A novel metalloprotease from Vipera lebetina venom induces human endothelial cell apoptosis☆
Introduction
Apoptosis is a programmed physiological mode of cell suicide. The main characteristic morphological features (changes) of apoptosis include cellular shrinkage, membrane blebbing, nuclear condensation and DNA fragmentation, and disassembly of the cell into membrane-enclosed vesicles (apoptotic bodies). Apoptosis takes place during embryogenesis, during development of the nervous system and the immune system. Apoptosis is implicated in the homeostatic cell balance in normal adult tissues. Animals use apoptosis to eliminate extraneous or dangerous cells. Defects in the coupling of cell death and multiplication result in pathologies such as tumors or functional deficiencies (Golstein, 1998). Studies on eukaryote (human) cells have shown that a number of related proteases (family of caspases) are involved in cell apoptosis. Proteases are usually synthesized as precursors that have little, if any, catalytic activity. The precursor is converted into an active enzyme by proteolytic processing. Activated caspases destroy particular intracellular proteins that protect living cells from apoptosis and also reorganize cellular structures indirectly by cleaving several proteins involved in cytoskeleton regulation, thereby killing the cell (Thornberry and Lazebnik, 1998).
Endothelial cells play a critical role in vascular homeostasis such as blood coagulation, vascular contraction, and the control of vascular permeability. Human umbilical vein endothelial cells (HUVEC) express different integrins such as α2β1, α3β1, α5β1, α6β1 and αvβ3 on the surface of their membranes. The endothelial cell integrins interact with extracellular matrix (ECM) proteins. These interactions are important for vascular integrity, permeability and angiogenesis. Extracellular matrix proteins (e.g. fibronectin, vitronectin, fibrinogen, etc.) as well as snake venom metalloprotease/disintegrin components contain the RGD (or other) sequence motif that recognizes integrin receptors involved in cell–matrix interactions (Luscinskas and Lawler, 1994).
Snake venoms contain different proteins (l-amino acid oxidase, metalloproteases, disintegrins, etc.) affecting apoptosis of normal (e.g. endothelial) and cancer cells (Ali et al., 2000, Araki et al., 1993, Suhr and Kim, 1996; Torii et al., 1997, Torii et al., 2000; Masuda et al., 1998, Masuda et al., 2000, Masuda et al., 2001). Snake venom metalloproteases are classified into four major groups by their protein domain structures or cDNA sequences (Hite et al., 1994, Matsui et al., 2000). All four groups share homologous signal peptide, pro-domains and protease domain. Class P-I enzymes consist of protease domains only. Class P-II enzymes consist of protease and disintegrin domains. The disintegrin domain might be released autocatalytically or by the action of other proteases. Fibrinolytic enzyme lebetase from Vipera lebetina venom is the representative of this class (Siigur et al., 1996). Lebetase purified from the venom lacks the disintegrin domain. Class P-III enzymes comprise protease, disintegrin and cysteine-rich domains. Class P-IV metalloproteases have additionally disulfide linked C-type lectin-like domains. Factor X activator from V. lebetina venom (Siigur et al., 2001, Siigur et al., 2004) belongs to the P-IV class. Mammalian proteins homologous to snake venom metalloproteases are classified into a disintegrin and metalloprotease (ADAM) family. ADAMs are involved in several physiological processes such as fertilization, cell differentiation and shedding of receptors. Both ADAM and snake venom metalloproteases belong to the reprolysin protein family of metalloproteases (Fox and Long, 1998). Recently, different snake venom metalloproteases such as halysase from Gloydius halys venom (You et al., 2003), agkistin from Agkistrodon halys (Wang et al., 2003), HV1 from Trimeresurus flavoviridis venom (Masuda et al., 1998, Masuda et al., 2001), VAP-1 from Crotalus atrox venom (Masuda et al., 2000), and graminelysin I from Trimeresurus gramineus venom (Wu et al., 2001) were reported to induce apoptosis of human endothelial cells. The molecular mechanism of apoptosis is different in case of these snake venom metalloproteases. It has been shown that the metalloprotease and disintegrin-like domains of halysase cooperatively induce apoptotic cell death of endothelial cells (You et al., 2003). HUVEC cell apoptosis induced by graminelysin I is completely inhibited by EDTA. Graminelysin I does not contain disintegrin part (Wu et al., 2001).
In this study, we report the purification, proteolytic specificity and cDNA cloning of a novel snake venom metalloprotease from V. lebetina venom, designated as VLAIP, since it is capable of inducing apoptosis of human umbilical vein endothelial cells. We showed that VLAIP inhibits the endothelial cell adhesion to extracellular matrix proteins: fibrinogen, fibronectin, vitronectin, collagen I and collagen IV.
Section snippets
Materials
Vipera lebetina snake venom was collected from different districts of Central Asia and purchased from Tashkent Integrated Zoo Plant (Uzbekistan). Sephadex G-100 (superfine) was from Pharmacia (Uppsala, Sweden), CM-52 cellulose, fibronectin, vitronectin, collagen I, collagen IV, laminin, fibrinogen, insulin B-chain, streptomycin, heparin, MTT were from Sigma (St Louis, MO, USA), FCS from PAA Laboratories (Austria), ECGS from Upstate (USA). DNA ladder marker was from Fermentas (Vilnius,
Purification and characterization of VLAIP
A novel metalloprotease VLAIP was isolated from the snake venom of V. lebetina. The purification scheme was principally the same as in case of purification of factor X activator (Siigur et al., 2001) with additional rechromatography on TSK-DEAE HPLC column. The crude venom was initially fractionated by gel filtration in a column of Sephadex G-100 (sf). Endothelial cells affecting component was eluted in I–II fraction with different high molecular weight components including factor X activator
Acknowledgements
The authors are grateful for the financial support from Estonian Ministry of Education and Research, Estonian Science Foundation Grant No. 5554 (JS) and Howard Hughes Medical Institute International Research Fellow (PK). PK is an International Senior Research Fellow of the Wellcome Trust (UK). Mrs Gunilla Rönnholm is acknowledged for skilful technical assistance and especially for performing all the LC–MS/MS analyses. We thank Dr Lee Tammemäe and Dr Ferenc Szirko with their colleagues from
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The sequence data of VLAIP-A and VLAIP-B reported in this paper have been submitted to the GenBank under accession nos. AY835996 and AY835997, respectively.