Skip to main content
SpringerLink
Log in

Isolation by anion-exchange of immunologically and enzymatically active human islet glutamic acid decarboxylase 65 overexpressed in Sf9 insect cells

  • Originals
  • Published:
Diabetologia Aims and scope Submit manuscript

Summary

The enzymel-glutamic acid decarboxylase is a major autoantigen of the beta cell. Autoantibodies against this enzyme are observed before the onset of insulin-dependent diabetes mellitus (IDDM) in man and may be of predictive value. There is evidence that this enzyme is involved in the development of autoimmune diabetes in animals. In order to facilitate the investigation of the role ofl-glutamine acid decarboxylase in IDDM, we expressed the 65 kDa isoform of human isletl-glutamic acid decarboxylase in insect cells using a baculovirus-based vector. The material was expressed at high levels (up to 50 mg/l of cells). Partially purified metabolically labelledl-glutamic acid decarboxylase bound to immunoglobulins in the sera from 20 of 49 subjects with newly-diagnosed IDDM. The enzyme was isolated in high yields (up to 26 mg/l cell culture) with fully maintained enzymatic activity by either ion-exchange chromatography or immunoaffinity chromatography. Purifiedl-glutamic acid decarboxylase inhibited the binding of radioactivel-glutamic acid decarboxylase, prepared by in vitro translation of mRNA, to immunoglobulins in the sera of subjects with IDDM. Recombinant human isletl-glutamic acid decarboxylase, isolated from Sf9 cells, is a suitable material for the large scale investigation of the utility of this enzyme in the prediction and prevention of autoimmune diabetes. [Diabetologia (1995) 38: 14–23

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

IDDM:

Insulin-dependent diabetes mellitus

GAD:

L-glutamic acid decarboxylase

ICA:

islet cell cytoplasmic antibodies

w/v:

weight/volume

PCR:

polymerase chain reaction

DTT:

1,4-dithiothreitol

HEPES:

N-2-hydroxyethylenepiperazine-N′-ethansulphonic acid

DEAE:

dietylaminoethyl

References

  1. Baekkeskov S, Aanstoot H-J, Christgau S et al. (1990) Identification of the 64k autoantigen in insulin-dependent diabetes as the GABA-synthetizing enzyme glutamic acid decarboxylase. Nature 347: 151–156

    Article  CAS  PubMed  Google Scholar 

  2. Erlander MG, Tillakaratne NJK, Feldblum S, Patel N, Tobin AJ (1991) Two genes encode distinct glutamate decarboxylases. Neuron 7: 91–100

    Article  CAS  PubMed  Google Scholar 

  3. Koch Michelsen B, Petersen JS, Boel E, Moeldrup A, Dyrberg T, Dragsbaek Madsen O (1991) Cloning, characterisation and autoimmune recognition of rat islet glutamic decarboxylase in insulin-dependent diabetes mellitus. Proc Natl Acad Sci USA 88: 8754–8758

    Google Scholar 

  4. Karlsen AE, Hagopian WA, Grubin CE et al. (1991) Cloning and primary structure of a human isoform of glutamic acid decarboxylase from chromosome 10. Proc Natl Acad Sci USA 88: 8337–8341

    CAS  PubMed  Google Scholar 

  5. Cram DS, Barnett LD, Joseph JL, Harrison LC (1991) Cloning and partial nucleotide sequence of human glutamic acid decarboxylase cDNA from brain and pancreatic islets. Biochem Biophys Res Commun 176: 1239–1244

    Article  CAS  PubMed  Google Scholar 

  6. Huang W-M, Reed-Fourquet L, Wu E, Wu J-Y (1990) Molecular cloning and amino acid sequence of brainl-glutamate decarboxylase. Proc Natl Acad Sci USA 87: 8491–8495

