Molecular cloning and expression of structural domains of bothropasin, a P-III metalloproteinase from the venom of Bothrops jararaca.
Mature P-III snake metalloproteinases are soluble venom components which belong to the Reprolysin sub family and are structurally related to the mammalian membrane-bound A Disintegrin And Metalloproteinase (ADAMs). Here we present the molecular cloning of bothropasin, a metalloproteinase with hemorrhagic and myonecrotic activities isolated from the venom of Bothrops jararaca. The full-length cDNA encoding the bothropasin precursor was cloned by immunoscreening and its authenticity was confirmed by the amino acid sequence of internal fragments obtained from an autolyzed sample of native bothropasin. The predicted bothropasin precursor is comprised of the elements of a P-III venom metalloproteinase: signal sequence, pro-, metalloproteinase, disintegrin-like and cysteine-rich domains. In the autolysis process of native bothropasin, the disintegrin-like and cysteine-rich domains remained intact while the metalloproteinase domain was cleaved at different sites. The attempts made to obtain the recombinant precursor form of bothropasin using bacterial, yeast and mammalian cell expression systems failed to produce it in an amount sufficient to analyze the activation of the zymogen. Nevertheless, the study of the expression of the individual domains of bothropasin using a bacterial system resulted in the production of recombinant pro-and disintegrin-like+cysteine-rich domains but not the metalloproteinase domain. These results along with the autolysis pattern of the native protein suggest a role for the metalloproteinase domain in the structural stability of bothropasin.
Assakura
Marina T
MT
Laboratório de Bioquímica e Biofísica, Instituto Butantan, Av. Vital Brasil 1500, CEP 05503-900, São Paulo, SP, Brazil.
Silva
Carlos A
CA
Mentele
Reinhard
R
Camargo
Antonio C M
AC
Serrano
Solange M T
SM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017837:Bothrops / Q000502:physiology*; D019556:COS Cells; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D019483:Disintegrins / Q000235:genetics; D008666:Metalloendopeptidases / Q000145:classification / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D003435:Crotalid Venoms; D018076:DNA, Complementary; D019483:Disintegrins; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C472493:bothropasin, Bothrops jararaca