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. Author manuscript; available in PMC: 2010 Dec 25.
Published in final edited form as: J Biol Chem. 2009 Oct 26;284(52):36223–36233. doi: 10.1074/jbc.M109.058354

MOLECULAR BASIS FOR GALACTOSYLATION OF CORE FUCOSE RESIDUES IN INVERTEBRATES: IDENTIFICATION OF CAENORHABDITIS ELEGANS N-GLYCAN CORE α1,6-FUCOSIDE β1,4-GALACTOSYLTRANSFERASE GALT-1 AS A MEMBER OF A NOVEL GLYCOSYLTRANSFERASE FAMILY

Alexander Titz 1,#, Alex Butschi 2,#, Bernard Henrissat 3, Yao-Yun Fan 1, Thierry Hennet 4, Ebrahim Razzazi-Fazeli 5, Michael O Hengartner 2, Iain BH Wilson 6, Markus Künzler 1, Markus Aebi 1,*
PMCID: PMC2794738  EMSID: UKMS29250  PMID: 19858195

Abstract

Galectin CGL2 from the ink cap mushroom Coprinopsis cinerea displays toxicity towards the model nematode Caenorhabditis elegans. A mutation in a putative glycosyltransferase-encoding gene resulted in a CGL2-resistant C. elegans strain characterized by N-glycans lacking the β1,4-galactoside linked to the α1,6-linked core fucose. Expression of the corresponding GALT-1 protein in insect cells was used to demonstrate a manganese dependent galactosyltransferase activity. In vitro, the GALT-1 enzyme showed strong selectivity for acceptors with α1,6-linked N-glycan core fucosides and required Golgi-dependent modifications on the oligosaccharide antennae for optimal synthesis of the Gal-β1,4-Fuc structure. Phylogenetic analysis of the GALT-1 protein sequence identified a novel glycosyltransferase family (GT92) with members widespread among eukarya but absent in mammals.

Introduction

Carbohydrate-binding proteins or lectins are found in all domains of life. They are the key mediators between carbohydrate signals and biological processes (1). Galectins constitute a distinct family of lectins with a characteristic fold (sandwich of two antiparallel β-sheets) and a conserved signature of β-galactoside-coordinating residues (2). Galectins have been implicated in various cellular and extracellular processes (3) such as apoptosis, cancer, cell adhesion, infection and innate immunity (4).

We have identified three closely related galectin-like proteins (CGL1, CGL2 and CGL3) from the ink cap mushroom C. cinerea, primarily expressed in the fruiting body developmental stage (5-9). Simultaneous silencing of cgl1 and cgl2 genes by small hairpin RNA did not alter the fruiting body formation, making an essential function in fruiting body development unlikely (10). The carbohydrate binding specificity of recombinant CGL2 is specific for β-galactosides (8,9) and the structure of CGL2 in complex with various β-galactosides was determined by X-ray crystallography (7).

The 16 kDa galectin CGL2 is toxic for the model nematode C. elegans in a carbohydrate-dependent manner (11), suggesting that galectins might have a direct role as effectors in defense against predators, parasites or pathogens and that such a lectinmediated defense, known primarily from bacteria (12,13) and plants (14), may be present in fungi as well. In a forward genetic screen, we identified several CGL2 resistant C. elegans mutant strains. The mutations conferring resistance to the nematode are related to GDP-Fuc biosynthesis and N-glycan biosynthesis. Mutation in either the fut-8 gene or in ORF M03F8.4 show a resistant phenotype, whereas deficiency in FUT-1 activity (i.e., core α1,3-fucosylation (15)) does not result in CGL2 resistant worms (11). Interestingly, Hanneman et al. (16) identified β-1,4-galactose linked to core α-1,6 fucose at the reducing end GlcNAc in N-glycans. Since CGL2 binds β-galactosides and FUT-8 (17) is responsible for the core α1,6-fucosylation of N-glycans, we hypothesized that ORF M03F8.4 might encode the galactosyltransferase to generate the core β-galactoside absent in the corresponding deletion strains (11).

Here we show that the C. elegans ORF M03F8.4 encodes a manganese dependent UDP-galactose galactosyltransferase that adds β-galactose to position 4 of α-1,6 linked fucose at the reducing end GlcNAc in N-glycan cores. Therefore, the protein encoded by M03F8.4 was termed GALT-1.

Experimental procedures

Chemicals

UDP-Gal was obtained from VWR International and Sigma, UDP-Glc, UDP-GlcNAc, UDP-GalNAc (all Sigma), UDP-14C-Gal (GE Healthcare), GnGnF6 (Dextra Laboratories, UK). dabsyl-GEN[GnGnF6]R (18), dabsyl-GEN[MMF6]R (19), dabsyl-GEN[MMF3]R (19) and dansyl-N[GnGnF6]ST (20) were obtained according to previously published methods.

