Abstract
Disease-associated HLA-DR molecules, which may present autoantigens, constitute the greatest genetic risk factor for rheumatoid arthritis (RA) and antibiotic-refractory Lyme arthritis (LA). The peptides presented by HLA-DR molecules in synovia have not previously been defined. Using tandem mass spectrometry, rigorous database searches, and manual spectral interpretation, we identified 1,427 HLA-DR-presented peptides (220-464 per patient) from the synovia of four patients, two diagnosed with RA and two diagnosed with LA. The peptides were derived from 166 source proteins, including a wide range of intracellular and plasma proteins. A few epitopes were found only in RA or LA patients. However, two patients with different diseases who had the same HLA allele had the largest number of epitopes in common. In one RA patient, peptides were identified as originating from source proteins that have been reported to undergo citrullination under other circumstances, yet neither this post-translational modification nor anti-cyclic citrullinated peptide antibodies were detected. Instead, peptides with the post-translational modification of S-cysteinylation were identified. We conclude that a wide range of proteins enter the HLA-DR pathway of antigen-presenting cells in the patients' synovial tissue, and their HLA-DR genotype, not the disease type, appears to be the primary determinant of their HLA-DR-peptide repertoire. New insights into the naturally presented HLA-DR epitope repertoire in target tissues may allow the identification of pathogenic T cell epitopes, and this could lead to innovative therapeutic interventions.
Highlights
Rheumatoid arthritis (RA),1 the most common form of chronic inflammatory arthritis, is an autoimmune disease of
This region of the molecule is thought to be important in the specificity of peptide binding, and it seems to be a critical factor for defining a person’s HLA-DR-peptide repertoire. These same RA-associated HLA-DR alleles and the DRB5*0101 allele, which bind an epitope of B. burgdorferi outer surface protein A (OspA[161–175]), occur more frequently in patients with antibiotic-refractory LA than in those with antibiotic-responsive LA [7]. It is unclear how these HLA-DR molecules are involved in autoimmune arthritis [11]: these DR molecules may present specific arthritogenic autoantigens in the joint; they may fail to present specific self-peptides during ontogeny, resulting in the survival of certain autoreactive T cells; or they may compatibility complex, type II, encoded by human leukocyte antigen; LA, Lyme arthritis; CCP, cyclic citrullinated peptide; UPLC, Ultra Performance LC; OMSSA, Open Mass Spectrometry Search Algorithm
Using LC-MS/MS, we identified 1,427 HLA-DR-presented peptides (220 – 464 per patient) derived from 166 source pro
Summary
Study Patients—The Human Investigations Committee at Massachusetts General Hospital approved the study, and patients gave written informed consent. Peptides were eluted by 3 ϫ 5-min incubations at RT with 0.5 ml of 10% acetic acid followed by centrifugation for 1 min at 800 ϫ g to collect eluates. All spectra files were recalibrated using high confidence peptides identified during preliminary database searches. Analysis of post-translational modifications was conducted using accurate precursor mass measurements and collision-induced dissociation on an LTQ-Orbitrap Discovery mass spectrometer (ThermoFisher Scientific, Waltham, MA) that was fitted with a TriVersa NanoMate nanoelectrospray source (Advion, Ithaca, NY) and used the nanoACQUITY UPLC system with the columns and solvent gradient described above. Search Result Filtering by Consensus Matching—Each spectrum was searched against the International Protein Index database using three search programs: Mascot, OMSSA, and X!Tandem. A Microsoft Access query was run to associate peptide-to-spectrum matches from the three programs to searched spectra.
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