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. 1997 Aug;6(8):1643–1652. doi: 10.1002/pro.5560060805

Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes.

L F Jerva 1, G Sullivan 1, E Lolis 1
PMCID: PMC2143775  PMID: 9260277

Abstract

Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP-2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP-2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP-2 exhibits a highly ordered secondary structure consistent with the alpha/beta structures of other chemokines. Recombinant MIP-2 is chemotactic for human and murine neutrophils and up-regulates cell surface expression of Mac-1. MIP-2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP-2 to the human types A and B IL-8 receptors and the murine homologue of the IL-8 receptor. MIP-2 displays low-affinity binding to the type A IL-8 receptor (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity binding to the IL-8 type B receptor are used to identify an extended N-terminal surface that interacts with this receptor. Two mutants of MIP-2 establish that this region is also involved in binding and activating the murine homologue of the IL-8 receptor. Differences in the sequence between IL-8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL-8 for binding to the type A receptor.

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Selected References

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  1. Ahuja S. K., Lee J. C., Murphy P. M. CXC chemokines bind to unique sets of selectivity determinants that can function independently and are broadly distributed on multiple domains of human interleukin-8 receptor B. Determinants of high affinity binding and receptor activation are distinct. J Biol Chem. 1996 Jan 5;271(1):225–232. doi: 10.1074/jbc.271.1.225. [DOI] [PubMed] [Google Scholar]
  2. Baron E. J., Proctor R. A. Elicitation of peritoneal polymorphonuclear neutrophils from mice. J Immunol Methods. 1982 Mar 26;49(3):305–313. doi: 10.1016/0022-1759(82)90130-2. [DOI] [PubMed] [Google Scholar]
  3. Bleul C. C., Farzan M., Choe H., Parolin C., Clark-Lewis I., Sodroski J., Springer T. A. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature. 1996 Aug 29;382(6594):829–833. doi: 10.1038/382829a0. [DOI] [PubMed] [Google Scholar]
  4. Bozic C. R., Gerard N. P., Gerard C. Receptor binding specificity and pulmonary gene expression of the neutrophil-activating peptide ENA-78. Am J Respir Cell Mol Biol. 1996 Mar;14(3):302–308. doi: 10.1165/ajrcmb.14.3.8845182. [DOI] [PubMed] [Google Scholar]
  5. Bozic C. R., Gerard N. P., von Uexkull-Guldenband C., Kolakowski L. F., Jr, Conklyn M. J., Breslow R., Showell H. J., Gerard C. The murine interleukin 8 type B receptor homologue and its ligands. Expression and biological characterization. J Biol Chem. 1994 Nov 25;269(47):29355–29358. [PubMed] [Google Scholar]
  6. Bozic C. R., Kolakowski L. F., Jr, Gerard N. P., Garcia-Rodriguez C., von Uexkull-Guldenband C., Conklyn M. J., Breslow R., Showell H. J., Gerard C. Expression and biologic characterization of the murine chemokine KC. J Immunol. 1995 Jun 1;154(11):6048–6057. [PubMed] [Google Scholar]
  7. Cerretti D. P., Kozlosky C. J., Vanden Bos T., Nelson N., Gearing D. P., Beckmann M. P. Molecular characterization of receptors for human interleukin-8, GRO/melanoma growth-stimulatory activity and neutrophil activating peptide-2. Mol Immunol. 1993 Mar;30(4):359–367. doi: 10.1016/0161-5890(93)90065-j. [DOI] [PubMed] [Google Scholar]
  8. Charo I. F., Myers S. J., Herman A., Franci C., Connolly A. J., Coughlin S. R. Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2752–2756. doi: 10.1073/pnas.91.7.2752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chaudhuri A., Zbrzezna V., Polyakova J., Pogo A. O., Hesselgesser J., Horuk R. Expression of the Duffy antigen in K562 cells. Evidence that it is the human erythrocyte chemokine receptor. J Biol Chem. 1994 Mar 18;269(11):7835–7838. [PubMed] [Google Scholar]
  10. Choe H., Farzan M., Sun Y., Sullivan N., Rollins B., Ponath P. D., Wu L., Mackay C. R., LaRosa G., Newman W. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell. 1996 Jun 28;85(7):1135–1148. doi: 10.1016/s0092-8674(00)81313-6. [DOI] [PubMed] [Google Scholar]
