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Insufficient (Sub-native) Helix Content in Soluble/Solid Aggregates of Recombinant and Engineered Forms of IL-2 Throws Light on How Aggregated IL-2 is Biologically Active

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An Erratum to this article was published on 21 June 2015

Abstract

Interleukin 2 (IL-2) is an extremely aggregation-prone, all-alpha helical cytokine. In its receptor-bound state, ~72 % of the polypeptide chain adopts helical structure and there is no beta sheet content whatsoever. In the past, recombinant IL-2 has been formulated and used therapeutically in humans, following production in E. coli. Therapeutic IL-2 consists entirely of functionally-active soluble aggregates with ~30 subunits per aggregate particle. Side-effects attributed to aggregation resulted in discontinuation of usage over a decade ago. Structurally, and biochemically, activity in IL-2 aggregates can potentially be explained in one of two ways : (a) individual IL-2 chains exist in sterically-accessible, receptor binding-competent (native) structures, allowing aggregates to bind directly to IL-2 receptors (IL-2R); alternatively, (b) IL-2 chains dissociate from aggregates, become free to adopt native structure, and then bind to IL-2R. We produced native IL-2 and numerous engineered forms in E. coli with the objective of obtaining insights into these possibilities. Each IL-2 variant was subjected to size exclusion chromatography, circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). All forms produced and studied (including those with native IL-2 sequences) turned out to aggregate and also display less than ~50 % helix content as well as significant beta sheet content. No conditions were found that obviate aggregation. Aggregated IL-2 is thus insufficiently native-like to bind to IL-2R. Activity in aggregates thus probably owes to adoption of receptor binding-competent structures by chains that have already dissociated from aggregates.

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Abbreviations

IL-2:

Interleukin 2

CD:

Circular dichroism

FTIR:

Fourier transform infrared spectroscopy

CHO:

Chinese hamster ovary

IL-2R:

Interleukin 2 Receptor

Ni–NTA:

Nickel–Nitroloacetic acid

UV:

Ultraviolet

DTT:

Dithiothreitol

References

  1. Araujo DM, Lapchak PA, Collier B, Quirion R (1989) Brain Res 498:257–266

    Article  CAS  Google Scholar 

  2. Atkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G, Margolin K, Abrams J, Sznol M, Parkinson D, Hawkins M, Paradise C, Kunkel L, Rosenberg SA (1999) J Clin Oncol 17:2105–2116

    CAS  Google Scholar 

  3. Baccarini M, Schwinzer R, Lohmann-Matthes ML (1989) Protein. J Immunol 142:118–125

    CAS  Google Scholar 

  4. Bazan JF (1992) Science 257:410–413

    Article  CAS  Google Scholar 

  5. Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, Caughey WS (1991) Biochemistry 30:7672–7680

    Article  CAS  Google Scholar 

  6. Cerretti DP, McKereghan K, Larsen A, Cantrell MA, Anderson D, Gillis S, Cosman D, Baker PE (1986) Proc Natl Acad Sci USA 83:3223–3227

    Article  CAS  Google Scholar 

  7. Cherenova LV, Logunov DY, Shashkova EV, Shmarov MM, Verkhovskaya LV, Neugodova GL, Kazansky DB, Doronin KK, Naroditsky BS (2004) Virus Res 100:257–261

    Article  CAS  Google Scholar 

  8. Conradt HS, Nimtz M, Dittmar KE, Lindenmaier W, Hoppe J, Hauser H (1989) J Biol Chem 264:17368–17373

    CAS  Google Scholar 

  9. Curatolo L, Valsasina B, Caccia C, Raimondi GL, Orsini G, Bianchetti A (1997) Cytokine 9:734–739

    Article  CAS  Google Scholar 

  10. Dauphinee MJ, Kipper SB, Wofsy D, Talal N (1981) J Immunol 127:2483–2487

    CAS  Google Scholar 

  11. Dennert G (1980) Nature 287:47–49

    Article  CAS  Google Scholar 

  12. Devos R, Plaetinck G, Cheroutre H, Simons G, Degrave W, Tavernier J, Remaut E, Fiers W (1983) Nucleic Acids Res 11:4307–4323

