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Purification and characterization of a membrane protein (gp45-70) that is a cofactor for cleavage of C3b and C4b.

The Journal of Experimental Medicine, 01 Apr 1986, 163(4):837-855
https://doi.org/10.1084/jem.163.4.837 PMID: 3950547 PMCID: PMC2188078

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Abstract 

Based on preliminary evidence indicating that a cell-associated protein of U937 (a human monocyte-like cell line) possessed cofactor activity and was not the C3b/C4b receptor, we sought to further characterize this protein. A sequential four-column purification procedure was devised that includes C3(H2O) affinity chromatography to isolate in reasonable yields and purity a cell-associated protein of U937 and several other human cell lines. Based on its pattern and Mr on SDS-PAGE, acidic pI, and ligand specificity, it is identical to a recently described C3(H2O) or C3b-binding membrane glycoprotein of human PBL and cell lines; having no presently identified function, it was termed gp45-70. After purifying this protein, we determined its functional capabilities and compared them to those of the other complement proteins with regulatory activity directed at components comprising the C3 convertases. This protein was the most efficient (50 times that of H) yet-described cofactor for the I-mediated first cleavage of C3b. It also was a cofactor for the first cleavage of C4b, but was not as efficient as C4bp. The second cleavage of C3b and C4b was not efficiently mediated. It had no ability to accelerate decay in the classical or alternative pathway C3 convertases. Based on this unique activity profile and ability to be surface labeled, we have renamed this molecule membrane cofactor protein (MCP). We suggest that this protein plays a major role in preventing autologous complement activation.

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J Exp Med. 1986 Apr 1; 163(4): 837–855.
PMCID: PMC2188078
PMID: 3950547

Purification and characterization of a membrane protein (gp45-70) that is a cofactor for cleavage of C3b and C4b

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Abstract

Based on preliminary evidence indicating that a cell-associated protein of U937 (a human monocyte-like cell line) possessed cofactor activity and was not the C3b/C4b receptor, we sought to further characterize this protein. A sequential four-column purification procedure was devised that includes C3(H2O) affinity chromatography to isolate in reasonable yields and purity a cell-associated protein of U937 and several other human cell lines. Based on its pattern and Mr on SDS- PAGE, acidic pI, and ligand specificity, it is identical to a recently described C3(H2O) or C3b-binding membrane glycoprotein of human PBL and cell lines; having no presently identified function, it was termed gp45- 70. After purifying this protein, we determined its functional capabilities and compared them to those of the other complement proteins with regulatory activity directed at components comprising the C3 convertases. This protein was the most efficient (50 times that of H) yet-described cofactor for the I-mediated first cleavage of C3b. It also was a cofactor for the first cleavage of C4b, but was not as efficient as C4bp. The second cleavage of C3b and C4b was not efficiently mediated. It had no ability to accelerate decay in the classical or alternative pathway C3 convertases. Based on this unique activity profile and ability to be surface labeled, we have renamed this molecule membrane cofactor protein (MCP). We suggest that this protein plays a major role in preventing autologous complement activation.

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Selected References

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  • Lachmann PJ, Hughes-Jones NC. Initiation of complement activation. Springer Semin Immunopathol. 1984;7(2-3):143–162. [Abstract] [Google Scholar]
  • Porter RR, Reid KB. The biochemistry of complement. Nature. 1978 Oct 26;275(5682):699–704. [Abstract] [Google Scholar]
  • Pangburn MK, Müller-Eberhard HJ. The alternative pathway of complement. Springer Semin Immunopathol. 1984;7(2-3):163–192. [Abstract] [Google Scholar]
  • Fujita T, Nussenzweig V. The role of C4-binding protein and beta 1H in proteolysis of C4b and C3b. J Exp Med. 1979 Aug 1;150(2):267–276. [Europe PMC free article] [Abstract] [Google Scholar]
  • Fearon DT. Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5867–5871. [Europe PMC free article] [Abstract] [Google Scholar]
  • Iida K, Nussenzweig V. Complement receptor is an inhibitor of the complement cascade. J Exp Med. 1981 May 1;153(5):1138–1150. [Europe PMC free article] [Abstract] [Google Scholar]
  • Nicholson-Weller A, Burge J, Fearon DT, Weller PF, Austen KF. Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system. J Immunol. 1982 Jul;129(1):184–189. [Abstract] [Google Scholar]
  • Nagasawa S, Stroud RM. Mechanism of action of the C3b inactivator: requirement for a high molecular weight cofactor (C3b-C4bINA cofactor) and production of a new C3b derivative (C3b'). Immunochemistry. 1977 Nov-Dec;14(11-12):749–756. [Abstract] [Google Scholar]
  • Cole JL, Housley GA, Jr, Dykman TR, MacDermott RP, Atkinson JP. Identification of an additional class of C3-binding membrane proteins of human peripheral blood leukocytes and cell lines. Proc Natl Acad Sci U S A. 1985 Feb;82(3):859–863. [Europe PMC free article] [Abstract] [Google Scholar]
  • Schneider RJ, Kulczycki A, Jr, Law SK, Atkinson JP. Isolation of a biologically active macrophage receptor for the third component of complement. Nature. 1981 Apr 30;290(5809):789–792. [Abstract] [Google Scholar]
  • Dixit R, Schneider R, Law SK, Kulczycki A, Jr, Atkinson JP. Ligand binding specificity of a rabbit alveolar macrophage receptor for C3b. J Biol Chem. 1982 Feb 25;257(4):1595–1597. [Abstract] [Google Scholar]
  • Wong WW, Fearon DT. p65: A C3b-binding protein on murine cells that shares antigenic determinants with the human C3b receptor (CR1) and is distinct from murine C3b receptor. J Immunol. 1985 Jun;134(6):4048–4056. [Abstract] [Google Scholar]
  • Takahashi K, Nagasawa S, Koyama J. The NH-2-terminal sequences of a subunit of the first component of human complement, C1s, and its activated form, C1s. FEBS Lett. 1975 Feb 15;50(3):330–333. [Abstract] [Google Scholar]
  • Nagasawa S, Stroud RM. Purification and characterization of a macromolecular weight cofactor for C3b-inactivator, C4bC3bINA-cofactor, of human plasma. Mol Immunol. 1980 Nov;17(11):1365–1372. [Abstract] [Google Scholar]
  • Nagasawa S, Stroud RM. Cleavage of C2 by C1s into the antigenically distinct fragments C2a and C2b: demonstration of binding of C2b to C4b. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2998–3001. [Europe PMC free article] [Abstract] [Google Scholar]
  • Volanakis JE, Schrohenloher RE, Stroud RM. Human factor D of the alternative complement pathway: purification and characterization. J Immunol. 1977 Jul;119(1):337–342. [Abstract] [Google Scholar]
  • Seya T, Nagasawa S, Matsukura M, Hasegawa H, Atkinson JP. Generation of C3d,g and C3d by urokinase-treated plasma in association with fibrinolysis. Complement. 1985;2(2-3):165–174. [Abstract] [Google Scholar]
  • Seya T, Holers VM, Atkinson JP. Purification and functional analysis of the polymorphic variants of the C3b/C4b receptor (CR1) and comparison with H, C4b-binding protein (C4bp), and decay accelerating factor (DAF). J Immunol. 1985 Oct;135(4):2661–2667. [Abstract] [Google Scholar]
  • Seya T, Nagasawa S. Limited proteolysis of the third component of human complement, C3, by heat treatment. J Biochem. 1981 Feb;89(2):659–664. [Abstract] [Google Scholar]
  • 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 Aug;125(2):578–582. [Abstract] [Google Scholar]
  • Nagasawa S, Mizuguchi K, Ichihara C, Koyama J. Limited chymotryptic cleavage of human C4-binding protein: isolation of a carbohydrate-containing core domain and an active fragment. J Biochem. 1982 Oct;92(4):1329–1332. [Abstract] [Google Scholar]
  • Seya T, Nagasawa S. Limited proteolysis of complement protein C3b by regulatory enzyme C3b inactivator: isolation and characterization of a biologically active fragment, C3d,g. J Biochem. 1985 Jan;97(1):373–382. [Abstract] [Google Scholar]
  • Seya T, Nagasawa S. A fluorometric method for determination of C3b inactivator. Clin Chim Acta. 1982 Mar 12;119(3):189–196. [Abstract] [Google Scholar]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [Abstract] [Google Scholar]
  • Kumar BV, Lakshmi MV, Atkinson JP. Fast and efficient method for detection and estimation of proteins. Biochem Biophys Res Commun. 1985 Sep 16;131(2):883–891. [Abstract] [Google Scholar]
  • Dykman TR, Hatch JA, Atkinson JP. Polymorphism of the human C3b/C4b receptor. Identification of a third allele and analysis of receptor phenotypes in families and patients with systemic lupus erythematosus. J Exp Med. 1984 Mar 1;159(3):691–703. [Europe PMC free article] [Abstract] [Google Scholar]
  • Kulczycki A, Jr, Krause V, Killion CC, Atkinson JP. Improved cell surface radioiodination of macrophages. J Immunol Methods. 1980;37(2):133–138. [Abstract] [Google Scholar]
  • Tal M, Silberstein A, Nusser E. Why does Coomassie Brilliant Blue R interact differently with different proteins? A partial answer. J Biol Chem. 1985 Aug 25;260(18):9976–9980. [Abstract] [Google Scholar]
  • Medof ME, Kinoshita T, Nussenzweig V. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J Exp Med. 1984 Nov 1;160(5):1558–1578. [Europe PMC free article] [Abstract] [Google Scholar]
  • Pangburn MK, Schreiber RD, Müller-Eberhard HJ. Deficiency of an erythrocyte membrane protein with complement regulatory activity in paroxysmal nocturnal hemoglobinuria. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5430–5434. [Europe PMC free article] [Abstract] [Google Scholar]
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