Cloning, expression and functional characterization of the putative regeneration and tolerance factor (RTF/TJ6) as a functional vacuolar ATPase proton pump regulatory subunit with a conserved sequence of immunoreceptor tyrosine-based activation motif

Yael Babichev1, Ami Tamir1, Meeyoug Park2, Shmuel Muallem2 and Noah Isakov1

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


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In an attempt to identify new immunoreceptor tyrosine-based activation motif (ITAM)-containing human molecules that may regulate hitherto unknown immune cell functions, we BLAST searched the National Center for Biotechnology Information database for ITAM-containing sequences. A human expressed sequence tag showing partial homology to the murine TJ6 (mTJ6) gene and encoding a putative ITAM sequence has been identified and used to clone the human TJ6 (hTJ6) gene from an HL-60-derived cDNA library. hTJ6 was found to encode a protein of 856 residues with a calculated mass of 98 155 Da. Immunolocalization and sequence analysis revealed that hTJ6 is a membrane protein with predicted six transmembrane-spanning regions, typical of ion channels, and a single putative ITAM (residues 452–466) in a juxtamembrane or hydrophobic intramembrane region. hTJ6 is highly homologous to Bos taurus 116-kDa subunit of the vacuolar proton-translocating ATPase. Over-expression of hTJ6 in HEK 293 cells increased H+ uptake into intracellular organelles, an effect that was sensitive to inhibition by bafilomycin, a selective inhibitor of vacuolar H+ pump. Northern blot analysis demonstrated three different hybridizing mRNA transcripts corresponding to 3.2, 5.0 and 7.3 kb, indicating the presence of several splice variants. Significant differences in hTJ6 mRNA levels in human tissues of different origins point to possible tissue-specific function. Although hTJ6 was found to be a poor substrate for tyrosine-phosphorylating enzymes, suggesting that its ITAM sequence is non-functional in protein tyrosine kinase-mediated signaling pathways, its role in organellar H+ pumping suggests that hTJ6 function may participate in protein trafficking/processing.

Keywords: cellular proliferation, immune regulation, protein kinases/phosphatases, tolerance/suppression/anergy


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Binding of extracellular ligands to their cognate surface receptors regulates a plethora of cell functions in a wide range of cell types. They do so by activating receptor-linked signal transduction pathways that function as ‘on-or-off’ molecular switches, which regulate gene transcription and translation as well as post-translational processes. These signaling pathways therefore regulate cell replication, differentiation, maturation and acquisition of specific effector functions.

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.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Materials
PHA, histopaque-1077, aprotinin, leupeptin and Triton X-100 were from Sigma Co. 4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF) was from ICN Biomedicals, Inc. (Aurora, OH, USA). Human recombinant IL (rIL)-2 was a gift from Hoffman LaRoche. Nitrocellulose membranes were from Schleicher & Schuell (Keene, NH, USA); ECL, [{gamma}-32P]ATP (3000 Ci mmol–1), [{gamma}-32P]dCTP (3000 Ci mmol–1) and protein A sepharose were from Amersham Pharmacia Biotech. The Myc 1-9E102 hybridoma producing anti-human Myc IgG1 mAb (21) was obtained from the American Type Tissue Collection (ATCC) (CRL 1729) and ascites were prepared in Balb/c mice. Anti-phosphotyrosine mAb (4G10) was from Upstate Biotechnology Inc. Anti-ZAP-70 polyclonal antiserum was raised in rabbits against a glutathione-S-transferase fusion protein containing amino acids 255–345 of human ZAP-70 (22). A TCR{zeta}-chain-specific rabbit polyclonal antiserum was a gift from Larry Samelson (National Institutes of Health, MD, USA) (23). ddNTPs, T4 DNA ligase and DNA-labeling beads were from Promega (Madison, WI, USA). Pfu DNA polymerase was from Stratagene (La Jolla, CA, USA). Restriction enzymes were from New England Biolabs (Beverly, MA, USA). pEF4/Myc-His was from Invitrogen (Carlsbad, CA, USA). QIAquick gel extraction and DNA purification kits were from Qiagen, and oligonucleotides were from MWG Biotech AG (Ebersberg, Germany).

Molecular cloning of hTJ6
A search of the protein data bank and expressed sequence tag (EST) database for proteins with the YXXL/IX(6–8)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 ml–1) 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 ml–1 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 ml–1 penicillin, 50 µg ml–1 streptomycin (all from Biological Industries, Beit Haemek, Israel) and 5 x 10–6 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 ml–1) 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 ml–1) 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 30–45% CD25+ (IL-2R{alpha}+) cells. The cells were activated with 5 µg ml–1 PHA or 100 ng ml–1 phorbol myristate acatate (PMA) plus 200 ng ml–1 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 ml–1 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 Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 10 µg ml–1 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 Tris–HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 10 mM ß-mercaptoethanol, 10 µg ml–1 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 14–19-mer synthetic peptides. Five different peptides (TJ6-1 to TJ6-5, corresponding to hTJ6 residues 80–92, 140–153, 252–269, 684–701 and 843–856, respectively, each with an N-terminal Cys) were synthesized by Research Genetics (Huntsville, AL, USA) and conjugated to keyhole limpet hemocyanin (Sigma–Aldrich) 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 ml–1 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.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Sequence analyses of hTJ6
ITAM-bearing proteins represent a selected group of immunoreceptors, which transduce activation signals in immunocytes, and are critical for the initiation of early activation events leading to efficient responses of both cell-mediated and humoral immune systems. To identify additional ITAM-containing proteins, which may be involved in the early activation response of immunocytes, we used the BLAST program to search the protein data bank for entries predicted to possess a conserved ITAM, followed by search of human EST sequences in the GenBank database. A human EST clone (NCBI accession no. C05950) was identified, that possesses a high degree of sequence similarity with a region on the mTJ6 gene and complete matching with the ITAM sequence YXXLX(7)YXXL. This EST clone was used for cloning of the hTJ6 gene (GenBank accession no. AF112972) from an HL-60 human pro-myelocytic leukemia (ATCC CCL 240)-derived cDNA library. The hTJ6 gene is predicted to encode a polypeptide of 856-amino acid residues (accession no. NP_036595) with a calculated molecular mass of 98 155 Da.

