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-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.

TNF-α is a 17-kDa glycoprotein containing lectin-like domains capable of binding with the glycomoiety of THP [14]. Many authors have reported that carbohydrate side chains in THP consist mainly of N-linked glycans with high mannose sequences carried by Asn251-Man6GlcNAc2 and Man5GluNAc2 that mediate interactions with type-1 fimbriated Escherichia coli [20][21][22][23]. O-linked chains in THP molecules are responsible for interactions with different proteins [24]. TNF-α may play an important role in chronic inflammatory diseases such as rheumatoid arthritis [25,26]. Hession et al. [27] found that THP might act as a unique renal regulatory glycoprotein via binding to a number of potent circulatory cytokines including TNF-α and IL-1β. In clinical practice, anti-TNF-α biological therapy could rapidly suppress rheumatoid activity better than conventional disease-modified anti-rheumatic drugs (DMARDs) therapy [28,29]. However, the molecular basis of the structure-binding relationship between THP and TNF-α remains unclear. In this study, we explored the binding-structure foundation by using lectin-binding ELISA, enzyme digestion, and monosaccharide inhibition tests. These findings may yield a novel therapeutic strategy for rheumatoid arthritis.

Non-Specific THP Binding to Serum Proteins and Proinflammatory Cytokines
THP binds non-specifically to a broad spectrum of protein molecules [11,16]. To confirm this property, microwells were coated with 100 μL of 20 μg/mL BSA, human IgG, human recombinant TNF-α, IFN-, IL-6, or IL-1β and incubated at 37 °C for 2 h and 4 °C overnight. THP (100 μL at 10 μg/mL) was then added to the microwells and incubated at 37 °C for 2 h. HRP-conjugated anti-uromucoid antibodies were added to detect THP binding to different proteins. We thus confirmed THP purified from normal human urine non-specifically bound different protein molecules with diverse capacities (Figure 1). Maximum binding occurred between THP and TNF-α; minimum binding occurred between THP and IFN-γ.

Dose-Dependent Binding Between THP and TNF-α
To verify binding between THP and TNF-α, Western blots (2 to 16 μg/mL THP; Figure 2A) and ELISA (0.5 to 6 μg/mL TNF-α; Figure 2B) were performed. Binding between THP and TNF-α was clearly dose-dependent. Moonen et al. [30] argued that native TNF-α and IL-1β do not bind THP because TNF-α in liquid phase does not competitively inhibit THP binding to TNF-α-coated microtiter plates. However, Hession et al. [27] argued that THP is a renal ligand for cytokines in vivo. Why THP only reacts with denatured, not native, TNF-α, and whether TNF-α and IL-1β are denatured in vivo requires further investigation.

Low Binding Affinity with K d =1.4 −1.7 × 10 −6 M between THP and TNF-α
It is believed that the binding affinity between two proteins is less than antigen-antibody or ligand-receptor interactions. We estimated the THP-TNF-α binding affinity as described by Katanick et al. [31] with some modifications. The specific binding vs. TNF-α concentrations in two experiments are plotted in Figure 3A (Experiment 1) and 3C (Experiment 2). The Y transformed curves derived from the specific binding data are plotted in 3B (Experiment 1) and 3D (Experiment 2). B max and K d values were calculated by Scatchard plot analysis shown in Figure 3. The K d values of the two experiments were 1.368 × 10 −6 M ( Figure 3B) and 1.647 × 10 −6 M ( Figure 3D). However, Rhodes et al. [32] demonstrated two binding affinities between sheep THP and IgG; a high-affinity K d of 10 −8 -10 −9 M and a low-binding affinity K d of 10 −6 -10 −7 M. Although we did not measure the K d of human THP-IgG binding, it was 20% lower than human THP-TNF-α binding as shown in the ELISA results ( Figure 1). Experiments to measure the binding affinity of different mammalian THPs with human TNF-α are underway.

Carbohydrate Compositions of THP and THP-Binding Proteins
Sherblom et al. [14] demonstrated a lectin-like interaction between human recombinant TNF-α and uromodulin. We hypothesized that THP molecules may play dual roles in protein binding as their high carbohydrate content (25%-30%) are targets of lectin-like domains of TNF-α. Conversely, some THP domain structures such as the zona pellucina (ZP) domain may possess adhesive molecule-like or even lectin-like properties and bind to carbohydrate components in TNF-α molecules. We assessed the carbohydrate compositions of THP and THP-binding molecules. Lectin-binding ELISA method was performed with 5 lectins with specific sugar moieties to detect the sugar compositions of BSA, IgG, TNF-α, IFN-γ, and THP ( Figure 4). IFN-γ contained minimal glycomoieties, consistent with its minimal binding to THP. We noted that BSA, IgG, and TNF-α contain abundant β(1,4)-GlcNAc oligomers and GlcNAc/branched mannose. These glycomoieties are also present in THP. Muchmore et al. [33] found that high-mannose glycopeptides [Man5(6)GluNAc 2 -Asn] THP interact with recombinant TNF-α and IL-1β. It is possible the high-mannose glycans in human THP are carried by Asn251 [21,23]. IL-2 also exhibits lectin-like properties specific for high-mannose glycopeptides capable of binding THP [13,33]. Lucas et al. [34] demonstrated that trypanosome-TNF-α interaction was inhibited by N,N′-diacetylchitobiose. This domain structure also possessed lectin-like affinity for TNF-α. These results suggest that sugar-lectin-sugar interactions are one of the mechanisms for THP-protein binding.

