Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability

Lee,Eun  Hee; Park,Jung  Eun; Park,Jong  Woo; Lee,Jung  Sup

doi:10.3892/ijmm.2014.1864

Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability

Authors:
- Eun Hee Lee
- Jung Eun Park
- Jong Woo Park
- Jung Sup Lee
View Affiliations

Affiliations: Department of Biomedical Science and BK21-Plus Research Team for Bioactive Control Technology, College of Natural Sciences, Chosun University, Gwangju 501-759, Republic of Korea
Published online on: July 24, 2014 https://doi.org/10.3892/ijmm.2014.1864
Pages: 1180-1190

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

In the present study, a novel fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom was purified and characterized in terms of enzyme kinetics and substrate specificity. The purified enzyme [termed snake venom metalloprotease-Macrovipera mauritanica (SVMP‑MM)] was composed of a single polypeptide with an apparent molecular weight of 27 kDa, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminus of the enzyme was composed of NH2-QRFAPRYIEL-COOH, as determined by N-terminal sequencing. The Aα- and the Bβ-chains of fibrinogen were completely cleaved by SVMP-MM within 20 and 480 min, respectively. However, the γ-chain was much more resistant to digestion by the enzyme. The enzyme also exhibited proteolytic activity, cleaving the α-α polymer of cross-linked fibrin, but did not effectively digest the γ-γ polymer. To determine the kinetic parameters for SVMP-MM, a fluorescence-quenching peptide (termed o-aminobenzoic acid-HTEKLVTS-2,4-dinitrophenyl‑NH2) containing a K-L sequence for SVMP-MM cleavage was designed and synthesized. The optimal pH and temperature for the enzyme activity were found to be 5.5 and 37˚C, respectively, when the fluorogenic substrate was synthesized and used as a substrate. Among the various divalent cations tested, Ni2+ and Cu2+ showed strong inhibitory effects on enzyme activity, with an average of 69.6% inhibition. The enzyme activity was also inhibited by treatment with 1,10-phenanthroline, ethylenediaminetetraacetic acid and glycol-bis-(2‑aminoethylether)-N,N,N',N'-tetraacetic acid, but not with aprotinin, tosyl-lysine chloromethyl ketone and tosyl-phenylalanyl chloromethyl ketone, suggesting that SVMP-MM is a metalloprotease and not a serine protease. The enzymatic parameters, including the KM, kcat, and kcat/KM values were estimated to be 0.015 mM, 0.031 sec-1, and 20.67 mM-1sec-1, respectively. SVMP-MM induced vascular permeability by digesting type IV collagen. The results obtained in our study demonstrate that SVMP-MM is a fibrin(ogen)olytic P-I class metalloprotease, which can induce a hemorrhagic reaction in vivo.

