Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro

Abstract

Complement factor H (FH) inhibits complement activation and interacts with glomerular endothelium via its complement control protein domains 19 and 20, which also recognize heparan sulfate (HS).In conclusion, our study shows that FH and FH19 -20 binding to glomerular endothelial cells is differentially mediated by HS but not other GAGs. Furthermore, we describe a novel, patient serum-independent competition assay for pathogenicity screening of FH19 -20 mutants.The complement system, which consists of the classical, lectin, and alternative pathway, initiates and amplifies inflammatory responses, including proliferative glomerulonephritis (1-3). The three pathways converge in the activation of complement component C3 and lead to the formation of membrane attack complexes that lyse the affected cells. The alternative pathway is initiated by spontaneous hydrolysis of C3 (4), eventually leading to covalent attachment of C3b to both adjacent host and non-host cell surfaces (5) and the release of the proinflammatory anaphylatoxin C3a (6). Deposited C3b binds complement factor B and, after proteolytic cleavage by factor D, forms C3 convertases (C3bBb) on the cell surface, providing localized feed-forward amplification of complement activation (7). To protect host cells from complement-mediated damage, several regulatory proteins disrupt the complement cascade, including the plasma proteins complement factor H (FH) 2 and FH-like protein 1, and membrane-bound regulators like complement receptor 1 (CD35), membrane cofactor protein (CD46), and decay accelerating factor (CD55) (8 -12).FH, a 155-kDa glycoprotein, is the major inhibitor of the alternative pathway both in the fluid phase and on cellular surfaces (13-15). It competes with factor B for C3b (16), acts as a cofactor for complement factor I-mediated proteolytic inactivation of C3b (14), and promotes the dissociation of C3bBb convertases (17). FH consists of 20 complement control protein (CCP, also called short consensus repeats) domains of ϳ60 amino acids each (18).

Disturbed sialic acid recognition on endothelial cells and platelets in complement attack causes atypical hemolytic uremic syndrome

“…These results are consistent with earlier data on hemolysis 12 and on C3b deposition on glomerular endothelial cells. 27 Importantly, our new data and recently published functional data by others 12,27 were observed to be not parallel with FH19-20 binding to either heparin or C3b. 12,13 Instead, the functional data paralleled that for FH19-20 binding to C3b-bearing erythrocytes and endothelial cells ( Figures 4G-I and 5D-E surfaces.…”

“…Binding of FH19-20 to kidney glomerulus sections has indeed been shown to decrease after a treatment with heparinase, 10 and binding of FH19-20 to glomerular endothelial cells was recently reported to decrease on removal of heparan sulfate, although only by 15%. 27 However, if heparan sulfate was significantly involved in FH function, then FH19-20 mutants L1189R and E1198A having higher affinity for heparin and for glomerular endothelial cells 12,13 would be expected to display increased ability to antagonize FH function on heparan sulfate-bearing cells. Our observations were exactly the opposite: the L1189R and E1198A mutations impaired the ability of FH19-20 to inhibit FH binding to human glomerular endothelial cells bearing C3b deposition ( Figure 5E).…”

“…In line with this, the mutation E1198A was recently reported to abolish FH19-20 binding in a murine glomerular endothelial cell model. 27 Furthermore, based on structural data, the mutations L1189R and E1198A were predicted to impair certain interactions between FH19-20 and sialic acid, 17 explaining the reduced binding of the FH19-20 mutants L1189R and E1198A to C3b-bearing cells. Moreover, even though the FH6-8 construct has been reported to display higher affinity for heparin than FH19-20, FH6-8 was not able to compete with FH in binding to C3b-bearing endothelial cells ( Figure 5C).…”

“…All in all, our results point to sialic acid being more relevant than heparan sulfate for FH function on endothelium. However, because removal of heparan sulfate from endothelial cells has been shown to affect FH binding, 27 a role of heparan sulfate in FH function cannot be completely ruled out. This negatively charged glycosaminoglycan could, for example, assist in attracting FH onto endothelium.…”

The murine factor H-related protein FHR-B promotes complement activation

“…The protective role of FH binding to endothelial cells and extracellular matrices is known from functional analyses of disease-associated FH mutations and FH autoantibodies, in vitro experiments and mouse models of diseases (12,(45)(46)(47)(48)(49)(50)(51)(52). All these data point to a delicate balance of complement activation and inhibition necessary for homeostasis and prevention of harmful inflammation.…”