Reconstitution of C5 convertase of the alternative complement pathway with isolated C3b dimer and factors B and D.
C5 convertase of the alternative complement pathway is a trimolecular complex consisting of two molecules of C3b and one molecule of Bb. We previously proposed a model of the alternative pathway C5 convertase in which the second C3b molecule binds covalently to the first C3b molecule bearing Bb, and the C5 molecule binds to each C3b molecule of the covalently linked C3b dimer, resulting in its appropriate presentation to the catalytic site on Bb. In the present study, we purified the covalently linked C3b dimer and reconstituted the C5 convertase with the C3b dimer and factors B and D to obtain evidence in support of this model. An insoluble glucan, OMZ-176, was incubated with human serum to activate the alternative pathway and to allow formation of the alternative C5 convertase on the surface of the glucan, and the glucan bearing the C5 convertase was then solubilized by incubation with glucosidases. In this way, the covalently linked C3b dimer was obtained in solution without using a detergent. The C3b dimer was then separated from enzymes, C3b monomer, C3b oligomer, and other materials by chromatographies. SDS-PAGE analysis demonstrated that the purified C3b dimer had intact alpha'-chains. Alternative pathway C5 convertase was reconstituted when the isolated C3b dimer was incubated with factors B and D. The presence of P enhanced C5 convertase formation threefold. These results support the notions that the formation of the covalently linked C3b dimer is a general phenomenon associated with activation of the alternative pathway and that the C3b dimer acts as a part of the C5 convertase.
Hong
K
K
Department of Bacteriology, Osaka University Medical School, Japan.
Kinoshita
T
T
Pramoonjago
P
P
Kim
Y U
YU
Seya
T
T
Inoue
K
K
D050577:Complement C3-C5 Convertases / Q000302:isolation & purification / Q000378:metabolism*; D003179:Complement C3b / Q000302:isolation & purification / Q000502:physiology*; D011415:Complement Factor B / Q000502:physiology*; D011416:Complement Factor D / Q000502:physiology*; D003170:Complement Pathway, Alternative / Q000502:physiology*; D003910:Dextranase; D005936:Glucans; D006026:Glycoside Hydrolases; D006801:Humans; D013295:Streptococcus mutans
D005936:Glucans; D003179:Complement C3b; D006026:Glycoside Hydrolases; D003910:Dextranase; C021780:exo-1,3-alpha-glucanase; D050577:Complement C3-C5 Convertases; C493488:CFD protein, human; D011416:Complement Factor D; D011415:Complement Factor B