Biochemical and Functional Studies of Lymphoid-Specific Tyrosine Phosphatase (Lyp) Variants S201F and R266W

et al. (2012) Biochemical and Functional Studies of Lymphoid-Specific Tyrosine Phosphatase (Lyp) Variants S201F and R266W. PLoS ONE 7(8): e43631. doi:10.1371/journal.pone.0043631 Biochemical and Functional Studies of Lymphoid- Specific Tyrosine Phosphatase (Lyp) Variants S201F and R266W Jing Liu 0 Ming Chen 0 Rong Li 0 Fan Yang 0 Xuanren Shi 0 Lichao Zhu 0 Hong-Mei Wang 0 Wei Yao 0 Qiji Liu 0 Fan-Guo Meng 0 Jin-Peng Sun 0 Qi Pang 0 Xiao Yu 0 Veerle Janssens, University of Leuven (KU Leuven), Faculty of Medicine, Belgium 0 1 Key Laboratory for Experimental Teratology of the Ministry of Education, Shandong University School of Medicine , Jinan, Shandong, China, 2 The 309 The Lymphoid specific tyrosine phosphatase (Lyp) has elicited tremendous research interest due to the high risk of its missense mutation R620W in a wide spectrum of autoimmune diseases. While initially characterized as a gain-of-function mutant, R620W was thought to lead to autoimmune diseases through loss-of-function in T cell signaling by a recent study. Here we investigate the biochemical characters and T cell signaling functions of two uncharacterized Lyp variants S201F and R266W, together with a previously characterized Lyp variant R263Q, which had reduced risk in several autoimmune diseases, including systemic lupus erythematosus (SLE), ulcerative colitis (UC) and rheumatoid arthritis (RA). Our kinetic and functional studies of R263Q polymorphism basically reproduced previous findings that it was a loss-of-function mutant. The other variant S201F reduced Lyp phosphatase activity moderately and decreased Lyp function in T cell slightly, while R266W severely impaired phosphatase activity and was a loss-of-function variant in T cell signaling. A combined kinetic and structure analysis suggests that the R266W variant may decrease its phosphatase activity through perturbing either the Qloop or the WPD loop of Lyp. As both R266W and R263Q significantly change their phosphatase activity and T cell functions, future work could be considered to evaluate these mutants in a broader spectrum of autoimmune diseases. - Funding: This work was supported by grants from the National Natural Science Foundation of China (31000362, 31100580), the Foundation for Excellent Young Scientists of Shandong University (2010JQ014), and the Eleventh Five-year Plan for Medical Science Development of PLA (10MA017). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Protein tyrosine phosphorylations regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) are essential signal transduction events mediating the immune response [1,2]. Disregulation of either PTKs or PTPs leads to the abnormal immune response and correlates to human disease development [3,4]. Most importantly, PTPN22, which encodes lymphoid-specific tyrosine phosphatase (Lyp), attracts tremendous attentions due to the linkage of its R620W single nucleotide polymorphism (SNP) to many autoimmune diseases, including Type 1 Diabetes [5,6], rheumatoid arthritis [7,8], and systemic lupus erythematosus [9,10]. Therefore, intensive efforts have been input to investigate Lyps cellular function and its underlying mechanism in autoimmune diseases [11,12,13,14,15,16]. Human Lyp was first identified in 1999, with 90% homology to murine phosphatase PEP in its phosphatase catalytic domain [17]. Lyp and its murine homologue PEP are negative regulators in T cell signaling through direct dephosphorylation of Lck and ZAP70 kinases [17,18,19,20]. Lyp also associates with CSK, an Lck negative regulator, through the interaction of its first C-terminal poly-proline (P1) region with SH3 domain of CSK [21,22,23]. The disease related mutation R620W, which resides in the P1 region, disrupts this interaction and is firstly reported as a gain-of-function mutation in regulating T cell function [5]. It was observed that less interleukin-2 was secreted from T cells with R620W allele. These studies indicate selectively inhibiting Lyp activity may be considered to develop new treatment for autoimmune diseases [5,12,13]. A specific salicylic acid-based inhibitor was identified through our previous biochemical studies, and it could rescue impaired B cell signaling in Lyp620W- expressing B cell [12,24]. Conversely, recent research argues that R620W decreased Lyp expression level and causes disease through an impaired T cell function, raised the question whether Lyp can be a therapeutic target [16]. Besides Lyp R620W mutation, human genomics studies have identified several missense polymorphisms which did not display significant correlation to cause immune diseases [25,26]. One of the variants, R263Q, which was identified as a loss-of-function mutation, was found to reduce the risk of several autoimmune diseases, including systemic lupus erythematosus, ulcerative colitis and rheumatoid arthritis, but increase susceptibility to infectious disease like pulmonary tuberculosis (PT) [25,27,28,29]. These results suggest the important role of Lyp polymorphism in different autoimmune diseases. Besides R620W and R263Q variants, genetics and clinical studies have accumulated more Lyp polymorphisms with few investigations on their autoimmune disease relationship. A deeper insight of Lyp polymorphism effects on its activity and function will improve our understanding of its potential relationship to autoimmune diseases. In our previous study, we purified the catalytic domain of Lyp and solved its crystal structure together with either a specific inhibitor or peptide substrates [12,14]. The crystal structure revealed that Lyp catalytic domain assumed a classic tyrosine phosphatase folding with a specific insert at N-terminal, which was a determinant of Lyp substrate specificity. In this work, we biochemically characterized two new Lyp polymorphisms, S201F and R266W, together with a previously characterized variant R263Q. Basically, we reproduced the previous biochemical and cellular phenotype of the R263Q polymorphism [25]. In addition, we found that R266W significantly decreased its phosphatase activity toward several substrates, including the small artificial substrate pNPP, the Lck phosphor-peptide 394, and the phosphorSrc protein catalytic domain. Another Lyp variant, S201F did decrease the activity toward pNPP, but only slightly impaired its activity towards the Lck phosphor-peptide and the purified phosphor-Src protein. In consistent with these biochemical results, R266W impaired Lyp function significantly in negatively regulating T cell signaling in cells while S201F displayed a moderate decreased effect. Future work could be considered to evaluating the association of these Lyp polymorphisms with a broad spectrum of autoimmune diseases, and see how they relate to ou (...truncated)