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208 Targeting of the mouse protein C inhibitor gene
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POSTER PRESENTATIONS: Interrelation between coagulation and flbrinolysis
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207 Purification and molecular cloning of salmobin, a thrombin-like enzyme from korean salmosa snake (Agkistrodon halys) *CHUNG KH, **KOH YS, ***YUN YD, *JANG YS, *CHOSH, and **KIM DS *Cardiovascular Research Institute, College of Medicine, **Department of Biochemistry and Bioproducts Research Center, Yonsei University, Seoul 120- 752 and ***Mogam Biotechnology Research Institute, 341 Pojung-ri, Koosung-myon, Yongin 449-910, Korea Salmobin, a thrombin-like proteinase has been identified and purified to homogeneity from the venom of Agkistrodon halys (Korean Salmosa snake). It is a single chain glycoprotein with an apparent molecular weight of 43,000 on SDSPAGE and 38,320 by MALDI-mass spectrometry, and an isoelectric point of pH7.6. Salmobin acted on fibrinogen to form fibrin with a specific acyivity of 267 NIH units/mg. It predominantly cleaved the A? chain of fibrinogen. The activity of salmobin was readily inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, antipain and 2-mercaptoethanol, indicating it is a serine protease and disulfide bonds will be critical for maintaining its activity. However, the enzyme was not inhibited by aprotinin, himdin, soybean trypsin inhibitor, a2-antiplasmin and antithrombin Ill. cDNA library was prepared from the venom glands of A. halys and was screened with oligonucleotide
probes designed on the basis of the N-terminal and the internal peptide sequences of salmobin. The deduced complete amino acid sequence of salmobin indicates that the mature salmobin protein is synthesized as a pre-zymogen of 260 amino acids, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acid residues. The cDNA sequence indicates that the mature salmobin protein is composed of 236 amino acids and contains two potential N-glycosylation site, Asn-X-Ser/Thr at Ash99 and Ash 132.The sequence of salmobin exhibits a high degree of sequence identity with other snake venom proteases: 78% with gyroxin, 75% with calobin, 67% with ancrod and flaboxobin, 65% with batroxobin, and 62% with RVV-V. Like other thrombin-like enzymes, salmobin contains 12 cysteine residues and it could be predicted that they are all involved in the formation of six disulfide bonds. The homology of salmobin with venom thrombin-like enzymes indicated that the amino acid residues which form the catalytic triad are His43, AspsS, and Ser t82.
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208 Targeting of the mouse protein C inhibitor gene *UHRIN P, *HILPERTM, **DEWERCHINM, **CARMELIETP, *ZECHME1STER-MACHHARTM, **COLLEND, *GEIGER M, and *BINDER BR *Department of Vascular Biology and Thrombosis Research, University of Vienna, Vienna, Austria, ** Flanders lnteruniversity Institutefor Biotechnology, Centerfor Transgene Technology & Gene Therapy, Leuven, Belgium Protein C inhibitor (PCI) is a serine protease inhibitor that inhibits a variety of plasmatic and extravascular serine proteases by forming enzymatically inactive stable 1:1 complexes. Serine proteases inhibited by PCI include the anticoagulatory enzyme activated protein C, clotting factors Xa, Xla,-plasma kallikrein and thrombin, the plasminogen activators urokinase and tissue plasminogen activtor, as well as the trypsin-like, extravascular proteases tissue kalikrein, prostate specific antigen and acrosin. So far, however, the physiological role of PCI has not been elucidated completely. Therefore we aim to create transgenic mice with null PCI alleles and to study the resulting phenotype and physiological consequences of PCI targeting.
A targeting vector, pPNT.PCI consisting of 9.2 kb homologous sequence of mouse PCI gene, a positive selection marker (neomycine resistance gene) and a negative selection marker (thymidine kinase) gene was prepared. Electroporation of RI embryonic stem (ES) cells with the linearized pPNT.PCI yielded five targeted clones with correct homologous recombination at the 5'- and 3'-ends, as confirmed by Southern blot analysis of purified genomic DNA with appropriate restriction enzymes and probes. These targeted clones are currently being used to generate PCI deficient mice. The animals will be tested with respect to the tissue damage, hemostatic sysem, kallikrein - kinin system and the reproduction system, as well. Because of the anticipated important role of PCI in the inhibition of the activities of serine proteases, investigation of mice with null PCI alleles should further elucidate the role of this protein.
