The Background Of Protein Tyrosine Phosphatases

Published Date: 02 Nov 2017

Protein tyrosine phosphatases (PTPs) as mediators of immunity homeostasis are important candidates for carrying disease-related SNPs and more than half of the known PTPs in the human genome are expressed in T cells.A dynamic balance of tyrosine phosphorylation process is regulated by the coordinated action of protein tyrosine kinases (PTKs) and PTPs. PTPs play key roles in the regulation of numerous physiological processes by selectively dephosphorylating their substrates。The tyrosine dephosphorylation phenomena is critical for negtive regulating T-cell Receptor(TCR)sinalling pathways by inactivating TCR-associated kinases and their substrates and preventing spontaneous T-cell activation. Moreover,specific PTPs are targets for development of novel anticancer drug. SNPs of PTPs have been proved to involve in several autoimmune diseases.

LYP/CSK and TCR

The human genome contains at least 107 genes for families of PTPs,one of which is the PTPN22 located on chromosome 1p13.3–13.1that encodes the lymphoid-specific tyrosine phosphatase (LYP) which has received enormous attention because of its splice variants and point mutation. Human LYP belonging to non-receptor classical PTPs was first identified in 1999, with 90% homology to murine pest-enriched phosphatase（PEP）,encoded by PTPN8 in its phosphatase catalytic domain.The first complete resequencing of the human PTPN22 gene was completed by the Begovich group in 2005。LYP is a 110-kDa protein consisting of an ∼300-amino acid N-terminal phosphatase domain and a long noncatalytic ∼200-amino acid C-terminal segment with several Prorich motifs(termed P1-P4). The catalytic domain and the C-terminal domain are separated by an ∼300-amino acid region called "the interdomain". LYP/PEP physically associates through its with the（Src homology 3）SH3 domain of the CSK kinase8, an important suppressor of the Src family kinases. In T cells, numerous studies have shown that the P1 motif of LYP/PEP and the SH3 domain of CSK is a high stoichiometry complex in TCR signaling by a series mechanisms.

LYP has generated tremendous research interest in the structure and biochemical mechanism that directly regulate its function. Yu et al. demonstrated that LYP can be exclusively phosphorylated by PKC on Ser-35, the LYP specific region S35TKYKADK42 that harbors a PKC phosphorylation site,which impairs the LYP-mediated substrate dephosphorylation and signaling in T cell. Moreover，an intramolecular regulation mechanism of LYP phosphatase activity is mediated by an interaction between the interdomain of the molecule and the catalytic domain. Early in 1999, Wingren et al. have been reported that truncation of mouse PEP at amino acid 290 increases the phosphatase activity. Nunzio et al.in 2009 reported that truncation of LYP at amino acid 300 immediately C-terminal to the catalytic domain causes a dramatic increase in phosphatase activity and the 20 amino acids immediately proximal to the catalytic domain are mainly responsible for the effects by directly interacts with the catalytic domain and reduces its activity. Between human LYP and mouse PEP，the whole interdomain (amino acids 301-600 of LYP and amino acids 301-599 of PEP) showed 52% identity and 65.1% similarity. Authors also refered that the primary structure couldnot explain the mechanism of inhibition. However,Knapp et al. found LYP as well as SHP2,BDP1,SHP1,TCPTP,MEG2,PTPBAS,DEP1,PEST and GLEPP1 has a secondary substrate-binding pocket analogous to that found in PTP1B which harbors a basic residue corresponding to Arg24，through analyzing the structural topology and residue characteristics of the secondary substrate-binding pocket of other PTPs.

