A novel endo-beta-galactosidase from Clostridium perfringens that liberates the disaccharide GlcNAcalpha 1-->Gal 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 GlcNAcalpha1-->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 NMR spectroscopy, the structure of the disaccharide released from porcine gastric mucin by this enzyme was established to be GlcNAcalpha1-->4Gal. The specificity of this enzyme as an endo-beta-galactosidase was established by analyzing the liberation of GlcNAcalpha1-->4Gal from GlcNAcalpha1-->4Galbeta1-->4GlcNAcbeta1-->6(GlcNAcalpha1--> 4Galbeta1-->3)GalNAc-ol by mass spectrometry. Because this novel endo-beta-galactosidase specifically releases the GlcNAcalpha1-->4Gal moiety from porcine gastric mucin, we propose to call this enzyme a GlcNAcalpha1-->4Gal-releasing endo-beta-galactosidase (Endo-beta-Gal(GnGa)). Endo-beta-Gal(GnGa) was found to remove the GlcNAcalpha1-->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 GlcNAcalpha1-->4Gal epitope.
Ashida
H
H
Department of Biochemistry, Tulane University Health Sciences Center School of Medicine, New Orleans, Louisiana 70112, USA.
Anderson
K
K
Nakayama
J
J
Maskos
K
K
Chou
C W
CW
Cole
R B
RB
Li
S C
SC
Li
Y T
YT
D000230:Adenocarcinoma / Q000378:metabolism; D000645:Ammonium Sulfate / Q000378:metabolism; D000818:Animals; D002846:Chromatography, Affinity; D003016:Clostridium perfringens / Q000201:enzymology*; D018076:DNA, Complementary / Q000378:metabolism; D004187:Disaccharides / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005753:Gastric Mucosa / Q000378:metabolism*; D006026:Glycoside Hydrolases; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015151:Immunoblotting; D007150:Immunohistochemistry; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009077:Mucins / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013274:Stomach Neoplasms / Q000196:embryology; D013379:Substrate Specificity; D013552:Swine; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000737:chemistry* / Q000378:metabolism
D018076:DNA, Complementary; D004187:Disaccharides; D000939:Epitopes; D009077:Mucins; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase; D000645:Ammonium Sulfate