Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

Department of Physics, IBILCE/UNESP, CP 136, Sao José de Rio Preto, CEP 15054-000, Brazil.
Protein Science (Impact Factor: 2.85). 11/2003; 12(10):2273-81. DOI: 10.1110/ps.03102403
Source: PubMed

ABSTRACT BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C(164)I(165)M(166), which characterize the "metzincin" superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four alpha-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single alpha-helix and several loops. The catalytic zinc ion is coordinated by the N(epsilon 2) nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.

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    • "The crystal structures for various SVMPs of the P-I class have been reported and showed remarkable similarity in terms of folding, despite the fact that there are different disulfide bond patterns observed among these SVMPs (Gomis-Ruth et al., 1993; Huang et al., 2002; Watanabe et al., 2003; Zhang et al., 1994). Interestingly, the determination of crystal structures of P-III class SVMPs also showed that the folding of the metalloproteinase domain is similar between these enzymes and those of the P-I class (Igarashi et al., 2007; Muniz et al., 2008; Takeda et al., 2006, 2007). "
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    ABSTRACT: By catalyzing limited proteolysis or extensive degradation, proteolytic enzymes determine the fate of most proteins in an organism. In the evolutionary process of snake venoms, genes encoding proteinases were tailored to generate potent toxins to target key physiological proteins and thereby play a critical role in prey capture, immobilization and defense against predators. In Bothrops jararaca, metalloproteinases and serine proteinases are among the most abundant toxins both in newborn and adult venoms. In this review, we examine the proteinase-rich venom proteome of B. jararaca and how the proteinases act in a complex and heterogeneous fashion to exert their deleterious local and systemic effects.
    Toxin Reviews 06/2014; 33(4). DOI:10.3109/15569543.2014.922581 · 0.84 Impact Factor
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    • "Its sequence revealed high identity with others SVMPs (P-I class). Two consensus sequences of this class of enzymes were identified: (H170E171XXH174XXG177XXH180 e A124Q125L126L127T128), as well as conserved residues (145,185, 187,192, 209, 225), which can be involved in the disulfide bonds formation (Watanabe et al., 2003; Akao et al., 2010). BpMPI showed azocaseinolytic dose dependence activity (Fig. 3A). "
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    ABSTRACT: Snake Venom Metalloproteinases (SVMPs) are the most abundant components present in Viperidae venom. They are important in the induction of systemic alterations and local tissue damage after envenomation. In the present study, a metalloproteinase named BpMPI was isolated from Bothropoides pauloensis snake venom and its biochemical and enzymatic characteristics were determined. BpMPI was purified in two chromatography steps on ion exchange CM-Sepharose Fast flow and Sephacryl S-300. This protease was homogeneous on SDS-PAGE and showed a single chain polypeptide of 20kDa under non reducing conditions. The partial amino acid sequence of the enzyme showed high similarity with other SVMPs enzymes from snake venoms. BpMPI showed proteolytic activity upon azocasein and bovine fibrinogen and was inhibited by EDTA, 1,10 phenanthroline and β-mercaptoethanol. Moreover, this enzyme showed stability at neutral and alkaline pH and it was inactivated at high temperatures. BpMPI was able to hydrolyze glandular and tissue kallikrein substrates, but was unable to act upon factor Xa and plasmin substrates. The enzyme did not induce local hemorrhage in the dorsal region of mice even at high doses. Taken together, our data showed that BpMP-I is in fact a fibrinogenolytic metalloproteinase and a non hemorrhagic enzyme.
    Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 02/2012; 161(2):102-9. DOI:10.1016/j.cbpb.2011.10.002 · 1.90 Impact Factor
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    • "Among several SVMPs isolated from B. asper venom (Angulo and Lomonte, 2009), BaP1 is a 22.7 kDa P-I SVMP comprising a single chain of 202 amino acids that shows highest sequence identity with SVMP isolated from the venoms of snakes of the subfamily Crotalinae. The amino acid sequence and the crystal structure of this enzyme have been described (Watanabe et al., 2003; Lingott et al., 2009). BaP1 exerts multiple tissuedamaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema (Gutiérrez et al., 1995; Rucavado et al., 1995, 1998; Jiménez et al., 2008). "
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    ABSTRACT: BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (K(d)), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The K(d)s of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.
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