    CAS  PubMed  Google Scholar 

  7. Bjork E, Kampe O, Karlsson FA et al. (1992) Glucose regulation of the autoantigen GAD65 in human pancreatic islets. J Clin End Metab 75: 1574–1576

    CAS  Google Scholar 

  8. Petersen JS, Russel S, Marshall MO et al. (1993) Differential expression of glutamic decarboxylase in rat and human islets. Diabetes 42: 484–495

    CAS  PubMed  Google Scholar 

  9. Christgau S, Aanstoot H-J, Schierbeck H-J et al. (1992) Membrane anchoring of the autoantigen GAD65 to microvesicles in pancreatic beta cells by palmitoylation in the NH2-terminal domain. J Cell Biol 118: 309–320

    Article  CAS  PubMed  Google Scholar 

  10. Reetz A, Solimena A, Matteoli M, Folli F, Takei K, De Camillo P (1991) GABA and pancreatic beta cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like vesicles suggests their role in GABA storage and secretion. EMBO J 10: 1275–1284

    CAS  PubMed  Google Scholar 

  11. Velloso LA, Kampe O, Eizirik DL, Hallberg A, Andersson A, Karlsson FA (1993) Human autoantibodies react with glutamic acid decarboxylase antigen in human and rat but not mouse islets. Diabetologia 36: 39–46

    Article  CAS  PubMed  Google Scholar 

  12. Petersen JS, Dyrberg T, Markholst H, Karlson A, Madsen O, Michelsen B (1993) Analysis of GAD65 antibodies in NOD mice and GAD expression in the pancreas: evidence for low antibody frequency and low expression. 12th International Immunology and Diabetes Workshop 1 (Abstract)

  13. Kim J, Richter W, Aanstoot HJ et al. (1994) Differential expression of GAD(65) and GAD(67) in human, rat and mouse pancreatic islets. Diabetes 42: 1799–1808

    Google Scholar 

  14. Thivolet CH, Tappaz M, Durand A et al. (1992) Glutamic acid decarboxylase (GAD) autoantibodies are additional markers of type I (insulin dependent) diabetes mellitus in high risk individuals. Diabetologia 35: 570–576

    Article  CAS  PubMed  Google Scholar 

  15. De Aizpurua HJ, Wilson YM, Harrison LC (1992) Glutamic acid decarboxylase autoantibodies in preclinical insulindependent diabetes. Proc Natl Acad Sci USA 89: 9841–9845

    PubMed  Google Scholar 

  16. Bjork E, Kampe O, Andersson A, Karlsson FA (1992) Expression of the 64kDa/glutamic acid decarboxylase rat islet cell autoantigen is influenced by the rate of insulin secretion. Diabetologia 32: 490–493

    Google Scholar 

  17. Barmeir H, McCulloch DK, Neifing JL et al. (1991) Risk for developing type I (insulin-dependent) diabetes and the presence of islet 64K antibodies. Diabetologia 34: 727–733

    Google Scholar 

  18. Rowley MJ, Mackay IR, Chen Q-Y, Knowles WJ, Zimmet PZ (1992) Antibodies to glutamic acid decarboxylase discriminate major types of diabetes mellitus. Diabetes 41: 548–551

    CAS  PubMed  Google Scholar 

  19. Serjeantson SW, Kohonen MRJ, Rowley MJ, Mackay IR, Knowles W, Zimmet P (1992) Antibodies to glutamic acid decarboxylase are associated with HLA-DR genotypes in both Australians and Asians with type 1 (insulin dependent) diabetes mellitus. Diabetologia 35: 996–1001

    Article  CAS  PubMed  Google Scholar 

  20. Kaufman DL, Erlander MG, Clare-Salzer M, Atkinson MA, Maclaren NK, Tobin AJ (1992) Autoimmunity to two forms of glutamate decarboxylase in insulin dependent diabetes mellitus. J Clin Invest 89: 283–292

