Molecular cloning and characterisation of a neutrophil chemotactic protein from porcine platelets.
In our search for novel chemoattractant factors, we have purified a heparin-binding protein from porcine platelets which is a potent chemoattractant for human neutrophils. The protein has 80 amino acids and a molecular mass of 8597.5Da as measured by electrospray mass spectrometry. It has been characterised by amino acid sequencing and shown to have highest identity to members of the human platelet basic-protein-family. Its N-terminal sequence is intermediate in length between the human connective-tissue-activating polypeptide III (CTAP-III) and neutrophil-activating polypeptide-2 (NAP-2). The porcine NAP-2/CTAP-III shows the classic CXC cysteine spacing found towards the N-terminus in the chemokine alpha family and contains the ELR motif which has been shown to be essential for neutrophil chemotaxis. We have isolated mRNA from porcine platelets and constructed a cDNA library containing 1.0 x 10(6) independent clones. Using probes based on the protein sequence we have isolated a full length-clone for this gene, with an open reading frame containing 119 amino acids. Despite overall similarity between the human and porcine proteins, the N-terminal region is almost completely different between the two species, with only two identical amino acids. The proteolytic cleavage sites required for processing of human platelet basic protein are completely missing in the porcine homologue, implying a different processing pathway or mechanism. The porcine protein is capable of agonizing certain effects of both NAP-2 and CTAP-III when incubated with human cells indicating that the same porcine protein may be involved in both processes.
Power
C A
CA
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Proudfoot
A E
AE
Magnenat
E
E
Bacon
K B
KB
Wells
T N
TN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001779:Blood Coagulation Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D001792:Blood Platelets / Q000737:chemistry*; D002630:Chemotactic Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D002634:Chemotaxis, Leukocyte; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006636:Histamine Release; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D013552:Swine; D001620:beta-Thromboglobulin
D001779:Blood Coagulation Factors; D002630:Chemotactic Factors; D018076:DNA, Complementary; C061615:PPBP protein, human; D010455:Peptides; D001620:beta-Thromboglobulin; C009862:connective tissue-activating peptide; C018329:low affinity platelet factor 4