ABSTRACT
Snake venom metalloproteases (SVMPs) embody zinc-dependent multidomain enzymes responsible for a relevant pathophysiology in envenomation, including local and systemic hemorrhage. The molecular features responsible for hemorrhagic potency of SVMPs have been associated with their multidomains structures which can target these proteins them to several receptors of different tissues and cellular types. BjussuMP-I, a SVMP isolated from the Bothrops jararacussu venom, has been characterized as a P-III hemorrhagic metalloprotease. The complete cDNA sequence of BjussuMP-I with 1641bp encodes open reading frames of 547 amino acid residues, which conserve the common domains of P-III high molecular weight hemorrhagic metalloproteases: (i) pre-pro-peptide, (ii) metalloprotease, (iii) disintegrin-like and (iv) rich cysteine domain. BjussuMP-I induced lyses in fibrin clots and inhibited collagen- and ADP-induced platelet aggregation. We are reporting, for the first time, the primary structure of an RGD-P-III class snake venom metalloprotease. A phylogenetic analysis of the BjussuMP-I metalloprotease/catalytic domain was performed to get new insights into the molecular evolution of the metalloproteases. A theoretical molecular model of this domain was built through folding recognition (threading) techniques and refined by molecular dynamics simulation. Then, the final BjussuMP-I catalytic domain model was compared to other SVMPs and Reprolysin family proteins in order to identify eventual structural differences, which could help to understand the biochemical activities of these enzymes. The presence of large hydrophobic areas and some conserved surface charge-positive residues were identified as important features of the SVMPs and other metalloproteases.
Subject(s)
Bothrops/genetics , Bothrops/metabolism , Crotalid Venoms/chemistry , Crotalid Venoms/genetics , Metalloendopeptidases/chemistry , Metalloendopeptidases/genetics , Amino Acid Sequence , Animals , Base Sequence , Bothrops/classification , Catalytic Domain/genetics , Computer Simulation , Crotalid Venoms/classification , Crotalid Venoms/toxicity , DNA, Complementary/genetics , Fibrinolysis/drug effects , In Vitro Techniques , Metalloendopeptidases/classification , Metalloendopeptidases/toxicity , Models, Molecular , Molecular Sequence Data , Phylogeny , Platelet Aggregation/drug effects , Rabbits , Sequence Homology, Amino Acid , Static Electricity , ThermodynamicsABSTRACT
MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA(2)s or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA(2) homologues and, together with additional strategies, supports the concept of the presence of others "bioactive sites" distinct from the catalytic site in snake venom myotoxic PLA(2)s.
Subject(s)
Bothrops , Phospholipases A/pharmacology , Acetic Anhydrides/chemistry , Acetophenones/chemistry , Amino Acid Sequence , Animals , Base Sequence , Candida albicans/drug effects , Candida albicans/growth & development , Creatine Kinase/blood , Cyanogen Bromide/chemistry , Edema/chemically induced , Escherichia coli/drug effects , Escherichia coli/growth & development , Humans , Jurkat Cells , Leishmania/drug effects , Male , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Neurotoxins/chemistry , Neurotoxins/genetics , Neurotoxins/pharmacology , Nitrobenzenes/chemistry , Phospholipases A/chemistry , Phospholipases A/genetics , Schistosoma mansoni/drug effects , Schistosoma mansoni/pathogenicity , Sequence Analysis, DNAABSTRACT
Anemopaegma arvense (Vell.) Stellfeld ex de Souza is a medicinal species also known as Catuaba. All commercially available formulations of Catuaba have been produced from wild crafting, the gathering of plant biomass from its native habitat. Nodal segments were used as explants and 4.8 new buds per explant were induced on MS media supplemented with 4.4 microM of kinetin in 30 days. The percentage of in vitro rooting was low, although acclimatization of unrooted plants into soil was successfully achieved. For germplasm maintenance, cultures of A. arvense were stored on 4 % (w/v) sorbitol and maintained viable at low growth rate without subcultures for six months.