POSTER PRESENTATIONS: Interrelation between coagulation and flbrinolysis
m
207 Purification and molecular cloning of salmobin, a thrombin-like enzyme from korean salmosa snake (Agkistrodon halys) *CHUNG KH, **KOH YS, ***YUN YD, *JANG YS, *CHOSH, and **KIM DS *Cardiovascular Research Institute, College of Medicine, **Department of Biochemistry and Bioproducts Research Center, Yonsei University, Seoul 120- 752 and ***Mogam Biotechnology Research Institute, 341 Pojung-ri, Koosung-myon, Yongin 449-910, Korea Salmobin, a thrombin-like proteinase has been identified and purified to homogeneity from the venom of Agkistrodon halys (Korean Salmosa snake). It is a single chain glycoprotein with an apparent molecular weight of 43,000 on SDSPAGE and 38,320 by MALDI-mass spectrometry, and an isoelectric point of pH7.6. Salmobin acted on fibrinogen to form fibrin with a specific acyivity of 267 NIH units/mg. It predominantly cleaved the A? chain of fibrinogen. The activity of salmobin was readily inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, antipain and 2-mercaptoethanol, indicating it is a serine protease and disulfide bonds will be critical for maintaining its activity. However, the enzyme was not inhibited by aprotinin, himdin, soybean trypsin inhibitor, a2-antiplasmin and antithrombin Ill. cDNA library was prepared from the venom glands of A. halys and was screened with oligonucleotide
probes designed on the basis of the N-terminal and the internal peptide sequences of salmobin. The deduced complete amino acid sequence of salmobin indicates that the mature salmobin protein is synthesized as a pre-zymogen of 260 amino acids, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acid residues. The cDNA sequence indicates that the mature salmobin protein is composed of 236 amino acids and contains two potential N-glycosylation site, Asn-X-Ser/Thr at Ash99 and Ash 132.The sequence of salmobin exhibits a high degree of sequence identity with other snake venom proteases: 78% with gyroxin, 75% with calobin, 67% with ancrod and flaboxobin, 65% with batroxobin, and 62% with RVV-V. Like other thrombin-like enzymes, salmobin contains 12 cysteine residues and it could be predicted that they are all involved in the formation of six disulfide bonds. The homology of salmobin with venom thrombin-like enzymes indicated that the amino acid residues which form the catalytic triad are His43, AspsS, and Ser t82.
m
208 Targeting of the mouse protein C inhibitor gene *UHRIN P, *HILPERTM, **DEWERCHINM, **CARMELIETP, *ZECHME1STER-MACHHARTM, **COLLEND, *GEIGER M, and *BINDER BR *Department of Vascular Biology and Thrombosis Research, University of Vienna, Vienna, Austria, ** Flanders lnteruniversity Institutefor Biotechnology, Centerfor Transgene Technology & Gene Therapy, Leuven, Belgium Protein C inhibitor (PCI) is a serine protease inhibitor that inhibits a variety of plasmatic and extravascular serine proteases by forming enzymatically inactive stable 1:1 complexes. Serine proteases inhibited by PCI include the anticoagulatory enzyme activated protein C, clotting factors Xa, Xla,-plasma kallikrein and thrombin, the plasminogen activators urokinase and tissue plasminogen activtor, as well as the trypsin-like, extravascular proteases tissue kalikrein, prostate specific antigen and acrosin. So far, however, the physiological role of PCI has not been elucidated completely. Therefore we aim to create transgenic mice with null PCI alleles and to study the resulting phenotype and physiological consequences of PCI targeting.
A targeting vector, pPNT.PCI consisting of 9.2 kb homologous sequence of mouse PCI gene, a positive selection marker (neomycine resistance gene) and a negative selection marker (thymidine kinase) gene was prepared. Electroporation of RI embryonic stem (ES) cells with the linearized pPNT.PCI yielded five targeted clones with correct homologous recombination at the 5'- and 3'-ends, as confirmed by Southern blot analysis of purified genomic DNA with appropriate restriction enzymes and probes. These targeted clones are currently being used to generate PCI deficient mice. The animals will be tested with respect to the tissue damage, hemostatic sysem, kallikrein - kinin system and the reproduction system, as well. Because of the anticipated important role of PCI in the inhibition of the activities of serine proteases, investigation of mice with null PCI alleles should further elucidate the role of this protein.