cd NA Purif ication, cd NA cloning and sequence analysis of thrombin2like enzyme from Gloydi us saxatilis

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1 49 (6) : A cta Zoologica S inica cd NA 3 ( ) Purif ication cd NA cloning and sequence analysis of thrombin2like enzyme from Gloydi us saxatilis SUN De2J un 3 YAN G Chun2Wei YAN G Tong2Shu YAN Wei2Qun WAN G Wei ( Instit ute of Frontier Medical Science Jili n U niversity Changchun Chi na) Abstract Thrombin2like enzyme has great medical application in treating thrombus. A thrombin2like enzyme from Gloy2 dius saxatilis snake venom was isolated and purified to homogeneity by a rapid and effective method using ion2exchange chromatography on DEAE2Sepharose and affinity chromatography on heparin2sepharose. SDS2polyacrylamide elec2 trophoresis under reducing condition revealed that the purified enzyme had a single protein band and its molecular weight was dalton. Total RNAs were extracted from the venom gland of the G. saxatilis snake. Using degenerate primers we amplified the cdna of the thrombin2like enzyme gene in the venom gland of G. saxatilis using the reverse transcription2polymerase chainreaction ( RT2PCR) method. The cdna fragment was inserted into p GEM T vector cloned and its nucleotide sequence was determined. Its open reading frame is composed of 774 nucleotides and codes a protein pre2 zymogen of 258 amino acids including a putative secretory signal peptide of 18 amino acids and a proposed pro2peptide of 6 amino acid residues. It contains 12 cysteine residues. The sequence analysis indicates that the deduced amino acid se2 quence of the cdna fragment shares high identity with the thrombin2like enzyme genes of other snakes in the gene bank. The query sequence exhibits strong amino acid sequence homology of 88 % 88 % and 86 % to the serine proteas of T. gramineus thrombin2like defibrase of D. acutus and serine protease catroxase of C. at rox respectively. Based on the amino acid sequences of other thrombin2like enzymes the catalytic residues and disulfide bridges of this thrombin2like enzyme are deduced as follows : catalytic residues His 65 Asp 110 Ser 204 ; and six disulfide bridges Cys 31 2Cys 163 Cys 50 2 Cys 66 Cys 98 2Cys 256 Cys 142 2Cys 210 Cys 174 2Cys 189 and Cys 200 2Cys 225. According to the possible linked glycosylation sites N2X2T (Asn2X2Thr) or N2X2S (Asn2X2Ser) its possible glycosylation sites are N 44 2S 45 2T 46 and N 251 2T 252 2T 253 residues [ Acta Zoologica Sinica 49 (6) : ]. Key words Gloydius saxatilis Thrombin2like enzyme cdna clone Sequence analysis cdna ( Gloydius sax atilis) al. 1983) Itoh et al. (1987) 231 ( Thrombin2like enzyme) 6 His ( Itoh et al. Asp Ser Huang et al ; Pirkle et al ; Shieh et al. 1988) (1994) ; Hahn et al. (1996) A. caliginosus calobin 238 Pan et al. (Latallo et (1999) acutin (Corresponding author). E2mail : edu. cn : ν 2003 Acta Zoologica Sinica

2 6 : cdna Yang et al. (2002) T 1 A TGGTGCT2 GA TCA GA GTGCTG; T 2 TTACGGG DNA PCR p GEM2T DNA D EA E2Sp harose FF Heparin2 Sepharose CL26B Pharmacia DNA (DEPC) Sigma TaqDNA DNA cdna ; 211 mrna p GEM2T Promega Biotools g 100 ml 0105 mol/ L Tris2HCl ( p H 714 ) DEAE2Sepharose FF 015 mol/ L NaCl NaCl ; 50 g ml 0105 mol/ L TrisHCl (p H 714) g 10 min dal dal 500 ml ; DEAE2 Sepharose FF 012 ; Heparin2Sepharose CL26B 0115 mol/ L NaCl ( 9701 ) RNA 1 ( 2) Chomezynski et al. (1987) RNA GGGCAA GTCAC PCR min 55 1 min min min PCR Sambrook et al. 2 (1989) 13 ( 1) mol/ L NaCl 1 DEAE2Sepharose FF Fig11 The crude venom was applied mol/ L NaCl to a column ( cm) A : 0105 mol/ L Tris2HCl (A : 0105 mol/ L Tris2 HCl was used as balance eluent) B : mol/ L NaCl (B : gradient of mol/ L NaCl was used as eluent. Peak posesses throm2 bin2like enzyme activity) ( 3) 600 ml mrna cdna SDS2

