina eggs by reductive β-elimination. Biochimie. 2002;84:611–624. doi: 10.1016/s0300-9084(02)01432-3. doi:10.1016/S0300-9084(02)01432-3. [DOI] [PubMed] [Google Scholar]
Gagneux P, Varki A. Evolutionary considerations in relating oligosaccharide diversity to biological function. Glycobiology. 1999;9:747–755. doi: 10.1093/glycob/9.8.747. doi:10.1093/glycob/9.8.747. [DOI] [PubMed] [Google Scholar]
Guerardel Y, Chang LY, Maes E, Huang CJ, Khoo KH. Glycomic survey mapping of zebrafish identifies unique sialylation pattern. Glycobiology. 2006;16:244–257. doi: 10.1093/glycob/cwj062. doi:10.1093/glycob/cwj062. [DOI] [PubMed] [Google Scholar]
Inoue S, Iwasaki M, Ishii K, Kitajima K, Inoue Y. Isolation and structures of glycoprotein-derived free sialooligosaccharides from the unfertilized eggs of Tribolodon hakonensis, a dace. Intracellular accumulation of a novel class of biantennary disialooligosaccharides. J Biol Chem. 1989;264:18520–18526. [PubMed] [Google Scholar]
Iwasaki M, Inoue S. Structures of the carbohydrate units of polysialoglycoproteins isolated from the eggs of four species of salmonid fishes. Glycoconjugate J. 1985;2:209–228. doi:10.1007/BF01049269. [Google Scholar]
Iwasaki M, Inoue S, Kitajima K, Nomoto H, Inoue Y. Novel oligosaccharide chains on polysialoglycoproteins isolated from rainbow trout eggs. A unique carbohydrate sequence with a sialidase-resistant sialyl group, dGa1NAcβ1→ 4(NeuGc2→3) dGa1NAc. Biochemistry. 1984;23:305–310. doi: 10.1021/bi00297a020. doi:10.1021/bi00297a020. [DOI] [PubMed] [Google Scholar]
Iwasaki M, Seko A, Kitajima K, Inoue Y, Inoue S. Fish egg glycophosphoproteins have species-specific N-linked glycan units previously found in a storage pool of free glycan chains. J Biol Chem. 1992;267:24287–24296. [PubMed] [Google Scholar]
Joziasse DH, Shaper NL, Kim D, Van den Eijnden DH, Shaper JH. Murine α1,3-galactosyltransferase. A single gene locus specifies four isoforms of the enzyme by alternative splicing. J Biol Chem. 1992;267:5534–5541. [PubMed] [Google Scholar]
Kaplan HA, Woloski BM, Hellman M, Jamieson JC. Studies on the effect of inflammation on rat liver and serum sialyltransferase. Evidence that inflammation causes release of Galβ1→4GlcNAc α2→6 sialyltransferase from liver. J Biol Chem. 1983;258:11505–11509. [PubMed] [Google Scholar]
Khoo KH, Nieto A, Morris HR, Dell A. Structural characterization of the N-glycans from Echinococcus granulosus hydatid cyst membrane and protoscoleces. Mol Biochem Parasitol. 1997;86:237–248. doi: 10.1016/s0166-6851(97)00036-4. doi:10.1016/S0166-6851(97)00036-4. [DOI] [PubMed] [Google Scholar]
Kondo A, Suzuki J, Kuraya N, Hase S, Kato I, Ikenaka T. Improved method for fluorescence labeling of sugar chains with sialic acid residues. Agric Biol Chem. 1990;54:2169–2170. [PubMed] [Google Scholar]
Lo NW, Shaper JH, Pevsner J, Shaper NL. The expanding β4-galactosyltransferase gene family: Messages from the databanks. Glycobiology. 1998;8:517–526. doi: 10.1093/glycob/8.5.517. doi:10.1093/glycob/8.5.517. [DOI] [PubMed] [Google Scholar]
Maes E, Florea D, Delplace F, Lemoine J, Plancke Y, Strecker G. Structural analysis of the oligosaccharide-alditols released by reductive β-elimination from oviducal mucins of Rana temporaria. Glycoconj J. 1997;14:127–146. doi: 10.1023/a:1018577302255. doi:10.1023/A:1018577302255. [DOI] [PubMed] [Google Scholar]
Mindell DP, Sorenson MD, Huddleston CJ, Miranda HC, Knight A, Sawchuk SJ, Yuri T. Phylogenetic relationships among and within select avian orders based on mitochondrial DNA. In: Mindell DP, editor. Avian Molecular Evolution and Systematics. San Diego (CA): Academic Press; 1997. pp. 213–247. [Google Scholar]
Moriguchi K, Takemoto T, Aoki T, Nakakita S, Natsuka S, Hase S. Free oligosaccharides with Lewis x structure expressed in the segmentation period of zebrafish embryo. J Biochem (Tokyo) 2007;142:213–227. doi: 10.1093/jb/mvm128. [DOI] [PubMed] [Google Scholar]
Mourad R, Morelle W, Neveu A, Strecker G. Diversity of O-linked glycosylation patterns between species. Characterization of 25 carbohydrate chains from oviducal mucins of Rana ridibunda. Eur J Biochem. 2001;268:1990–2003. doi: 10.1046/j.1432-1327.2001.02071.x. doi:10.1046/j.1432-1327.2001.02071.x. [DOI] [PubMed] [Google Scholar]
Nakagawa H, Kawamura Y, Kato K, Shimada I, Arata Y, Takahashi N. Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography. Anal Biochem. 1995;226:130–138. doi: 10.1006/abio.1995.1200. doi:10.1006/abio.1995.1200. [DOI] [PubMed] [Google Scholar]
Sarnesto A, Kohlin T, Thurin J, Blaszczyk-Thurin M. Purification of H gene-encoded β-galactoside α1→2fucosyltransferase from human serum. J Biol Chem. 1990;265:15067–15075. [PubMed] [Google Scholar]
Shimamura M, Endo T, Inoue Y, Inoue S. A novel neutral oligosaccharide chain found in polysialoglycoproteins isolated from Pacific salmon eggs. Structural studies by secondary ion mass spectrometry, proton nuclear magnetic resonance spectroscopy, and chemical methods. Biochemistry. 1983;22:959–963. doi: 10.1021/bi00273a039. doi:10.1021/bi00273a039. [DOI] [PubMed] [Google Scholar]
Sibley CG, Ahlquist JE. Phylogeny and Classification of Birds: A Study in Molecular Evolution. New Haven (CT): Yale University Press; 1990. [Google Scholar]
Slomiany A, Slomiany BL, Horowitz MI. Structural study of the blood group A active glycolipids of hog gastric mucosa. J Biol Chem. 1974;249:1225–1230. [PubMed] [Google Scholar]
Suzuki N, Khoo KH, Chen CM, Chen HC, Lee YC. N-Glycan structures of pigeon IgG: A major serum glycoprotein containing Galα1-4Gal termini. J Biol Chem. 2003;278:46293–46306. doi: 10.1074/jbc.M307132200. doi:10.1074/jbc.M307132200. [DOI] [PubMed] [Google Scholar]
Suzuki N, Khoo KH, Chen HC, Johnson JR, Lee YC. Isolation and characterization of major glycoproteins of pigeon egg white: Ubiquitous presence of unique N-glycans containing Galα1-4Gal. J Biol Chem. 2001;276:23221–23229. doi: 10.1074/jbc.M101379200. doi:10.1074/jbc.M101379200. [DOI] [PubMed] [Google Scholar]
Suzuki N, Laskowski M, Jr., Lee YC. Phylogenetic expression of Galα1-4Gal on avian glycoproteins: Glycan differentiation inscribed in the early history of modern birds. Proc Natl Acad Sci USA. 2004;101:9023–9028. doi: 10.1073/pnas.0402822101. doi:10.1073/pnas.0402822101. [DOI] [PMC free article] [PubMed] [Google Scholar]
