Izabela Lenart

Tapasin dependence of major histocompatibility complex class I molecules correlates with their conformational flexibility

Major histocompatibility complex (MHC) class I molecules present cell internally derived peptides at the plasma membrane for surveillance by cytotoxic T lymphocytes. The surface expression of most class I molecules at least partially depends on the endoplasmic reticulum protein, tapasin, which helps them to bind peptides of the right length and sequence. To determine what makes a class I molecule dependent on support by tapasin, we have conducted in silico molecular dynamics (MD) studies and laboratory experiments to assess the conformational state of tapasin‐dependent and ‐independent class I molecules. We find that in the absence of peptide, the region around the F pocket of the peptide binding groove of the tapasin‐dependent molecule HLA‐B∗44:02 is in a disordered conformational state and that it is converted to a conformationally stable state by tapasin. This novel chaperone function of tapasin has not been described previously. We demonstrate that the disordered state of class I is caused by the presence of two adjacent acidic residues in the bottom of the F pocket of class I, and we suggest that conformational disorder is a common feature of tapasin‐dependent class I molecules, making them essentially unable to bind peptides on their own. MD simulations are a useful tool to predict such conformational disorder of class I molecules.—Garstka, M. A., Fritzsche, S., Lenart, I., Hein, Z., Jankevicius, G., Boyle, L. H., Elliott, T., Trowsdale, J., Antoniou, A. N., Zacharias, M., Springer, S. Tapasin dependence of major histocompatibility complex class I molecules correlates with their conformational flexibility. FASEB J. 25, 3989–3998 (2011). www.fasebj.org

HLA-B27 homodimers and free H chains are stronger ligands for leukocyte Ig-like receptor B2 than classical HLA class I.

Possession of HLA-B27 (B27) strongly predisposes to the development of spondyloarthritis. B27 forms classical heterotrimeric complexes with β2-microglobulin (β2m) and peptide and (β2m free) free H chain (FHC) forms including B27 dimers (termed B272) at the cell surface. In this study, we characterize the interaction of HLA-B27 with LILR, leukocyte Ig-like receptor (LILR)B1 and LILRB2 immune receptors biophysically, biochemically, and by FACS staining. LILRB1 bound to B27 heterotrimers with a KD of 5.3 ± 1.5 μM but did not bind B27 FHC. LILRB2 bound to B272 and B27 FHC and B27 heterotrimers with KDs of 2.5, 2.6, and 22 ± 6 μM, respectively. Domain exchange experiments showed that B272 bound to the two membrane distal Ig-like domains of LILRB2. In FACS staining experiments, B27 dimer protein and tetramers stained LILRB2 transfectants five times more strongly than B27 heterotrimers. Moreover, LILRB2Fc bound to dimeric and other B27 FHC forms on B27-expressing cell lines more strongly than other HLA-class 1 FHCs. B27-transfected cells expressing B27 dimers and FHC inhibited IL-2 production by LILRB2-expressing reporter cells to a greater extent than control HLA class I transfectants. B27 heterotrimers complexed with the L6M variant of the GAG KK10 epitope bound with a similar affinity to complexes with the wild-type KK10 epitope (with KDs of 15.0 ± 0.8 and 16.0 ± 2.0 μM, respectively). Disulfide-dependent B27 H chain dimers and multimers are stronger ligands for LILRB2 than HLA class I heterotrimers and H chains. The stronger interaction of B27 dimers and FHC forms with LILRB2 compared with other HLA class I could play a role in spondyloarthritis pathogenesis.

Novel detection of in vivo HLA–B27 conformations correlates with ankylosing spondylitis association

OBJECTIVE The class I major histocompatibility complex (MHC) molecule HLA-B27 exhibits a strong association with the autoimmune inflammatory arthritis disorder ankylosing spondylitis (AS) and with other related spondylarthropathies. In the absence of both a defined autoimmune response and a target autoantigen(s), the propensity of HLA-B27 to misfold has been hypothesized to be a major parameter in disease pathogenesis. We undertook this study to test the hypothesis that HLA-B27 misfolding is due to exposure of cysteine residues within the heavy chain to the oxidizing environment of the endoplasmic reticulum. METHODS A rapid acidification and alkylation modification method was used to examine cysteine residue exposure and accessibility within AS-associated and non-AS-associated HLA-B27 subtypes. RESULTS This novel approach to probing in vivo class I MHC structure revealed that the HLA-B27 heavy chain adopts conformations not previously described. Furthermore, amino acid residues specific to subtypes HLA-B*2706, B*2709, and B*2704 can have an impact on these novel conformations and on cysteine residue exposure. CONCLUSION HLA-B27 can adopt novel conformations, resulting in differential accessibility of cysteine residues, which can explain the propensity to misfold. Cysteine exposure in the HLA-B27 heavy chain is also affected by residues within the 114 and 116 regions, thereby providing a potential biochemical basis for the association of HLA-B27 subtypes with AS.

