Original ArticlesSubstitution of aspartic acid at β57 with alanine alters MHC class II peptide binding activity but not protein stability: HLA-DQ (α1∗0201, β1∗0302) and (α1∗0201, β1∗0303)☆
Introduction
Through extensive population studies, the incidence of insulin-dependent diabetes mellitus (IDDM) has been shown to be strongly correlated with the presence of particular class II major histocompatibility complex (MHC) HLA-DQ genes 1, 2, 3, 4. In general, DQ alleles that correlate with IDDM protection have Asp at β57, whereas susceptibility alleles have Ala, Val, or Ser at β57. The NOD (non-obese diabetes) mouse shows strong susceptibility to spontaneous disease development [5] and is a model system for IDDM. The DQ homologue carried by the NOD mouse is I-Ag7, which has serine at position β57. Mutation of the Serβ57 residue to Asp in I-Ag7 reduces the incidence of murine IDDM, but does not prevent insulitis, sialadenitis, or the development of insulin and nuclear autoantibodies [6]. Despite the clear connection between particular MHC alleles and susceptibility to autoimmune disease, the mechanism by which the residue at β57 position influences disease development is not understood.
All murine and human class II MHC proteins for which structures have been determined have aspartic acid at position β57 7, 8, 9, 10, 11, 12, 13, 14, 15. In each case, the aspartic acid side chain makes a salt bridge with the side chain of a conserved arginine at position 76 in the α chain. The interaction bridges the α- and β-subunit helices underneath the peptide in the vicinity of the P9 pocket 7, 15. The structural consequences of disruption of this salt bridge in alleles with Ala, Val, or Ser at position β57 are not known. If the salt bridge plays an important role in the energetics of subunit association or protein folding, loss of the interaction could destabilize the protein. Alternately, substitution of Aspβ57 could affect the peptide-binding specificity through direct interactions in the P9 pocket region, or through conformational changes induced by this substitution. Because there are no structures of class II MHC proteins with residues other than Asp at position β57, no direct information is available on possible structural effects of this substitution.
Several attempts have been made to address the issue of relative protein stability in IDDM protective and susceptible alleles. In one approach, DQ- or IA-peptide complexes were evaluated for their ability to resist αβ chain dissociation in a non-boiled sample on SDS-PAGE 16, 17, 18, 19, 20. This property is often referred to as the “SDS-stability” of a particular complex 21, 22. Complexes of DQ8 (α1∗0301, β1∗0302) with endogenous peptides are more susceptible to SDS-induced chain dissociation than those of DQ9 (α1∗0301, β1∗0303), which is identical to DQ8 except in having Asp rather than Ala at β57 16, 17. I-Ag7 (Serβ57) is also sensitive to SDS, whereas other murine alleles, such as I-Ab (Aspβ57) and I-Ak (Aspβ57), are stable 18, 19. An I-Ag7 mutant (Hisβ56Pro, Serβ57Asp), however, also showed sensitivity to SDS, even though the salt bridge was supposedly reinserted into the structure [19].
The physiological relevance of SDS-stability is not clear. Some SDS-sensitive empty class II MHC molecules are stable against denaturation and chain dissociation in the absence of SDS, at temperatures up to 60°C 23, 24. In addition, several fully antigenic SDS-sensitive class II MHC-peptide complexes are stable to thermal denaturation [25] or chain dissociation in the absence of SDS 26, 27. The mechanism of SDS-induced chain dissociation may involve SDS binding into open pockets or interstitial spaces, a process that would not be active in the absence of SDS [27]. In general, the structural and functional correlates of SDS-sensitivity are not known. In one study, DQ6 (α1∗0102, β1∗0602) and DQ8 (α1∗0301, β1∗0302) were shown to persist on cells for similar lifetimes despite the difference in their SDS-stability—the majority of DQ6 (Aspβ57) is SDS-stable, while DQ8 (Alaβ57) exists in both SDS-sensitive and stable populations [16]. I-Ag7 has also been reported to display normal cell surface lifetime in similar experiments [16]. However, another study has reported reduced lifetimes [19]. These results suggest that SDS-stability may not be an accurate measure of overall stability of one complex versus another and that more detailed analysis must be performed in the absence of SDS to address this question.
Several groups have observed that DQ8 and I-Ag7 (Alaβ57 and Serβ57, respectively) show a unique binding preference for peptides with acidic residues near the C-terminal P9 pocket 16, 18, 28, 29, 30, 31, 32, 33. Presumably this preference is due a possible interaction with the positively charged Argα76 residue, which would be left unpaired in the absence of an acidic residue at β57. However, other residues within the P9 pocket may be responsible for the specificity, as modeling of non-Aspβ57 alleles shows that Argα76 may be too far from the P9 side chain to influence peptide preferences at this position [34]. Additionally, other IDDM-susceptibility alleles carrying residues other than Asp at position β57, such as DQ2 (α1∗0501, β1∗0201), do not show such a preference for acidic residues [35].
Thus, the role played by the HLA-DQ β57 polymorphism in HLA-DQ structure and peptide-binding specificity, and its importance to the mechanism of IDDM etiology, are still unclear. In an attempt to determine a structural mechanism for IDDM development stemming from DQ susceptibility alleles, we analyzed the thermodynamic stability of empty and peptide-loaded MHC complexes for two alleles that differ only at position β57. DQ(α1∗0201, β1∗0302) has Alaβ57, and DQ(α1∗0201, β1∗0303) has Aspβ57. In this work these proteins will be referred to as DQAlaβ57 and DQAspβ57. This pair has a different DQα chain but the same DQβ chains as DQ8 (α1∗0301, β1∗0302) and DQ9 (α1∗0301, β1∗0303), another pair that has been used to investigate the Asp/Alaβ57 polymorphism 16, 17. We find that DQAspβ57 and DQAlaβ57 are similarly stable, but have somewhat different secondary structures and peptide-binding activities.
