Rapid Publications Recombinant Glutamic Acid Decarboxylase (Representing the Single Isoform Expressed in Human Islets) Detects ID DM-Associated 64,000-Mr Autoanti bodies ALLAN E. KARLSEN, WILLIAM A. HAGOPIAN, JACOB S. PETERSEN, ESPER BOEL, THOMAS DYRBERG, CATHERINE E. GRUBIN, BIRGITTE K. MICHELSEN, OLE D. MADSEN, AND AKE LERNMARK

GAD is an autoantigen in IDDM. Molecular cloning and specific antibodies allowed us to demonstrate that only the lower Mr GAD64 isoform is expressed in human islets, in contrast to human brain, rat islets, and rat brain, all of which express both GAD64 and GAD67. Expression of the human islet GAD64 isoform in COS-7 and BHK cells resulted in an enzymatically active rGAD64, which is immunoreactive with diabetic sera comparable with that of the islet 64,000-Afr autoantigen. Immunoprecipitation analyses showed that 21/28 (75%) IDDM sera had rGAD64 antibodies compared with only 1/59 (1.7%) of the healthy control sera. In immunoblot analyses, an SMS serum—but only 1/10 randomly selected IDDM sera—recognized the blotted rGAD64 without relation to immunoprecipitation titers. In conclusion, only the GAD64 isoform is expressed in human islets, in contrast to rat islets, which also express the GAD67 isoform. The immunological properties of human rGAD64 are comparable with the native 64,000-Mr islet autoantigen, allowing further studies of the immunopathogenesis of IDDM. Diabetes 41:1355-59, 1992

From the Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden; The Hagedorn Research Institute, Gentofte, Denmark; The R.H. Williams Laboratory, Department of Medicine, University of Washington, Seattle; and Bioscience and Diabetes Research, Novo Nordisk A/S, Bagsvaerd, Denmark. Please address correspondence and reprint requests to Allan E. Karlsen, The Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark. Received for publication 8 May 1992 and accepted in revised form 9 July 1992. IDDM, insulin-dependent diabetes mellitus; GAD, glutamic acid decarboxylase; BHK cells, baby hamster kidney cells; rGAD, recombinant glutamic acid decarboxylase; SMS, stiff-man syndrome; GABA, "y-aminobutyric acid; CNS, central nervous system.

DIABETES, VOL. 41, OCTOBER 1992

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he 64,000-/Wr protein (1), recently identified as GAD (2), is the major antigen for autoantibodies in sera from IDDM patients (3-7). GAD catalyzes the formation of GABA, a major neuroinhibitor in the CNS. GAD also is expressed in the islets of Langerhans, although the significance of islet GAD and GABA is unclear (8). Antibodies against the 64,000-Mr autoantigen or GAD are found in sera in 75-96% of patients at the time of clinical onset (3-7,9), and have been demonstrated up to 8 yr before onset (3,6,9). In contrast, GAD antibodies are found in

Recombinant glutamic acid decarboxylase (representing the single isoform expressed in human islets) detects IDDM-associated 64,000-M(r) autoantibodies.

GAD is an autoantigen in IDDM. Molecular cloning and specific antibodies allowed us to demonstrate that only the lower M(r) GAD64 isoform is expressed...
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