تولید و بررسی ویژگیهای آنزیم نوترکیب ریبونوکلئاز پانکراس گوسفند

نوع مقاله : علمی پژوهشی- ژنتیک و اصلاح دام و طیور

نویسندگان

1 گروه علوم دامی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران.

2 گروه علوم دامی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران

3 گروه علوم دامی، دانشکده کشاورزی، دانشگاه گیلان، گیلان، ایران

چکیده

همان‌طور‌که تحقیقات گوناگون نشان داده‌اند، در سال‌های اخیر، میزان مرگ‌ومیر براثرِ سرطان رشد چشمگیری داشته است؛ به‌همین‌دلیل است که محققان درپیِ تولید داروهای جدید، به‌ویژه داروهایی با اثرات جانبی کمتر، هستند. برهمین‌اساس، آنزیم ریبونوکلئاز پانکراس گوسفند نیز یکی از بهترین اورتولوگ‌ها برای آنزیم ریبونوکلئاز پانکراس گاو است که آن را در القای مرگ سلول‌های سرطانی به‌کار می‌گیرند. آنزیم گوسفندی تنها در 4 اسیدآمینۀ A19S، K37Q، V46F و N103E با آنزیم گاوی تفاوت دارد. در مطالعۀ پیش‌رو، ابتدا شکل سه‌بعدی آنزیم ریبونوکلئاز پانکراس گوسفند پیش‌بینی و سپس از‌نظر پایداری بررسی شد. درادامه، مشخصات فیزیکوشیمیایی این آنزیم با برنامه پرات پارام نیز مورد بررسی قرار گرفت.  همچنین، با استفاده ازروش‌های بیوانفورماتیکی شبیه‌سازی و با‌توجه ‌به RMSF، RMSD و Gyration analyses میزان پایداری پروتئین نیز پیش‌بینی گردید. مطالعات آزمایشگاهی نیز از ‌قبیل بررسی فرار این آنزیم از RI، میزان مقاومت آن درمقابل پپسین، غلظت پروتئین تولیدی، قابلیت القای مرگ سلولی در سلول‌های سرطانی و نیز میزان فعالیت این آنزیم، انجام گرفت. نتایج این تحقیق نشان می‌دهد که آنزیم مدنظر پس از 22 ساعت انکوبه در مجاورت پپسین تا 90% دست‌نخورده باقی می‌مانَد. میزان غلظت پروتئین تولیدی نیز mg/ml 4.78 بود که با استفاده از روش برادفورد تخمین زده شد. در این مطالعه فعالیت آنزیم ریبونوکلئاز طبیعی پانکراس گوسفند 558±22 U/mol برآورد شد. همان‌طورکه در تحقیقات گذشته نشان داده شده است، انواعِ RNaseA گاو و گوسفند نمی‌توانند وارد سلول شوند و درنتیجه هیچ‌گونه سمیت سلولی نشان نمی‌دهند، ولی در این مطالعه با استفاده از لیپوفکتامین، این آنزیم‌ها وارد سلول شدند و مرگ سلولی را به سلول‌های سرطانی Hella القا کردند. میزان زنده‌مانی سلول‌ها نیز به‌روش MTT assay  اندازه‌گیری شد. براساس این تحقیق، مشخص شد که آنزیم ریبونوکلئاز پانکراس گوسفندی فعالیت آنزیمی و توکسیتی مشابهی با آنزیم ریبونوکلئاز پانکراس گاوی دارد و برای استفاده به‌عنوان توکسین در تولید ایمونوتوکسین‌ها جانشین مناسبی است.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Production of ovine Pancreatic Ribonuclease and investigation of enzyme characteristics

نویسندگان [English]

  • mahsa Zabetian 1
  • Mohammadreza Nassiry 2
  • Ali Javadmannesh 1
  • Shahrokh ghovati 3
1 Department Animal Science, Faculty of agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
2 Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
3 Faculty of Agriculture, Animal Science Department, University of Guilan, Iran.
چکیده [English]

