Characterization of two different endo-α-N-acetylgalactosaminidases from probiotic and pathogenic enterobacteria, Bifidobacterium longum and Clostridium perfringens

Abstract

Endo-α-N-acetylgalactosaminidase (endo-α-GalNAc-ase) catalyzes the hydrolysis of the O-glycosidic bond between α-GalNAc at the reducing end of mucin-type sugar chains and serine-threonine of proteins to release oligosaccharides. Previously, we identified the gene engBF encoding endo-α-GalNAc-ase from Bifidobacterium longum, which specifically released the disaccharide Galβ1-3GalNAc (Fujita K, Oura F, Nagamine N, Katayama T, Hiratake J, Sakata K, Kumagai H, Yamamoto K. 2005. Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-α-N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem. 280:37415-37422). Here we cloned a similar gene named engCP from Clostridium perfringens, a pathogenic enterobacterium, and characterized the gene product EngCP. Detailed analyses on substrate specificities of EngCP and EngBF using a series of p-nitrophenyl-α-glycosides chemically synthesized by the di-tert-butylsilylene-directed method revealed that both enzymes released Hex/HexNAcβ1-3GalNAc (Hex = Gal or Glc). EngCP could also release the core 2 trisaccharide Galβ1-3(GlcNAcβ1-6)GalNAc, core 8 disaccharide Galα1-3GalNAc, and monosaccharide GalNAc. Our results suggest that EngCP possesses broader substrate specificity than EngBF. Actions of the two enzymes on native glycoproteins and cell surface glycoproteins were also investigated. © The Author 2008. Published by Oxford University Press. All rights reserved.