Isolation of C. elegans cDNA and expression of GALT-1

Isolation of C. elegans M03F8.4 cDNA

Methods for culturing C. elegans have been described (21). The wild type Bristol N2 strain was grown at 20 °C on standard NGM agar plates seeded with Escherichia coli OP50. A C. elegans mixed culture was harvested from one standard NGM agar plate and washed twice in sterile M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 85 mM NaCl, 1 mM MgSO4). Total RNA was extracted using the NucleoSpin® RNA II RNA isolation kit (MACHEREY-NAGEL AG). cDNA synthesis was performed with 0.5 μg total RNA using the First-strand cDNA synthesis step of the SuperScript™ III Platinum Two-Step qRT-PCR Kit (Invitrogen AG).

Construction of the pFastBac1 donor plasmid for recombinant gene expression in Sf9 insect cells

galt-1 cDNA was amplified from cDNA by PCR using Phusion High-Fidelity DNA Polymerase (Finnzymes). For construction of an untagged version forward and reverse primers flanked with SalI and XbaI restrictions sites, respectively, were used. The resulting fragment was digested with the appropriate restriction enzymes and cloned into the pFastBac1 donor plasmid (Invitrogen). For construction of an N-terminally FLAG tagged version, a forward primer lacking the start codon was used. The resulting fragment was cloned into a pFastBac1 donor plasmid containing a N-terminal FLAG sequence (22).

Recombinant baculoviruses containing the C. elegans galt-1 cDNA (with and without N-terminal FLAG-tag) and a vector control were generated according to the manufacturer’s instructions (Invitrogen). After infection of 2 × 106 Spodoptera frugiperda (Sf9) adherent insect cells with recombinant baculoviruses and incubation for 72 h at 28 °C, cells were lysed with shaking (4 °C, 15 min) in 150 μL Tris-buffered saline (pH 7.4) containing 2% (w/v) Triton-X100 and protease inhibitor cocktail (Roche, complete EDTA-free). The lysis mixtures were centrifuged (2000 × g, 5 min), the postnuclear supernatant was recovered and used for all further enzymatic studies.

Denaturing gel electrophoretic analysis and immunoblotting

Infected Sf9 cells (2 × 106 cells, see above) were lyzed in 200 μL reducing sample buffer (0.0625 M Tris x HCl pH 6.8, 2% SDS [v/w], 5% β-mercaptoethanol [v/v], 10% glycerol [v/v], 0.01% bromophenolblue [w/v]) and proteins were denatured by heating (95 °C, 5 min), prior to SDS-PAGE (12% acrylamide, 120 V) and subsequent analysis by either silver staining or immunoblotting on nitrocellulose. After blocking the membrane (5% BSA in PBST, PBST contained 136 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, and 0.1% Tween-20 [v/v]), immunodetection was performed by incubation with anti-FLAG antibody M2 (SIGMA, dilution 1:2000 in PBST, supplemented with 1% BSA) followed by anti-mouse-IgG coupled to horse radish peroxidase (HRP) (Santa Cruz Biotechnology, dilution 1:10000 in PBST, supplemented with 1% BSA). After extensive washing (PBST), HRP activity was detected using the ECL reagent kit (Pierce) and exposure to photographic film.

Glycosyltransferase assays

Enzymatic activity using different carbohydrates or glycoconjugate acceptors was assessed using 0.5 μL (0.1 μL only in case of kinetics analysis) of the raw extract of Sf9 cells (transfected with either the vector bacmid, GALT-1 expressing bacmid or a FLAG-tagged GALT-1 expressing bacmid) in 2.5 μL final volume of MES buffer (pH 6.5, 40 μM) containing manganese(II) chloride (10 μM), UDP-galactose (1 mM) and the acceptor fucoside (glycan or glycopeptide, 40 μM). Glycosylation reactions were run for 2 h at room temperature, unless noted otherwise. For donor specificity analysis, UDP-galactose was replaced by equal amounts of UDP-Glc, UDP-GlcNAc or UDP-GalNAc respectively. For co-factor specificity analysis, MnCl2 was replaced by equal concentrations of the various metal chlorides or Na2EDTA. To quantify the incorporation of galactose into the acceptor glycans, UDP-Gal was doped with UDP-14C-Gal (GE Healthcare) to a final specific activity of 28.5 mCi/mmol. Excess substrate was removed by loading the reaction mixture (quenched with 100 μL H2O) onto a column of anion exchange resin (AG1-X8, Cl− form, Bio-Rad Laboratories, 200 mg) and elution of the uncharged products with H2O (900 μL).

Analysis of the reaction products was performed either by direct MALDI-TOF mass spectrometry, HPLC analysis of fluorescently labeled glycopeptides for donor specificity or scintillation counting of radio-labeled product. Thin layer chromatography was performed on Cellulose F TLC plates (Merck, Germany) with MeCN/H2O (65/35). Radioactivity on TLC plates was detected with a TLC plate reader ‘System 200 imaging scanner’, Bioscan (Washington, DC).

Structural analysis of oligosaccharide products

After exposing dabsyl-GEN[GnGnF6]R to galactosylation conditions, the resulting crude mixture was adjusted to 50 mM sodium citrate and pH 4.5, digested with Aspergillus oryzae β-galactosidase (27 mU) (23) for 2 days at 37 °C. The samples were analyzed by MALDI-TOF mass spectrometry (vide infra).

HPLC analysis

Dansyl-N[GnGnF6]ST acceptor substrate was separated from the reaction product using an isocratic solvent system (flow rate of 1.5 mL/min, 8.5% MeCN (v/v) in 0.05% aqueous TFA (v/v)) on a reversed phase Hypersil ODS C18 column (4 × 250 mm, 5 μm) and fluorescence detection (excitation at 315 nm, emission detected at 550 nm) at room temperature. The Shimadzu HPLC system consisted of a SCL-10A controller, two LC10AP pumps and a RF-10AXL fluorescence detector controlled by a personal computer using Class-VP software (V6.13SP2). Dansyl-N[GnGnF6]ST eluted at a retention time of 9.09 min and the galactosylated reaction product at 8.06 min.

Mass spectrometry

Glycans were analyzed by MALDI-TOF mass spectrometry on a BRUKER Ultraflex TOF/TOF machine using α-cyano-4-hydroxycinnamic acid as matrix. A peptide standard mixture (Bruker) was used for external calibration. Mass spectra were analyzed using Bruker software and the mMass V2.4 software package (24).

Scintillation Counting

The eluates of the anion exchange resin column were thoroughly mixed with scintillation fluid (Irga-Safe Plus, Packard, 4 mL) and measured with a Perkin Elmer Tri-Carb 2800TR.

Use of M03F8.4::mCherry fusion for localization of GALT-1 expression

Transgenic strains (opEx and opIs alleles) were generated by microparticle bombardment of constructed strain unc-119(ed3);galt-1(op497) using a Biolistic Particle Delivery System (PDS-1000, Bio-Rad). The protocol was carried out as previously described (25) using unc-119(ed3) as a transformation marker. Resulting strains containing integrated arrays were unc-119(ed3);galt-1(op497);opIs444 and unc-119(ed3);galt-1(op497);opIs445. Transgenic animals were analyzed for expression of mCherry by fluorescence microscopy and for rescue of sensitivity towards CGL2-mediated developmental arrest.

For construction of the extrachromosomal and integrated arrays for localization of galt-1 a region comprising 1.8 kb upstream of the predicted start codon and the complete coding region without the stop codon of galt-1 were PCR amplified from genomic DNA obtained from worm lysates using Phusion® High-Fidelity DNA Polymerase (Finnzymes). The resulting 4.3 kb galt-1 fragment was verified by DNA sequencing and cloned in frame upstream of the mCherry coding sequence and the let-858 transcriptional terminator in the bombardment vector pLN022 (kindly provided by Lukas Neukomm) containing the unc-119(ed3) gene as transformation marker. The final galt-1::mCherry::let-858 fusion construct (pAB15) was verified by DNA sequencing. Primers used are shown in the supplemental information (table 1S).

A plate assay was devised to examine the toxicity of wild type and mutant CGL2 towards C. elegans. NGM plates containing 1mM IPTG and 50 μg/mL kanamycin were seeded with E. coli BL21(DE3) expressing either wild type CGL2 or mutant CGL2(W72G) as described above. As a control, plates were seeded with E. coli BL21(DE3) containing vector DNA. The plates were incubated overnight at 37 °C seeded with synchronized populations of C. elegans (26) for the developmental assay. Quantitative data on the effect of CGL2 on C. elegans development was acquired by placing 50 to 100 newly hatched L1 larvae of the indicated genotypes on the plates. After 72 h, the fraction of animals that reached L4 stage was determined.

Differential interference contrast (DIC) and fluorescence microscopy of nematodes

For general worm handling, a Leica MZ 12.5 stereomicroscope was used. To analyze transgenic lines for expression of the galt-1-mCherry construct, we used a Leica MZ 16 FA stereomicroscope equipped with appropriate filtersets (DsRed). Pictures were taken with a Nikon coolpix 990 digital camera. For fluorescence microscopy, worms were placed on 2% agarose pads in M9 (27), anaesthetized with levamisole (3-5 mM, Sigma) and mounted under a coverslip for observation using a Leica DM-RA or Zeiss Axiovert 200 microscope equipped with DIC (Nomarski) optics and standard epifluorescence with a DsRed filterset for detection of mCherry. Pictures were taken with a Hamamatsu ORCA-ER camera. Images were false-colored using OpenLab software.

Bioinformatics

Amino acid sequences were retrieved from the National Center for Biotechnology Information (NCBI) by iterative BLAST searches starting from the C. elegans sequence with accession NP_504545. Sequences were aligned using MUSCLE (28). The aligned sequences were clustered using the SECATOR algorithm (29), which relies on BIONJ (30) to build a tree from the multiple-sequence alignment, and subsequently collapses the branches from subtrees after identification of the nodes joining different subtrees (29). The neighbor-joining tree was made from the resulting distance matrix using Blosum62 substitution parameters (31). Visualization of the tree was done with Dendroscope (32). The multiple sequence alignment was displayed using ESPript (33).

Abbreviations

BSA: bovine serum albumin; FLAG: FLAG peptide: DYKDDDDK; HPLC: high performance liquid chromatograph; HRP: horse radish peroxidase; MALDI: matrix assisted laser desorption ionization; MES: morpholino-N-ethane sulfonic acid; MS: mass spectrometry; MS/MS: tandem MS; NGM: nematode growth medium; PBST: phosphate buffered saline supplemented with 0.1% (v/v) Tween-20; TFA: trifluoroacetic acid; TOF: time of flight; UDP: uridine diphosphate; Complex glycans are abbreviated according to the Schachter(34) nomenclature: GnGnF6: GlcNAcβ1,2Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAc; MMF6: Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAc; MMF3: Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4(Fucα1,3)GlcNAc;

Results

Expression of GALT-1 in baculovirus infected insect cells

The cDNA encoding the putative nematode galactosyltransferase GALT-1 was isolated and the protein coding region was cloned into the pFastBac1 vector for expression of both the authentic protein and the N-terminally FLAG-tagged variant in baculovirus infected Sf9 insect cells. The latter protein was produced for detection of its expression by immunostaining. After incorporation of the corresponding vectors into the bacmid, Sf9 cells were transfected and viruses amplified. After the third passage, protein expression was analysed by SDS-PAGE of whole cell extracts and detection of the FLAG-tag by immunoblotting. Two strong signals close to the 50 kDa marker band were detected in extracts derived from cells infected with FLAG-GALT-1 virus (figure 1). Since the protein sequence of GALT-1 contains two putative N-glycosylation sites (Asn109, Asn152), whole cell extracts derived from FLAG-GALT-1 expressing virus infection were treated with PNGaseF. This resulted in a mobility shift and the detection of a single protein species upon SDS-PAGE and immunoblotting (data not shown). We concluded that these cells expressed the C. elegans protein and that the partially glycosylated glycosyltransferase domain was oriented towards the lumen of the secretory apparatus.

Figure 1.

Figure 1

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Expression analysis of GALT-1 in baculovirus infected insect cells. Sf9 cells were transfected with bacmids containing vector DNA (lanes 1 & 2), bacmids containing N-terminally FLAG-tagged galt-1 DNA (lanes 3-5) and bacmids containing untagged galt-1 DNA (lanes 6-8). Extracts were prepared, separated by SDS-PAGE, and transferred to nitrocellulose. The blot was probed with FLAG-tag specific antibodies. The position of the molecular weight markers in kDa is given at the left.

Enzymatic characterization of GALT-1

Extracts of insect cells expressing GALT-1 were tested for galactosyltransferase activity. Considering that the galt-1 mutant strain lacks galactose modification on core α1,6-fucosylated N-glycans (11), an N-terminally dabsylated glycopeptide bearing a core α1,6-fucosylated biantennary asialo agalacto N-glycan (referred to as GnGnF6) was used as a substrate in the presence of manganese ions, UDP-galactose and the GALT-1 expressing insect cell extracts. Incorporation of an additional hexose (mass increase of 162 Da) into this acceptor glycopeptide was observed by MALDI mass spectrometry of the reaction products (figure 2A). The same activity was observed in extracts containing recombinant forms of either the N-terminally FLAG-tagged (data not shown) or non-tagged GALT-1. When extracts of the baculovirus infected insect cells carrying only the vector DNA were tested, no activity was detected. Experiments with functional GALT-1 but in absence of UDP-galactose did not change the mass of the acceptor glycopeptide, indicating a specific transfer of a galactose residue from UDP-galactose by GALT-1. Product analysis by tandem mass spectrometry (figure 2B) revealed addition of the galactose moiety to the fucose at the reducing terminal GlcNAc of the N-glycan core. The indicatory secondary ion with m/z 1273 corresponded to the glycopeptide bearing a Hex-dHex-HexNAc sugar structure and was only observed after fragmentation of the galactosylated glycopeptide (parent ion m/z 2369) and not with the acceptor GnGnF6-glycopeptide (parent ion m/z 2207). Treatment of substrate and product with Aspergillus β-galactosidase led to identical mass spectra indicating that the nature of the additional hexose is a β-linked galactoside bound to the core fucose of the N-glycan (figure 2C). In addition, permethylation of the reaction product, released by PNGaseF and subsequent MALDi-MS/MS analysis revealed the same characteristic y fragmentation ion with m/z 678 as reported for the native Galβ1,4Fuc structure (figure S3) (16).

Figure 2.

Figure 2

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MALDI-MS based galactosyltransferase assays and determination of position and linkage of the galactose residue resulting from GALT-1 catalysis. (A) Dabsylated GnGnF6 glycopeptide was incubated with: UDP-Gal and insect cell extract derived from infection with galt-1 containing baculovirus (upper trace), UDP-Gal and insect cell extract derived from infection with vector DNA containing baculovirus (middle trace) or insect cell extract derived from infection with galt-1 containing baculovirus in absence of UDP-Gal (lower trace). (B) The reaction products of experiment (A, upper trace) and experiment (A, middle trace) were analyzed by MS/MS for determination of regioisomers (B, upper and lower trace, respectively). (C) The reaction products of experiment (A, upper trace) and experiment (A, middle trace) were analyzed for linkage anomericity by enzymatic reaction with Aspergillus β-galactosidase followed by MS (C, upper and lower trace, respectively). Laser breakdown peaks are indicated with an asterisk (*) and peaks from residual hexosaminidase activity (23) originating from the insect cell extracts are marked with the pound sign (#).

The donor specificity of GALT-1 was addressed in an HPLC based assay with detection of dansylated GnGnF6 glycopeptides by fluorescence (figure 3A). The retention time of the acceptor glycopeptide (tR = 9.09 min) shifted when the glycosylation reaction was performed in presence of UDP-galactose (tR = 8.06 min). We concluded that the galactosylated product eluted with a retention time of 8.06 min. In case of UDP-GalNAc, UDP-GlcNAc or the controls no change was observed, whereas with UDP-glucose a minor peak (< 10%) with the same retention time of the galactosylated product (tR = 8.06 min) became visible. This quantitative detection of the reaction product was used to determine the kinetic parameters for the UDP-Gal substrate in the galactosyltransferase reaction. Visualization of the obtained data in a Hanes plot (figure 3C) gave a graphically determined apparent Km value of 84 μM for UDP-Gal, consistent with values for nucleotide sugar donors of other ER and Golgi localized glycosyltransferases (35). Fucose and 4-nitrophenyl-α-L-fucoside were not accepted by GALT-1 as substrates (data not shown). Due to limited amounts of the oligosaccharide substrate, we were unable to determine the kinetic parameters for this acceptor.

Figure 3.

Figure 3

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The donor specificity (A) of GALT-1 was analyzed by HPLC separation of reactions of dansyl-N[GnGnF6]ST glycopeptide with insect cell extract derived from infection with galt-1 containing baculovirus and different donor substrates (UDP-Gal, UDP-GalNAc, UPD-Glc, UDP-GlcNAc), in absence of a donor substrate (w.o. donor) and in presence of UDP-Gal and insect cell extract derived from infection with vector DNA containing baculovirus (e.v., UDP-Gal). (B) Kinetics of GALT-1 (B and C) for UDP-Gal was analyzed by HPLC (turnover describes the ratio of the peak areas of product to the sum of product and reactant multiplied by 100%). (C) Hanes plot of the data obtained in (B) for graphical determination of the Km value for GALT-1 and UDP-Gal. Two independent data sets are shown (black and white squares), the linear regression is derived from the average values.

We determined the specificity of GALT-1 for different acceptor glycans by product analysis using mass spectrometry (figure 4). Dabsylated glycopeptides bearing the GnGnF6 carbohydrate structure were completely galactosylated in 2 h reaction time as observed from the complete disappearance of the peak corresponding to the acceptor mass (m/z 2207) and the appearance of the peak of the galactosylated product (m/z 2369). Interestingly, when the non-reducing terminal GlcNAc on the antenna were removed (MMF6), only a partial turnover from the acceptor (m/z 1801) into the galactosylated product (m/z 1963) was observed. The positional isomer of the latter structure, bearing an α1,3-fucose instead of the α1,6-fucose at the innermost GlcNAc (MMF3) remained unchanged when exposed to GALT-1 under standard galactosylation conditions.

Figure 4.

Figure 4

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Analysis of the acceptor specificity of GALT-1 by MALDI-MS. Dabsylated tetrapeptides with varying glycan structure (GnGnF6, MMF6, MMF3) were incubated with UDP-Gal and insect cell extract derived from infection with galt-1 containing baculovirus and the reaction products were analyzed by MS. Laser breakdown peaks are indicated with an asterisk (*).

For quantification of a possible metal co-factor dependence of GALT-1, the acceptor substrate GnGnF6 was used as an untagged free glycan. Galactosylation under standard conditions was performed using radiolabelled UDP-14C-Gal. The specific incorporation of 14C-galactose into GnGnF6 was visualized after separation of the reaction mixture on cellulose TLC plates and quantification of the reaction product (36). The specificity of incorporation into the acceptor glycan could be visualized after separation by thin layer chromatography and the resulting radioactive nonasaccharide was retained on the cellulose stationary phase through carbohydrate-carbohydrate interactions to a higher extent than both starting material and the unspecific hydrolysis product. The thin layer chromatogram (figure 5A) showed the incorporation of radioactive galactose into GnGnF6 by GALT-1 as additional slow migrating peak (32 mm) being absent when vector control extracts were used. Excess UDP-14C-Gal migrated at 60 mm and the fast migrating peak (78 mm) most likely resulted from substrate hydrolysis due to extended reaction time. For the quantification of the metal co-factor specificity of GALT-1, excess donor was removed from the reaction mixture by anion exchange chromatography and a reduced incubation time was chosen to obtain a minimum of substrate hydrolysis (figure 5B). Addition of EDTA prevented the transfer of galactose. Bivalent alkaline earth metals and transition metals were tested and highest turnover was observed with manganese, iron and cobalt, consistent with results obtained for other glycosyltransferases (37). In comparison, when earth alkaline metals (Mg, Ca, Sr, Ba) or nickel were added, the activity was slightly increased as compared to the control with addition of water (figure 5B column aq.). The presence of copper or zinc ions inhibited incorporation of 14C-galactose into GnGnF6.

Figure 5.

Figure 5

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Determination of the co-factor dependence of GALT-1. (A) GnGnF6 was incubated with 14C-UDP-Gal and insect cell extract derived from infection with galt-1 containing baculovirus (GALT-1) or insect cell extract derived from infection with vector DNA containing baculovirus (e.v.) for 12 h and the reaction products were separated by TLC on cellulose-F plates and analyzed by counting with a TLC plate reader. (B) The reaction was performed for 2 h as described for (A) in presence of various bivalent metals, EDTA, residual co-factors from the insect cell extract (aq.) or insect cell extract derived from infection with vector DNA containing baculovirus (e.v.) and the reaction products were analyzed by scintillation counting after removal of excess substrate using anion exchange resin. Activities are shown relative to the highest average activity (corresponds to cpm) which was arbitrarily set to 100%; error bars refer to standard deviation of 3-7 independent experiments.

Expression of GALT-1 in nematodes

For in vivo localization of the expression of GALT-1, a CGL2-resistant C. elegans unc-119(ed3);M03F8.4(op497) mutant was transformed with a galt-1::mCherry translational fusion. The fusion construct comprised 1843 bp of the genomic DNA upstream of the translational start codon. Rescue of the inability to form Dauer stages associated with the unc-119(ed3) mutation was used to select for low-copy integrants (25), which were examined both for CGL2-sensitivity and for fluorescence in comparison to the untransformed unc-119(ed3) single and unc-119(ed3);M03F8.4(op497) double mutant strains. The observed rescue of CGL2-sensitivity by the transformed fusion construct suggested that the encoded fusion protein was functional (figure 6A). Examination of these integrants by fluorescence microscopy revealed specific expression of galt-1 in the intestine and in coelomocytes (figure 6B). Upon magnifying the fluorescence image at altered contrast, expression of GALT-1 appears to be localized in vesicular structures or organelles within the coelomocytes.

Figure 6. Localization of a functional galt-1(M03F8.4)-mCherry fusion protein in C. elegans.

Figure 6

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Transgenic worms carrying the integrated galt-1(M03F8.4)-mCherry fusion construct were generated by biolistic transformation of unc-119(ed3);galt-1(op497). The resulting strains unc-119(ed3);galt-1(op497);opIs444/opIs445 were analyzed for rescue of CGL2-sensitivity of the CGL2-resistant galt-1 (M03F8.4) mutant strain (A) and red fluorescence (B). For the rescue (A), C. elegans of the indicated genotypes were analyzed for development from L1 to L4. Columns represent the average of 3 replicates. Error bars indicate standard deviations. For the localization (B), the expression of the galt-1 reporter construct was examined by differential interference contrast (DIC) (panels 1, 3, 5) and fluorescence microscopy (panels 2, 4, 6) of unc-119(ed3);galt-1(op497) (panels 1 and 2) vs. unc-119(ed3);galt-1(op497);opIs444/opIs445 worms (panels 3 to 6). Asterisks indicate the pronounced expression of the galt-1 reporter construct in the three coelomocyte pairs along the stained intestine. Panels 5 and 6 show a magnification of the frontal coelomocyte pair. A Leica MZ 16 FA stereomicroscope equipped with a DsRed filterset was used in panels 1 to 4 and a Leica DM-RA microscope equipped with DIC (Nomarski) optics and standard epifluorescence with an appropriate filterset for higher magnification in panels 5 and 6.

GALT-1 as a member of a new glycosyltransferase family

Database searching with the sequence of C. elegans GALT-1 resulted in identification of 115 proteins of previously unknown function. On the basis of the presented data, these proteins can now be classified as a new family of glycosyltransferases in the Carbohydrate-Active Enzymes database (38) (figure 7), namely as family GT92. This family can be phylogenetically clustered into three main subgroups. The subgroup containing GALT-1 features homologues from various species, among which are vertebrates (Xenopus, Danio rerio), invertebrates (Caenorhabditis, Drosophila) and protozoa (Cryptosporidium). While the second subgroup consists of homologues exclusively from C. elegans and C. briggsae, the third one comprises proteins of plant origin (e.g., from Arabidopsis, Oryzae, Physcomitrella). The sequence alignment of the homologous domain of the entire family is exemplified with three selected proteins of each branch (figure 8).

Figure 7.

Figure 7

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Secator-BIONJ tree of the new glycosyltransferase family (GT 92). The GenBank accession numbers are indicated along with abbreviated species names. The boxed number shows the enzyme studied here (GALT-1, C. elegans NP_504545).

Figure 8.

Figure 8

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Representative alignment of the homologous domain of 9 selected sequences (3 of each branch, A,B,C and D,E,F and G,H,I) of the phylogenetic tree according to the entire new enzyme family (full alignment in supporting information). Among the entire family highly conserved amino acids are boxed. A: GALT-1 (Caenorhabditis elegans NP_504545), B: Cryptosporidium parvum EAK88153, C: Drosophila melanogaster ABW09323, D: Caenorhabditis elegans AAC17772, E: Caenorhabditis briggsae CAP30593, F: Caenorhabditis elegans AAB37068, G: Vitis vinifera CAO66312, H: Physcomitrella patens EDQ50855, I: Arabidopsis thaliana CAB79017. Sequence numbering refers to GALT-1 (A).

Discussion

Our previous study (11) revealed the necessity of a functional gene in C. elegans ORF M03F8.4 for the presence of the galactosyl-β1,4-fucosyl-α1,6 moiety (16) at the reducing end GlcNAc in N-glycans in vivo. To determine the biochemical activity of the GALT-1 protein encoded by this gene and displaying sequence homology to other glycosyltransferases, the corresponding cDNA was isolated and expressed in baculovirus infected insect cells. GALT-1 is likely to be a Golgi glycosyltransferase due to its N-terminal signal sequence targeting it to the secretory pathway. With a D289X290D291 sequence, it shares with many other Golgi glycosyltransferases a DxD motif (39) often associated with a requirement for manganese (or occasionally magnesium) for coordination of the phosphates of the activated sugar during catalysis; the subsequent enzymatic characterization of GALT-1 was compatible with such a requirement for bivalent metal ions.

Three types of enzymatic assay methods (based on MALDI-TOF MS, HPLC and radioactivity) indicated transfer of galactose by GALT-1 to core α1,6-fucosylated N-glycans. This preference was absolute and an isomeric core α1,3-fucosylated glycopeptide was not galactosylated by this enzyme. Despite the presence of other hexose modifications on the core α1,3-fucoses on the distal and proximal GlcNAc residues of the core region of C. elegans N-glycans (16,40), these epitopes are probably not synthesized by GALT-1 in vivo. Therefore, the action of GALT-1 as a bifunctional enzyme also acting on N-glycans with different remote (i.e., on the non-reducing ends) sugar structures or on core α1,3-fucose cannot be fully excluded, but seems rather unlikely. Considering the preference of GALT-1 for UDP-Gal, the sensitivity of its enzymatic product to a β1,4-specific galactosidase, our detailed product analysis and the lack of galactosylated core α1,6-fucosylated glycans in the galt-1 mutant, we postulated that this enzyme is a functional β1,4-galactosyltransferase necessary for the transfer of galactose to core α1,6-fucose in C. elegans.

