Autoantigen In Insulin-dependent Diabetes Mellitus Research Articles

Expression of a Glutamate Transporter cDNA in Human Pancreatic Islets

A number of neuron-specific molecules have been found to be also expressed in pancreatic islets. Some of them might play a major role in the pathogenesis of an autoimmune reaction against the insulin-secreting beta-cells resulting in beta-cell destruction and the manifestation of insulin-dependent diabetes mellitus (IDDM). In ongoing studies our goal was to determine the role of other neuron-specific molecules in beta-cells function and in insulitis. We cloned a L-glutamate transporter from a highly purified fraction of human islet cells. The expression of that specific mRNA was marked in various regions of the brain; weak expression was detected in human islet cells, while no expression in any other tissue was observed. Sequence analysis revealed identity to the transcripts isolated from human brain. The cloned novel cDNA from human islets encodes a key molecule of the excitatory neurotransmission pathway. The techniques used provide a model for the cloning and expression of molecules expressed both in endocrine and neuronal tissue. With characterization of its structure the possible role of the L-glutamate transporter as autoantigen in IDDM is now under investigation.

Expression and Function of IA-2 Family Proteins, Unique Neuroendocrine-specific Protein-tyrosine Phosphatases

IA-2 (also known as islet cell antigen ICA-512) and IA-2 beta (also known as phogrin, phosphatase homologue in granules of insulinoma) are major autoantigens in insulin-dependent diabetes mellitus (IDDM). Autoantibodies against both proteins are expressed years before clinical onset, and they become predictive markers for high-risk subjects. However, the role of these genes in the IDDM pathogenesis has been reported fairly negative by recent studies. IA-2 and IA-2 beta are type I transmembrane proteins that possess one inactive protein-tyrosine phosphatase (PTP) domain in the cytoplasmic region, and act as one of the constituents of regulated secretory pathways in various neuroendocrine cell types including pancreatic beta-cells. Existence of IA-2 homologues in different species suggests a fundamental role in neuroendocrine function. Studies of knockout animals have shown their involvement in maintaining hormone content, however, their specific steps in the secretory pathway IA-2 functions as well as their molecular mechanisms in the hormone content regulation are still unknown. More recent studies have suggested a novel function showing that they contribute to pancreatic beta-cell growth. This review attempts to show the possible biological functions of IA-2 family, focusing on their expression and localization in the neuroendocrine cells.

Diabetic sera react with the glutamic acid decarboxylase molecule in a dimeric-oligomeric form.

Glutamic acid decarboxylase (GAD) is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). This was initially identified as a 64-65 kD molecule according to migration in gels after immunoprecipitation from pancreatic islets. We studied the antigenicity of two different radiolabelled preparations of GAD, derived either by affinity purification from porcine brain and known to contain GAD 65 and GAD 67, or by expression from a cDNA for human GAD 65 by rabbit reticulocyte lysate (RRL). Radiolabelled immunoprecipitated pellets from the reaction of potent antisera to GAD from patients with IDDM were examined by autoradiography after SDS-PAGE under reducing or non-reducing conditions. Also, preparations of porcine brain GAD were "depleted' of GAD by exposure to antisera, and then similarly re-examined. Autoradiography of radiolabelled GAD either affinity purified from porcine brain, or expressed by RRL, showed that the immunoprecipitated protein migrated under non-reducing conditions according to a M(r) of approximately 110-130 kD, corresponding to dimeric forms of monomeric GAD of approximately 55-65 kD. Depletion by immunoprecipitation of this minor higher M(r) component from preparations of GAD left, in the supernatant, an abundance of GAD of M(r) 64-65 kD corresponding to monomer that was completely non-reactive with potent IDDM sera. We conclude that IDDM sera react with the GAD molecule in a dimeric (or oligomeric) form. Our findings have general connotations for self-tolerance and autoimmunity.

