The Binding Affinity and Molecular Basis of the Structure-Binding Relationship between Urinary Tamm-Horsfall Glycoprotein and Tumor Necrosis Factor-α

In a previous study we noted significant THP binding to TNF-alpha, but did not explore the molecular basis of the structure-binding relationship. In this study, we used lectin-binding ELISA to assess the carbohydrate compositions of THP, BSA, IgG, TNF-alpha, and IFN-gamma. We identified beta(1,4)-N-acetylglucosamine oligomers (GlcNAc) and GlcNAc/branched mannose in BSA, IgG, TNF-alpha, and THP, but not in IFN-gamma. These carbohydrate moieties mediated binding with THP. Small amounts of Sia alpha(2,3)Gal/GalNAc, Sia(2,6) Gal/GalNAc, and mannose residues were also present in THP and TNF-alpha. Binding affinity (K-d) between THP and TNF-alpha by Scatchard plot analysis was 1.4-1.7 x 10(-6) M, lower than antigen-antibody or ligand-receptor binding affinities. To elucidate the structure-binding relationship of THP-TNF-alpha, THP was digested with neuraminidase, beta-galactosidase, O-sialoglycoprotein endopeptidase, carboxypeptidase Y, or proteinase K. beta-galactosidase increased binding capacity of THP for TNF-alpha. Monosaccharide inhibition suggested that alpha-methyl-D-mannoside, GlcNAc, and GalNAc, but not sialic acid, suppress THP-TNF-alpha binding as detected by ELISA. We conclude that sugar-lectin and sugar-protein interactions between cognate sites in THP and TNF-alpha mediate their binding.