Binding of Tamm‐Horsfall protein to complement 1q and complement 1, including influence of hydrogen‐ion concentration

Abstract

SummaryThe goal of this study was to further characterize the interaction between an abundant urinary glycoprotein, Tamm-Horsfall protein, and complement 1q to determine the robustness of this reaction under different environmental conditions (particularly pH) and to begin to determine the specificity of this reaction. The influence of pH coupled with ionic strength was evaluated with an ELISA that demonstrated immobilized TammHorsfall protein bound complement 1q strongly with a K D in the nmol/L range from pH 9 to pH 5.5. Increasing the ionic strength from 10 mmol/L sodium chloride (NaCl) to 154 mmol/L NaCl decreased the affinity of TammHorsfall protein for complement 1q slightly (2-7-fold) at pH 9 to pH 6.5. A resonant mirror biosensor was also utilized to evaluate the binding of Tamm-Horsfall protein to complement 1q at different pH values (pH 8.2-5.8). These studies indicated that, compared to at pH 8.2, Tamm-Horsfall protein bound complement 1q at pH 5.8 with an almost two-fold higher affinity (pH 8.2, K D = 5.1 nmol/L vs at pH 5.8, K D = 2.8 nmol/L) due to a faster association rate (pH 8.2 k ass = 1.6 × 10 6 L/mol per s vs pH 5.8 k ass = 2.9 × 10 6 L/mol per s). Surprisingly, the capacity of Tamm-Horsfall protein for complement 1q decreased significantly at pH 5.8, suggesting that a site for complement 1q binding to Tamm-Horsfall protein may be lost at the acidic pH. Biosensor studies also showed that Tamm-Horsfall protein bound the entire complement 1 complex with binding affinities and association rates similar to those obtained for complement 1q individually. This suggested that Tamm-Horsfall protein bound complement 1q at a site other than the region of its collagenous tail where C1r 2 and C1s 2 bind. By western blot analysis, it was demonstrated that Tamm-Horsfall protein bound preferentially to the C chain of complement 1q.

Intact protein core structure is essential for protein-binding, mononuclear cell proliferating, and neutrophil phagocytosis-enhancing activities of normal human urinary Tamm–Horsfall glycoprotein

“…However, Moonen et al [37] reported that THP could not bind with native cytokines including TNF-α. In addition, the binding of THP to complement 1 and complement 1q depends on the electrostatic evens as demonstrated by the influence of hydrogen concentration and ionic-strength on THP-C1q binding affinity [16,38]. In this study, we found that enzymatic digestion of THP with neuraminidase or β-galactosidase did not significantly affect the binding activity with different protein molecules, although a tendency of decrease was shown in Figs.…”

“…The daily excretion of THG ranges from 50-200 mg in humans [32]. The glycoprotein can interact with viral proteins [1], bacterial structure components [2], bacterial exotoxin [33], immunoglobulin light chains [14,15], complement component 1 and 1q [16], proinflammatory cytokine IL-1 [12], IL-2 [34] and TNF-α [12,13], and surface membrane proteins on human PMN, lymphocytes and monocytes [17][18][19][20][21]. Immunologically, THG not only activates PMN through binding to a single class of sialic acid-specific cell surface receptors [17] but enhances the release of IL-1, IL-6, and TNF-α from monocytes responsible for B and T lymphocytes proliferation [20,21].…”

Uromodulin Facilitates Neutrophil Migration Across Renal Epithelial Monolayers

“…Specifically, uromodulin has been shown to induce pro-inflammatory cytokine release from human whole blood [8], to activate myeloid dendritic cells (DC) to acquire a fully mature DC phenotype [9], and to activate monocytes [8]. Precisely how uromodulin orchestrates these diverse immunological properties is unknown, however the protein has been shown to bind with high affinity to a number of immuno-proteins including the complement factors C1, C1q and C3 [10,11], IgG [12,13] and cytokines such as TNF alpha, IL-1 beta and IL-8 [14]. It is thought that these diverse but potent pro-inflammatory properties of uromodulin may signal tubular damage and repair [8,9].…”

Tamm-Horsfall Protein Regulates Circulating and Renal Cytokines by Affecting Glomerular Filtration Rate and Acting as a Urinary Cytokine Trap

“…The trapping role played by urinary THP may not be limited to cytokines. Previous in vitro studies suggested that THP also binds to complement C1 and C1q (61)(62)(63). It is possible that THP is a promiscuous urinary trap not only for cytokines, but also for other immunological effectors.…”