Ne on the most important ion peaks in the full MS scan
Ne in the major ion peaks inside the complete MS scan, with mz 558.33, was compatible using the [M 2H]2 species in the oxidized form of MRDHTITLL. Its correct assignment was confirmed by comparison with all the MSMS spectrum with the corresponding synthetic peptide in its oxidized type (Fig. 3). This result demonstrates the Hepcidin/HAMP Protein Species endogenous processing and presentation by HLA-B27 of the predicted chlamydial epitope NQRA(330 38) in NQRA transfectant cells. This is the second HLA-B27-restricted T-cell epitope with demonstrated relevance in Chlamydiainfected ReA patients which has been shown to become generated in live cells. DNAP–The unfractionated HLA-B27-bound peptide pool from C1R-B27:05 transfected using the EGFP-DNAP(90 50) fusion protein (38) was subjected to MSMS evaluation in an LTQOrbitrap mass spectrometer and searched against a compact databaseincluding the chlamydial DNAP fusion protein sequence. A parental ion of mz 508.62, compatible with DNAP(21123) (RRFKEGGRGGKYI) was NKp46/NCR1 Protein supplier identified (Fig. 4A). This peptide was two residues longer than 1 previously found from this protein, DNAP(21121) (Table 1). Both sequences show higher homology having a natural ligand of HLA-B27, arising in the endogenous processing on the HLA-B27 heavy chain, B27(309 20) (RRKSSGGKGGSY) (62). To confirm the tentative assignment from the Orbitrap evaluation, a targeted search for this peptide (Fig. 1D) was carried out in the HPLC-fractionated B27-bound peptide pool from the DNAP transfectant, focusing on the mz values corresponding towards the [M H] , [M 2H]2 , and [M 3H]3 forms of DNAP(21123). The analysis revealed the presence of this peptide because the charge variants [M 3H]3 (mz 508.62) (Fig. 4A) and [M 2H]2 (mz 762.43) (Fig. 4B), whose identity was confirmed by comparison together with the MSMS spectra in the synthetic peptide. Higher Homology involving the ClpC and NQRA-derived HLAB27 Ligands and Human Sequences–To explore the doable molecular mimicry in between the B27-restricted peptides from C. trachomatis located in this study and putative self-derived HLAB27 ligands, we looked for human sequences showing high homology to ClpC(20311) and NQRA(330 38). The search was performed against the human proteome, looking for sequences containing 50 amino acid identity together with the bacterial peptides and also the primary binding motif of HLA-B27 ligands, R2. Only human sequences with residues present amongst recognized HLA-B27 ligands (63, 64) having a frequency of 1 in the anchor P1, P3, and P positions had been thought of. Numerous human sequences homologous to the ClpC- and NQRA-derived peptides had been identified (Table two). The majority of the sequences showed predictive scores compatible with proteasomeimmunoproteasome cleavage at their C-terminal residue ( 0.five). MD Simulation of Chlamydial DNAP and Homologous Human-derived HLA-B27 Ligands–To discover the similarity of DNAP(21121) and DNAP(21123) with B27(309 20) in the three-dimensional level, comparative MD simulation of their interaction in complex with B27:05 was carried out. The initial, energy-minimized, three-dimensional structures on the complexes involving the 3 peptides, all built by homology modeling, and pVIPR(400 408) in its canonical conformation have been subjected to MD simulations for 30 ns. Right after this time, the stability in the trajectories was analyzed. Both the mean C RMSD as well as the mean RMSF for the B27:05 heavy chain and 2m were similar amongst the three complexes (Fig. 5, A and B). In contrast, the mean RMSD and RMSF values for the peptides had been a lot more variable, spreading from 0.58 to.
