Complement Factor H-Related Protein 3 Serum Levels Are Low Compared to Factor H and Mainly Determined by Gene Copy Number Variation in CFHR3

Abstract

The major human complement regulator in blood, complement factor H (FH), has several closely related proteins, called FH-related (FHR) proteins. As all FHRs lack relevant complement regulatory activity, their physiological role is not well understood. FHR protein 3 (FHR-3) has been suggested to compete with FH for binding to Neisseria meningitidis, thereby affecting complement-mediated clearance. Clearly, the in vivo outcome of such competition greatly depends on the FH and FHR-3 concentrations. While FH levels have been established, accurate FHR-3 levels were never unequivocally reported to date. Moreover, CFHR3 gene copy numbers commonly vary, which may impact the FHR-3 concentration. Hence, we generated five anti-FHR-3 mAbs to specifically measure FHR-3 in human healthy donors of which we determined the gene copy number variation at the CFH/CFHR locus. Finally, we examined the acute-phase response characteristics of FHR-3 in a small sepsis cohort. We determined FHR-3 levels to have a mean of 19 nM and that under normal conditions the copy number of CFHR3 correlates to a very large extent with the FHR-3 serum levels. On average, FHR-3 was 132-fold lower compared to the FH concentration in the same serum samples and FHR-3 did not behave as a major acute phase response protein.

Complement Regulator FHR-3 Is Elevated either Locally or Systemically in a Selection of Autoimmune Diseases

“…An immunoassay for FHR-3 quantification from human samples was established, using mAb RETC-2 antibody as capture antibody and biotin-labeled anti-FHR-3.4 mAb (which reacts with FH and FHR-3) as detection antibody (6) (Figures 3A,D).…”

“…The specific detection of proteins depends mostly on distinct binding of antibodies to the target antigen. Due to the high sequence identities among the FH protein family, the majority of previously generated antibodies showed cross-reactivity with other FH-family members (Figure S5 in Supplementary Material) (6, 44), with the exception of mAb HSL1 (19). HSL1 binds to SCR4–5 of FHR-3 and was used to determine binding of FHR-3 to pathogens.…”

“…Besides a previous estimation on FHR-3 levels (5), there has been only one other report using an immunoassay to measure FHR-3 levels in healthy individuals, reporting systemic FHR-3 levels of 0.7 μg/mL on average, and a major influence of copy number variation in cfhr3 on the variation of the FHR-3 levels within a population (6). According to the high specificity of mAb RETC-2, we confirmed the FHR-3 concentration in human serum probes derived from healthy individuals in a concentration ranged between 0.41 and 2.49 μg/mL (excluding cfhr3 -deficient donors).…”

“…Recombinant FHR-4A and FHR-4B were purified as previously described (6). FH and C3b were ordered from Complement Technology (Tyler, TX, USA).…”

“…A312, San Diego, CA, USA), the mouse mAb anti-FH was a kind gift of S. Berra (Università degli Studi di Milano, Milano, Italy) (21), and mouse mAb anti-FHR-3.1 and anti-FHR-3.4 were described previously (6). Rabbit anti-Iba1 polyclonal antibody was ordered from Wako Chemicals (cat.…”