RESUMO
In this paper, we present a cDNA sequence encoding a full-length precursor form of a new member (ACLD) of the metalloproteinase-disintegrin-like protein family from the venom glands of Agkistrodon contortrix laticinctus (broad-banded copperhead) snake. Comparison of the deduced amino acid sequence of ACLD with those of other members of the metalloproteinase-disintegrin protein family from both mammalian and snake venom origin suggests that some conserved residues may be involved in processing of the disintegrin domain.
Assuntos
Venenos de Crotalídeos/química , Cisteína/química , DNA Complementar/análise , Desintegrinas/química , Metaloendopeptidases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Venenos de Crotalídeos/genética , Cisteína/genética , DNA Complementar/isolamento & purificação , Desintegrinas/genética , Humanos , Metaloendopeptidases/genética , Dados de Sequência Molecular , Família Multigênica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
A cDNA clone (ACLPREMT1) for a K49 phospholipase A2 (PLA2) myotoxin from Agkistrodon contortrix laticinctus snake venom was isolated from a venom gland library and sequenced. The ACLPREMT1 cDNA is 734 bp in length and has an open reading frame of 414 bp. It codes for a K49 phospholipase A2 with 121 amino acid residues. The sequence of the first 20 amino acid residues of the predicted mature protein matches exactly with the N-terminal sequence of the purified myotoxin. Comparison of the ACLPREMT1 cDNA sequence with PLA2 cDNAs from Viperidae snakes shows that it has a similar organization: highly conserved 5' and 3' untranslated regions, a sequence encoding a 16-amino acid signal peptide, and the mature protein coding region. Comparison of the predicted sequence of ACL myotoxin and other K49 and D49 PLA2 myotoxins shows that, despite the homology (85-97%) at the nucleotide level, K49 PLA2 myotoxins are distinct from the D49 PLA2s and form a highly conserved protein family. In addition to the substitution of D49K, K49 myotoxins have several invariant residues not found in the D49 group, including K7, K78, K80, K115, and K116. There are also some conserved residues (E12, T13, K16, and N17) in all myotoxic proteins, including some neurotoxic and myotoxic PLA2s. Molecular modeling of ACL myotoxin shows that these residues are close together on the surface of one side of the molecule which suggests a potential site for binding to membranes and/or induction of toxicity.
Assuntos
Agkistrodon , Venenos de Crotalídeos/enzimologia , DNA Complementar/genética , Neurotoxinas/genética , Fosfolipases A/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/isolamento & purificação , Dados de Sequência Molecular , Neurotoxinas/isolamento & purificação , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Alinhamento de SequênciaRESUMO
The cDNA sequences of two related genes coding for metalloproteinases from a venom gland library of Agkistrodon contortrix laticinctus have been determined. The ACLPREH cDNA codes for a 220-amino acid zinc-dependent hemorrhagic metalloproteinase, and the predicted sequence is in agreement with the N-terminal sequence of the previously purified protein. ACLPREF cDNA sequence predicts a 222-amino acid molecule having strong similarity to fibrolase, a fibrinolytic enzyme isolated from A. contortrix contortrix venom that is reported to be devoid of hemorrhagic activity. Both cDNAs present the same pattern of domain organization as other members of the metalloproteinase/disintegrin gene family. They code for short toxins which do not have the disintegrin domain and have three-disulfide bonds in the metalloproteinase domain. Both cDNA sequences have the highly conserved 5' and 3' untranslated regions that have been described for the metalloproteinase and phospholiphase A2 snake venom gene families. ACLPREH and ACLPREF cDNAs share a higher degree of homology in the untranslated regions and proenzyme domain than in the mature protein domain. The amino acid sequences of hemorrhagic and/or fibrinolytic metalloproteinases demonstrate that a few substitutions may result in different enzymatic activities. Also, some of these toxins have valine instead of isoleucine in the CIM Met-turn consensus sequence. From our data, we can suggest that snake venom fibrinolytic metalloproteinase genes belong to the metalloproteinase/disintegrin gene family. This is the first report of cDNA sequences of small snake venom metalloproteinases having three disulfide bonds in the metalloproteinase domain.
Assuntos
Agkistrodon/genética , Agkistrodon/metabolismo , DNA Complementar/genética , Metaloendopeptidases/genética , Venenos de Serpentes/enzimologia , Venenos de Serpentes/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , Primers do DNA/genética , Metaloendopeptidases/química , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Hyperbaric oxygen therapy (HBOT) at 1, 2, and 2.75 atmospheres absolute (ATA) was used to treat rattlesnake (Crotalus atrox) venom-induced tissue damage and edema in thigh muscles of mice. Tissue damage was evaluated by double-blind histopathologic examination: tissue edema was determined by measuring tissue water content. A total of 10 intermittent exposures to oxygen over a period of 4 days at 2 and 2.75 ATA did not influence the resolution of venom-induced tissue edema, whereas tissue damage was significantly ameliorated as compared to air-treated envenomated controls. HBOT also promoted healing in the venom-injected mice as evidenced by the presence of regenerating muscle cells. It is concluded that HBOT may limit rattlesnake venom-induced myonecrosis and promote healing in a dose-response relationship without reducing venom-induced edema.