IA-2 and IA-2beta are members of the protein tyrosine phosphatase family and major autoantigens in insulin-dependent diabetes mellitus (IDDM). Autoantibodies to these molecules appear years before the development of clinical disease and can serve as predictive markers for identifying individuals at high risk of becoming diabetic. Individuals with autoantibodies to both IA-2 and glutamic acid decarboxylase (another important autoantigen in IDDM) have a 50% or greater liklihood of developing IDDM within 5 years. Relatively little is still known about the biological and immunogenic properties of the IA-2 molecule. Over the last year we have studied the expression and processing of IA-2 in the baculovirus system. Our studies showed that IA-2 is expressed as a 120 kDa glycosylated protein. The IA-2 protein is rapidly processed into a 64 kDa doublet and then into several smaller fragments. From these and other studies we conclude that IA-2 is not only the precursor of the 64 kDa fragment, which is probably the major naturally occurring cleavage product of IA-2, but also the precursor of several of the lower molecular weight fragments that are sometimes immunoprecipitated when a beta cell lysate is incubated with IDDM serum. Studies in the early 1990s from Europe showed that the treatment of a beta cell lysate with trypsin and immunoprecipitation with IDDM sera produced fragments of 37 and 40 kDa in size. The identity of these tryptic fragments, which reacted with a high percentage of IDDM sera, was unknown. Last year, using recombinant proteins, we showed that IA-2 is the precursor of the 40 kDa fragment and IA-2beta is the precursor of the 37 kDa fragment. The location of these tryptic fragments within the IA-2 and IA-2beta molecules, however, remained elusive. Over the last year we determined the precise location of these fragments. The 40 kDa tryptic fragment is 326 amino acids in length and begins at position 653 of IA-2. The 37 kDa fragment is 307 amino acids in length and begins at position 679 of IA-2beta. Both fragments extend to the end of their respective molecules. Of particular importance was the demonstration that the epitopes on IA-2 and IA-2beta are largely conformational. This was determined by reducing the disulfide bonds and alkylating them to prevent refolding. When the reduced and alkylated molecules were tested, the ability of IA-2 and IA-2beta to be recognized by IDDM autoantibodies was largely lost. We conclude from these studies that maintaining the conformational integrity of IA-2 and IA-2beta is essential for the detection of autoantibodies. These findings also suggest that it is highly likely that T cells, which react with linear peptides, will recognize very different antigenic determinants on IA-2 and IA-2beta than those recognized by autoantibodies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Intramural Research (Z01)
Project #
1Z01DE000423-13
Application #
6104597
Study Section
Special Emphasis Panel (OIIB)
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
1998
Total Cost
Indirect Cost
Name
National Institute of Dental & Craniofacial Research
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Cai, Tao; Hirai, Hiroki; Fukushige, Tetsunari et al. (2009) Loss of the transcriptional repressor PAG-3/Gfi-1 results in enhanced neurosecretion that is dependent on the dense-core vesicle membrane protein IDA-1/IA-2. PLoS Genet 5:e1000447
Kim, Soo Mi; Theilig, Franziska; Qin, Yan et al. (2009) Dense-core vesicle proteins IA-2 and IA-2{beta} affect renin synthesis and secretion through the {beta}-adrenergic pathway. Am J Physiol Renal Physiol 296:F382-9
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Notkins, Abner Louis (2007) New predictors of disease. Molecules called predictive autoantibodies appear in the blood years before people show symptoms of various disorders. Tests that detected these molecules could warn of the need to take preventive action. Sci Am 296:72-9
Mett, Vadim; Shamloul, Abdel-Moneim; Hirai, Hiroki et al. (2007) Engineering and expression of the intracellular domain of insulinoma-associated tyrosine phosphatase (IA-2ic), a type 1 diabetes autoantigen, in plants. Transgenic Res 16:77-84
Harashima, S-I; Harashima, C; Nishimura, T et al. (2007) Overexpression of the autoantigen IA-2 puts beta cells into a pre-apoptotic state: autoantigen-induced, but non-autoimmune-mediated, tissue destruction. Clin Exp Immunol 150:49-60
Li, Na; Shigihara, Toshikatsu; Tzioufas, Athanasios G et al. (2007) Human chorionic gonadotropin prevents Sjogren's syndrome-like exocrinopathy in mice. Arthritis Rheum 56:2211-5
Kubosaki, Atsutaka; Nakamura, Shinichiro; Clark, Anne et al. (2006) Disruption of the transmembrane dense core vesicle proteins IA-2 and IA-2beta causes female infertility. Endocrinology 147:811-5
Harashima, Shin-ichi; Clark, Anne; Christie, Michael R et al. (2005) The dense core transmembrane vesicle protein IA-2 is a regulator of vesicle number and insulin secretion. Proc Natl Acad Sci U S A 102:8704-9
Kubosaki, Atsutaka; Nakamura, Shinichiro; Notkins, Abner Louis (2005) Dense core vesicle proteins IA-2 and IA-2beta: metabolic alterations in double knockout mice. Diabetes 54 Suppl 2:S46-51

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