title: cDNA cloning, structural, and functional analyses of venom phospholipases A₂ and a Kunitz-type protease inhibitor from steppe viper Vipera ursinii renardi.
abstract: Snake venom phospholipases A₂ (PLA₂s) display a wide array of biological activities and are each characteristic to the venom. Here, we report on the cDNA cloning and characterization of PLA₂s from the steppe viper Vipera ursinii renardi venom glands. Among the five distinct PLA₂ cDNAs cloned and sequenced, the most common were the clones encoding a basic Ser-49 containing PLA₂ (Vur-S49). Other clones encoded either ammodytin analogs I1, I2d and I2a (designated as Vur-PL1, Vur-PL2 and Vur-PL3, respectively) or an ammodytoxin-like PLA₂ (Vurtoxin). Additionally, a novel Kunitz-type trypsin inhibitor for this venom species was cloned and sequenced. Comparison of these PLA₂ and Kunitz inhibitor sequences with those in the sequence data banks suggests that the viper V. u. renardi is closely related to Vipera ammodytes and Vipera aspis. Separation of V. u. renardi venom components by gel-filtration and ion-exchange chromatography showed the presence of many PLA₂ isoforms. Remarkably, the most abundant PLA₂ isolated was Vur-PL2 while Vur-S49 analog was in very low yield. There are great differences between the proportion of cDNA clones and that of the proteins isolated. Two Vur-PL2 isoforms (designated as Vur-PL2A and Vur-PL2B) indistinguishable by masses, peptide mass fingerprinting, N-terminal sequences and CD spectroscopy were purified from the pooled venom. However, when rechromatographed on cation-exchanger, Vur-PL2A showed only one peak corresponding to Vur-PL2B, suggesting the existence of conformers for Vur-PL2. Vur-PL2B was weakly cytotoxic to rat pheochromocytoma PC12 cells and showed both strong anticoagulant and anti-platelet activities. This is the first case of a strong anticoagulating ammodytin I analog in Vipera venom.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, P. O. Box 23-106, Taipei, Taiwan.
authors.forename: Inn-Ho
authors.initials: IH
authors.lastname: Tsai
authors.forename: Ying-Ming
authors.initials: YM
authors.lastname: Wang
authors.forename: An Chun
authors.initials: AC
authors.lastname: Cheng
authors.forename: Vladislav
authors.initials: V
authors.lastname: Starkov
authors.forename: Alexey
authors.initials: A
authors.lastname: Osipov
authors.forename: Ilya
authors.initials: I
authors.lastname: Nikitin
authors.forename: Yana
authors.initials: Y
authors.lastname: Makarova
authors.forename: Rustam
authors.initials: R
authors.lastname: Ziganshin
authors.forename: Yuri
authors.initials: Y
authors.lastname: Utkin
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D008969:Molecular Sequence Data; D016716:PC12 Cells; D054467:Phospholipases A2 / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D051381:Rats; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D014757:Viper Venoms / Q000201:enzymology*; D017819:Viperidae
chemical_list: D000925:Anticoagulants; D018076:DNA, Complementary; D010975:Platelet Aggregation Inhibitors; D014361:Trypsin Inhibitors; D014757:Viper Venoms; D054467:Phospholipases A2