Identification of different forms of human C4b-binding protein lacking beta-chain and protein S binding ability.
Human C4b-binding protein (C4BP) is a multimeric regulatory component of the complement system that circulates in plasma either as a free protein or in a noncovalent complex with the vitamin K-dependent protein S. The major form of C4BP is composed of seven identical alpha-chains (70 kDa) and one beta-chain (45 kDa). C4BP was purified from human plasma after barium citrate adsorption using anti-C4BP monoclonal antibody affinity chromatography. C4BP-high and low Mr forms were both obtained from the barium citrate precipitate and the supernatant. C4BP-high and low forms from the barium citrate precipitate were separated by sodium dodecylsulfate polyacrylamide slab gel electrophoresis and extracted with Triton X-100. Both forms contained the beta-chain as was demonstrated on sodium dodecylsulfate polyacrylamide slab gel electrophoresis under reduced conditions after silver-staining and with Western-blotting using monoclonal antibodies specific for the beta-chain. The C4BP-high and low forms demonstrated similar protein S binding affinity (KA: 3.18 x 10(8) and 3.21 x 10(8) M-1, respectively) in a C4BP-protein S binding assay and a protein S ligand blot using a peroxidase-conjugated monoclonal anti-protein S antibody. The barium citrate supernatant contained two forms of C4BP-high and one form of C4BP-low. One form of C4BP-high did contain the beta-chain and was capable of protein S binding (KA: 4.35 x 10(8) M-1). The two other forms of C4BP lacked the beta-chain and were unable to bind protein S.
Hessing
M
M
Department of Haematology, University Hospital Utrecht, The Netherlands.
Kanters
D
D
Hackeng
T M
TM
Bouma
B N
BN
D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D015935:Complement C4b / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D015394:Molecular Structure; D008970:Molecular Weight; D017293:Protein S; D011951:Receptors, Complement / Q000378:metabolism*
D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D017293:Protein S; D011951:Receptors, Complement; D015935:Complement C4b