Abstract
Connective tissue-activating peptide III (CTAP-III) and neutrophil-activating peptide-2 (NAP-2) are both derived from a common precursor, platelet basic protein (PBP), which is stored in the α-granules of platelets and released upon their activation. CTAP-III is an 85-residue peptide which is converted to NAP-2 by enzymic removal of the 15 amino-terminal residues. Both peptides play a role in the early stages of wound healing and inflammation through different activities. We have cloned the cDNA for PBP and expressed constructs coding for the CTAP-III and NAP-2 polypeptides in Escherichia coli. We have purified and renatured these recombinant proteins. The integrity of the recombinant proteins has been ascertained by in vitro bioassays. CTAP-III causes 51% histamine release from the basophilic cell lin KU812 at 10−7 M, whereas NAP-2 only causes 28% release at the same concentration. In assays on human neutrophils, NAP-2 had an EC 50 of 2×10−8 M in chemotaxis, an EC 50 of 3×10−8 M for shape change, and could displace IL-8 from neutrophils with a K d of 7.5×10−9 M. CTAP-III had no activity in these assays. The disulfide bonds have been identified by peptide mapping and sequence analysis, and are in the positions predicted by homology to interleukin-8 and platelet factor 4. Measurement of the molecular mass at physiologic concentrations by gel permeation chromatography has shown that CTAP-III forms predominantly tetramers and dimers, whereas NAP-2 is only dimetric. SDS/PAGE analysis of samples cross-linked with disuccinimidyl suberate support these topologies. We postulate a mechanism for tetramer formation based on the interaction of the amino-terminal extension in CTAP-III involving a helix–helix interaction that could stabilize the association of two CTAP-III dimers.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
REFERENCES
Allet, B., Payton, M., Mattaliano, R. J., Gronenborn, A. M., Clore, M. C., and Wingfield, P. W. (1988). Purification and characterization of the DNA binding protein Ner of bacteriophage Mu, Gene (Amst.) 65, 259-268.
Alouani, S., Gaertner, H., Mermod, J.-J., Power, C., Bacon, K. B., Wells, T. N. C., and Proudfoot, A. E. I. (1995). A fluorescent interleukin-8 receptor probe produced by targetted labelling at the amino terminus, Eur. J. Biochem. 227, 328-334.
Baggiolini, M., Dewald, B., and Moser, B. (1994). Interleukin-8 and related chemotactic cytokines—CXC and CC chemokines, Adv. Immunol. 55, 97-179.
Bairoch, A., and Boeckmann, B. (1992). The SWISS-PROT protein sequence data bank, Nucleic Acids Res. 20, 2019-2022.
Baldwin, E. T., Weber, I. T., St Charles, R., Xuan, J. C., Appella, E., Yamada, M., Matsushima, K., Edwards, B. F. P., Clore, G. M., Gronenborn, A. M., and Wlodawer, A. (1991). Crystal structure of interleukin 8, symbiosis of NMR and crystallography. Proc. Natl. Acad. Sci. USA 88, 502-506.
Begg, G. S., Pepper, D. S., Chesterman, C. N., and Morgan, F. J. (1978). Complete covalent structure of human β-thromboglobulin, Biochemistry 17, 1739-1744.
Bock, P. E., Luscombe, M., Marshall, S. E., Pepper, D. S., and Holbrook, J. J. (1980). The multiple complexes formed by the interaction of platelet factor 4 with heparin, Biochem. J. 191, 769-776.
Brandt, E., and Flad, H.-D. (1992). Structure and function of platelet-derived cytokines of the β-thromboglobulin/interleukin-8 family, Plaetlets 3, 295-305.
Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S., and Karplus, M. (1983). CHARMM, A program for macromolecular energy, minimization and dynamics calculation, J. Comp. Chem. 4, 187-217.
