Potential autoantigens in IDDM. Expression of carboxypeptidase-H and insulin but not glutamate decarboxylase on the beta-cell surface.
Insulin, carboxypeptidase-H (CP-H), and glutamate decarboxylase (GAD) have been identified as potential autoantigens in insulin-dependent diabetes mellitus (IDDM). Previous studies have described immunoreactive insulin as a surface molecule on the plasma membrane of rat islet cells and suggested that cell-surface insulin was derived during exocytosis by the fusion of insulin secretory granules with the beta-cell plasma membrane. These findings predict that insulin and other secretory granule-derived proteins such as the putative autoantigen CP-H may be colocalized with insulin at specific sites of exocytosis on the beta-cell surface. In studies to test this hypothesis, cell-surface staining of dispersed rat islet cells occurred in a granule-like pattern with antibodies for CP-H and insulin. The specificity of the CP-H antiserum was confirmed by immunoblotting and indicated that the antiserum was essentially monospecific for CP-H. Confocal laser microscopy confirmed that immunoreactive staining for CP-H and insulin was confined to the beta-cell surface. Colocalization of CP-H and insulin on the cell surface of beta-cells was demonstrated by double staining with antibodies to CP-H and insulin, and the percentage of beta-cells positive for both of these autoantigens increased twofold with increases in insulin secretion. In contrast, islet cells failed to reveal cell-surface staining for GAD65, another putative autoantigen in IDDM, under either basal or insulin stimulatory conditions or following exposure of islet cells to the cytokines interleukin-1 beta, tumor necrosis factor-alpha, and recombinant human interferon-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)
Aguilar-Diosdado
M
M
Department of Pediatrics, Washington University School of Medicine, Saint Louis, Missouri 63110.
Parkinson
D
D
Corbett
J A
JA
Kwon
G
G
Marshall
C A
CA
Gingerich
R L
RL
Santiago
J V
JV
McDaniel
M L
ML
D000818:Animals; D001324:Autoantigens / Q000032:analysis*; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000276:immunology*; D002462:Cell Membrane / Q000276:immunology; D003921:Diabetes Mellitus, Experimental / Q000276:immunology*; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D005968:Glutamate Decarboxylase / Q000276:immunology*; D015151:Immunoblotting; D007328:Insulin / Q000276:immunology*; D007515:Islets of Langerhans / Q000276:immunology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
D001324:Autoantigens; D005456:Fluorescent Dyes; D007328:Insulin; D002268:Carboxypeptidases; D043423:Carboxypeptidase H; D005968:Glutamate Decarboxylase; D016650:Fluorescein-5-isothiocyanate