Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1980 Oct 1;152(4):1102–1114. doi: 10.1084/jem.152.4.1102

Relation of putative thioester bond in C3 to activation of the alternative pathway and the binding of C3b to biological targets of complement

PMCID: PMC2185963  PMID: 6903192

Abstract

The reaction of [14C]methylamine with native human C3 led to the stoichiometric incorporation of methylamine, loss of hemolytic activity, and the concomitant exposure of a sulfhydryl group that could be labeled with [14C]iodoacetamide. Both labeled sites were located in the C3d portion of the alpha-chain, which is known to contain the metastable binding of C3b. The methylamine-modified C3 [C3(CH3NH2)] was shown to exhibit many of the functional properties of C3b, although the C3a portion of the molecule remained covalently attached. C3(CH3NH2) bound Factor B and beta 1H, and could be cleaved by C3b inactivator in the presence of beta 1H. C3(CH3NH2) added to human serum caused activation of the alternative pathway and consumption of C3. In presence of Factors B and D and Mg++, C3(CH2NH2) formed a C3 convertase. The convertase-forming material could be removed from solution by anti-C3a Sepharose and the preformed convertase was completely inhibited by purified antibody to C3a. This antibody did not affect the function of the C3 convertase that contained C3b. Similar functional properties were exhibited by C3 exposed for short periods of time to relatively low concentrations of chaotropic reagents, such as KSCN or guanidine. These results suggest that the initial C3 convertase of the alternative pathway may be formed from native C3, without proteolysis, by the attack of a variety of nucleophiles including water. The C3 convertase formed from this altered C3 then generates by proteolytic cleavage the initial metastable C3b that is capable of attaching to receptive surfaces. Conversion of C3 to C3b exposes one sulfhydryl residue as does modification of C3 with methylamine. When the C3d portion of C3b bound to zymosan particles via the metastable binding site was treated with radiolabeled methylamine, the fragment was released from the particles in radiolabeled form. These findings are consistent with the concept that native C3 contains an active carbonyl group, probably in the form of a thioester, which can either react with water to form functionally C3b-l;ike C3 or, upon enzymatic conversion of C3 to C3b, allows C3b to form an ester bond with hydroxyl groups on the target surface.

Full Text

The Full Text of this article is available as a PDF (770.8 KB).

Selected References

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

  1. Cooper N. R., Müller-Eberhard H. J. The reaction mechanism of human C5 in immune hemolysis. J Exp Med. 1970 Oct 1;132(4):775–793. doi: 10.1084/jem.132.4.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dalmasso A. P., Müller-Eberhard H. J. Hemolytic activity of lipoprotein-depleted serum and the effect of certain anions on complement. J Immunol. 1966 Nov;97(5):680–685. [PubMed] [Google Scholar]
  3. Fearon D. T., Austen K. F. Initiation of C3 cleavage in the alternative complement pathway. J Immunol. 1975 Nov;115(5):1357–1361. [PubMed] [Google Scholar]
  4. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 1978 Feb 28;80(4):849–857. doi: 10.1016/0006-291x(78)91322-0. [DOI] [PubMed] [Google Scholar]
  5. Götze O., Medicus R. G., Müller-Eberhard H. J. Alternative pathway of complement: nonenzymatic, reversible transition of precursor to active properdin. J Immunol. 1977 Feb;118(2):525–532. [PubMed] [Google Scholar]
  6. Hatefi Y., Hanstein W. G. Solubilization of particulate proteins and nonelectrolytes by chaotropic agents. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1129–1136. doi: 10.1073/pnas.62.4.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hugli T. E. Human anaphylatoxin (C3a) from the third component of complement. Primary structure. J Biol Chem. 1975 Nov 10;250(21):8293–8301. [PubMed] [Google Scholar]
  8. Hugli T. E., Müller-Eberhard H. J. Anaphylatoxins: C3a and C5a. Adv Immunol. 1978;26:1–53. doi: 10.1016/s0065-2776(08)60228-x. [DOI] [PubMed] [Google Scholar]
  9. Lachmann P. J., Halbwachs L. The influence of C3b inactivator (KAF) concentration on the ability of serum to support complement activation. Clin Exp Immunol. 1975 Jul;21(1):109–114. [PMC free article] [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Law S. K., Levine R. P. Interaction between the third complement protein and cell surface macromolecules. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2701–2705. doi: 10.1073/pnas.74.7.2701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Law S. K., Lichtenberg N. A., Levine R. P. Evidence for an ester linkage between the labile binding site of C3b and receptive surfaces. J Immunol. 1979 Sep;123(3):1388–1394. [PubMed] [Google Scholar]
  13. Lesavre P. H., Hugli T. E., Esser A. F., Müller-Eberhard H. J. The alternative pathway C3/C5 convertase: chemical basis of factor B activation. J Immunol. 1979 Aug;123(2):529–534. [PubMed] [Google Scholar]
  14. Lesavre P. H., Müller-Eberhard H. J. Mechanism of action of factor D of the alternative complement pathway. J Exp Med. 1978 Dec 1;148(6):1498–1509. doi: 10.1084/jem.148.6.1498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. MULLER-EBERHARD H. J. Isolation and description of proteins related to the human complement system. Acta Soc Med Ups. 1961;66:152–170. [PubMed] [Google Scholar]
  16. Medicus R. G., Götze O., Müller-Eberhard H. J. Alternative pathway of complement: recruitment of precursor properdin by the labile C3/C5 convertase and the potentiation of the pathway. J Exp Med. 1976 Oct 1;144(4):1076–1093. doi: 10.1084/jem.144.4.1076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Medicus R. G., Schreiber R. D., Götze O., Müller-Eberhard H. J. A molecular concept of the properdin pathway. Proc Natl Acad Sci U S A. 1976 Feb;73(2):612–616. doi: 10.1073/pnas.73.2.612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Müller-Eberhard H. J., Fjellström K. E. Isolation of the anticomplementary protein from cobra venom and its mode of action on C3. J Immunol. 1971 Dec;107(6):1666–1672. [PubMed] [Google Scholar]
  19. Müller-Eberhard H. J., Schreiber R. D. Molecular biology and chemistry of the alternative pathway of complement. Adv Immunol. 1980;29:1–53. doi: 10.1016/s0065-2776(08)60042-5. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Nicol P. A., Lachmann P. J. The alternate pathway of complement activation. The role of C3 and its inactivator (KAF). Immunology. 1973 Feb;24(2):259–275. [PMC free article] [PubMed] [Google Scholar]
  22. Pangburn M. K., Müller-Eberhard H. J. Complement C3 convertase: cell surface restriction of beta1H control and generation of restriction on neuraminidase-treated cells. Proc Natl Acad Sci U S A. 1978 May;75(5):2416–2420. doi: 10.1073/pnas.75.5.2416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pangburn M. K., Schreiber R. D., Müller-Eberhard H. J. Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution. J Exp Med. 1977 Jul 1;146(1):257–270. doi: 10.1084/jem.146.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schreiber R. D., Götze O., Müller-Eberhard H. J. Alternative pathway of complement: demonstration and characterization of initiating factor and its properdin-independent function. J Exp Med. 1976 Oct 1;144(4):1062–1075. doi: 10.1084/jem.144.4.1062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schreiber R. D., Medicus R. G., Gïtze O., Müller-Eberhard H. J. Properdin- and nephritic factor-dependent C3 convertases: requirement of native C3 for enzyme formation and the function of bound C3b as properdin receptor. J Exp Med. 1975 Sep 1;142(3):760–772. doi: 10.1084/jem.142.3.760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schreiber R. D., Pangburn M. K., Lesavre P. H., Müller-Eberhard H. J. Initiation of the alternative pathway of complement: recognition of activators by bound C3b and assembly of the entire pathway from six isolated proteins. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3948–3952. doi: 10.1073/pnas.75.8.3948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Swenson R. P., Howard J. B. Characterization of alkylamine-sensitive site in alpha 2-macroglobulin. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4313–4316. doi: 10.1073/pnas.76.9.4313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tack B. D., Prahl J. W. Third component of human complement: purification from plasma and physicochemical characterization. Biochemistry. 1976 Oct 5;15(20):4513–4521. doi: 10.1021/bi00665a028. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES