Factor H-Related (FHR)-1 and FHR-2 Form Homo- and Heterodimers, while FHR-5 Circulates Only As Homodimer in Human Plasma

Abstract

The complement factor H-related (FHR) proteins are hypothesized to fine-tune the regulatory role of complement factor H (FH) in the alternative pathway of the complement system. Moreover, FHR-1, FHR-2, and FHR-5 have been proposed to be dimers, which further complicates accurate analysis. As FHRs are highly similar among themselves and toward FH, obtaining specific reagents for quantification of serum levels and functional analysis is challenging. In this study, we generated antibodies and developed ELISAs to measure FHR-1, FHR-2, and FHR-5 in serum. We used both recombinant and serum-derived proteins to show that four dimers occur in human circulation: homodimers of FHR-1, FHR-2, and FHR-5, as well as FHR-1/FHR-2 heterodimers. Heterodimers containing FHR-5 were not found. In individuals with homozygous CFHR1 deletions or compound heterozygous CFHR2 missense/nonsense mutations identified in this study, the respective FHR-1 and FHR-2 homo- and heterodimers were absent. Using FRET, we found that recombinant FHR dimers exchange monomers rapidly. This was confirmed ex vivo, using FHR-1- and FHR-2-deficient sera. Of all FHR dimers, FHR-5/5 homodimers demonstrated strong binding affinity toward heparin. Specific ELISAs demonstrated that serum levels of FHR-1/1, FHR-1/2, FHR-2/2, and FHR-5/5 dimers were low compared to FH, which circulates at a 10- to 200-fold molar excess. In summary, FHR-1, FHR-2, and FHR-5 homodimerize, with FHR-1 and FHR-2 forming heterodimers as well, and equilibrate quickly in plasma.

The role of the alternative pathway of complement activation in glomerular diseases

“…The CFHR5 ability to form heterodimers is controversial. In the recent study performed by van Beek et al, CFHR1-CFHR5 and CFHR2-CFHR5 heterodimers were absent in human plasma [42]. It has to be underlined that the results of this report are in opposition to the former (based on in vitro assays) suggesting that dimerization confers avidity for tissue-bond complement fragments and thus may enhance complement deregulation [38].…”

“…Secondly, CFHR5 may deregulate complement indirectly by competing with CFH in binding to other than C3b physiological ligands, including pentraxin PTX3, CRP and extracellular matrix [36]. Also, CFHR4 and CFHR5 have been reported to directly promote C3 convertase formation by binding to C3b, with stronger ability to assemble C3bBb on CFHR4, and with C3bBb-CFHR4 complex being more resistant to CFH cleavage in comparison with C3bBb [36,41,42].…”

“…In case of CFHR-1 and CFHR-2, they also form CFHR1-CFHR2 heterodimers. Since CFHR1, CFHR2, and CFHR5 monomers were not detected in human sera, they are suspected of creating dimers directly after being released to plasma [42]. The CFHR5 ability to form heterodimers is controversial.…”

“…According to the latter study, serum levels of CFHR-1-CFHR1, CFHR-1-CFHR2, CFHR-2-CFHR2, and CFHR-5-CFHR5 dimers were low compared to CFH, which circulates at a 10-to [45], modified 200-fold molar excess. Therefore, these dimers are not supposed to compete efficiently with CFH [42].…”

Insights into the role of C3d dimers in B cell activation and Staphylococcal immune evasion

“…Following the swap, the altered placement of the swapped helix α1 could enhance its susceptibility to further degradation and lead to the formation of C3dt, a more compact dimer, as suggested by the previously-published structure of a truncated rat dimer which lacks the exact region of C3d involved in helix swapping (M1/994 -G16/1009) 34 . On a broader perspective, this newly-discovered N-terminal helix swap of dimeric C3d is unique as this relatively rare structural event has not been described in C3d or a complement protein previously, although 3D domain swapping has been documented in a number of disparate proteins 40,43 and various complement components such as FH-related proteins (FHRs) 44,45 are known to exist in a dimeric form. To the best of our knowledge this is also the first report of a domain swap facilitated by the binding of a ligand.…”