Primary Structure of a Hemorrhagic Metalloproteinase, HT-2, Isolated from the Venom of Crotalus ruber ruber1

Abstract

Crotalidae and Viperidae snake venoms contains several kinds of metalloproteinases which cause localized hemorrhage by direct action on blood vessel walls. We report here the entire amino acid sequence and the disulfide bridge locations of HT-2, one of the hemorrhagic toxins isolated from the venom of Crotalus ruber ruber (red rattlesnake). The non-reduced protein was first cleaved at methionine residues to provide a set of 8 fragments, which covered the entire sequence of HT-2. The disulfide bridge locations of HT-2 were also determined by using these primary fragments. The unambiguous sequence for the whole protein was then established by conventional methods using lysyl endopeptidase and thermolysin digests. HT-2 consisted of 202 amino acid residues with two disulfide bridges, which were assigned to Cys-117-Cys-197 and Cys-157-Cys-164. HT-2 had a typical zinc-chelating sequence His-Glu-X-X-His (residues 142-146) found in thermolysin, and its overall sequence showed, respectively, 50, 52, and 53% identities to those of HR2a, H2-proteinase, and the metalloproteinase domain of HR1B. However, the disulfide bridge locations of HT-2 were different from those in the other metalloproteinases. The primary structure of HT-2 was more closely related to that of Ht-d from Crotalus atrox recently determined (81% identity). From the structural comparison with five metalloproteinases so far elucidated, six conservative amino acid residues, which may possibly be related to the induction of the hemorrhagic activity, were suggested to be present in these toxins.

Structure-function relationship of human neutrophil collagenase: identification of regions responsible for substrate specificity and general proteinase activity.

“…The third ligand is felt to be the conserved cysteine in the propeptide domain consistent with the attractive "cysteine switch" model in which the propeptide blocks the active site (29). The identity of the fourth ligand of the active-site zinc has been more speculative; however, the remarkable homology in HEXX-HXXGXXH sequence (located at residues 217-227 in NC) seen throughout the MMP family and also seen in the other families of metalloendopeptidases (30)(31)(32) although other residues may be involved including the acidic amino acids aspartic and glutamic acid.…”

Crystal structure of a non-hemorrhagic fibrin(ogen)olytic metalloproteinase complexed with a novel natural tri-peptide inhibitor from venom of Agkistrodon acutus

“…VlF from Vipera lebetina (Gasmi et al, 2000); Atroxase from Crotalus Atrox (Tu et al, 1996); Neuwiedase from Bothrops neuwiedi (Rodrigues et al, 2000). Hemorrhagic SVMPs: Lebetase from Vipera lebetina (Siigur et al, 1998); Acutolysin A from A. acutus (Gong et al, 1998); HT-2 from Crotalus ruber rubber (Takeya et al, 1990); atrolysin C from Crotalus atrox (Zhang et al, 1994); BaP1 from Bothrops asper (Watanabe et al, 2003); LHFII from Lachesis muta muta (Sanchez et al, 1991); ACLPREH from Agkistrodon contortrix laticinctus (Selistre de Araujo and Ownby, 1995); TM-3 from Taiwan Habu (Huang et al, 2002a,b). Sequences were aligned using CLUSTAL W (1.82).…”

Families of metalloendopeptidases and their relationships

“…The homology of MME-d with other members in the matrixin family ranges from 33% (MMP-7) to 48% (MMP-3a and MMP-3d) [24]. Snake venom metalloproteinases all share approximate 50% identity [30]. By contrast, there is no significant homology present in the overall sequences for the members from different families.…”