β-galactosidase-digested THP Enhanced THP-TNF-α Binding
THP contains complex carbohydrate side chains around the protein core structure; thus, it is necessary to determine whether the side chain glycomoiety or protein-core structure mediates THP-TNF-α binding. We used carbohydrate-degrading enzymes (neuraminidase, β-galactosidase), protein-degrading enzyme (proteinase K), and glycoprotein-degrading enzymes (O-sialoglycoprotein endopeptidase, carboxy-peptidase Y) to digest intact THP as shown in Figure 5A. Digested THP products were then immunoblotted with TNF-α (1° binder) and HRP-anti-TNF antibody (2° binder). As shown in Figure 5B, only β-galactosidase-digested THP products reacted with TNF-α on Western blots. Thus, galactoside residues in the THP side chain prevent THP-TNF-α binding. Because the amount of THP (4 g) used in Figure 5 was insufficient for reacting with TNF-α as shown in Figure 2, it is difficult to conclude whether the protein-core structure is involved in THP-TNF-α binding. We suggest the ZP domain responsible for polymerization of THP into supra-molecular structures mediates homologous THP-THP binding and heterogeneous THP-TNF-α binding. Genetic manipulation to delete the ZP domain is in progress.

Figure 5.
Binding capacity of intact THP and enzyme-digested products with TNF-α, was detected by Western blotting. (A) The molecular size of intact THP, TNF-α, and the different enzyme-digested THP products was identified by Coomasie blue staining. (B) The binding capacity of intact THP and enzyme-digested THP products was detected by Western blot using TNF-α (1° binder) and HRP-anti-TNF-α antibody (2° binder) as indicators.

Reagents and Antibodies
Purified human IgG, BSA, neuraminidase, β-galactosidase, proteinase K, and carboxypeptidase Y were purchased from Sigma-Aldrich (

Purification of THP from Normal Human Urine
Twenty-four-hour urine was collected in a clean glass bottle from a normal individual. We followed the purification procedures described in our previous report [20]. THP purity and relative molecular weight were detected by 10% SDS-PAGE. Identification of THP was confirmed by Western blotting with anti-uromucoid antibody (The Binding Site Ltd, University of Birmingham Research Institute, Birmingham, UK). This study was approved by the IRB and Medical Ethics Committee, Taipei Veterans General Hospital, Taiwan (VGH IRB NO: 94-07-27A). All participants provided signed, informed consent.

ELISA
THP (100 μL at 20 μg/mL) was placed in microtiter wells and incubated for 24 h at 4 °C. TNF-α (0.5~6.0 μg/mL) was added and incubated overnight at 4 °C. After several washes, HRP-conjugated anti-TNF-α antibody was added and incubated at room temperature for 2 h; color development and absorption were measured as OD450.

Scatchard Plot Analysis and K d Calculations
A Scatchard plot was used to analyze the binding affinities of THP and TNF-α. We followed the method described by Kananick et al. [31] with modifications: radioisotope was replaced by HRP-conjugated-TNF-α in the ELISA. Briefly, recombinant human TNF-α (0, 500, 1,000, 1,500, 2,000, and 2,500 pg/mL) were added to THP-coated microwells and incubated at room temperature for 2 h with continuous shaking. The mixture was spun at 450 g for 20 min. Unbound THF-α in the supernatant and thrice-washed aspirates were collected. Bound TNF-α in the microwells was lysed with 200 µL lysis buffer containing 50 mM borate, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 25 mM phenylmethylsulphonyl fluoride, pH 8.0. Bound and unbound TNF-α were measured by ELISA. A Scatchard plot was drawn and analyzed by the "Prism" statistical program provided by GraphPad Software [36]

Microwell-Coated THP Was Preincubated with Different Monosaccharides
THP-coated microwells were preincubated with 10 μg of α-MDM, GalNAc, or GlcNAc overnight at 4 °C. TNF-α (2 μg/mL) was added and incubated overnight at 4 °C. HRP-anti-TNF-α antibody was added and incubated at room temperature. The binding of TNF-α to THP was detected by OD450 nm.

Statistical Analysis
Results are reported as mean ± S.D. Continuous variables were assessed by non-parametric Wilcoxon rank-sum test using commercially available software (Stata/SE8.0 Windows). Statistical significance was indicated by p < 0.05.

Conclusions
We have explored the molecular basis of the structure-binding relationship between THP and TNF-α. At least four original findings are reported here: (1) The K d of THP-TNF-α binding was around 1.5 × 10 −6 M, less than antigen-antibody or ligand-receptor binding. (2) THP and TNF-α contain high amounts of β(1,4)-GlcNAc oligomers and GlcNAc/branched mannose, which are relevant to THP-TNF-α binding. (3) Galactoside in THP carbohydrate side-chains hindered THP-TNF-α binding via sugar (in the THP molecule)-lectin (in the TNF-α molecule)-sugar (in the TNF-α molecule)-ZP domain (in the THP molecule) interactions. (4) Many monosaccharides such as α-methyl-D-mannoside, N-acetylglucosamine, or N-acetylgalactosamine, but not sialic acid, mediate THP-TNF-α binding. TNF-α is a crucial proinflammatory cytokine in many inflammatory disorders including rheumatoid arthritis, ankylosing spondylitis, psoriasis/psoriatic arthropathy, or inflammatory bowel diseases. Identification of the THP domain structure(s) responsible for TNF-α binding may yield a novel therapeutic strategy for acute and chronic inflammatory diseases.