Abstract
A high-molecular-weight (approximately 3.5 x 10(3) kDa) peanut agglutinin (PNA)-binding glycoprotein was previously identified as a tumour marker in pancreatic cancer sera. In the present study the density of this glycoprotein has been estimated and further analysis performed using sequential lectin blotting combined with serial mild acid hydrolysis followed by repeated Smith degradation. This has allowed partial in-situ characterization of the carbohydrate side-chains. The PNA-binding glycoprotein variably expresses the blood group H and sialylated Lewis a antigens. H and sialyl Lea expression disappeared after mild acid hydrolysis and T (PNA acceptor) disappeared after the first Smith degradation step. T (beta gal(1-3)alpha galNAc) expression reappeared after a second Smith degradation, suggesting that the backbone regions of the mucin side-chains with this core sequence are built up by the further addition of at least 2 monosaccharides. A model has been constructed showing the minimum variations in side chain structure that would explain the results of the serial degradation and lectin blotting. Purification and caesium chloride density gradient centrifugation show that the intact PNA-binding glycoprotein has a density of greater than 1.48 g/ml, characteristic of a mucin. The development of assays using a panel of monoclonal antibodies (MAbs) or lectins directed against the different carbohydrate epitopes expressed on this mucin may provide better diagnostic accuracy for pancreatic cancer than current marker assays which rely on detection of a single epitope.
Published Version
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