Abstract
BackgroundEpidemiological evidence and animal models suggest that exposure to low and non-neurotoxic concentrations of mercury may contribute to idiosyncratic autoimmune disease. Since defects in function and signaling in B cells are often associated with autoimmunity, we investigated whether mercury exposure might alter B cell responsiveness to self-antigens by interfering with B cell receptor (BCR) signal transduction. In this study we determined the effects of mercury on the protein tyrosine kinase SYK, a critical protein involved in regulation of the BCR signaling pathway.MethodsPhosphorylation sites of murine SYK were mapped before and after treatment of WEHI cell cultures with mercury, or with anti-IgM antibody (positive control) or pervanadate (a potent phosphatase inhibitor). Phosphopeptides were enriched by either titanium dioxide chromatography or anti-phosphotyrosine immunoaffinity, and analyzed by liquid chromatography-mass spectrometry. Select SYK phosphosite cluster regions were profiled for responsiveness to treatments using multiple reaction monitoring (MRM) methodology.ResultsA total of 23 phosphosites were identified with high probability in endogenous SYK, including 19 tyrosine and 4 serine residues. For 10 of these sites phosphorylation levels were increased following BCR activation. Using MRM to profile changes in phosphorylation status we found that 4 cluster regions, encompassing 8 phosphosites, were activated by mercury and differentially responsive to all 3 treatments. Phosphorylation of tyrosine-342 and -346 residues were most sensitive to mercury exposure. This cluster is known to propagate normal BCR signal transduction by recruiting adaptor proteins such as PLC-γ and Vav-1 to SYK during formation of the BCR signalosome.ConclusionsOur data shows that mercury alters the phosphorylation status of SYK on tyrosine sites known to have a role in promoting BCR signals. Considering the importance of SYK in the BCR signaling pathway, these data suggest that mercury can alter BCR signaling in B cells, which might affect B cell responsiveness to self-antigen and have implications with respect to autoimmunity and autoimmune disease.
Highlights
Epidemiological evidence and animal models suggest that exposure to low and non-neurotoxic concentrations of mercury may contribute to idiosyncratic autoimmune disease
We have shown that mercury alters the phosphorylation status of LYN regulatory sites which are involved in B cell receptor (BCR) signaling [8]
Phosphorylation profile of murine SYK The two main goals of this work are to determine i) all the potential phosphosites of murine SYK, and ii) how the phosphorylation status of these sites are altered in response to mercury exposure
Summary
Epidemiological evidence and animal models suggest that exposure to low and non-neurotoxic concentrations of mercury may contribute to idiosyncratic autoimmune disease. Since defects in function and signaling in B cells are often associated with autoimmunity, we investigated whether mercury exposure might alter B cell responsiveness to self-antigens by interfering with B cell receptor (BCR) signal transduction. Between 50 and 75% of newly produced immature B cells are autoreactive, and many are removed by tolerance mechanisms in the bone marrow before migrating to the spleen where a different set autoantigens are presented [3]. Immature B cells with the lowest avidity to self-antigens mature and eventually migrate to germinal centers or marginal zones where they are activated by foreign antigen. These B cells eventually differentiate into antibody-producing plasma cells or memory cells
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