Tamm‐Horsfall Protein and Properdin Biochemical and Functional Interactions

Tamm‐Horsfall protein (THP), also known as uromodulin, is an abundant protein in normal mammalian urine that interacts with numerous cells and molecules of the immune system, including various complement proteins such as C1q and factor H. Properdin (factor P), which potentiates complement activation by stabilizing the alternative pathway C3 convertase, previously has been shown to bind to THP. In the present study, an enzyme‐linked immunosorbent assay (ELISA) was used to examine the effect of ionic strength, pH, and heparin on the binding between these two oppositely charged proteins. Additionally, since THP has been shown to act as a cofactor, like factor H, in factor I‐mediated C3b degradation, we investigated whether properdin altered this cofactor activity of THP. All ELISA buffers contained 1 mM MgCl 2 and 1 mM CaCl 2 because earlier studies showed a requirement for divalent cations in this reaction. As measured by ELISA, binding between THP and properdin was similar (K~ 10 −8 M) in 20 mM, 60 mM, and 90 mM NaCl solutions, but no significant binding was detected in 154 mM NaCl buffers. There was a reduction in binding capacity, but not overall affinity in 120 mM NaCl buffers. Binding of properdin to immobilized THP in 60 mM NaCl was similar at pH 6.0, 7.5, and 8.5. The THP/properdin interaction was very sensitive to heparin, with 0.008 units of heparin/ml producing a 50% inhibition of binding of 2 μg/ml properdin to immobilized THP. In vitro cofactor assays analyzed on silver‐stained SDS‐PAGE gels demonstrated that while 300 ng of properdin inhibited by more than 50% the ability of 1 ng factor H to serve as a cofactor in factor‐I mediated C3b degradation, properdin appeared to increase by about 25% the ability of THP to serve as a cofactor in factor I‐mediated C3b degradation. Properdin, by itself, was not able to act as a cofactor in factor I‐mediated C3b degradation. In conclusion, THP and properdin bind more readily in lower ionic strength buffers than in higher ionic strength solutions, but these interactions are stable over a range of pH conditions typically found in urine. Heparin is very effective at inhibiting the binding between properdin to THP. On a functional level, properdin may augment THP's ability to act as a cofactor in C3b degradation, while it inhibits factor H's cofactor activity. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .