Differential detection of rat islet and brain glutamic acid decarboxylase (GAD) isoforms with sequence-specific peptide antibodies.

Abstract

We studied the distribution of the M(r) 65,000 and M(r) 67,000 isoforms of glutamic acid decarboxylase, GAD65 and GAD67, in rat islets and brain by immunocytochemistry. Synthetic peptides representing selected GAD65 or GAD67 sequences were used to produce sequence-specific antibodies, allowing differential immunocytochemical detection of the two isoforms. GAD-specific reactivity of each peptide antiserum was confirmed by ELISA, immunoblotting, and immunoprecipitation. Immunostaining specificity was verified by displacement with either immunizing or irrelevant peptide. Dual immunostaining with GAD isoform-specific antibodies and polyclonal antibodies to glucagon showed that GAD65 was primarily detected in rat pancreatic islet beta-cells, whereas alpha-cells had weak GAD65 staining. In contrast, GAD67 was detected primarily in alpha-cells. In rat brain, GAD65 and GAD67 were present in neuron cell bodies and processes. These data demonstrate that antibodies raised against the N-terminus of GAD allow differential immunocytochemical identification of GAD67 and GAD65. Differential expression of GAD isoforms within islet alpha- and beta-cells supports the role of GAD65 in autoimmune diabetes and stiff-man syndrome.

Glial Fibrillary Acidic Protein (GFAP)-like Immunoreactivity in Rat Endocrine Pancreas

“…Moreover, even though restricted to glucagon-secreting elements, GFAP expression should be added to an already long list of markers shared by the endocrine and nervous systems (Le Douarin 1988;Teitelman 1991;Larsson 1998). In particular, glucagon cells constantly or transiently express molecules that have been also reported in the nervous system, such as glucagon-like peptide-1 and glucagon-like peptide-2 (Eissele et al 1994;Larsen et al 1997), tyrosine hydroxylase (Alpert et al 1988), neuron-specific enolase (Lloyd et al 1984), the 67-kD isoform of the glutamic acid decarboxylase (Li et al 1995), chromogranins (Schmid et al 1994), synaptophysin (Bouwens et al 1997), insulin (Larsson 1998), and PYY (Larsson 1998). However, we emphasize that by the use of several different technical approaches, such as the removal of the entire ectoderm from embryonic rats (Pictet et al 1976) or the generation of chimeric chick-quail embryos (Andrew 1976;Fontaine and Le Douarin 1977), the hypothesis that pancreatic endocrine precursors reside in the neural crest appears less likely (Le Douarin 1988).…”

Conformation-dependent GAD65 autoantibodies in diabetes

“…To analyse GAD65Ab epitopes sensitive to the GAD65-E517P mutation and to define possible interactive sequences in other GAD65 regions, we analysed the binding of rabbit polyclonal antisera (Group C, Table 1) directed against GAD65-specific peptides [23]. As shown in Figure 1, the E517P point mutation reduced GAD65Ab binding to the C-terminal and middle regions, but not to the N terminus (amino acid residues 4-91) or to the GAD65 enzymat- -418), the GAD65-E517P mutation induced a marked reduction in antibody binding (Fig.…”

“…Group B included standard sera from ten children (aged 11-16 years) newly diagnosed with Type 1 diabetes, which were obtained at the clinical onset of disease [22]. Group C included 14 rabbit antisera previously generated by immunisation with synthetic peptides specific for human GAD65 [23]. Antibody specificities are shown in Table 1.…”

Does immune-tolerance treatment with Alum-formulated GAD65 protect insulin-production in the pancreatic islet β cells?

“…20 GAD65 is present mainly in pancreatic beta cells and neuron cells. 21,22 This enzyme produces GABA, which is stored in small neurotransmitter vesicles. 22 Two isoforms are known: GAD65 and GAD67.…”