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

Abstract

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-γ. We identified β(1,4)-N-acetylglucosamine oligomers (GlcNAc) and GlcNAc/branched mannose in BSA, IgG, TNF-α, and THP, but not in IFN-γ. 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 (Kd) 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.

Tamm–Horsfall Protein is a Potent Immunomodulatory Molecule and a Disease Biomarker in the Urinary System

“…Wu et al [ 61 ] indicated that the intact protein core structure of THP is essential for this protein’s binding with other proteins and immune cells. The same group measured the binding affinity ( K d ) between THP and TNF-α by Scatchard plot analysis and revealed that the binding affinity of THP and TNF-α was 1.4–1.7 × 10 −6 M, which is lower than the antigen–antibody or ligand–receptor binding affinity [ 62 ]. They also identified that β(1,4)- N -acetylglucosamine oligomer (GluNAc) and GluNAc/branched mannose in bovine serum albumin, IgG, and TNF-α are essential for binding with THP.…”

Glycoproteomic Analysis of Human Urinary Exosomes

“…The precise function of this protein is not well understood but is thought to be important for kidney and renal function , and is annotated as exosomal. Glycosylation on this protein has been shown to control protein recognition between THP/uromodulin and other clinically relevant proteins . THP glycosylation also functions in cellular recognition and has been shown to modulate immune cell activation. , Additionally, this protein also blocks against infection through the effective “coating” of pathogens’ surfaces, thus preventing renal infections .…”

CP-25 Attenuates the Activation of CD4+ T Cells Stimulated with Immunoglobulin D in Human

“…The intrinsic binding affinity between the fluorescence-labeled IgD antibody and IgD on IgDR in CD4 + T cells was evaluated by fluorescence-based receptor binding assays ( Wu et al, 2012 ; McCall et al, 2014 ). IgD binding for detection by flow cytometry was performed as follows.…”