Download PDF
Thromb Haemost 1990; 64(02): 245-250
DOI: 10.1055/s-0038-1647294
Original Article
Schattauer GmbH Stuttgart

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

Martin Hessing
The Department of Haematology, University Hospital Utrecht, Utrecht, The Netherlands
,
Deon Kanters
The Department of Haematology, University Hospital Utrecht, Utrecht, The Netherlands
,
Tilman M Hackeng
The Department of Haematology, University Hospital Utrecht, Utrecht, The Netherlands
,
Bonno N Bouma
The Department of Haematology, University Hospital Utrecht, Utrecht, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 01 March 1990

Accepted after revision04 May 1990

Publication Date:
25 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

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 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 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 (KA: 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 (KA: 4.35 × 108 M-1). The two other forms of C4BP lacked the β-chain and were unable to bind protein S.

 

  • References

  • 1 Scharfstein J, Ferreira A, Gigli I, Nussenzweig V. Human C4-binding protein. I. Isolation and characterization. J Exp Med 1978; 148: 207-222
    CrossrefPubMedSearch in Google Scholar
  • 2 Fujita T, Gigli I, Nussenzweig V. Human C4-binding protein. II. Role in proteolysis of C4b by C3b-inactivator. J Exp Med 1978; 148: 1044-1051
    CrossrefPubMedSearch in Google Scholar
  • 3 Gigli I, Fujita T, Nussenzweig V. Modulation of the classical pathway C3 convertase by plasma proteins C4 binding protein and C3b inactivator. Proc Natl Acad Sci USA 1979; 76: 6596-6600
    CrossrefPubMedSearch in Google Scholar
  • 4 Nagasawa S, Ichihara C, Stroud RM. Cleavage of C4b by C3b inactivator: production of a nicked form of C4b, C4b\ as an intermediate cleavage product of C4b by C3b inactivator. J Immunol 1980; 125: 578-582
    PubMedSearch in Google Scholar
  • 5 Dahlback B, Stenflo J. High molecular weight complex in human plasma between vitamin K-dependent protein S and complement C4b-binding protein. Proc Natl Acad Sci USA 1981; 78: 2412-2416
    CrossrefPubMedSearch in Google Scholar
  • 6 Walker FJ. Regulation of activated protein C by a new protein. A possible function for bovine protein S. J Biol Chem 1980; 255: 5521-5524
    PubMedSearch in Google Scholar
  • 7 Walker FJ. Regulation of activated protein C by protein S. J Biol Chem 1981; 256: 11128-11131
    PubMedSearch in Google Scholar
  • 8 Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood 1984; 63: 486-489
    PubMedSearch in Google Scholar
  • 9 Koedam JA, Meijers JC M, Sixma JJ, Bouma BN. Inactivation of human factor VIII. J Clin Invest 1988; 82: 1236-1243
    CrossrefPubMedSearch in Google Scholar
  • 10 Fujita T, Nussenzweig V. The role of C4-binding protein and β1H in proteolysis of C4b and C3b. J Exp Med 1979; 150: 267-276
    CrossrefPubMedSearch in Google Scholar
  • 11 Dahlbäck B. Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S. Biochem J 1983; 209: 847-856
    CrossrefPubMedSearch in Google Scholar
  • 12 Comp PC, Esmon CT. Recurrent venous thromboembolism in patients with partial deficiency of protein S. N Engl J Med 1984; 311: 1525-1528
    CrossrefPubMedSearch in Google Scholar
  • 13 Dahlbäck B. Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein. J Biol Chem 1986; 261: 12022-12027
    PubMedSearch in Google Scholar
  • 14 Reid KB M, Gagnon J. Human C4-binding protein: N-terminal amino acid sequence analysis and limited proteolysis by trypsin. FEBS Lett 1982; 137: 75-79
    CrossrefPubMedSearch in Google Scholar
  • 15 Dahlbäck B, Muller-Eberhard HJ. Visualisation of C4b-binding protein fragments formed by limited proteolysis using chymotrypsin. J Biol Chem 1984; 259: 11631-11634