  11. Clackson T., Wells J. A. A hot spot of binding energy in a hormone-receptor interface. Science. 1995 Jan 20;267(5196):383–386. doi: 10.1126/science.7529940. [DOI] [PubMed] [Google Scholar]
  12. Clark-Lewis I., Dewald B., Geiser T., Moser B., Baggiolini M. Platelet factor 4 binds to interleukin 8 receptors and activates neutrophils when its N terminus is modified with Glu-Leu-Arg. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3574–3577. doi: 10.1073/pnas.90.8.3574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Clark-Lewis I., Dewald B., Loetscher M., Moser B., Baggiolini M. Structural requirements for interleukin-8 function identified by design of analogs and CXC chemokine hybrids. J Biol Chem. 1994 Jun 10;269(23):16075–16081. [PubMed] [Google Scholar]
  14. Clark-Lewis I., Schumacher C., Baggiolini M., Moser B. Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities. J Biol Chem. 1991 Dec 5;266(34):23128–23134. [PubMed] [Google Scholar]
  15. Clore G. M., Appella E., Yamada M., Matsushima K., Gronenborn A. M. Three-dimensional structure of interleukin 8 in solution. Biochemistry. 1990 Feb 20;29(7):1689–1696. doi: 10.1021/bi00459a004. [DOI] [PubMed] [Google Scholar]
  16. Cocchi F., DeVico A. L., Garzino-Demo A., Arya S. K., Gallo R. C., Lusso P. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells. Science. 1995 Dec 15;270(5243):1811–1815. doi: 10.1126/science.270.5243.1811. [DOI] [PubMed] [Google Scholar]
  17. Combadiere C., Ahuja S. K., Murphy P. M. Cloning and functional expression of a human eosinophil CC chemokine receptor. J Biol Chem. 1995 Jul 14;270(28):16491–16494. doi: 10.1074/jbc.270.28.16491. [DOI] [PubMed] [Google Scholar]
  18. Deng H., Liu R., Ellmeier W., Choe S., Unutmaz D., Burkhart M., Di Marzio P., Marmon S., Sutton R. E., Hill C. M. Identification of a major co-receptor for primary isolates of HIV-1. Nature. 1996 Jun 20;381(6584):661–666. doi: 10.1038/381661a0. [DOI] [PubMed] [Google Scholar]
  19. Feng L., Xia Y., Yoshimura T., Wilson C. B. Modulation of neutrophil influx in glomerulonephritis in the rat with anti-macrophage inflammatory protein-2 (MIP-2) antibody. J Clin Invest. 1995 Mar;95(3):1009–1017. doi: 10.1172/JCI117745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Feng Y., Broder C. C., Kennedy P. E., Berger E. A. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 1996 May 10;272(5263):872–877. doi: 10.1126/science.272.5263.872. [DOI] [PubMed] [Google Scholar]
  21. Gao J. L., Murphy P. M. Cloning and differential tissue-specific expression of three mouse beta chemokine receptor-like genes, including the gene for a functional macrophage inflammatory protein-1 alpha receptor. J Biol Chem. 1995 Jul 21;270(29):17494–17501. doi: 10.1074/jbc.270.29.17494. [DOI] [PubMed] [Google Scholar]
  22. Gayle R. B., 3rd, Sleath P. R., Srinivason S., Birks C. W., Weerawarna K. S., Cerretti D. P., Kozlosky C. J., Nelson N., Vanden Bos T., Beckmann M. P. Importance of the amino terminus of the interleukin-8 receptor in ligand interactions. J Biol Chem. 1993 Apr 5;268(10):7283–7289. [PubMed] [Google Scholar]
  23. Geiser T., Dewald B., Ehrengruber M. U., Clark-Lewis I., Baggiolini M. The interleukin-8-related chemotactic cytokines GRO alpha, GRO beta, and GRO gamma activate human neutrophil and basophil leukocytes. J Biol Chem. 1993 Jul 25;268(21):15419–15424. [PubMed] [Google Scholar]
  24. Hammond M. E., Shyamala V., Siani M. A., Gallegos C. A., Feucht P. H., Abbott J., Lapointe G. R., Moghadam M., Khoja H., Zakel J. Receptor recognition and specificity of interleukin-8 is determined by residues that cluster near a surface-accessible hydrophobic pocket. J Biol Chem. 1996 Apr 5;271(14):8228–8235. doi: 10.1074/jbc.271.14.8228. [DOI] [PubMed] [Google Scholar]
  25. Holmes W. E., Lee J., Kuang W. J., Rice G. C., Wood W. I. Structure and functional expression of a human interleukin-8 receptor. Science. 1991 Sep 13;253(5025):1278–1280. doi: 10.1126/science.1840701. [DOI] [PubMed] [Google Scholar]