View Figures

View References

1	Gutiérrez JM, Escalante T and Rucavado A: Experimental pathophysiology of systemic alterations induced by Bothrops asper snake venom. Toxicon. 54:976–987. 2009.
2	Rucavado A, Soto M, Escalante T, Loria GD, Arni R and Gutiérrez JM: Thrombocytopenia and platelet hypoaggregation induced by Bothrops asper snake venom. Toxins involved and their contribution to metalloproteinase-induced pulmonary hemorrhage. Thromb Haemost. 94:123–131. 2005.PubMed/NCBI
3	Takeda S, Takeya H and Iwanaga S: Snake venom metalloproteinases: Structure, function and relevance to the mammalian ADAM/ADAMTS family proteins. Biochim Biophys Acta. 1824:164–176. 2012. View Article : Google Scholar : PubMed/NCBI
4	White J: Snake venoms and coagulopathy. Toxicon. 45:951–967. 2005. View Article : Google Scholar : PubMed/NCBI
5	Matsui T, Fujimura Y and Titani K: Snake venom proteases affecting hemostasis and thrombosis. Biochim Biophys Acta. 1477:146–156. 2000. View Article : Google Scholar : PubMed/NCBI
6	Markland FS Jr and Swenson S: Snake venom metalloproteinases. Toxicon. 62:3–18. 2013. View Article : Google Scholar
7	Anderson SG and Ownby CL: Systemic hemorrhage induced by proteinase H from Crotalus adamanteus (eastern diamondback rattlesnake) venom. Toxicon. 35:1301–1313. 1997. View Article : Google Scholar : PubMed/NCBI
8	Nagy JA, Benjamin L, Zeng H, Dvorak AM and Dvorak HF: Vascular permeability, vascular hyperpermeability and angiogenesis. Angiogenesis. 11:109–119. 2008. View Article : Google Scholar : PubMed/NCBI
9	Tu AT, Baker B, Wongvibulsin S and Willis T: Biochemical characterization of atroxase and nucleotide sequence encoding the fibrinolytic enzyme. Toxicon. 34:1295–1300. 1996. View Article : Google Scholar : PubMed/NCBI
10	Berger M, Pinto AF and Guimarães JA: Purification and functional characterization of bothrojaractivase, a prothrombin-activating metalloproteinase isolated from Bothrops jararaca snake venom. Toxicon. 51:488–501. 2008. View Article : Google Scholar
11	Kress LF and Catanese J: Enzymatic inactivation of human antithrombin III. Limited proteolysis of the inhibitor by snake venom proteinases in the presence of heparin. Biochim Biophys Acta. 615:178–186. 1980. View Article : Google Scholar
12	Garrigues T, Dauga C, Ferquel E, Choumet V and Failloux A-B: Molecular phylogeny of Vipera Laurenti, 1768 and the related genera Macrovipera (Reuss, 1927) and Daboia (Gray, 1842), with comments about neurotoxic Vipera aspis aspis populations. Mol Phylogenet Evol. 35:35–47. 2005.
13	Lago NR, de Adolfo Roodt R, Archundia I, et al: Local damage produced by Vipera and Macrovipera venoms and some immunochemical characteristics. Toxicon. 60:2272012.
14	Archundia IG, de Roodt AR, Ramos-Cerrillo B, et al: Neutralization of Vipera and Macrovipera venoms by two experimental polyvalent antisera: A study of paraspecificity. Toxicon. 57:1049–1056. 2011.PubMed/NCBI
15	Samel M, Subbi J, Siigur J and Siigur E: Biochemical characterization of fibrinogenolytic serine proteinases from Vipera lebetina snake venom. Toxicon. 40:51–54. 2002. View Article : Google Scholar : PubMed/NCBI
16	Siigur J, Samel M, Tõnismägi K, Subbi J, Siigur E and Tu AT: Biochemical characterization of lebetase, a direct-acting fibrinolytic enzyme from Vipera lebetina snake venom. Thromb Res. 90:39–49. 1998. View Article : Google Scholar : PubMed/NCBI
17	Trummal K, Vija H, Subbi J and Siigur J: MALDI-TOF mass spectrometry analysis of substrate specificity of lebetase, a direct-acting fibrinolytic metalloproteinase from Vipera lebetina snake venom. Biochim Biophys Acta. 1476:331–336. 2000. View Article : Google Scholar
18	Swenson S and Markland FS Jr: Snake venom fibrin(ogen)olytic enzymes. Toxicon. 45:1021–1039. 2005. View Article : Google Scholar : PubMed/NCBI
19	Leonardi A, Fox JW, Trampus-Bakija A and Krizaj I: Ammodytase, a metalloprotease from Vipera ammodytes venom, possesses strong fibrinolytic activity. Toxicon. 49:833–842. 2007.
20	Markland FS Jr: Snake venom fibrinogenolytic and fibrinolytic enzymes: an updated inventory. Registry of Exogenous Hemostatic Factors of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost. 79:668–674. 1998.
21	Buschek S, Ignjatovic V, Summerhayes R and Lowe R: The effect of different snake venoms and anti-venoms on thrombin clotting time in human plasma. Thromb Res. 125:e149–e152. 2010. View Article : Google Scholar : PubMed/NCBI
22	Park JW, Park JE, Choi HK, Jung TW, Yoon SM and Lee JS: Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata. Process Biochem. 48:979–987. 2013. View Article : Google Scholar
23	Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T₄. Nature. 227:680–685. 1970. View Article : Google Scholar : PubMed/NCBI
24	Chang AK, Kim HY, Park JE, et al: Vibrio vulnificus secretes a broad-specificity metalloprotease capable of interfering with blood homeostasis through prothrombin activation and fibrinolysis. J Bacteriol. 187:6909–6916. 2005. View Article : Google Scholar
25	Miles AA and Miles EM: Vascular reactions to histamine, histamine-liberator and leukotaxine in the skin of guinea-pigs. J Physiol. 118:228–257. 1952. View Article : Google Scholar : PubMed/NCBI
