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

Abstract

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...

Functional Interaction of the Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 1 Polymorphism and HLA-B27 in Vivo

“…Presumably, this mechanism might largely bypass tapasinmediated peptide editing, leading to fast folding, albeit with a suboptimal peptide cargo. Indeed, B*27:06, B*27:07, B*27:09, and B*1403 did not misfold significantly in the ER (30,35). The lower stability of B*27:04 in WE-I is not obviously due to altered hydrophobicity of its peptide binding site, because the molecule itself is not altered; rather, presumably, it is due to the inability of the ERAP1 variant to generate an optimal peptide repertoire.…”

“…Only one experiment could be performed with JSL. The data concerning C1R04, except for one additional experiment included here, were reported previously (35) and are shown here only for comparison.…”

“…Second, although the influence of ERAP-1 on HLA-B27 folding was not examined, the decreased molecular stability of HLA-B27 observed in WE-I indicates that certain ERAP1 variants containing a number of AS-protective polymorphisms generate suboptimal B27 peptidomes. We have shown previously that high thermostability is a feature of three AS-associated subtypes (B*27:02, B*27:04, and B*27:05) expressed on C1R cells that is not shared by the non-ASassociated B*27:06 and B*27:09 subtypes or the AS-associated B*27:07 and B*1403 (30,35). The thermostability of B*27:04 in WE-I was closer to that of the latter subtypes, indicating that ERAP1 polymorphism protective against AS can approach the molecular stability phenotype of an ASassociated B27 subtype to that shown by non-AS-associated ones.…”

“…6). We previously reported that B*27:04 expressed on C1R cells shows very high thermostability, a feature that was shared with two other AS-associated subtypes (B*27:02 and B*27:05) but not with the non-AS-associated B*27:06 and B*27:09 (35). An equally high thermostability of B*27:04 was observed in JSL, and it was nearly as high in KNE, but WE-I showed lower thermostability even at the 4 h chase time, which best reflects the steady state of mature HLA-B27.…”

Tapasin Facilitation of Natural HLA-A and -B Allomorphs Is Strongly Influenced by Peptide Length, Depends on Stability, and Separates Closely Related Allomorphs

“…The amino acid occupancy at positions 114 and/or 116 have been shown to be of crucial importance for the degree of tapasin dependence among different sets of HLA-I molecules (17,20). Different amino acid occupancy at position 59 or 134 also separates tapasin-dependent allomorphs from less tapasin-dependent ones (21,49). This indicates an important role for several specific positions in the peptide-binding groove and might explain tapasin dependency to some degree.…”