Structure prediction and expression of modified rCTLA4-Ig as a blocker for B7 molecules

Document Type : Research article

Authors

1 Department of Biology, Science and Research branch, Islamic Azad University, Tehran, Iran.

2 Chemical Injuries Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.

3 Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.

Abstract

Aims: CTLA4-Ig (Abatacept) has been produced to suppress immune response by inhibition of T cells functions in autoimmune disease. A new drug, which is called belatacept, has recently been developed that is more efficient. The development has been occurred by two substitutions (A29Y, L104E) in the extracellular domain of CTLA4. In the present study, the bioinformatics analysis was used in order to make a new structure that has a better function in comparison with belatacept.
Methods: Firstly, eight different structures were designed. Thereafter, the secondary and 3D structures, mRNA structure, docking of chimeric proteins with CD80/CD86, antigenicity and affinity of designed chimeric molecules were predicted. Based on the criteria, a new candidate molecule was selected and its gene synthesized. The gene was cloned and expressed in E. coli BL21 (DE3) successfully. The purified rCTLA4-Ig was analyzed by SDS-PAGE, western blotting and ELISA. Circular dichroism analysis (CD analysis) was used for characterization of the rCTLA4-Ig. Affinity of rCTLA4-Ig was also evaluated by the flow cytometry method. Finally, its biological activity was determined by T cell inhibition test.
Results: The results showed rCTLA4-Ig and the belatacept protein have some similarities in structure and function. In addition rCTLA4-Ig was able to bind CD80/CD86 and inhibit T cell function.
Conclusion: Although flow cytomery results showed that the standard protein (CTLA4-Ig), represented better affinity than rCTLA4-Ig, the recombinant protein was able to inhibit T cell proliferation as well as CTLA4-Ig.

Graphical Abstract

Structure prediction and expression of modified rCTLA4-Ig as a blocker for B7 molecules

