title: Hemorrhagin VaH4, a covalent heterodimeric P-III metalloproteinase from Vipera ammodytes ammodytes with a potential antitumour activity.
abstract: In the envenomation caused by a bite of Vipera ammodytes ammodytes, the most venomous snake in Europe, hemorrhage is usually the most severe consequence in man. Identifying and understanding the hemorrhagic components of its venom is therefore particularly important in optimizing medical treatment of patients. We describe a novel high molecular mass hemorrhagin, VaH4. The isolated molecule is a covalent dimer of two homologous subunits, VaH4-A and VaH4-B. Complete structural characterization of A and partial characterization of B revealed that both belong to the P-III class of snake venom metalloproteinases (SVMPs), comprising a metalloproteinase, a disintegrin-like domain and a cysteine-rich domain. However, neither VaH4-A nor VaH4-B possess the Cys174 involved in the inter-subunit disulphide bond of P-III SVMPs. A three-dimensional model of the VaH4 dimer suggests that Cys132 serves this function. This implies that dimers in the P-III class of SVMPs can be formed either between their Cys132 or Cys174 residues. The proteolytic activity and stability of VaH4 depend on Zn²⁺ and Ca²⁺ ions and the presence of glycosaminoglycans, which indicates physiological interaction of VaH4 with the latter element of the extracellular matrix (ECM). The molecular mass of VaH4, determined by MALDI/TOF mass spectrometry, is 110.2 kDa. N-deglycosylation reduced the mass of each monomer by 8.7 kDa. The two possible N-glycosylation sites in VaH4-A are located at completely different positions from those in homodimeric P-IIIc VaH3 from the same venom, however, without any evident functional implications. The hemorrhagic activity of this slightly acidic SVMP is ascribed to its hydrolysis of components of the ECM, particularly fibronectin and nidogen, and of some blood coagulation proteins, in particular the α-chain of fibrinogen. VaH4 is also significant medically as we found it cytotoxic against cancer cells and due to its substantial sequence similarity to ADAM/ADAMTS family of physiologically very important human proteins of therapeutic potential.
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova cesta 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Adrijana
authors.initials: A
authors.lastname: Leonardi
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova cesta 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Tamara
authors.initials: T
authors.lastname: Sajevic
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova cesta 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Lidija
authors.initials: L
authors.lastname: Kovačič
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova cesta 39, SI-1000 Ljubljana, Slovenia.
authors.forename: Jože
authors.initials: J
authors.lastname: Pungerčar
authors.affiliation: Research and Development Department, Institute of Immunology, Inc., Rockefellerova 10, HR-10 000 Zagreb, Croatia.
authors.forename: Maja
authors.initials: M
authors.lastname: Lang Balija
authors.affiliation: University of Zagreb, Centre for Research and Knowledge Trasfer in Biotechnology, Rockefellerova 10, HR-10000 Zagreb, Croatia.
authors.forename: Beata
authors.initials: B
authors.lastname: Halassy
authors.affiliation: Medical Centre Ljubljana, Division of Pediatrics, SI-1525 Ljubljana, Slovenia.
authors.forename: Alenka
authors.initials: A
authors.lastname: Trampuš Bakija
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova cesta 39, SI-1000 Ljubljana, Slovenia; Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva cesta 5, SI-1000 Ljubljana, Slovenia; Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Jamova cesta 39, SI-1000 Ljubljana, Slovenia. Electronic address: igor.krizaj@ijs.si.
authors.forename: Igor
authors.initials: I
authors.lastname: Križaj
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects; D002448:Cell Adhesion / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003094:Collagen; D019281:Dimerization; D004338:Drug Combinations; D005470:Fluorometry; D006367:HeLa Cells; D006470:Hemorrhage / Q000139:chemically induced*; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007797:Laminin; D045726:Metalloproteases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011509:Proteoglycans; D011517:Prothrombin Time; D017422:Sequence Analysis, DNA; D013997:Time Factors; D017819:Viperidae / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen; D045726:Metalloproteases
keywords: 2D-PAGE; ACN; ADAM; ADAM with thrombospondin type-1 motif; ADAMTS; Antitumour; BM; BSA; CNBr; CR; DMEM; DSF; DTT; Dis; Dulbecco's Modified Eagle Medium; ECM; EDTA; FPLC; FSB; FX; GAG; HA; HC; HPLC; Hemorrhagin; M12B clan of zinc metalloproteinases; MEROPS; MP; MS; Metalloproteinase; PNGF; PT; PT time; PTT; PVDF; RMSD; RP-HPLC; SVMP; Snake venom; Structure; TT; V. a. ammodytes; VaH1-4; VaH4-A/B; Vipera ammodytes ammodytes; Vipera ammodytes hemorrhagin 1-4; a disintegrin and metalloproteinase; acetonitrile; basement membrane; bovine serum albumin; cyanogen bromide; cysteine-rich; differential scanning fluorimetry; disintegrin-like; dithiothreitol; ethylenediaminetetraacetic acid; extracellular matrix; factor X; fast protein liquid chromatography; fetal bovine serum; glycosaminoglycan; heavy chain; high performance liquid chromatography; hydroxyapatite; mass spectrometry; metalloproteinase; peptide N-glycosidase F; polyvinylidenedifluoride; prothrombin; reversed phase-HPLC; root-mean-square deviation; snake venom metalloproteinase; subunit A/B of VaH4; thrombin time; two-dimensional PAGE