    CAS  PubMed  Google Scholar 

  21. Velloso LA, Kampe O, Hallberg A, Christmanson L, Betsholtz C, Karlsson FA (1993) Demonstration of GAD-65 as the main immunogenic isoform of glutamate decarboxylase in type I diabetes and determination of autoantibodies using a radioligand produced by eukaryote expression. J Clin Invest 91: 2084–2090

    CAS  PubMed  Google Scholar 

  22. Hagopian WA, Michelsen B, Karlson AE et al. (1993) Autoantibodies in IDDM primarily recognise the 65,000 Mr rather than the 67,000 Mr isoform of glutamic acid decarboxylase. Diabetes 42: 1–3

    Google Scholar 

  23. De Aizpurua HJ, Harrison LC, Cram DS (1992) An ELISA for antibodies to recombinant glutamic acid decarboxylase in IDDM. Diabetes 41: 1182–1187

    Google Scholar 

  24. Petersen JS, Hejnaes KR, Moody AJ et al. (1994) Detection of GAD65 antibodies in diabetes and other autoimmune diseases using a simple radioligand assay. Diabetes 43: 459–467

    CAS  PubMed  Google Scholar 

  25. Chrisie MR, Daneman D, Champagne P, Delovitch TL (1990) Persistence of serum antibodies to 64000 Mr islet cell proteins after onset of type 1 diabetes. Diabetes 39: 653–656

    Google Scholar 

  26. Kawasaki E, Moriuchi R, Takino H et al. (1992) Autoantibodies to 64,000/Mr islet cell protein in long-term type 1 (insulin-dependent) diabetic patients. Diabetologia 35: 748–752

    CAS  PubMed  Google Scholar 

  27. Tuomi T, Groop LC, Zimmet PZ, Rowley MJ, Knowles W, Mackay IR (1993) Antibodies to glutamic acid decarboxylase reveal latent autoimmune diabetes mellitus in adults with non-insulin dependent onset of diabetes. Diabetes 42: 359–362

    CAS  PubMed  Google Scholar 

  28. Hagopian WA, Karlsen AE, Gottsater A et al. (1993) Quantitative assay using recombinant human islet glutamic acid decarboxylase (GAD65) shows that 64K autoantibody positivity at onset predicts diabetes type. J Clin Invest 91: 368–374

    CAS  PubMed  Google Scholar 

  29. Solimena M, Folli F, Apari R, Pozza G, De Camilli P (1990) Autoantibodies to gaba-ergic neurons and pancreatic beta cells in Stiff-Man syndrome. N Eng J Med 322: 1555–1560

    CAS  Google Scholar 

  30. Atkinson MA, Kaufman DL, Cambell L et al. (1992) Response of peripheral-blood mononuclear cells to glutamate decarboxylase in insulin-dependent diabetes. Lancet 339: 458–459

    Article  CAS  PubMed  Google Scholar 

  31. Honeyman MC, Cram DS, Harrison LC (1993) Glutamic acid decarboxylase 67-reactive T cells: a marker of insulin dependent diabetes. J Exp Med 177: 535–540

    Article  CAS  PubMed  Google Scholar 

  32. Tisch R, Yang XD, Singer SM, Liblau RS, Mcdevitt HO (1993) Immune-response to glutamic-acid decarboxylase correlates with insulitis in nonobese diabetic mice. Nature 366: 72–75

    Article  CAS  PubMed  Google Scholar 

  33. Kaufman DL, Clare Salzler M, Tian JD et al. (1993) Spontaneous loss of T-cell tolerance to glutamic-acid decarboxylase in murine insulin-dependent diabetes. Nature 366: 69–72

    Article  CAS  PubMed  Google Scholar 

  34. Mauch L, Seisler J, Haubruck H et al. (1993) Baculovirusmediated expression of human 65 kDa and 67 kDa glutamic acid decarboxylase in SF9 insect cells and their relevance in diagnosis of insulin dependent diabetes mellitus. J Biochem 113: 699–704

