RESUMO
An insulin granule membrane protein, phogrin (phosphatase homologue of granules from rat insulinoma), with homology to islet cell antigen (ICA) 512/IA-2 has recently been cloned from an insulinoma cDNA expression library with antigranule membrane sera. We have developed a radioimmunoassay for detecting antiphogrin autoantibodies using in vitro transcribed and translated phogrin and have established the sensitivity and specificity of this assay. Thirty-two of 57 (56%) new-onset patients with type I diabetes and 26 of 44 (59%) first-degree relatives followed to diabetes had anti-phogrin antibody levels exceeding the 99th percentile of 108 normal control subjects. Levels of antiphogrin autoantibodies correlated with ICA512/IA-2 autoantibodies (r = 0.82, P < 0.0001), but minimally with insulin autoantibodies (r = 0.20, P = 0.05) and not with GAD65 autoantibodies (r = 0.16, P = 0.12). Ninety-eight percent (57 of 58) of patients positive for anti-phogrin autoantibodies were also positive for autoantibodies against ICA512/IA-2. Nine percent (9 of 101) of new-onset patients and relatives followed to diabetes were ICA512/IA-2 autoantibody-positive but anti-phogrin autoantibody-negative. Preincubation of sera with recombinant ICA512/IA-2 protein completely for the majority and partially for a minority inhibited binding to in vitro translated phogrin. In three relatives in which ICA512/IA-2 autoantibodies converted to positivity with sequential follow-up, anti-phogrin autoantibodies developed at the same time. These results suggest that anti-phogrin and ICA512/IA-2 autoantibodies are related subsets of anti-islet autoantibodies.
Assuntos
Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas de Membrana/imunologia , Proteínas de Neoplasias/imunologia , Estado Pré-Diabético/imunologia , Proteínas Tirosina Fosfatases/imunologia , Adolescente , Adulto , Fatores Etários , Animais , Especificidade de Anticorpos , Autoantígenos , Criança , Pré-Escolar , DNA Complementar , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/genética , Feminino , Glutamato Descarboxilase/imunologia , Humanos , Lactente , Insulinoma , Ilhotas Pancreáticas/imunologia , Masculino , Pessoa de Meia-Idade , Núcleo Familiar , Neoplasias Pancreáticas , Estado Pré-Diabético/sangue , Estado Pré-Diabético/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Ratos , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores , Proteínas Recombinantes/imunologia , Valores de Referência , Sensibilidade e EspecificidadeRESUMO
An insulin granule membrane protein-tyrosine phosphatase (PTP) homologue, phogrin, was cloned by expression screening of a rat insulinoma cDNA library. The 3723-base pair cDNA encoded a transmembrane glycoprotein of 1004 amino acids (Mr 111876) that underwent post-translational proteolysis to 60-64-kDa products after a 30-min delay. The kinetics of proteolytic conversion (t1/2 = 45 min) and turnover (t1/2 = 12 h) were consistent with sorting and conversion in a late compartment of the secretory pathway. Studies on the native beta-cell protein suggested that the COOH-terminal PTP domain was on the cytosolic face of the secretory granule. The lumenal segment was comprised of a protease-resistant globular domain of around 25 kDa. Its localization and topology is thus consistent with a transmembrane receptor function related to granule biogenesis, exocytosis, or subsequent membrane recovery, and it should prove to be a useful cell biological marker for the granule membrane. High expression of the mRNA (5.4 kilobases) and protein was evident in islets, pancreatic alpha- and beta-cell tumor lines, brain cells, and other cells of neuroendocrine lineage. It is closely related to the diabetic autoantigen ICA512 (IA-2) (42% identity overall; 80% in the 260-amino acid PTP domain) and thus a potential target of autoimmunity in diabetes mellitus.