Characterization of two different endo-alpha-N-acetylgalactosaminidases from probiotic and pathogenic enterobacteria, Bifidobacterium longum and Clostridium perfringens.
Endo-alpha-N-acetylgalactosaminidase (endo-alpha-GalNAc-ase) catalyzes the hydrolysis of the O-glycosidic bond between alpha-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-alpha-GalNAc-ase from Bifidobacterium longum, which specifically released the disaccharide Gal beta 1-3GalNAc (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-alpha-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-alpha-glycosides chemically synthesized by the di-tert-butylsilylene-directed method revealed that both enzymes released Hex/HexNAc beta 1-3GalNAc (Hex = Gal or Glc). EngCP could also release the core 2 trisaccharide Gal beta 1-3(GlcNAc beta 1-6)GalNAc, core 8 disaccharide Gal alpha 1-3GalNAc, 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.
Ashida
Hisashi
H
Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan. ashida@lif.kyoto-u.ac.jp
Maki
Riichi
R
Ozawa
Hayato
H
Tani
Yasushi
Y
Kiyohara
Masashi
M
Fujita
Masaya
M
Imamura
Akihiro
A
Ishida
Hideharu
H
Kiso
Makoto
M
Yamamoto
Kenji
K
D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics; D004755:Enterobacteriaceae / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D006023:Glycoproteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D019936:Probiotics / Q000378:metabolism; D013379:Substrate Specificity; D048809:alpha-N-Acetylgalactosaminidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D006023:Glycoproteins; D008562:Membrane Glycoproteins; D048809:alpha-N-Acetylgalactosaminidase