Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Nov 15;288(Pt 1):261–266. doi: 10.1042/bj2880261

A recombinant hybrid anaphylatoxin with dual C3a/C5a activity.

W Bautsch 1, T Kretzschmar 1, T Stühmer 1, A Kola 1, M Emde 1, J Köhl 1, A Klos 1, D Bitter-Suermann 1
PMCID: PMC1132107  PMID: 1445269

Abstract

By site-directed mutagenesis of a human complement factor C5a cDNA clone, we have designed a hybrid anaphylatoxin in which three amino acid residues in the C-terminal sequence of human C5a were exchanged to create the native C-terminal human C3a (hC3a) sequence Leu-Gly-Leu-Ala-Arg. This hybrid anaphylatoxin rC5a-(1-69)-LGLAR exhibited true C3a and C5a activity when tested in the guinea pig ileum contraction assay. Quantitative measurements of ATP release from guinea pig platelets revealed about 1% intrinsic C3a activity for this hybrid, while the C5a activity was essentially unchanged. Competitive binding assays confirmed that the rC5a-(1-69)-LGLAR mutant was able to displace radioiodinated rhC5a with a KI of approx. 40 nM and hC3a with a KI of approx. 3.7 microM from guinea pig platelets. Since the C-termini of both human C3a and C5a anaphylatoxins are known to interact with their respective receptors, we conclude that the same peptidic sequence, LGLAR, is able to bind to and activate two different receptors, the C3a receptor as well as the C5a receptor. This clone provides a novel tool for the identification of further receptor-binding residues in both anaphylatoxins, since any mutants may be tested for altered C3a and C5a activity simultaneously.