Multimerization of the Protein-tyrosine Phosphatase (PTP)-like Insulin-dependent Diabetes Mellitus Autoantigens IA-2 and IA-2β with Receptor PTPs (RPTPs): INHIBITION OF RPTPα ENZYMATIC ACTIVITY

Most receptor-type protein-tyrosine phosphatases (RPTPs) contain two tandem PTP domains. For some RPTPs the enzymatically inactive membrane-distal phosphatase domains (D2) were found to bind enzymatically active membrane proximal PTP (D1) domains, and oligomerization has been proposed as a general regulatory mechanism. The RPTP-like proteins IA-2 and IA-2beta, major autoantigens in insulin-dependent diabetes mellitus, contain just a single enzymatically inactive PTP-like domain. Their physiological role is as yet enigmatic. To investigate whether the catalytically inactive cytoplasmic domains of IA-2 and IA-2beta are involved in oligomerization, we exploited interaction trap assay in yeast and glutathione S-transferase pull-down and co-immunoprecipitation strategies on lysates of transfected COS-1 cells. The results show that IA-2 and IA-2beta are capable of homo- and heterodimerization to which both the juxtamembrane region and the phosphatase-like segment can contribute. Furthermore, they can form heterodimers with some other RPTP members, most notably RPTPalpha and RPTPepsilon, and down-regulate RPTPalpha enzymatic activity. Thus, in addition to homo-dimerization, the enzymatic activity of receptor-type PTPs can be regulated through heterodimerization with other RPTPs, including the catalytically inactive IA-2 and IA-2beta.

Expression, characterization, processing and immunogenicity of an insulin-dependent diabetes mellitus autoantigen, IA-2, in Sf-9 cells.

Autoantibodies to a 64-kD protein and a 40-kD tryptic fragment from pancreatic islets have been detected at high frequency in the sera of patients with insulin-dependent diabetes mellitus (IDDM). IA-2, a newly isolated transmembrane protein tyrosine phosphatase, is a major islet cell autoantigen in IDDM and the precursor of a 40-kD tryptic fragment. To express large quantities of recombinant IA-2 protein and analyse post-translational modifications we expressed full-length human IA-2 in baculovirus-infected Sf-9 cells. IA-2 expression was analysed by Western blot and by immunoprecipitation of 35S-methionine-radiolabelled proteins with rabbit antisera or IDDM sera. A 120-kD IA-2 protein was detected during the early, but not the late, phase of the infection. Pulse-chase experiments showed that the 120-kD protein was processed into fragments of 64 kD and smaller fragments of approximately 50 kD, 38 kD and 32 kD. The 64-kD fragment appeared as a doublet. Tunicamycin and PNGase F treatment down-shifted the 120-kD protein and the 64-kD doublet into lower molecular weight bands, suggesting that both were glycosylated. Trypsin treatment converted the 120-kD protein and the 64-kD doublet into a 40-kD fragment. Baculovirus-expressed IA-2 was as sensitive or slightly more sensitive than in vitro translated IA-2 in detecting autoantibodies to IA-2: 66% of sera from newly diagnosed IDDM patients reacted with baculovirus-expressed IA-2 compared with 59% of the same sera which reacted with in vitro translated IA-2. It is concluded that baculovirus-expressed IA-2 is a good source of autoantigen and that a number of lower molecular weight fragments with which IDDM autoantibodies react are derived from the 120-kD full-length IA-2 molecule.

IL-12 administration reveals diabetogenic T cells in genetically resistant I-Ealpha-transgenic nonobese diabetic mice: resistance to autoimmune diabetes is associated with binding of Ealpha-derived peptides to the I-A(g7) molecule.

