Identification of Different Forms of Human C4b-Binding Protein Lacking β-Chain and Protein S Binding Ability

Summary

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 α-chains (70 kDa) and one β-chain (45 kDa). C4BP was purified from human plasma after barium citrate adsorption using anti-C4BP monoclonal antibody affinity chromatography. C4BP-high and low M_r 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 β-chain as was demonstrated on sodium dodecylsulfate polyacrylamide slab gel electrophoresis under reduced conditions after silverstaining and with Western-blotting using monoclonal antibodies specific for the β-chain. The C4BP-high and low forms demonstrated similar protein S binding affinity (K_A: 3.18 × 10⁸ and 3.21 × 10⁸ 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 β-chain and was capable of protein S binding (K_A: 4.35 × 10⁸ M^-1). The two other forms of C4BP lacked the β-chain and were unable to bind protein S.

• References

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