title: VaSP1, catalytically active serine proteinase from Vipera ammodytes ammodytes venom with unconventional active site triad.
abstract: VaSP1, a serine proteinase from Vipera ammodytes ammodytes venom, is a glycosylated monomer of 31.5 kDa, as determined by MALDI mass spectrometry, showing multiple isoelectric points between pH 6.5 and pH 8.5. Partial amino acid sequencing of VaSP1 by Edman degradation and MS/MS analysis identified sequences which allowed its classification among the so-called snake venom serine proteinase homologues, members of the peptidase S1 family, however being devoid of the canonical catalytic triad. Only few representatives of this group have been identified so far with just two of them characterised in detail at the protein level. Despite substitution of His57 with Arg, VaSP1 possesses proteolytic activity which can be inhibited by Pefabloc, benzamidine, Zn²⁺ ions, DTT and trypsin inhibitor II, a Kunitz/BPTI group member. It hydrolyses N(α)-benzoyl-Phe-Val-Arg-p-NA, exhibiting Michaelis-Menten behaviour with K(m) = 48.2 μM and V(m) = 0.019 nM s⁻¹. The pH for optimal activity on tested substrate is around 9.0. VaSP1 also cleaves insulin B-chain, digesting it at positions His¹⁰-Leu¹¹, Ala¹⁴-Leu¹⁵ and Tyr¹⁶-Leu¹⁷. Furthermore, the novel serine proteinase is active towards wide array of proteins involved in haemostasis where its degradation of fibrinogen, fibrin, prothrombin, factor X and plasminogen in vivo probably results in depletion of coagulation factors in blood circulation. The possibility that VaSP1 possesses anticoagulant properties has been further indicated by its ability to prolong prothrombin time and activated partial thromboplastin time.
authors.affiliation: University of Zagreb, Centre for Research and Knowledge Transfer in Biotechnology, Rockefellerova 10, 10 000 Zagreb, Croatia. Electronic address: tihana.kurtovic@unizg.hr.
authors.forename: Tihana
authors.initials: T
authors.lastname: Kurtović
authors.affiliation: University of Zagreb, Centre for Research and Knowledge Transfer in Biotechnology, Rockefellerova 10, 10 000 Zagreb, Croatia.
authors.forename: Marija
authors.initials: M
authors.lastname: Brgles
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
authors.forename: Adrijana
authors.initials: A
authors.lastname: Leonardi
authors.affiliation: Institute of Immunology, Rockefellerova 10, 10 000 Zagreb, Croatia.
authors.forename: Maja
authors.initials: M
authors.lastname: Lang Balija
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
authors.forename: Tamara
authors.initials: T
authors.lastname: Sajevic
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia; Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva cesta 5, 1000 Ljubljana, Slovenia; Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Jamova cesta 39, 1000 Ljubljana, Slovenia.
authors.forename: Igor
authors.initials: I
authors.lastname: Križaj
authors.affiliation: Institute for Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, 1060 Vienna, Austria.
authors.forename: Günter
authors.initials: G
authors.lastname: Allmaier
authors.affiliation: Institute for Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, 1060 Vienna, Austria.
authors.forename: Martina
authors.initials: M
authors.lastname: Marchetti-Deschmann
authors.affiliation: University of Zagreb, Centre for Research and Knowledge Transfer in Biotechnology, Rockefellerova 10, 10 000 Zagreb, Croatia.
authors.forename: Beata
authors.initials: B
authors.lastname: Halassy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001550:Benzamidines / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects; D020134:Catalytic Domain / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011517:Prothrombin Time; D020539:Sequence Analysis, Protein; D057057:Serine Proteases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013450:Sulfones / Q000494:pharmacology; D014757:Viper Venoms / Q000201:enzymology*; D017819:Viperidae / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
chemical_list: D001550:Benzamidines; D015842:Serine Proteinase Inhibitors; D013450:Sulfones; D014757:Viper Venoms; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D057057:Serine Proteases; D015032:Zinc; C032157:benzamidine
keywords: Mass spectrometry; Non-canonical catalytic triad; Snake venom serine proteinase; Vipera ammodytes