Nonobese diabetic (NOD) and NOD-DRalpha transgenic (tg) mice, expressing Aalpha(d):Abeta(g7) and Aalpha(d):Abeta(g7) plus DRalpha:Ebeta(g7) class II molecules, respectively, both develop insulin-dependent diabetes mellitus (IDDM), whereas NOD-Ealpha tg mice expressing Aalpha(d):Abeta(g7) plus Ealpha:Ebeta(g7) are protected. We show that IL-12 administration induces rapid IDDM onset in NOD-DRalpha but fails to provoke insulitis and diabetes in NOD-Ealpha tg mice. Nevertheless, T cells from IL-12-treated NOD-Ealpha tg mice secrete IFN-gamma and transfer IDDM to NOD-SCID and NOD-Ealpha-SCID recipients, demonstrating the presence of peripheral diabetogenic Th1 cells in the protected mice. Surprisingly, regulatory cells were undetectable. Moreover, Ealpha:Ebeta(g7) could substitute for DRalpha:Ebeta(g7) in Ag presentation, arguing against mechanisms of protection involving capture of diabetogenic I-A(g7)-restricted epitopes by Ealpha:Ebeta(g7)molecules. Interestingly, the expression of naturally processed epitopes derived from DRalpha- and Ealpha-chains bound to I-A(g7) is different in the two strains of tg mice, and the difference is enhanced by IL-12 administration. I-A(g7) molecules from both NOD-DRalpha and NOD-Ealpha tg mice present the conserved DRalpha/Ealpha 52-68 sequence, at high and low levels, respectively. In addition, only IDDM-resistant NOD-Ealpha tg mice possess APCs bearing Ealpha65-77/I-A(g7) complexes, which tolerize the specific T cells. This is associated with the selective inhibition of the response to insulinoma-associated protein 2 (IA-2), an autoantigen in IDDM. Our results support protective mechanisms based on I-A(g7) blockade by peptides unique to the Ealpha-chain, such as Ealpha65-77 and/or tolerance of diabetogenic T cells cross-reactive with Ealpha-peptide/I-A(g7) complexes.

Importance of GAD65 peptides and I-Ag7 in the development of insulitis in nonobese diabetic mice.

Insulin-dependent diabetes mellitus (IDDM) develops in nonobese diabetic (NOD) mice through the destruction of the B cells in pancreatic Langerhans islets by islet autoantigen-specific T cells. The islet autoantigen glutamic acid decarboxylase 65 (GAD65) is thought to be a major target autoantigen in IDDM. In the present report, we established GAD65-specific T-cell clones using overlapping peptides that cover the amino acid sequences of mouse GAD65. T-cell epitopes of GAD65 were characterized by proliferation and binding assays using various analogue peptides and wild-type or mutant I-Ag7 transfectants. The efficacy of the peptide vaccine in IDDM was determined by administering T-cell epitope peptides to NOD mice and evaluating the histopathology of their insulitis. We obtained two types of T-cell clone, one specific for peptide p316-335 and another specific for p531-545 of GAD65. The p531-545 site has already been identified, but we report the p316-335 site for the first time. T-cell clones recognized those peptides in the wild-type I-Ag7 but not in the mutant I-Ag7 in which the serine at position 57 of the beta-chain was replaced by an aspartic acid. Both the p316-335 and p531-545 peptides bound weakly to I-Ag7. Some peptides with amino acid substitutions had antagonistic activity, and administration of a large amount of wild-type peptide reduced the severity of insulitis in NOD mice. Our results suggest that peptide vaccine therapy may be useful in autoimmune diseases, including IDDM.

Inhibition of Mitochondrial Oxidative Phosphorylation Induces Hyper-Expression of Glutamic Acid Decarboxylase in Pancreatic Islet Cells

It has been hypothesised that mitochondrial dysfunction in pancreatic beta cells could produce hyper-expression of glutamic acid decarboxylase (GAD), a major autoantigen in insulin-dependent diabetes mellitus (IDDM) (Degli Esposti, M. and Mackay, I.R. Diabetologia 40: 352–356, 1997). Here we report that specific inhibition of mitochondrial respiration enhances the expression of GAD in both foetal mouse pancreatic tissue and hamster HIT-TI5 cells. Inhibitors of NADH-ubiquinone oxidoreductase (complex I) seem to be particularly effective in increasing the expression of GAD in both foetal mouse pancreas and HIT-T15 hamster beta cells, especially in the presence of nutrients such as arginine and glucose. These results represent the first evidence that GAD expression is enhanced under conditions that are toxic to pancreatic beta cells, and establish a link between mitochondrial dysfunction and expression of IDDM autoantigens.