Suzuki N, Laskowski M, Jr, Lee YC. Tracing the history of Galα1-4Gal on glycoproteins in modern birds. Biochim Biophys Acta. 2006;1760:538–546. doi: 10.1016/j.bbagen.2005.10.005. [DOI] [PubMed] [Google Scholar]
Suzuki N, Lee YC. Glycophylogeny of Galα1-4Gal in avian egg glycoproteins. In: Kamerling JP, Boons G-J, Lee YC, Suzuki A, Taniguchi N, Voragen AGJ, editors. Comprehensive Glycoscience: From Chemistry to Systems Biology. Amsterdam (Boston): Elsevier; 2007. pp. 237–251. [Google Scholar]
Suzuki N, Su TH, Wu SW, Yamamoto K, Khoo KH, Lee YC. Structural analysis of N-glycans from gull egg white glycoproteins and egg yolk IgG. Glycobiology. 2009;19:693–706. doi: 10.1093/glycob/cwp025. doi:10.1093/glycob/cwp025. [DOI] [PMC free article] [PubMed] [Google Scholar]
Suzuki N, Yamamoto K. Molecular cloning of pigeon UDP-galactose: β-d-galactoside α1,4-galactosyltransferase and UDP-galactose:β-d-galactoside β1,4-galactosyltransferase, two novel enzymes catalyzing the formation of Galα1-4Galβ1-4Galβ1-4GlcNAc sequence. J Biol Chem. 2010;285:5178–5187. doi: 10.1074/jbc.M109.018663. doi:10.1074/jbc.M109.018663. [DOI] [PMC free article] [PubMed] [Google Scholar]
Taguchi T, Iwasaki M, Muto Y, Kitajima K, Inoue S, Khoo KH, Morris HR, Dell A, Inoue Y. Occurrence and structural analysis of highly sulfated multiantennary N-linked glycan chains derived from a fertilization-associated carbohydrate-rich glycoprotein in unfertilized eggs of Tribolodon hakonensis. Eur J Biochem. 1996;238:357–367. doi: 10.1111/j.1432-1033.1996.0357z.x. doi:10.1111/j.1432-1033.1996.0357z.x. [DOI] [PubMed] [Google Scholar]
Taguchi T, Seko A, Kitajima K, Inoue S, Iwamatsu T, Khoo KH, Morris HR, Dell A, Inoue Y. Structural studies of a novel type of tetraantennary sialoglycan unit in a carbohydrate-rich glycopeptide isolated from the fertilized eggs of Indian Medaka fish, Oryzias melastigma. J Biol Chem. 1993;268:2353–2362. [PubMed] [Google Scholar]
Taguchi T, Seko A, Kitajima K, Muto Y, Inoue S, Khoo KH, Morris HR, Dell A, Inoue Y. Structural studies of a novel type of pentaantennary large glycan unit in the fertilization-associated carbohydrate-rich glycopeptide isolated from the fertilized eggs of Oryzias latipes. J Biol Chem. 1994;269:8762–8771. [PubMed] [Google Scholar]
Takahashi N, Khoo KH, Suzuki N, Johnson JR, Lee YC. N-Glycan structures from the major glycoproteins of pigeon egg white: Predominance of terminal Galα(1-4)Gal. J Biol Chem. 2001;276:23230–23239. doi: 10.1074/jbc.M101380200. doi:10.1074/jbc.M101380200. [DOI] [PubMed] [Google Scholar]
Tomiya N, Awaya J, Kurono M, Endo S, Arata Y, Takahashi N. Analyses of N-linked oligosaccharides using a two-dimensional mapping technique. Anal Biochem. 1988;171:73–90. doi: 10.1016/0003-2697(88)90126-1. doi:10.1016/0003-2697(88)90126-1. [DOI] [PubMed] [Google Scholar]
Ui N, Tarutani O. Purification of hog thyroglobulin. J Biochem (Tokyo) 1961;50:508–518. doi: 10.1093/oxfordjournals.jbchem.a127483. [DOI] [PubMed] [Google Scholar]
Loss of T-synthase (uridine diphosphate galactose:N-acetylgalactosaminyl-α1-Ser/Thr β3galactosyltransferase), a key enzyme required for the formation of mucin-type core 1 O-glycans, is observed in several human diseases, including cancer, Tn syndrome and IgA nephropathy, but current methods to assay the enzyme use radioactive substrates and complicated isolation of the product. Here we report the development of a novel fluorescent assay to measure its activity in a variety of tumor cell lines. Deficiencies in T-synthase activity correlate with mutations in the gene encoding the molecular chaperone Cosmc that is required for folding the T-synthase. This new high-throughput assay allows for facile screening of tumor specimens and other biological material for T-synthase activity and could be used diagnostically.
The Tn antigen is a normal endogenous substrate for several enzymes, most notably the T-synthase (uridine diphosphate galactose:N-acetylgalactosaminyl-α1-Ser/Thr β3galactosyltransferase), a β3-galactosyltransferase that transfers d-galactose (Gal) from uridine diphosphate galactose (UDP-Gal) to the Tn antigen to form the core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr (Ju, Brewer et al. 2002; Ju and Cummings 2005; Ju, Lanneau et al. 2008). A unique feature of vertebrate T-synthase is its requirement for a specific molecular chaperone, termed Cosmc, to promote correct folding in vivo (Ju and Cummings 2002; Ju, Aryal et al. 2008). Deficiencies of the T-synthase activity have been linked to acquired, somatic mutations in the X-linked Cosmc (Ju and Cummings 2005; Ju, Lanneau et al. 2008).
Typical assays of glycosyltransferases are difficult, since they commonly utilize radioactive nucleotide sugar donors. T-synthase activity is commonly assayed using radiolabeled UDP-Gal by measuring the 3H- or 14C-Gal incorporated into acceptor glycopeptides, phenyl-α-GalNAc or benzyl-α-GalNAc, followed by separation techniques to remove unreacted nucleotide sugars (Mendicino et al. 1982; Furukawa and Roth 1985; Granovsky et al. 1994; Ju, Cummings et al. 2002; Ju and Cummings 2010). Such approaches are time-consuming and impractical, especially for high-throughput screening. To address this problem, we have developed a sensitive fluorescent method for assessing T-synthase activity and show its utility to quantify T-synthase activity in a variety of biological samples. We also genetically characterize several human Jurkat leukemic cell lines with regard to mutations in the Cosmc gene and the effects on T-synthase activity.
Results
GalNAc-α-(4-methylumbelliferone) as acceptor for T-synthase
The potential assay method for T-synthase utilizes GalNAc-α-(4-methylumbelliferone) (GalNAc-α-(4-MU)) as its acceptor substrate and UDP-Gal as a donor to form Galβ1-3GalNAc-α-(4-MU) (Figure 1A). The reaction product is cleaved by endo-α-N-acetylgalactosaminidase (O-glycosidase) to release free 4-MU, which is highly fluorescent (Figure 1A). This enzyme is specific for the release of the Galβ1-3GalNAc disaccharide from O-glycans linked to aglycones (Kobata 1979). The experimental procedure requires three simple steps (Figure 1B).
