Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1993 May;91(5):2084–2090. doi: 10.1172/JCI116431

Demonstration of GAD-65 as the main immunogenic isoform of glutamate decarboxylase in type 1 diabetes and determination of autoantibodies using a radioligand produced by eukaryotic expression.

L A Velloso 1, O Kämpe 1, A Hallberg 1, L Christmanson 1, C Betsholtz 1, F A Karlsson 1
PMCID: PMC288207  PMID: 8486775

Abstract

Plasmids containing cDNA for the rat 67- and 65-kD isoforms of glutamate decarboxylase (GAD-67 and GAD-65) were expressed in COS-cells, and lysates of [35S]methionine-labeled cells were used for immunoprecipitations. Sera from 38 patients with type 1 (insulin-dependent) diabetes mellitus, which precipitated a 64-kD antigen from rat islets, reacted with recombinant GAD-65 in relation to their anti-64-kD titers. The eight strongest sera also precipitated recombinant GAD-67, suggesting that certain epitopes are common to both isoforms. Subsequently, [35S]methionine-labeled GAD-65 was purified from COS cell lysates and employed in a binding assay with 50 sera of patients with recent onset of type 1 diabetes mellitus. 38 sera (76%) precipitated labeled GAD-65 with titers that correlated with islet cell antibodies (ICA), determined in a standard immunofluorescence assay. 2 sera were GAD positive but ICA negative, 4 were positive only for ICA, and 6 were negative for both GAD and ICA, as were the sera of 20 controls. The data illustrate that antibodies against GAD-65 are present in a majority of patients with type 1 diabetes mellitus and that autoantibodies against other islet cell antigens also exist. The radioligand-binding assay, which is convenient and sensitive for detecting GAD antibodies, will facilitate the screening of individuals with autoimmune islet cell disease.

