Purification and immunohistochemistry of Griffonia simplicifolia agglutinin-II-binding mucus glycoprotein in rat stomach.

Abstract

Gastric mucus is thought to protect the gastric wall from mechanical trauma, desiccation, pathogenic microorganisms, acid and proteases. We purified Griffonia simplicifolia agglutinin-II (GSA-II)-binding mucus glycoprotein (GMG) from rat gastric mucosa by solubilization in a guanidine-containing buffer, gel permeation chromatography, Ricinus communis agglutinin-I (RCA-I)-affinity chromatography and GSA-II-affinity chromatography. Rat GMG showed high molecular weight on a Sephacryl S-1000 column, and a single band in 0.5% agarose-2% polyacrylamide composite gels and blots. A protein of approximately 60 kDa was contained in the GMG preparation. GMG was deglycosylated with trifluoromethanesulphonic acid treatment. An antibody was raised against deglycosylated GMG (deGMG). The antibody recognized deGMG, GMG, periodic acid-treated deGMG and O-glycanase-digested deGMG, but did not react to trypsin-digested deGMG. These results suggest that the antibody recognizes proteinase-sensitive region or peptide backbone of GMG. In immunohistochemistry, the mucous gel layer of the stomach luminal surface was stained with antibody. The antibody recognized not only gastric mucous neck cells and pyloric gland cells, but also gastric surface mucous cells, mucous cells in the duodenal gland, and goblet cells in the small intestine and colon. These results indicate that GMG is a component of rat gastric mucus, and that the antibody recognizes mucous-secreting cells in rat stomach and intestine.