Elsevier

Human Immunology

Volume 60, Issue 12, December 1999, Pages 1227-1236
Human Immunology

Original Articles
Substitution 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)

https://doi.org/10.1016/S0198-8859(99)00120-2Get rights and content

Abstract

In class II major histocompatibility complex (MHC) proteins, residue β57 is usually aspartic acid. Alleles carrying serine, valine, or alanine at this position are strongly correlated with the development of insulin-dependent diabetes mellitus (IDDM). Aspβ57 participates in a conserved salt bridge that bridges the α and β subunits in the peptide-binding site. It has been proposed that the correlation between IDDM and MHC alleles lacking Aspβ57 may be due to an instability of the protein caused by loss of this salt bridge. Using a pair of HLA-DQ proteins (α1∗0201, β1∗0302) and (α1∗0201, β1∗0303) differing only in having aspartic acid or alanine at position β57, we show that the polymorphism does not have a significant effect on protein stability for either the empty or peptide-loaded forms. However, the circular dichroism spectra indicate that empty and peptide-loaded Alaβ57 proteins display slightly different secondary structures relative to their Aspβ57 counterparts. A set of three peptides shows different binding affinities for DQ(α1∗0201, β1∗0302) relative to DQ(α1∗0201, β1∗0303). We propose that substitution of Aspβ57 residue causes a local rearrangement within the DQ peptide-binding site that alters the peptide-binding specificity. This rearrangement may help to explain the previously observed differences in SDS stability between Asp and non-Aspβ57 DQ proteins.

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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