Functional Analysis of the Amino- and Carboxyl-Termini of Streptokinase

Summary

Streptokinase (SK) is a 414 amino acid bacterial protein that activates human plasminogen. Streptokinase fragments derived from the central portion of the protein bind plasminogen, but are inactive, indicating that the amino- and/or carboxyl-termini are required for normal plasminogen activator activity. To better define the function of the N- and C-termini of SK we generated and characterized 21 N-terminal and 20 C-terminal deletion mutants. All mutants lacking ≥18 N-terminal or ≥51 C-terminal amino acids exhibited markedly reduced plasminogen activator activity, while mutants lacking ≤12 N-terminal or ≤40 C-terminal residues were fully active. The decrease in SK activity with N-terminal deletion appeared to result not from loss of plasminogen binding capacity, but rather from increased susceptibility of deletion mutants to degradation by plasmin. Point mutations at positions 13 (SK V13D) or 20 (SK V20D) produced functional abnormalities similar to those observed in N-terminal deletion mutants, with SK V13D exhibiting delayed amidolytic activity and SK V20D exhibiting only 1% plasminogen activator activity and marked sensitivity to degradation by plasmin. C-terminal deletion mutants lacking ≥51 amino acids also bound plasminogen, but did not induce significant amidolytic activity in plasminogen or activator activity in plasmin. Prevention of cleavage at position 59 of SK had no effect on plasminogen activator activity, suggesting that the rapid hydrolysis of this bond that occurs after SK-plasminogen complex formation is not required for normal function of the N-terminus. These results suggest that residues within or near positions 13-20 of SK are important determinants of its capacity to generate amidolytic activity and are a critical determinant of the stability of SK, while residues within or near position 364-374 are required for generating amidolytic activity and for conferring plasminogen activator activity to plasmin(ogen). These results also suggest that SK fragments significantly smaller than SK 13-374 are unlikely to be effective thrombolytic agents.

• References

• 1 Reddy KN. Streptokinase – biochemistry and clinical applications. Enzyme 1988; 40: 79-89.
• 2 Bajaj AP, Castellino FJ. Activation of human plasminogen by equimolar levels of streptokinase. J Biol Chem 1977; 252: 492-8.
• 3 Teuten AJ, Broadhurst RW, Smith RA, Dobson CM. Characterization of structural and folding properties of streptokinase by n.m.r. spectroscopy. Biochem J 1993; 290: 313-9.
• 4 Radek JT, Castellino FJ. Conformational properties of streptokinase. J Biol Chem 1989; 264: 9915-22.
• 5 Parrado J, Conejero-Lara F, Smith RAG, Marshall JM, Ponting CP, Dobson CM. The domain organization of streptokinase: Nuclear magnetic resonance, circular dichroism, and functional characterization of proteolytic fragments. Protein Sci 1996; 5: 693-704.
• 6 Rodriguez P, Fuentes P, Barro M, Alvarez JG, Munoz E, Collen D, Lijnen HR. Structural domains of streptokinase involved in the interaction with plasminogen. Eur J Biochem 1995; 229: 83-90.
• 7 Reed GL, Lin LF, Parhami-Seren B, Kussie P. Identification of a plasminogen binding region in streptokinase that is necessary for the creation of a functional streptokinase-plasminogen activator complex. Biochemistry 1995; 34: 10266-71.
• 8 Siefring GE, Castellino FJ. Interaction of streptokinase with plasminogen. J Biol Chem 1976; 251: 3913-20.
• 9 Shi GY, Chang BI, Chen SM, Wu DH, Wu HL. Function of streptokinase fragments in plasminogen activation. Biochem J 1994; 304: 235-41.
• 10 McClintock DK, Englert ME, Dziobkowski C, Snedeker EH, Bell PH. Two distinct pathways of the streptokinase-mediated activation of highly purified human plasminogen. Biochemistry 1974; 13: 5334-44.
• 11 Brockway WJ, Castellino FJ. A characterization of native streptokinase and altered streptokinase isolated from a human plasminogen activator complex. Biochem 1974; 13: 2063-70.
• 12 Young KC, Shi GY, Chang UF, Chang BI, Chang LC, Lai MD, Chuang WJ, Wu HL. Interaction of streptokinase and plasminogen: studies with truncated streptokinase peptides. J Biol Chem 1995; 270: 29601-6.
• 13 Deutsch D, Mertz E. Plasminogen: Purification from human plasma by affinity chromatography. Science 1970; 170: 1095-6.
• 14 Harlow E, and Lane D. Antibodies. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1988
• 15 Sherman PM, Lawrence DA, Yang AY, Vandenberg ET, Paielli D, Olson ST, Shore JD, Ginsburg D. Saturation mutagenesis of the plasminogen activator inhibitor-1 reactive center. J Biol Chem 1992; 267: 7588-95.
• 16 Estrada MP, Hernandez L, Perez A, Rodriguez P, Serrano R, Rubiera R, Pedraza A, Padron G, Antuch W, de la Fuente J, Herrera L. High level expression of streptokinase in Escherichia coli. BioTechnology 1992; 10: 1138-42.
• 17 Henikoff S. Ordered deletions by DNA sequencing and in vitro mutagenesis by polymerase extension and exonuclease III gapping of circular templates. Nucleic Acids Res 1990; 18: 2961-6.
• 18 Kim IC, Kim JS, Lee SH, Byun SM. C-terminal peptide of streptokinase, Met369-Pro373, is important in plasminogen activation. Biochem Mol Biol Int 1996; 40: 939-95.
• 19 Jackson KW, Tang J. The amino-terminal sequence of streptokinase and its functional implications in plasminogen activation. Thromb Res 1978; 13: 693-9.
• 20 Rodriguez P, Fuentes D, Munoz E, Rivero D, Orta D, Alburquerque S, Perez S, Besada V, Herrera L. The streptokinase domain responsible for plasminogen binding. Fibrinolysis 1994; 8: 276-85.
• 21 Lee BR, Park SK, Kim JH, Byun SM. Site-specific alterations of Gly-24 in streptokinase: Its effect on plasminogen activation. Biochem Biophys Res Commun 1989; 165: 1085-90.
• 22 Marder VJ. Recombinant streptokinase: opportunity for an improved agent. Blood Coagul Fibrinolysis 1993; 4: 1039-40.
• 23 Brugemann J, van der Meer J, Bom VJ, van der Schaaf W, de Graeff PA, Lie KI. Antistreptokinase antibodies inhibit fibrinolytic effects of anistreplase in acute myocardial infarction. Am J Cardiol 1993; 72: 462-4.
• 24 Lew AS, Neer T, Rodriguez L, Geft IL, Shah PK, Ganz W. Clinical failure of streptokinase due to an unsuspected high titer of antistreptokinase antibody. J Am Coll Cardiol 1984; 4: 183-185.
• 25 Hoffmann JJML, Fears R, Bonnier JJRM, Standring R, Ferres H, De Swart JBRM. Significance of antibodies to streptokinase in coronary thrombolytic therapy with streptokinase or APSAC. Fibrinolysis 1988; 2: 203-210.
• 26 Parhami-Seren B, Lynch M, White HD, Reed GL. Mapping the antigenic regions of streptokinase in humans before and after streptokinase therapy. Mol Immunol 1995; 32: 717-24.
• 27 Reed GL, Kussie P, Parhami-Seren B. A functional analysis of the antigenicity of streptokinase using monoclonal antibody mapping and recombinant streptokinase fragments. J Immunol 1993; 150: 4407-15.