Interestingly, non-reducing terminal GlcNAcs on the antenna had an important role on the rate of galactosylation by GALT-1. The GlcNAc bearing glycopeptide (GnGnF6) was completely transformed into the corresponding galactoside. Under identical reaction conditions, but in absence of these terminal GlcNAcs, the MMF6 glycopeptide was only partially galactosylated. Thereby, our in vitro results are compatible with the observed accumulation of glycans carrying both Galβ1,4Fuc and non-reducing terminal GlcNAc in the hex-2 hexosaminidase-deficient worm strain (23).

A similar influence of non-reducing GlcNAc is described for FUT-8, responsible for the addition of the core α1,6-fucose (41). FUT-8 requires one remote GlcNAc on the antenna of the N-glycan for addition of the α1,6-fucose, but in turn its activity is inhibited by the presence of a bisecting GlcNAc on the β-mannose or an α1,3-fucose on the reducing end GlcNAc. We concluded that the prior action of GlcNAc-TI was an important pre-requisite for formation of the Galβ1,4Fuc epitope during biosynthesis of C. elegans N-glycans (42), despite the absence of the non-reducing terminal GlcNAc in many of the wild-type N-glycans. Thus, our data also partly account for the less complex N-glycome of nematodes lacking functional forms of all three GlcNAc-TI genes in C. elegans (43) and are compatible with the premise that the Golgi hexosaminidases in the worm act after GALT-1.

Using the sequence of GALT-1 for a search of homologous proteins present in the databases, an entire new family of glycosyltransferases was revealed which was classified as GT92 in the CAZy database. Three subfamilies were identified in the tree, one of which included the C. elegans enzyme studied here, and two others that are more distantly related. Interestingly, while all GT92 members are of eukaryotic origin, no mammalian sequence was identified. Furthermore, a large recent expansion of one of the subfamilies in C. elegans and C. briggsae was observed. With only two exceptions out of over 100 family members, all sequences bear the conserved DxD motif commonly used in glycosyltransferases for manganese coordination. As observed for eukaryotic Golgi glycosyltransferases, family GT92 glycosyltransferases appear to have a single pass transmembrane N-terminal of the catalytic domain (in some instances the N-terminal transmembrane domain is lacking probably due to incomplete protein models).

Although the β-1,4-galactose epitope linked to core α-1,6 fucose at the reducing end GlcNAc in N-glycans is not only observed in C. elegans (16) but was first discovered as a feature of octopus rhodopsin (44) and later on N-glycans of squid rhodopsin (45) and keyhole limpet haemocyanin (46), at present (06/15/09) no GALT-1 related sequences are found among the published EST database of these species. Due to relatively high sequence divergence with GALT-1, it is presently impossible to predict the substrate specificity of the distant relatives (members of the other two subfamilies). It is interesting to note that none of the newly identified plant sequences were predicted by Hansen and co-workers (47) in a bioinformatics approach to identify novel glycosyltransferase sequences, showing that experimental investigation is still essential for enzyme discovery.

The observed intestinal localization of GALT-1 was in accordance with the reported localization of the CGL2-ligand to the intestinal epithelium and the observed damage of the C. elegans intestine upon CGL2 intoxication (11). The significance of the initial binding event between CGL2 and its Galβ1,4Fuc ligand at the intestinal epithelium and the expression of galt-1 in coelomocytes, cells implicated in C. elegans innate immunity (48), for the mechanism of CGL2-mediated nematotoxicity remain to be clarified. Furthermore, the molecular basis for the transfer of previously identified hexose modifications to core α1,3-fucose of C. elegans N-glycans, as well as the subsequent modification of the Galβ1,4Fuc epitope by additional hexoses (16,40) remains to be resolved. It is possible that the distantly related sub-family consisting exclusively of C. elegans and C. briggsae genes is responsible for the biogenesis of these further epitopes. Considering the enzymological and structural complexity of such glycosylation events, these and other aspects of the unusual modifications of the core region of nematode N-glycans, as well as the function of GALT-1 homologues in other species, represent challenges for future studies.

Supplementary Material

1

Acknowledgements

The authors would like to thank Katharina Nöbauer, Drs. Martin Badertscher and Belinda Schegg for technical assistance. Financial support from the Swiss National Science Foundation (grant no. 3100A3-116827 to M.K., M.J., and M.A. and grant no. 31003A-105541 to M.A.), the Roche Research Foundation (to A.T.), EMBO (ASTF-377.00-2008 to A.T.), the ETH Zurich and the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (grant P18447 to I.B.H.W.) are gratefully acknowledged.

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