ISSN:
1573-4943
Keywords:
CTAP-III/NAP-2
;
recombinant protein
;
disulfide bridge assignment
;
quaternary structure
;
molecular modeling
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1026390811336
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