Genomic Structure and Promoter Sequence of the Insulin-Dependent Diabetes Mellitus Autoantigen, IA-2 (PTPRN)

IA-2 is a transmembrane protein tyrosine phosphatase, expressed in neuroendocrine cells, and a major autoantigen in insulin-dependent diabetes mellitus. In the present study we elucidated the structure of the IA-2 gene (HGMW-approved symbol PTPRN) and its promoter sequence. A 40-kb genomic clone covering the whole IA-2 coding sequence and 4 kb proximal 5′-upstream sequence was isolated and mapped. The IA-2 gene encompasses approximately 20 kb with 23 exons ranging from 34 bp to more than 650 bp. The extracellular domain is encoded by exons 1–12, the transmembrane region by exon 13, and the intracellular domain by exons 14–23. The transcriptional start site(s) of the IA-2 gene was mapped by 5′ rapid amplification of cDNA ends to 97 bp upstream of the translational start site. A 3-kb 5′-upstream region was sequenced, revealing a GC-rich region and TATA-less sequence containing several potential transcription-regulating sites (i.e., Sp1, CREB, GATA-1, and MZF). Functional promoter activity was confirmed by transient transfection of U87MG cells with deletion mutants linked to a luciferase reporter gene.

IgG subclass antibodies to glutamic acid decarboxylase and risk for progression to clinical insulin-dependent diabetes

Pancreatic islet beta cell destruction leading to insulin-dependent diabetes mellitus (IDDM) is believed to be mediated by a T-helper 1 (T H1) lymphocyte response to islet antigens. In the mouse, T H1 (IL-2, IFN-γ) and T H2 (IL-4, -5, -6, -10) responses are associated with the generation of IgG2a and IgG1 subclasses, respectively. The equivalent human subclasses have not been defined. Because the IgG subclass response to an antigen may be a potentially useful marker of T H1/T H2 immune balance we measured IgG subclass antibodies to glutamic acid decarboxylase (GAD), a major islet autoantigen in IDDM, in 34 newly-diagnosed IDDM patients and in 28 at-risk, first-degree relatives of people with IDDM. In the newly-diagnosed patients, total IgG antibodies to GAD were detected in 74% (25/34); IgG1 and/or IgG3 were significantly more frequent than IgG4 or IgG4/IgG2 (14/34 versus 5/34, p = 0.01). GAD antibody-negative patients were significantly younger ( p = 0.01). In 15 at-risk relatives who had not progressed to clinical diabetes after a median of 4.5 years, 10 had IgG2 and/or IgG4 antibodies compared to only 3/13 progressors ( p = 0.02). Total IgG and IgG2 antibodies were higher in non-progressors. Non-progressors were older than progressors ( p = 0.01), and relatives with IgG2 and/or IgG4 responses were also older ( p = 0.01). These results suggest that IgG subclass antibodies to GAD may contribute to diabetes risk assessment in islet antibody relatives.

Identification of mimicry peptides based on sequential motifs of epitopes derived from 65-kDa glutamic acid decarboxylase.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease with a predominantly non-hereditary etiology that results in a destruction of pancreatic beta cells by autoaggressive T lymphocytes. Neither the mechanism of initial stimulation of these T cells nor the nature of the environmental factors implicated in the disease have so far been identified. However, both issues are taken into account by the hypothesis of initial T cell activation by viral or bacterial mimicry peptides with sequence similarities to pancreatic self antigens. We determined sequential epitope motifs to search for mimicry peptides stimulating T cell lines specific for two epitopes derived from the IDDM autoantigen 65-kDa glutamic acid decarboxylase (GAD65). These were GAD65 (88-99), presented by HLA-DRB1*0101, and GAD65 (248-257), presented by HLA-DRB5*0101. T cell stimulation by peptides with substitutions in HLA anchor or T cell contact positions was analyzed to establish degenerate epitope motifs for database searching. Out of 28 tested candidate mimicry peptides derived from bacterial, viral and human proteins, 3 stimulated T cell lines and a T cell clone specific for epitope GAD65 (248-257). Our results demonstrate that mono- and polyclonal GAD65-specific T cells from IDDM patients can be stimulated by viral and bacterial peptides with little apparent sequence homology with autoantigenic epitopes. Moreover, in a synopsis with related published studies, our findings suggest that simple degenerate search motifs comprising principal T cell contacts plus HLA class II binding motifs may suffice to identify most mimicry peptides.

Cow milk feeding induces antibodies to insulin in children--a link between cow milk and insulin-dependent diabetes mellitus?

Exposure to cow milk (CM)-based formulas in early infancy has been associated with an increased risk of insulin-dependent diabetes mellitus (IDDM), but studies on the possible pathogenic mechanism(s) linking CM and IDDM are contradicting. We hypothesized that if CM formulas contained bovine insulin (BI), exposure to them could lead to immunization against insulin, which is the only known beta-cell-specific autoantigen in IDDM. We measured immunoglobulin G (IgG) antibodies by enzyme immunoassay (EIA) to BI and human insulin (HI) in children who received, during the first 9 months of life, either a formula containing whole CM proteins or a formula containing hydrolyzed casein (HC) peptides. BI was detectable by radioimmunoassay (RIA) and immunoblotting in the CM-based formula. At 6 months of age the children who received CM formula had higher levels of IgG antibodies to BI than children who received either HC formula or children who were exclusively breast-fed (median levels 0.480 versus 0.185, P = 0.04; and 0.480 versus 0.160, P = 0.04; respectively). Also, at 9 months of age, children in the CM group differed from the HC group (0.403 versus 0.230; P = 0.02). Antibodies to BI and HI showed a positive correlation and cross-reacted in inhibition studies. The high incidence of insulin-binding antibodies in young children with IDDM may be explained by oral immunization to BI present in CM. Exposure to BI, which differs from HI only by three amino acids, may break the tolerance to insulin.

The GM2-1 ganglioside islet autoantigen in insulin-dependent diabetes mellitus is expressed in secretory granules and is not beta-cell specific.

The pancreatic islet monosialo-ganglioside (GM2-1), an autoantigen in insulin-dependent diabetes mellitus (IDDM) recently shown to be the target of autoantibodies associated with diabetes development in relatives of IDDM patients, is islet specific within the pancreas, and its expression is metabolically regulatable. In the present study we sought to establish 1) whether GM2-1 is beta-cell specific, and 2) its intracellular localization. To this end, we analyzed the pattern of ganglioside expression in highly purified beta- and non-beta-cells isolated from rat islets. In addition, ganglioside levels were determined in subcellular fractions of a rat beta-cell line (INS). No qualitative or quantitative difference was found in the pattern of ganglioside expression between beta and non-beta rat islet cells, with GM3, GM2-1, and GD3 gangliosides expressed in both cell populations. Within INS cells, GM2-1 ganglioside was expressed in the fraction containing secretory granules and, to a lesser extent, in plasma membranes; GM3 was expressed in secretory granules, whereas GD3 was found only in plasma membranes. These data indicate that the GM2-1 autoantigen is not beta-cell specific within the islets, in accordance with the observation that this molecule is a target of islet cell autoantibodies that bind to the whole pancreatic islet. Interestingly, this autoantigen is present in secretory granules similarly to other autoantigens in IDDM (insulin, carboxypeptidase H, 38-kDa protein, etc.), suggesting that the autoimmunity to the components of this organelle may be central to the pathogenesis of the disease.

The GM2-1 Ganglioside Islet Autoantigen in Insulin-Dependent Diabetes Mellitus Is Expressed in Secretory Granules and Is Not -Cell Specific

The pancreatic islet monosialo-ganglioside (GM2-1), an autoantigen in insulin-dependent diabetes mellitus (IDDM) recently shown to be the target of autoantibodies associated with diabetes development in relatives of IDDM patients, is islet specific within the pancreas, and its expression is metabolically regulatable. In the present study we sought to establish 1) whether GM2-1 is β-cell specific, and 2) its intracellular localization. To this end, we analyzed the pattern of ganglioside expression in highly purified β- and non-β-cells isolated from rat islets. In addition, ganglioside levels were determined in subcellular fractions of a rat β-cell line (INS). No qualitative or quantitative difference was found in the pattern of ganglioside expression between β and non-β rat islet cells, with GM3, GM2-1, and GD3 gangliosides expressed in both cell populations. Within INS cells, GM2-1 ganglioside was expressed in the fraction containing secretory granules and, to a lesser extent, in plasma membranes; GM3 was expressed in secretory granules, whereas GD3 was found only in plasma membranes. These data indicate that the GM2-1 autoantigen is not β-cell specific within the islets, in accordance with the observation that this molecule is a target of islet cell autoantibodies that bind to the whole pancreatic islet. Interestingly, this autoantigen is present in secretory granules similarly to other autoantigens in IDDM (insulin, carboxypeptidase H, 38-kDa protein, etc.), suggesting that the autoimmunity to the components of this organelle may be central to the pathogenesis of the disease.

