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-α, 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-α, and IFN-g. We identified β(1,4)-N-acetylglucosamine oligomers (GlcNAc) and GlcNAc/branched mannose in BSA, IgG, TNF-α, and THP, but not in IFN-g. These carbohydrate moieties mediated binding with THP. Small amounts of Siaα(2,3)Gal/ GalNAc, Sia(2,6)Gal/GalNAc, and mannose residues were also present in THP and TNF-α. Binding affinity (K(d)) between THP and TNF-α by Scatchard plot analysis was 1.4-1.7 × 10⁻⁶ M, lower than antigen-antibody or ligand-receptor binding affinities. To elucidate the structure-binding relationship of THP-TNF-α, THP was digested with neuraminidase, β-galactosidase, O-sialoglycoprotein endopeptidase, carboxypeptidase Y, or proteinase K. β-galactosidase increased binding capacity of THP for TNF-α. Monosaccharide inhibition suggested that α-methyl-D-mannoside, GlcNAc, and GalNAc, but not sialic acid, suppress THP-TNF-α binding as detected by ELISA. We conclude that sugar-lectin and sugar-protein interactions between cognate sites in THP and TNF-α mediate their binding.
Institute of Clinical Medicine, National Taiwan University College of Medicine and National Taiwan University Hospital, No.7 Chung-Shan South Road, Taipei 100, Taiwan.
Cheng-Han
CH
Wu
Ko-Jen
KJ
Li
Sue-Cien
SC
Siao
Yu-Hsuan
YH
Chen
Tsai-Hung
TH
Wu
Chang-Youh
CY
Tsai
Chia-Li
CL
Yu
D002241:Carbohydrates / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009005:Monosaccharides / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D059748:Proteolysis; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000378:metabolism*; D058949:Uromodulin / Q000737:chemistry* / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
D002241:Carbohydrates; D009005:Monosaccharides; D014409:Tumor Necrosis Factor-alpha; D058949:Uromodulin; D001616:beta-Galactosidase