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.