A novel endo-beta-galactosidase from Clostridium perfringens that liberates the disaccharide GlcNAc alpha 1 -> 4Gal from glycans specifically expressed in the gastric gland mucous cell-type mucin

We found that commercially available sialidases prepared from Clostridium p erfringens ATCC10543 were contaminated with an endoglycosidase capable of r eleasing the disaccharide GlcNAc alpha1->4Gal from glycans expressed in the gastric gland mucous cell-type mucin. We have isolated this enzyme in elec trophoretically homogeneous form from the culture supernatant of this organ ism by ammonium sulfate precipitation followed by affinity chromatography u sing a Sephacryl S-200 HR column. The enzyme was specifically retained by a nd eluted from the column with methyl-alpha -Glc. By spectroscopy, the stru cture of the disaccharide released from porcine gastric mucin by this enzym e was established to be GlcNAc alpha1-->4Gal. The specificity of this enzym e as an endo-beta -galactosidase was established by analyzing the liberatio n of GlcNAc alpha1-->4Gal from GlcNAc alpha1--->4Gal beta1-->4GlcNAc beta1- ->6(GlcNAc alpha1-->4Gal beta1-3)GalNAc-ol by mass spectrometry. Because th is novel endo-beta -galactosidase specifically releases the GlcNAc alpha1-- >4Gal moiety from porcine gastric mucin, we propose to call this enzyme a G lcNAc alpha1-->4Gal-releasing endo-beta -galactosidase (Endo-beta -Gal(GnGa )). Endo-beta -Gal(GnGa) was found to remove the GlcNAc alpha1-->4Gal epito pe expressed in gastric adenocarcinoma AGS cells transfected with alpha1,4- N-acetylglucosaminyltransferase cDNA. Endo-beta -Gal(GnGa) should become us eful for studying the structure and function of glycoconjugates containing the terminal GlcNAc alpha1-->4Gal epitope.