Properdin Pattern Recognition on Proximal Tubular Cells Is Heparan Sulfate/Syndecan-1 but Not C3b Dependent and Can Be Blocked by Tick Protein Salp20

Abstract

Introduction: Proteinuria contributes to progression of renal damage, partly by complement activation on proximal tubular epithelial cells. By pattern recognition, properdin has shown to bind to heparan sulfate proteoglycans on tubular epithelium and can initiate the alternative complement pathway (AP). Properdin however, also binds to C3b(Bb) and properdin binding to tubular cells might be influenced by the presence of C3b(Bb) on tubular cells and/or by variability in properdin proteins in vitro. In this study we carefully evaluated the specificity of the properdin-heparan sulfate interaction and whether this interaction could be exploited in order to block alternative complement activation. Methods: Binding of various properdin preparations to proximal tubular epithelial cells (PTEC) and subsequent AP activation was determined in the presence or absence of C3 inhibitor Compstatin and properdin inhibitor Salp20. Heparan sulfate proteoglycan dependency of the pattern recognition of properdin was evaluated on PTEC knocked down for syndecan-1 by shRNA technology. Solid phase binding assays were used to evaluate the effectivity of heparin(oids) and recombinant Salp20 to block the pattern recognition of properdin. Results: Binding of serum-derived and recombinant properdin preparations to PTECs could be dose-dependently inhibited (P < 0.01) and competed off (P < 0.01) by recombinant Salp20 (IC50: ∼125 ng/ml) but not by Compstatin. Subsequent properdin-mediated AP activation on PTECs could be inhibited by Compstatin (P < 0.01) and blocked by recombinant Salp20 (P < 0.05). Syndecan-1 deficiency in PTECs resulted in a ∼75% reduction of properdin binding (P = 0.057). In solid-phase binding assays, properdin binding to C3b could be dose-dependently inhibited by recombinant Salp20> heparin(oid) > C3b. Discussion: In this study we showed that all properdin preparations recognize heparan sulfate/syndecan-1 on PTECs with and without Compstatin C3 blocking Lammerts et al. C3b-Independent Properdin Binding to PTECs conditions. In contrast to Compstatin, recombinant Salp20 prevents heparan sulfate pattern recognition by properdin on PTECs. Both complement inhibitors prevented properdin-mediated C3 activation. Binding of properdin to C3b could also be blocked by heparin(oids) and recombinant Salp20. This work indicates that properdin serves as a docking station for AP activation on PTECs and a Salp20 analog or heparinoids may be viable inhibitors in properdin mediated AP activation.

Initial properdin binding contributes to alternative pathway activation at the surface of viable and necrotic cells

“…Binding of properdin to viable cells has also been demonstrated for renal proximal tubular epithelial cells, where heparan sulfate was identified as a ligand to which properdin binds [ 16 , 17 ]. Therefore, we compared different polysaccharides to determine their inhibitory effect on the binding of properdin to different viable and dead cells.…”

“…Properdin is able to bind to proximal tubular epithelial cells and it has been shown that heparan sulfates are involved in this interaction [ 12 , 16 , 17 ]. We also investigated the properdin binding to viable DCs and macrophages and observed a properdin binding to both pro‐ and anti‐inflammatory macrophages (Fig.…”

“…A total of 10 μg/mL purified properdin was preincubated with various concentrations of unfractionated heparin (LEO Pharma, Ballerup, Denmark), with either 0.1, 1, or 10 μg/mL O ‐sulfated and unsulfated K5 or with 10 μg/mL of polysaccharide derivatives: heparin, low‐molecular weight heparin, O ‐sulfated and unsulfated K5, fucoidan, dextran sulfate, unsulfated dextran T40, N ‐desulfated/reacetylated heparin, periodate oxidized/reduced heparin, chondroitin sulfate C, chondroitin sulfate B, and heparin sulfate from bovine kidney [ 17 ]. Mixtures were added to the cells (1 h, 4˚C).…”

“…Our group recently showed properdin deposition in the glomeruli of C3 KO mice with induced acute antiglomerular basement membrane disease [ 14 ], providing evidence of a C3b‐independent binding of properdin. The binding of properdin to tubular epithelial cells was shown to be mediated by heparan sulfates expressed on the cell surface [ 15 , 16 , 17 ].…”

The susceptibility of the kidney to alternative pathway activation—A hypothesis

“…On the contrary, consumption of complement proteins in the glomeruli, such as in patients with glomerulonephritis, reduces the concentration of complement proteins entering the tubulointerstitium 29–31 . Experiments have also suggested that properdin can bind to tubular epithelial cells, potentially initiating AP activation on the surface of this specific kidney cell type 32 . Properdin can form non‐physiologic multimers during handling, however, so experiments with purified properdin need to be interpreted cautiously.…”