A 64 kDa antigen/glutamic acid decarboxylase (GAD) in fetal pig pro-islets: co-precipitation with a 38 kDa protein and recognition by T cells in humans at risk for insulin-dependent diabetes.
The development of insulin-dependent diabetes mellitus (IDDM) is associated with circulating antibodies to a pancreatic islet protein of MW 64,000 (64 kDa), reported to be glutamic acid decarboxylase (GAD). To investigate the antigenic properties of the 64 kDa antigen/GAD in IDDM we employed fetal pig pro-islets, a convenient source of islet antigens and an alternative to adult human islets for transplantation. Pro-islets contained a 64 kDa protein precipitated by antibodies in the sera of 9/14 (64%) at-risk IDDM, 12/33 (36%) recent-onset IDDM and 4/12 (33%) established IDDM patients and in 1/18 (5%) healthy control subjects. In addition, a 38 kDa protein was co-precipitated with the 64 kDa protein by 6/14 (43%) at-risk IDDM, 5/33 (12%) recent-onset IDDM, 1/12 (8%) established IDDM patient sera and 1/18 (5%) control subject serum. Both 64 kDa and 38 kDa antigens were specific to pro-islets; neither was detected in fetal pig thyrocytes, hepatocytes or splenocytes. The majority of the 64 kDa protein was co-precipitated with GAD enzymatic activity from pro-islets by either IDDM sera or a sheep anti-GAD serum. Previously, we showed that peripheral blood mononuclear cells (PBMC) from over half the subjects defined as being at risk for IDDM proliferate in response to fetal pig pro-islets. Proliferative responses of PBMC from pre-diabetic subjects to a crude extract of pro-islets were therefore measured before and after depletion of GAD by adsorption of the extract against GAD-1 monoclonal antibody. In 5/10 at-risk subjects, T cell stimulation indices were greater than the control mean + 2 SD and decreased in four by > 50% after depletion of GAD. In summary, a 64 kDa protein with the properties of GAD is present in fetal pig pro-islets and is recognized by both antibodies and T cells in a significant proportion of subjects at risk for early IDDM. Some subjects with anti-64 kDa antibodies also have antibodies that co-precipitate a 38 kDa pro-islet protein. Prospective studies of T cell reactivity in at-risk IDDM subjects are required to delineate the role of GAD and other candidate autoantigens in the initiation and progression of beta cell destruction.
DeAizpurua
H J
HJ
Burnet Clinical Research Unit, Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, Australia.
Honeyman
M C
MC
Harrison
L C
LC
D000293:Adolescent; D000328:Adult; D000818:Animals; D000941:Antigens / Q000302:isolation & purification; D001323:Autoantibodies; D001324:Autoantigens; D002648:Child; D003922:Diabetes Mellitus, Type 1 / Q000209:etiology / Q000276:immunology*; D005260:Female; D005333:Fetus / Q000276:immunology; D005968:Glutamate Decarboxylase / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D066298:In Vitro Techniques; D007515:Islets of Langerhans / Q000201:enzymology / Q000276:immunology*; D008213:Lymphocyte Activation; D008297:Male; D008875:Middle Aged; D013552:Swine; D013601:T-Lymphocytes / Q000276:immunology*
D000941:Antigens; D001323:Autoantibodies; D001324:Autoantigens; C075362:islet 64k antigen; D005968:Glutamate Decarboxylase