Antigenic and structural features of goblet-cell mucin of human small intestine.
With the use of a newly developed solid-phase radioimmunoassay method, the major antigenic determinants of human small-intestinal goblet-cell mucin were investigated and related to the overall tertiary structure of the mucin. Preliminary hapten inhibition studies with various oligosaccharides of known sequence and structure suggested that the determinants did not reside in carbohydrate. Exhaustive thiol reduction, however, almost abolished antigenicity, caused breakdown of the mucin into small heterogeneous glycopeptides, and liberated a 'link' peptide of Mr 118000. Western 'blots' of reduced mucin from polyacrylamide gels on to nitrocellulose sheets showed that a small amount of residual antigenicity remained in large-Mr glycopeptides (Mr greater than 200000). The 'link' peptide was not antigenic. Timed Pronase digestion of native mucin resulted in a progressive loss of antigenic determinants. Gel electrophoresis revealed that after 8h of digestion the 118000-Mr peptide had disappeared, whereas antigenicity, which was confined to large-Mr glycopeptides, was destroyed much more slowly with time (70% by 24h, 100% by 72h). Despite the loss of antigenicity, 72h-Pronase-digested glycopeptides retained all of the carbohydrate of the native mucin. Therefore the antibody to human small-intestinal mucin appears to recognize a 'naked' (non-glycosylated and Pronase-susceptible) peptide region(s) of mucin glycopeptides. For full antigenicity, however, disulphide bonds are required to stabilize a specific three-dimensional configuration of the 'naked' region.
D000596:Amino Acids / Q000276:immunology; D002241:Carbohydrates / Q000276:immunology; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D007421:Intestine, Small / Q000032:analysis* / Q000276:immunology; D008956:Models, Chemical; D009077:Mucins / Q000032:analysis / Q000276:immunology; D011402:Pronase; D011863:Radioimmunoassay; D013438:Sulfhydryl Compounds
D000596:Amino Acids; D002241:Carbohydrates; D000939:Epitopes; D009077:Mucins; D013438:Sulfhydryl Compounds; D011402:Pronase