Detection of pancreatic islet 64,000 M(r) autoantigens in insulin-dependent diabetes distinct from glutamate decarboxylase.

Patients with insulin-dependent diabetes (IDDM) possess antibodies to islet proteins of M(r)-64,000. Potential autoantigens of this M(r) include glutamate decarboxylase (GAD) and 65 kD heat shock protein. We have detected two distinct antibody specificities in IDDM that bind 50,000 M(r) or 37,000/40,000 M(r) proteolytic fragments of 64,000 M(r) proteins. In this study, we investigated relationships of these proteolytic fragments to GAD and heat shock proteins. Polyclonal antibodies to GAD bound 50,000 M(r) fragments of islet antigen. Recombinant GAD65, but not GAD67, blocked binding to this antigen, suggesting that 50,000 M(r) fragments are derived from islet GAD65. In contrast, GAD antibodies did not recognize 37,000/40,000 M(r) fragments, and neither GAD isoforms blocked autoantibody binding to precursors of these fragments. The 37,000/40,000 M(r) fragments, but not the 50,000 M(r) fragments, were detected after trypsin treatment of immunoprecipitates from insulinoma cells that lacked expression of major GAD isoforms. Antibodies in IDDM did not bind native or trypsinized islet heat shock proteins. Thus, IDDM patients possess antibodies to GAD, but also distinct antibodies to a 64,000 M(r) protein that is not related to known GAD isoforms or heat shock proteins.

Isolation and characterization of cDNA encoding the alpha subunit of Cap Z(36/32), an actin-capping protein from the Z line of skeletal muscle.

cDNA encoding the alpha chain of Cap Z has been isolated by screening a lambda gt11 library with affinity-purified antibodies. A single cDNA insert (designated CE2) of 2153 base pairs (bp) contains an open reading frame of 836 bp, which is incomplete at its 5' end. The technique of "rapid amplification of cDNA ends" has been used to extend the 5' end of this open reading frame to a potential transcription initiation site that is preceded by 320 bp of an apparently untranslated region. The protein predicted by the resulting nucleotide sequence has a Mr of 32,960 and contains four regions that show close homology with four alpha-chymotryptic digestion fragments of the alpha chain. The amino acid composition of the alpha chain of Cap Z and the predicted protein are also similar. Northern blot analysis of whole chicken embryos shows two mRNA species of 1.9 and 2.4 kilobases, respectively, that hybridize with CE2. Three potential polyadenylylation signals in two regions of CE2 460 bp apart are identified, suggesting that the two messages may result from the use of alternative polyadenylylation sites. Comparison of the sequence data with that of other known actin-capping and severing proteins shows no significant homologies, suggesting that Cap Z may be a member of a unique group of capping, nonsevering proteins.

cDNAs encoding the beta subunit of cap Z, the actin-capping protein of the Z line of muscle.

Three cDNAs encoding the beta chain of Cap Z have been isolated from lambda gt11 chicken libraries screened with antibodies. The coding region, which is identical among the cDNAs, contains 831 basepairs encoding a protein with 277 amino acid residues and Mr = 31,352. The predicted protein sequence contains eight regions that match perfectly the NH2-terminal sequence of eight peptides isolated from muscle Cap Z beta. The amino acid compositions of the protein predicted from the cDNA sequence and Cap Z beta are similar. Comparison of the cDNA sequence and the predicted protein sequence with those of other actin-binding proteins and all sequences in the GenBank and NBRF Protein databases shows no remarkable similarities. Two of the three cDNAs contain the complete coding region. Both coding regions begin with a consensus translation start site; however, their 5'-untranslated regions are different. Northern analysis of whole chicken embryos and adult chicken tissues shows two mRNA species. The embryo and non-muscle tissues have transcripts of 1.35 and 2.00 kilobases, and the muscle tissues have transcripts of 2.15 and 1.45 kilobases. Southern analysis of chicken genomic DNA shows that there are probably two related genes, one more similar to the cDNA than the other.