Molecular cloning and functional expression of contortrostatin, a homodimeric disintegrin from southern copperhead snake venom.
Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Through antagonism of integrins alphaIIbbeta3, alpha5beta1, alphavbeta3, and alphavbeta5, contortrostatin inhibits platelet aggregation and disrupts cancer cell adhesion and invasion. We cloned cDNA from a library made from the venom gland cells of Agkistrodon contortrix contortrix using polymerase chain reaction. We found that the contortrostatin gene is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. The precursor cDNA is 2027 bp with a 1449-bp open reading frame. The disintegrin domain is 195 bp encoding 65 amino acids. Like other members of the disintegrin family, each subunit of contortrostatin has an RGD site, and the cysteine alignment is conserved. The disintegrin domain of the cDNA has been expressed in a eukaryotic expression system as a homodimeric fusion protein with an immunoglobulin. The recombinant protein is recognized by an antiserum against native contortrostatin in Western blot. Both the native and recombinant proteins bind to integrins alphavbeta3 and alphavbeta5. Like native contortrostatin, the recombinant fusion protein inhibits platelet aggregation, blocks cancer cell adhesion to fibronectin and vitronectin, and prevents invasion of cancer cells through a Matrigel barrier. The success of functional expression not only validates the cDNA cloning of this disintegrin, but also provides adequate material for functional studies of contortrostatin.
Zhou
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Q
Department of Biochemistry, Norris Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles, California, 90033, USA.
Hu
P
P
Ritter
M R
MR
Swenson
S D
SD
Argounova
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S
Epstein
A L
AL
Markland
F S
FS
D017836:Agkistrodon / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D019483:Disintegrins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009361:Neoplasm Invasiveness / Q000473:pathology; D010974:Platelet Aggregation / Q000187:drug effects; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019030:Receptors, Vitronectin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D003435:Crotalid Venoms; D018076:DNA, Complementary; D019483:Disintegrins; D016023:Integrins; D011498:Protein Precursors; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins; C403494:contortrostatin; C064660:integrin alphaVbeta5; D008666:Metalloendopeptidases