Background/Purpose . In all current hypotheses for the association of B27 with SpA, the B27 peptide repertoire (peptidome) is likely to play a key role. ERAP1 directly influences the MHC-I peptide repertoire and has strong genetic association with AS. The B27 peptidome has previously been reported only in cell lines. We used mass spectrometry to characterize the in vivo B27 peptidome in spleen cells from rats transgenic (TG) for B27 and human beta-2-microglobulin (hb2m).

Methods . B27 TG rats that develop SpA (males only), the protective Dazl-knockdown (kd) transgene, and rats TG for a B27 C67S mutant have been described (A&R 54,1317, 2006; 64:2518, 2012). ERAP1 knockout (ko) rats were produced by microinjection of LEW rat zygotes with a zinc finger nuclease targeting ERAP1. In ERAP1-ko spleen, absence of ERAP1 protein was confirmed by immunoblotting, and ERAP1 mRNA abundance was 18±3% of wild type. One B27/hb2m/ERAP1-ko male was available for peptide analysis. Frozen spleens were solubilized with 1% octyl-glucoside, B27 molecules were immunoaffinity-purified, and peptides were dissociated in 0.1% TFA and isolated by capillary reverse–phase chromatography. Peptides were analyzed by Orbitrap tandem mass spectrometry and data analyzed for peptide identities and relative intensities by MaxQuant, Sequest and Mascot software.

Results . A total of 20,096 unique peptides fitting the B27 peptide motif were identified, of which 10,587 were shared by all genotypes. The data are summarized in the table. The C67S mutant rats showed fewer B27-motif peptides and had a higher proportion of unique peptides, compared with the groups with wild type B27. The Dazl-kd rats, which have no disease manifestations, carried fewer unique peptides than the other groups.

B27-bound peptides isolated from male rat spleens

EO = epididymo-orchitis; *phenotype not yet known

The peptidome from the ERAP1 ko rat was skewed toward longer peptides, compared with the young wild type and Dazl-kd rats (Figure).

Conclusion .  HLA-B27 in TG rat spleen carries large numbers of peptides conforming to the B27 peptide motif identified in human cell lines. Rats with disease show more unique B27-bound peptides than healthy rats, suggesting that disease itself alters the B27 peptidome. Whether either specific peptides or the peptidome as a whole play a role is disease initiation is not yet clear, but the data are consistent with alterations in the peptidome playing a role in disease perpetuation. ERAP1 deletion leads to binding of longer peptides, and the effect of the deletion on disease should be known soon.