Engagement of the TCR leads to the activation of LCK and FYN, the Src family protein tyrosine kinases (PTKs) which provides docking sites for the SH2 domains of ZAP-70, a Syk family PTK to allow ZAP-70 to be phosphorylated and activated. Then ZAP-70 phosphorylates the adaptor proteins SLP-76 and LAT, the nucleate signalling complexes, leading to the phosphorylation and activation of multiple downstream effectors. When T cells recognize autoantigens and bind autoantigens, it leads to autoimmune diseases such as rheumatoid arthritis, type 1 diabetes and lupus erythematosus because of the negtive TCR process. The LCK, FYN, ZAP-70, as well as ITAMs of the TCR/CD3 complex, Vav, valosin-containing protein and other key signaling molecules are the substrate of LYP/PEP to be dephosphated. In TCR signaling pathway, the activation state of the Src family kinases (SFKs) is balanced between the activities of CSK/CD45 complex and CSK/LYP complex. CSK and CD45 respectively phosphorylate and dephosphorylate the negative regulatory tyrosine in the inhibitory C-terminus of LCK (residue Tyr505) and FYN, while a number of protein tyrosine phosphatases (PTPs), including LYP/PEP, SHP1, PTPH1 and PTP-MEG1 and perhaps CD45 and PTP-PEST dephosphorylate and inactive the positive regulatory site in the catalytic domain of LCK(residue Tyr394) and FYN.

Based on above, LYP and Csk are thought to act in a cooperative manner when forming a complex as the most TCR-proximal mechanisms for downregulation. Supported by analyses of mice genetically engineered to lack LYP4, studies of the PEP suggested that LYP and Csk worked together to inhibit the excessive T-cell activation. When LYP interacts with CSK, the inhibitory function of it on TCR transduction is enhanced. In contrast,it shows that CSK may restrict the ability of LYP to regulate TCR signaling negatively. Moreover,Vang et al. recently provide new insights indicating that the catalytic activity of human LYP is interfered when bound to Csk and that it becomes more active in negtive regulating TCR sinalling pathways when it is released，which is associated with several autoimmune diseases,including type I diabetes, rheumatoid arthritis, Graves disease, and systemic lupus erythematosus.

R620W:gain or loss of function

A major advancement in the study of LYP is the finding of a disease-causing SNP C1858T,a C-to-T substitution at position 1858 in PTPN22, which changes amino acid residue 620 from Arg (R) to Trp (W) that resides in the P1 region of the C terminus in LYP.The R620W variant interrupts the interaction of LYP with the Csk. The initial hypothesis on the cause of an increased risk of developing autoimmune diseases with the R620W variant is an excess of T-cell activation based on the theory of LYP and Csk working together to inhibit TCR signaling pathways.However,many statistical evidence using unmanipulated primary human cells shows that the 620W allele could result in less efficient T-cell regulatory action and may cause a predisposition to autoimmune disease.

Studies of R620W allele in primary human cells and prior overexpression studies in Jurkat cells suggest that the variant impairs TCR signaling pathways. The PTPN22 R620W allele demonstrated a more potent inhibition of TCR signaling than wild type,implying a gain of function. The expression of IL-2 is a well-established marker for TCR stimulation. The absence of IL-2 turn an acute, self-limited disease to a chronic and progressive disease. The first evidence showed that the R620W allele is a more potent negative regulator with less IL-2 and higher catalytic activity of phosphatase. Another group has studied the R620W variant directly in primary human T cells from heterozygous and homozygous carriers is associated with reduced responsiveness of CD4+ memory T cells, expression of CD25, calcium mobilization, and IL-10 production upon TCR stimulation. T cells from T1D patients carrying the T1858 allele were characterized by decreased T-cell activation evidenced by reduced TCR-induced IL-2 secretion, calcium mobilization and proliferation in CD4+ T cells by alterations of the intracellular signalling events. Peilin Zheng et al.showed the first functional study of LYP R620W in vivo within a model of type 1 diabetes, and the data also did not support a loss of function for the R620W variant. These results suggest that the R620W variant results in decreased TCR signaling, implying a gain-of-function variant.

However,it was not studied in the context of Csk to clarify the effect of the polymorphism on the synergistic inhibition of TCR signaling in some studies. Without assessed in the context of Csk, previous studies in Jurkat cells have failed to identify fold overexpression or dose response. Moreover,several other studies using primary human cells from patients with autoimmune disease are also more difficult to interpret. Zikherman et al.reported that calcium mobilisation in Jurkat cells coexpressing W620 Csk and Phospho-Erk (extracellular signal–regulated kinase) were significantly increased than R620 and Csk. Another exception is a single report about myasthenia gravis, where the R620W allele associated with increased IL-2 production upon TCR stimulation. These results suggest that when the mutant phosphatase co-expressed with Csk,it increased TCR signalling,implying a loss-of-function variant. The differences in these studies could be in part due to the different indicators and readouts used to measure TCR responses and the different stimuli used to initiate signalling. The mechanism that how the R620W variant affect the immune system remains to be further established.

