Abstract
C4b-binding protein (C4BP) is a soluble, 570 kDa large glycoprotein, present in plasma at a concentration of approximately 200 mg/L. C4BP is the main inhibitor of the classical and lectin pathways of complement, where it controls C4b-mediated reactions. Here, we describe a method for purification of C4BP from human plasma, which is based on barium chloride precipitation, anion exchange chromatography, and gel filtration. We also describe a functional assay, in which C4BP’s cofactor activity to factor I, in the degradation of C4b, can be assessed.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Dahlbäck B (1983) Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S. Biochem J 209:847–856
Scharfstein J, Ferreira A, Gigli I et al (1978) Human C4b-binding protein, isolation and characterization. J Exp Med 148:207–222
Hillarp A, Hessing M, Dahlbäck B (1989) Protein S binding in relation to the subunit composition of human C4b-binding protein. FEBS Lett 259:53–56
Sanchez Corral P, Criado Garcia O, Rodriguez de Cordoba S (1995) Isoforms of human C4b-binding protein. I. Molecular basis for the C4BP isoform pattern and its variations in human plasma. J Immunol 155:4030–4036
Kask L, Hillarp A, Ramesh B et al (2002) Structural requirements for the intra-cellular subunit polymerization of the complement inhibitor C4b-binding protein. Biochemistry 41:9349–9357
Dahlbäck B, Smith CA, Müller Eberhard HJ (1983) Visualization of human C4b-binding protein and its complexes with vitamin K-dependent protein S and complement protein C4b. Proc Natl Acad Sci USA 80:3461–3465
Happonen KE, Sjoberg AP, Morgelin M et al (2009) Complement inhibitor C4b-binding protein interacts directly with small glycoproteins of the extracellular matrix. J Immunol 182:1518–1525
Hillarp A, Dahlbäck B (1988) Novel subunit in C4b-binding protein required for protein S binding. J Biol Chem 263:12759–12764
Härdig Y, Dahlbäck B (1996) The amino-terminal module of the C4b-binding protein beta-chain contains the protein S binding site. J Biol Chem 271:20861–20867
Webb JH, Blom AM, Dahlbäck B (2002) Vitamin K-dependent protein S localizing complement regulator C4b-binding protein to the surface of apoptotic cells. J Immunol 169:2580–2586
Trouw LA, Nilsson SC, Goncalves I et al (2005) C4b-binding protein binds to necrotic cells and DNA, limiting DNA release and inhibiting complement activation. J Exp Med 201:1937–1948
Barnum SR, Dahlbäck B (1990) C4b-binding protein, a regulatory component of the classical pathway of complement, is an acute-phase protein and is elevated in systemic lupus erythematosus. Complement Inflamm 7:71–77
Garcia de Frutos P, Alim RI, Härdig Y et al (1994) Differential regulation of alpha and beta chains of C4b-binding protein during acute-phase response resulting in stable plasma levels of free anticoagulant protein S. Blood 84:815–822
Dahlback B (1986) Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein. J Biol Chem 261:12022–12027
Zöller B, Garcia de Frutos P, Dahlbäck B (1995) Evaluation of the relationship between protein S and C4b-binding protein isoforms in hereditary protein S deficiency demonstrating type I and type III deficiencies to be phenotypic variants of the same genetic disease. Blood 85:3524–3531
Simmonds RE, Zöller B, Ireland H et al (1997) Genetic and phenotypic analysis of a large (122-member) protein S-deficient kindred provides an explanation for the familial coexistence of type I and type III plasma phenotypes. Blood 89:4364–4370
Gigli I, Fujita T, Nussenzweig V (1979) Modulation of the classical pathway C3 convertase by plasma protein C4b binding and C3b inactivator. Proc Natl Acad Sci USA 76:6596–6600
Seya T, Nakamura K, Masaki T et al (1995) Human factor H and C4b-binding protein serve as factor I cofactors both encompassing inactivation of C3b and C4b. Mol Immunol 32:355–360
Blom AM, Kask L, Dahlbäck B (2003) CCP1-4 of the C4b-binding protein α-chain are required for Factor I mediated cleavage of C3b. Mol Immunol 39:547–556
Spijkers PP, Denis CV, Blom AM et al (2008) Cellular uptake of C4b-binding protein is mediated by heparan sulfate proteoglycans and CD91/LDL receptor-related protein. Eur J Immunol 38:809–817
Blom AM, Hallstrom T, Riesbeck K (2009) Complement evasion strategies of pathogens-acquisition of inhibitors and beyond. Mol Immunol 46:2808–2817
Blom AM, Webb J, Villoutreix BO et al (1999) A cluster of positively charged amino acids in the N-terminal modules of the C4BP α-chain is crucial for C4b binding and factor I cofactor function. J Biol Chem 274:19237–19245
Persson J, Lindahl G (2005) Single-step purification of human C4b-binding protein (C4BP) by affinity chromatography on a peptide derived from a streptococcal surface protein. J Immunol Methods 297:83–95
Greenwood FC, Hunter WM, Glover JS (1963) The preparation of 125I-labelled human growth hormone of high specific activity. Biochem J 89:114–123
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Mohlin, F.C., Blom, A.M. (2014). Purification and Functional Characterization of C4b-Binding Protein (C4BP). In: Gadjeva, M. (eds) The Complement System. Methods in Molecular Biology, vol 1100. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-724-2_14
Download citation
DOI: https://doi.org/10.1007/978-1-62703-724-2_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-723-5
Online ISBN: 978-1-62703-724-2
eBook Packages: Springer Protocols