The association of different B27 subtypes with the peptide loading complex

Background

The molecular basis for the strong association of HLA-B27 with ankylosing spondylitis (AS) has not been elucidated. A number of B27 alleles including B*2704 and B*2705 are associated with increased susceptibility to AS; in contrast, the alleles B*2706 and B*2709 are not associated with AS. These alleles differ in amino acid regions that have been shown to influence the interaction with tapasin, an accessory molecule that plays a critical role in incorporating HLA class I into the peptide loading complex (PLC). Our aim was to determine whether B27 subtypes differ in their association with the PLC.

Methods

The .221 line that is negative for HLA class I alleles A, B and C was transfected with B*2704, 05, 06 and 09 expression constructs, and stable long-term transfectants with equivalent HLA-B27 expression were selected. The PLC was immunoprecipitated using the anti-TAP antibody 148.3 (provided by R Tampe) and magnetic anti-immunoglobulin microbeads. Non-specific binding of HLA class I was determined by immunoprecipitation with an isotype control mAb. Following reducing SDS-PAGE and western blotting of the eluted proteins, the co-precipitation of HLA-B27 was detected by probing the immunoblots with the anti-HLA class I mAb, HC10, followed by HRP-linked secondary antibodies and visualized using electro-chemiluminescence. Detection of TAP and tapasin was achieved by stripping bound mAbs and reprobing the immunoblots with RING4C (provided by P Cress-well) or 'Giles' (provided by T Elliott) polyclonal antisera, respectively.

Results

Only HLA-B*2704, 05 and 09 were found to be readily detectable above background on the immunoblot; suggesting that they were co-precipitated by the anti TAP mAb and therefore incorporated into the PLC. Despite expressing equivalent quantities of HLA class I heavy chain to the other B27 subypes, HLA-B*2706 was unique in that it was not detectable above background levels. The inability to detect B*2706 was not due to different efficiencies in immunoprecipitation of the PLC since equivalent quantities of TAP and tapasin were immunoprecipitated from the cell lysates of all B27 subtypes.

Conclusions

We have shown that B*2706 differs from the other B27 subtypes in that it cannot be detected within the PLC. B*2706 differs from B*2704 at amino acid positions 114 and 116, and we suggest that these amino acid differences confer the PLC independence of this allele. PLC independence may play a role in contributing protection from arthritis by a number of potential mechanisms such as conferring an inability to bind an athritogenic peptide that is only efficiently bound in the context of the PLC. Alternatively, lack of association with the PLC could also modify an event downstream of peptide loading that may protect from disease susceptibility.