Structure and Bioactivity of Recombinant Human CTAP-III and NAP-2 |
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Authors: | Amanda E. I. Proudfoot Manuel C. Peitsch Christine A. Power Bernard Allet Jean-Jacques Mermod Kevin Bacon Timothy N. C. Wells |
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Affiliation: | (1) Geneva Biomedical Research Institute, Glaxo-Wellcome S. A., 1228 Plan-les-Ouates, Switzerland;(2) Present address: DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304-1104, USA |
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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 -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 EC50 of 2×10–8 M in chemotaxis, an EC50 of 3×10–8 M for shape change, and could displace IL-8 from neutrophils with a Kd 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. |
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Keywords: | CTAP-III/NAP-2 recombinant protein disulfide bridge assignment quaternary structure molecular modeling |
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