sodium-dodecyl-sulfate and Diabetes-Mellitus--Type-1

Topics: Amino Acid Sequence; Animals; Antigen-Presenting Cells; Diabetes Mellitus, Type 1; Histocompatibility Antigens Class II; Humans; Mice; Mice, Inbred NOD; Molecular Sequence Data; Peptides; Sodium Dodecyl Sulfate; Time Factors

Low-grade intestinal inflammation and alterations of gut barrier integrity are found in patients affected by extraintestinal autoimmune diseases such as type 1 diabetes (T1D), but a direct causal link between enteropathy and triggering of autoimmunity is yet to be established. Here, we found that onset of autoimmunity in preclinical models of T1D is associated with alterations of the mucus layer structure and loss of gut barrier integrity. Importantly, we showed that breakage of the gut barrier integrity in

Topics: Animals; Bacteria; Blood Glucose; Colitis; Diabetes Mellitus, Type 1; Disease Models, Animal; Female; Gastrointestinal Microbiome; Gene Expression; Humans; Intestinal Mucosa; Islets of Langerhans; Mice; Mice, Inbred NOD; Mice, Transgenic; Permeability; Receptors, Antigen, T-Cell; Sodium Dodecyl Sulfate; Survival Analysis; T-Lymphocytes; Transgenes

Studies of the stability of HLA-DQ have revealed a correlation between SDS stability of MHC class II alphabeta dimers and insulin-dependent diabetes mellitus (IDDM) susceptibility. The MHC class II alphabeta dimer encoded by HLA-DQA1*0102/DQB1*0602 (DQ0602), which is a dominant protective allele in IDDM, exhibits the greatest SDS stability among HLA-DQ molecules in EBV-transformed B-lymphoblastoid cells and PBLs. DQ0602 is also uniquely SDS stable in the HLA-DM-deficient cell line, BLS-1. We addressed the molecular mechanism of the stability of DQ0602 in BLS-1. A panel of mutants based on the polymorphic differences between HLA-DQA1*0102/DQB1*0602 and HLA-DQA1*0102/DQB1*0604 were generated and expressed in BLS-1. An Asp at beta57 was found to be critical for SDS stability, whereas Tyr at beta30, Gly at beta70, and Ala at beta86 played secondary roles. Furthermore, the level of class II-associated invariant chain peptide bound to HLA-DQ did not correlate with SDS stability, suggesting that class II-associated invariant chain peptide does not play a direct role in the unique SDS stability of DQ0602. These results support a role for DQB1 codon 57 in HLA-DQ alphabeta dimer stability and IDDM susceptibility.

Topics: Alleles; Amino Acid Substitution; Antigens, Differentiation, B-Lymphocyte; Aspartic Acid; Cell Line, Transformed; Cell-Free System; Diabetes Mellitus, Type 1; Dimerization; Genetic Predisposition to Disease; Histocompatibility Antigens Class II; HLA-DQ alpha-Chains; HLA-DQ Antigens; HLA-DQ beta-Chains; Humans; Membrane Glycoproteins; Mutagenesis, Site-Directed; Protein Binding; Sodium Dodecyl Sulfate

Structural and functional studies of murine MHC class II I-A molecules have been limited by the low yield and instability of soluble, recombinant heterodimers. In the murine autoimmune diseases experimental autoimmune encephalomyelitis and collagen-induced arthritis, MHC class II molecules I-Au and I-Aq present peptides derived from myelin basic protein and type II collagen, respectively, to autoreactive T cells. To date, systems for the expression of these two I-A molecules in soluble form for use in structure-function relationship studies have not been reported. In the present study, we have expressed functional I-Au and I-Aq molecules using a baculovirus insect cell system. The chain pairing and stability of the molecules were increased by covalently linking the antigenic peptides to beta-chains and adding carboxyl-terminal leucine zippers. Peptide:I-Aq complex quantitatively formed an SDS-stable dimer, whereas peptide:I-Au formed undetectable amounts. However, the two complexes did not show any significant difference in their response to thermal denaturation as assessed by circular dichroism analyses. The autoantigen peptide:I-A complexes were highly active in stimulating cognate T cells to secrete IL-2 and inducing Ag-specific apoptosis of the T cells. Interestingly, the T cells were stimulated by these soluble molecules in the apparent absence of experimentally induced cross-linking of TCRs, indicating that they may have therapeutic potential in autoimmune disease models.

Topics: Animals; Baculoviridae; Cell Line; Circular Dichroism; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Dimerization; Histocompatibility Antigens Class II; Macromolecular Substances; Mice; Recombinant Proteins; Sodium Dodecyl Sulfate; Solubility; Spodoptera; Structure-Activity Relationship

Sequence variability in MHC class II molecules plays a major role in genetically determined susceptibility to insulin-dependent diabetes mellitus (IDDM). It is not yet clear whether MHC class II polymorphism allows selective binding of diabetogenic peptides or regulates some key intracellular events associated with class II-restricted Ag presentation. In this study, we have employed gene transfer techniques to analyze the intracellular events that control peptide acquisition by the unique class II molecule expressed by nonobese diabetic mice (I-Ag7). This structurally unique class II molecule fails to demonstrate stable binding to antigenic peptides and fails to undergo the conformational change associated with stable peptide binding to class II molecules. The experiments reported here demonstrate that I-Ag7 can productively associate with two protein cofactors important in class II-restricted Ag presentation, invariant chain (Ii) and DM. DM participates in the removal of the Ii-derived class II-associated Ii chain peptide and the p12 degradation product from the I-Ag7 molecule. In addition, I-Ag7 undergoes a conformational change when DM is expressed within the APC. Finally, DM can mediate accumulation of peptide/class II complexes on the surface of APCs. Collectively, our experiments indicate that the failure of the I-Ag7 molecule to stably bind peptide cannot be attributed to a failure to interact with the DM or Ii glycoproteins.

