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. 1983 Feb 15;210(2):567–576. doi: 10.1042/bj2100567

Enzymic assay of C3b receptor on intact cells and solubilized cells.

E Sim, R B Sim
PMCID: PMC1154258  PMID: 6222733

Abstract

The C3b receptor of human erythrocytes is known to act as a cofactor for the cleavage of the complement protein C3b by the serine proteinase C3b/C4b Ina. The same cofactor activity is shown to be present on human tonsil B-lymphocytes. The cofactor activity of the C3b receptor can be assayed, on intact cells or in solubilized extracts of cells, by determining the rate of C3b cleavage in the presence of fixed concentrations of C3b and of C3b/C4b Ina. This assay method was used to compare the characteristics and relative quantities of C3b receptors on erythrocytes and lymphocytes. The cofactor activities associated with these two cell types resemble each other, but are distinct from the serum cofactor proteins, C4bp and Factor H, in antigenicity and in pH- and ionic-strength-dependence, and are distinct from Factor H in substrate specificity. Assay of cofactor activity in intact cells indicates that there are about 80-fold more receptors per cell on the lymphocyte surface than on erythrocytes. Assays with cells made permeable by detergent show that, whereas essentially all of the receptors on erythrocytes are on the cell surface, B-lymphocytes contain a large internal receptor pool, which makes up more than 80% of the total cofactor activity of the cell.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arnaout M. A., Melamed J., Tack B. F., Colten H. R. Characterization of the human complement (c3b) receptor with a fluid phase C3b dimer. J Immunol. 1981 Oct;127(4):1348–1354. [PubMed] [Google Scholar]
  2. Barrett A. J. Alpha 2-macroglobulin. Methods Enzymol. 1981;80(Pt 100):737–754. doi: 10.1016/s0076-6879(81)80056-0. [DOI] [PubMed] [Google Scholar]
  3. Bolotin C., Morris S., Tack B., Prahl J. Purification and structural analysis of the fourth component of human complement. Biochemistry. 1977 May 3;16(9):2008–2015. doi: 10.1021/bi00628a039. [DOI] [PubMed] [Google Scholar]
  4. Christensson B., Biberfeld P. Distribution of Fc and complement receptors in spleen sections. I. Comparison of various erythrocyte indicator systems. J Immunol Methods. 1978;19(1):13–28. doi: 10.1016/0022-1759(78)90003-0. [DOI] [PubMed] [Google Scholar]
  5. Crossley L. G., Porter R. R. Purification of the human complement control protein C3b inactivator. Biochem J. 1980 Oct 1;191(1):173–182. doi: 10.1042/bj1910173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
  7. DiScipio R. G. The conversion of human complement component C5 into fragment C5b by the alternative-pathway C5 convertase. Biochem J. 1981 Dec 1;199(3):497–504. doi: 10.1042/bj1990497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dobson N. J., Lambris J. D., Ross G. D. Characteristics of isolated erythrocyte complement receptor type one (CR1, C4b-C3b receptor) and CR1-specific antibodies. J Immunol. 1981 Feb;126(2):693–698. [PubMed] [Google Scholar]
  9. Fearon D. T. Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte. J Exp Med. 1980 Jul 1;152(1):20–30. doi: 10.1084/jem.152.1.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fearon D. T., Kaneko I., Thomson G. G. Membrane distribution and adsorptive endocytosis by C3b receptors on human polymorphonuclear leukocytes. J Exp Med. 1981 Jun 1;153(6):1615–1628. doi: 10.1084/jem.153.6.1615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fearon D. T. 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. doi: 10.1073/pnas.76.11.5867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ferreira A., Nussenzweig V. Mouse C4 (Ss): three-step purification of its C4c fragment and production of a monospecific antiserum. J Immunol. 1979 Feb;122(2):490–493. [PubMed] [Google Scholar]
  13. Fujita T., Gigli I., Nussenzweig V. Human C4-binding protein. II. Role in proteolysis of C4b by C3b-inactivator. J Exp Med. 1978 Oct 1;148(4):1044–1051. doi: 10.1084/jem.148.4.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gerdes J., Naiem M., Mason D. Y., Stein H. Human complement (C3b) receptors defined by a mouse monoclonal antibody. Immunology. 1982 Apr;45(4):645–653. [PMC free article] [PubMed] [Google Scholar]
  15. Hammer C. H., Wirtz G. H., Renfer L., Gresham H. D., Tack B. F. Large scale isolation of functionally active components of the human complement system. J Biol Chem. 1981 Apr 25;256(8):3995–4006. [PubMed] [Google Scholar]
  16. Harrison R. A., Lachmann P. J. The physiological breakdown of the third component of human complement. Mol Immunol. 1980 Jan;17(1):9–20. doi: 10.1016/0161-5890(80)90119-4. [DOI] [PubMed] [Google Scholar]
  17. Hsiung L., Barclay A. N., Brandon M. R., Sim E., Porter R. R. Purification of human C3b inactivator by monoclonal-antibody affinity chromatography. Biochem J. 1982 Apr 1;203(1):293–298. doi: 10.1042/bj2030293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Iida K., Mornaghi R., Nussenzweig V. Complement receptor (CR1) deficiency in erythrocytes from patients with systemic lupus erythematosus. J Exp Med. 1982 May 1;155(5):1427–1438. doi: 10.1084/jem.155.5.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Iida K., Nussenzweig V. Complement receptor is an inhibitor of the complement cascade. J Exp Med. 1981 May 1;153(5):1138–1150. doi: 10.1084/jem.153.5.1138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaplan J. Polypeptide-binding membrane receptors: analysis and classification. Science. 1981 Apr 3;212(4490):14–20. doi: 10.1126/science.6259730. [DOI] [PubMed] [Google Scholar]
  21. Lambris J. D., Dobson N. J., Ross G. D. Release of endogenous C3b inactivator from lymphocytes in response to triggering membrane receptors for beta 1H globulin. J Exp Med. 1980 Dec 1;152(6):1625–1644. doi: 10.1084/jem.152.6.1625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Müllerèberhard H. J., Dalmasso A. P., Calcott M. A. The reaction mechanism of beta-1C-globulin (C'3) in immune hemolysis. J Exp Med. 1966 Jan 1;123(1):33–54. doi: 10.1084/jem.123.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nagasawa S., Stroud R. M. 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. doi: 10.1016/0019-2791(77)90345-7. [DOI] [PubMed] [Google Scholar]
  24. Press E. M., Gagnon J. Human complement component C4. Structural studies on the fragments derived from C4b by cleavage with C3b inactivator. Biochem J. 1981 Nov 1;199(2):351–357. doi: 10.1042/bj1990351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reid K. B., Porter R. R. The proteolytic activation systems of complement. Annu Rev Biochem. 1981;50:433–464. doi: 10.1146/annurev.bi.50.070181.002245. [DOI] [PubMed] [Google Scholar]
  26. Salisbury J. L., Condeelis J. S., Maihle N. J., Satir P. Calmodulin localization during capping and receptor-mediated endocytosis. Nature. 1981 Nov 12;294(5837):163–166. doi: 10.1038/294163a0. [DOI] [PubMed] [Google Scholar]
  27. Sim E., Sim R. B. Binding of fluid-phase complement components C3 and C3b to human lymphocytes. Biochem J. 1981 Sep 15;198(3):509–518. doi: 10.1042/bj1980509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sim E., Wood A. B., Hsiung L. M., Sim R. B. Pattern of degradation of human complement fragment, C3b. FEBS Lett. 1981 Sep 14;132(1):55–60. doi: 10.1016/0014-5793(81)80426-7. [DOI] [PubMed] [Google Scholar]
  29. Sim R. B., DiScipio R. G. Purification and structural studies on the complement-system control protein beta 1H (Factor H). Biochem J. 1982 Aug 1;205(2):285–293. doi: 10.1042/bj2050285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sim R. B., Sim E. Autolytic fragmentation of complement components C3 and C4 under denaturing conditions, a property shared with alpha 2-macroglobulin. Biochem J. 1981 Jan 1;193(1):129–141. doi: 10.1042/bj1930129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sim R. B. The first component of human complement--C1. Methods Enzymol. 1981;80(Pt 100):6–16. doi: 10.1016/s0076-6879(81)80004-3. [DOI] [PubMed] [Google Scholar]
  32. Sim R. B., Twose T. M., Paterson D. S., Sim E. The covalent-binding reaction of complement component C3. Biochem J. 1981 Jan 1;193(1):115–127. doi: 10.1042/bj1930115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stein H., Siemssen U., Lennert K. Complement receptor subtypes C3b and C3d in lymphatic tissue and follicular lymphoma. Br J Cancer. 1978 Apr;37(4):520–529. doi: 10.1038/bjc.1978.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Theofilopoulos A. N., Bokisch V. A., Dixon F. J. Receptor for soluble C3 and C3b on human lymphoblastoid (RAJI) cells. Properties and biologocal significance. J Exp Med. 1974 Mar 1;139(3):696–711. doi: 10.1084/jem.139.3.696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Whaley K., Ruddy S. Modulation of the alternative complement pathways by beta 1 H globulin. J Exp Med. 1976 Nov 2;144(5):1147–1163. doi: 10.1084/jem.144.5.1147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wilson A. B., Prichard-Thomas S., Gurner B. W., Lachmann P. J., Coombs R. R. Complement components on human lymphocytes. Clin Immunol Immunopathol. 1982 Jan;22(1):118–127. doi: 10.1016/0090-1229(82)90028-9. [DOI] [PubMed] [Google Scholar]
  37. von Zabern I., Nolte R., Vogt W. Treatment of human complement components C4 and C3 with amines or chaotropic ions. Evidence of a functional and structural change that provides uncleaved C4 and C3 with properties of their soluble activated froms, C4b and C3b. Scand J Immunol. 1981;13(5):413–431. doi: 10.1111/j.1365-3083.1981.tb00152.x. [DOI] [PubMed] [Google Scholar]

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