Molecular cloning and characterization of a highly basic protein, IA-4, expressed in pancreatic islets and brain.

A substraction library was constructed from mouse insulinoma (betaTC-1) and glucagonoma (alphaTC-1) cell lines. Differential screening and sequencing revealed a novel cDNA clone, IA-4, which was expressed in the islets of Langerhans and the brain. IA-4 cDNA is 1,007 bp in length and predicts a protein of 187 amino acids with a molecular mass of 19,940 D. Examination of the amino acid sequence showed a high content of arginine (18.7%), proline (14.4%), alanine (16.0%), leucine (13.4%) and glycine (9.6%). The deduced pI value is 12.5 indicating a highly basic protein. Northern blot analysis revealed a 1-kb mRNA highly expressed in brain, trigeminal ganglia and cell lines of neuroendocrine origin. Rabbit polyclonal antiserum raised against a synthetic IA-4 peptide, designated Pep-1, not only reacted with IA-4 recombinant protein, but also immunostained the islets of Langerhans and large neurons of the hippocampus, cerebral cortex, spinal cord, dorsal ganglia and Purkinje cells of the cerebellum. The high expression of IA-4 protein in neuroendocrine cells and its unique amino acid sequence suggest that IA-4 may have an important, but still undetermined, function in these special cell types.

Autoantigens in insulin-dependent diabetes mellitus: molecular cloning and characterization of human IA-2 beta.

In this study, we describe the isolation, expression, and characterization of a new member of the transmembrane protein tyrosine phosphatase family from human brain, designated IA-2 beta. The 3853-bp cDNA encodes 986 amino acids with a molecular mass of 108,044 daltons (a predicted pI value of 5.8). The intracellular domain of human IA-2 beta is 74% identical to human IA-2. Northern blot analysis showed that IA-2 beta cDNA recognized two transcripts (approximately 5.0 kb and 4.0 kb) in four of five human insulinomas, one glucagonoma, and in normal human brain, pituitary, and pancreas, but not in a variety of other normal tissues. Rabbit antiserum, raised against the intracellular domain of IA-2 beta, reacted with pancreatic islets. Treatment of in vitro-translated full-length IA-2 beta protein with trypsin converted it into a 37-kD fragment. Using recombinant human IA-2 beta, we developed a radioimmunoprecipitation assay to measure autoantibodies in the sera of patients with insulin-dependent diabetes mellitus (IDDM). Seventy-six new-onset IDDM patients were tested. Thirty-seven percent (28 of 76) of the IDDM sera-but less than 1% of the control sera (1 of 174)-reacted with IA-2 beta. The same IDDM sera tested for autoantibodies to IA-2 and glutamic acid decarboxylase (GAD65) showed that 64% (49 of 76) and 57% (43 of 76), respectively, were positive. All but two of the IA-2 beta autoantibody-positive sera also reacted with IA-2, supporting the close sequence similarity between the two molecules. Combination of any two markers, such as IA-2 beta and IA-2, or IA-2 beta and GAD65, or IA-2 and GAD65, revealed that 67%, 74%, and 87% of IDDM sera were positive for autoantibodies, respectively. Blocking of IDDM sera with recombinant IA-2, IA-2 beta, or GAD65 resulted in marked inhibition of reactivity of IDDM sera with pancreatic islet sections as measured by islet cell autoantibody immunofluorescence. This result suggests that these three autoantigens are the major targets of islet-cell autoantibody reactivity.

Molecular characterization of a pancreas-specific protein disulfide isomerase, PDIp.

A novel tissue-specific cDNA, PDIp, was previously isolated from human pancreas. It encodes a protein that is structurally and functionally related to protein disulfide isomerase (PDI). To compare the expression pattern of PDI and PDIp in human pancreas and liver tissues, we prepared rabbit polyclonal antiserum against a recombinant glutathione-S-transferase-coupled PDIp fusion protein. Western blot analysis revealed that pancreas expresses both PDI and PDIp, whereas liver only expresses PDI. Rabbit antiserum raised against recombinant PDIp immunostained specifically to the acinar cells of human pancreas. Treatment of PDIp with peptide:N-glycosidase F caused PDIp down shift in the NaDodSO4-PAGE gel, indicating that PDIp is a glycoprotein. A 2.0-kb message was detected from mouse pancreas using a human PDIp cDNA probe. Similarly, PDIp glycoprotein was detected in mouse pancreas extract by anti-human PDIp antiserum, suggesting that PDIp is highly conserved in human and mouse pancreas. From these studies, we conclude that the pancreas expresses two members of PDI and that PDIp is a glycoprotein specifically expressed in pancreatic acinar cells.

Characterization and chromosomal localization of a new protein disulfide isomerase, PDIp, highly expressed in human pancreas.

Protein disulfide isomerase (PDI) catalyzes protein folding and thiol-disulfide interchange reactions. The enzyme is localized in the lumen of endoplasmic reticulum (ER) and is abundant in secretory cells of various tissues. In this study we describe the isolation and characterization from human pancreas of a new protein, PDIp, that is structurally and functionally related to PDIs. PDIp cDNA is 1,659 bp in length and predicts a protein with an open reading frame of 511 amino acids. PDIp amino acid sequence shows 46% identity and 66% similarity to that of human PDI. PDIp possesses two thioredoxin-like active sites (WCGHCQ and WCTHCK) and an endoplasmic reticulum retention signal sequence, KEEL, at the carboxyl terminus. Northern analysis of normal human tissues and various human tumor cell lines revealed PDIp mRNA (2.0 kb) expression only in the normal pancreas. Recombinant PDIp protein catalyzed reductive cleavage of insulin and renaturation of reduced RNaseA. Somatic cell genetics and fluorescence in situ hybridization localized the PDIp gene to the short arm of human chromosome 16. It is concluded that PDIp is a new member of the PDI family and is highly expressed in human pancreas.

Tumour formation and insulin secretion by clonal RINm5F cells following repeated subcutaneous transplantation in NEDH rats.

The effects of repeated s.c. transplantation of clonal insulin-secreting RINm5F cells in NEDH rats on tumour morphology, insulin-glucose homeostasis and the function of RINm5F cells re-established in culture were examined after maintenance in vivo for periods of up to 308 days. Transplantation of RINm5F cells for ten consecutive passages consistently produced a single encapsulated vascularized tumour associated with the development in recipient rats of hyperinsulinaemia, hypoglycaemia and eventual neuroglycopenic coma. At the tenth passage, tumour-bearing rats exhibited a markedly enhanced rate of insulin-stimulated glucose uptake by 19 days, with evidence of a large and variable insulin response to i.p. glucose. Survival was 22 +/- 2 days, and resected RINm5F cell tumours exhibited weak immunostaining for insulin in scattered cells, with strands of fibrous tissue separating clusters of tumour cells many of which had distinct polarity. There was no detectable immunostaining for glucagon, somatostatin or pancreatic polypeptide. The insulin content and insulin secretory output of RINm5F cells re-established in culture after 20, 146, 259 or 308 days propagation in vivo were generally enhanced compared with non-passaged RINm5F cells. The magnitude of the effect was not appreciably affected by the duration of maintenance in vivo, but it was critically dependent upon the subsequent period of culture in vitro. Thus, whereas 2-day cultured RINm5F cells from the eighth tumour passage exhibited a greater than 100-fold increment of insulin content and release, with enhanced secretory responsiveness to leucine, arginine, theophylline, K+ (25 mmol/l) or Ca2+ (7.6 mmol/l), RINm5F cells cultured for a further 19 days had almost completely lost the attributes resulting from 259 days of maintenance in vivo. The results indicate substantial enhancement of the functional capabilities of RINm5F cells in vivo, and suggest that the resulting tumours afford a novel model in NEDH rats of responsive trabecular-type insulinomas.