Binding studies of α-GaINAc-specific lectins to the α-GaINAc (Tn-antigen) form of porcine submaxillary mucin and its smaller fragments

Isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements demonstrate that a chemically and enzymatically prepared form of porcine submaxillary mucin that possesses a molecular mass of similar to 10(6) daltons and similar to 2300 alpha-GalNAc residues (Tn-PSM) binds to the soybean agglutinin (SBA) with a K-d of 0.2 nM, which is similar to 10(6) -fold enhanced affinity relative to GalNAc alpha 1-O-Ser (Tn), the pancarcinoma carbohydrate antigen. The enzymatically derived 81 amino acid tandem repeat domain of Tn-PSM containing similar to 23 alpha-GalNAc residues binds with similar to 10(3) -fold enhanced affinity, while the enzymatically derived 38/40 amino acid cleavage product(s) of Tn-PSM containing similar to 11-12 alpha-GaINAc residues shows similar to 10(2) -fold enhanced affinity. A natural carbohydrate decorated form of PSM (Fd-PSM) containing 40% of the core 1 blood group type A tetrasaccharide, and 58% peptide-linked GalNAc alpha 1-O-Ser/Thr residues, with 45% of the peptide-linked a-GaINAc residues linked alpha-(2,6) to N-glycolylneuraminic acid, shows similar to 10(4) enhanced affinity for SBA. Vatairea macrocarpa lectin (VML), which is also a GalNAc binding lectin, displays a similar pattern of binding to the four forms of PSM, although there are quantitative differences in its affinities as compared with SBA. The higher affinities of SBA and VML for Tn-PSM relative to Fd-PSM indicate the importance of carbohydrate composition and epitope density of mucins on their affinities for lectins. The higher affinities of SBA and VML for Tn-PSM relative to its two shorter chain analogs demonstrate that the length of a mucin polypeptide and hence total carbohydrate valence determines the affinities of the three Tn-PSM analogs. The results suggest a binding model in which lectin molecules "bind and jump" from alpha-GaINAc residue to alpha-GalNAc residue along the polypeptide chain of Tn-PSM before dissociating. The complete thermodynamic binding parameters for these mucins including their binding stoichiometries are presented. The results have important implications for the biological activities of mucins including those expressing the Tn cancer antigen.