It is fundamental to know the role of PTPN22 in regulating immune cell responses in order to understand the functions of it as a general autoimmune susceptibility locus. An imbalance in the immune response process causes autoimmune disorders. Regulatory T cells (Tregs) can inhibit effector T cell responses so that reduce damaging and proinflammatory responses, which result in less effective at immunosuppression. Tregs may help in the treatment of autoimmune diseases. The TCR-dependent increase in the extent of integrin-mediated adhesion is critical for Treg function through the activation of Rap1,the guanosine triphosphatase (GTPase), whereas TCR signals, such as ERK and Ca2+ flux, are entirely dispensable. Either the number or the function of Tregs are critical for the mechanism of the autoimmune diseases. Genetic studies have no strong evidence to clearly identify either a recessive or dominant effect of the polymorphism in RA ( homozygous odds ratio 3.32; heterozygous odds ratio 1.98 ).

Christian J. Maine et al. demonstrsted that the Het with no significatly higher suppressive activity (similar CD 25 with the WT mice) had more increased numbers of Tregs and increased ratio of Tregs to effector T cells in thymus than the WT mice, which significantly developed more disease than the Het and KO mice. And depleting the Tregs of KO mouse by anti-CD25 Ab could increase the opportunity and severity of autoimmune disorder.Nevertheless, in T1D the numbers of Tregs are increased and the function of them are impaired partly on account of changes in the CD25R–IL-2 pathway. Another group investigated PTPN22 implicated in autoimmunity in human and mice and found Rap1 phosphorylation was increased in the absence of PTPN22. Data showed that PTPN22 deficient mice did not develop autoimmunity though they had more potent effector T cells, because they also had Tregs with greater immunosuppressive function than those of wild-type mice. PTPN22-deficient Tregs produced more immunosuppressive cytokine interleukin-10 and enhanced adhesive properties than wildtype Tregs, preventing autoimmunity. Julie Zikherman et al. demonstrated that the R620W human risk allele can cooperate with hyperresponsive CD45 E613R B cells to provoke autoantibody production and a lupus-like autoimmune disease to break tolerance in mice. Loss of PTPN22 in the NOD model did not increase the risk of autoimmune diabetes with an increse in regulatory T cells. Pep-deficient mice provide a support for a negative regulatory role in TCR signaling in vivo. More researches are needed to elucidate the involvement of the PTPN22 mutation in Treg functions.

As an intracellular enzyme, LYP was initially thought to be expressed mainly in the cytoplasm of the lymphocytes of human tissues (spleen, thymus, ovary, testis, prostate, small intestine and colon) specifically and predominately and in dendritic cells, granulocytes, monocytes and a number of myeloid cell lines, raising the possibility of uncharacterized effects of the R620W variant on the immune response.

One group assessed whether the R620W allele affects the removal of developing autoreactive B cells. They found that compared with those from wild carriers, new emigrant and mature naive B cells from the PTPN22 risk allele contained high frequencies of autoreactive clones. They demonstrate that the R620W variant interferes with the removal of developing autoreactive B cells and increase the expression of genes such as CD40, IRF5, and TRAF1, which encode proteins that promote B cell activation. Thus,early B cell tolerance defects common to autoimmune diseases such as SLE, RA, and T1D may result from specific polymorphisms and precede the onset of these autoimmune diseases,rather result from chronic ongoing inflammation processes. Individuals with the R620W allele have fewer memory B cells and diminish the BCR process directly studied in primary human T cells.

