Binding of Tamm‐Horsfall Protein to Properdin in Various Ionic Environments

Tamm‐Horsfall protein (THP), also known as uromodulin, is produced in the kidney and is the most abundant protein excreted into urine. It is an ~86kD acidic glycoprotein (pI≈3) with complex N‐linked carbohydrates often capped with sialic acid. THP may act as an immunomodulatory protein since it binds to IgG, complement 1q (C1q), complement 3b (C3b), and factor H (fH); inhibits the classical complement pathway; and acts as a cofactor for factor I in C3b degradation. THP also binds properdin which is a basic protein (pI > 9.5) consisting of multiple, identical 53 kD subunits. Properdin is the only known positive regulator of complement, stabilizing C3b. The purpose of this study was to investigate the effect of different ionic conditions on the interaction between THP and properdin, which are oppositely charged proteins. Previous studies of the interaction between THP and properdin had utilized Falcon Pro Bind microtiter plates that are no longer available. Hence, the first studies compared the remaining Falcon plates to three other microtiter plates. For these and all subsequent ELISAs, wells were coated with THP purified from the urine of one healthy male. Multiple concentrations of properdin (Complement Technologies) were added to THP‐coated wells, and the bound properdin was detected with goat anti‐properdin serum and rabbit anti‐goat IgG‐alkaline phosphatase. Of the plates tested, the Corning Costar plates acted in a manner most similar to Falcon plates and were used in the remaining studies. The binding buffer used in all studies was 20 mM Tris/1 mM MgCl 2 /1 mM CaCl 2 /0.05% Tween 20 (pH ~7.6) with concentrations of NaCl varying from 20 mM to 154 mM. These studies showed that the optimal NaCl concentration was dependent on the properdin concentration, with maximal binding of properdin to THP occurring at 70–80 mM NaCl for low properdin concentrations (0.25 and 0.5 μg/ml) and maximal binding of properdin to THP occurring at 20 mM NaCl with high properdin concentrations (5 μg/ml). The lowest amount of THP/properdin binding consistently was seen in the 154 mM NaCl buffer. These results differ somewhat from our very early, much more limited experiments with the Falcon microtiter plates, where THP binding to properdin was higher at all concentrations of properdin in a 90 mM ionic strength buffer. It is noteworthy that if the interaction between THP and properdin was purely electrostatic in nature, binding would be consistently higher in low ionic strength buffers, which was not what the present study showed. Further studies, not only on the effect of ionic strength but also on the effect of pH on the THP/properdin interaction are warranted since urine, which is where THP and properdin most likely could interact, varies considerably in these parameters. Support or Funding Information Supported by the PNWU Research Funds.