Identification of a variant form of tyrosine phosphatase LYP
© Wang et al; licensee BioMed Central Ltd. 2010
Received: 13 July 2010
Accepted: 2 November 2010
Published: 2 November 2010
Protein tyrosine phosphatases (PTPs) are important cell signaling regulators with major pathological implications. LYP (also known as PTPN22) is an intracellular enzyme initially found to be predominately expressed in lymphocytes. Importantly, an allelic R620W variant of LYP is strongly associated with multiple autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and autoimmune thyroid disease.
In this study, we isolated a novel isoform of LYP designated LYP3. LYP3 differs from LYP1, the known isoform of LYP, in that it lacks a 28 amino acid segment right after the R620W site embedded in a proline-rich protein-protein interaction motif. Genomic sequence analysis revealed that LYP3 resulted from alternative splicing of the LYP gene located on chromosome 1p 13.3-13.1. Reverse transcription PCR analyses of 48 human tissues demonstrated that both LYP1 and LYP3 are predominantly expressed in primary and secondary lymphoid tissues but the relative expression levels of the two isoforms varies in different human tissues and individuals.
We thus identified a new variant form of LYP and conducted a comprehensive analysis of LYP tissue expressions. Considering the pathogenesis of LYP R620W, we believe that the expression of LYP3 may have an important role in regulating activity and function of LYP and may be implicated in autoimmune diseases.
Protein tyrosine phosphatases (PTPs) act in a coordinated manner with protein tyrosine kinases to control cell signaling thereby regulating various physiological processes . Malfunctioning of these enzymes has major pathological implications. One of the best known examples is the allelic variant of the lymphoid tyrosine phosphatase LYP (PTPN22) which is associated with multiple autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and autoimmune thyroid disease [2, 3].
LYP is a cytoplasmic enzyme belonging to the PEST group of non-receptor classical PTPs . It contains 807 amino acid residues. The murine ortholog of LYP is called PEP . LYP and PEP share 89% and 61% sequence identity in their PTP domains and noncatalytic portions, respectively. The N-terminal part of LYP/PEP contains the catalytic domain conserved in all classical PTPs. The structure of the sequence following the catalytic domain is largely undefined. The last 200 amino acid segment contains 4 proline-rich sequence motifs (P1-P4) which presumably provides docking sites for SH3 domain-containing signaling proteins. The first of these motifs, P1, is known to bind with relatively high stoichiometry to the SH3 domain of the Csk tyrosine kinase, an important negative regulator of T-cell antigen receptor signaling [6–8]. LYP/PEP and Csk appear to have complementary functions. While Csk phosphorylates the negative regulatory tyrosine residue at the C-terminal end of Lck and Fyn, LYP/PEP dephosphorylates the positive regulatory site in the middle of these two tyrosine kinases . Both actions result in inhibition of TCR signaling. LYP/PEP also negatively regulates T cell signaling by dephosphorylating Zap-70 and the tyrosyl residues within the immunoreceptor tyrosine-based activation motifs in CD3 ζ-chains, which are phosphorylated by Src family tyrosine kinases . Further studies by using a substrate-trapping mutant of LYP in combination with mass spectrometry identified the following substrates: Lck (at Y394), ZAP-70 (at Y493), Vav, valosin-containing protein, and immunoreceptor tyrosine-based activation motifs in CD3 ζ-chains . LYP/PEP is predominately expressed in lymphocytes, and it is generally defined as a key inhibitor of T-cell activation.
A major advancement in the study of LYP is the finding of a single nucleotide polymorphism (SNP), namely, C1858T, which generates an R620W amino acid substitution (reviewed in 2, 3). This SNP is a common risk factor for many autoimmune diseases, including type I diabetes , systemic lupus erythematosus , rheumatoid arthritis [14, 15], and Graves' disease . The 620 amino acid residue is located within the first protein-rich motif P1, which interacts with the SH3 domain of Csk [6–8]. The R620W amino acid substitution presumably interrupts the interaction of P1 with the SH3 domain of Csk . However, a later study revealed that the R620W substitution is actually a gain-of-function mutation that generates a more active PTP that acts as a more effective inhibitor of T-cell signaling than the wild-type enzyme . The mechanism by which this R620W substitution in the non-catalytic segment leads to activation of the phosphatase remains to be defined. It was postulated that this activating mutation in LYP may cause a predisposition to autoimmune disease either by failure to delete auto-reactive T cells or due to insufficient activity of regulatory T cells . In any event, the C-terminal part of LYP has a crucial role in regulating the catalytic activity of LYP and its physiological functions. In this study, we isolated a new variant form of LYP designated LYP3 which differs from LYP1, the known isoform of LYP, in the C-terminal region right after the R620W substitution site. We found that the expression level of LYP3 relative to LYP1 varies in different tissues and among different individuals.
Total RNAs were isolated from de-identified human peripheral blood samples by using the Trizol reagent (Invitrogen) and were then treated with RQ1 RNase-free DNase (Promega) to remove contaminated genomic DNAs. First-strand cDNA was synthesized by using the iScript cDNA Synthesis Kit (Bio-Rad) with random primers. Human Major Tissue qPCR Array containing first strand DNAs from 48 tissues was purchased from OriGene.
Molecular cloning of LYP
Based on the coding sequence of LYP in GenBank database, two PCR primers, namely, Lyp5' (5'-GACATGCCCTCCCTCAACCTACTTA) and Lyp3' (5'-TGCAGGTGTACTTGCAGCCCATATTA) were synthesized. They correspond to the 5' and 3' ends of the coding sequence of LYP1, respectively. Single-strand cDNAs synthesized with total RNAs purified from human peripheral blood samples were used as templates. The PCR was run for 35 cycles with Phusion polymerase (Finnzymes) at 94°C for 30 sec, 65°C for 30 sec, and 72°C for 2 min. The PCR products were ligated with T4 DNA ligase into the pBluescript KS vector which was opened by digestion with EcoRV. Clones were selected based on blue-white selection, and plasmid DNAs were isolated for sequencing analyses with T3 and T7 primers by using the automatic DNA sequencing facility at the Oklahoma Medical Research Foundation.
Determination of tissue distributions of LYP1, LYP2, and LYP3
To determine the expression of LYP1, LYP2, and LYP3 in various human tissues, we employed the Human Major Tissue qPCR Array (OriGene) containing first strand DNAs from 48 tissues. The PCR primers were L1 (5'-CTAGCAACTGCTCCAAGGATAGATGA), L1r (5'-GCTTGTTTGGTGGGCAAGAATTACA), L2r (5'-GGGCTAAATGTCATCTAAAGCCAAG), and L2 (5'- TCCTCAGCTGTGAAGAGTGTAAAACTC). These primers were derived from different exons on the LYP gene and are expected to give rise to distinct PCR products for LYP1, LYP2, and LYP3 cDNAs. L1 and L1r produce 505 and 421 bp fragments from LYP1 and LYP3, respectively; L1 and L2r amplify a 284 bp fragment from LYP2 only; L2 and L1r specifically yield a 301 bp fragment of LYP3. The PCR was run for 35-45 cycles, and the conditions were 94°C for 20 sec, 63°C for 20 sec, and 72°C for 1 min. The products were analyzed on 1.5% agarose gels stained with ethidium bromide or on 8% acrylamide gels visualized by using silver staining.
Results and Discussion
Identification of LYP3, a variant form of LYP
LYP3 is an alternative splicing product
Distribution of three isoforms of LYP in human tissues
DNA sequence variations play a fundamental role in human phenotypic variability, including susceptibility to diseases. SNPs represent the most extensively studied sequence variations. Non-synonymous SNPs change the amino acid sequence and presumably alter protein functions. The C1858T SNP that leads to the R620W substitution in LYP is a typical example. On the other hand, alternative splicing of mRNA may generate protein isoforms of different sizes with different biological properties such as protein/protein interaction, subcellular localization, and catalytic activity [19, 20]. We believe that this should be the case for LYP2 and LYP3. To ensure correct cellular functions, gene expression is finely regulated both spatially and temporally, and the importance of aberrant RNA processing in diseases has been generally recognized. Pre-mRNA splicing is a tightly regulated process affected by many factors including SNPs . In this regard, it should not be a surprise if the C1858T SNP affects expression of alternative spliced forms of LYP. The C1858T SNP is present mainly in Caucasian populations and is essentially absent in African and Asian populations . It is not known if other DNA variations in LYP affect autoimmune diseases in the latter populations which have no fewer incidences of these diseases. Our data indicate that the relative ratios of LYP isoforms vary among different individuals. It will be important to find if aberrant expression of LYP isoforms cause any human diseases, particularly autoimmune diseases.
To conclude, extensive literatures exist to support the association between LYP and multiple autoimmune diseases. However, the underlying molecular mechanism is still elusive. We have now identified a novel isoform of LYP designated as LYP3. This allows us to look at the problem from a new angle.
This work was supported by grants HL079441 and HL094591 from the National Institutes of Health, a grant from Oklahoma Center for the Advancement of Science & Technology, and a Boyou fund from China Soong Ching Ling Foundation (to ZJ Zhao).
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