Cloning, characterization, and autoimmune recognition of rat islet glutamic acid decarboxylase in insulin-dependent diabetes mellitus.

A 64-kDa islet protein is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). Autoantibodies against the 64-kDa protein were recently shown to immunoprecipitate glutamic acid decarboxylase (GAD; L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from brain and from islets. We present evidence that the autoantisera also recognize a hydrophilic islet protein of approximately 67 kDa in addition to the amphiphilic 64-kDa form. We have isolated a full-length rat islet GAD cDNA encoding a hydrophilic 67-kDa protein, which appears to be identical to rat brain 67-kDa GAD. A partial sequence of human insulinoma 67-kDa GAD was identical to human brain 67-kDa GAD. Allelic variations were observed in rat as well as in human 67-kDa GAD sequences. The expressed rat islet 67-kDa GAD protein is functional and is immunoprecipitated by IDDM sera; it comigrates electrophoretically with the 67-kDa islet autoantigen. The hydrophilic 67-kDa form of GAD in islets is an additional autoantigen in IDDM and is recognized by a different subset of autoantibodies than the 64-kDa autoantigen. Thus, mammalian cell lines expressing functionally active, recombinant GAD may become important tools to study the nature and the role of GAD autoreactivity in IDDM.     

Double-Label Immunofluorescence Study of Glutamic Acid Decarboxylase in the Fetal and Adult Ovine Pancreas by Light and Confocal Microscopy: Evidence for Predominant Beta-Cell Coexpression

Glutamic acid decarboxylase (GAD) is present in the central nervous system and in several nonneuronal tissues including the pancreatic islets. There are two isoforms with molecular weights of 65 kDa (GAD65) and 67 kDa (GAD67). The cellular specificity of the two molecular forms of GAD and their levels within the mammalian islets may be species-dependent, being coexpressed in both beta and in non-beta cells. We have examined the ovine pancreas, from the adult and fetal stages of late gestation, for the expression of GAD65 within the islet cells by double-label immunofluorescence light and confocal microscopy. In the adult tissue, GAD65 was colocalized in a majority of the beta cells (>95%), with only a few glucagon and somatostatin cells (<5%) showing immunolocalization. During the fetal stages GAD65 also showed a similar predominant beta-cell coexpression. The enzyme was also detected in a few fetal glucagon (<5%) but not somatostatin cells. In the degenerating large fetal islets, GAD65 was also observed in the majority of the residual beta cells. These results demonstrate that in the ovine pancreas GAD65 is expressed during fetal development and is predominantly beta-cell-restricted. This pattern of expression is maintained during adult life. However, the physiological role of pancreatic GAD and/or its biosynthetic product, γ-aminobutyric acid, in islet function in the sheep and in other ruminants remains unclear.