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. 1993 Sep;2(9):1391–1399. doi: 10.1002/pro.5560020904

Site-specific mutations in the N-terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor.

D F Carney 1, T E Hugli 1
PMCID: PMC2142461  PMID: 8401225

Abstract

C5a is an inflammatory mediator that evokes a variety of immune effector functions including chemotaxis, cell activation, spasmogenesis, and immune modulation. It is well established that the effector site in C5a is located in the C-terminal region, although other regions in C5a also contribute to receptor interaction. We have examined the N-terminal region (NTR) of human C5a by replacing selected residues in the NTR with glycine via site-directed mutagenesis. Mutants of rC5a were expressed as fusion proteins, and rC5a was isolated after factor Xa cleavage. The potency of the mutants was evaluated by measuring both neutrophil chemotaxis and degranulation (beta-glucuronidase release). Mutants that contained the single residue substitutions Ile-6-->Gly or Tyr-13-->Gly were reduced in potency to 4-30% compared with wild-type rC5a. Other single-site glycine substitutions at positions Leu-2, Ala-10, Lys-4, Lys-5, Glu-7, Glu-8, and Lys-14 showed little effect on C5a potency. The double mutant, Ile-6-->Gly/Tyr-13-->Gly, was reduced in potency to < 0.2%, which correlated with a correspondingly low binding affinity for neutrophil C5a receptors. Circular dichroism studies revealed a 40% reduction in alpha-helical content for the double mutant, suggesting that the NTR contributes stabilizing interactions that maintain local secondary or tertiary structure of C5a important for receptor interaction. We conclude that the N-terminal region in C5a is involved in receptor binding either through direct interaction with the receptor or by stabilizing a binding site elsewhere in the intact C5a molecule.

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Selected References

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  1. Bernauer W., Hahn F., Nimptsch P., Wissler J. Studies on heart anaphylaxis. V. Cross-desensitisation between antigen, anaphylatoxin, and compound 48-80 in the guinea-pig papillary muscle. Int Arch Allergy Appl Immunol. 1972;42(1):136–151. [PubMed] [Google Scholar]
  2. Chenoweth D. E., Erickson B. W., Hugli T. E. Human C5a-related synthetic peptides as neutrophil chemotactic factors. Biochem Biophys Res Commun. 1979 Jan 30;86(2):227–234. doi: 10.1016/0006-291x(79)90856-8. [DOI] [PubMed] [Google Scholar]
  3. Ember J. A., Hugli T. E. Characterization of the human neutrophil response to sex pheromones from Streptococcus faecalis. Am J Pathol. 1989 Apr;134(4):797–805. [PMC free article] [PubMed] [Google Scholar]
  4. Ember J. A., Johansen N. L., Hugli T. E. Designing synthetic superagonists of C3a anaphylatoxin. Biochemistry. 1991 Apr 16;30(15):3603–3612. doi: 10.1021/bi00229a003. [DOI] [PubMed] [Google Scholar]
  5. Ember J. A., Sanderson S. D., Taylor S. M., Kawahara M., Hugli T. E. Biologic activity of synthetic analogues of C5a anaphylatoxin. J Immunol. 1992 May 15;148(10):3165–3173. [PubMed] [Google Scholar]
  6. Franke A. E., Andrews G. C., Stimler-Gerard N. P., Gerard C. J., Showell H. J. Human C5a anaphylatoxin: gene synthesis, expression, and recovery of biologically active material from Escherichia coli. Methods Enzymol. 1988;162:653–668. doi: 10.1016/0076-6879(88)62107-0. [DOI] [PubMed] [Google Scholar]
  7. Gerard C., Showell H. J., Hoeprich P. D., Jr, Hugli T. E., Stimler N. P. Evidence for a role of the amino-terminal region in the biological activity of the classical anaphylatoxin, porcine C5a des-Arg-74. J Biol Chem. 1985 Mar 10;260(5):2613–2616. [PubMed] [Google Scholar]
  8. Gerard N. P., Gerard C. Construction and expression of a novel recombinant anaphylatoxin, C5a-N19, as a probe for the human C5a receptor. Biochemistry. 1990 Oct 2;29(39):9274–9281. doi: 10.1021/bi00491a024. [DOI] [PubMed] [Google Scholar]
  9. Grant J. A., Dupree E., Goldman A. S., Schultz D. R., Jackson A. L. Complement-mediated release of histamine from human leukocytes. J Immunol. 1975 Mar;114(3):1101–1106. [PubMed] [Google Scholar]
  10. Greer J. Model structure for the inflammatory protein C5a. Science. 1985 May 31;228(4703):1055–1060. doi: 10.1126/science.3992245. [DOI] [PubMed] [Google Scholar]
  11. Huber R., Scholze H., Pâques E. P., Deisenhofer J. Crystal structure analysis and molecular model of human C3a anaphylatoxin. Hoppe Seylers Z Physiol Chem. 1980 Sep;361(9):1389–1399. doi: 10.1515/bchm2.1980.361.2.1389. [DOI] [PubMed] [Google Scholar]
  12. Hugli T. E., Gerard C., Kawahara M., Scheetz M. E., 2nd, Barton R., Briggs S., Koppel G., Russell S. Isolation of three separate anaphylatoxins from complement-activated human serum. Mol Cell Biochem. 1981 Dec 4;41:59–66. doi: 10.1007/BF00225297. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Hugli T. E. Structure and function of the anaphylatoxins. Springer Semin Immunopathol. 1984;7(2-3):193–219. doi: 10.1007/BF01893020. [DOI] [PubMed] [Google Scholar]
  15. Hugli T. E. The structural basis for anaphylatoxin and chemotactic functions of C3a, C4a, and C5a. Crit Rev Immunol. 1981 Feb;1(4):321–366. [PubMed] [Google Scholar]
  16. Kurimoto Y., de Weck A. L., Dahinden C. A. Interleukin 3-dependent mediator release in basophils triggered by C5a. J Exp Med. 1989 Aug 1;170(2):467–479. doi: 10.1084/jem.170.2.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mandecki W., Powell B. S., Mollison K. W., Carter G. W., Fox J. L. High-level expression of a gene encoding the human complement factor C5a in Escherichia coli. Gene. 1986;43(1-2):131–138. doi: 10.1016/0378-1119(86)90016-8. [DOI] [PubMed] [Google Scholar]
  18. Melewicz F. M., Plummer J. M., Spiegelberg H. L. Comparison of the Fc receptors for IgE on human lymphocytes and monocytes. J Immunol. 1982 Aug;129(2):563–569. [PubMed] [Google Scholar]
  19. Mollison K. W., Mandecki W., Zuiderweg E. R., Fayer L., Fey T. A., Krause R. A., Conway R. G., Miller L., Edalji R. P., Shallcross M. A. Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis. Proc Natl Acad Sci U S A. 1989 Jan;86(1):292–296. doi: 10.1073/pnas.86.1.292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schorlemmer H. U., Davies P., Allison A. C. Ability of activated complement components to induce lysosomal enzyme release from macrophages. Nature. 1976 May 6;261(5555):48–49. doi: 10.1038/261048a0. [DOI] [PubMed] [Google Scholar]
  21. Schröder J. M., Mrowietz U., Morita E., Christophers E. Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity. J Immunol. 1987 Nov 15;139(10):3474–3483. [PubMed] [Google Scholar]
  22. Stimler N. P., Hugli T. E., Bloor C. M. Pulmonary injury induced by C3a and C5a anaphylatoxins. Am J Pathol. 1980 Aug;100(2):327–348. [PMC free article] [PubMed] [Google Scholar]
  23. Williamson M. P., Madison V. S. Three-dimensional structure of porcine C5adesArg from 1H nuclear magnetic resonance data. Biochemistry. 1990 Mar 27;29(12):2895–2905. doi: 10.1021/bi00464a002. [DOI] [PubMed] [Google Scholar]

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