Schematic illustration of the fluorescent assay for T-synthase activity. (A) The principle of the method: T-synthase utilizes GalNAc-α-(4-MU) as its acceptor substrate and UDP-Gal as a donor to form Galβ1-3GalNAc-α-(4-MU), which is subsequently quantitatively cleaved by O-glycosidase to release highly fluorescent 4-MU. The fluorescence intensity of 4-MU represents the amount of T-synthase product. (B) The procedure of the fluorescent T-synthase assay: reaction mixtures including the acceptor, donor or H2O, divalent cation, detergent, buffer and O-glycosidase are prepared and aliquotted into the 96-well black plate, and cell extracts or sera are added to the corresponding wells. The reaction is incubated at 37°C for a certain period of time, the stop solution is added, the fluorescence of 4-MU is measured and the activity of T-synthase is directly calculated in terms of pmol product over time and per protein concentration.
To define the feasibility of this assay, we tested reaction mixtures containing GalNAc-α-(4-MU) as an acceptor and either purified human recombinant T-synthase co-expressed with Cosmc in Hi-5 cells or extracts from these cells (Figure 2A). The fluorescent intensity in the reaction is high in the presence of all four critical components (acceptor, donor, T-synthase and O-glycosidase) and in the presence of ∼1500 pmol/h purified enzyme, as expected, generated much higher relative fluorescence units (RFU; ∼900,000) than that in the presence of ∼500 pmol/h enzyme (∼300,000) from the cell extracts. Importantly, the signal from the blank (no UDP-Gal) is very low and the background fluorescence levels (reactions without O-glycosidase, without enzyme or with enzyme alone) are also very low. These results show that GalNAc-α-(4-MU) is an acceptor for T-synthase and that the signal of 4-MU is derived from the enzymatic hydrolysis of the T-synthase product.
GalNAc-α-(4-MU) can serve as an acceptor for T-synthase. (A) Fluorescent intensity: 4-MU fluorescence from the reactions, blanks and backgrounds using both Hi-5 cell extracts containing 500 pmol/h human recombinant T-synthase and 1500 pmol/h purified recombinant T-synthase as enzyme source. The experiments were performed in duplicate. The inset shows the graph of RFU up to 9000. (B–G) LC-MS data of the acceptor and the T-synthase product: T-synthase reactions with and without the donor UDP-Gal were set-up in the absence of O-glycosidase and incubated overnight at 37°C. The reactions were chromatographed on C18 cartridges, and the bound material was eluted and dried, then redissolved in 50 μL of H2O and analyzed on LC-MS equipped with a C18 column. (B) Acceptor GalNAc-α-(4-MU) standard; (C) the C18 cartridge-purified material from the blank reaction (without UDP-Gal); (D) the standard 4-MU; (E) sample from (C) treated with O-glycosidase; (F) the C18 cartridge-purified material from the T-synthase reaction (with all of the components); the inset shows an expansion of the three major peaks; (G) sample from (F) treated with O-glycosidase.
To further characterize the reaction, all compounds from the overnight reactions in the absence of O-glycosidase were purified on C18 cartridges, and then analyzed on liquid chromatography-mass spectrometry (LC-MS). The standard GalNAc-α-(4-MU) was eluted as a single peak at 9.14 min (Figure 2B). The C18-purified material from the blank reaction without UDP-Gal gave a major peak with elution time at 9.14 and minor peak at 10.75 min, representing the acceptor substrate and 4-MU, respectively (Figure 2C), as confirmed by the 4-MU standard (Figure 2D). The free 4-MU likely results from the hydrolysis of GalNAc-α-(4-MU) by endogenous lysosomal α-N-acetylhexosaminidase overnight incubation, which is consistent with the observation (Figure 2A) that the reaction using Hi-5 cell extracts gave a higher background than the purified enzyme. The monosaccharide GalNAc [N-acetylgalactosamine (2-acetamido-2-deoxy-Gal)] was lost during purification using C18 cartridge, because it does not bind to the C18 cartridge. Since those compounds were detected by LC-MS, it is difficult to precisely quantify each of them; therefore, we use the terms “major” or “minor” to refer to their abundance. These results indicate that the acceptor GalNAc-α-(4-MU) is stable except for very minor hydrolysis by the presumed endogenous lysosomal glycosidase (Figure 2C). O-Glycosidase treatment did not change the profile of this material, indicating that this O-glycosidase lacks significant exoglycosidase activity (Figure 2E). The material purified from the reaction in the presence of UDP-Gal gave a major peak at elution time 8.88 min, which was the expected product of T-synthase, Galβ1-3GalNAc-α-(4-MU) (Figure 2F), and two other peaks, the unreacted acceptor and 4-MU (Figure 2C). Furthermore, after treatment with O-glycosidase, the product peak Galβ1-3GalNAc-α-(4-MU) disappeared, and one new peak emerged at 0.63 min corresponding to Galβ1-3GalNAc, which is hydrophilic, and was eluted in the void volume, along with increased free 4-MU and unchanged acceptor (Figure 2G). O-Glycosidase specifically cleaves core 1 O-glycan to release Galβ1-3GalNAc from glycopeptides, glycoproteins and artificial carriers (Kobata 1979; Fujita et al. 2005; Goda et al. 2008; Suzuki et al. 2009; Willis et al. 2009). Thus, the disaccharide structure Galβ1-3GalNAc of the product is based on both the co-chromatography with standard disaccharide and the specificity of the O-glycosidase. Taken together, these results demonstrate that GalNAc-α-(4-MU) is an acceptor substrate for T-synthase and the assay is specific for T-synthase.
Product characterization
Although the results from Figure 2 showed that the product from the T-synthase reaction was Galβ1-3GalNAc-α-(4-MU), as indicated by the LC-MS profile and specific cleavage by O-glycosidase, we also sequenced the products by electrospray ionization-mass spectrometry (ESI-MS)/MS. The profile of standard GalNAc-α-(4-MU) displayed a peak of 380 Da representing the intact molecule, as well as fragments with smaller mass such as 43 Da (acetyl group), 204.2 Da (acetylgalactosaminyl group) and others (Figure 3A). The material from the blank reaction (without UDP-Gal) regardless of treatment with O-glycosidase showed the same profiles (Figure 3B and C) as the standard (Figure 3A). The product purified from T-synthase reaction appeared with the expected size of 542.0 Da, corresponding to Galβ1-3GalNAc-α-(4-MU) (Figure 3D). The MS/MS profile gave 380, 366.0 and 177.0 Da fragments, which are presumably the acceptor, dehydrated disaccharide and 4-MU respectively. All other fragments from the GalNAc-α-(4-MU) were also seen, comparable with Figure 3A–C. After treatment of O-glycosidase, the 542.0 Da product disappeared, and the disaccharide (384 Da) and 4-MU (177.0 Da) appeared. The MS/MS confirmed that this 384 Da peak was Galβ1-3GalNAc with the correct fragmentation pattern, including the GalNAc residue (222.2 Da), dehydrated GalNAc residue (204.2 Da) and even double dehydrated GalNAc (186.2 Da; Figure 3E). The 177.0 Da peak corresponds to 4-MU (Figure 3F) with the expected fragmentation pattern. The galactose residue from the product was not seen by MS/MS, probably because free galactose is not ionized well in MS, unlike its amino sugar galactosamine. These results demonstrate that T-synthase synthesizes Galβ1-3GalNAc-α-(4-MU) from the acceptor GalNAc-α-(4-MU) and donor UDP-Gal and that O-glycosidase specifically cleaves the product, but not GalNAc-α-(4-MU).
