Study Of HLA-B-bound Peptides On The Pathogenesis Of Trichloroethylene-induced Hypersensitivity Dermatitis

Trichloroethylene (TCE)-induced hypersensitivity dermatitis is one of the most critical occupational diseases among workers in the southeast coastal areas of China. With a significant association with the polymorphism of human leukocyte antigen (HLA), the pathogenesis of TCE-induced hypersensitivity dermatitis is complicated, and possibly caused by the interactions of different risk factors including both genetic and environmental ones. The mechanism of TCE-induced hypersensitivity dermatitis remained unknownOur previous studies showed that hypersensitivity dermatitis induced by TCE has a strong genetic linkage to HLA-B* 13:01 allele. These results suggested that HLA-B* 13:01 molecule is a participant in the pathogenesis of the disease. HLAs are highly polymorphic proteins that initiat immune function by presenting pathogen-derived peptides to T cells. Therefore, we proposed a hypothesis that the HLA-B* 13:01 molecule are involved in the pathogenesis of TCE-induced hypersensitivity dermatitis by presenting specific peptides.An experiment proposal was made to verify the hypothesis.1) HLA-B*13:02-bound peptides were selected as control to analyze the relations of the specificity of HLA-B* 13:01-bound peptides with the pathogenesis of TCE-induced hypersensitivity dermatitis. There are only three different amino acids between HLA-B*13:01 and HLA-B*13:02 molecules, and HLA-B*13:02 allele is not associated with TCE-induced hypersensitivity dermatitis. Therefore, HLA-B*13:02-bound peptides would be a perfect control group to explore HLA-B*13:01-bound peptides,2) The human B-lymphoblastoid cell line Hmy2.ClR transfected with HLA-B* 13:01 and HLA-B* 13:02 allele were selected. Immunoaffinity purification procedures were used to isolate and purify the HLA class I complex. Peptides were separated from the HLA complexes by acidification and Amicon ultra centrifugal Filters. HLA-B* 13:01-bound peptides were sequenced using liquid chromatography-tandem mass spectrometry (LC-MS). We analyzed the characteristics of the motifs of HLA-B* 13:01/HLA-B* 13:02-bound peptides and compared differences of two types pepetides.3) Some HLA-B*13:01/HLA-B* 13:02-bound peptides were synthesized according to the high frequency of amino acids of peptides in anchor residues P2 and PQ. The activity of peptides to reconstitute HLA-B complexes was tested by HLA class I peptide-binding assay. To understand mechanism of TCE induces the disease by altering binding between HLA molecules and peptides, alterations of the activity of peptides with the presence of TCE or Trichloroethanol (TCOH) compare to the absence of TCE or TCOH were observed.The results of the research are as follows:1) The Computational analysis of HLA-bound peptidesA total of 57 HLA-B* 13:01-bound peptides and 88 HLA-B*13:02-bound peptides were identified. In the study, for HLA-B* 13:01-bound peptides, the anchor residues were leucine (L) (39%) and glutamine (Q) (33%) at P2, L(35%), phenylalanine (F) (24%), (I) (15%) and tryptophan (W) (11%) at PQ; for HLA-B*13:02-bound peptides, the anchor residues were L (26%) and Q (16%) at P2. The preferred amino acids at P2 were L and Q, which was the same in both HLA-B* 13:01-bound peptides and HLA-B*13:02-bound peptides. However, the amino acids (L, V and I) of HLA-B*13:02-bound peptides at PQ were different from of HLA-B* 13:01-bound peptides (L, F, I, W) except L. Only three residues were different between HLA-B*13:01 and HLA-B*13:02. The different residues were I94I95R97 (HLA-B* 13:01) versus T94W95T97 (HLA-B* 13:02). F pocket including 95 and 97 amino acids of HLA molecules was binding with side of amino acid of peptides at PQ. The types of amino acids sides were determined by the shape and characteristics of F pockets. These suggest that the differences in amino acids at PQ between HLA-B* 13:01-bound peptides and HLA-B*13:02-bound peptides were related to the polymorphism of F pocket of HLA.2) The functional analysis of peptidesAccording to HLA class ? peptide-binding assay, the activity of the HLA-B*13:01-bound peptides RQDDPSYRSF (Q-F), AGDGTFQKW (G-W), ALLNLAERL (L-L), KLYEEKTGNAW (L-W), LFDHAVSKF (F-F), RMDEEFTKI (M-I) and FLSKIEEKLT(L-T) were 54%?50%?45%?44%?36%? 36% and 32%, respectively; KLKGEENTV (L-V) has strong affinities with HLA-B* 13:02 molecules and the activity was 73%. The activities of two peptides Q-F and G-W with HLA-B* 13:02 were decreased to 24% and 0.09%, indicating that these peptides were specific to HLA-B* 13:01-bound peptides; the activity of HLA-B* 13:02-bound peptide L-V with HLA-B* 13:01 were decreased to 27%, indicating that these peptides were specific to HLA-B* 13:02-bound peptides.3) The effect of TCE or TCOH on the activity between HLA and peptides:For HLA-B* 13:01-bound peptides, the activities of Q-F, L-T and G-W with HLA-B* 13:01 which could be altered with the presence of TCE were decreased from 54%,32% and 50% to 11%,11% and 32% with the presence of TCE, respectively. TCOH could also alter the activity of Q-F and L-W with HLA-B* 13:01, decreased from 55% and 44% to 11% and 11% respectively. Interestingly, activity of peptides with HLA-B* 13:02 could not be altered with the presence of TCE or TCOH. The results indicated that HLA-B* 13:01 molecule are involved in the pathogenesis of TCE-induced hypersensitivity dermatitis by presenting specific peptides.In conclusion,1) the main differences of motifs are P? between HLA-B*13:01-bound peptides and HLA-B* 13:02-bound peptides, which bind with pocket F of HLA-B molecule, indicating that the pathogenesis of TCE-induced hypersensitivity dermatitis is related to the polymorphism of pocket F of HLA-B* 13:01.2) TCE or TCOH could decrease the activity of peptide binding in the HLA-B* 13:01 specific assays to alter preferred amino acids of HLA-B* 13:01-bound peptides but not for HLA-B* 13:02 specific assays. When the PQ is F, the affinity is more susceptible to TCE or TCOH, suggesting that TCE or TCOH could alter HLA-B*13:01-bound peptides to induce hypersensitivity dermatitis by presenting specific peptides.The study states the mechanism of association between HLA-B* 13:01 and TCE-induced hypersensitivity dermatitis. The reaearch not only enrich the database of peptide repertoire, but also provide an experimental basis for the further study.