    CAS  PubMed  Google Scholar 

  35. Worsaae A, Ludvigsson J, Pociot F et al. (1993) Human T cell reactivity to human islet GAD65. 12th International Immunology and Diabetes Workshop (Abstract)

  36. Bottazzo GF, Gleichmann H (1986) Immunology and diabetes workshops: report of the first international workshop on the standardisation of cytoplasmic islet cell antibodies. Diabetologia 29: 125–126

    Article  Google Scholar 

  37. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685

    Article  CAS  PubMed  Google Scholar 

  38. Denner LA, Wei SC, Lin HS, Lin C-T, Wu J-Y (1987) Brainl-glutamate decarboxylase: purification and subunit structure. Proc Natl Acad Sci USA 84: 668–672

    CAS  PubMed  Google Scholar 

  39. Tabor S, Richardson CC (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84: 4767–4771

    CAS  PubMed  Google Scholar 

  40. Felgner PL, Gadek TR, Holm M et al. (1987) Lipofectin: a highly efficient, lipid-mediated DNA transfection procedure. Proc Natl Acad Sci USA 84: 7413–7417

    CAS  PubMed  Google Scholar 

  41. Waldmann H, Cobbold S, Lefkovits (1987) I. Lymphocytes, a practical approach, IRL Press. Oxford, pp 163–188

    Google Scholar 

  42. Maxbac, A (1992) manual of methods for baculovirus vectors and insect cell culture procedures, Invitrogen Corporation San Diego, pp 26

  43. Bordier C (1981) Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 256: 1604–1607

    CAS  PubMed  Google Scholar 

  44. Bohlen P, Stein S, Daiman W, Idenfriend S (1973) Fluorometric assay of proteins in the nanogram range. Arch Biochem Biophys 155: 213–220

    CAS  PubMed  Google Scholar 

  45. Spink DC, Wu SJ, Martin DL (1983) Multiple forms of glutamate decarboxylase in porcine brain. J Neurochem 40: 1113–1119

    CAS  PubMed  Google Scholar 

  46. Blindermann J-M, Maitre M, Mandel P (1978) Purification and some properties ofl-glutamate decarboxylase from human brain. Eur J Biochem 86: 143–152

    Article  CAS  PubMed  Google Scholar 

  47. Mouillac B, Caron M, Bonin H, Dennis M, Bouvier M (1991) Agonist-modulated palmitoylation of beta 2-adrenergic receptor in Sf9 cells. J Biol Chem 267: 21733–21737

    Google Scholar 

  48. Han KK, Martinage A (1992) Possible relationship between coding recognition amino acid sequence motif or residues(s) and post-translational chemical modification of proteins. Int J Biochem 24: 1349–1363

    Article  CAS  PubMed  Google Scholar 

  49. Karlsen AE, Hagopian WA, Petersen JS et al. (1992) Recombinant glutamic acid decarboxylase (representing the single isoform expressed in human islets) detects IDDM associated 64000-Mr autoantibodies. Diabetes 41: 1355–1359

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Diabetes Care Division, Novo Nordisk A/S, Novo Alle, 2880, Bagsværd, Denmark

    A. J. Moody, K. R. Hejnæs, M. O. Marshall, F. S. Larsen, E. Boel, I. Svendsen, E. Mortensen & T. Dyrberg

Authors
  1. A. J. Moody
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. K. R. Hejnæs
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. O. Marshall
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F. S. Larsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. E. Boel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. I. Svendsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. E. Mortensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. T. Dyrberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Moody, A.J., Hejnæs, K.R., Marshall, M.O. et al. Isolation by anion-exchange of immunologically and enzymatically active human islet glutamic acid decarboxylase 65 overexpressed in Sf9 insect cells. Diabetologia 38, 14–23 (1995). https://doi.org/10.1007/BF02369348

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02369348

Key words

Search

Navigation