α-N-Acetylgalactosaminidase from Infant-associated Bifidobacteria Belonging to Novel Glycoside Hydrolase Family 129 Is Implicated in Alternative Mucin Degradation Pathway

Masashi Kiyohara,Takashi Nakatomi,Shin Kurihara,Shinya Fushinobu,Hideyuki Suzuki,Tomonari Tanaka,Shin-Ichiro Shoda,Motomitsu Kitaoka,Takane Katayama,Kenji Yamamoto,Hisashi Ashida

doi:10.1074/jbc.m111.277384

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.

Highlights

The degradation pathway of the intestinal mucin by bifidobacteria is poorly understood
We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo␣-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides
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

Summary

Background

The degradation pathway of the intestinal mucin by bifidobacteria is poorly understood. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo␣-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. Novel ␣-N-Acetylgalactosaminidase from Bifidobacteria bifidobacteria can release the core-1 disaccharide from mucin glycoproteins through the action of the extracellular membrane-bound EngBF, incorporate it into the cytosol through a specific ATP-binding cassette-type transporter on the plasma membrane (11–13), and phosphorolyze it by a specific phosphorylase in the cytosol (14). These findings suggest that bifidobacteria selectively grow in the intestines by assimilating mucin-type oligosaccharides utilizing a unique degradation pathway. Its putative homologs were conserved in infant-associated bifidobacteria, and they were found to form a novel GH family

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION