1 Department of Microbiology and Immunology, Cancer Research Center, Faculty of Health Sciences, Ben Gurion University of the Negev, PO Box 653, Beer Sheva 84105, Israel
2 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
Correspondence to: N. Isakov; E-mail: noah{at}bgumail.bgu.ac.il
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Abstract |
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Keywords: cellular proliferation, immune regulation, protein kinases/phosphatases, tolerance/suppression/anergy
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Introduction |
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A unique group of surface receptors, which specializes in transmitting extracellular signals in immunocytes, includes the T cell antigen receptors (TCRs), B cell antigen receptors (BCRs), MHC class-I-specific activating receptors on NK cells (i.e. CD94/NKG2C-associated DAP12) and selected receptors for Fc (FcR) (1). While differing in ligand specificity, these receptors share a stretch of amino acid sequence in their cytoplasmic tails, which is critical for the transduction of cell activation signals. This sequence, termed immunoreceptor tyrosine-based activation motif (ITAM), is highly conserved both in content and position of its tyrosine (Y) and leucine (L) or isoleucine (I) residues. All ITAMs possess a duplicate of the sequence YXXL/I (where X is any amino acid), with six to eight intervening residues. Mutating a single tyrosine or leucine/isoleucine residue, or changing the distances between them, completely abolishes their ability to transduce activation signals (25). The conserved physical structure of the ITAMs is mandatory for their function as temporal scaffolds for specific molecules that control the assembly of multiple signaling molecules at the receptor site, on the inner side of the plasma membrane. Thus, engagement of an immunoreceptor leads to the phosphorylation of its ITAMs' tyrosine residues that serve as transient docking sites for specific Src homology 2 (SH2)-containing effector molecules. The phosphorylated ITAMs promote the assembly of multimolecular complexes at the receptor site and the formation of subcellular structures, such as the supramolecular activation cluster (6) or the immunological synapse (79), which are assumed to be critical for cell activation.
In an attempt to identify additional ITAM-containing receptor molecules, which may also regulate activation events in immunocytes, we performed a BLAST search of the National Center for Biotechnology Information (NCBI) database and found that a protein product of the murine TJ6 (mTJ6) gene possesses a putative ITAM sequence (10). mTJ6 has been implicated in immune cell regulation because of its ability to inhibit an in vitro allogeneic mixed lymphocyte reaction (10). In addition, this protein was found to increase IL-10 production by T cells (11), be involved in apoptotic processes in lymphocytes (12) and play a role in pregnancy-induced tolerance induction against allogeneic embryos (1315). Increased expression levels of TJ6 were observed in CD19+ B cells from pregnant, but not from non-pregnant, women (13) and in NK cells from pregnant women with spontaneous recurrent abortions (14). Furthermore, expression of human TJ6 (hTJ6) was observed in B cell chronic lymphocytic leukemia (16), and increased hTJ6 levels were detected in lymphocytes from HIV-seropositive patients (17, 18). However, the cellular and molecular functions of hTJ6 are unknown.
An additional gene product, T cell immune response cDNA7 protein (TIRC7), was recently identified and found to be homologous to mTJ6 (52% total amino acid identity). This protein, termed also vacuolar proton-translocating ATPase 116-kDa subunit a isoform 3, possesses a partially conserved ITAM sequence and plays a role in the induction of T cell activation (19, 20). The TIRC7 is expressed on the surface of human T cells in an activation-dependent manner and participates in immune-mediated cellular responses to alloantigens. Thus, anti-TIRC7 antibodies were shown to down-regulate T cell proliferation and IL-2 secretion in vitro, and to inhibit the acute renal allograft rejection in vivo.
The present work describes the molecular cloning and cDNA sequencing of hTJ6, analysis of its expression in human peripheral blood T cells and in different human tissues and organs, assessment of the potential role of its ITAM sequence in receptor-coupled protein tyrosine kinase (PTK)-mediated signaling pathways and analysis of the biological role of hTJ6 in organellar H+ pumping.
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Methods |
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Molecular cloning of hTJ6
A search of the protein data bank and expressed sequence tag (EST) database for proteins with the YXXL/IX(68)YXXL/I consensus ITAM sequence was performed using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). This yielded the identification of an EST clone (NCBI accession no. C05950), which is highly homologous to a sequence found in the mTJ6 gene, and possesses a consensus ITAM sequence. Cloning of the hTJ6 cDNA from an HL-60 human pro-myelocytic leukemia (ATCC clone CCL 240)-derived cDNA library was performed according to standard procedures.
Expression vector and northern blotting
The hTJ6 expression construct was generated by PCR amplification, using the hTJ6 cDNA as a template and primers containing KpnI (5'-AGGTACCATATCATGGGGTCCCTGTTCC-3') and EcoRV (5'-TCTGCAGATATCGTGCCACACTGTCGTCGTTATT-3') sites. The PCR was carried out with Pfu polymerase (Stratagene) and the amplified product and the pEF4/Myc-His (Invitrogen) eukaryotic expression vector were digested with EcoRV and KpnI and ligated with a T4 DNA ligase (Promega) to create the expression vector, pEF-hTJ6-Myc. The sequence of pEF-hTJ6-Myc has been verified by sequencing extended regions on both sides of the restriction sites.
Analysis of hTJ6 gene expression was performed in Jurkat T cells and in rested or reactivated peripheral blood lymphocyte (PBL) T cells. Cells were washed twice in cold PBS and total RNA was isolated using the TriReagent (MRC, Inc., Cincinnati, OH, USA). Samples of total RNA (20 µg) were size fractionated by electrophoresis on 1% agarose and 0.66% formaldehyde gels, and then blotted onto nylon filters (Schleicher & Schuell, Inc.). Filters were hybridized with a cDNA probe corresponding to the entire coding sequence of hTJ6, which was 32P labeled using the random primer method (24). Overnight hybridization was performed with 32P-labeled cDNA probe (106 counts per minute ml1) at 65°C in 10% dextran sulfate, 4x saline-sodium citrate buffer (SSC), 7 mM Tris (pH 7.6), 0.8x Denhardt's solution, 0.02 mg ml1 salmon sperm DNA and 10% SDS. Blots were washed once in 2x SSC and 0.1% SDS for 20 min at room temperature, and twice at 65°C in 0.2x SSC, 0.1% SDS. Membranes were developed using either a phosphorimager (BioRad) or BioMax X-ray films (Eastman Kodak Company, Rochester, NY, USA) in the presence of an intensifying screen at 80°C.
Hybond northern blot filters containing multiple poly A+ mRNA samples from human tissues and organs were obtained from Amersham Pharmacia Biotech (7-lane Human b filter, #RPN 4802, and 12-lane Human d filter, #RPN 4800). Hybridization of filters was performed in RapidHyb solution (Amersham Pharmacia Biotech) according to the manufacture's guide. Washings of the blots and autoradiography were performed as detailed above.
Cell culture, stimulation and transfection
Human leukemic Jurkat T cells were maintained at a logarithmic growth phase in complete RPMI [RPMI-1640 supplemented with 5% heat-inactivated FCS, 2 mM L-glutamine, 50 units ml1 penicillin, 50 µg ml1 streptomycin (all from Biological Industries, Beit Haemek, Israel) and 5 x 106 M ß-mercaptoethanol (Sigma)] in 75-cm2 growth-area tissue culture flasks (Cell-Cult, Sterilin Limited, Feltham, UK) in an atmosphere of 5% CO2, at 37°C. Treatment of Jurkat cells was performed at 37°C by exposure to hydrogen peroxide (5 min, 10 mM) or pervanadate (30 min, 100 µM Na3VO4 + 0.01% H2O2).
PBLs were obtained by histopaque gradient centrifugation of heparinized blood from healthy volunteers. An enriched population of pre-activated and rested PBL T cells was obtained by the culture of freshly isolated PBLs (1 x 106 ml1) in 10% FCS-containing complete RPMI in the presence of 0.5% (v/v) PHA in 75-cm2 growth-area tissue culture flasks (50 ml per flask). Human rIL-2 (20 units ml1) was added after 72 h of culture and after 3 more days the cells were washed and cultured for 24 h in the absence of PHA or rIL-2. At this stage, the culture contained >90% CD3+ and 3045% CD25+ (IL-2R+) cells. The cells were activated with 5 µg ml1 PHA or 100 ng ml1 phorbol myristate acatate (PMA) plus 200 ng ml1 ionomycin for 24 h.
Cos-7 and HEK 293 cells were grown in complete DMEM. For transfection and cell staining, the cells were removed from plates by trypsin treatment, washed three times in supplement-free DMEM, re-suspended at 7.5 x 106 cells ml1 in supplement-free medium and aliquoted into 0.4-cm-gap Gene Pulser cuvettes (BioRad) (3 x 106 cells per 400 µl per cuvette). Plasmid DNA (10 µg per cuvette) was added and the cells were electroporated using a BioRad Gene Pulser (250 V, 950 µF). The cells were then cultured on glass cover slips or in 75-cm2 tissue culture flasks in complete DMEM for 48 h.
Preparation of cell lysates and immunoprecipitation
Cell lysates were prepared by re-suspension of cells in a lysis buffer containing 25 mM TrisHCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 10 µg ml1 each of leupeptin and aprotinin, 2 mM AEBSF and 1% Triton X-100, followed by a 20-min incubation on ice. Lysates were centrifuged at 13 000 x g for 30 min at 4°C and the nuclear-free supernatants were used for immunoprecipitation studies, or mixed with equal volumes of 2x SDS sample buffer, vortexed, incubated at 100°C for 5 min and analyzed by SDS-PAGE. Cytosol and particulate fractions were prepared by re-suspending the cells in buffer A (20 mM TrisHCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 10 mM ß-mercaptoethanol, 10 µg ml1 each of leupeptin and aprotinin and 2 mM AEBSF), and repeatedly aspirating them through a 1-ml syringe with a 26-gauge needle for 20 s. Cell lysates were centrifuged at 400 x g for 5 min, and nuclear pellets were removed and lysates were re-centrifuged at 13 000 x g. Supernatants (cytosolic fractions) were transferred to a second set of microcentrifuge tubes, Triton X-100 was added up to a 1% final concentration and samples were either mixed with 5x SDS sample buffer (4 : 1, v/v) or used for immunoprecipitation. Pellets were washed once in buffer A, re-suspended in buffer A plus 1% Triton X-100 (in the original volume used for the lysis), incubated for 30 min on ice and centrifuged at 13 000 x g for 20 min. Supernatants (particulate fractions) were either mixed with 5x SDS sample buffer (4 : 1, v/v) or used for immunoprecipitation.
Preparation of anti-hTJ6 rabbit antisera
Selected regions on hTJ6, representing hydrophilic sequences with a high surface probability, were identified using PSORT II (http://psort.nibb.ac.jp) and selected for the preparation of 1419-mer synthetic peptides. Five different peptides (TJ6-1 to TJ6-5, corresponding to hTJ6 residues 8092, 140153, 252269, 684701 and 843856, respectively, each with an N-terminal Cys) were synthesized by Research Genetics (Huntsville, AL, USA) and conjugated to keyhole limpet hemocyanin (SigmaAldrich) using the sulfo-maleimidobenzoyl-N-hydoxysuccinimide ester cross-linker (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. The peptide-carrier antigens were emulsified in CFA and used for immunization of five individual rabbits. Animals were boosted three times, at 3-week intervals, and bled 12 days after the last immunization. ELISA-based antisera screen was performed against the respective peptide, as well as non-specific peptide, followed by western blot analysis of Jurkat cell-derived whole cell lysate. Anti-TJ6-2 antiserum was found to specifically react with the relevant peptide, as well as the endogenous hTJ6 protein, and therefore selected for further studies.
Confocal laser scanning microscopy
Transfected and untransfected Cos-7 cells were seeded on 20-mm2 glass cover slips in culture dishes, washed with cold PBS and fixed with 3.7% PFA at room temperature for 20 min. After washing, the cells were permeabilized with PBS containing 3% BSA and 0.05% saponin for 20 min. Cells were then incubated (1 h at room temperature) with a pre-determined optimal concentration of anti-Myc mAb, followed by incubation with CyTM3-conjugated AffiniPure Goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.), and subsequent washings (x4) with 1% BSA in PBS. After the final wash, the cells were mounted onto glass slides using a drop of Immu-Mount (Shandon, PA, USA). Confocal analysis at x100 magnification was performed on an Axiovert 100M microscope equipped with an LSM510 confocal laser imaging system.
Measurement of vacuolar H+ pump activity
Control and hTJ6 transfected HEK 293 cells were released by trypsinization and washed twice in a medium composed of 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES (pH 7.4 with NaOH), 10 mM glucose and 10 mM pyruvate (solution A). The cells were counted, re-suspended at a density of 5 x 106 cells ml1 and placed on ice until use. H+ uptake was initiated by the addition of 2.5 x 105 cells (50 µl) to pre-warmed 1.45 ml solution A containing 10 µM acridin orange (AO) and placed in a thermostated cuvette. The cell suspension was continuously steered with a pedal stirrer and AO fluorescence was measured at an excitation wavelength of 495 nm and an emission wavelength of 540 nm.
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Results |
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Alignment of the deduced amino acid sequence of hTJ6 with proteins in the GenBank database demonstrated various degrees of homology to proteins that are related to the vacuolar proton pump (VPP) ATPases. hTJ6 is highly homologous to the Bos taurus 116-kDa subunit of the vacuolar proton-translocating ATPase (25) and to the mTJ6 protein (10), with 92 and 91% identity, respectively, and 95% similarity with both proteins at the amino acid composition (see Fig. 1). Furthermore, hTJ6 shows 50% total amino acid identity with the human TIRC7, that functions as an inducible cell surface activation receptor on T lymphocytes (19). NCBIBLASTP (http://prodes.toulouse.inra.fr/prodom/current/html/home.php) (ProDom release CG42) search revealed multiple alignments of four distinct regions within the hTJ6 protein with V-type VPP protein-derived sequences (Fig. 2, upper panel). The N-terminal 363-amino acid sequence of hTJ6 and a sequence between residues 527 and 612 are highly homologous to regions within a subunit of VPP, a variant of an ion glycoprotein hydrogen transporter (CG149936 and CG145575, respectively). In addition, a sequence between residues 367 and 475 and another sequence between residues 725 and 839 are homologous to regions within the subunit of a V-type transmembrane hydrogen ion transport ATP synthase (CG2645066 and CG001977).
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Many secretory and integral membrane eukaryotic proteins are directed to the endoplasmic reticulum (ER) membrane by special amino-terminal leader sequences that are found in the precursor protein, but are usually absent from its mature form. We therefore searched for a potential signal peptide in hTJ6 utilizing the SignalP V2.0.ß2 program (http://www.cbs.dtu.dk/services/SignalP-2.0/). A putative signal peptide sequence was identified at the very first 22 amino acids of hTJ6 (at a probability of 0.957) with a cleavage site between Ala22 and Tyr23 (at a probability of 0.889) (29). We suggest therefore that the mature hTJ6 protein is 834-amino acid long and possesses a theoretical molecular mass of 95 782 Da.
Gene expression of hTJ6
Northern blot analysis using the EST clone, C05950, and the hTJ6 full-length cDNA as probes demonstrated the existence of three different species of hybridizing mRNA (corresponding to 3.2, 5.0 and 7.3 kb) in a preparation of total RNA from Jurkat T cells. The 3.2-kb hybridizing transcript was the most prevalent, supporting the assumption that the obtained sequence of hTJ6 includes the entire ORF.
To further analyze hTJ6 expression in normal human T cells and test whether its expression is affected by extracellular signals leading to cell proliferation, we have obtained freshly isolated human peripheral blood cells and prepared a long-term culture of enriched T cells by the inclusion of PHA and rIL-2 in the culture medium. After 6 days of culture, the cells were washed and re-stimulated with PHA or PMA plus ionomycin, for 24 h. Cells were harvested and total RNA was prepared and subjected to northern blot analysis. Results demonstrated that resting human PBL T cells express two major transcripts of hTJ6 mRNA, with a relative ratio of 1 : 1 (Fig. 3). Re-activation of T cells with PHA, which stimulate their proliferation, resulted in 5-fold increase in hTJ6 RNA levels. In contrast, PMA plus ionomycin, which are poor mitogens for purified T cells, did not affect hTJ6 RNA levels. Preliminary studies demonstrated increased expression of the hTJ6 protein in PHA-stimulated peripheral blood T cells, supporting the assumption that hTJ6 levels increase in activated and/or proliferating T cells.
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To verify that this protein band corresponds to the protein product of hTJ6, we prepared a Myc-tagged hTJ6 expression vector (pEF-hTJ6-Myc) and expressed it in Cos-7 cells. Two days later, the cells were lysed and anti-Myc immunoreactive proteins were immunoprecipitated from the cell lysate and subjected to SDS-PAGE and immunoblotting. Sequential incubation of the nitrocellulose membrane with anti-Myc mAbs and anti-hTJ6 antiserum revealed that the two antibodies reacted with the same 100-kDa protein band. The fact that the anti-hTJ6 antiserum reacted with the pEF-hTJ6-Myc gene product, and recognized a Jurkat cell-derived endogenous protein possessing a similar molecular mass demonstrated its specificity toward the hTJ6 protein.
To determine the relative subcellular distribution of hTJ6 in situ, we transfected Cos-7 cells with pEF-hTJ6-Myc and cultured them for 48 h on 20-mm2 glass cover slips in culture dishes. The cells were then fixed with PFA, stained with rabbit anti-Myc mAb and CyTM3-conjugated AffiniPure Goat anti-mouse IgG and analyzed by confocal laser scanning microscopy. Figure 5(A and B) shows that only transfected cells reacted with the antibody, indicating high antibody specificity due to the absence of Cos-7 cell endogenous proteins that immunoreact with the anti-Myc antibodies. Staining of transfected cells demonstrated that hTJ6 is localized at distinct structures within the cytoplasm, which included the Golgi and/or part of the ER that extended to selective areas at the plasma membrane. The staining results further indicate that hTJ6 is a membrane-embedded protein and that the expression pattern is consisted with its being a subunit of a vacuolar H+ pump.
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We then tested whether tyrosine residues within the hTJ6 ITAM region serve as potential in vivo substrates for PTKs and thereby enable hTJ6 to interact with SH2-containing proteins. Jurkat T cells were incubated with hydrogen peroxide or pervanadate, pharmacological agents that non-specifically activate PTKs and inhibit the activity of PTPases, and tested whether under these conditions hTJ6 will undergo tyrosine phosphorylation. We found that anti-hTJ6 antiserum immunoprecipitated a major protein band of 100 kDa from the Jurkat cell lysate, which was recognized in the immunoblot with the anti-hTJ6 antiserum (Fig. 6B). hTJ6 from resting Jurkat T cells was not phosphorylated on tyrosine residues and pervanadate induced only a weak tyrosine phosphorylation signal (Fig. 6A). In contrast, a TCR polypeptide, which possesses three functional ITAMs, exhibited, under similar activation conditions, a very strong response when immunoreacted with anti-phosphotyrosine antibodies (Fig. 6C). The reason for immunoprecipitation of the TCR
by using ZAP-70-specific antibodies is that anti-TCR
antibodies precipitate very well the non-phosphorylated TCR
, but much less efficiently the tyrosine phosphorylated TCR
(Fig. 6E). On the other hand, the tyrosine phosphorylated TCR
co-immunoprecipitates very well with the ZAP-70 PTK from activated T cells (Fig. 6C). Under the assay conditions used, ZAP-70 also underwent a high level of tyrosine phosphorylation (Fig. 6C and D). These results, together with the predicted hydrophobic nature of the hTJ6 ITAM sequence, suggest that this region is not a preferred target for phosphorylation by PTKs, and is therefore not likely to function as a PTK-dependent regulatory domain.
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Discussion |
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The hTJ6 gene appears to be expressed in a wide range of human tissues and organs. It shows the highest homology to a Bos taurus gene encoding the 116-kDa subunit of the vacuolar proton-translocating ATPase (25), and to the mTJ6 (10), representing a family of 116-kDa V-type ATPases isoform a2. Furthermore, hTJ6 shares amino acid homology with additional proteins that grouped in Pfam (http://pfam.wustl.edu) under accession no. PF01496. mTJ6 has been implicated in multiple immune-related phenomena and was found to be involved in the regulation of lymphocyte responses to alloantigens, cytokine production, induction of apoptosis and immune tolerance (10). Such multiple effects are consistent with a role for hTJ6 in a fundamental cellular activity that affects many functions, such as the control of organellar acidification.
An additional member of this group includes the TIRC7, which was found to play a role in T cell proliferation and IL-2 secretion, to selectively regulate type-1 cytokine secretion and to be involved in the immune rejection response against allogeneic tissue grafts (19). Despite the fact that all these proteins show sequence similarity to different subunits of VPPs, most have not been functionally characterized, and in analogy to the mTJ6 and the human TIRC7 proteins, they may possess additional or different biological roles [reviewed in (38)]. It is worthwhile mentioning that TIRC7 was found in two different isoforms: a long one, which is expressed predominantly in osteoclastomas, and a shorter version, which is expressed predominantly in the thymus. It is possible therefore that other genes encoding vacuolar H+ pump subunit-like proteins may yield alternatively spliced forms that are involved in distinct biological functions.
hTJ6 was found to possess the sequence, YXXLX(7)YXXL (452466 amino acids), which is identical with a conserved sequence comprising the ITAM domain. However, the hTJ6 protein is structurally distinct from most ITAM-containing molecules, including the TCR subunits (,
,
and
), the BCR subunits (
and ß), the common FcR-
subunit, as well as the DAP12 (1). Thus, hTJ6 is predicted to include six transmembrane regions, in contrast to a single transmembrane region in most of the proteins indicated above (1). The only known exception, thus far, among the ITAM-containing proteins is the Fc
RI-ß subunit, which is predicted to include four transmembrane regions.
ITAMs play important roles in transmitting signals from cell surface receptors since engagement of the corresponding receptors leads to their tyrosine phosphorylation, followed by recruitment and binding of multiple SH2-containing effector molecules to the phosphorylated ITAMs. Clustering of these molecules at the receptor site is required for activating biochemical cascades that transmit signals to the nucleus and other subcellular compartments. Nevertheless, the extent of tyrosine phosphorylation of the relevant ITAMs following receptor ligation varies tremendously, suggesting that some ITAMs play more active roles than others in signal transduction and amplification. We found that hTJ6 is a poor in vivo substrate for PTKs in pervanadate or hydrogen peroxide-treated Jurkat cells. This finding, the hydrophobic nature of the hTJ6 ITAM, and the predicted location of the ITAM adjacent to/or within a transmembrane region suggest that the hTJ6 ITAM, unlike most ITAM sequences, is not a functional activation domain and is not involved in linking the hTJ6 to a PTK-dependent downstream signaling pathway.
Studies of the murine form of TJ6 suggested that the mature protein undergoes post-translational modification and proteolytic cleavage that yields an 50-kDa transmembrane protein and a short protein, of
1820 kDa, which is secreted from the cells and exhibits immunoregulatory properties (39, 40). More recent work has identified a soluble factor termed ShIF (secreted short form of immune suppressor factor), of
27 kDa, which is secreted from stroma cells and can support the in vitro growth of Ba/F3-derived S21 mutant cells (33, 41). In contrast, it inhibited the growth of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner (33). Incidentally, ShIF was found to be identical with a portion of the mTJ6 molecule, but in contrast to the soluble mTJ6, which is derived from its amino terminus (39), ShIF is derived from the carboxy terminus of mTJ6 (33). It has been suggested that ShIF may act as an antagonist to the ATPase complex, and thereby decrease the ability of HUVEC to proliferate. While ShIF represents an accidental, chemical mutagenesis-originated mutant gene product, its effects on cell growth together with the reported findings of the soluble form of mTJ6 support the suggestion that TJ6 proteins have multiple forms, which can affect different cell types, including cells of the immune system. We now aim at the identification of putative alternatively spliced forms and/or proteolytic products of hTJ6 and define their immunoregulatory and other biological activities in different experimental systems.
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Acknowledgements |
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Abbreviations |
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AEBSF | 4-(2-aminoethyl)-benzenesulfonyl fluoride |
ATCC | American Type Tissue Collection |
AO | acridin orange |
BCR | B cell antigen receptor |
ER | endoplasmic reticulum |
EST | expressed sequence tag |
HUVEC | human umbilical vein endothelial cell |
hTJ6 | human TJ6 |
ITAM | immunoreceptor tyrosine-based activation motif |
mTJ6 | murine TJ6 |
NCBI | National Center for Biotechnology Information |
PBL | peripheral blood lymphocyte |
PMA | phorbol myristate acatate |
PTK | protein tyrosine kinase |
SH2 | Src homology 2 |
ShIF | secreted short form of immune suppressor factor |
SSC | saline-sodium citrate buffer |
TCR | T cell antigen receptor |
TIRC7 | T cell immune response cDNA7 protein |
VPP | vacuolar proton pump |
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Notes |
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Received 3 May 2005, accepted 15 July 2005.
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References |
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