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Phosphorylation of plasminogen activators
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86
POSTER PRESENTATIONS P-l : Plasminogen and Plasminogen Activators
PHOSPHORYLATION OF PLASMINOGEN ACTIVATORS. Barlati S., Bona C. and De Petro G. Div. of Biology and Genetics, Dept. Sciences and of Biomedical University of Biotechnologies, Brescia, Italy. The main aim of this study was to verify the presence of P-Tyr, P-Ser and P-Thr residues in t-PA, u-PA and PG. The data obtained show that human t-PA (purified from human melanoma cells) and human plasminogen (purified from contain P-Tyr residues human plasma) recognition (by immunological whose monoclonal anti-P-Tyr antibodies) was specifically inhibited by the addition of 20 mM P-Tyr, but not by 20 mM P-Ser HPLC Moreover the or P-Thr. analysis of acid hydrolysed u-PA, t-PA
87 CONSTRUCTION AND FUNCTIONAL ANALYSIS OF STAPHYLOKINASE VARIANTS. Ueshima S, Okada K, Hagiya Y, Matsumoto H, Yuasa H, Nonaka T, Fukao H and Matsuo 0 Dept. of Physiology, Kinki University School of Medicine, Osaka, Japan. Staphylokinase (SAK), produced by Staphylococcus aureus, is a profibrinolytic protein with molecular weight 15,500 consisting of 136 amino acids. SAK expressed plasminogen activator activity by forming the complex with plasminogen. In order to produce the SAK variant deleted the Fist 2.5 amino acid residues ( A25SAK), the DNA fragment which encoded the amino acid sequence of Met26-Lys136 of SAK was synthesized and inserted into the pMALc2 expression vector. Although AZSSAK was expressed in E. CM as the fusion protein with maltose binding protein (MBP), MBP was removed by Xa-Sepharose and A25SAK was purified by anti-SAK IgG-Sepharose. NH2-terminal amino acid
88
NEW ASPECTS OF THE INTERACTION BETWEEN STAPHYLOKINASE AND PLASMINOGEN K.-H. Guehrs, E. Birch-Hirschfeld, A. Gase, M. Hartmann and B. Schlott Institute of Molecular Biotechnology Dept. of Biochemistry PF 100813 D-07708 Jena Germany Staphylokinase (Sak) is a plasminogen activator originally expressed by different strains of Staphylococcus aureus. The protein activates plasminogen indirectly via formation of an active staphylokinaselplasminogen complex. Plasminogen activation by Sak under plasma conditions is only possible in presence of fibrin because a2-antiplasmin which efficiently inhibits the staphylokinaselplasmin complex is ,lot active in a fibrin-rich environment (1). Binding of Sak onto plasmin(ogen) occurs at the protease domain of the latter apparently without any contribution of the kringle domains (2). Several parts of the Sak molecule play an essential role in complex formation with plasmin(ogen) or in the exposition of the
and PG and the immunoblotting with anti-P-Ser or anti-P-Thr antibodies showed that: 1) P-Ser residues are u-PA present in in and t-PA; 2) P-Thr in U-PA and PG; 3) P-Tyr in u-PA, t-PA and PG. These data show that, in addition to u-PA (FEBS, 266: 109-114, 1990; FEBS, 281: 137140, 1991), also t-PA and PG are phosphorylated and contain may P-Tyr, P-Ser and/or P-Thr residues. Work supported by C.N.R. Target Projects, ACRO, Biotechnology and Bioinstrumentation, Genetic EngineerAIRC and by MURST. ing, by
sequence of purified protein revealed a sequence Met-ValAsn-Val-Thr-, corresponding to the A 25SAK sequencee. SAK variant deleted the first 26 amino acids residues( A 26SAK) was generated by treatment of recombinant mature SAK with CNBr which cleaved the Met26-Va127 peptide bond in SAK. Hydrolysis of S-2251 by the low concentration of mature SAK-plasminogen was observed with the initial lag phase. At the high concentration of A 26SAK, hydrolysis of S-2251 was occured rapidly. Conversion of plasminogen to two-chain plasmin by both A 25SAK and A 26SAK, as revealed by reduced SDS-PAGE, was less than that by mature SAK. Although the mature SAK showed the lysis band on enzymography, both A25SAK and A26SAK did not show the clear lysis bands. These results suggest that the NH2- terminal region of SAK may play an important role to express plasminogen activator activity.
