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 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-M(r) autoantigen. Immunoprecipitation analyses showed that 21/28 (75%) IDDM sera had rGA D64 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 GA D64 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-M(r) islet autoantigen, allowing further studies of the immunopathogenesis of IDDM.
Karlsen
A E
AE
Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden.
Hagopian
W A
WA
Petersen
J S
JS
Boel
E
E
Dyrberg
T
T
Grubin
C E
CE
Michelsen
B K
BK
Madsen
O D
OD
Lernmark
A
A
D000818:Animals; D001323:Autoantibodies / Q000032:analysis*; D001324:Autoantigens / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D001921:Brain / Q000201:enzymology* / Q000276:immunology; D003001:Cloning, Molecular; D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology / Q000276:immunology*; D005968:Glutamate Decarboxylase / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D006801:Humans; D007330:Insulin Antibodies / Q000032:analysis*; D007515:Islets of Langerhans / Q000201:enzymology* / Q000276:immunology; D007527:Isoenzymes / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D008970:Molecular Weight; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012016:Reference Values
D001323:Autoantibodies; D001324:Autoantigens; D007330:Insulin Antibodies; D007527:Isoenzymes; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase