A Novel Endo-β-galactosidase from Clostridium perfringens that Liberates the Disaccharide GlcNAcα1→4Gal from Glycans Specifically Expressed in the Gastric Gland Mucous Cell-type Mucin

Abstract

We found that commercially available sialidases prepared from Clostridium perfringens ATCC10543 were contaminated with an endoglycosidase capable of releasing the disaccharide GlcNAcα1→4Gal from glycans expressed in the gastric gland mucous cell-type mucin. We have isolated this enzyme in electrophoretically homogeneous form from the culture supernatant of this organism by ammonium sulfate precipitation followed by affinity chromatography using a Sephacryl S-200 HR column. The enzyme was specifically retained by and eluted from the column with methyl-α-Glc. By NMR spectroscopy, the structure of the disaccharide released from porcine gastric mucin by this enzyme was established to be GlcNAcα1→-4Gal. The specificity of this enzyme as an endo-β-galactosidase was established by analyzing the liberation of GlcNAcα1→4Gal from GlcNAcα1→4Galβ 1→4GlcNAcβ1→6(GlcNAcα1→ 4Galβ1→3)GalNAc-ol by mass spectrometry. Because this novel endo-β-galactosidase specifically releases the GlcNAcα1→4Gal moiety from porcine gastric mucin, we propose to call this enzyme a GlcNAcα1→4Gal-releasing endo-β-galactosidase (Endo-β-GalGnGa). Endo-β-Gal GnGa was found to remove the GlcNAcα1→4Gal epitope expressed in gastric adenocarcinoma AGS cells transfected with α1,4-N-acetylglucosaminyltransferase cDNA. Endo-β-GalGnGa should become useful for studying the structure and function of glycoconjugates containing the terminal GlcNAcα1→4Gal epitope.