Improved in Planta Expression of the Human Islet Autoantigen Glutamic Acid Decarboxylase (GAD65)
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The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH2-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD671-87/GAD6588-585 molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes.
- Arakawa T and Langridge WH (1998) Plants are not just passive creatures! Nat Med 5: 550–551.
- Arakawa T, Yu J, Chong DKX, Hough J, Engen PC and Langridge WHR (1998) A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes. Nature Biotech 16: 934–938.
- Baulcombe DC, Chapman S and Santa Cruz S (1995) Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 7: 1045–1053.
- Chong DK, Roberts W, Arakawa T, Illes K, Bagi G, Slattery CW et al. (1997) Expression of the human milk protein beta-casein in transgenic potato plants. Trans Res 6: 289–296.
- Daniell H, Streatfield SJ and Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 5: 219–226.
- Datla RS, Hammerlindl JK, Panchuk B, Pelcher LE and Keller W (1992) Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 122: 383–384.
- Dieryck W, Pagnier J, Poyart C, Marden MC, Gruber V, Bournat P et al. (1997) Human haemoglobin from transgenic tobacco. Nature 386: 29–30.
- Endl J, Otto H, Jung G, Dreisbusch B, Donié F, Stahl P et al. (1997) Identification of naturally processed T cell epitopes from glutamic acid decarboxylase presented in the context of HLADR alleles by T lymphocytes of recent onset IDDM patients. J Clin Invest 99: 2405–2415.
- Falorni A, Ackefors M, Carlberg C, Daniels T, Persson B, Robertson J et al. (1996) Diagnostic sensitivity of immunodominant epitopes of glutamic acid decarboxylase (GAD65) autoantibodies in childhood IDDM. Diabetologia 39: 1091–1098.
- Falorni A, Örtqvist E, Persson B and Lernmark Å (1995) Radioimmunoassays for glutamic acid decarboxylase (GAD65) and GAD65 autoantibodies using 35S or 3H recombinant human ligands.J Immunol Meth 186: 89–99.
- Farres J, Holmberg N, Schlattner U, Bailey JE, Wallimann T and Kallio P (2002) Expressing creatine kinase in transgenic tobacco – a first step towards introducing an energy buffering system in plants. Transgenic Res 11: 49–59.
- Gallie DR (1998) Controlling gene expression in transgenics. Curr Opin Plant Biol 2: 166–172.
- Giddings G (2001) Transgenic plants as protein factories. Curr Opin Biotechnol 12: 450–454.
- Karlsen AE, Hagopian WA, Grubin CE, Dube S, Disteche CM, Adler DA et al. (1991) Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10. Proc Natl Acad Sci USA 88: 8337–8341.
- Kaufman DL, Clare-Salzler M, Tian J, Forsthuber T, Ting GSP, Robinson P et al. (1993) Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 366:69–72.
- Kavanagh T, Goulden M, Santa Cruz S, Chapman S, Barker I and Baulcombe DC (1992) Molecular analysis of a resistancebreaking strain of Potato Virus X. Virology 189: 609–617.
- Kong Q, Richter L, Fang Yang Y, Arntzen CJ, Mason HS and Thanavala Y (2001) Oral immunization with hepatitis B surface antigen expressed in transgenic plants. Proc Natl Acad Sci USA 98: 11539–11544.
- Kooter JM, Matzke MA and Meyer P (1999) Listening to the silent genes: transgene silencing, gene regulation and pathogen control. Trends Plant Sci 9: 340–347.
- Kusnadi AR, Nikolov ZL and Howard JA (1997) Production of recombinant proteins in transgenic plants: practical considerations. Biotechnol Bioeng 56: 473–484.
- Lernmark Å and Falorni A (1997) Immunology of insulindependent diabetes mellitus. In: Pickup J, Williams C (eds), Textbook of Diabetes. (pp. 15.1–15.23) Blackwell, Oxford.
- Li YQ, Faleri C, Geitmann A, Zhang HQ and Cresti M (1995) Immunogold localization of arabinogalactan proteins, unesterified and esterified pectin in pollen grains and pollen tubes of Nicotiana tabacum. Protoplasma 189: 26–36.
- Lohmann T, Leslie RD and Londei M (1996) T cell clones to epitopes of glutamic acid decarboxylase 65 raised from normal subjects and patients with insulin-dependent diabetes. J Autoimmunity 9: 385–389.
- Lomonossoff G (2001) Growing medicines using plant viruses. Biologist 6: 263–267.
- Ma SW, Zhao DL, Yin ZQ, Mukherjee R, Singh B, Qin HY et al. (1997) Transgenic plants expressing autoantigens fed to mice to induce oral immune tolerance. Nature Med 3: 793–796.
- Mason HS, Ball JM, Shi J-J, Jiang X, Estes MK and Arntzen CJ (1996) Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc Natl Acad Sci USA 93: 5335–5340.
- McCormick AA, Kumagai MH, Hanley K, Turpen TH, Hakim I, Grill LK et al. (1999) Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants. Proc Natl Acad Sci USA 96: 703–708.
- Michelsen BK, Peterson JS, Boel E, Moeldrup A, Dyrberg T and Madsen D (1991) Cloning, characterization and autoimmune recognition of rat islet glutamic acid decarboxylase in insulin-dependent diabetes melilitus. Proc Natl Acad Sci USA 88: 8754–8758.
- Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15: 473–497.
- Namchuk M, Lindsay L, Tusck CW, Kanaani J and Baekkeskov S (1997) Phosphorylation of serin residues 3, 6, 10 and 13 distinguishes membrane anchored from soluble glutamic acid decarboxylase 65 and is restricted to glutamic acid decarboxylase 65α. J Biol Chem 272: 1548–1557.
- Petersen JS, Karlsen AE, Markholst H, Worsaae A, Dyrberg T and Michelsen B (1994) Neonatal tolerization with glutamic acid decarboxylase but not with bovine serum albumin delays the onset of diabetes in NOD mice. Diabetes 43: 1478–1484.
- Porceddu A, Falorni A, Ferradini N, Cosentino A, Calcinaro F, Faleri C et al. (1999) Transgenic plants expressing human glutamic acid decarboxylase (GAD65), a major autoantigen in insulin-dependent diabetes mellitus. Mol Breed 5: 553–560.
- Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn, Cold Spring Harbor Press, New York.
- Schloot NC, Roep BO, Wegmann DR, Yu L, Wang TB and Eisenbarth GS (1997) T-cell reactivity to GAD65 peptide sequences shared with coxsackie virus protein in recent-onset IDDM, post-onset IDDM patients and control subjects. Diabetologia 40: 332–338.
- Shen WJ and Forde BG (1989) Efficient transformation of Agrobacterium spp by high voltage electroporation. Nucl Acids Res 17: 83–85.
- Solimena M, Aggujaro D, Muntzel C, Dirkx R, Butler M, De Camilli P et al. (1993) Association of GAD-65, but not of GAD-67, with the Golgi complex of transfected Chinese hamster ovary cells mediated by the N-terminal region. Proc Natl Acad Sci USA 90: 3073–3077.
- Takase K and Hagiwara K (1998) Expression of human alpha-lactalbumin in transgenic tobacco. J Biochem 123: 440–444.
- Tian J, Atkinson MA, Clare-Salzler M, Herschenfeld A, Forsthuber T, Lehmann PV et al. (1996) Nasal administration of glutamate decarboxylase (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes. J Exp Med 183: 1561–1567.
- Tirlapur KU, Cai G, Faleri C, Moscatelli A, Scali M, Del Casino C et al. (1995) Confocal imaging and immunogold electron microscopy of changes in distribution of myosin during pollen hydration, germination and pollen tube growth in Nicotiana tabacum. Eur J Cell Biol 67: 209–217.
- Tisch R, Yang XD, Singer SM, Liblau RS, Fugger L and McDevitt HO (1993) Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature 366: 72–75.
- Weiner HL and Mayer LF (eds) (1996) Oral Tolerance: Mechanisms and Applications. Ann NY Acad Sci 778.
- Yoon JW, Yoon CS, Lim HW, Huang QQ, Kang Y, Pyun KH et al. (1999) Control of autoimmune diabetes in NOD mice by GAD expression or suppression in beta cells. Science 284: 1183–1187.
- Zhang ZJ, Davidson L, Eisenbarth G and Weiner HL (1991) Suppression of diabetes in nonobese diabetic mice by oral administration of porcine insulin. Proc Natl Acad Sci USA 88: 10252–10256.
- Improved in Planta Expression of the Human Islet Autoantigen Glutamic Acid Decarboxylase (GAD65)
Volume 12, Issue 2 , pp 203-212
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- diabetes mellitus
- oral tolerance
- transgenic plants
- transient expression
- Industry Sectors
- Author Affiliations
- 1. Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada le Grazie 15, 37134, Verona, Italy
- 2. Dipartimento di Medicina Interna, Sezione di Medicina Interna e Scienze Endocrine e Metaboliche, Università degli Studi di Perugia, Perugia, Italy
- 3. ENEA, Centro Ricerche Trisaia UTS BIOTEC, Matera, Italy
- 4. Istituto di Ricerche sul Miglioramento Genetico Piante Foraggere del CNR, Perugia, Italy
- 5. Dipartimento di Scienze Ambientali, Università di Siena, Italy