Full text

PDF
261

Selected References

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

  1. Bautsch W., Emde M., Kretzschmar T., Köhl J., Suckau D., Bitter-Suermann D. Human C5a anaphylatoxin: gene cloning and expression in Escherichia coli. Immunobiology. 1992 Jun;185(1):41–52. doi: 10.1016/S0171-2985(11)80316-5. [DOI] [PubMed] [Google Scholar]
  2. Bitter-Suermann D., Burger R. Guinea pigs deficient in C2, C4, C3 or the C3a receptor. Prog Allergy. 1986;39:134–158. [PubMed] [Google Scholar]
  3. Caporale L. H., Tippett P. S., Erickson B. W., Hugli T. E. The active site of C3a anaphylatoxin. J Biol Chem. 1980 Nov 25;255(22):10758–10763. [PubMed] [Google Scholar]
  4. Cheng Y., Prusoff W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973 Dec 1;22(23):3099–3108. doi: 10.1016/0006-2952(73)90196-2. [DOI] [PubMed] [Google Scholar]
  5. Chenoweth D. E., Hugli T. E. Demonstration of specific C5a receptor on intact human polymorphonuclear leukocytes. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3943–3947. doi: 10.1073/pnas.75.8.3943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Fukuoka Y., Hugli T. E. Demonstration of a specific C3a receptor on guinea pig platelets. J Immunol. 1988 May 15;140(10):3496–3501. [PubMed] [Google Scholar]
  10. Gennaro R., Simonic T., Negri A., Mottola C., Secchi C., Ronchi S., Romeo D. C5a fragment of bovine complement. Purification, bioassays, amino-acid sequence and other structural studies. Eur J Biochem. 1986 Feb 17;155(1):77–86. doi: 10.1111/j.1432-1033.1986.tb09460.x. [DOI] [PubMed] [Google Scholar]
  11. Gerard N. P., Hodges M. K., Drazen J. M., Weller P. F., Gerard C. Characterization of a receptor for C5a anaphylatoxin on human eosinophils. J Biol Chem. 1989 Jan 25;264(3):1760–1766. [PubMed] [Google Scholar]
  12. Gerardy-Schahn R., Ambrosius D., Casaretto M., Grötzinger J., Saunders D., Wollmer A., Brandenburg D., Bitter-Suermann D. Design and biological activity of a new generation of synthetic C3a analogues by combination of peptidic and non-peptidic elements. Biochem J. 1988 Oct 1;255(1):209–216. [PMC free article] [PubMed] [Google Scholar]
  13. Gerardy-Schahn R., Ambrosius D., Saunders D., Casaretto M., Mittler C., Karwarth G., Görgen S., Bitter-Suermann D. Characterization of C3a receptor-proteins on guinea pig platelets and human polymorphonuclear leukocytes. Eur J Immunol. 1989 Jun;19(6):1095–1102. doi: 10.1002/eji.1830190620. [DOI] [PubMed] [Google Scholar]
  14. Hammerschmidt D. E. Clinical utility of complement anaphylatoxin assays. Complement. 1986;3(3):166–176. doi: 10.1159/000467893. [DOI] [PubMed] [Google Scholar]
  15. Hemsley A., Arnheim N., Toney M. D., Cortopassi G., Galas D. J. A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res. 1989 Aug 25;17(16):6545–6551. doi: 10.1093/nar/17.16.6545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hoffmann T., Böttger E. C., Baum H. P., Messner M., Hadding U., Bitter-Suermann D. In vivo effects of C3a on neutrophils and its contribution to inflammatory lung processes in a guinea-pig model. Clin Exp Immunol. 1988 Mar;71(3):486–492. [PMC free article] [PubMed] [Google Scholar]
  17. Hugli T. E., Erickson B. W. Synthetic peptides with the biological activities and specificity of human C3a anaphylatoxin. Proc Natl Acad Sci U S A. 1977 May;74(5):1826–1830. doi: 10.1073/pnas.74.5.1826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johnson A. R., Hugli T. E., Müller-Eberhard H. J. Release of histamine from rat mast cells by the complement peptides C3a and C5a. Immunology. 1975 Jun;28(6):1067–1080. [PMC free article] [PubMed] [Google Scholar]
  19. Kawai M., Quincy D. A., Lane B., Mollison K. W., Or Y. S., Luly J. R., Carter G. W. Structure-function studies in a series of carboxyl-terminal octapeptide analogues of anaphylatoxin C5a. J Med Chem. 1992 Jan 24;35(2):220–223. doi: 10.1021/jm00080a004. [DOI] [PubMed] [Google Scholar]
  20. Klos A., Ihrig V., Messner M., Grabbe J., Bitter-Suermann D. Detection of native human complement components C3 and C5 and their primary activation peptides C3a and C5a (anaphylatoxic peptides) by ELISAs with monoclonal antibodies. J Immunol Methods. 1988 Jul 22;111(2):241–252. doi: 10.1016/0022-1759(88)90133-0. [DOI] [PubMed] [Google Scholar]
  21. Kola A., Klos A., Bautsch W., Kretzschmar T., Köhl J. Functional activities of synthetic anaphylatoxic peptides in widely used biological assays. Clin Exp Immunol. 1992 May;88(2):368–372. doi: 10.1111/j.1365-2249.1992.tb03090.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kretzschmar T., Kahl K., Rech K., Bautsch W., Köhl J., Bitter-Suermann D. Characterization of the C5a receptor on guinea pig platelets. Immunobiology. 1991 Nov;183(5):418–432. doi: 10.1016/S0171-2985(11)80526-7. [DOI] [PubMed] [Google Scholar]
  23. Köhl J., Casaretto M., Gier M., Karwath G., Gietz C., Bautsch W., Saunders D., Bitter-Suermann D. Reevaluation of the C3a active site using short synthetic C3a analogues. Eur J Immunol. 1990 Jul;20(7):1463–1468. doi: 10.1002/eji.1830200709. [DOI] [PubMed] [Google Scholar]
  24. Lundwall A. B., Wetsel R. A., Kristensen T., Whitehead A. S., Woods D. E., Ogden R. C., Colten H. R., Tack B. F. Isolation and sequence analysis of a cDNA clone encoding the fifth complement component. J Biol Chem. 1985 Feb 25;260(4):2108–2112. [PubMed] [Google Scholar]
  25. Maina C. V., Riggs P. D., Grandea A. G., 3rd, Slatko B. E., Moran L. S., Tagliamonte J. A., McReynolds L. A., Guan C. D. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene. 1988 Dec 30;74(2):365–373. doi: 10.1016/0378-1119(88)90170-9. [DOI] [PubMed] [Google Scholar]
  26. Meuer S., Ecker U., Hadding U., Bitter-Suermann D. Platelet-serotonin release by C3a and C5a: two independent pathways of activation. J Immunol. 1981 Apr;126(4):1506–1509. [PubMed] [Google Scholar]
  27. Meuer S., Hadding U., Andreatta R., Bitter-Suermann D. Synthetic C3a analogs as specific inhibitors of C3a activity. Immunopharmacology. 1981 Sep;3(3):275–280. doi: 10.1016/0162-3109(81)90009-6. [DOI] [PubMed] [Google Scholar]
  28. Meuer S., Hugli T. E., Andreatta R. H., Hadding U., Bitter-Suermann D. Comparative study on biological activities of various anaphylatoxins (C4a, C3a, C5a). Investigations on their ability to induce platelet secretion. Inflammation. 1981 Dec;5(4):263–273. doi: 10.1007/BF00911092. [DOI] [PubMed] [Google Scholar]
  29. Mollison K. W., Fey T. A., Krause R. A., Mandecki W., Fox J. L., Carter G. W. High-level C5a gene expression and recovery of recombinant human C5a from Escherichia coli. Agents Actions. 1987 Aug;21(3-4):366–370. doi: 10.1007/BF01966518. [DOI] [PubMed] [Google Scholar]
  30. Mollison K. W., Fey T. A., Krause R. A., Miller L., Edalji R. P., Conway R. G., Mandecki W., Shallcross M. A., Kawai M., Or Y. S. C5a structural requirements for neutrophil receptor interaction. Agents Actions Suppl. 1991;35:17–21. [PubMed] [Google Scholar]
  31. 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]
  32. Morgan E. L., Thoman M. L., Weigle W. O., Hugli T. E. Anaphylatoxin-mediated regulation of the immune response. II. C5a-mediated enhancement of human humoral and T cell-mediated immune responses. J Immunol. 1983 Mar;130(3):1257–1261. [PubMed] [Google Scholar]
  33. Morgan E. L., Thoman M. L., Weigle W. O., Hugli T. E. Human C3a-mediated suppression of the immune response. I. Suppression of murine in vitro antibody responses occurs through the generation of nonspecific Lyt-2+ suppressor T cell. J Immunol. 1985 Jan;134(1):51–57. [PubMed] [Google Scholar]
  34. Morgan W. T., Vallota E. H., Müller-Eberhard H. J. Circular dichroism of C5a anaphylatoxin of porcine complement. Biochem Biophys Res Commun. 1974 Apr 8;57(3):572–577. doi: 10.1016/0006-291x(74)90584-1. [DOI] [PubMed] [Google Scholar]
  35. Müller-Eberhard H. J. Molecular organization and function of the complement system. Annu Rev Biochem. 1988;57:321–347. doi: 10.1146/annurev.bi.57.070188.001541. [DOI] [PubMed] [Google Scholar]
  36. Nettesheim D. G., Edalji R. P., Mollison K. W., Greer J., Zuiderweg E. R. Secondary structure of complement component C3a anaphylatoxin in solution as determined by NMR spectroscopy: differences between crystal and solution conformations. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5036–5040. doi: 10.1073/pnas.85.14.5036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Olsen U. B., Selmer J., Kahl J. U. Complement C5a receptor antagonism by protamine and poly-L-Arg on human leukocytes. Complement. 1988;5(3):153–162. doi: 10.1159/000463049. [DOI] [PubMed] [Google Scholar]
  38. Or Y. S., Clark R. F., Lane B., Mollison K. W., Carter G. W., Luly J. R. Improvements in the minimum binding sequence of C5a: examination of His-67. J Med Chem. 1992 Jan 24;35(2):402–406. doi: 10.1021/jm00080a030. [DOI] [PubMed] [Google Scholar]
  39. Plummer T. H., Jr, Hurwitz M. Y. Human plasma carboxypeptidase N. Isolation and characterization. J Biol Chem. 1978 Jun 10;253(11):3907–3912. [PubMed] [Google Scholar]
  40. Rüther U., Müller-Hill B. Easy identification of cDNA clones. EMBO J. 1983;2(10):1791–1794. doi: 10.1002/j.1460-2075.1983.tb01659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  42. Valone F. H., Goetzl E. J. Immunologic release in the rat peritoneal cavity lipid chemotactic and chemokinetic factors for polymorphonuclear leukocytes. J Immunol. 1978 Jan;120(1):102–108. [PubMed] [Google Scholar]
  43. Webster R. O., Hong S. R., Johnston R. B., Jr, Henson P. M. Biologial effects of the human complement fragments C5a and C5ades Arg on neutrophil function. Immunopharmacology. 1980 Jun;2(3):201–219. doi: 10.1016/0162-3109(80)90050-8. [DOI] [PubMed] [Google Scholar]
  44. Zuiderweg E. R., Nettesheim D. G., Mollison K. W., Carter G. W. Tertiary structure of human complement component C5a in solution from nuclear magnetic resonance data. Biochemistry. 1989 Jan 10;28(1):172–185. doi: 10.1021/bi00427a025. [DOI] [PubMed] [Google Scholar]
  45. di Guan C., Li P., Riggs P. D., Inouye H. Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene. 1988 Jul 15;67(1):21–30. doi: 10.1016/0378-1119(88)90004-2. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES