Strong association of HLA-B27 heavy chain with β2-microglobulin

Journal of Biological Chemistry

2010

Molecular polymorphism influences the strong association of HLA-B27 with ankylosing spondylitis through an unknown mechanism. Natural subtypes and site-directed mutants were used to analyze the effect of altering the peptide-binding site of this molecule on its stability, interaction with tapasin, folding, and export. The disease-associated subtypes B*2705, B*2702, and B*2704 showed higher thermostability at 50°C than all other subtypes and mutants, except some mimicking B*2702 polymorphism. The lowest values were found among pocket B mutants, most of which interacted strongly with tapasin, but otherwise there was no correlation between thermostability and tapasin interaction. Mutants resulting in increased hydrophobicity frequently acquired their maximal thermostability faster than those with increased polarity, suggesting that this process is largely driven by the thermodynamics of peptide binding. Folding, export, and tendency to misfold were influenced by polymorphism all along the peptide-binding site and were not specifically dependent on any particular region or structural feature. Frequent uncoupling of thermostability, folding/misfolding, and export can be explained by the distinct effect of mutations on the acquisition of a folded conformation, the optimization rate of B27-peptide complexes, and their quality control in the endoplasmic reticulum, all of which largely depend on the ways in which mutations alter peptide binding, without excluding additional effects on interactions with tapasin or other proteins involved in folding and export. The similarity of the generally disease-associated B*2707 to nondisease-associated subtypes in all the features analyzed suggests that molecular properties other than antigen presentation may not currently explain the relationship between HLA-B27 polymorphism and ankylosing spondylitis.HLA-B27 is the major susceptibility factor for a group of chronic inflammatory diseases known as spondyloarthropathies, whose prototype is ankylosing spondylitis (AS).2 This is a disorder characterized by inflammation of the entheses and joints of the axial skeleton, followed by pathological new bone formation, ultimately leading to ankylosis (1). HLA-B27 is involved in triggering the inflammatory process, but the mechanism is unknown. Four main hypotheses, each based on a particular property of HLA-B27, are currently being investigated. The arthritogenic peptide hypothesis (2) is based on the antigen-presenting specificity of HLA-B27 and assumes that molecular mimicry between microbial and self-derived B27 ligands would trigger autoimmune T cell cross-reaction and tissue injury. The misfolding hypothesis (3) is based on the slow folding and tendency to misfold of HLA-B*2705 (4) and assumes that accumulation of misfolded B27 heavy chain (HC) could trigger inflammation by eliciting the unfolded protein response and activation of NFB. The surface homodimer hypothesis (5, 6) is based on the expression of HC homodimers at the cell surface, following dissociation of HLA-B27-peptide complex...

Section: Discussionmentioning

confidence: 99%

Mutational Analysis Reveals a Complex Interplay of Peptide Binding and Multiple Biological Features of HLA-B27

Galocha

Journal of Biological Chemistry

2010

“…However, a limitation of this approach is that quantitative removal of HLA class I-bound peptides by acid washing can only be indirectly assessed by flow cytometry, and it is difficult to rule out that a small amount of peptides may resist acid removal, especially in HLA-B27 whose association with ␤ 2 -microglobulin is particularly strong (41,42). This might complicate the assignment of proteasome inhibitor-resistant ligands among those sequenced from acidwashed cells.…”

mentioning

confidence: 99%

Proteasome-independent HLA-B27 Ligands Arise Mainly from Small Basic Proteins

Marcilla

Cragnolini

Molecular & Cellular Proteomics

2007

Many of the constitutive peptide ligands of HLA-B27, a molecule strongly associated with spondyloarthritis, are proteasome-independent. Stable isotope tagging, mass spectrometry, and epoxomicin-mediated inhibition were used to determine their percentage, structural features, and parental proteins. Of 104 molecular species examined, 29.8% were proteasome-independent, paralleling the level of HLA-B27 re-expression in the presence of epoxomicin after acid stripping. Proteasome-dependent and -independent ligands differed little in peptide motifs, flanking sequences, and cellular localization of the parental proteins. In contrast, whereas the former set arose from proteins whose size and isoelectric point distribution largely reflected those in the human proteome, proteasome-independent ligands, other than a few matching signal sequences, were almost totally derived from small (about 6 -16.5 kDa) and basic proteins, which account for only 6.6% of the human proteome. Thus, a non-proteasomal proteolytic pathway with strong preference for small proteins is responsible for a significant fraction of the HLA-B27-bound peptide repertoire. Molecular & Cellular Proteomics 6:923-938, 2007.

“…Free heavy chains probably arise on the cell surface following dissociation of MHC-peptide complexes (3), since migration of ␤ 2 m-free heavy chains from the ER to the cell surface has not been demonstrated and is impaired by quality control mechanisms (4). The molecular features that determine the extent of dissociation of different class I MHC allotypes are not well defined, but are probably related to their peptide-binding specificity, the overall affinity of their constitutive peptide repertoires, and perhaps also to intrinsic structural features of the heavy chain that influence the stability of the ␣/␤ 2 m heterodimer (5,6).…”

mentioning

confidence: 99%

Similar cell surface expression of β₂‐microglobulin–free heavy chains by HLA–B27 subtypes differentially associated with ankylosing spondylitis

Vázquez

2005

Arthritis & Rheumatism

Objective. To determine whether the cell surface features of HLA-B27 subtypes reported to be differentially associated with ankylosing spondylitis (AS) differ in a way that correlates with disease susceptibility.Methods. Human cell transfectants expressing or lacking the transporter associated with antigen processing were used to determine the cell surface expression of B27 subtypes by flow cytometry with antibodies recognizing the B27 heterodimer or ␤ 2 -microglobulin (␤ 2 m)-free heavy chains.Results. In lymphoid cells with an intact peptideloading complex, all B27 subtypes, irrespective of their association with disease, showed similar ratios of free heavy chain to heterodimer, suggesting similar surface stability. A substantial decrease in dissociated heavy chains, which never reached 100%, was observed upon addition of a B27 ligand, with no significant differences among subtypes. This is compatible with similar surface expression of irreversible ␤ 2 m-free heavy chain forms among subtypes differentially associated with disease. In cells lacking the transporter associated with antigen processing, both disease-associated and non-diseaseassociated subtypes expressed a population of heterodimers at 26°C that was less stable than the population expressed at 37°C. In the presence of exogenous peptide, the expression of heterodimers increased, without a concomitant decrease in ␤ 2 m-free heavy chains. This suggests that in these cells, and for all subtypes tested, most of the dissociated heavy chains at the cell surface are in irreversible forms. At 37°C, the expression of ␤ 2 m-free B27 heavy chains was very low on T2 transfectant cells. Conclusion. HLA-B27 subtypes showing differential associations with AS are similar in their extent of ␤ 2 m dissociation and surface expression of free heavy chains.Class I major histocompatibility complex (MHC) proteins are heterodimers consisting of a polymorphic heavy chain that is noncovalently bound to ␤ 2 -microglobulin (␤ 2 m), a nonpolymorphic polypeptide. Assembly of MHC molecules takes place in the endoplasmic reticulum (ER), where they constitutively bind peptides. These arise mainly from proteasomal degradation in the cytosol, are transported into the lumen of the ER by means of the transporter associated with antigen processing (TAP), and bind, sometimes after trimming, to the class I molecule in a process involving several proteins collectively known as the peptide-loading complex (1). Peptides bound with sufficient affinity stabilize the native conformation of the nascent class I molecule. When this happens, the peptide-MHC complex is released from the ER and traffics to the cell surface. In cells lacking TAP, the peptide supply into the ER and the expression of class I MHC are drastically reduced. However, at 26°C, MHC molecules that are presumably devoid of peptide can be sufficiently stable to leave the ER and reach the cell surface (2).The ␤ 2 m-free class I MHC polypeptides are found at the cell surface. This can be revealed, for example, by cell surface stain...