Introduction Obviously, the recent decades strategy in cancer therapy, anticancer drug discovery and drug improvement is to characterize, distinguish and validate the most promising cancer-related molecular targets to which new drugs can be designed. The unique features of the pancreatic RNase (RNase A) such as high activity, stability, lack of a cofactor, and small molecular size have made it the most popular enzyme in the ribonuclease family. Specifically, RNase A is involved in endonucleolytic cleavage of 3'-phosphomononucleotides and 3'- phosphooligonucleotides ending in C-P or U-P with 2', 3’- cyclic phosphate intermediates. RNase A was purified from the Bovidae family including bovine, ovine, bison, eland, goat and gnu. Although phylogenetic analyses of RNase A revealed high similarity among members of the Bovidae family, some functional mutations were also found.
Several studies showed that the RNA hydrolyzing action of ribonuclease is able to induce apoptosis and cell death in cancer cells, independently. This effect could be enhanced thousands of times when ribonuclease is linked to antibodies. These enzymes show potent cytotoxic activity on cell internalization but do not show sensible immunogenicity or non-specific toxicity toward normal cells. Ovine pancreatic ribonuclease enzyme is a member of super family RNase A, it can be a good candidate as a toxin for designing new drugs. The objective of this study was to produce ovine pancreatic ribonuclease enzyme in E. coli and characterize its activity.
Materials and methods All structures needed for this study were downloaded from the Protein Data Bank (PDB) website (http://www.rcsb.org/). PDB files (accession numbers: 10.2210 /pdb1YV6/pdb and 10.2210/pdb3SNF/pdb) related to natural Bovine Pancreatic RNase were selected. Gene synthesis and production of recombinant protein were conducted by using the pelB signal sequence at the beginning of the structure for periplasmic protein production. The native ovine RNase and bovine RNase A were optimized for E. coli host by GenRay codon optimization service and sent to GenRay Biotechnology (Shanghai, China) for synthesis. The target genes in pGH vector were sub-cloned in pUC19 and then cloned into the pET21b (+) vector between XbaI and HindIII sites. After transformation, E. coli cells containing recombinant pET21b (+) were cultivated in LB broth medium containing ampicillin. To extract the proteins, osmotic shock methods were applied. After that Q-sepharose chromatography was used to extract the target protein. Finally, Bradford analysis was used to determine the protein concentration. The ribonucleolytic activity of the recombinant native ovine RNase A was compared with native bovine RNase A following Tripathy et al (2013) method. To investigate the antitumor activity of recombinant proteins, HeLa cells were prepared for seeding in a 96-well flask and incubated at 37°C in 5% CO2 for 72 h.
Results and discussion The production of native ovine and bovine RNase A was confirmed by SDS PAGE. Protein purification was successfully performed using osmotic shock in the Q-Sepharose column. Although our findings confirmed protein expression, no detectable proteins other than RNase A was observed in the LB medium, indicating that almost all the proteins were expressed either inside the bacterial cell or secreted into the periplasmic area. According to the Bradford analysis, the concentration of the recombinant proteins extracted was 4.78 ovine RNase A. Based on our results, it was shown that the ribonucleolytic activity of native ovine RNase A was 558±22. The results showed that RNase A exhibited resistance to pepsin degradation during the whole incubation process (22 h), in the course of which 90% of RNase A remained undamaged. To determine the cytotoxic effect of ovine RNase on the HeLa cell line, MTT assay was done following incubation with ovine RNase A. The commercial RNase A was used as control. The results showed that ovine RNase A and bovine RNase A had no cytotoxic effect on HeLa cells. When RNase treatment was done by the lipofectamine 3000 (Thermo, USA), the cytotoxicity effect was observed. Several studies have shown that some ribonucleases such as onconase, bovine seminal ribonuclease and bovine pancreatic ribonuclease have a great promise as cancer immunotherapeutic agents and cause a significant reduction in the protein synthesis of tumor cells after internalization into cytosol. 
Conclusion Our findings demonstrate that ovine RNase similar to bovine RNase has a great potential for use in drug design industry. We revealed that the native ovine RNase A was more stable than the native bovine RNase. In future work, we intend to fuse the engineered ovine-RNase A to dedicated recombinant antibodies for cancer therapy and investigation of engineered immuno-ribonuclease potency and cell killing effects as a fusion protein.
 