    PubMedSearch in Google Scholar
  • 16 Nagasawa S, Unno H, Ichihara C, Koyama J, Koide T. Human C4b-binding protein, C4BP. FEBS Lett 1983; 164: 135-138
    CrossrefPubMedSearch in Google Scholar
  • 17 Perkins SJ, Chung LP, Reid KB M. Unusual ultrastructure of complement-component-C4b-binding protein of human complement by synchroton X-ray scattering and hydrodynamic analysis. Biochem J 1986; 233: 799-807
    CrossrefPubMedSearch in Google Scholar
  • 18 Dahlbäck B, Smith CA, Müller-Eberhard HJ. Visualization of human C4b-binding protein and its complexes with vitamin K-depen-dent protein S and complement protein C4b. Proc Natl Acad Sci USA 1983; 80: 3461-3465
    CrossrefPubMedSearch in Google Scholar
  • 19 Fujita T, Kamato T, Tamura N. Characterization of functional properties of C4-binding protein by monoclonal antibodies. J Immunol 1985; 134: 3320-3324
    PubMedSearch in Google Scholar
  • 20 Hillarp A, Dahlbäck B. The protein S-binding site localized to the central core of C4b-binding protein. J Biol Chem 1987; 262: 11300-11307
    PubMedSearch in Google Scholar
  • 21 Suzuki K, Nishioka J. Binding site for vitamin K-dépendent protein S on complement C4b-binding protein. J Biol Chem 1988; 263: 17034-17039
    PubMedSearch in Google Scholar
  • 22 Hillarp A, Dahlbäck B. Novel subunit in C4b-binding protein required for protein S binding. J Biol Chem 1988; 263: 12759-12764
    PubMedSearch in Google Scholar
  • 23 Hillarp A, Hessing M, Dahlbäck B. Protein S binding in relation to the subunit composition of human C4b-binding protein. FEBS Lett 1989; 259: 53-56
    CrossrefPubMedSearch in Google Scholar
  • 24 Chung LP, Gagnon J, Reid KB M. Amino acid sequence studies of human C4b-binding protein: N-terminal sequence analysis and alignment of the fragments produced by limited proteolysis with chymotrypsin and the peptides produced by cyanogen bromide treatment. Mol Immunol 1985; 22: 427-435
    CrossrefPubMedSearch in Google Scholar
  • 25 Chung LP, Bentley DR, Reid KB M. Molecular cloning and characterization of the cDNA coding for C4b-binding protein, a regulatory protein of the classical pathway of the human complement system. Biochem J 1985; 230: 133-141
    CrossrefPubMedSearch in Google Scholar
  • 26 Nagasawa S, Mizuguchi K, Ichihara C, Koyama J. Limited chymotryp-tic cleavage of human C4-binding protein: Isolation of a carbohydrate-containing core domain and an active fragment. J Biochem (Tokyo) 1982; 92: 1329-1331
    CrossrefPubMedSearch in Google Scholar
  • 27 Hessing M, Vlooswijk RA A, Hackeng TM, Kanters D, Bouma BN. The localization of heparin-binding fragments on human C4b-binding protein. J Immunol 1990; 144: 204-208
    PubMedSearch in Google Scholar
  • 28 Di Scipio RG, Davie EW. Characterization of protein S, a γ-carboxy-glutamic acid containing protein from bovine and human plasma. Biochemistry 1979; 18: 899-904
    CrossrefPubMedSearch in Google Scholar
  • 29 Little JR, Donahue H. Spectral properties of proteins and small molecules of immunological interest. Methods Immunol Immunochem 168 2: 343-368
    PubMed
  • 30 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
    CrossrefPubMedSearch in Google Scholar
  • 31 Morrisey JH. Silver stain for proteins in polacrylamide gels: a modified procedure with enhanced sensitivity. Anal Biochem 1981; 117: 307-310
    CrossrefPubMedSearch in Google Scholar
  • 32 Towbin H, Staehelin Th, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc Natl Acad Sci USA 1979; 76: 4350-4354
    CrossrefPubMedSearch in Google Scholar
  • 33 Nukatsuka M, Nagasawa S. Characterization of the interaction between human protein S and C4b-binding protein (C4bp). J Biochem (Tokyo) 1987; 102: 599-605
    CrossrefPubMedSearch in Google Scholar