other LYP variants and LYP splice isoforms

Besides PTPN22 R620W variant, human genomics studies have identified several other missense polymorphisms, such as R263Q(G788A, rs33996649), S201F(C602T, rs74163647) and R266W(C796T, rs72650670). R263Q is located within the catalytic domain exon 10 of LYP which alters the tridimensional configuration of the active domain leads to reduced phosphatase activity. High-resolution structural analysis laid in the molecular basis for its loss of function. Furthermore, the Q263 variant protect human from systemic lupus erythematosus, and is beneficial in human autoimmunity. PTPN22 R263Q is associated with a lower risk of RA and independent in association between PTPN22 R620W and RA of outpatient clinics from 6 different countries, but both variants have an additive effect on the risk of RA. Rencently, Xiao Yu et al. also demostrated that R263Q polymorphism was a loss-of-function variant by kinetic and functional studies. Moreover，they frist investigated the biochemical characters and T cell signaling functions of two new uncharacterized PTPN22 variants R266Wand S201F as well as R263Q, which had reduced the risk in several autoimmune diseases. The S201F variant reduced phosphatase activity of LYP moderately and decreased the function of LYP in T cell signaling slightly, while the R266W variant severely impaired phosphatase activity of LYP through perturbing either the Qloop or the WPD loop of LYP by kinetic and structure analysis.

In any event, the carboxy-terminal portion of LYP plays a critical role in regulating the catalytic activity and physiological functions of LYP. Alternative splicing is a process that the splice variants are translated into different protein isoforms by distinct mRNA transcripts generated from a single gene. During this process,different exons are connected together to form a mRNA. Thus the alternative isoforms share part of the DNA sequence but not all of it. The PTPN22 gene has 11 known spliced mRNAs, and LYP1 and LYP2 are translated into proteins. The LYP1 contains an amino-terminal PTP domain, a central region of unknown function, and a carboxy-terminal portion of approximately 200 amino acids containing four proline-rich motifs termed P1 to P4. The LYP2 has a shorter carboxyl terminus lacking the P2 to P4 motifs.The LYP3 differs from LYP1 as it misses 28 amino acid residues in between the P1 and P2 region. The C1858T SNP happens to cause the R620W substitution in P1 region.

Zhao et al.first performed PCR with primers of both LYP1 and LYP3. The data showed that both LYP1 and LYP3 were highly expressed in major lymphoid tissues and fat,lower expressed in many other tissues considered as the mucosa-associated lymphoid system and not expressed in major non-lymphoid tissues such as brain, heart, liver, and muscle. It is worth noting that lung expressed LYP1 at high levels and LYP3 is not present in the lung. The retina merely expressed LYP3. LYP2 existed in peripheral blood lymphocytes, thymus, and interestingly in prostate at very low levels.

Furthermore,the ratios between LYP1 and LYP3 vary in different tissues. Ronninger et al. have investigated the mRNA expression of the LYP1 and LYP2 splice forms in peripheral blood mononuclear cells collected from the Caucasian patients and normalcontrols. Both cells from RA patients and stimulated by interferon-γ showed pronounced lower levels of LYP2, higher levels of LYP1 and comparabal higher ratio of expression of the two isoforms (LYP1/LYP2) than normal controls, which might indicate an activated inflammatory process and interact with the proinflammatory conditions. Marcus Ronninger et al. proposed a similar hypothesis that there may have different expression of PTPN22 splice forms in patients and controls.The following research showed in Caucasian origin for patients the longer splice form was increased and the shorter was reduced and compared the ratio of the both, the effect was further enhanced for analysis of RA patients and controls. Conversely, the LYP2 had higher levels in multiple sclerosis (MS) patients compared with normal controls. Further studies are required to clarify the expression of LYP splice isoforms in different tissues and the regulatory mechanism to find the association between the LYP isoforms and human diseases, particularly autoimmune diseases.

LYP variants and autoimmune diseases

The initial seminal report of association of the R620W variant with an autoimmune disease came from a candidate gene study of two independent distinct populations with type 1 diabetes (T1D) by Bottini group in 2004. In contrary to Arg620 (allele 1858C),LYP with Trp620 (allele 1858T) does not bind the SH3 domain of Csk. Befor and after one year, the assiciation between PTPN22 and rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) was subsequently researched and replicated in a number of studies and widely extended to many additional autoimmune diseases such as immune thrombocytopenia, generalized vitiligo with concomitant autoimmune diseases, idiopathic inflammatory myopathies, Graves’ disease, juvenile idiopathic arthritis, myasthenia gravis, anti-neutrophil cytoplasmic antibody-associated vasculitis and Addison’s disease. By contrast, well-powered studies found the C1858T SNP had a negligible association with multiple sclerosis, some celiac disease, multiple sclerosis, psoriasis, ankylosing spondylitis, pemphigus vulgaris, primary sclerosing cholangitis, primary biliary cirrhosis, Crohn’s disease (CD), ulcerative colitis (UC)and acute anterior uveitis. Most autoimmune diseases show an insignificant association with PTPN22 C1858T manifest in skin, the gastrointestinal tract or in immune privileged sites. It may be speculated that PTPN22 nonassociated diseases associated with the presence of autoantibodies strongly had less importance of humoral components and less susceptible to inhibition by Tregs than PTPN22 associated diseases.

