The C-terminus of factor H: monoclonal antibodies inhibit heparin binding and identify epitopes common to factor H and factor H-related proteins

Abstract

We have generated monoclonal antibodies (mAbs) specific for the C-terminus of factor H that can be used as inhibitory antibodies for heparin binding and for the specific detection of factor H and factor H-related proteins (FHRs) in plasma and triacylglycerol-rich lipoproteins. Four distinct mAbs were established: IXF9 (IgG1), VD3 (IgG2a), VIG8 (IgG1) and IIC5 (IgG1). Each reacts specifically with FHR-1 and factor H (and also with FHR-2 in the case of VIG8), but none binds to the related FHR-3 and FHR-4 proteins nor to factor H-like protein 1. By the use of deletion mutants of factor H and by comparing the reactivity with FHR-1 and FHR-2, the binding epitopes of the mAbs were identified and localized to different short consensus repeats (SCRs): mAbs IXF9 and VD3 bind to related or even identical sites within SCR18 (factor H) and SCR3 (FHR-1) respectively. mAbs VIG8 and IIC5 bind to different epitopes located within SCRs 19 to 20 of factor H and SCRs 4 to 5 of FHR-1 respectively. Only mAb VIG8 reacts with the corresponding SCRs 3 to 4 of FHR-2. These antibodies are useful for the detection of the corresponding proteins in biological specimens such as fractions of lipoproteins. In addition, mAb VIG8 has the unique feature of inhibiting binding of factor H to heparin. Given the recent identification of a heparin- and a C3b-binding domain within the C-terminus of factor H, these mAbs should provide useful tools for functional analysis and for the precise localization of the domain(s) required for this interaction.

Each of the Three Binding Sites on Complement Factor H Interacts with a Distinct Site on C3b

“…Since the non-activating surfaces usually contain negatively charged surface structures (sialic acids and glycosaminoglycans), interactions between fH and polyanions have been intensively characterized. FH has been found to contain two or three binding sites for heparin and/or sialic acid on SCR7, SCR20, and possibly SCR12-14 (23)(24)(25)(26)(27)(28). It has been suggested that these heparin-binding sites have a role in the ability of AP to discriminate between activators and nonactivators; however, the physiological role of these sites is not yet fully understood.…”

“…Cloning, expression in the baculovirus system, and purification of most of the used recombinant fragments of fH have been described earlier (27,36,50). Also the additional fragments representing SCR8-11, SCR15-18 and SCR15-20 were generated using the pBSV-8His expression vector (50).…”

FHR-4A: a new factor H-related protein is encoded by the human FHR-4 gene

“…The structural similarity and the sequence identity of individual protein domains suggest overlapping function(s) of FHR proteins and activities shared with the complement and immune regulator Factor H. 7 FHRs and Factor H bind the same ligands, as demonstrated for C3b (FHR-1, FHR-3, FHR-4 and FHR-5), 8 -10 heparin (FHR-1 and FHR-3, FHR-5), 8,9,11,12 Creactive protein (FHR-3 and FHR-4) 13 and some subtypes of the streptococcal M protein (FHR-3). 14 FHR-1, FHR-2 and FHR-4 as well as Factor H are present in plasma lipoproteins and FHR-5 has been found in kidney glomerular complement deposits.…”

The Complement Regulator Factor H Binds to the Surface Protein OspE of Borrelia burgdorferi

“…Human C5 was purchased from Calbiochem (La Jolla, CA). Recombinant proteins FHL-1 (representing the N-terminal seven SCRs of factor H plus four unique additional amino acids), SCRs 8 -20 and SCR 15-20 of factor H, factor H-related proteins FHR-3 and FHR-4, and a construct SCR 1-4 of FHR-3 were expressed with the baculovirus expression system as described previously (31)(32)(33)(34). The recombinant proteins were purified by Ni 2ϩ -chelate chromatography as described (35).…”