Characterization of the product by ESI-MS/MS: the standard GalNAc-α-(4-MU) and purified material from T-synthase reactions as in Figure 2C, D, F and G were subjected to ESI-MS/MS analyses. (A) The fragmentation of standard acceptor GalNAc-α-(4-MU) (380.0 Da); (B) The fragmentation profile of the C18 cartridge-purified material (380.2 Da) from the blank reaction (without UDP-Gal); (C) the profile of sample in (B) treated with O-glycosidase; (D) the fragmentation profile of C18 cartridge-purified material from the T-synthase reaction (with all of the components); (E) the MS/MS data of disaccharide (384.0 Da) from the treatment of the product (542.0 Da) with O-glycosidase; (F) the MS/MS data of 4-MU (177.0 Da) from the product (542.0 Da) treated with O-glycosidase.
Determining amount of O-glycosidase required in the assay
The T-synthase activity is quantified by the fluorescent intensity of 4-MU released from its product by O-glycosidase; thus, the accuracy of this assay relies on the sufficiency of the O-glycosidase in the reaction. In the presence of different amounts of O-glycosidase, free 4-MU intensity was linear within a range of 0–200 units of O-glycosidase, indicating that 200 units O-glycosidase is insufficient to cleave all product of T-synthase (Figure 4A). With the amount of O-glycosidase above 400 units per reaction, the 4-MU intensity approached a plateau, which reflects the activity (∼400 pmol/h, calculated based on 1 pmol 4-MU = 600 RFU as determined below) of T-synthase. Therefore, 800 units of O-glycosidase were used as the standard assay, in which the T-synthase, range of 0–800 pmol/h can be measured accurately. Within the preparation of O-glycosidase, there are only traces of exo-α-N-acetylhexosaminidase activity, which does not disturb this assay. These results not only define the amount of O-glycosidase required for this method, but also demonstrate that the assay specifically reflects the activity of T-synthase.
Characterization of the method: (A) O-glycosidase curve: the T-synthase reactions were set-up in the presence of different amounts (0–800 U) of O-glycosidase. The reactions were incubated at 37°C for 60 min, and the stop solution was added and the fluorescence united was measured. RFU of 4-MU was plotted versus the O-glycosidase concentration. (B) Standard curve of 4-MU: 50 μL of a serial concentration of standard 4-MU solutions ranging from 10 to 20,000 nM in triplicate were pipetted into plates and incubated at 37°C for 60 min and 100 μL of stop solution was added. The RFU were measured and plotted with corresponding concentrations of 4-MU. (C) Enzyme concentration curve: a serial dilution of the cell extracts from Hi-5 cell co-expressing human recombinant T-synthase and wild-type Cosmc were incubated with the reaction mixture. The 4-MU fluorescence was measured after incubation at 37°C for 60 min and plotted with the enzyme concentration. (D) Time course: the reaction with Hi-5 cell extracts containing human recombinant T-synthase was set up in the same tube and 50 μL of reaction mixtures were aliquot into the fluorescent plate. Reactions were incubated at 37°C from 0 to 180 min until addition of stop solution as indicated. The fluorescence of 4-MU was measured at the same time and plotted with the incubation time (min). All of the experiments were performed in duplicates or triplicates.
4-MU standard curve
To determine the specific activity of 4-MU, the sensitivity of the fluorescent method, and the linear range of 4-MU, serial concentrations of 4-MU were measured for their RFU. The lowest detectable concentration of 4-MU in the current assay condition was 20 nM (1 pmol), and the RFU was linear with 4-MU concentration up to 20,000 nM (1000 pmol), giving a specificity of 4-MU around 600 RFU/pmol (Figure 4B, insets).
Kinetics of T-synthase reaction
We also determined the linear range for the time and enzyme concentration. Under the defined assay conditions, the reaction product formation was linear with respect to enzyme concentration from 500 to 60,000 pmol/h-mL (Figure 4C), through 3 h of incubation (Figure 4D).
Comparison to UDP-[3H]-Gal method and sequencing Cosmc in Jurkat I 2.1 and I 9.2 cells
To substitute the common radioactive methods with this new fluorescent assay, we sought to compare the sensitivity of these two approaches using purified T-synthase, renatured and refolded T-synthase by Cosmc in vitro and T-synthase in cell extracts by the two methods in parallel. The purified recombinant T-synthase had comparable activity measured by both methods (Figure 5A). We observed that the radioactive method gave slightly higher activity of this recombinant T-synthase. This difference could be due to the high amount of enzyme or activity that was used in these assays in which any technical variation could result in this difference. Furthermore, by means of this fluorescent assay, thermally denatured recombinant T-synthase lost more than 60% activity; after renaturation by incubating with purified Cosmc, the T-synthase activity was recovered significantly (Figure 5B), consisting with our earlier observation (Aryal et al. 2010). These results indicate that the fluorescent method is a suitable replacement method for the radiochemical approach for assaying T-synthase activity.
Application of the fluorescent method for assaying T-synthase activity: (A) purified recombinant T-synthase: approximately 0.25 µg of purified T-synthase co-expressed with Cosmc in Hi-5 insect cells was assayed for its activity by using the 4-MU fluorescent method and the radioactive method. The experiments were carried out in five replicates and averaged, and the activity of T-synthase (mean ± SD) obtained from both methods was compared. (B) The 4-MU method was used for studying in vitro reconstitution of denatured T-synthase: purified recombinant T-synthase was thermally denatured at 54°C for ∼2 min. Reconstitution was carried out with or without addition of Cosmc and T-synthase activity was measured. The experiment was performed in four replicates and averaged. (C) The activity of T-synthase in the cell extracts from cell lines: the cell extracts were made from the cell lines indicated and assayed for T-synthase activity in triplicates by the 4-MU fluorescent method and the radioactive method. The specific activity of T-synthase from the same cell lines by those two methods was compared side-by-side on bar graphs. (D) Comparison of the Sequence of Cosmc: the genomic DNA from HL60, Jurkat E6.1 and Jurkat clones I 2.1 and I 9.2 were isolated from the cells and the open reading frame of Cosmc was amplified by PCR. The PCR product was purified and subjected to direct sequencing. The mutated region within the Cosmc sequence is shown.
To evaluate the utilization of this fluorescent method for assaying T-synthase activity in mammalian cell extracts, we chose a variety of cell lines with different levels of T-synthase activity. There was good agreement in the results between the two assay approaches, especially for cells containing a moderate level of T-synthase activity, such as Cosmc-transfectants STn positive cell clone from colon adenocarcinoma cells LS174T (LSC)-Cosmc, Jurkat-Cosmc and human melanoma cell line (LOX)-Cosmc cells, as well as HL60 and FEMX-I cells (Figure 5C). Mock-transfected Jurkat E6.1, human colorectal carcinoma LSC and human melanoma LOX cells have little to no T-synthase activity due to mutations in Cosmc resulting in an inactive T-synthase (Ju and Cummings 2002; Ju, Lanneau et al. 2008). Both methods gave similar sensitivity, detecting as low as 1–2 pmol/h of enzyme. Introduction of wild-type Cosmc into these cells restored the T-synthase activity, shown in both methods. However, there were some differences noted in cell lines with very high activity of T-synthase, such as the human colorectal carcinoma cells, STn negative cell clone from colon adenocarcinoma cells LS174T (LSB) and NCI-87. Since the radioactive assays by necessity are performed in lower nucleotide sugar concentrations compared with the fluorescent assay, differences between these cell lines in nucleotide sugar stability and/or product stability could account for some of these differences. In any case, it is important when attempting to define that total T-synthase activity by either method to re-measure activities after an appropriate dilution.
Interestingly, both methods revealed that Jurkat clones I2.1 and I9.2 had very low T-synthase activity, as seen in Jurkat clone E6.1 which has a mutated Cosmc resulting in an inactive T-synthase in these cells in our previous studies (Ju and Cummings 2002). To explore whether clones I2.1 and I9.2 had a mutation in Cosmc and whether the mutation was congruent to the E6.1 clone, we performed polymerase chain reaction (PCR) and sequencing of Cosmc. Although HL60 cells contain wild-type Cosmc, both Jurkat I2.1 and I9.2 cells contained the same mutation in Cosmc as in Jurkat E6.1 (mock-transfected), which has a T-deletion at 478 in its nucleotide sequence (Figure 5D). This mutation results in truncated Cosmc with little chaperone activity for T-synthase as shown in our earlier study (Ju and Cummings 2002). This is the first evidence that all three Jurkat cell clones tested here carry the identical mutation in Cosmc.
To readily enable such measurements, we have developed the fluorescent method for measuring T-synthase activity, which can replace the cumbersome and the expensive radioactive assays. Our results show that T-synthase efficiently utilizes GalNAc-α-(4-MU) as its acceptor substrate and transfers Gal from UDP-Gal to form Galβ1-3GalNAc-α-(4-MU), which can then be specifically and quantitatively hydrolyzed by O-glycosidase to release the highly fluorescent 4-MU (Figure 1A and B). This method is linear over a wide range of enzyme concentrations and reaction time and its sensitivity is comparable with the assay using the radiochemical method and can be used to assay the activity of the enzyme in cell extracts.
Several different methods for T-synthase activity have been used by many groups, but most involve radioactive substrates or donors and/or high performance liquid chromatography (HPLC) or complex separation technologies (Mendicino et al. 1982; Furukawa and Roth 1985; Granovsky et al. 1994; Leppanen et al. 1999; Ju, Cummings et al. 2002; Ju and Cummings 2010). Compared with other methods for measuring T-synthase activity, this fluorescent assay has many significant advantages. The fluorescent assay is more accurate, since the activity is reflected by the enzyme product; although the radiochemical method also measures the product, the calculation is based on the concentration of donor UDP-Gal; therefore, the accuracy of the donor concentration and the quality of both UDP-Gal and UDP-[3H]-Gal are important. This new assays is also much simpler and only requires three steps (Figure 1B). Other methods, such as radioactive methods, require numerous steps involving manipulating the radiolabeled compounds. This new assay is cheaper, since GalNAc-α-(4-MU) is a relatively inexpensive chemical compound, and O-glycosidase is a recombinant bacterial enzyme. In contrast, UDP-[3H or 14C]-Gal is expensive, and the assay requires C18 columns, scintillation vials, scintillation cocktail and radioactive waste disposal. This new assay is also comparable in sensitivity to the radioactive assays, generates little waste, utilizes a plate-based format that is suitable for high-through assays and can be easily performed in typical clinical laboratory conditions. Finally, this new assay might be useful for enzymatically quantifying UDP-Gal in biological fluids, if recombinant T-synthase is provided in the reaction system and endogenous T-synthase is inactivated, e.g. boiling or chemical treatment.
A unique finding in our study was the evidence that several different clones of Jurkat cells, which are commonly used in many types of immunological studies (Binstadt et al. 2000; Abraham and Weiss 2004), all lack T-synthase activity. In particular, we examined Jurkat clones I2.1 (FADD deficient) and I9.2 (caspase-8 deficient), which are cell lines originally established from a wild-type subclone termed Jurkat A3, used for apoptosis studies (Juo et al. 1998, 1999). Using this new assay, as well as the traditional radiochemical assay method, we showed that both I2.1 and I9.2 clones have very low T-synthase activity, similar to what is seen in the originally ATCC-deposited Jurkat clone E6.1. The study of T-synthase activity and Cosmc in these Jurkat clones is important on several levels. Cosmc is encoded by a single exon gene on Xq24 and functions as an endoplasmic reticulum (ER) localized molecular chaperone to prevent aggregation and subsequent proteasomal degradation of newly synthesized T-synthase (Ju, Aryal et al. 2008). We have shown that tumor antigens Tn and sialyl Tn (STn) arise from the mutations in Cosmc in human tumor cell lines, including Jurkat E6.1 and primary human cervical cancer samples (Ju, Lanneau et al. 2008). However, we considered the possibility that the mutation in Cosmc from Jurkat cell clone E6.1 might have resulted spontaneously over the years of in vitro cell culturing, rather than being a mutation in cells from the original patient. Our results demonstrate that all Jurkat cell clones have similar low T-synthase activity and carry the same mutation in Cosmc. The results strongly suggest that the mutation in Cosmc seen in all Jurkat clones may have been present originally in the first established leukemic cell line from the patient. The new assay described here will facilitate rapid screening of all T cell lines, including Jurkat, for T-synthase activity and potential mutations in Cosmc.
Sugar derivatives of 4-MU have been used to assay many exoglycosidases, such as α-glucosidase A (Fensom et al. 1976), α-l-iduronidase (Isemura et al. 1978; Minami et al. 1980), α-galactosidase (Hultberg et al. 1975), α-mannosidase (Ockerman 1969) and α-l-fucosidase (Gramer et al. 1994). 4-MU is advantageous because it is highly fluorescent at pH 10, but derivatives with modified 7-OH groups are nonfluorescent, including derivatives having sugars linked via a glycosidic bond. There have been several assays that utilize 4-MU derivatives as acceptors, such as the use of 4-MU-xylose as an acceptor for galactosyltransferase-I activity assay (Higuchi et al. 1994). However, such assays have not exploited the highly fluorescent nature of free 4-MU. In our study, we show that GalNAc-α-(4-MU) serves as an excellent acceptor for T-synthase to form the product Galβ1-3GalNAc-α-(4-MU) (Figure 2). The O-glycosidase (Fujita et al. 2005; Goda et al. 2008; Suzuki et al. 2009; Willis et al. 2009) specifically and efficiently cleaves the product of T-synthase, which makes the fluorescent assay ideal for assessing T-synthase activity. Interestingly, the recombinant O-glycosidase cloned from Enterococcus faecalis and expressed in Escherichia coli (Koutsioulis et al. 2008) can hydrolyze both unsubstituted core 1 and 3 (GlcNAcβ1-3GalNAcα1-Ser/Thr) O-glycans. However, since UDP-Gal is the only donor substrate supplied in our assay system, Galβ1-3GalNAc-α-(4-MU), the product of T-synthase will be specifically synthesized and subsequently hydrolyzed by the O-glycosidase. Nevertheless, this same assay system could be used to assay the core 3 β1-3 N-acetylglucosaminyltransferase (Core 3 GnT), if UDP-GlcNAc, rather than UDP-Gal is supplied. Our assay may presage the development of additional fluorescent-based assays for other hydrolytic enzymes, such as N-glycanase, and other glycosyltransferases, such as GnT-I through -VI, fucosyltransferases and sialyltransferases, if specific endoglycosidases can be identified to cleave the products and release free 4-MU. In fact, the production of disaccharide products from sugar-4-MU acceptors could be useful in identifying such unique endoglycosidases from various microbes. Moreover, this assay could be adapted to include recombinant, exogenous T-synthase along with the acceptor GalNAc-α-(4-MU), so that the assay could be used to measure O-glycosidase activity and could also be used to measure UDP-Gal levels in biological fluids, such as cytoplasm, Golgi apparatus, ER and sera.
The specificity of the assay for the T-synthase using UDP-Gal as the donor is remarkable. This conclusion is supported by the results from Figure 5C in which three cell line extracts, LOX, LSC and Jurkat were lacking T-synthase activity due to the mutations in Cosmc in these cells. Human colorectal carcinoma LSC cells contain a mutated Cosmc with a T-insertion at position 53 resulting in a 28 amino acid polypeptide with no chaperone function, whereas the melanoma LOX cell line lacks the transcript for Cosmc because of the deletion of its promoter. The activity in those cell lines with a dysfunctional Cosmc was restored only by transfection of wild-type Cosmc (Ju, Lanneau et al. 2008). Conveniently, the O-glycosidase works efficiently during the reaction of the T-synthase at nearly neutral pH, without further purifying the product, changing buffer pH or adding other components to the reaction, which made this assay method even easier, simpler and suitable for high-throughput assays of large quantities of samples. To our knowledge, this is the first report on a fluorescent assay of glycosyltransferase activity that uses a sugar-(4-MU) as an acceptor to generate a fluorescent product. This work will not only facilitate the research and clinical application of assessing the T-synthase activity in biological samples for potential diagnostic purpose, but also promote the development for high-throughput assays using fluorescence measurements for other glycosyltransferases for screening and diagnosis of CDG.
Materials and methods
Materials
UDP-Gal was purchased from Calbiochem (San Diego, CA). GalNAc-α-(4-MU) was purchased from Carbosynth Limited (Berkshire, UK). 7-Hydroxy-(4-MU) was obtained from Acros Organics (Geel, Belgium). Benzyl-α-GalNAc was purchased from Sigma-Aldrich (St Louis, MO). C18 cartridges were obtained from Millipore Corp. (Billerica, MA). Flexigene DNA Kit and QIAquick Gel Extraction Kit were purchased from Qiagen Inc. (Valencia, CA). O-Glycosidase (40,000,000 units/mL) and the Phusion™ High Fidelity PCR Kit were obtained from New England Biolabs (New England, MA).
Cell lines and culture
Human T-lymphoid Jurkat cells [Clone E6-1 (ATCC TIB-152™), I 2.1 (CRL-2572™) and I 9.2 (CRL-2571™)], NCI-N87 and Hi-5 insect cells were originally purchased from ATCC (Manassas, VA). Human melanoma LOX cells were kindly provided by the group of Dr. Oystein Fodstad at the Norwegian Radium Hospital Research Foundation (Oslo, Norway). Human colorectal carcinoma cells, LSC and LSB, were a gift from Dr. Steve Itzkowitz (Mount Sinai School of Medicine, New York). Mammalian cells were cultured in RPMI1640 media containing 10% fetal bovine serum (FBS) in 5% CO2 at 37°C. Jurkat, LOX, LSC and LSB cells were transfected with either empty vector pcDNA3.1(+) or pcDNA3.1(+) expressing human wild-type Cosmc. Cosmc-transfected LOX cells were sorted with fluorescein isothiocyanate (FITC)-labeled peanut agglutinin (PNA) (Sigma-Aldrich) after desialylation on a FACSorter (Becton Dickinson) and maintained in complete media in 5% CO2 at 37°C.
Reaction system
The acceptor GalNAc-α-(4-MU) was dissolved in dimethyl sulfoxide (DMSO) at 40 mM concentration and then adjusted to 10 mM in 0.5 M 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH (pH 6.8) as a stock stored at 4°C. The UDP-Gal was dissolved in water at 10 mM as a stock at −20°C. The O-glycosidase was diluted 1:50 in 25 mM MES-NaOH (pH 6.8) at concentration of 800 units/μL. The 50 μL reaction system containing 1000 μM GalNAc-α-4-(MU), 500 μM UDP-Gal, 20 mM MnCl2, 0.2% Triton X-100, 800 units of O-glycosidase, in 50 mM MES-NaOH buffer (pH 6.8), and an appropriate amount of enzyme was place in a 96-well black plate suitable for fluorescence assay. The blank reaction was set-up by replacing the donor UDP-Gal with H2O in the 50 μL reaction system. The background was set up with everything except O-glycosidase and/or enzyme. The reactions were incubated at 37°C for 60 min or the time indicated. Then, 100 μL of 1.0 M glycine-NaOH (pH 10.0) was added to each well to stop the reaction, and the relative fluorescence intensity or RFU were measured on a Victor Multiple-Label Counter (PerkinElmer, Waltham, MA) using umbelliferone mode, e.g. Ex 355 nM and Em 460 nm. For the concentration of enzyme, the cell extracts from Hi-5 cells expressing human recombinant T-synthase and wild-type Cosmc were serially diluted with tris-buffered saline (TBS) containing 0.5% Triton X-100 and protease inhibitor cocktail. For the time course experiment, the reaction was set up in the same tube and 50 μL reactions were aliquotted into the plate. Stop solution was added to the time zero wells immediately, and the plate was incubated at 37°C. At time points of 15, 30, 45, 60, 90, 120, 150 and 180 min, the stop solution was added to the corresponding wells, and the RFU for all time points was determined.
O-Glycosidase concentration dependence
The T-synthase reactions were set up as described in the Reaction system section in the time course, except for the different amounts of O-glycosidase (0–800 units) added. The reactions were incubated at 37°C for 60 min, the stop solution was added and the fluorescence was measured.
Standard curve for 4-MU
4-MU was dissolved in DMSO at 1.0 mM, and the concentrations of 10, 50, 100, 200, 500, 1000, 2000, 5000 10,000 and 20,000 nM were made by dilution with 50 mM MES-NaOH (pH 6.8). Then, 50 μL of 4-MU of each concentration was transferred into plates in triplicates and 100 μL of stop solution was added. The fluorescence units were measured on the Victor Multiple-Label Reader.
Preparation of cell extracts
Cell pellets of Hi-5 cells coexpressing human recombinant T-synthase and Cosmc, mock-transfected LSC, Cosmc-transfected LSC, LSB and NCI-N87 cells, mock-transfected and Cosmc-transfected LOX cells, mock-transfected and Cosmc-transfected Jurkat cells (Clone E6-1) and Jurkat (clones I2.1 and I9.2) cells were suspended in an appropriate volume (1:8, v:v) of TBS containing Complete-Mini protease inhibitor cocktail and sonicated on ice using the micro-tip for 3 s five times. The cell post-nuclear supernatant was obtained by centrifugation at 1000 × g at 4°C for 5 min. The cell extracts were prepared by adding Triton X-100 0.5% (final concentration), vortexed well and solubilized on ice for 20 min. The extracts were ready for T-synthase activity and protein concentration assay.
Preparation of the product Galβ1-3GalNAc-α-(4-MU)
Standard reactions for T-synthase activity assay in 1.5 mL microtubes with and without UDP-Gal were set up with 10 nmol/h recombinant T-synthase and incubated for 16 h. Then, 0.5 mL of H2O was added into the reactions, which were loaded onto two C18 cartridges (50 mg) pre-activated with methanol and washed with H2O. After washing with 1.2 mL of H2O six times, the bound materials were eluted with 80% acetonitrile and dried in a speed-vac system. The dried materials were dissolved in 50 μL of H2O. A 10 μL aliquot was transferred into 500-μL microtubes, 1 μL of O-glycosidase (800 units) in 25 mM MES was added and 1 μL of 25 mM MES was added to the untreated tube. After incubation at 37°C for 8 h, the materials were analyzed by LC-MS and MS/MS.
