C3b2-IgG complexes retain dimeric C3 fragments at all levels of inactivation.
C3b2-IgG complexes are formed during complement activation in serum by attachment of two C3b molecules (the proteolytically activated form of C3) to one IgG heavy chain (IgG HC) via ester bonds. Because of the presence of two C3b molecules, these complexes are very efficient activators of the alternative complement pathway. Likewise, dimeric C3b is known to enhance complement receptor 1-dependent phagocytosis, and dimeric C3d (the smallest thioester-containing fragment of C3) linked to a protein antigen facilitates CR2-dependent B-cell proliferation. Because the efficiency of all these interactions depends on the number of C3 fragments, we investigated whether C3b2-IgG complexes retained dimeric structure upon physiological inactivation. We used two-dimensional SDS-PAGE and Western blot to study the arrangement of the C3b molecules by analyzing the fragmentation pattern after cleavage of the ester bonds. Upon inactivation with factors H and I, a 185-kDa band was generated under reducing conditions. It released IgG HC and the 65-kDa fragment of C3b alpha' chain after hydrolysis of the ester bonds with hydroxylamine. The two C3b molecules were not 65-kDa-to-40-kDa linked, because neither ester-bonded 65 kDa HC nor 65 kDa-40 kDa fragments were observed, nor was a 40-kDa peptide released after hydroxylamine cleavage. Factor I and CR1 cleaved the C3b2-IgG molecule to its final physiological product, C3dg2-IgG, which migrated as a 133-kDa fragment in reduced form. This fragment released exclusively C3dg (the final physiological product of C3b inactivation by factor I) and IgG HC. C3dg2-HC appeared as a double band on SDS-PAGE only at low gel porosity, suggesting the presence of two conformers of the same composition. Our results suggest that, upon physiological inactivation, C3b2-IgG complexes retain dimeric inactivated C3b and C3dg, which allows bivalent binding to the corresponding complement receptors.
Jelezarova
Emiliana
E
Institute of Biochemistry, Swiss Federal Institute of Technology, CH-8093 Zurich, Switzerland. jelezarova@bc.biol.ethz.ch
Luginbuehl
Alexander
A
Lutz
Hans U
HU
D001769:Blood / Q000276:immunology; D003167:Complement Activation; D003179:Complement C3b / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004952:Esters; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011951:Receptors, Complement / Q000378:metabolism
D004952:Esters; D007074:Immunoglobulin G; D010446:Peptide Fragments; D011951:Receptors, Complement; C045477:complement C3d,g; D003179:Complement C3b