کلیدواژه‌ها [English]

  • Bioinformatics
  • Biological activity
  • Cytotoxicity
  • Ovine pancreatic ribonuclease
  1. Ardelt, W., K. Shogen, and Z. Darzynkiewicz. 2008. Onconase and amphinase, the antitumor ribonucleases from Rana pipiens oocytes. Current Pharmaceutical Biotechnology, 9(3):215-225.
  2. Barnard, E. 1969. Biological Function of Pancreatic Ribonuclease. Nature, 221: 340- 344.
  3. Becker, R. R., J. L. Halbrook, and C. Hirs.. 1973. Isolation and Characterization of Ovine Ribonuclease A, B, and C from Pancreatic Secretion. Journal of Biological Chemistry, 248:7826-7832.
  4. Breukelman, H. J., N. Munnik, and R. Kleineidam. 1998. Secretory ribonuclease genes and pseudogenes in true ruminants. Gene, 212(2):259-68.
  5. J. J., and J. M. van der Laan. 1986. Comparison of the structure of turtle pancreatic ribonuclease with those of mammalian ribonuclease. The Federation of European Biochemical Societies, 194(2):338–343.
  6. Claudi, M., M. Cuchillo, R. Victòria Nogués, and T. Raines. 2011. Bovine Pancreatic Ribonuclease: 50 Years of the First Enzymatic Reaction Mechanism. The Journal of Biochemistry, 50(37):7835–7841.
  7. De Lorenzo, C., A. Nigro, R. Piccoli, and G. D'Alessio. 2002. A new RNase-based immunoconjugate selectively cytotoxic for ErbB2-overexpressing cells. The Federation of European Biochemical Societies, 516(1-3):208-212.
  8. De Lorenzo, C., A. Arciello, R. Cozzolino, D. B. Palmer, P. Laccetti, R. Piccoli, and G. D'Alessio. 2004. A fully human antitumor immunoRNase selective for ErbB-2-positive carcinomas. Cancer Research, 64(14):4870-4874.
  9. DelCardayr, S. B., M. Ribio, E. M. Yokel, D. J. Quirk, W. J. Rutter and R. T. Ruines. 1995. Engineering ribonuclease A: Production, purification and characterization of wild-type enzyme and mutants at Glnll. Protein Engineering, 18:261-273.
  10. Forouharmehr, A., M. Nassiri, S. Ghovvati, and A. Javadmanesh. 2020. Production and introduction of a novel immunotoxin based on mutant RNase A for inducing death to Her1‐positive cell lines. Journal of Cell Physiology, doi: 10.1002/jcp.29346
  11. Forouharmehr, A., M. Nassiri, S. Ghovvati, and A. Javadmanesh, A. 2018. Different Signal Peptides for Secretory Production of Recombinant Bovine Pancreatic Ribonuclease A in Gram Negative Bacterial System: An In silico Study. Current Proteomics, 15: 24-31
  12. R. F., and J. Atkins. 1993. The RNA World. Cold SpringHarbor LaboratoryPress Plainview, NY.
  13. Ghovvati, S. 2014. Engineering ScFv-FC antibody bound to RNase to target HER2 antigen. Doctoral dissertation, Ferdowsi University. (In Persian)
  14. He, F. 2011. Bradford Protein Assay. Biotechnology Innovation Organization, 101:e45. 10.21769.45.
  15. Jaydarian, A., N. Nazifi, and A. Forouharmehr. 2020. Computational design of a novel multi-epitope vaccine against Coxiella burnetii. Human Immunology, 10:10-16.
  16. Leich, F., N. Stohr, A. Rietz, R. Ulbrich-Hofmann, and U. Arnold. 2007. Endocytotic internalization as a crucial factor for the cytotoxicity of ribonucleases. Journal of Biological Chemistry, 282(38):27640-27646.