The polymorphic allele present most in Scandinavia, more frequent (around 12.5%) in Northern than in Southern Europeans and essentially absent in African and Asian populations. In Chinese populations it was higher than it was in Japanese(0.2%, Ikari et al., 2006) and African (0,Begovich et al.，2004)populations. A US group reported association of the R620W variant with RA in 2005 which was followed by numerous other studies in populations of European descent, including UK, Swedish, Dutch Spanish, Finnish, German, and Canadian populations. There had significant variance of the R620 mutation in 15 Chinese populations owing to the migration.

Studies in Japanese and Russian population of RA could not test for association with R620W variant on account of the very low minor allele frequency. However, Zhebrun et al. found a significant association in Russian population between PTPN22 1858 C/T SNP and T1D and GD. 1858T/T genotype was also observed more frequently in T1D and GD patients. Rasha Tarif Hamza et al. found that R620W variant was not a major risk allele for SLE susceptibility in Egyptian SLE patients but was associated with autoimmune thyroid disease in SLE patients of Egyptian (p=0.002).

To explain whether the PTPN22 had association with T1D in Asian populations, Eiji Kawasaki et al. showed in the Japanese population and Korean population the -1123G>C(rs2488457) promoter SNP was associated with acute-onset but not slow-onset T1D and the association was stronger in -1123G>C compared to +1858C>T(rs2476601; R620W) in T1D from multiplex families of European. And -1123G>C and +2740C>T SNP of PTPN22 were in strong linkage disequilibrium.In conclusion, the promoter -1123G>C SNP is a more likely causative variant independent or in linkage disequilibrium with the +1858C>T polymorphism in PTPN22. Moreover, Taniyama M et al. also revealed that in the Japanese population the allele of rs1310182 other than R620W was either frequent or absent in two different rare haplotype in T1D patients and differed significantly between T1D patients and controls. Some other PTPN22 gene polymorphisms (rs1310182, rs2488457and rs3789604) inverstigated recently were not involved in ocular Behcet’s disease in two Chinese Han populations.

Vijay Baranathan et al. first described R620W variant may provide protection for patients with BD in the UK, which was not certain for Middle Eastern patients because they had a greatly reduced prevalence compared to whom in UK. Taken together with the very low prevalence in Africans,

it implied that the R620W variant spread at the same time as

migration into western Europe under some novel evolutionary pressure possibly. Morver, R620W variant had no association with either UK patients with retinal vasculitis or their healthy UK counterparts. Wagenleiter et al had revealed no association of the R620W allele with CD in 146 patients of Northern German origin which were in agreement with previous studies of Criswell et al, Prescott et al in British subjects, Martin et al in Spanish subjects and van Oene et al in Canadian patients. Whereas, Sfar et al. showed an association of inflammatory bowel disease (IBD) susceptibility with the PTPN22 R620W allele in Tunisian patients, without significant association found between the frequencies of allele and the CD and UC. Barrett JC et al. in a large study suggested R620W might be a protective factor in CD. Futhermore, recently, Lina-Marcela Diaz-Gallo et al. found the R620W variant was significantly associated with a reduced risk of developing CD other than UC and the R263Q variant associated with UC. A hypothesis could be that IBD may cause an autoreactive

response to the commensal microbiota in a genetically predisposed

host. Different gene alles like R620W and R263Q with opposite effects in different related diseases may balance the susceptibility alleles in humans.

Rencently, María Carmen Cénit et al. performed a case-control study for the first which suggest that the R620W and R263Q of the PTPN22 gene analyzed in isolation and in combination do not play an important role in the pathophysiology of endogenous non-anterior uveitis from a Spanish population.