Identification and Molecular Cloning of a Novel Glycoside Hydrolase Family of Core 1 Type O-Glycan-specific Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum

Abstract

We found endo-␣-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl ␤1,3-N-acetyl-Dgalactosamine (Gal␤1,3GalNAc) ␣-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590 -1381 amino acid residues) exhibited 31-53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Gal␤1,3GalNAc disaccharide from Gal␤1,3GalNAc␣1pNP and asialofetuin, but did not release GalNAc, Gal␤1,3(GlcNAc␤1,6)GalNAc, GlcNAc␤1,3GalNAc, and Gal␤1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by 1 H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono-and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-␣-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).Mucin-type oligosaccharides (O-glycans) are involved in several important biological events including cell-to-cell communication in higher eukaryotes, bacterial adhesion to host cells, and so on (1). Eight core-type structures of O-glycans have been proposed, and among them, galactosyl ␤1,3-N-acetyl-D-galactosamine (Gal␤1,3GalNAc) 2 attached to the serine or threonine residue via an ␣-linkage (Core 1, also referred to as T antigen) is one of the most abundant core structures found in mucin glycoproteins that are widely distributed in intestinal tracts of human and animals. This immunogenic structure is normally masked by further glycosylation such as sialylation (sialyl T antigen) and N-acetylglucosaminylation (Core 2 structure). However, it has been shown that T antigen is frequently exposed on the cell surface of carcinoma and T-cell lymphoma, and this unmasked form is thought to be involved in tumor cell adhesion and tissue invasion (2, 3). In addition, incomplete synthesis of the Core 1 structure associates with several autoimmune diseases including IgA nephropathy (4), Tn-syndrome (5), and Henoch-Schönlein purpura (6).Endo-␣-N-acetylgalactosaminidase (EC 3.2.1.97; endo-␣-GalNAcase, glycopeptide ␣-N-acetylgalactosaminidase) is a unique enzyme that hydrolyzes Core 1-type O-glycan from glycoproteins. This enzyme also serves as a powerful tool for elucidating the presence of O-glycan...

Genome analysis of Bifidobacterium bifidum PRL2010 reveals metabolic pathways for host-derived glycan foraging

“…Given the diversity and complexity of mucin structures found within the gut (12,29), specific strategies for deconstructing these molecules must be inherent features in the genomes of mucinusing bacteria. The B. bifidum PRL2010 genome encodes various glycosyl hydrolases putatively implicated in degradation of mucinderived oligosaccharides, including a predicted cell wall-anchored endo-α-N-acetylgalactosaminidase (BBPR_0264), an enzyme that has been shown previously to catalyze the hydrolysis of the O-glycosidic α-linkage between GalNAc and serine/threonine residues of various mucin-type glycoproteins (30)(31)(32). Moreover, the genome of B. bifidum PRL2010 encodes a putative 1,2-α-Lfucosidase (BBPR_0193), as well as a predicted 1,3/4-α-L-fucosidase (BBPR_1360), which releases various α-linked L-fucoses from the oligosaccharide core of the mucin structure (33)(34)(35).…”

Characterization of a Novel β-l-Arabinofuranosidase in Bifidobacterium longum

“…Previously, we characterized an endo-␣-N-acetylgalactosaminidase (BLLJ_0168) from B. longum JCM 1217, which releases Gal-␤1,3-GalNAc (GNB) disaccharide from core-1 mucin-type O-glycans (18). Kitaoka et al (19,20) proposed a metabolic pathway for GNB from core-1 mucin-type O-glycans and Gal-␤1,3-GlcNAc (LNB) from human milk oligosaccharides based on the characterization of the genes encoded in the GNB/LNB operon (BLLJ_1620-BLLJ_1626) of B. longum.…”

New Structural Insights on Carbohydrate-active Enzymes

“…The catalytic residue indirectly affecting the substrate via water, catalytic base in inverting GHs and supposedly acid base in retaining ones seems to have less strict positional requirement. A number of CAZy families, including GH93, 74) GH95, 75) GH101 76) and PL15, 77) have been created after discovery of new enzymes unrelated to previously known CAZymes or for other reasons. Determination of the structural representatives of GH families, which display a variety of folds, provides valuable insights from the standpoint of structural biology, and can sometimes shed light on possible evolutionary relationships between apparently unrelated enzyme families.…”