The MHC Class I Heavy Chain Structurally Conserved Cysteines 101 and 164 Participate in HLA-B27 Dimer Formation

AIMS The human leukocyte antigen (HLA)-B27 is strongly associated with a group of inflammatory arthritic disorders known as the spondyloarthropathies (SpAs). The unusual biochemistry of HLA-B27 has been proposed to participate in disease development, especially the enhanced ability of HLA-B27 to form several heavy chain-dimer populations. HLA-B27 possesses three unpaired cysteine (C) residues at position 67, 308, and 325, in addition to the four conserved cysteine residues at p101, 164, 203, and 259. C67 was proposed to participate in dimer formation of recombinant HLA-B27 protein and in vivo heavy chain-dimers. However, the structurally conserved C164 was demonstrated to participate in endoplasmic reticulum (ER) resident heavy chain-dimer formation. We therefore wanted to determine whether these aggregates involve cysteines other than C164 and the basis for the difference between the observed heavy chain-dimer species. RESULTS We determined that C164 and C101 can form distinct dimer structures and that the heterogenous nature of heavy chain-dimer species is due to differences in both redox status and conformation. Different HLA-B27 dimer populations can be found in physiologically relevant cell types derived from HLA-B27-positive patients with inflammatory arthritis. In addition, HLA-B27 dimer formation can be correlated with cellular stress induction. INNOVATION The use of both mutagenesis and manipulating cellular redox environments demonstrates that HLA-B27 dimerization requires both specific cysteine?cysteine interactions and conformations with differing redox states. CONCLUSION HLA-B27 heavy chain-dimerization is a complex process and these findings provide an insight into HLA-B27 misfolding and a potential contribution to inflammatory disease development.

Pathogenicity of Misfolded and Dimeric HLA-B27 Molecules

The association between HLA-B27 and the group of autoimmune inflammatory arthritic diseases, the spondyloarthropathies (SpAs) which include ankylosing spondylitis (AS) and Reactive Arthritis (ReA), has been well established and remains the strongest association between any HLA molecule and autoimmune disease. The mechanism behind this striking association remains elusive; however animal model and biochemical data suggest that HLA-B27 misfolding may be key to understanding its association with the SpAs. Recent investigations have focused on the unusual biochemical structures of HLA-B27 and their potential role in SpA pathogenesis. Here we discuss how these unusual biochemical structures may participate in cellular events leading to chronic inflammation and thus disease progression.

Endoplasmic Reticulum Degradation–Enhancing α‐Mannosidase–like Protein 1 Targets Misfolded HLA–B27 Dimers for Endoplasmic Reticulum–Associated Degradation

HLA–B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR‐induced ER‐associated degradation (ERAD) pathway in the disposal of HLA–B27 dimeric conformers.

The Oxidative Folding and Misfolding of Human Leukocyte Antigen-B27

The major histocompatibility complex class I molecule human leukocyte antigen (HLA)-B27 is strongly associated with a group of inflammatory arthritic disorders known as the spondyloarthropathies. Many autoimmune diseases exhibit associations with major histocompatibility complex molecules encoded within the class II locus with defined immune responses either mediated by T or B-lymphocytes. Despite the association being known for over 30 years, no defined immune response and target autoantigens have been characterized for the spondyloarthropathies. Thus, the mechanism and role of HLA-B27 in disease pathogenesis remains undetermined. One hypothesis that has recently received much attention has focused around the enhanced propensity for HLA-B27 to misfold and the increased tendency of the heavy chain to dimerize. The misfolding of HLA-B27 has been associated with its redox status and this is postulated to be involved in disease development. Here we discuss the impact of the redox status on HLA-B27 biosynthesis and function.

Rapid acidification and alkylation: redox analysis of the MHC class I pathway.

The technique of rapid acidification and alkylation can be used to characterise the redox status of oxidoreductases, and to determine numbers of free cysteine residues within substrate proteins. We have previously used this method to analyse interacting components of the MHC class I pathway, namely ERp57 and tapasin. Here, we have applied rapid acidification/alkylation as a novel approach to analysing the redox status of MHC class I molecules. This analysis of the redox status of the MHC class I molecules HLA-A2 and HLA-B27, which is strongly associated with a group of inflammatory arthritic disorders referred to as Spondyloarthropathies, revealed structural and conformational information. We propose that this assay provides a useful tool in the study of in vivo MHC class I structure.

EDEM1 targets misfolded HLA-B27 dimers for endoplasmic reticulum associated degradation

HLA–B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR‐induced ER‐associated degradation (ERAD) pathway in the disposal of HLA–B27 dimeric conformers.

Salmonella exploits HLA-B27 and host unfolded protein responses to promote intracellular replication

Objective Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells. Methods Isogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S. enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane. Results S. enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism. Conclusions HLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention.