BIOCHEMICAL-ANALYSIS OF A BLADDER-CANCER-ASSOCIATED MUCIN - STRUCTURAL FEATURES AND EPITOPE CHARACTERIZATION

Three monoclonal antibodies (mAbs), M344, M300 and M75, were shown to define a unique tumour-associated antigen (TAA) of superficial bladder tumours. The antigenic determinants are expressed on a very-high-mole cular-mass component and, in about 50% of the positive samples, one de terminant is also detected on a 62 kDa molecular species, observed onl y under reducing conditions. The objectives of the present study were to characterize further this TAA by analysing (1) the biochemical natu re of the epitopes recognized by the three mAbs, and (2) the biochemic al and structural features of the molecule bearing them. The antigenic ity was resistant to heat denaturation, trypsin and alpha-chymotrypsin treatments but highly sensitive to papain and Pronase digestion. NaIO 4 oxidation decreased reactivity to mAbs M344 and M300 but enhanced re activity to mAb M75. The three determinants were insensitive to beta-g alactosidase and alpha-L-fucosidase but were sensitive to Vibrio chole rae neuraminidase. None of the three mAbs reacted with ovine, bovine o r porcine submaxillary mucins. Deglycosylation with O-glycosidase or t rifluoromethanesulphonic acid completely abolished the reactivity of t he mAbs whereas N-glycosidase F deglycosylation had no appreciable eff ect. The presence on the molecule of cryptic Gal beta(1 --> 3)GalNAc a s a major core disaccharide was demonstrated by a heterologous sandwic h assay using mAb M75 and peanut agglutinin. Thiol reduction using bet a-mercaptoethanol increased mobility of the high-molecular-mass compon ent in polyacrylamide gels. We thus conclude that mAbs M344 and M300 r eact with sialylated carbohydrate epitopes, and mAb M75 reacts with a partially cryptic and periodate-resistant sialylated epitope expressed on a typical secreted high-molecular-mass oligomeric mucin which we n amed MAUB for mucin antigen of the urinary bladder.