Characterization of the T-synthase product by ESI-MS/MS
The substrate and the product were analyzed in a positive mode by LC-MS/MS, QTRAP 5500 from Applied Biosystems (Foster City, CA). For the LC-MS experiment, the sample was diluted using solution (water/acetonitrile/formic acid, 98/2/0.1, v/v/v) to 50 fmol/µL and 10 µL for analysis was injected on an Ultra Aqueous C18 (3 µm, 50 × 2.1 mm) from Resteck (Bellefonte, PA). The mobile phase was methanol with 0.05% formic acid. The sample was eluted using a gradient from 2% methanol to 92% methanol over 20 min at a flow rate of 0.25 mL/min. For the MS/MS, the samples were dissolved or diluted in acetonitrile/water/formic acid (50/50/0.25, v/v/v) at a final concentration of 1 pmol/µL by infusion. The mass spectrometry parameters are as follows: the infusion flow rate is 10 µL/min; curtain gas: 25.0; ion spray: 5500.00; temperature: 350°C; nebulizer gas: 30.0; heater gas: 30.0; collisionally activated dissociation: medium; declustering potential: 90.0; entrance potential: 10.0. The peak of interest on the first MS was subjected to secondary MS and the data were collected.
Additional methods
T-synthase activity assays using UDP-[3H]-Gal were also conducted as described previously (Ju, Cummings et al. 2002). A soluble version of HPC4-tagged recombinant human T-synthase was expressed by coexpressing wild-type Cosmc and was directly purified from the media as described previously (Ju, Aryal et al. 2008). A soluble version of recombinant Cosmc was purified. Heat denaturing and reconstitution experiments were carried out as described (Aryal et al. 2010). The genomic DNA preparation and PCR of Cosmc from cell lines were performed as reported previously (Ju and Cummings 2002). The protein concentration in cell extracts was determined by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL) following the manufacturer's instructions with bovine serum albumin as a standard. PCR was carried out with Phusion™ High Fidelity PCR Kit (New England Biolabs) as described previously (Ju, Lanneau et al. 2008). The PCR product was purified using the QIAquick Gel Extraction Kit (Qiagen) following the manufacturer's protocol and sequenced.
Funding
This work was supported by the National Institutes of Health (RO1GM068559 to R.D.C and RO1DK80876 to T.J.).
We thank Drs. Jamie Heimburg-Molinaro, David F. Smith, Xuezheng Song and Anthony Luyai, as well as Connie Arthur for their helpful suggestions on this manuscript.
References
Abraham RT, Weiss A. Jurkat T cells and development of the T-cell receptor signalling paradigm. Nat Rev Immunol. 2004;4:301–308. doi: 10.1038/nri1330. doi:10.1038/nri1330. [DOI] [PubMed] [Google Scholar]
Allen AC, Topham PS, Harper SJ, Feehally J. Leucocyte beta 1,3 galactosyltransferase activity in IgA nephropathy. Nephrol Dial Transplant. 1997;2:701–706. doi: 10.1093/ndt/12.4.701. doi:10.1093/ndt/12.4.701. [DOI] [PubMed] [Google Scholar]
Binstadt BA, Williams BL, Abraham RT, Leibson PJ. Use of mutant Jurkat T cell lines to define human natural killer cell surface receptor signaling pathways. Methods Mol Biol. 2000;121:297–304. doi: 10.1385/1-59259-044-6:297. [DOI] [PubMed] [Google Scholar]
Blanchard B, Nurisso A, Hollville E, Tetaud C, Wiels J, Pokorna M, Wimmerova M, Varrot A, Imberty A. Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I. J Mol Biol. 2008;383:837–853. doi: 10.1016/j.jmb.2008.08.028. doi:10.1016/j.jmb.2008.08.028. [DOI] [PubMed] [Google Scholar]
Cartron JP, Nurden AT. Galactosyltransferase and membrane glycoprotein abnormality in human platelets from Tn-syndrome donors. Nature. 1979;282:621–623. doi: 10.1038/282621a0. doi:10.1038/282621a0. [DOI] [PubMed] [Google Scholar]
Crew VK, Singleton BK, Green C, Parsons SF, Daniels G, Anstee DJ. New mutations in C1GALT1C1 in individuals with Tn positive phenotype. Br J Haematol. 2008;142:657–667. doi: 10.1111/j.1365-2141.2008.07215.x. doi:10.1111/j.1365-2141.2008.07215.x. [DOI] [PubMed] [Google Scholar]
Fu J, Gerhardt H, McDaniel JM, Xia B, Liu X, Ivanciu L, Ny A, Hermans K, Silasi-Mansat R, McGee S, et al. Endothelial cell O-glycan deficiency causes blood/lymphatic misconnections and consequent fatty liver disease in mice. J Clin Invest. 2008;118:3725–3737. doi: 10.1172/JCI36077. doi:10.1172/JCI36077. [DOI] [PMC free article] [PubMed] [Google Scholar]
Fujita K, Oura F, Nagamine N, Katayama T, Hiratake J, Sakata K, Kumagai H, Yamamoto K. Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-α-N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem. 2005;280:37415–37422. doi: 10.1074/jbc.M506874200. doi:10.1074/jbc.M506874200. [DOI] [PubMed] [Google Scholar]
Fukuda M, Carlsson SR. Leukosialin, a major sialoglycoprotein on human leukocytes as differentiation antigens. Med Biol. 1986;64:335–343. [PubMed] [Google Scholar]
Furukawa K, Roth S. Co-purification of galactosyltransferases from chick-embryo liver. Biochem J. 1985;227:573–582. doi: 10.1042/bj2270573. [DOI] [PMC free article] [PubMed] [Google Scholar]
Goda HM, Ushigusa K, Ito H, Okino N, Narimatsu H, Ito M. Molecular cloning, expression, and characterization of a novel endo-alpha-N-acetylgalactosaminidase from Enterococcus faecalis. Biochem Biophys Res Commun. 2008;375:441–446. doi: 10.1016/j.bbrc.2008.08.065. [DOI] [PubMed] [Google Scholar]
Gramer MJ, Schaffer DV, Sliwkowski MB, Goochee CF. Purification and characterization of alpha-L-fucosidase from Chinese hamster ovary cell culture supernatant. Glycobiology. 1994;4:611–616. doi: 10.1093/glycob/4.5.611. doi:10.1093/glycob/4.5.611. [DOI] [PubMed] [Google Scholar]
Granovsky M, Bielfeldt T, Peters S, Paulsen H, Meldal M, Brockhausen J, Brockhausen I. UDPgalactose:glycoprotein-N-acetyl-d-galactosamine 3-β-d-galactosyltransferase activity synthesizing O-glycan core 1 is controlled by the amino acid sequence and glycosylation of glycopeptide substrates. Eur J Biochem. 1994;221:1039–1046. doi: 10.1111/j.1432-1033.1994.tb18822.x. doi:10.1111/j.1432-1033.1994.tb18822.x. [DOI] [PubMed] [Google Scholar]
Higuchi T, Tamura S, Takagaki K, Nakamura T, Morikawa A, Tanaka K, Tanaka A, Saito Y, Endo M. A method for determination of galactosyltransferase I activity synthesizing the proteoglycan linkage region. J Biochem Biophys Methods. 1994;29:135–142. doi: 10.1016/0165-022x(94)90049-3. doi:10.1016/0165-022X(94)90049-3. [DOI] [PubMed] [Google Scholar]
