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. 2012 Jan 2;287(1):693-700.
doi: 10.1074/jbc.M111.277384. Epub 2011 Nov 16.

α-N-acetylgalactosaminidase from infant-associated bifidobacteria belonging to novel glycoside hydrolase family 129 is implicated in alternative mucin degradation pathway

Masashi Kiyohara  1 , Takashi Nakatomi  2 , Shin Kurihara  2 , Shinya Fushinobu  3 , Hideyuki Suzuki  4 , Tomonari Tanaka  4 , Shin-Ichiro Shoda  5 , Motomitsu Kitaoka  6 , Takane Katayama  7 , Kenji Yamamoto  1 , Hisashi Ashida  8
Affiliations

α-N-acetylgalactosaminidase from infant-associated bifidobacteria belonging to novel glycoside hydrolase family 129 is implicated in alternative mucin degradation pathway

Masashi Kiyohara et al. J Biol Chem. .

Abstract

Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo-α-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Galβ1-3GalNAcα1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core-3-type O-glycans (GlcNAcβ1-3GalNAcα1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel α-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAcα1-Ser/Thr). Sialyl and galactosyl core-3 (Galβ1-3/4GlcNAcβ1-3(Neu5Acα2-6)GalNAcα1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as α-sialidase (SiaBb2), lacto-N-biosidase (LnbB), β-galactosidase (BbgIII), and β-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129.

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Figures

FIGURE 1.
FIGURE 1.
Substrate specificity of NagBb. Substrates (250 μm for pNP-glycosides; 3 mm for the others) were incubated with recombinant NagBb (14 ng/μl for pNP-glycosides; 50 ng/μl for the others) and analyzed by TLC. Gn and GNB, standard GalNAc and Galβ1–3GalNAc; Tn, GalNAcα1-Ser; sTn, Neu5Acα2–6GalNAcα1-Ser; UDP-Gn, GalNAcα1-UDP; A tri, GalNAcα1–3(Fucα1–2)Gal; Gn-Lac, GalNAcα1–3Galβ1–4Glc; Core-1–Core-8, pNP derivatives of each core glycan; Gn-pNP, GalNAcα1-pNP. Plus and minus, presence and absence of NagBb; asterisk, contaminant in the core-4-pNP sample.
FIGURE 2.
FIGURE 2.
Stereochemical analysis of hydrolysis catalyzed by NagBb. GalNAcα1-pNP (2 mm) was incubated with NagBb in 20 mm sodium acetate buffer (pH 5.0) at 37 °C for the indicated time period. The reaction mixtures were immediately analyzed by normal-phase HPLC.
FIGURE 3.
FIGURE 3.
Transglycosylation activity of NagBb. NagBb (35 ng/μl) was incubated with 3 mm GalNAcα1-pNP as a donor in the presence of 100 mm Ser as an acceptor in 50 mm sodium acetate buffer (pH 5.0) at 37 °C. The reaction mixture was analyzed by the Prominence amino acid analyzer equipped with a postcolumn fluorescence labeling system using o-phthalaldehyde and a Shim-pack Amino-Na column. A, heat-inactivated enzyme; B, active enzyme; C, standard GalNAcα1-Ser.
FIGURE 4.
FIGURE 4.
Phylogenetic analysis of NagBb homologs and GH101 endo-α-N-acetylgalactosaminidases. A tree was constructed by the ClustalW program using the neighbor-joining method. For GH101 enzymes, the conserved GH101 domains were used: EndoEF, amino acids 309–971; EngBF, amino acids 345–1089; and EngCP, amino acids 492–1145.
FIGURE 5.
FIGURE 5.
Two distinct pathways for assimilation of mucin-type O-glycans in B. bifidum. A, a pathway for core-1-type O-glycan in gastroduodenal mucin. EngBF, endo-α-N-acetylgalactosaminidase; GL-BP, galacto-N-biose (Galβ1–3GalNAc)/lacto-N-biose I (Galβ1–3GlcNAc)-binding protein, a solute-binding protein of ATP-binding cassette transporter; GLNBP, galacto-N-biose/lacto-N-biose I phosphorylase. B, a proposed pathway for sialyl and galactosyl core-3-type O-glycan in intestinal mucin. LnbB, lacto-N-biosidase; BbgIII, β-galactosidase; BbhI and BbhII, β-N-acetylhexosaminidase I and II; SiaBb2, α-sialidase 2.
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