Binding and complement activation by C-reactive protein via the collagen-like region of C1q and inhibition of these reactions by monoclonal antibodies to C-reactive protein and C1q.
Ligand-complexed C-reactive protein (CRP), like aggregated or complexed IgG, can react with C1q and activate the classical C pathway. Whereas IgG is known to bind to the globular region and not to the collagen-like region (CLR) of C1q, the site of interaction of C1q with CRP has not been defined. CRP-trimers were prepared by cross-linking and found to bind to C1q and to activate the C system. Heat-aggregated IgG (Agg-IgG) did not block the binding of CRP-trimers to C1q, nor did CRP-trimers block binding of Agg-IgG to C1q, suggesting that CRP and IgG bind at different sites. ELISA and Western blot analysis showed that CRP-trimers bound to the CLR, whereas Agg-IgG bound only to the globular region; similarly, anti-CLR mAb inhibited binding of CRP-trimers to C1q whereas anti-globular region mAb did not. Reactivity with CRP-trimers as well as with Agg-IgG was retained after reduction/alkylation and SDS treatment of C1q. A group of 22 anti-CRP mAb directed against at least six distinct native-CRP epitopes and eight distinct neo-CRP epitopes was tested for ability to inhibit the CRP-CLR interaction; one mAb, anti-native CRP mAb 8D8, with strong inhibitory activity was identified. Fab' of 8D8 blocked binding of CRP-trimers to intact C1q as well as CLR, and also inhibited CRP (CRP-trimers and CRP-protamine complexes) induced C activation, but had no effect on C1q binding or C activation by Agg-IgG. These results indicate that a conformation-determined region on CRP binds to a sequence-determined region on the CLR of C1q in an interaction which leads to C activation. Anti-CRP and anti-C1q mAb that specifically inhibit this interaction are described.
Jiang
H X
HX
Department of Immunology/Microbiology, Rush Medical College, Chicago, IL 60612.
Siegel
J N
JN
Gewurz
H
H
D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002097:C-Reactive Protein / Q000378:metabolism*; D003167:Complement Activation / Q000187:drug effects; D015922:Complement C1q / Q000737:chemistry / Q000378:metabolism*; D003171:Complement Pathway, Classical; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G / Q000378:metabolism; D066298:In Vitro Techniques; D011479:Protamines / Q000494:pharmacology; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011479:Protamines; D015922:Complement C1q; D002097:C-Reactive Protein