Tamm-Horsfall Glycoproteins Interaction with Clq

Rhodes, Diana

Abstract

Tamm-Horsfall glycoprotein (THP) is produced solely within the thick ascending limb tubules of the kidney and is excreted in large quantities into the urine. It recently has been discovered that THP binds C1q, a subunit of the first component of the classical complement pathway. THP in the tubular fluid and urine normally will not encounter C1q; however, in various diseases of the kidney and lower urinary tract, THP gains access to the interstitium where it may bind C1q. The long-term goal of this project is to determine the physiological significance of THP's binding to C1q; is this interaction beneficial or detrimental to the host as the diseased tissue attempts to limit damage and initiate repair? This proposal specifically will test the following hypothesis. Hypothesis #1: THP binds C1q with an affinity that varies depending on the THP sample and the ionic strength and pH of the reaction mixture. THP from ten individuals will be evaluated for its ability to bind C1q in a solid phase assay using buffers of differing pH and ionic strengths. Hypothesis #2: THP binds to a discrete site on C1q that can be localized to one of C1q's various domains. THP will be tested for its ability to bind the collagen-like region and globular region of C1q by the solid phase assay and fir its ability to bind chains A, B, and C of C1q by Western blotting techniques. Hypothesis #3: THP's carbohydrate moieties are critical for binding to C1q. After removal of THP's sugars by N-Glycanase, deglycosylated THP's binding to C1q will be evaluated by Western blot analyses and solid phase binding assays. Hypothesis #4: C1q's activity is altered by binding THP. THP's ability to activate C1q and hence the classical complement pathway will be monitored by the consumption of C4 and generation of C3d. THP's affect on C1q- mediated hemolysis will also be evaluated.