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

Abstract

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...

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

“…That subtypes with lower thermostability presented faster folding and export rates than B*2705 suggests that they exit the ER with less stable peptide cargoes, which might favor dissociation at the cell surface. However, this was not observed (34,56), suggesting that under physiological conditions these subtypes might be sufficiently stable as to show no increased expression of unfolded HC at the cell surface. Nevertheless, the lower thermostability of B*2706 and B*2709 relative to most AS-associated subtypes does not support the ␤ 2 m deposition hypothesis for the pathogenesis of AS (11), because a prediction of this hypothesis was that disease-associated subtypes should be less stable than subtypes not associated with AS, which is the opposite of what was found in our analysis.…”

Disparate folding and stability of the ankylosing spondylitis–associated HLA–B*1403 and B*2705 proteins

“…Cell surface expression of HLA-B27 heavy-chain homodimers occurs upon endosomal recycling (22), presumably following dissociation of the canonical heterodimers. In B*2705 C1R cells, the surface expression of ␤ 2 m-free heavy chains is ϳ5% of the heterodimer (40). Thus, we reasoned that if HLA-B14-peptide complexes were less stable than HLA-B27 and dissociated to a larger extent after exiting the ER, the surface expression of HC10-reactive molecules should be higher in B*1403 and B*1402 than in B*2705.…”

“…Although not to the extent of other HLA class I molecules (44), both the nature and significant stability of the B*2705-bound peptide repertoire are tapasin-dependent (16,45). Thus, at the cell surface of tapasin-proficient cells, such as C1R, only low amounts of ␤ 2 m-free heavy chain are detected (40).…”

The interplay between HLA-B27 and ERAP1/ERAP2 aminopeptidases: from anti-viral protection to spondyloarthritis

“…So far, this matter has not been explored in an in vivo setting in which cells from AS patients are compared with the controls expressing the different B27 alleles. However, no significant difference in cell surface expression of FHC among several B27 subtypes expressed on C1R cells has been reported [38]. Moreover, one report highlighted that B*2706, unlike B*2709, expressed the highest surface level of FHC, probably reflecting the poor tapasin dependence during the assembly process which produces more dissociation-prone heterotrimeric complexes [39].…”