Human urinary bladder carcinoma glycoconjugates expressing T-(Gal beta(1-3)GalNAc alpha 1-O-R) and T-like antigens: a comparative study using peanut agglutinin and poly- and monoclonal antibodies.
T- and T-like antigens on glycoproteins and glycolipids were examined in extracts of human urinary bladder tumors and normal tissue by Western blot analysis and reagent binding to thin layer chromatograms. Three different anti-T-reagents were used: peanut (Arachis hypogaea) lectin (PNA) and mono- and polyclonal antibodies specific for T-antigen (Gal beta(1-3)GalNAc alpha 1-O-R). Immunodetection with the T-specific reagents in nitrocellulose replicas of bladder tumor glycoproteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, demonstrated tumor-specific T-antigen-bearing glycoproteins compared to normal urothelial glycoproteins. In addition, a remarkable difference in binding was found between the immunological reagents and PNA lectin. PNA showed major reactivity to a 28-kD glycoprotein extracted from tumors. Monoclonal anti-T-antibody (clone HH8) showed major reactivity with an M(r) 34,000 glycoprotein, and polyclonal anti-T-antibody showed major reactivity with an M(r) 36,000 glycoprotein. PNA agarose column affinity-purified tumor glycoproteins did not bind the antibodies. Glycoproteins, M(r) 28,000 and 34,000, were shown to be O-linked by stepwise deglycosylation. In solid phase monosaccharide inhibition tests, galactose followed by N-acetyl-galactosamine were the most potent monosaccharides inhibiting binding to immobilized bladder tumor glycoproteins. None of the anti-T-reagents reacted with glycolipids extracted from tumor tissue. It is concluded that PNA lectin, in addition to the T-disaccharide, reacts with other protein-anchored carbohydrate structures in carcinomas.
Langkilde
N C
NC
Department of Experimental Clinical Oncology, Danish Cancer Society, Aarhus.
Wolf
H
H
Clausen
H
H
Orntoft
T F
TF
D000911:Antibodies, Monoclonal / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000276:immunology; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000276:immunology* / Q000378:metabolism; D001667:Binding, Competitive; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002277:Carcinoma / Q000276:immunology*; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D004187:Disaccharides / Q000737:chemistry / Q000276:immunology*; D006001:Glycoconjugates / Q000276:immunology*; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000378:metabolism; D019887:Peanut Agglutinin; D013329:Structure-Activity Relationship; D001743:Urinary Bladder / Q000276:immunology*; D001749:Urinary Bladder Neoplasms / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D015295:Antigens, Tumor-Associated, Carbohydrate; D004187:Disaccharides; D006001:Glycoconjugates; D006023:Glycoproteins; D037102:Lectins; D009005:Monosaccharides; D019887:Peanut Agglutinin; C016467:Thomsen-Friedenreich antigen