Serum Proteins Binding to Tamm‐Horsfall Protein

Tamm‐Horsfall protein (THP), also called uromodulin, is an approximately 86kD, heavily glycosylated, single‐chain acidic protein (estimated pI=3). THP is produced in the kidney within the thick ascending limb of the loop of Henle and is excreted in milligram quantities daily into the urine. Despite its abundance, the normal physiologic function of THP remains enigmatic. One theory is that THP is an immunomodulatory molecule, due to its ability to bind various immune proteins such as IL‐1, TNF‐α, IgG, complement 1q (C1q), and factor H (fH). The purpose of the present study was to determine if other serum proteins could be shown to bind to immobilized THP. Multiple attempts to couple THP (purified from the urine of one healthy female) to a commercially available N‐hydroxysuccinimide (NHS)‐activated agarose resin (Thermo Scientific TM Pierce ® ) failed, at least partially because of solubility issues with THP, a protein that readily forms large aggregates in solution. As an alternative approach, purified THP was coated to microtiter plate wells and normal human serum diluted 1:5 with 20 mM Tris/20 mM NaCl/0.05% Tween 20, pH 7.5 was incubated in THP‐coated or non‐THP‐coated wells. After rinsing to remove unbound protein, the proteins bound to the wells were extracted by addition of 1X sample buffer containing 5% β‐mercaptoethanol, and the recovered samples were analyzed by SDS‐PAGE. IgG in serum did not appear to bind THP, even though binding of purified IgG to THP could be detected by this method. Among the proteins that specifically bound to the THP‐coated wells was a prominent protein with an estimated molecular weight of about 78 kD. Based on its molecular weight and abundance, transferrin seemed to be a likely candidate for this protein. Purified human transferrin (Sigma) and the protein recovered from THP‐coated microtiter plates had identical mobilities on SDS‐PAGE gels. Direct binding of THP to purified transferrin was analyzed by ELISA, incubating human transferrin (from 4 mg/ml to 100 μg/ml) in THP‐coated and non‐THP‐coated microtiter plate wells and then detecting bound transferrin with goat anti‐transferrin antiserum and rabbit anti‐goat IgG‐alkaline phosphatase. This ELISA demonstrated that transferrin bound to immobilized THP in a dose‐dependent manner. In summary, these studies indicated that immobilized THP does not bind to IgG within diluted serum, even though THP binds to purified IgG (in the absence of diluted serum), suggesting that a component of serum inhibits the THP/IgG interaction. These studies also provided evidence for an interaction between THP and transferrin, the significance of which remains to be elucidated. Support or Funding Information Funded by the PNWU Research Seed Program.