An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. II. Cloning of the cDNA and expression.
Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP), that specifically blocks collagen-mediated platelet aggregation (Connolly, T. M., Jacobs, J. W., and Condra, C. (1992) J. Biol. Chem. 267, 6893-6898). Degenerate oligonucleotides whose sequences were derived from two short peptides from V8 digests of the native LAPP were used as primers to generate a polymerase chain reaction (PCR) product which contains the cDNA region coding for the sequence between these two peptides. Using this PCR product as a hybridization probe, phage containing cDNA clones were isolated containing the entire deduced amino acid sequence for LAPP. Computer analysis of the amino acid sequence predicts a peptidase cleavage site between a 21-residue pre-peptide and a mature protein of 126 amino acids. A DNA insert to express the predicted mature LAPP protein was generated by PCR amplification using phage-derived cDNA clones as a substrate. This insert encoded a fusion protein with the leader sequence of the yeast alpha mating factor and the mature LAPP cDNA. These PCR products were cloned into the yeast expression vector pKH4 alpha 2. A KEX 2 Lys-Arg endopeptidase cleavage site was placed NH2-terminal to the predicted mature protein. This vector transfected into the yeast Saccharomyces cerevisiae directs expression of a secreted mature protein at levels up to 200 mg of LAPP/liter of culture medium. The recombinant protein was comparable to native LAPP in its electrophoretic mobility, its reactivity with anti-LAPP antisera, and its biological activity including inhibition of collagen-stimulated platelet aggregation and the adhesion of platelets to collagen. Availability of significant quantities of recombinant LAPP opens the way to further biochemical structure/function studies and to studies on the effects of an inhibitor of collagen-stimulated platelet aggregation in vivo.
Keller
P M
PM
Department of Cellular and Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Schultz
L D
LD
Condra
C
C
Karczewski
J
J
Connolly
T M
TM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002448:Cell Adhesion / Q000187:drug effects; D003001:Cloning, Molecular; D003094:Collagen / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D004247:DNA / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D007865:Leeches / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D015539:Platelet Activation / Q000187:drug effects*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012469:Salivary Glands / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000235:genetics* / Q000494:pharmacology
C073760:LAPP protein, Haementeria officinalis; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; D003094:Collagen; D004247:DNA