26	Stroka A, Donato JL, Bon C, Hyslop S and de Araújo AL: Purification and characterization of a hemorrhagic metalloproteinase from Bothrops lanceolatus (Fer-de-lance) snake venom. Toxicon. 45:411–420. 2005. View Article : Google Scholar
27	Gasmi A, Srairi N, Karoui H and El Ayeb M: Amino acid sequence of VlF: identification in the C-terminal domain of residues common to non-hemorrhagic metalloproteinases from snake venoms. Biochim Biophys Acta. 1481:209–212. 2000. View Article : Google Scholar : PubMed/NCBI
28	Watanabe L, Shannon JD, Valente RH, et al: Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities. Protein Sci. 12:2273–2281. 2003. View Article : Google Scholar : PubMed/NCBI
29	Okamoto DN, Kondo MY, Oliveira LC, et al: P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: Insights into substrate selectivity and kinetic behavior. Biochim Biophys Acta. 1844:545–552. 2014.
30	Rodrigues VM, Soares AM, Guerra-Sá R, Rogrigues V, Fontes MR and Giglio JR: Structural and functional characterization of neuwiedase, a nonhemorrhagic fibrin(ogen)olytic. Arch Biochem Biophys. 38:213–224. 2000. View Article : Google Scholar : PubMed/NCBI
31	Siigur E, Aaspõllu A, Tu AT and Siigur J: cDNA cloning and deduced amino acid sequence of fibrinolytic enzyme (lebetase) from Vipera lebetina snake venom. Biochem Biophys Res Commun. 224:229–236. 1996. View Article : Google Scholar : PubMed/NCBI
32	Sajevic T, Leonardi A, Kovačič L, et al: VaH3, one of the principal hemorrhagins in Vipera ammodytes ammodytes venom, is a homodimeric P-IIIc metalloproteinase. Biochimie. 95:1158–1170. 2013.PubMed/NCBI
33	Huang KF, Hung CC, Pan FM, Chow LP, Tsugita A and Chiou SH: Characterization of multiple metalloproteinases with fibrinogenolytic activity from the venom of Taiwan habu (Trimeresurus mucrosquamatus): protein microsequencing coupled with cDNA sequence analysis. Biochem Biophys Res Commun. 216:223–233. 1995. View Article : Google Scholar : PubMed/NCBI
34	Xiao R, Li QW, Perrett S and He RQ: Characterisation of the fibrinogenolytic properties of the buccal gland secretion from Lampetra japonica. Biochimie. 89:383–392. 2007. View Article : Google Scholar : PubMed/NCBI
35	Doolittle RF, Watt KWK, Cottrell BA, Strong DD and Riley M: The amino acid sequence of the alpha-chain of human fibrinogen. Nature. 280:464–468. 1979. View Article : Google Scholar : PubMed/NCBI
36	Araujo MC, Melo RL, Cesari MH, Juliano MA, Juliano L and Carmona AK: Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme. Biochemistry. 39:8519–8525. 2000. View Article : Google Scholar : PubMed/NCBI
37	Baker AH, Edwards DR and Murphy G: Metalloproteinase inhibitors: biological actions and therapeutic opportunities. J Cell Sci. 115:3719–3727. 2002. View Article : Google Scholar : PubMed/NCBI
38	Norris LA: Blood coagulation. Best Pract Res Clin Obstet Gynaecol. 17:369–383. 2003. View Article : Google Scholar
39	Yamazaki Y, Nakano Y, Imamura T and Morita T: Augmentation of vascular permeability of VEGF is enhanced by KDR-binding proteins. Biochem Biophys Res Commun. 355:693–699. 2007. View Article : Google Scholar : PubMed/NCBI
40	Gasmi A, Srairi N, Guermazi S, Dekhil H, Karoui H and El Ayeb M: Amino acid structure and characterization of a heterodimeric disintegrin from Vipera lebetina venom. Biochim Biophys Acta. 1547:51–56. 2001. View Article : Google Scholar : PubMed/NCBI
41	Pereira AL, Fritzen M, Faria F, Motta G and Chudzinski-Tavassi AM: Releasing or expression modulating mediator involved in hemostasis by Berythractivase and Jararhagin (SVMPs). Toxicon. 47:788–796. 2006. View Article : Google Scholar : PubMed/NCBI
42	Randolph A, Chamberlain SH, Chu HL, Retzios AD, Markland FS Jr and Masiarz FR: Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom. Protein Sci. 1:590–600. 1992. View Article : Google Scholar : PubMed/NCBI
43	Aaspõllu A, Siigur J and Siigur E: cDNA cloning of a novel P-I lebetase isoform Le-4. Toxicon. 46:591–594. 2005.PubMed/NCBI
44	Oyama E, Kitagawa Y and Takahashi H: Primary structure and characterization of a non hemorrhagic metalloproteinase with fibrinolytic activity, from the snake venom of Protobothrops tokarensis (Tokara-habu). Toxicon. 70:153–161. 2013. View Article : Google Scholar : PubMed/NCBI
45	Wahby AF, Mahdy el SM, El-Mezayen HA, Salama WH, Abdel-Aty AM and Fahmy AS: Egyptian horned viper Cerastes cerastes venom hyaluronidase: purification, partial characterization and evidence for its action as a spreading factor. Toxicon. 60:1380–1389. 2012.
46	Bernardes CP, Menaldo DL, Camacho E, et al: Proteomic analysis of Bothrops pirajai snake venom and characterization of BpirMP, a new P-I metalloproteinase. J Proteomics. 80:250–267. 2013.
47	Bernardes CP, Santos-Filho NA, Costa TR, et al: Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom. Toxicon. 51:574–584. 2008. View Article : Google Scholar : PubMed/NCBI
48	Sun MZ, Liu S and Greenaway FT: Characterization of a fibrinolytic enzyme (ussurenase) from Agkistrodon blomhoffii ussurensis snake venom: insights into the effects of Ca²⁺ on function and structure. Biochim Biophys Acta. 1764:1340–1348. 2006.PubMed/NCBI
49	Rubinstein I, Nadel JA, Graf PD and Caughey GH: Mast cell chymase potentiates histamine-induced wheal formation in the skin of ragweed-allergic dogs. J Clin Invest. 86:555–559. 1990. View Article : Google Scholar : PubMed/NCBI