title: IgG naturally occurring antibodies stabilize and promote the generation of the alternative complement pathway C3 convertase.
abstract: Normal human IgG contains naturally occurring anti-C3 antibodies (anti-C3 NAbs) that have been proposed to regulate complement amplification. Here, we report a novel procedure for anti-C3 NAb purification. Pooled human IgG was fractionated on a DEAE column prior to affinity chromatography on IgG and then on C3. Anti-C3 NAbs co-purified with anti-F(ab')2 NAbs. In a refined protocol, IgG fractions were absorbed on Fc, F(ab')2, and C3, which allowed to isolate the directly accessible NAbs and to remove IgG hinge-region-specific NAbs. Since a substantial fraction of total anti-C3 NAbs in whole IgG pre-existed as complexes, IgG that did not bind to the three affinity columns was treated with urea and the affinity chromatography repeated to collect the dissociated NAbs. The urea-accessible anti-F(ab')2 NAbs were rather pure but anti-C3 NAbs yet contained substantial amounts of anti-F(ab')2 NAbs. Anti-C3 NAbs showed up to 400-fold and anti-F(ab')2 NAbs, up to 30-fold enrichment as compared to pooled normal human IgG. Anti-C3 NAb preparations exhibited nephritic factor activity that was up to 60 times stronger than that of total IgG from a patient with membranoproliferative glomerulonephritis type 2. In addition, anti-C3 NAbs promoted C3 convertase generation, when added to the convertase precursor or during convertase assembly, suggesting a non-nephritic-factor mechanism. Factors H and I reduced the overall level of activity but had no influence on the NAb dose-response curve meaning that NAbs did not interfere with factor H binding. Convertase promoting activity during assembly correlated with the content of anti-C3 NAbs in NAb complexes. In conclusion, anti-C3 NAbs associated with framework-specific anti-idiotypic NAbs stabilize C3 convertase and promote its generation but their activity is compensated for in whole IgG.
authors.affiliation: Institute of Biochemistry, Swiss Federal Institute of Technology, ETH Hoenggerberg, HPM D14, Schafmattstrasse 18, CH-8093 Zurich, Switzerland.
authors.forename: Emiliana
authors.initials: E
authors.lastname: Jelezarova
authors.forename: Hans U
authors.initials: HU
authors.lastname: Lutz
mesh_terms: D000888:Antibodies, Anti-Idiotypic / Q000302:isolation & purification / Q000378:metabolism; D003176:Complement C3 / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D050577:Complement C3-C5 Convertases / Q000096:biosynthesis / Q000378:metabolism*; D017242:Complement Factor H / Q000378:metabolism / Q000494:pharmacology; D003170:Complement Pathway, Alternative; D004795:Enzyme Stability; D005340:Fibrinogen / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007113:Immunity, Innate; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism*; D066298:In Vitro Techniques
chemical_list: D000888:Antibodies, Anti-Idiotypic; C493495:CFH protein, human; D003176:Complement C3; D007074:Immunoglobulin G; D017242:Complement Factor H; D005340:Fibrinogen; D050577:Complement C3-C5 Convertases