active site. The cleavage of the N-terminal 10 amino acids of mature staphylokinase which occurs simultaneously with active site formation is a prerequisite for the formation of a complex efficient in plasminogen activation. The region around methionine at position 26 is critical for functional activity of staphylokinase (3). Maintenance of activity is only possible by substitution of this residue by leucine or cysteine whereas introduction of a alanine moiety at this position results in a completely inactive protein. Interestingly, the exchange of methionine against several other amino acids yields proteins inducible by traces of plasmin with increased reactivity towards Lys-plasminogen in comparison to Glu-plasminogen. This behaviour support the suggestion that the region around methionine 26 takes place in a specific hydrophobic interaction. References: (1) H.R. Lijnen et al., J. Biol. Chem., 266 (lQQl), 11826-l 1832 (2) H. R. Lijnen et al. Eur. J. Biochem., 211 (1993), 91-97 (3) B. Schlott et al., Biochim. Biophys. Acta (1994) in press
86
POSTER PRESENTATIONS P-l : Plasminogen and Plasminogen Activators
PHOSPHORYLATION OF PLASMINOGEN ACTIVATORS. Barlati S., Bona C. and De Petro G. Div. of Biology and Genetics, Dept. Sciences and of Biomedical University of Biotechnologies, Brescia, Italy. The main aim of this study was to verify the presence of P-Tyr, P-Ser and P-Thr residues in t-PA, u-PA and PG. The data obtained show that human t-PA (purified from human melanoma cells) and human plasminogen (purified from contain P-Tyr residues human plasma) recognition (by immunological whose monoclonal anti-P-Tyr antibodies) was specifically inhibited by the addition of 20 mM P-Tyr, but not by 20 mM P-Ser HPLC Moreover the or P-Thr. analysis of acid hydrolysed u-PA, t-PA
87 CONSTRUCTION AND FUNCTIONAL ANALYSIS OF STAPHYLOKINASE VARIANTS. Ueshima S, Okada K, Hagiya Y, Matsumoto H, Yuasa H, Nonaka T, Fukao H and Matsuo 0 Dept. of Physiology, Kinki University School of Medicine, Osaka, Japan. Staphylokinase (SAK), produced by Staphylococcus aureus, is a profibrinolytic protein with molecular weight 15,500 consisting of 136 amino acids. SAK expressed plasminogen activator activity by forming the complex with plasminogen. In order to produce the SAK variant deleted the Fist 2.5 amino acid residues ( A25SAK), the DNA fragment which encoded the amino acid sequence of Met26-Lys136 of SAK was synthesized and inserted into the pMALc2 expression vector. Although AZSSAK was expressed in E. CM as the fusion protein with maltose binding protein (MBP), MBP was removed by Xa-Sepharose and A25SAK was purified by anti-SAK IgG-Sepharose. NH2-terminal amino acid
88
NEW ASPECTS OF THE INTERACTION BETWEEN STAPHYLOKINASE AND PLASMINOGEN K.-H. Guehrs, E. Birch-Hirschfeld, A. Gase, M. Hartmann and B. Schlott Institute of Molecular Biotechnology Dept. of Biochemistry PF 100813 D-07708 Jena Germany Staphylokinase (Sak) is a plasminogen activator originally expressed by different strains of Staphylococcus aureus. The protein activates plasminogen indirectly via formation of an active staphylokinaselplasminogen complex. Plasminogen activation by Sak under plasma conditions is only possible in presence of fibrin because a2-antiplasmin which efficiently inhibits the staphylokinaselplasmin complex is ,lot active in a fibrin-rich environment (1). Binding of Sak onto plasmin(ogen) occurs at the protease domain of the latter apparently without any contribution of the kringle domains (2). Several parts of the Sak molecule play an essential role in complex formation with plasmin(ogen) or in the exposition of the
and PG and the immunoblotting with anti-P-Ser or anti-P-Thr antibodies showed that: 1) P-Ser residues are u-PA present in in and t-PA; 2) P-Thr in U-PA and PG; 3) P-Tyr in u-PA, t-PA and PG. These data show that, in addition to u-PA (FEBS, 266: 109-114, 1990; FEBS, 281: 137140, 1991), also t-PA and PG are phosphorylated and contain may P-Tyr, P-Ser and/or P-Thr residues. Work supported by C.N.R. Target Projects, ACRO, Biotechnology and Bioinstrumentation, Genetic EngineerAIRC and by MURST. ing, by
sequence of purified protein revealed a sequence Met-ValAsn-Val-Thr-, corresponding to the A 25SAK sequencee. SAK variant deleted the first 26 amino acids residues( A 26SAK) was generated by treatment of recombinant mature SAK with CNBr which cleaved the Met26-Va127 peptide bond in SAK. Hydrolysis of S-2251 by the low concentration of mature SAK-plasminogen was observed with the initial lag phase. At the high concentration of A 26SAK, hydrolysis of S-2251 was occured rapidly. Conversion of plasminogen to two-chain plasmin by both A 25SAK and A 26SAK, as revealed by reduced SDS-PAGE, was less than that by mature SAK. Although the mature SAK showed the lysis band on enzymography, both A25SAK and A26SAK did not show the clear lysis bands. These results suggest that the NH2- terminal region of SAK may play an important role to express plasminogen activator activity.
active site. The cleavage of the N-terminal 10 amino acids of mature staphylokinase which occurs simultaneously with active site formation is a prerequisite for the formation of a complex efficient in plasminogen activation. The region around methionine at position 26 is critical for functional activity of staphylokinase (3). Maintenance of activity is only possible by substitution of this residue by leucine or cysteine whereas introduction of a alanine moiety at this position results in a completely inactive protein. Interestingly, the exchange of methionine against several other amino acids yields proteins inducible by traces of plasmin with increased reactivity towards Lys-plasminogen in comparison to Glu-plasminogen. This behaviour support the suggestion that the region around methionine 26 takes place in a specific hydrophobic interaction. References: (1) H.R. Lijnen et al., J. Biol. Chem., 266 (lQQl), 11826-l 1832 (2) H. R. Lijnen et al. Eur. J. Biochem., 211 (1993), 91-97 (3) B. Schlott et al., Biochim. Biophys. Acta (1994) in press