Autoantigenic Reactivity of Diabetes Sera with a Hybrid Glutamic Acid Decarboxylase GAD67–65 Molecule GAD67(1–101)/GAD65(96–585)

Glutamic acid decarboxylase (GAD) is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). Two GAD isoforms exist, GAD65 and GAD67, which differ mostly in the first 100 ainino acids of the amino terminus. IDDM sera are predominantly reactive with GAD65 but autoepitopes have been localised only to regions of GAD65 highly homologous with GAD67. In this study we investigated the contribution of the amino terminus to the IDDM epitope on GAD65, in order to test whether this region of GAD could explain the difference in reactivity between GAD65 and GAD67. A recombinant hybrid GAD molecule consisting of amino acids 1–101 of GAD67 and 96–585 of GAD65 was constructed and a truncated GAD65 was also constructed consisting of amino acids 98–585 of GAD65. The reactivity with the hybrid GAD molecule, GAD65 and GAD67, and truncated GAD65 was examined by radioimmunoprecipitation using 50 IDDM sera with known reactivity to purified porcine brain GAD. Over 90% of the IDDM sera were reactive with the hybrid GAD molecule confirming that the amino terminus of GAD65 does not contribute to the autoepitope and that the IDDM epitope is localised to the middle and carboxyl terminal domains of GAD65. Furthermore, evidence is presented that autoantibodies to GAD65 in IDDM sera react with an epitope formed on a dimeric configuration of the molecule.

Assignment of Ptprn2, the gene encoding receptor-type protein tyrosine phosphatase IA-2β, a major autoantigen in insulin-dependent diabetes mellitus, to mouse chromosome region 12F

The receptor-type protein tyrosine phosphatase IA-2β gene (mouse gene symbol Ptprn2) encodes a major autoantigen in insulin-dependent diabetes mellitus. We physically mapped Ptprn2 by fluorescence in situ hybridization to band F of mouse chromosome 12, a region that lacks diabetes susceptibility loci. The mapping confirms the proposed synteny of mouse 12F with band q36 of human chromosome 7.

Expression of neuronal growth associated protein gene in sensitive period cat visual cortex and LGN

Polymorphism of the genes coding for the human histocompatibility leukocyte antigen class II DR and DQ molecules makes the single largest genetic contribution to the risk of developing insulin-dependent diabetes mellitus (IDDM) and can be associated with highly elevated as well as decreased disease frequency. The mechanism of IDDM risk modification by HLA polymorphism is likely to involve differential presentation of autoantigenic peptides by HLA class II proteins. We have generated T cell lines (TCL) with specificity for the IDDM autoantigen 65 kDa glutamic acid decarboxylase (GAD65) from lymphocytes of two patients carrying HLA class II alleles associated with distinct risk of IDDM (DRB1*0101/0401 and 1302/1501). For both patients, TCL generated at various time points all recognized single epitopes mapped as GAD 88–99 and 248–257, respectively. These epitopes are presented by the DRB1*0101 and DRB5*0101, HLA class II molecules associated with a moderately elevated risk of IDDM, or carried in a strongly protective haplotype, respectively. In an HLA/peptide binding assay, epitope GAD 248–257 was shown to possess high affinity for DRB5*0101. This epitope overlaps with a central GAD peptide binding to the high risk allele DQB1*0302 and containing a Coxsackie P2C-identical mimicry sequence, raising the possibility of competition of DRB5*0101 and DQB1*0302 for binding of a central GAD65 fragment.

Effects of external cation concentrations on the induction of long-term potentiation at visual cortical inhibitory synapses

Polymorphism of the genes coding for the human histocompatibility leukocyte antigen class II DR and DQ molecules makes the single largest genetic contribution to the risk of developing insulin-dependent diabetes mellitus (IDDM) and can be associated with highly elevated as well as decreased disease frequency. The mechanism of IDDM risk modification by HLA polymorphism is likely to involve differential presentation of autoantigenic peptides by HLA class II proteins. We have generated T cell lines (TCL) with specificity for the IDDM autoantigen 65 kDa glutamic acid decarboxylase (GAD65) from lymphocytes of two patients carrying HLA class II alleles associated with distinct risk of IDDM (DRB1*0101/0401 and 1302/1501). For both patients, TCL generated at various time points all recognized single epitopes mapped as GAD 88–99 and 248–257, respectively. These epitopes are presented by the DRB1*0101 and DRB5*0101, HLA class II molecules associated with a moderately elevated risk of IDDM, or carried in a strongly protective haplotype, respectively. In an HLA/peptide binding assay, epitope GAD 248–257 was shown to possess high affinity for DRB5*0101. This epitope overlaps with a central GAD peptide binding to the high risk allele DQB1*0302 and containing a Coxsackie P2C-identical mimicry sequence, raising the possibility of competition of DRB5*0101 and DQB1*0302 for binding of a central GAD65 fragment.

Autoantibodies to IA-2 and IA-2 beta in insulin-dependent diabetes mellitus recognize conformational epitopes: location of the 37- and 40-kDa fragments determined.

IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabetes mellitus (IDDM) and the precursors, respectively, of a 40-and 37-kDa tryptic fragment that reacts with IDDM sera. In the present study, by amino acid sequencing of recombinant IA-2 and IA-2 beta, we determined the tryptic cleavage sites involved in the generation of these fragments. Both cleavage sites are immediately after an arginine residue at position 653 for IA-2 and position 679 for IA-2 beta. The resulting tryptic fragments are 326 and 307 amino acids in length and retain their ability to react with IDDM sera. In contrast to IA-2 and IA-2 beta, other members of the protein tyrosine phosphatase (PTP) family (i.e., RPTP kappa, RPTPmu, NU-3, SHP, and 3CH134) are completely susceptible to digestion by trypsin. Sequence analysis revealed five conserved cysteine residues in IA-2 and IA-2 beta that are not present in other PTPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant proteins resulted in loss of both resistance to digestion by trypsin and reactivity with autoantibodies in IDDM sera. It is concluded that disulfide bond formation plays a critical role in the maintenance of antigenic structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.

High Affinity Presentation of an Autoantigenic Peptide in Type I Diabetes by an HLA Class II Protein Encoded in a Haplotype Protecting From Disease

Polymorphism of the genes coding for the human histocompatibility leukocyte antigen class II DR and DQ molecules makes the single largest genetic contribution to the risk of developing insulin-dependent diabetes mellitus (IDDM) and can be associated with highly elevated as well as decreased disease frequency. The mechanism of IDDM risk modification by HLA polymorphism is likely to involve differential presentation of autoantigenic peptides by HLA class II proteins. We have generated T cell lines (TCL) with specificity for the IDDM autoantigen 65kDa glutamic acid decarboxylase (GAD65) from lymphocytes of two patients carrying HLA class II alleles associated with distinct risk of IDDM (DRB1*0101/0401 and 1302/1501). For both patients, TCL generated at various time points all recognized single epitopes mapped as GAD 88–99 and 248–257, respectively. These epitopes are presented by the DRB1*0101 and DRB5*0101, HLA class II molecules associated with a moderately elevated risk of IDDM, or carried in a strongly protective haplotype, respectively. In an HLA/peptide binding assay, epitope GAD 248–257 was shown to possess high affinity for DRB5*0101. This epitope overlaps with a central GAD peptide binding to the high risk allele DQB1*0302 and containing a Coxsackie P2C-identical mimicry sequence, raising the possibility of competition of DRB5*0101 and DQB1*0302 for binding of a central GAD65 fragment.