Section snippets
Preparation of HLA-DQ molecules
Soluble DQAlaβ57 (α1∗0201, β1∗0302) and DQAspβ57 (α1∗0201, β1∗0303) were produced in insect cells using separate baculovirus recombinants for α (EDIVAD … IPAPMS) and β (RDSPED … WRAQSE) subunits as described in Stern et al. [22] and Raddrizzani et al. [36]. For protein production, High Five (BTI-TN-5B1-4) insect cells were grown in 6-liter suspension cultures in Gibco BRL Sf900-II serum-free medium and were co-infected at a multiplicity of infection (MOI) between 5 and 10. This optimal MOI was
Production of soluble DQAlaβ57 and DQAspβ57 for biochemical analysis
To investigate the role of β57 polymorphism to DQ structure, stability, and peptide-binding activity, we prepared soluble versions of DQAlaβ57 (α1∗0201, β1∗0302) and DQAspβ57 (α1∗0201, β1∗0303) by recombinant expression in insect cells, as previously described for DR1 [22]. These alleles differ only at position β57, with DQAlaβ57 β-chain associated with IDDM susceptibility and that of DQAspβ57 neutral with respect to IDDM protection. DQ α and β subunits were assembled and secreted efficiently,
Discussion
Since the original publications citing β57 as the key residue for susceptibility to IDDM, the relationship between the Asp β57 polymorphism in DQ alleles and disease development has plagued the field of diabetes immunology 1, 2. In the initial biochemical analyses of DQ8 from EBV-transformed B-cells, the majority of the isolated complexes were susceptible to SDS-induced chain dissociation on SDS-PAGE [16], similar to results obtained with the murine NOD-associated I-Ag7 16, 19. This led to a
Acknowledgements
We would like to thank Laura Raddrizzani for help with HLA-DQ production and assay, Mia Rushe for advice on baculovirus expression, Jennifer Zarutskie for help with CD workup and critical reading of the manuscript, Laura Santambrogio for general advice on autoimmunity, and Daniel DeOliveira for peptide synthesis help.
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Identification and characterization of a novel recessive KCNQ1 mutation associated with Romano-Ward Long-QT syndrome in two Iranian families
2017, Journal of ElectrocardiologyCitation Excerpt :Amino acid 566 to 569 have also been reported as pathogenic and likely pathogenic variants [15,34,35]. Moreover, replacing a negatively charged Aspartic acid which is critical for the proper conformation of α-helix with a Glycine belonging to a group of small non-polar amino acids including Alanine [36] and the substitution of conserved Aspartic acid with Alanine which is reported to have impact on the function of another protein [37] revealed that the variant p.D564G might be a pathogenic mutation altering this conserved part of the protein. Furthermore, the altered amino acid was predicted to have a probable pathogenic effect due to a potential alteration of RNA splicing (Table 1).
The importance of the Non Obese Diabetic (NOD) mouse model in autoimmune diabetes
2016, Journal of AutoimmunityCitation Excerpt :NOD mice express MHC-II molecules I–Ag7 (ortholog of HLA-DQ), no I-E (ortholog of HLA-DR) and MHC-I H-2KdDb (MHCI), of which the I–Ag7 contributes significant susceptibility to developing diabetes. I–Ag7 has a polymorphism [65,66], where the charged aspartate residue at position 57 of the beta chain is substituted with serine, a change which is also found in human HLA-DQA10201/B10302 (DQ8) [67] (alanine substitution for the aspartate). Substitution of the serine at amino acid position 57 in the beta chain of I–Ag7 to aspartate, the amino acid found in most other mouse strains, protected NOD mice from disease [68].
Use of nonobese diabetic mice to understand human type 1 diabetes
2010, Endocrinology and Metabolism Clinics of North AmericaCitation Excerpt :DQB alleles with serine (Ser), alanine (Ala), or valine (Val) at amino acid residue 57 are associated with T1D susceptibility, whereas those alleles containing an aspartic acid (Asp) residue are considered protective. The non-Asp-containing alleles cause a local rearrangement within the peptide-binding site, which alters the peptide-binding specificity,27 resulting in altered T-cell recognition and thymic selection.28 Likewise, susceptibility has also been linked to specific HLA-A and HLA-B class I alleles29; however, little is known about the role of the disease-associated variants in T1D.
Substitution of Aspartic Acid at Position 57 of the DQβ1 Affects Relapse of Autoimmune Pancreatitis
2008, GastroenterologyCitation Excerpt :Sequencing the amino acids from the known HLA class II second exon, it was revealed that a single nucleotide substitution at position 57 of DQβ1 solely affects the relapse of AIP by alignment analysis (Figure 1). Because the peptide binding preference of the DQβ1 57 may be dependent on the presence of aspartic or nonaspartic acid (Ala, Val, or Ser) at this position,30–32 the amino acid was divided into 2 amino acid groups (nonaspartic vs aspartic acid). In this analysis, the nonaspartic acid residue at DQβ1 57 was significantly associated with relapse of AIP (nonrelapse group [n = 8/27], 29.6%; relapse group [n = 13/13], 100%; P = .00003; odds ratio, 3.38; 95% confidence interval, 1.9–6.0) (Table 2).
Directed evolution of soluble single-chain human class II MHC molecules
2004, Journal of Molecular Biology
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Supported by a grant from the NHS to L.J.S. (RO1-AI38996).