title: Expression and characterization of a recombinant C4b-binding protein lacking the beta-chain.
abstract: C4b-binding protein (C4BP) is a high-molecular-mass glycoprotein which contains binding sites for complement component C4b, anti-coagulant vitamin K-dependent protein S and serum amyloid P component (SAP). The major form of C4BP in plasma is composed of seven identical alpha-chains and a single beta-chain. We have expressed full-length cDNA for the alpha-chain in a eukaryotic expression system and characterized functional properties of non-beta-chain-containing C4BP. During synthesis, recombinant alpha-chains polymerized into two different high-molecular-mass C4BP forms which were composed of seven or eight alpha-chains. Recombinant C4BP bound C4(H2O) (used instead of C4b) equally as well as native C4BP, functioned equally as well as factor I cofactor in the degradation of C4(H2O) and bound to SAP. In contrast, the recombinant C4BP did not bind protein S and therefore did not inhibit the ability of protein S to function as a cofactor to activated protein C. Tunicamycin treatment of the transfected cells prevented N-linked glycosylation, but did not affect polymerization of the alpha-chains into a high-molecular-mass C4BP. The non-glycosylated C4BP had comparable properties to glycosylated C4BP in several functional assays. These results demonstrate polymerization of C4BP alpha-chains to be independent both of the beta-chain and of the N-linked carbohydrates. Moreover, N-linked carbohydrates and the beta-chain were neither required for the ability of C4BP to bind C4b and to function as factor I cofactor nor for the interaction with SAP.
authors.affiliation: Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
authors.forename: Y
authors.initials: Y
authors.lastname: Härdig
authors.forename: P
authors.initials: P
authors.lastname: García de Frutos
authors.forename: B
authors.initials: B
authors.lastname: Dahlbäck
mesh_terms: D001667:Binding, Competitive; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003169:Complement Inactivator Proteins; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D005786:Gene Expression Regulation; D006023:Glycoproteins; D006031:Glycosylation; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D017293:Protein S / Q000378:metabolism; D011951:Receptors, Complement / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D000683:Serum Amyloid P-Component / Q000378:metabolism; D014162:Transfection / Q000235:genetics; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D001798:Blood Proteins; D003169:Complement Inactivator Proteins; D018076:DNA, Complementary; D006023:Glycoproteins; D017293:Protein S; D011951:Receptors, Complement; D011994:Recombinant Proteins; D000683:Serum Amyloid P-Component; D014415:Tunicamycin; D005340:Fibrinogen