Utilization of Natural Fucosylated Oligosaccharides by Three Novel α-l-Fucosidases from a Probiotic Lactobacillus casei Strain

Abstract

Three putative α-l-fucosidases encoded in the Lactobacillus casei BL23 genome were cloned and purified. The proteins displayed different abilities to hydrolyze natural fucosyloligosaccharides like 2′-fucosyllactose, H antigen disaccharide, H antigen type II trisaccharide, and 3′-, 4′-, and 6′-fucosyl-GlcNAc. This indicated a possible role in the utilization of oligosaccharides present in human milk and intestinal mucosa.

l-Fucose is one of the most common monosaccharides occurring at the nonreducing end of many glycans on mammalian cell surfaces, intestinal mucin, blood group antigens, and human milk oligosaccharides (HMO) (2). α-l-Fucosidases (EC 3.2.1.51), which are exoglycosidases capable of cleaving α-linked l-fucose residues from fucosyloligosaccharides, play important roles in the adaptation of bacteria to particular niches. Therefore, infant intestinal bacteria such as bifidobacteria are able to use HMO (4, 9).

The genome of Bifidobacterium longum strains carries gene clusters related to the utilization of these substrates (8), which contain the necessary activities to degrade all of their glycosidic linkages, including α-l-fucosidases. In Bifidobacterium bifidum, two α-l-fucosidases, AfcA and AfcB, have been characterized as belonging to glycoside hydrolase (GH) families 95 and 29, respectively, and degrade α-(1,2)- and α-(1,3/4)-fucosylated HMO, respectively (1, 3).

However, there are no reports of α-l-fucosidases in lactobacilli, another important group of probiotic bacteria which are common inhabitants of the human intestine. Genome analysis of 25 Lactobacillus species reveals that only the Lactobacillus casei-Lactobacillus rhamnosus group encodes putative α-l-fucosidases (6).

Cloning and purification of three putative α-l-fucosidases from L. casei BL23.

Analysis of the L. casei BL23 genome sequence (GenBank accession no. FM177140) (5) showed the presence of three genes coding for putative α-l-fucosidases of GH family 29: LCABL_20390 (which was already automatically annotated as α-l-fucosidase A [alfA]), LCABL_28270, and LCABL_29340 (hereafter alfB and alfC, respectively). Alignment of the deduced amino acid sequences of the three putative α-l-fucosidases from L. casei BL23 showed low homology (21% identity), suggesting that they can have different substrate specificities.

alfA, alfB, and alfC were amplified by PCR using specific oligonucleotides and chromosomal DNA from BL23 as the template. They were cloned into the pQE80 vector (Qiagen) for expression as 6×His-tagged proteins in Escherichia coli. E. coli DH5α containing pQE80 derivatives carrying alfA, alfB, and alfC was grown in 500 ml of LB with 100 μg/ml ampicillin, and when the cultures reached an optical density at 550 nm of 0.6 to 0.8, expression of the proteins was induced with 0.1 mM isopropyl-β-d-thiogalactopyranoside at 25°C for 5 h. Bacterial cells were lysed by sonication, the cleared extracts were directly loaded onto Ni-nitrilotriacetic acid agarose (1 ml) columns (Qiagen), and His-tagged proteins were purified according to the supplier's recommendations. The 6×His-tagged AlfA enzyme did not bind the Ni-nitrilotriacetic acid agarose under native conditions, and it was finally purified by adding 4 M urea to all purification buffers. Subsequently, the urea was removed by sequential dialysis in buffer with 3, 2, 1, 0.5, and 0 M urea, respectively. The three enzymes were finally dialyzed against 100 mM HEPES (pH 7)-20% glycerol-0.5 mM dithiothreitol and stored at −80°C. Figure 1 A shows a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified enzymes. AlfA presented degradation products that originated during the five dialysis steps, while high-purity preparations were obtained for AlfB and AlfC. The native molecular weight of the proteins was estimated by gel filtration using a Superdex 200 10/30GE column, showing that all three enzymes probably have the form of tetramers (around 160 kDa) (Fig. 1B).