Topics: Animals; Antigen-Presenting Cells; Cell Line; Diabetes Mellitus, Type 1; Dimerization; Electrophoresis, Polyacrylamide Gel; Histocompatibility Antigens Class II; HLA-D Antigens; Macromolecular Substances; Mice; Mice, Inbred NOD; Peptides; Protein Conformation; Sodium Dodecyl Sulfate

HLA-DQ alleles are closely associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM) but the immunologic mechanisms involved are not understood. Structural studies of the IDDM-susceptible allele, HLA-DQA1*0301/DQB1*0302, have classified it as a relatively unstable dimer, particularly at neutral pH. This is reminiscent of studies in the nonobese diabetic mouse, in which I-A(g7) is relatively unstable, in contrast to other murine I-A alleles, suggesting a correlation between unstable MHC class II molecules and IDDM susceptibility. We have addressed this question by analysis of dimer stability patterns among various HLA-DQ molecules. In EBV-transformed B-lymphoblastoid cell lines and PBL, the protein encoded by the IDDM-protective allele HLA-DQA1*0102/DQB1*0602 was the most SDS stable when compared with other HLA-DQ molecules, including HLA-DQA1*0102/DQB1*0604, a closely related allele that is not associated with protection from IDDM. Expression of six different HLA-DQ allelic proteins and three different HLA-DR allelic proteins in the bare lymphocyte syndrome cell line, BLS-1, revealed that HLA-DQA1*0102/DQB1*0602 is SDS stable even in the absence of HLA-DM, while other HLA class II molecules are not. These results suggest that the molecular property of HLA-DQ measured by resistance to denaturation of the alphabeta dimer in SDS may play a role in IDDM protection.

Topics: Alleles; B-Lymphocytes; Cell Line, Transformed; Diabetes Mellitus, Type 1; Dimerization; Genetic Predisposition to Disease; Herpesvirus 4, Human; HLA-DQ alpha-Chains; HLA-DQ Antigens; HLA-DQ beta-Chains; Humans; Protein Denaturation; Sodium Dodecyl Sulfate

The human HLA-DQ8 (A1*0301/B1*0302) allelic product manifests a strong association with insulin-dependent diabetes mellitus (IDDM). Previous biochemical studies of the major histocompatibility complex (MHC) class II I-A(g)7 protein of IDDM-prone non-obese diabetic mice produced controversial results. To better define the biochemical properties of IDDM-associated MHC class II molecules, we analyzed DQ8 proteins, in comparison to other DQ allelic products, by partially denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We now report that DQ8 proteins have a normal peptide occupancy and lifespan in cells. Similar to I-A(g)7, DQ8 proteins formed only a minor fraction of SDS-stable complexes with peptides. Although this phenotype was not unique to DQ8, some DQ allelic products such as IDDM-protective DQ6 proteins were SDS resistant. The DQ9 allelic product, differing from DQ8 only at position (P) beta 57, was SDS stable, suggesting that non-Asp residues at beta 57 might decrease the SDS stability of DQ proteins. We identified a single peptide which specifically induced an SDS-stable conformation in DQ8 as well as in I-A(g)7 molecules. The residues at anchor P1 in this peptide were found to influence the SDS stability of both molecules. Together with our previous observation of similar binding motifs of I-A(g)7 and DQ8, these results demonstrate an overall biochemical similarity of mouse and human diabetes-associated MHC class II molecules. This similarity might contribute to a common immunological mechanism of IDDM in both species.

Topics: Alleles; Animals; Cell Line, Transformed; Diabetes Mellitus, Type 1; Disease Susceptibility; Electrophoresis, Polyacrylamide Gel; Genes, MHC Class II; Histocompatibility Antigens Class II; HLA-DQ Antigens; Humans; Mice; Mice, Inbred NOD; Protein Denaturation; Sodium Dodecyl Sulfate; Species Specificity; Surface-Active Agents

The class II molecules of the diabetes-prone NOD mice, I-Ag7, showed very limited amounts of stable form when analyzed by SDS-PAGE. We included the analysis of spleen B cells and B lymphoma cells transfected with I-Ag7 genes. Early during bio-synthesis there was invariant chain binding to the alpha beta-chains. Examination of APCs from F1 mice (NOD x C57BL/6) indicated that the same APC expressed high levels of unstable I-Ag7 and normal amounts of stable class II molecules compared with the other haplotype (I-Ab). The half-life of I-Ag7-peptide complexes on the cell surface of APC was significantly shorter than that of other class II haplotypes. Direct biochemical demonstration of peptide interactions with I-Ag7 was difficult to demonstrate. In T cell assays, the immunogenic peptides, including the diabetogenic Ag, were rapidly lost when peptide-pulsed APCs were washed free of peptide. We hypothesize that the weak and unstable peptide-binding property of I-Ag7 molecules does not favor the elimination or inactivation of autoreactive T cells.

Topics: Amino Acid Sequence; Animals; Antigen Presentation; Autoantigens; Autoimmune Diseases; Base Sequence; Crosses, Genetic; Diabetes Mellitus, Type 1; Disease Models, Animal; Female; Genes, MHC Class II; Haplotypes; Histocompatibility Antigens Class II; Hybridomas; Islets of Langerhans; Isoantigens; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Inbred NOD; Mice, SCID; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments; Sodium Dodecyl Sulfate