Abstract
Snake venom metalloproteinases (SVMPs) play key biological roles in prey immobilization and digestion. The majority of these activities depend on the hydrolysis of relevant protein substrates in the tissues. Hereby, we describe several isoforms and a cDNA clone sequence, corresponding to PII SVMP homologues from the venom of the Central American pit viper Bothriechis lateralis, which have modifications in the residues of the canonical sequence of the zinc-binding motif HEXXHXXGXXH. As a consequence, the proteolytic activity of the isolated proteins was undetectable when tested on azocasein and gelatin. These PII isoforms comprise metalloproteinase and disintegrin domains in the mature protein, thus belonging to the subclass PIIb of SVMPs. PII SVMP homologues were devoid of hemorrhagic and in vitro coagulant activities, effects attributed to the enzymatic activity of SVMPs, but induced a mild edema. One of the isoforms presents the characteristic RGD sequence in the disintegrin domain and inhibits ADP- and collagen-induced platelet aggregation. Catalytically-inactive SVMP homologues may have been hitherto missed in the characterization of snake venoms. The presence of such enzymatically-inactive homologues in snake venoms and their possible toxic and adaptive roles deserve further investigation.
Highlights
Snake venom metalloproteinases (SVMPs) are abundant components in the venoms of advanced snakes of the superfamily Colubroidea and play key roles in venom toxic and digestive actions [1,2,3]
Fraction II of the latter chromatography was devoid of hemorrhagic activity, but had sequences characteristic of SVMPs by mass spectrometric analysis
Metalloproteinase metalloproteinase metalloproteinase metalloproteinase metalloproteinase metalloproteinase metalloproteinase metalloproteinase metalloproteinase metalloproteinase/disintegrin disintegrin disintegrin disintegrin metalloproteinase metalloproteinase metalloproteinase metalloproteinase metalloproteinase metalloproteinase metalloproteinase metalloproteinase metalloproteinase metalloproteinase/disintegrin disintegrin disintegrin disintegrin disintegrin disintegrin a Confidence: MALDI/DE NOVO and EDMAN (MV, manual validation); nESI/DE NOVO (PEAKS 8 De Novo average local confidence); b Error: error of the precursor ion relative to the proposed sequence; c Method: MALDI/DE NOVO: MALDI-TOF-TOF and manual de novo sequencing; EDMAN: N-terminal sequencing; nESI/DE NOVO: nLC-nanoelectrospray MS/MS and automatic de novo sequencing using PEAKS 8 software; * peptides generated by Glu-C endoproteinase digestion; ** peptides generated by chymotrypsin digestion
Summary
Snake venom metalloproteinases (SVMPs) are abundant components in the venoms of advanced snakes of the superfamily Colubroidea and play key roles in venom toxic and digestive actions [1,2,3]. Together with the ADAMs and ADAMTs, SVMPs are classified within the M12 family of metalloproteinases (subfamily M12B or reprolysin) [1], which, in turn, belong to the metzincin superfamily of these proteinases This superfamily is characterized by a canonical zinc-binding sequence (HEXXHXXGXXH) followed by a Met-turn [4]. Bothriechis lateralis, a species distributed in Central America [14], a fraction containing internal sequences characteristic of SVMPs, but being devoid of proteinase and hemorrhagic activities, was isolated The characterization of this fraction and the cDNA cloning of a related isoform showed the presence of modifications in the residues constituting the zinc-binding motif characteristic of the reprolysin group of metalloproteinases. SVMP variants and discusses some biological implications of their presence in snake venoms
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