    Article  CAS  Google Scholar 

  13. Eitan S, Zisling R, Cohen A, Belkin M, Hirschberg DL, Lotan M, Schwartz M (1992) Proc Natl Acad Sci USA 89:5442–5446

    Article  CAS  Google Scholar 

  14. Eitan S, Schwartz M (1993) Science 261:106–108

    Article  CAS  Google Scholar 

  15. Ferro TJ, Johnson A, Everitt J, Malik AB (1989) J Immunol 142:1916–1921

    CAS  Google Scholar 

  16. Fleischmann JD, Wentworth D, Valencic F, Imbembo AL, Koehler KA (1988) Biochem Biophys Res Commun 152:879–885

    Article  CAS  Google Scholar 

  17. Fyfe G, Fisher RI, Rosenberg SA, Sznol M, Parkinson DR, Louie AC (1995) J Clin Oncol 13:688–696

    CAS  Google Scholar 

  18. Henney CS, Kuribayashi K, Kern DE, Gillis S (1981) Nature 291:335–338

    Article  CAS  Google Scholar 

  19. Hora MS, Nandini K, Laderman KA (1992) US Patent 5078997

  20. Hora M c/o Chiron corporation (2005) European Patent EP1688146

  21. Huising MO, Kruiswijk CP, Flik G (2006) J Endocrinol 189:1–25

    Article  CAS  Google Scholar 

  22. Ju G, Collins L, Kaffka KL, Tsien WH, Chizzonite R, Crowl R, Bhatt R, Kilian PL (1987) J Biol Chem 262:5723–5731

    CAS  Google Scholar 

  23. Kaplan DR (1994) J Chromatogr B Biomed Appl 662:315–323

    Article  CAS  Google Scholar 

  24. Karpusas MA, Whitty A, Runkel L, Hochman P (1998) Cell Mol Life Sci 54:1203–1216

    Article  CAS  Google Scholar 

  25. Kashima T, Morishita A, Iwata H, Maeda K, Inoue T (1999) J Vet Med Sci 61:171–173

    Article  CAS  Google Scholar 

  26. Kunitani M, Johnson D, Snyder LR (1986) J Chromatogr 371:313–333

    Article  CAS  Google Scholar 

  27. Kuo LM, Robb RJ (1986) J Immunol 137:1538–1543

    CAS  Google Scholar 

  28. Landgraf B, Cohen FE, Smith KA, Gadski R, Ciardelli TL (1989) J Biol Chem 264:816–822

    CAS  Google Scholar 

  29. Liang SM, Thatcher DR, Liang CM, Allet B (1986) J Biol Chem 261:334–337

    CAS  Google Scholar 

  30. Liang SM, Lee N, Zoon KC, Manischewitz JF, Chollet A, Liang CM, Quinnan GV (1988) J Biol Chem 263:4768–4772

    CAS  Google Scholar 

  31. Liu Y, Xiao XY, Sun M, Hu YH, Ou-Yang KQ, Cai SX, Hua ZC (2006) Appl Biochem Biotechnol 133:77–86

    Article  CAS  Google Scholar 

  32. McKay DB (1992) Science 257:412–413

    Article  CAS  Google Scholar 

  33. Milburn MV, Hassell AM, Lambert MH, Jordan SR, Proudfoot AE, Graber P, Wells TN (1993) Nature 363:172–176

    Article  CAS  Google Scholar 

  34. Morgan DA, Ruscetti FW, Gallo R (1976) Science 193:1007–1008

    Article  CAS  Google Scholar 

  35. Mier JW, Gallo RC (1980) Proc Natl Acad Sci USA 77:6134–6138

    Article  CAS  Google Scholar 

  36. Mule JJ, Shu S, Rosenberg SA (1985) J Immunol 135:646–652

    CAS  Google Scholar 

  37. Nelson BH, Willerford DM (1998) Adv Immunol 70:1–81

    Article  CAS  Google Scholar 

  38. Oberg K, Chrunyk BA, Wetzel R, Fink AL (1994) Biochemistry 33:2628–2634

    Article  CAS  Google Scholar 

  39. O’Garra A (1989) Lancet 1:943–947

    Google Scholar 

  40. O’Garra A (1989) Lancet 1:1003–1005

    Article  Google Scholar 

  41. Oppenheim MH, Lotze MT (1994) Oncology 51:154–169

    Article  CAS  Google Scholar 

  42. Prummer O (1997) Biotherapy 10:15–24

    Article  CAS  Google Scholar 

  43. Robb RJ, Kutny RM, Panico M, Morris HR, Chowdhry V (1984) Proc Natl Acad Sci USA 81:6486–6490

    Article  CAS  Google Scholar 

  44. Robb RJ (1985) Behring Inst Mitt 77:56–67

    Google Scholar 

  45. Robb RJ, Mayer PC, Garlick R (1985) J Immunol Methods 81:15–30

    Article  CAS  Google Scholar 

  46. Rosenberg SA, Grimm EA, McGrogan M, Doyle M, Kawasaki E, Koths K, Mark DF (1984) Science 223:1412–1414

    Article  CAS  Google Scholar 

  47. Rosenberg SA, Lotze MT, Muul LM, Chang AE, Avis FP, Leitman S, Linehan WM, Robertson CN, Lee RE, Rubin JT, Seipp CA, Simpson CG, White DE (1987) N Engl J Med 316:889–897

    Article  CAS  Google Scholar 

  48. Schimpl A, Berberich I, Kneitz B, Kramer S, Santner-Nanan B, Wagner S, Wolf M, Hunig T (2002) Cytokine Growth Factor Rev 13:369–378

    Article  CAS  Google Scholar 

  49. Shena M, Siua S, Byrda S, Edelmanna KH, Patela N, Ketchema RR, Mehlina C, Arnettb HA, Hasegawaa H (2011) Exp Cell Res 317:976–993

    Article  Google Scholar 

  50. Siegel JP, Puri RK (1991) J Clin Oncol 9:694–704

    CAS  Google Scholar 

  51. Smith KA (1988) Science 240:1169–1176

    Article  CAS  Google Scholar 

  52. Smith KA (2006) Med Immunol 5:3

    Article  Google Scholar 

  53. Stauber DJ, Debler EW, Horton PA, Smith KA, Wilson IA (2006) Proc Natl Acad Sci USA 103:2788–2793

    Article  CAS  Google Scholar 

  54. Taniguchi T, Matsui H, Fujita T, Takaoka C, Kashima N, Yoshimoto R, Hamuro J (1983) Nature 302:305–310

    Article  CAS  Google Scholar 

  55. Vita N, Magazin M, Marchese E, Lupker J, Ferrara P (1990) Lymph Res 9:67–79

    CAS  Google Scholar 

  56. Wang A, Lu SD, Mark DF (1984) Science 224:1431–1433

    Article  CAS  Google Scholar 

  57. Wang W, Wang, Nayar R, Shearer MA (2000) US Patent 6689353

  58. Weir MP, Sparks J (1987) Biochem J 245:85–91

    CAS  Google Scholar 

Download references

Acknowledgments

UF would like to thank the CSIR for her doctoral research fellowship and IISER Mohali for a research project assistantship. PG, BV and SK would like to thank IMTECH, Chandigarh, as well as the Department of Biotechnology (DBT), Govt. of India, for program-mode support of this work under research project GAP0035 at IMTECH, Chandigarh. Further, PG would like to thank IISER Mohali for research support for the completion of this work and for the experiments required in revision.

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Authors and Affiliations

  1. Institute of Microbial Technology, Sector 39-A, Chandigarh, 160036, India

    Uzma Fatima, Balvinder Singh, Karthikeyan Subramanian & Purnananda Guptasarma

  2. Indian Institute of Science Education and Research, Mohali, Sector 81, SAS Nagar, Manauli P.O., Punjab, 140306, India

    Uzma Fatima & Purnananda Guptasarma

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  1. Uzma Fatima
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  2. Balvinder Singh
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  3. Karthikeyan Subramanian
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Correspondence to Purnananda Guptasarma.

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An erratum to this article is available at http://dx.doi.org/10.1007/s10930-015-9617-y.

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Fatima, U., Singh, B., Subramanian, K. et al. Insufficient (Sub-native) Helix Content in Soluble/Solid Aggregates of Recombinant and Engineered Forms of IL-2 Throws Light on How Aggregated IL-2 is Biologically Active. Protein J 31, 529–543 (2012). https://doi.org/10.1007/s10930-012-9429-2

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