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Abstract Number: 607

The HLA-B27 Peptidome in Vivo in Transgenic Rats

Joel D. Taurog1, Yael Haimovich2, Eilon Barnea3, Michal Bassani-Sternberg3, Shira Yair-Sabag2, Martha L. Dorris1, Nimman Satumtira1, Mylinh Nguyen4, Robert E. Hammer4, Tri M. Tran5, Robert A. Colbert5 and Arie Admon3, 1Internal Medicine, Rheumatic Diseases Division, UT Southwestern Medical Center, Dallas, TX, 2Technion-Israel Institute of Technology, Haifa, Israel, 3Biology, Technion-Israel Institute of Technology, Haifa, Israel, 4Biochemistry, UT Southwestern Medical Center, Dallas, TX, 5NIAMS/NIH, Bethesda, MD

Meeting: 2014 ACR/ARHP Annual Meeting

Keywords: Animal models, human leukocyte antigens (HLA) and spondylarthritis

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Session Information

Session Title: Spondyloarthropathies and Psoriatic Arthritis - Pathogenesis, Etiology

Session Type: Abstract Submissions (ACR)

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

Group (all B27/hb2m TG)

Age (d)

Phenotype

No. spleens

No. B27 motif peptides

No. group specific peptides

Wild type B27, Dazl, ERAP1

72-109

EO

4

9,808

1,238

223-236

EO, SpA

4

10,029

Dazl-knockdown

72-109

healthy

4

9,911

360

223-236

healthy

4

9,994

B27 C67S mutant

163-218

EO

12

7,610

1,896

ERAP1 knockout

57

*

1

9,808

781

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.


Disclosure:

J. D. Taurog,
None;

Y. Haimovich,
None;

E. Barnea,
None;

M. Bassani-Sternberg,
None;

S. Yair-Sabag,
None;

M. L. Dorris,
None;

N. Satumtira,
None;

M. Nguyen,
None;

R. E. Hammer,
None;

T. M. Tran,
None;

R. A. Colbert,
None;

A. Admon,
None.

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