Keywords


(1) Walunas TL, Lenschow DJ, Bakker CY, Linsley PS, Freeman GJ, Green JM, Thompson CB and Bluestone JA. CTLA-4 can function as a negative regulator of T cell activation. Immunity. (1994) 1: 405-13.
      (2) Vaughan AN, Malde P, Rogers NJ, Jackson IM, Lechler RI and Dorling A. Porcine CTLA4-Ig lacks a MYPPPY motif, binds inefficiently to human B7 and specifically suppresses human CD4+ T cell responses costimulated by pig but not human B7. J. Immuno. (2000) 165: 3175-81.
      (3)  Walker LS and Sansom DM. The emerging role of CTLA4 as a cell-extrinsic regulator of T cell responses. Nat. Rev. Immunol. (2011) 11: 852-63.
      (4)  Mccoy KD and Le gros G. The role of CTLA‐4 in the regulation of T cell immune responses. Immunol. cell biol. (1999) 77: 1-10.
      (5)  Karandikar NJ, Vanderlugt CL, Walunas TL, Miller SD and Bluestone JA. CTLA-4: a negative regulator of autoimmune disease. J. Exp. Med. (1996) 184: 783-8.
      (6)  Mola EM, Balsa A, Taboada VM, Sanmartí R, Marenco JL, Sarabia FN, Gómez J, Álvaro J M, Ivorra JA and Lojo L. Abatacept use in rheumatoid arthritis: evidence review and recommendations. Reumatol. Clín. (2013) 9: 5-17.
      (7)  Cross AH, Girard T, Giacoletto K, Evans R, Keeling R, Lin R and Trotter R. Long-term inhibition of murine experimental autoimmune encephalomyelitis using CTLA-4-Fc supports a key role for CD28 costimulation. J. Clin. Investig. (1995) 95: 2783-7.
      (8)  Larsen CP, Pearson TC, Adams AB, Tso P, Shirasugi N, Strobert ME, Anderson D, Cowan S, Price K and Naemura J. Rational development of LEA29Y (belatacept), a high‐affinity variant of CTLA4‐Ig with potent immunosuppressive properties. Am. J. Transplant (2005) 5: 443-53.
      (9)  Vincenti F, Larsen C, Durrbach A, Wekerle T, Nashan B, Blancho G, Lang P, Grinyo J, Halloran PF and Solez K. Costimulation blockade with belatacept in renal transplantation. N. Engl. J. Med. (2005) 353: 770-81.
    (10)  Xu Z, Juan V, Ivanov A, Ma Z, Polakoff D, Powers DB, DuBridge RB, Wilson K and Akamatsu Y. Affinity and cross-reactivity engineering of CTLA4-Ig to modulate T cell costimulation. J. Immunol. (2012) 189: 4470-7.
    (11)  Peraino JS, Zhang H, Li G, Huang CA and Wang Z. Molecular basis of cross-species reactivities of human versus porcine CTLA-4. Hum. immunol. (2013) 74: 842-8.
    (12)  Gurpreet K and Pratap KP. In silico physicochemical characterization and topology analysis of Respiratory burst oxidase homolog (Rboh) proteins from Arabidopsis and rice. Bioinformatics (2018)14: 93-100.
    (13)  Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. (2003) 31: 3406-15.
    (14)  Gruber AR, Lorenz R, Bernhart SH, Neuböck R and Hofacker IL. The vienna RNA websuite. Nucleic Acids Res. (2008) 36: 70-4.
    (15)  Garnier J, Gibrat JF and Robson B. GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol. (1996) 266: 540-53.
    (16)  Xia F, Dou Y, Lei G and Tan Y. FPGA accelerator for protein secondary structure prediction based on the GOR algorithm. BMC Bioinform. (2011) 12: 5-8.
    (17)  Roy A, Kucukural A and Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. protocol. (2010) 5: 725-38.
    (18)  Zhang Y. I-TASSER server for protein 3D structure prediction. BMC Bioinform. (2008) 9: 1-8.
    (19)  Roy A, Yang J and Zhang Y. COFACTOR: an accurate comparative algorithm for structure-based protein function annotation. Nucleic Acids Res. (2012) 40: 372-6.
    (20)  Lovell SC, Davis IW, Arendall WB, Bakker PI, Word JM, Prisant MG, Richardson JS and Richardson DC. Structure validation by Cα geometry: ϕ, ψ and Cβ deviation. Proteins (2003) 50: 437-50.
    (21)  Ritchie DW. Recent progress and future directions in protein-protein docking. Current Protein and Peptide Science. (2008) 9: 1-15.
    (22)  Pierce BG, Hourai Y and Weng Z. Accelerating protein docking in ZDOCK using an advanced 3D convolution library. PloS one (2011) 6: 1-6.
    (23)  Yugandhar K and Gromiha MM. Protein-protein binding affinity prediction from amino acid sequence. Bioinformatics (2014) 26: 580-5.
    (24)  Yugandhar K and Gromiha MM. Response to the comment on ‘protein‐protein binding affinity prediction from amino acid sequence’. Bioinformatics (2015) 31: 978-87.
    (25)  Doytchinova IA and Flower DR. VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinform. (2007) 8: 1-7.
    (26)  Doytchinova IA and Flower DR. Identifying candidate subunit vaccines using an alignment-independent method based on principal amino acid properties. Vaccine. (2007) 25: 856-66.
    (27)  Kruger NJ. The Bradford method for protein quantitation. The protein protocols handbook (2009) 17-24.
    (28)  Teft WA, Kirchhof MG and Madrenas J. A molecular perspective of CTLA-4 function. Annu. Rev. Immunol. (2006) 24: 65-97.
    (29)  Qureshi OS, Zheng Y, Nakamura K, Attridge K, Manzotti C, Schmidt EM, Baker J, Jeffery LE, Kaur S and Briggs Z. Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4. Science (2011) 332: 600-3.
    (30)  Ruperto N, Lovell DJ, Quartier P, Paz E, Rubiopérez N, Silva CA, Abudmendoza C, burgosvargas R, Gerloni V and Melogomes JA. Abatacept in children with juvenile idiopathic arthritis: a randomised, double-blind, placebo-controlled withdrawal trial. Lancet. (2008) 372: 383-91.
    (31)  O’Rourke RW, Kang SM, Lower JA, Feng S, Ascher NL, Baekkeskov S and Stock PG. A denderitic cell line genetically modified to express CTLA4-Ig as a means to prolong islet allograft survival. Transplantation (2000) 69: 1440-6.
    (32)  Khraishi MM. Experience with subcutaneous abatacept for rheumatoid arthritis: an update for clinicians. Ther. Adv. Musculoskel. Dis. (2014) 6:159-68
    (33)  Genovese MC, Becker JC, Schiff  M, Luggen M, Sherrer Y, Kremer J, Birbara C, Box J, Natarajan K and Nuamah I. Abatacept for rheumatoid arthritis refractory to tumor necrosis factor α inhibition. N. Engl. J. Med. (2005) 353: 1114-23.
    (34)  Kremer JM, Genant HK, Moreland LW, Russell AS, Emery P, Abudmendoza C, Szechinski J, Li T, Gezandbecker JC. Effects of abatacept in patients with methotrexate-resistant active rheumatoid arthritis: a randomized trial. Ann. Intern. Med. (2006) 144: 865-76.
    (35)  Vincenti F, Blancho G, Durrbach A, Friend P, Grinyo J, Halloran PF, Klempnauer J, Lang P, Larsen CP and Mühlbacher F. Five-year safety and efficacy of belatacept in renal transplantation. J. Am. Soc. Nephrol. (2010) 21: 1587-96.
    (36)  Vincenti F, Rostaing L, Grinyo J, Rice K, Steinberg S, Gaite L, Moal MC, Mondragon GA, Kothari J and Polinsky MS. Belatacept and long-term outcomes in kidney transplantation. N. Engl. J. Med. (2016) 374: 333-43.
    (37)  Macdonald JT, Barnes C, Kitney RI, Freemont PS and Stan GBV. Computational design approaches and tools for synthetic biology. Integr. Biol. (2011) 3: 97-108.
    (38)  Wang Y, Xing J, Xu Y, Zhou N, Peng J, Xiong Z, Liu X, Luo X, Luo C and Chen K. In silico ADME/T modelling for rational drug design. Q. R. Biophysics (2015) 48: 488-515.
    (39)  Sheffield WP and Eltringhamsmith LJ. Incorporation of albumin fusion proteins into fibrin clots in-vitro and in-vivo: comparison of different fusion motifs recognized by factor XIIIa. BMC Biotechnol. (2011) 11: 1-14.
    (40)  Zhao S, Zhang Y, Tian H, Chen X, Cai D and  Yaowand X. Extending the serum half-life of G-CSF via fusion with the domain III of human serum albumin. BioMed Res. Int. (2013) 20:1-9.
    (41)  Kenanova VE, Olafsen T, Salazar FB, Williams LE, Knowles S and Wu AM. Tuning the serum persistence of human serum albumin domain III: diabody fusion proteins. Pro. Eng. (2010) 23: 789-98.
    (42)  Chen X, Zaro JL and Shen WC. Fusion protein linkers: property, design and functionality. Adv. Drug Deliv. Rev. (2013) 65: 1357-69.
    (43)  George RA and Heringa J. An analysis of protein domain linkers: their classification and role in protein folding. Pro. Eng. (2002) 15: 871-9.
    (44)  Wriggers W, Chakravarty S and Jennings PA. Control of protein functional dynamics by peptide linkers. Pept. Sci. (2005) 80: 736-46.
    (45)  Arai R, Ueda H, Kitayama A, Kamiya N and Nagamune T. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Pro. Eng. (2001) 14: 529-32.
    (46)  Shin IS, Choi EW, Chung JY, Hwang CY, Lee CW and Youn HY. Cloning, expression and bioassay of canine CTLA4Ig. Vet. Immunol. (2007) 118: 12-18.
    (47)  Gasteiger E, Hoogland C, Gattiker A, Wilkins MR, Appel RD and Bairoch A. Protein identification and analysis tools on the ExPASy server. The proteomics protocols handbook (2005) 571-607.
    (48)  Demain AL and Vaishnav P. Production of recombinant proteins by microbes and higher organisms. Biotechnol. Adv. (2009) 27: 297-306.
    (49)  Zhu L, Guo Q, Guo H, Liu T, Zheng Y, Gu P, Chen X, Wang H, Hou Y. Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry. MAbs. (2014) 6: 1474-85
    (50)  Solá RJ and Griebenow K. Effects of glycosylation on the stability of protein pharmaceuticals. J. Pharm. Sci. (2009) 98: 1223-45.
    (51)  Xiong Y, Karuppanan K, Bernardi A, Li Q, Kommineni V, Lebrilla CB, Dandekar AM, Faller R, McDonald KA and Nandi S. Effects of N-glycosylation on the structure, function, and stability of a plant-made Fc-fusion anthrax decoy protein. Fron. Plant Sci. (2019) 10: 230-5
    (52)  Mkhikian H, Grigorian A, Li CF, Chen HL, Newton B, Zhou RW, Beeton C, Torossian S, Tatarian GG and Lee SU. Genetics and the environment converge to dysregulate N-glycosylation in multiple sclerosis. Nat. Commun. (2011) 2: 1-13.
    (53)  Pereira MS, Alves I, Vicente M, Campar A, Silva MC, Padrão NA, Pinto V, Fernandes Â, Dias AM and Pinho SS. Glycans as Key Checkpoints of T Cell Activity and Function. Fron. Immunol. (2018) 9: 450-64.
    (54)  Hufton SE, Neer N, Beuken T, Desmet J, Sablon E and Hoogenboom HR. Development and application of cytotoxic T lymphocyte‐associated antigen 4 as a protein scaffold for the generation of novel binding ligands. FEBS lett. (2000) 475: 225-31.
    (55)  Darlington PJ, Kirchhof  MG, Criado G, Sondhi J and Madrenas J. Hierarchical regulation of CTLA-4 dimer-based lattice formation and its biological relevance for T cell inactivation. J. Immunol. (2005) 175: 996-04.
    (56)  Wan L, Zhu S, Li Y, Liu S, Yang H, Li S, Li Y, Cheng J and Lu X. Production and characterization of LEA29Y, a variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin, in Pichia pastoris. Appl. Microbiol. Biotechnol. (2011) 91: 543-51.