Full text

PDF
2084

Images in this article

2086Image
on p.2086
2086Image
on p.2086
2087Image
on p.2087
2087Image
on p.2087

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andersson A. Isolated mouse pancreatic islets in culture: effects of serum and different culture media on the insulin production of the islets. Diabetologia. 1978 Jun;14(6):397–404. doi: 10.1007/BF01228134. [DOI] [PubMed] [Google Scholar]
  2. Atkinson M. A., Maclaren N. K., Scharp D. W., Lacy P. E., Riley W. J. 64,000 Mr autoantibodies as predictors of insulin-dependent diabetes. Lancet. 1990 Jun 9;335(8702):1357–1360. doi: 10.1016/0140-6736(90)91241-2. [DOI] [PubMed] [Google Scholar]
  3. Baekkeskov S., Aanstoot H. J., Christgau S., Reetz A., Solimena M., Cascalho M., Folli F., Richter-Olesen H., De Camilli P., Camilli P. D. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature. 1990 Sep 13;347(6289):151–156. doi: 10.1038/347151a0. [DOI] [PubMed] [Google Scholar]
  4. Baekkeskov S., Landin M., Kristensen J. K., Srikanta S., Bruining G. J., Mandrup-Poulsen T., de Beaufort C., Soeldner J. S., Eisenbarth G., Lindgren F. Antibodies to a 64,000 Mr human islet cell antigen precede the clinical onset of insulin-dependent diabetes. J Clin Invest. 1987 Mar;79(3):926–934. doi: 10.1172/JCI112903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baekkeskov S., Nielsen J. H., Marner B., Bilde T., Ludvigsson J., Lernmark A. Autoantibodies in newly diagnosed diabetic children immunoprecipitate human pancreatic islet cell proteins. Nature. 1982 Jul 8;298(5870):167–169. doi: 10.1038/298167a0. [DOI] [PubMed] [Google Scholar]
  6. Baekkeskov S., Warnock G., Christie M., Rajotte R. V., Larsen P. M., Fey S. Revelation of specificity of 64K autoantibodies in IDDM serums by high-resolution 2-D gel electrophoresis. Unambiguous identification of 64K target antigen. Diabetes. 1989 Sep;38(9):1133–1141. doi: 10.2337/diab.38.9.1133. [DOI] [PubMed] [Google Scholar]
  7. Björk E., Kämpe O., Karlsson F. A., Pipeleers D. G., Andersson A., Hellerström C., Eizirik D. L. Glucose regulation of the autoantigen GAD65 in human pancreatic islets. J Clin Endocrinol Metab. 1992 Dec;75(6):1574–1576. doi: 10.1210/jcem.75.6.1464667. [DOI] [PubMed] [Google Scholar]
  8. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bottazzo G. F., Florin-Christensen A., Doniach D. Islet-cell antibodies in diabetes mellitus with autoimmune polyendocrine deficiencies. Lancet. 1974 Nov 30;2(7892):1279–1283. doi: 10.1016/s0140-6736(74)90140-8. [DOI] [PubMed] [Google Scholar]
  10. Bu D. F., Erlander M. G., Hitz B. C., Tillakaratne N. J., Kaufman D. L., Wagner-McPherson C. B., Evans G. A., Tobin A. J. Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2115–2119. doi: 10.1073/pnas.89.6.2115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Burman P., Mårdh S., Norberg L., Karlsson F. A. Parietal cell antibodies in pernicious anemia inhibit H+, K+-adenosine triphosphatase, the proton pump of the stomach. Gastroenterology. 1989 Jun;96(6):1434–1438. doi: 10.1016/0016-5085(89)90509-x. [DOI] [PubMed] [Google Scholar]
  12. Chang Y. C., Gottlieb D. I. Characterization of the proteins purified with monoclonal antibodies to glutamic acid decarboxylase. J Neurosci. 1988 Jun;8(6):2123–2130. doi: 10.1523/JNEUROSCI.08-06-02123.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Christgau S., Schierbeck H., Aanstoot H. J., Aagaard L., Begley K., Kofod H., Hejnaes K., Baekkeskov S. Pancreatic beta cells express two autoantigenic forms of glutamic acid decarboxylase, a 65-kDa hydrophilic form and a 64-kDa amphiphilic form which can be both membrane-bound and soluble. J Biol Chem. 1991 Nov 5;266(31):21257–21264. [PubMed] [Google Scholar]
  14. Christie M. R., Brown T. J., Cassidy D. Binding of antibodies in sera from Type 1 (insulin-dependent) diabetic patients to glutamate decarboxylase from rat tissues. Evidence for antigenic and non-antigenic forms of the enzyme. Diabetologia. 1992 Apr;35(4):380–384. doi: 10.1007/BF00401206. [DOI] [PubMed] [Google Scholar]
  15. Christie M. R., Vohra G., Champagne P., Daneman D., Delovitch T. L. Distinct antibody specificities to a 64-kD islet cell antigen in type 1 diabetes as revealed by trypsin treatment. J Exp Med. 1990 Sep 1;172(3):789–794. doi: 10.1084/jem.172.3.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Chu G., Hayakawa H., Berg P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 1987 Feb 11;15(3):1311–1326. doi: 10.1093/nar/15.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Genovese S., Bonifacio E., McNally J. M., Dean B. M., Wagner R., Bosi E., Gale E. A., Bottazzo G. F. Distinct cytoplasmic islet cell antibodies with different risks for type 1 (insulin-dependent) diabetes mellitus. Diabetologia. 1992 Apr;35(4):385–388. doi: 10.1007/BF00401207. [DOI] [PubMed] [Google Scholar]
  18. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  19. Karlsen A. E., Hagopian W. A., Grubin C. E., Dube S., Disteche C. M., Adler D. A., Bärmeier H., Mathewes S., Grant F. J., Foster D. Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8337–8341. doi: 10.1073/pnas.88.19.8337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaufman D. L., Erlander M. G., Clare-Salzler M., Atkinson M. A., Maclaren N. K., Tobin A. J. Autoimmunity to two forms of glutamate decarboxylase in insulin-dependent diabetes mellitus. J Clin Invest. 1992 Jan;89(1):283–292. doi: 10.1172/JCI115573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kaufman D. L., Houser C. R., Tobin A. J. Two forms of the gamma-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions. J Neurochem. 1991 Feb;56(2):720–723. doi: 10.1111/j.1471-4159.1991.tb08211.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kämpe O., Andersson A., Björk E., Hallberg A., Karlsson F. A. High-glucose stimulation of 64,000-Mr islet cell autoantigen expression. Diabetes. 1989 Oct;38(10):1326–1328. doi: 10.2337/diab.38.10.1326. [DOI] [PubMed] [Google Scholar]
  23. Lendrum R., Walker G., Cudworth A. G., Theophanides C., Pyke D. A., Bloom A., Gamble D. R. Islet-cell antibodies in diabetes mellitus. Lancet. 1976 Dec 11;2(7998):1273–1276. doi: 10.1016/s0140-6736(76)92033-x. [DOI] [PubMed] [Google Scholar]
  24. Michelsen B. K., Petersen J. S., Boel E., Møldrup A., Dyrberg T., Madsen O. D. Cloning, characterization, and autoimmune recognition of rat islet glutamic acid decarboxylase in insulin-dependent diabetes mellitus. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8754–8758. doi: 10.1073/pnas.88.19.8754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oertel W. H., Schmechel D. E., Tappaz M. L., Kopin I. J. Production of a specific antiserum to rat brain glutamic acid decarboxylase by injection of an antigen-antibody complex. Neuroscience. 1981;6(12):2689–2700. doi: 10.1016/0306-4522(81)90113-5. [DOI] [PubMed] [Google Scholar]
  26. Persson H., Pelto-Huikko M., Metsis M., Söder O., Brene S., Skog S., Hökfelt T., Ritzén E. M. Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells. Mol Cell Biol. 1990 Sep;10(9):4701–4711. doi: 10.1128/mcb.10.9.4701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rorsman P., Berggren P. O., Bokvist K., Ericson H., Möhler H., Ostenson C. G., Smith P. A. Glucose-inhibition of glucagon secretion involves activation of GABAA-receptor chloride channels. Nature. 1989 Sep 21;341(6239):233–236. doi: 10.1038/341233a0. [DOI] [PubMed] [Google Scholar]
  28. Rowley M. J., Mackay I. R., Chen Q. Y., Knowles W. J., Zimmet P. Z. Antibodies to glutamic acid decarboxylase discriminate major types of diabetes mellitus. Diabetes. 1992 Apr;41(4):548–551. doi: 10.2337/diab.41.4.548. [DOI] [PubMed] [Google Scholar]
  29. Seed B. An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. 1987 Oct 29-Nov 4Nature. 329(6142):840–842. doi: 10.1038/329840a0. [DOI] [PubMed] [Google Scholar]
  30. Sorenson R. L., Garry D. G., Brelje T. C. Structural and functional considerations of GABA in islets of Langerhans. Beta-cells and nerves. Diabetes. 1991 Nov;40(11):1365–1374. doi: 10.2337/diab.40.11.1365. [DOI] [PubMed] [Google Scholar]
  31. Thivolet C. H., Tappaz M., Durand A., Petersen J., Stefanutti A., Chatelain P., Vialettes B., Scherbaum W., Orgiazzi J. Glutamic acid decarboxylase (GAD) autoantibodies are additional predictive markers of type 1 (insulin-dependent) diabetes mellitus in high risk individuals. Diabetologia. 1992 Jun;35(6):570–576. doi: 10.1007/BF00400486. [DOI] [PubMed] [Google Scholar]
  32. Uibo R., Mackay I. R., Rowley M., Humphries P., Armstrong J. M., McNeilage J. Inhibition of enzyme function by human autoantibodies to an autoantigen pyruvate dehydrogenase E2: different epitope for spontaneous human and induced rabbit autoantibodies. Clin Exp Immunol. 1990 Apr;80(1):19–24. doi: 10.1111/j.1365-2249.1990.tb06435.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Velloso L. A., Kämpe O., Eizirik D. L., Hallberg A., Andersson A., Karlsson F. A. Human autoantibodies react with glutamic acid decarboxylase antigen in human and rat but not in mouse pancreatic islets. Diabetologia. 1993 Jan;36(1):39–46. doi: 10.1007/BF00399091. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES