Cleavage of Epitectin, a Mucin-Type Sialoglycoprotein, from the Surface of Human Laryngeal Carcinoma-Cells by a Glycoprotease from Pasteurella haemolytica

Abstract

In this study we have assessed the action of a novel glycoprotease, secreted by the bovine pneumonia pathogen Pasteurella haemolytica, on epitectin expressed on the surface of human laryngeal carcinoma (H.Ep.2) cells. Epitectin has been previously characterized as a high buoyant density glycoprotein of mass of over 350 kDa extensively glycosylated on serine and threonine by small oligosaccharides. Purified metabolically labeled epitectin was very effectively hydrolyzed by the glycoprotease. However, short- and long-term treatments yielded a complex mixture of products which could not be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or column chromatography, probably because of the heterogenity of the structure and the distribution of the saccharides. Treatment of H.Ep.2 cells with glycoprotease followed by flow cytometric analysis revealed a significant loss in the cell surface epitopes detected by the anti-epitectin Ca2 monoclonal antibody. The action of the glycoprotease on cell surface epitectin was blocked by anti-glycoprotease antisera and was absent in an extract of a glycoprotease- negative strain of P. haemolytica. When extracts of cells treated with glycoprotease for 4 h were subjected to SDS-PAGE followed by 125I-wheat germ agglutinin overlay and autoradiography, the intensity of the characteristic epitectin bands was found to be drastically reduced compared to controls. H.Ep.2 cells metabolically labeled with [ 3H]glucosamine were also incubated with or without the glycoprotease and the released products were fractionated and analyzed. The enzyme-released products were found to be enriched in mucin-type glycopeptides. Thus the P. haemolytica glycoprotease could be used to selectively degrade mucin glycoproteins on cancer cell surface.