STRUCTURE AND BIOACTIVITY OF RECOMBINANT HUMAN CTAP-III AND NAP-2

Connective tissue-activating peptide III (CTAP-III) and neutrophil-act ivating peptide-2 (NAP-2) are both derived from a common precursor, pl atelet basic protein (PBP), which is stored in the alpha-granules of p latelets and released upon their activation. CTAP-III is an 85-residue peptide which is converted to NAP-2 by enzymic removal of the 15 amin o-terminal residues. Both peptides play a role in the early stages of wound healing and inflammation through different activities. We have c loned the cDNA for PBP and expressed constructs coding for the CTAP-II I and NAP-2 polypeptides in Escherichia coli. We have purified and ren atured these recombinant proteins. The integrity of the recombinant pr oteins has been ascertained by in vitro bioassays. CTAP-III causes 51% histamine release from the basophilic cell lin KU812 at 10(-7)M, wher eas NAP-2 only causes 28% release at the same concentration. In assays on human neutrophils, NAP-2 had an EC(50) of 2 x 10(-8) M in chemotax is, an EC(50) of 3 x 10(-8) M for shape change, and could displace IL- 8 from neutrophils with a K-d of 7.5 x 10(-9) M. CTAP-III had no activ ity in these assays. The disulfide bonds have been identified by pepti de mapping and sequence analysis, and are in the positions predicted b y homology to interleukin-8 and platelet factor 4. Measurement of the molecular mass at physiologic concentrations by gel permeation chromat ography has shown that CTAP-III forms predominantly tetramers and dime rs, whereas NAP-2 is only dimetric. SDS/PAGE analysis of samples cross -linked with disuccinimidyl suberate support these topologies. We post ulate a mechanism for tetramer formation based on the interaction of t he amino-terminal extension in CTAP-III involving a helix-helix intera ction that could stabilize the association of two CTAP-III dimers.