  26. Hébert C. A., Vitangcol R. V., Baker J. B. Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding. J Biol Chem. 1991 Oct 5;266(28):18989–18994. [PubMed] [Google Scholar]
  27. Kasama T., Strieter R. M., Lukacs N. W., Lincoln P. M., Burdick M. D., Kunkel S. L. Interleukin-10 expression and chemokine regulation during the evolution of murine type II collagen-induced arthritis. J Clin Invest. 1995 Jun;95(6):2868–2876. doi: 10.1172/JCI117993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee J., Cacalano G., Camerato T., Toy K., Moore M. W., Wood W. I. Chemokine binding and activities mediated by the mouse IL-8 receptor. J Immunol. 1995 Aug 15;155(4):2158–2164. [PubMed] [Google Scholar]
  29. Lee J., Horuk R., Rice G. C., Bennett G. L., Camerato T., Wood W. I. Characterization of two high affinity human interleukin-8 receptors. J Biol Chem. 1992 Aug 15;267(23):16283–16287. [PubMed] [Google Scholar]
  30. Lodi P. J., Garrett D. S., Kuszewski J., Tsang M. L., Weatherbee J. A., Leonard W. J., Gronenborn A. M., Clore G. M. High-resolution solution structure of the beta chemokine hMIP-1 beta by multidimensional NMR. Science. 1994 Mar 25;263(5154):1762–1767. doi: 10.1126/science.8134838. [DOI] [PubMed] [Google Scholar]
  31. Lolis E., Sweet R. M., Cousens L. S., Tekamp-Olson P., Sherry B. A., Cerami A. Preliminary crystallographic analysis of murine macrophage inflammatory protein 2. J Mol Biol. 1992 Jun 5;225(3):913–915. doi: 10.1016/0022-2836(92)90411-c. [DOI] [PubMed] [Google Scholar]
  32. Lowman H. B., Slagle P. H., DeForge L. E., Wirth C. M., Gillece-Castro B. L., Bourell J. H., Fairbrother W. J. Exchanging interleukin-8 and melanoma growth-stimulating activity receptor binding specificities. J Biol Chem. 1996 Jun 14;271(24):14344–14352. doi: 10.1074/jbc.271.24.14344. [DOI] [PubMed] [Google Scholar]
  33. Malkowski M. G., Wu J. Y., Lazar J. B., Johnson P. H., Edwards B. F. The crystal structure of recombinant human neutrophil-activating peptide-2 (M6L) at 1.9-A resolution. J Biol Chem. 1995 Mar 31;270(13):7077–7087. doi: 10.1074/jbc.270.13.7077. [DOI] [PubMed] [Google Scholar]
  34. Murphy P. M., Tiffany H. L. Cloning of complementary DNA encoding a functional human interleukin-8 receptor. Science. 1991 Sep 13;253(5025):1280–1283. doi: 10.1126/science.1891716. [DOI] [PubMed] [Google Scholar]
  35. Neote K., DiGregorio D., Mak J. Y., Horuk R., Schall T. J. Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor. Cell. 1993 Feb 12;72(3):415–425. doi: 10.1016/0092-8674(93)90118-a. [DOI] [PubMed] [Google Scholar]
  36. Oberlin E., Amara A., Bachelerie F., Bessia C., Virelizier J. L., Arenzana-Seisdedos F., Schwartz O., Heard J. M., Clark-Lewis I., Legler D. F. The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1. Nature. 1996 Aug 29;382(6594):833–835. doi: 10.1038/382833a0. [DOI] [PubMed] [Google Scholar]
  37. Oppenheim J. J., Zachariae C. O., Mukaida N., Matsushima K. Properties of the novel proinflammatory supergene "intercrine" cytokine family. Annu Rev Immunol. 1991;9:617–648. doi: 10.1146/annurev.iy.09.040191.003153. [DOI] [PubMed] [Google Scholar]
  38. Power C. A., Meyer A., Nemeth K., Bacon K. B., Hoogewerf A. J., Proudfoot A. E., Wells T. N. Molecular cloning and functional expression of a novel CC chemokine receptor cDNA from a human basophilic cell line. J Biol Chem. 1995 Aug 18;270(33):19495–19500. doi: 10.1074/jbc.270.33.19495. [DOI] [PubMed] [Google Scholar]
  39. St Charles R., Walz D. A., Edwards B. F. The three-dimensional structure of bovine platelet factor 4 at 3.0-A resolution. J Biol Chem. 1989 Feb 5;264(4):2092–2099. [PubMed] [Google Scholar]
  40. Standiford T. J., Strieter R. M., Lukacs N. W., Kunkel S. L. Neutralization of IL-10 increases lethality in endotoxemia. Cooperative effects of macrophage inflammatory protein-2 and tumor necrosis factor. J Immunol. 1995 Aug 15;155(4):2222–2229. [PubMed] [Google Scholar]
  41. Villanueva G. B., Allen N., Walz D. Circular dichroism of platelet factor 4. Arch Biochem Biophys. 1988 Feb 15;261(1):170–174. doi: 10.1016/0003-9861(88)90115-4. [DOI] [PubMed] [Google Scholar]

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