Structure and bioactivity of recombinant human CTAP-III and NAP-2.
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 alpha-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 x 10(-8) M in chemotaxis, an EC50 of 3 x 10(-8) M for shape change, and could displace IL-8 from neutrophils with a Kd of 7.5 x 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.
Proudfoot
A E
AE
Geneva Biomedical Research Institute, Glaxo-Wellcome S. A., Plan-les-Ouates, Switzerland.
Peitsch
M C
MC
Power
C A
CA
Allet
B
B
Mermod
J J
JJ
Bacon
K
K
Wells
T N
TN
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D018925:Chemokines; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D003001:Cloning, Molecular; D006636:Histamine Release / Q000187:drug effects; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D011506:Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D017421:Sequence Analysis; D013329:Structure-Activity Relationship; D001620:beta-Thromboglobulin
D018925:Chemokines; C061615:PPBP protein, human; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D001620:beta-Thromboglobulin; C009862:connective tissue-activating peptide