Importance of carbohydrate in the interaction of Tamm‐Horsfall protein with complement 1q and inhibition of classical complement activation

Abstract

Tamm-Horsfall protein (THP) binds strongly to complement 1q (C1q), a key component of the classical complement pathway. The goals of this study were to determine whether THP altered the activation of the classical complement pathway and whether the carbohydrate portion of THP was involved in this glycoprotein's binding to C1q and alteration of complement activation. The ability of THP to prevent complement activation in diluted serum or plasma incubated at 37 degrees C was assessed using both a haemolytic assay with antibody-sensitized sheep RBC and a C4d ELISA. Both these methods showed that THP inhibited activation of the classical complement pathway in a dose-dependent manner. Glycosidases were used to remove most of the carbohydrate from THP. This partially deglycosylated THP bound human IgG with a higher affinity (KD1 = 1.4 nmol/L; KD2 = 0.31 micromol/L) than did intact THP (KD1 = 33.4 nmol/L; KD2 = 31.0 micromol/L). An ELISA showed that removal of carbohydrate from THP reduced, but did not eliminate, the ability of this protein to inhibit binding of C1q to intact THP. Haemolysis assays using antibody-sensitized sheep RBC showed that removal of THP carbohydrate eliminated the ability of THP to protect against complement activation. In conclusion, THP inhibited the activation of the classical complement pathway that occurred in diluted serum or plasma. The carbohydrate moieties of THP appeared to be important in this inhibitory activity.

Human Tamm-Horsfall protein, a renal specific protein, serves as a cofactor in complement 3b degradation

“…Deglycosylated THP (dTHP), THP that had approximately 88% of its carbohydrate moieties removed enzymatically,[21] was evaluated for C3b cofactor activity. As seen in Fig 10, THP (Lane D) and dTHP (Lane F) yielded a higher concentration of C3b α-chain fragments than were present in the C3b/CFI sample (Lane B).…”

“…Deglycosylated THP (dTHP), used in one set of C3b cofactor assays, was produced using a series of glycosidases on native THP where dTHP was separated from free carbohydrates as described previously. [21] It was estimated, by the change in mobility on SDS-PAGE, that about 88% of the carbohydrate was removed from dTHP and, by lectin blotting, that all of the sialic acid was removed. [21]…”

“…[21] It was estimated, by the change in mobility on SDS-PAGE, that about 88% of the carbohydrate was removed from dTHP and, by lectin blotting, that all of the sialic acid was removed. [21]…”

“…THP binds strongly to complement 1q (C1q), a key initiating protein in the classical complement pathway, and appears to prevent activation of this pathway. [19,20,21] Several in vivo studies suggest a linkage between THP and renal complement inhibition. Complement activation occurs during ischemia-reperfusion injury (IRI)[22,23] and organ transplantation.…”

Tamm-Horsfall Protein Regulates Circulating and Renal Cytokines by Affecting Glomerular Filtration Rate and Acting as a Urinary Cytokine Trap

“…The trapping role played by urinary THP may not be limited to cytokines. Previous in vitro studies suggested that THP also binds to complement C1 and C1q (61)(62)(63). It is possible that THP is a promiscuous urinary trap not only for cytokines, but also for other immunological effectors.…”