Hultberg B, Sjoblad S, Ockerman PA. Glycosidases in human skin fibroblast cultures. α-fucosidase, α-galactosidase, α-glucosidase, β-mannosidase, and N-acetyl-α-glucosaminidase. Acta Paediatr Scand. 1975;64:123–131. doi:10.1111/j.1651-2227.1975.tb04389.x. [PubMed] [Google Scholar]
Inoue T, Sugiyama H, Hiki Y, Takiue K, Morinaga H, Kitagawa M, Maeshima Y, Fukushima K, Nishizaki K, Akagi H, et al. Differential expression of glycogenes in tonsillar B lymphocytes in association with proteinuria and renal dysfunction in IgA nephropathy. Clin Immunol. 2010;136:447–455. doi: 10.1016/j.clim.2010.05.009. doi:10.1016/j.clim.2010.05.009. [DOI] [PubMed] [Google Scholar]
Isemura M, Kosaka H, Ikenaka T, Kido R, Yoshimura T. Fluorometric measurement of urinary α-L-iduronidase activity. J Biochem. 1978;84:627–632. doi: 10.1093/oxfordjournals.jbchem.a132167. [DOI] [PubMed] [Google Scholar]
Jaeken J, Matthijs G. Congenital disorders of glycosylation: A rapidly expanding disease family. Annu Rev Genomics Hum Genet. 2007;8:261–278. doi: 10.1146/annurev.genom.8.080706.092327. doi:10.1146/annurev.genom.8.080706.092327. [DOI] [PubMed] [Google Scholar]
Ju T, Aryal RP, Stowell CJ, Cummings RD. Regulation of protein O-glycosylation by the endoplasmic reticulum-localized molecular chaperone Cosmc. J Cell Biol. 2008;182:531–542. doi: 10.1083/jcb.200711151. doi:10.1083/jcb.200711151. [DOI] [PMC free article] [PubMed] [Google Scholar]
Ju T, Brewer K, D'Souza A, Cummings RD, Canfield WM. Cloning and expression of human core 1 β1,3-galactosyltransferase. J Biol Chem. 2002;277:178–186. doi: 10.1074/jbc.M109060200. doi:10.1074/jbc.M109060200. [DOI] [PubMed] [Google Scholar]
Ju T, Cummings RD. A unique molecular chaperone Cosmc required for activity of the mammalian core 1 β3-galactosyltransferase. Proc Natl Acad Sci U S A. 2002;99:16613–16618. doi: 10.1073/pnas.262438199. doi:10.1073/pnas.262438199. [DOI] [PMC free article] [PubMed] [Google Scholar]
Ju T, Cummings RD. Protein glycosylation: Chaperone mutation in Tn syndrome. Nature. 2005;437:1252. doi: 10.1038/4371252a. doi:10.1038/4371252a. [DOI] [PubMed] [Google Scholar]
Ju T, Cummings RD, Canfield WM. Purification, characterization, and subunit structure of rat core 1 β1,3-galactosyltransferase. J Biol Chem. 2002;277:169–177. doi: 10.1074/jbc.M109056200. doi:10.1074/jbc.M109056200. [DOI] [PubMed] [Google Scholar]
Ju T, Lanneau GS, Gautam T, Wang Y, Xia B, Stowell SR, Willard MT, Wang W, Xia JY, Zuna RE, et al. Human tumor antigens Tn and sialyl Tn arise from mutations in Cosmc. Cancer Res. 2008;68:1636–1646. doi: 10.1158/0008-5472.CAN-07-2345. doi:10.1158/0008-5472.CAN-07-2345. [DOI] [PubMed] [Google Scholar]
Juo P, Kuo CJ, Yuan J, Blenis J. Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade. Curr Biol. 1998;8:1001–1008. doi: 10.1016/s0960-9822(07)00420-4. doi:10.1016/S0960-9822(07)00420-4. [DOI] [PubMed] [Google Scholar]
Juo P, Woo MS, Kuo CJ, Signorelli P, Biemann HP, Hannun YA, Blenis J. FADD is required for multiple signaling events downstream of the receptor Fas. Cell Growth Differ. 1999;10:797–804. [PubMed] [Google Scholar]
Leppanen A, Mehta P, Ouyang YB, Ju T, Helin J, Moore KL, van Die I, Canfield WM, McEver RP, Cummings RD. A novel glycosulfopeptide binds to P-selectin and inhibits leukocyte adhesion to P-selectin. J Biol Chem. 1999;274:24838–24848. doi: 10.1074/jbc.274.35.24838. doi:10.1074/jbc.274.35.24838. [DOI] [PubMed] [Google Scholar]
Leppanen A, Yago T, Otto VI, McEver RP, Cummings RD. Model glycosulfopeptides from P-selectin glycoprotein ligand-1 require tyrosine sulfation and a core 2-branched O-glycan to bind to L-selectin. J Biol Chem. 2003;278:26391–26400. doi: 10.1074/jbc.M303551200. doi:10.1074/jbc.M303551200. [DOI] [PubMed] [Google Scholar]
Qin W, Zhong X, Fan JM, Zhang YJ, Liu XR, Ma XY. External suppression causes the low expression of the Cosmc gene in IgA nephropathy. Nephrol Dial Transplant. 2008;23:1608–1614. doi: 10.1093/ndt/gfm781. doi:10.1093/ndt/gfm781. [DOI] [PubMed] [Google Scholar]
Reed UC. Congenital muscular dystrophy. Part I: A review of phenotypical and diagnostic aspects. Arq Neuropsiquiatr. 2009a;67:144–168. doi: 10.1590/s0004-282x2009000100038. [DOI] [PubMed] [Google Scholar]
Reed UC. Congenital muscular dystrophy. Part II: A review of pathogenesis and therapeutic perspectives. Arq Neuropsiquiatr. 2009b;67:343–362. doi: 10.1590/s0004-282x2009000200035. [DOI] [PubMed] [Google Scholar]
Schietinger A, Philip M, Yoshida BA, Azadi P, Liu H, Meredith SC, Schreiber H. A mutant chaperone converts a wild-type protein into a tumor-specific antigen. Science. 2006;314:304–308. doi: 10.1126/science.1129200. doi:10.1126/science.1129200. [DOI] [PubMed] [Google Scholar]
Wang Y, Ju T, Ding X, Xia B, Wang W, Xia L, He M, Cummings RD. Cosmc is an essential chaperone for correct protein O-glycosylation. Proc Natl Acad Sci USA. 2010;107:9228–9233. doi: 10.1073/pnas.0914004107. doi:10.1073/pnas.0914004107. [DOI] [PMC free article] [PubMed] [Google Scholar]
Willis LM, Zhang R, Reid A, Withers SG, Wakarchuk WW. Mechanistic Investigation of the Endo-α-N-acetylgalactosaminidase from Streptococcus pneumoniae R6. Biochemistry. 2009;48:10334–10341. doi: 10.1021/bi9013825. [DOI] [PMC free article] [PubMed] [Google Scholar]
Xia L, Ju T, Westmuckett A, An G, Ivanciu L, McDaniel JM, Lupu F, Cummings RD, McEver RP. Defective angiogenesis and fatal embryonic hemorrhage in mice lacking core 1-derived O-glycans. J Cell Biol. 2004;164:451–459. doi: 10.1083/jcb.200311112. doi:10.1083/jcb.200311112. [DOI] [PMC free article] [PubMed] [Google Scholar]
Yamada K, Kobayashi N, Ikeda T, Suzuki Y, Tsuge T, Horikoshi S, Emancipator SN, Tomino Y. Down-regulation of core 1 β1,3-galactosyltransferase and Cosmc by Th2 cytokine alters O-glycosylation of IgA1. Nephrol Dial Transplant. 2010 doi: 10.1093/ndt/gfq325. Epub ahead of print June 15, 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
Yeh JC, Hiraoka N, Petryniak B, Nakayama J, Ellies LG, Rabuka D, Hindsgaul O, Marth JD, Lowe JB, Fukuda M. Novel sulfated lymphocyte homing receptors and their control by a Core1 extension β1,3-N-acetylglucosaminyltransferase. Cell. 2001;105:957–969. doi: 10.1016/s0092-8674(01)00394-4. doi:10.1016/S0092-8674(01)00394-4. [DOI] [PubMed] [Google Scholar]