3 DEAE2Sepharose Fig12 The crude venom was applied to DEAE2Sepharose A : 0105 mol/ L Tris2HCl (A : 0105 mol/ L Tris2 HCl was used as balance eluent) B : 012 mol/ L NaCl [B : 012 mol/ L NaCl was used as elu2 ent. The active thrombin2like enzyme peak (second peak) was col2 lected] 3 Fig13 The collected peak from DEAE2Sepharose column was applied to aff inity A : 0105 mol/ L Tris2HCl (A : 0105 mol/ L Tris2 HCl was used as balance eluent) B : 0115 mol/ LNaCl [B : 0115 mol/ L NaCl was used as eluent. The active thrombin2like enzyme peak (second peak) was collected] 32 kd ; 215 RNA PCR : Cys 31 2Cys 163 RNA RNA 18 s 28 srna PCR Cys 189 Cys 200 2Cys bp p GEM2T S/ T2X2N ( ser/ t hr2x2axn) S 68 2T 69 2N 70 T 89 2R 90 2N T 1 T 2 32 kd 4 20 % 30 % 774 bp 258 GeneBank : A Y NCB I % 917 % : ( T % 4014 % gramineus) 94 % ( D. acut us) 93 % ( C. at rox ) 93 % ( Gloydius halys) 90 % NCB I Biotool ( T. gramineus) 88 % ( C. a2 t rox) 88 % ( D. acut us) 86 % ; Cys 6 Cys 50 2Cys 66 Cys 98 2Cys 256 Cys 142 2Cys 210 Cys kd SDS Biotool His 65 Asp 110 Ser kd

4 6 : cdna Fig14 Complete open reading frame of cdna and the deduced amino acid sequence of thrombin2like enzyme of Gloydius saxatilis (Amino acids 1-18 are a signal peptide are a propeptide are a thrombin2like domain amino acids are thrombin2like domains. indicates glycosylation position 3 is the active position) S/ T2X2N S 68 2T 69 2N 70 T 89 2R % 12 N 91 ( His 65 Asp 110 Ser 204 ) cdna Asp

5 Fig15 Comparision of thrombin2like enzyme from Gloydius saxatilis with serine proteinase from other species Q : ( G. saxatilis thrombin2like enzyme) T : 88 % ( T. grami neus serine proteas. Identities = 226/ 258 = 88 %) D : 86 % ( D. acut us thrombin2like defibrase. Identities = 223/ 258 = 86 %) C : 88 % ( C. at rox serine protease catroxase. Identities = 227/ 258 = 88 %) ( References) Chomezynski P. and N. Sacchi 1987 Single2step method of RNA isolation by acid guanidinium thiocyanate2phenol2chloroform extrac2 Hahn B. tion. A nal. Biochem. 162 : S. K. Baek W. S. Kim C. S. Lee I. L. Chang and Y. S. Kim 1996 Purification and molecular cloning of calobin a thrombin2like enzyme from A gkist rodon caliginosus ( Korean viper). J. Biochem. ( Tokyo). 119 : Huang C. C. K. F. Huang and S. H. Chiou 1994 Characteri2 zation of one novel venom protease with beta2fibrinogenase activity from the Taiwan habu ( Tri meresurus m ucrosquamat us) : purifica2 tion and cdna sequence analysis. Bioche. Biophys. Res. Com2 m un. 205 : Itoh N. N. Tanaka S. Mihashi and I. Yamashina 1987 Molecu2 lar cloning and sequence analysis of cdna for Batroxobin a throm2 bin2like snake venom enzyme. J. Biol. Chem. 262 : Latallo Z. S Retrospective study on complications and ad2 verse effects of treatment with thrombin2like enzymes : a multicen2 tre trial. Thrombosis and Haemostasis 50 : Pan H. X. Du G. Yang Y. Zhou and X. Wu 1999 cdna cloning and expression of acutin. Biochem. Biophys. Res. Com2 m un. 255 : Pirkle H. I. Theodor and R. Lopez 1989 Catroxobin a weakly thrombin2like enzyme from the venom of Crotal us at rax NH 2 2ter2 minal and active site amino acid sequences. Tromb. Res. 56 : Shieh T. C. S. Kawabata H. Kihara M. Ohno and S. Iwanaga 1988 Amino acid sequence of a coagulant enzyme flavoxobin from Tri meresurus f lavovirids venom. J. Biochem ( Tokyo ). 103 : Yang Q. X. J. Hu X. M. Xu L. J. An X. D. Yuan and Z. G. Su 2002 Cloning expression and purification of gussurobin a thrombin2like enzyme from the snake venom of Gloydius ussurien2 sis. Acta. Biochem. Bophys. Si n. 34 : 6 10.