Car, B. D., Baggiolini, M., and Walz, A. (1991). Formation of neutrophil-activating peptide 2 from platelet-derived connective-tissue-activating peptide III by different tissue proteinases, Biochem. J. 275, 581-584.
Castor, C. W., Walz, D. A., Johnson, P. H., Hossler, P. A., Smith, E. M., Bignall, M. C., Aaron, B. P., Underhill, P., Lazar, J. M., Hudson, D. H., Cole, L. A., Perini, F., and Montjoy, K. (1990). Connective tissue activation XXXIV, Effects of proteolytic processing on the biologic activities of CTAP-III, J. Lab. Clin. Med. 116, 516-526.
Chen, M.-J., and Mayo, K. H. (1991). Human platelet factor 4 subunit association/dissociation thermodynamics and kinetics, Biochemistry 30, 6402-6411.
Chung, C. W., Cooke, R. M., Proudfoot, A. E. I., and Wells, T. N. C. (1995). The three-dimensional solution structure of RANTES, Biochemistry 34, 9307-9314.
Clore, G. M., Appella, E., Yamada, M., Matsushima, K., and Gronenborn, A. M. (1990). Three-dimensional structure of interleukin 8 in solution, Biochemistry 29, 1689-1696.
Erickson, J., Davies, L. E., Castor, C. W., Walz, D. A., and Anderson, B. E. (1990). A possible receptor-binding function for the N-terminus of connective tissue activating peptide III, Biochemistry 29, 4077-4080.
Holt, J. C., Harris, M. E., Holt, A. M., Lange, E., Henschen, A., and Niewiarowski, S. (1986). Characterisation of human platelet basic protein, a precursor form of low-affinity platelet factor 4 and β-thromboglobulin, Biochemistry 25, 1988-1996.
Huang, X., and Miller, M. (1991). A time-efficient, linear-space local similarity algorithm, Adv. Appl. Math. 12, 337.
Kuna, P., Reddigari, S. R., Schall, T. J., Rucinski, D., Sadick, M., and Kaplan, A. P. (1993). Characterisation of the human basophil response to cytokines, growth factors, and histamine releasing factors of the intercrine/chemokine family, J. Immunol. 150, 1932-1943.
Lodi, P. J., Garrett, D. S., Kuszewski, J., Tsang, M. L., Weatherbee, J. A., Leonard, W. J., Gronenborn, A. M., and Clore, G. M. (1994). High-resolution solution structure of the beta chemokine hMIP-1 beta by multidimensional NMR, Science 263, 1762-1767.
Lüthy, R., Bowie, J. U., and Eisenberg, D. (1992). Assessment of protein models with three-dimensional profies, Nature 356, 83-85.
Mayo, K. H. (1991). Low-affinity platelet factor 4 1H NMR derived aggregate equilibria indicate a physiologic preference for monomers over dimers and tetramers. Biochemistry 30, 925-934.
Miller, M. D., and Krangel, M. S. (1992). Biology and biochemistry of the chemokines, A family of chemotactic and inflammatory cytokines, Crit. Rev. Immunol. 12, 17-46.
Moore, S., Pepper, D. S., and Cash, J. D. (1975). The isolation and characterisation of platelet specific β-globulin and the detection of anti-urokinase and anti-plasmin released from thrombin-aggregated washed human platelets, Biochim. Biophys. Acta 379, 370-385.
Murphy, P. M. (1994). The molecular biology of leucocyte chemoattractant receptors, Annu. Rev. Immunol. 12, 593-633.
Murphy, P. M., and Tiffany, H. L. (1991). Cloning of complementary DNA encoding a functional human interleukin-8 receptor, Science 253, 1280-1282.
Niewiarowski, S., Walz, D. A., James, P., Rucinski, B., and Kueppers, F. (1980). Identification and separation of secreted platelet proteins by isoelectric focusing. Evidence that low-affinity platelet factor 4 is converted to β-thromboglobulin by limited proteolysis, Blood 55, 453-456.
Paolini, J. F., Willard, D., Consler, T., Luther, M., and Krangel, M. S. (1994). The chemokines IL-8, monocyte chemoattractant protein-1, and I-309 are monomers at physiologically relevant concentrations, J. Immunol. 153, 2704-2717.
Pearson, W. R., and Lipman, D. J. (1988). Improved tools for biological sequence comparison, Proc. Natl. Acad. Sci. USA 85, 2444-2448.
Peitsch, M. C., and Jongeneel, C. V. (1993). A 3D model for the CD40 ligand predicts that it is a compact trimer similar to the tumor necrosis factors, Int. Immunol. 5, 233-238.
Ponder, J. W., and Richards, F. M. (1987). Tertiary templates for proteins. Use of packing criteria in the enumeration of allowed sequences for different structural classes, J. Mol. Biol. 193, 775-791.
Power, C. A., Proudfoot, A. E. I., Magnenat, E., Bacon, K. B., and Wells, T. N. C. (1994). Molecular cloning and characterisation of a neutrophil chemotactic protein from porcine platelets, Eur. J. Biochem. 221, 713-719.
Proudfoot, A. E. I., Magnenat, E., Haley, T. M., Maione, T. E., and Wells T. N. C. (1995). The complete primary structure of glycosylated porcine platelet factor 4. Eur. J. Biochem. 228, 658-684.
Rucinski, B., Niewiarowski, S., James, P., Walz, D. A., and Budzynski, A. Z. (1979). Antiheparin proteins secreted by human platelets. Purification, characterisation and radioimmunoassay, Blood 53, 47-62.
Shigeta, O., Lu, W. Q., Holt, J. C., Edmunds, L. H., Jr., and Niewiarowski, S. (1991). Ovine platelet factor 4, purification, amino acid sequence, radioimmunoassay and comparison with platelet factor 4 of other species, Thromb. Res. 64, 509-520.
St. Charles, R., Walz, D. A., and Edwards, B. F. P. (1989). The three-dimensional structure of bovine platelet factor 4 at 3.0-A resolution, J. Biol. Chem. 264, 2092-2098.
Stuckey, J. A., St. Charles, R., and Edwards, B. F. P. (1992). A model of the platelet factor 4 complex with heparin, Proteins 14, 277-287.
Walz, A., and Baggiolini, M. (1990). Generation of the neutrophil-activating peptide 2 from platelet basic protein or connective tissue-activating peptide III through monocyte proteases, J. Exp. Med. 171, 449-454.
Walz, A., Dewald, B., von Tscharner, V., and Baggiolini, M. (1989). Effects of the neutrophil-activating peptide NAP-2, platelet basic protein, connective tissue-activating peptide III and platelet factor 4 on human neutrophils, J. Exp. Med. 170, 1745-1750.
Wenger, R. H., Wicki, A., Walz, A., Kieffer, N., and Clemetson, K. K. J. (1989). Cloning of cDNA for connective tissue activating peptide III from human platelet derived lambdagtII expression library, Blood 73, 1498-1503.
Yang, Y., Mayo, K. H., Daly, T. J., Barry, J. K., and La Rosa G. J. (1994). Subunit association and structural analysis of platelet basic protein and related proteins investigated by 1H NMR spectroscopy and circular dichroism, J. Biol. Chem. 269, 20110-20118.
Zhang, X., Chen, L., Bancroft, D. P., Lai, C. K., and Maione, T. E. (1994). Crystal structure of recombinant human platelet factor 4, Biochemistry 33, 8361-8366.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Proudfoot, A.E.I., Peitsch, M.C., Power, C.A. et al. Structure and Bioactivity of Recombinant Human CTAP-III and NAP-2. J Protein Chem 16, 37–49 (1997). https://doi.org/10.1023/A:1026390811336
Published:
Issue Date:
DOI: https://doi.org/10.1023/A:1026390811336