FIG. 1.

Purification of L. casei BL23 α-l-fucosidases AlfA, AlfB, and AlfC. (A) Analysis of 1 μg each of AlfA, AlfB, and AlfC α-l-fucosidases by 10% SDS-PAGE and staining with Coomassie blue. The values on the right are molecular masses (Mw) of protein standards. All α-l-fucosidases migrate as a single band with the expected molecular mass (45, 47, and 44 kDa, respectively). The AlfA enzyme preparation showed degradation products (*). (B) Gel filtration experiments showing the tetramer organization of all of the enzymes. The calculated molecular masses of the enzymes are as follows: AlfA, 165.6 kDa; AlfB, 178.58 kDa; and AlfC, 159.06 kDa. BSA, bovine serum albumin.

Enzymatic characteristics of purified AlfA, AlfB, and AlfC.

The purified enzymes were tested for hydrolysis of a range of p/o-nitrophenyl (NP) sugars at 5 mM by measuring changes in absorbance at 404 nm. The enzymes were unable to hydrolyze p-NP-α-d-galactopyranoside, p-NP-β-d-galactopyranoside, o-NP-β-d-galactopyranoside, p-NP-α-d-glucopyranoside, p-NP-β-d-glucopyranoside, and p-NP-N-acetyl-β-d-glucosaminide. Contrarily, the three enzymes showed activity on p-NP-α-l-fucopyranoside (pNP-fuc), thus confirming their α-l-fucosidase specificity. The optimal pH was determined using 100 mM phosphate-citrate buffer (pH 3 to 7) and 50 mM glycine-NaOH buffer (pH 7.5 to 9.5) in the presence of each purified enzyme (1 μg in 100 μl) and 2 mM pNP-fuc. The optimal reaction temperature was analyzed in the range of 15 to 55°C at the optimal pH determined as described above. The K_m and V_max parameters of each enzyme were calculated in 100 mM phosphate-citrate buffer (pH 7) at 37°C in the presence of 0.01 to 10 mM pNP-fuc. Table 1 summarizes the enzymatic characterization of the three enzymes. Several effectors, including CaCl₂, MgCl₂, and MnCl₂ at 5 and 10 mM, as well as NaCl at 50 and 150 mM, were assayed in 100 mM morpholineethanesulfonic acid (MES) buffer (pH 7), showing no effect on α-l-fucosidase activity, except for MnCl₂, which reduced AlfB activity by 35 and 40% at 5 and 10 mM, respectively.

TABLE 1.

Characterization of enzymes AlfA, AlfB, and AlfC with pNP-fuc as the substrate

Enzyme	Vmax (μmol/mg protein/min)	Km(mM)	Optimal pH	Optimal temp (°C)
AlfA	1.14	0.27	7.5	39
AlfB	6	2.9	7	41
AlfC	22.2	5.2	7	41

Substrate specificity of L. casei α-l-fucosidases.

