A comparative study on the activity and antigenicity of truncated and full-length forms of streptokinase

• Autorzy: Arabi R. , Roohvand F. , Norouzian D. , Sardari S. , Aghasadeghi M. R. , Khanahmad H. , Memarnejadian A. , Motevalli F.
• Języki publikacji: EN

Abstrakty

EN

Application of streptokinase (SK) as a common and cost-effective thrombolytic drug is limited by its antigenicity and undesired hemorrhagic effects. Prior structural/functional and epitope-mapping studies on SK suggested that removal of 59 N-terminal residues led to its fibrin dependency and identified SK antigenic regions, respectively. Following in silico analyses two truncated SK proteins were designed and compared for their fibrin specificity and antigenicity with the full-length SK. Computer-based modeling was used to predict the effect of vector (pET41a)-born protein tags on the conformation of SK fragments. SK60-386, SK143-386 and full-length SK (1–414) were separately cloned, expressed in BL21 E. coli cells and confirmed by Western-blotting. Functional activity of the purified proteins was evaluated with chromogenic and clot lysis assays and their antigenicity was tested by ELISA assay using rabbit anti-streptokinase antibody. As expected, chromogenic bioassay showed a major activity decline for SK60-386 and SK143-386 (83 and 91 percent, respectively), compared to SK1-414. However, in clot lysis assay, which is a fibrin-dependent pharmacopoeia-approved test, SK60-386 and SK143-386 were respectively 35 and 31 percent more active though lysed the clots slower than full-length SK. Antigenic analysis also indicated significant decrease in ELISA signals obtained for truncated proteins compared to SK1-414 (45 and 28 percent less reactivity for SK143-386 and SK60-386, respectively, p < 0.0001). The results of this study for the first time pointed to SK143-386 and SK60-386, as improved SK derivatives with increased fibrin-selectivity and decreased antigenicity as well as acceptable bioactivity profiles in a pharmacopoeia-based analysis, which deserve more detailed pharmacological studies.

Słowa kluczowe

EN

antigenicity; truncated form; full-length form; streptokinase; fibrin specificity; protein modelling; thrombolytic drug; immunoblotting; pharmacology

• Wydawca: -
• Czasopismo: Polish Journal of Microbiology
• Rocznik: 2011
• Tom: 60
• Numer: 3
• Opis fizyczny: p.243-251,fig.,ref.

Twórcy

autor

Arabi R.

• Hepatitis and AIDS Department and NRGB, Pasteur Institute of Iran, Pasteur Ave., Tehran 1316943551, Iran

autor

Roohvand F.

autor

Norouzian D.

autor

Sardari S.

autor

Aghasadeghi M. R.

autor

Khanahmad H.

autor

Memarnejadian A.

autor

Motevalli F.

Bibliografia

• Banerjee A., C. Chisti and U.C. Banerjee. 2004. Streptokinase – a clinically useful thrombolytic agent. Biotechnol. Adv. 22: 287–307.
• Baruah D.B., R.N. Dash, M.R. Chaundhari and S.S. Kadam. 2004. Plasminogen Activators: A comparison. Vascul. Pharmacol. 44: 1–9.
• Coffey J.A., K.R. Jennings and H. Dalton. 2001. New antigenic regions of streptokinase are identified by affinity directed mass spectrometry. Eur. J. Biochem. 268: 5215–5221.
• Conjero-Lara F., J. Parrado, A.I. Azuaga, C.M. Dobson and P.C. Paula. 1998. Analysis of the interactions between streptokinase domains and human plasminogen. Protein Sci. 7: 2190–2199.
• Couto L., J.L. Donato and G. Nucci. 2004. Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay. Brazil J. Med. Biol. Res. 37: 1889–1894.
• Estrada M., L. Hernández, A Pérez, P. Rodríguez, R. Serrano, R. Rubiera, A.Pedraza, G. Padrón, W. Antuch, J. de la Fuente and L. Herrera. 1992. High level expression of streptokinase in Escherichia coli. Biotechnol. Adv. 10: 1138–1142.
• Houba V. and I. Hana. 1966. The difference in immunological characteristics of two streptokinases. Immunol. 11: 387–397.
• Lovell S.C., I.W. Davis, W.B. Arendall, P.I.W. de Bakker, J.M. Word, M.G. Prisant, J.S. Richardson and D.C. Richardson. 2002. Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins: Struct. Funct. and Genet. 50: 437–450.
• Mundada L.V., M. Prorok, M.E. DeFord, M. Figuera, F.J. Castellino and W.P. Fay. 2003. Structure-function analysis of the streptokinase amino terminus (residues 1–59). J. Biol. Chem. 278: 24421–24427.
• Nihalani D., R. Kumar, K. Rajagopal and G. Shan. 1998. Role of the amino-terminal region of streptokinase in the generation of a fully functional plasminogen activator complex probed with synthetic peptides. Protein Sci. 7: 637–648.
• Parhami-Seren B., K. Keel and G.L. Reed. 1997. Sequences of antigenic epitopes of streptokinase identified via random peptide libraries displayed on phage. J. Mol. Biol. 271: 333–341.
• Parhami-Serena B., M. Lynch, H.D. Whites and G.L. Reed. 1995. Mapping the antigenic regions of streptokinase in humans before and after streptokinase therapy. Mol. Immunol. 32: 111–724.
• Parhami-Serena B., M. Seaveya, J. Krudysza and P. Tsantili. 2003. Structural correlates of a functional streptokinase antigenic epitope: serine 138 is an essential residue for antibody binding. J. Immunol. Methods. 272: 93–105.
• Reed G., P. Kussie and B. Parhami-Seren. 1993. A functional analysis of the antigenicity of streptokinase using monoclonal antibody mapping and recombinant streptokinase fragments.The J. Immunol. 150: 4407–4415.
• Reed G., B. Parhami-Seren, S. Wang and L. Hedstrom. 1999. A catalytic switch and the conversion of streptokinase to a fibrin targeted plasminogen activator. PANAS 96: 8879–8883.
• Rodriguez P., P. Fuentes, M. Barro, J.G. Alvarez, M. Mwozz, D.C. Collen and H.R. Lunen. 1995. Structural domains of streptokinase involved in the interaction with plasminogen. Eur. J. Biochem. 229: 83–90.
• Saksella O. 1981. Radial caseinolylis in agarose: a simple method for detection of plasminogen activator in the presence of inhibitory substances and serum. Anal. Biochem. 111: 276–282.
• Sazoana I.Y., R.A. Mcanmee, A.K. Houng, S.M. King, L. Hedstorm and G.L. Reed. 2009. Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator. J. Thromb. and Haemostasis 7: 1321–1328.
• Sazonova I.Y., B.R. Robinson, I.P. Gladysheva, F.J. Castellino and G.L. Reed. 2004. Alpha domain deletion converts streptokinase into a fibrin-dependent plasminogen activator through mechanisms akin to staphylokinase and tissue plasminogen activator. J. Biol. Chem. 279: 24994–5001.
• Shi G., B. Chang, S. Chen, D. Wu and H. Wu. 1994. Function of streptokinase fragments in plasminogen activation. Biochem. J. 304: 235–241.
• Torrèns I., A.G. Ojalvo, A. Seralena, O. Hayes and J. Fuente. 1999. A mutant streptokinase lacking the C-terminal 42 amino acids is less immunogenic. Immunol. Lett. 70: 213–218.
• Wang X., X. L i n, J.A. Lay, J. Tang and X.C. Zhang. 1998. Crystal structure of the catalytic domain of human plasmin complexed with streptokinase. Science 281: 1662–1665.