  17. Lomax, J. E., C. M. Bianchetti, A. Chang, G. N. Phillips, B. G. Fox, and R. T. Raines. 2014. Functional evolution of ribonuclease inhibitor: insights from birds and reptiles. Journal of Molecular Biology, 426(17):3041‐3056.
  18. Lu, L., J. Li, M. Moussaoui, and E. Boix. 2018. Immune Modulation by Human Secreted RNases at the Extracellular Space. Frontiers in Immunology, 9:1012.
  19. Menzel, C., T. Schirrmann, Z. Konthur, T. Jostock, and S. Dubel. 2008. Human antibody RNase fusion protein targeting CD30+ lymphomas. Blood, 111(7):3830-3837.
  20. Merlino, A., L. Mazzarella, A. Carannante, A. Di Fiore, A. Di Donato, E. Notomista, and F. Sica. 2005. The importance of dynamic effects on the enzyme activity: X-ray structure and molecular dynamics of onconase mutants. The Journal of Biological Chemistry, 6;280(18):17953-60.
  21. Pikkemaat, M. G., B. M. Linssen, J. C. Berendsen, and D. B. Janssen. 2002. Molecular dynamics 355 simulations as a tool for improving protein stability. Protein Engineering, Design and Selection, 15:185–192.
  22. Raines, R. T. 1998. Ribonuclease A. Chemical Review, 98:1045-1065.
  23. Riccio, G., C. D. Avino, R. T. Raines, C. D. Lorenzo. 2013. A novel fully human antitumor ImmunoRNase resistant to the RNase inhibitor. Protein Engineering, Design & Selection, 26:243–248.
  24. Rutkoski, T. J., and R. T. Raines. 2008. Evasion of ribonuclease inhibitor as a determinant of 360 ribonuclease cytotoxicity. Current Pharmaceutical Biotechnology, 9(3):185-9.
  25. Sells, , J. Li, and J. Chernoff. 1995. Delivery of proteins into cells using cationic liposomes. BioTechniques, 19(1):72-6.
  26. Stigh, E., G. Aqvist, and C. B. Anfinsen. 1958. The Isolation and Characterization of Ribonucleases from Sheep Pancreas. The Journal of Biological Chemistry, 234:1112-1117.
  27. Schirrmann, T., J. Krauss, M. A. Arndt, S. M. Rybak, and S. Dubel. 2009. Targeted therapeutic RNases (ImmunoRNases). Expert Opinion on Biological Therapy, 9(1):79-95.
  28. Schmid, F., and H. Blaschek. 1984. An early intermediate in the folding of ribonuclease A is protected against cleavage by pepsin. Biochemistry, 23(10):2128‐2133.
  29. Sundlass, N. K., and R. T. Raines. 2011. Arginine residues are more effective than lysine residues in eliciting the cellular uptake of onconase. Biochemistry, 50(47):10293-9.
  30. Turcotte, R. F., L. D. Lavis, and R. T. Raines. 2009. Onconase cytotoxicity relies on the distribution of its positive charge. The Federation of European Biochemical Societies, 276(14):3846-3857.
  31. Tripathy, D. R., A. K. Dinda, A. K., and Dasgupta, S. 2013. A simple assay for the ribonuclease activity of ribonucleases in the presence of ethidium bromide. Analytical Biochemistry, 437(2):126-9.
  32. http://www.uniprot.org
  33. Welling, G., Scheffer, A., and Beintema, J. 1974. The Primary structure of goat and sheep pancreatic ribonucleases. North-Holland Publishing Company – Amsterdam,41:58- 61.
  34. Zeiske, T., Stafford, K. A., and Palmer, A. G. 2016. Thermostability of Enzymes from Molecular Dynamics Simulations. Journal of Chemical Theory and Computation, 14;12(6):2489-92
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