Tamm‐Horsfall Protein Binds Properdin

Abstract

The purpose of this study was to determine if Tamm‐Horsfall protein (THP), an abundant urinary protein that interacts with various molecules of the immune system, binds to properdin (Factor P), a positive regulator of complement activation which stabilizes the alternative pathway C3 convertase. Enzyme‐linked immunosorbent assays (ELISA) were used to evaluate the binding of soluble human properdin to immobilized human THP. In low ionic strength buffers containing magnesium and calcium, THP bound properdin strongly (KD ~ 10‐8 M). Removing divalent cations from the buffer markedly reduced the ability of THP to bind properdin. Ionic strength also was important in this interaction. Properdin binding to THP was stronger in low ionic strength (20mM NaCl) and intermediate ionic strength buffers (90mM NaCl), than in physiologic strength (154mM NaCl) buffers. By ELISA, soluble THP effectively competed with immobilized THP for binding to properdin, with 0.1µM soluble THP inhibiting 50% of 0.05µM properdin's binding to immobilized THP. Western blot analysis demonstrated that soluble THP could bind to properdin after the properdin had been separated by SDS‐PAGE and transferred to nitrocellulose paper. In conclusion, this study shows that THP binds to properdin in the presence of divalent cations. This interaction is influenced by NaCl concentration, suggesting that electrostatic interactions between the two molecules are important. Properdin appears to recognize THP in solution, as well as immobilized THP, and THP binds to soluble properdin, as well as properdin that has been partially denatured by SDS during electrophoresis and Western blotting. The functional significance of this interaction remains to be evaluated. Funded by the PNWU Research Seed Program.