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

We found that commercially available sialidases prepared from Clostridium perfringens ATCC10543 were contaminated with an endoglycosidase capable of releasing the disaccharide GlcNAc alpha1->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-alpha -Glc. By spectroscopy, the structure of the disaccharide released from porcine gastric mucin by this enzyme was established to be GlcNAc alpha1-->4Gal. The specificity of this enzyme as an endo-beta -galactosidase was established by analyzing the liberation of GlcNAc alpha1-->4Gal from GlcNAc alpha1--->4Gal beta1-->4GlcNAc beta1-->6(GlcNAc alpha1-->4Gal beta1-3)GalNAc-ol by mass spectrometry. Because this novel endo-beta -galactosidase specifically releases the GlcNAc alpha1-->4Gal moiety from porcine gastric mucin, we propose to call this enzyme a GlcNAc alpha1-->4Gal-releasing endo-beta -galactosidase (Endo-beta -Gal(GnGa)). Endo-beta -Gal(GnGa) was found to remove the GlcNAc alpha1-->4Gal epitope expressed in gastric adenocarcinoma AGS cells transfected with alpha1,4-N-acetylglucosaminyltransferase cDNA. Endo-beta -Gal(GnGa) should become useful for studying the structure and function of glycoconjugates containing the terminal GlcNAc alpha1-->4Gal epitope.