A Monoclonal Antibody That Provides a Model for C3 Nephritic Factors

Abstract

Complement is a major innate defense system that protects the intravascular space from microbial invasion. Complement activation results in the assembly of C3 convertases, serine proteases that cleave complement protein C3, generating bioactive fragments C3a and C3b. The complement response is rapid and robust, largely due to a positive feedback regulatory loop mediated by alternative pathway (AP) C3 convertase. C3 nephritic factors (C3NEFs) are autoantibodies that stabilize AP convertase, resulting in uncontrolled C3 cleavage, which, in principle, can promote critical tissue injury similar to that seen in certain renal conditions. Investigations of C3NEFs are hampered by a challenging issue: each C3NEF is derived from a different donor source, and there is no method to compare one C3NEF to another. We have identified a widely available mouse anti-C3 mAb that, similar to many C3NEFs, can stabilize functional AP convertase in a form resistant to decay acceleration by multiple complement regulators. The antibody requires the presence of properdin to confer convertase stability, and hampers the activity of Salp20, a tic salivary protein that accelerates convertase dissociation by displacing properdin from the convertase complex. This mAb can serve as an urgently needed standard for the investigation of C3NEFs. This study also provides novel insights into the dynamics of AP convertase.

Keywords: C3 glomerulonephritis; C3 nephritic factor; alternative pathway; complement; convertase; properdin.

FIG. 1.

Identification of a C3NEF-like mAb. C3bBbP were assembled from FB, P, and FD on C3b-opsonized sheep red blood in Mg²⁺ EGTA buffer. (A) C3bBbP-bound cells were treated with various anti-C3 mAb in 40 mM EDTA buffer and incubated for 0 or 2 hours at 30°C before treatment with serum-EDTA. (B) C3bBbP-bound cells were treated with mAb 7C10 in 40 mM EDTA buffer and incubated for 0 or 2 hours at 30°C before addition of serum-EDTA. Data compiled from three experiments. (C) C3bBbP-bound cells were treated with FH in 40 mM EDTA buffer for 5 minutes at 4°C before addition of serum-EDTA. Data compiled from three experiments. Z = average lytic sites per cell. C3NEF, C3 nephritic factor; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; FB, factor B; FD, factor D; FH, factor H.

FIG. 2.

Anti-C3 mAb 7C10 confers FH resistance but is not stable to convertase preformed without properdin. C3bBb was assembled from FB and FD on C3b-opsonized sheep red blood cells in Mg²⁺ EGTA buffer. (A) C3bBb-bound cells were treated with FH in 40 mM EDTA buffer for 5 minutes at 4°C, before addition of serum-EDTA. (B) C3bBb-bound cells were treated with mAb 7C10 in 40 mM EDTA buffer for 5 minutes at 0°C and incubated for 0, 15, or 30 minutes at 30°C before the addition of serum-EDTA. Data from each panel were compiled from three experiments. Z = average number of lytic sites per cell.

FIG. 3.

Anti-C3 mAb 7C10 confers resistance to Salp20, DAF and sCR1. (A–C) C3bBbP was assembled from FB, P, and FD on C3b-opsonized sheep red blood in Mg²⁺ EGTA buffer. C3bBbP-bound cells were treated with sCR1, recombinant DAF, or Salp20, with or without anti-C3 mAb 7C10, in 40 mM EDTA buffer for 5 minutes at 4°C before addition of serum-EDTA. The data in panels A and B are compiled from three experiments. The data in panel C are compiled from five experiments. Z = average number of lytic sites per cell. DAF, decay-accelerating factor; sCR1, soluble CR1.

FIG. 4.

3D structure of C3bBb. The C3b:Bb interface, stabilized by Mg(2+), is distal to the TED domain at the 7C10 recognition site. Bb is blue, the C3b carboxy terminal domain is salmon, the TED (C3d) domain is green, and the thioester reactive moiety is labeled light blue. Mg(2+) is shown in red. Structure generated from PDB ID:2WIN with PyMOL Molecular Graphics System, Version 2.5.1 Schrödinger, LLC. TED, thioester-containing domain.