In order to analyze the abilities of AlfA, AlfB, and AlfC to degrade natural oligosaccharides, several fucosylated and nonfucosylated oligosaccharides were assayed. The reactions were carried out with 100 mM MES buffer (pH 7) with 4 mM each substrate and incubation at 37°C for different periods of time ranging from 5 to 180 min. The reaction mixtures were analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection in an ICS3000 chromatographic system (Dionex) with a CarboPac PA100 column (Dionex) and a combined gradient of 100 to 300 mM NaOH and 0 to 150 mM acetic acid. Reaction products were confirmed by comparing their retention times with those of standard mono- and oligosaccharides. The enzymes were unable to act on lactose, melibiose, lactulose, maltose, or maltotriose. To the contrary, as shown in Table 2, AlfA, AlfB, and AlfC were able to hydrolyze fucosylated substrates, presenting different activities that were dependent on the type of linkage and the oligosaccharide length. AlfA hydrolyzed only 6′-fucosyl-GlcNAc with a low specific activity compared to its V_max using pNP-fuc as the substrate. AlfB was able to hydrolyze the antigen H disaccharide, the antigen H type II trisaccharide, 3′-fucosyl-GlcNAc, 4′-fucosyl-GlcNAc, and the HMO 2′-fucosyllactose. The observed differences in specific activity suggest that 3′-fucosyl-GlcNAc is probably a natural substrate for this enzyme. It is remarkable that the linkage present in this substrate (fucose-α1-3) is also present in 3′-fucosyllactose and in the Lewis X antigen, which were not hydrolyzed by this enzyme, suggesting that disaccharides and not tri- or tetrasaccharides, are the preferred substrates for AlfB. In contrast, AlfB was also able to hydrolyze, albeit with low specific activities, the α1-2 linkages in both di- and trisaccharides. Finally, AlfC hydrolyzed 6′-fucosyl-GlcNAc with high specific activity, which was several orders of magnitude higher than that of other linkages, including the antigen H disaccharide, 3′-fucosyl-GlcNAc, and 4′-fucosyl-GlcNAc. AlfC only hydrolyzed the fucosidic bonds which were present in disaccharides.

TABLE 2.

Specific activities of enzymes AlfA, AlfB, and AlfC on natural fucosylated substrates

Oligosaccharide	Structure	Activity (μmol of fucose/mg enzyme/min)a
		AlfA	AlfB	AlfC
Antigen H disaccharide	Fucα1-2Gal	−	0.132	0.002
Antigen H type II trisaccharide	Fucα1-2Galβ1-4GlcNAc	−	0.033	−
2′-Fucosyllactose	Fucα1-2Galβ1-4Glc	−	0.072	−
3′-Fucosyllactose	Galβ1-4(Fucα1-3)Glc	−	−	−
Lewis X trisaccharide	Galβ1-4(Fucα1-3)GlcNAc	−	−	−
Lewis A trisaccharide	Galβ1-3(Fucα1-4)GlcNAc	−	−	−
Lewis B tetrasaccharide	Fucα1-2Galβ1-3(Fucα1-4)GlcNAc	−	−	−
3′-Fucosyl-GlcNAc	Fucα1-3GlcNAc	−	27.5	0.0004
4′-Fucosyl-GlcNAc	Fucα1-4GlcNAc	−	0.032	0.008
6′-Fucosyl-GlcNAc	Fucα1-6GlcNAc	0.440	−	13.62

^a−, no activity detected.

We have characterized α-l-fucosidases from a Lactobacillus species for the first time. The substrates identified in this work represent important molecules present at mucosal surfaces as glycoconjugates (e.g., 6′-fucosyl-GlcNAc is part of the core sugar in protein N-glycosylation and the H antigen is the precursor of the ABO blood antigens, which are highly expressed at mucosal epithelium) or are HMO (2′-fucosyllactose). Therefore, these enzymes can be of great importance for survival in complex habitats such as the infant and adult human gut mucosa, where fucosyloligosaccharides may be an important carbon and energy source (7, 9). Interestingly, while AfcA and AfcB from B. bifidum are extracellular enzymes (1), the L. casei α-l-fucosidases lacked recognizable N-terminal sequences for secretion, suggesting that they are intracellular enzymes. Similarly, the four α-l-fucosidases encoded in the B. longum genome are intracellular (8). It is therefore probable that these bacteria need to take up fucosyloligosaccharides and hydrolyze them inside the cell for assimilation. In agreement with this idea, α-l-fucosidases from L. casei BL23 were shown to act preferentially on short rather than long fucosyloligosaccharides and the genes encoding these enzymes are clustered in the genome with genes encoding putative sugar permeases. The participation of these permeases in fucosyloligosaccharide uptake and metabolism by this microorganism remains to be investigated.