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). NCBI–BLASTP (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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Fig. 1. Linear alignment of the deduced amino acid sequences of hTJ6 and m and a Bos taurus VPP2 gene. Sequence alignment was performed using the CLUSTALW multiple alignment program (http://www.ebi.ac.uk/clustalw) (26). Residues of hTJ6 and all non-conserved residues are in bold-face capital letters, the putative ITAM sequence is underlined and the conserved tyrosine and leucine/isoleucine residues within this region are shown on a gray background. Symbols under the aligned sequences represent identity (asterisk), strong similarity (colon), weak similarity (dot) and lack of similarity (dash).

 


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Fig. 2. hTJ6 structural domain homology and Kyte–Doolittle hydrophilicity/hydrophobicity plot. The hTJ6 amino acid sequence was compared with NCBI–BLASTP (http://prodes.toulouse.inra.fr/prodom/current/html/home.php) (ProDom release CG42) multiple alignments, using Matrix: BLOSUM62 (upper panel). hTJ6 was found to include four major protein domains with high homology to known proteins, all corresponding to V-type VPP proteins. The topology plot of hTJ6 hydrophilicity profile was obtained using the Kyte–Doolittle method with a window length of 17 amino acids (http://bioinformatics.weizmann.ac.il/hydroph/index.html) (lower panel). The x-axis corresponds to the protein residue number (starting from the N-terminus) and the y-axis corresponds to the relative hydropathy value obtained using a comparison algorithm according to Kyte–Doolittle, as described (27). Hydrophobic sequences representing the predicted membrane-embedded regions are marked with a gray color.

 
Hydropathy profile of hTJ6, using either the Tmpred (28) (http://www.ch.embnet.org/software/TMPRED_form.html) or Kyte–Doolittle program (27) (http://fasta.bioch.virginia.edu/fasta/grease.htm) with a window size of 17 amino acids, predicted seven hydrophobic regions, corresponding to transmembrane-spanning sequences (Fig. 2, lower panel). While six of the transmembrane regions are predicted at a high score, one region (fifth from the amino terminus) is predicted at a relatively low score, raising a possibility that hTJ6 possesses six, instead of seven, transmembrane regions. A six membrane span paradigm or multiplication thereof is found in many ion channels and transporters and thus is more consistent with a role of TJ6 in H+ pumping (see below). Both HMMTOP (http://www.enzim.hu/hmmtop) and TMHMM (v. 2.0) (http://www.cbs.dtu.dk/services/TMHMM) topology programs predicted that the long N-terminal tail of hTJ6 (~400 amino acids) is in a hydrophilic environment.

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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Fig. 3. Northern blot analysis of hTJ6 in human peripheral blood T cells. Northern blot analysis was performed on 5-µg samples of total RNA from resting or reactivated population of enriched peripheral blood T cells. The cells were reactivated with 5 µg ml–1 PHA or 100 ng ml–1 PMA plus 200 ng ml–1 ionomycin for 24 h. RNA samples were separated on an agarose gel and analyzed by hybridization with a full-length hTJ6 cDNA probe that was nick translated in the presence of [32P]dCTP. The sizes of the hTJ6 mRNA were estimated to be ~3.2 and ~7.3 kb. Numbers on the left indicate the position of RNA size markers.

 
We next determined the tissue and organ expression of hTJ6 using multiple tissue northern blot filters (from Amersham Pharmacia Biotech) representing 19 different human tissues or organs (Fig. 4). Results demonstrated abundant expression of hTJ6 mRNA in skeletal muscle and stomach, where three hybridizing transcripts of 3.2, 5.0 and 7.3 kb were observed. hTJ6 expression levels were less abundant in the heart, lymphoid organs (thymus, spleen and lymph node) and internal organs, such as kidney, liver and adrenal, with no detection in tissues of the nervous system, including brain and spinal cord. While the quantitative relative ratios between the three different hTJ6-hybridizing transcripts were equal in some tissues (i.e. stomach), in most tissues the 3.2-kb transcript was the most prevalent, and sometimes represented the only transcript observed (i.e. thymus and placenta). Nevertheless, the presence of multiple transcripts indicates possible expression of distinct splice variants in a tissue-, cell- and/or organelle-specific manner. It should be noted that the relative expression levels of hTJ6 mRNA in all tissues were significantly lower than those of actin (Fig. 4, panel B). Thus, the intensity of the radioactive signals obtained after 2 h of X-ray film exposure of the actin-hybridized membranes was significantly stronger than the signal obtained after 4 days of film exposure to the hTJ6-hybridized membranes.



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Fig. 4. Tissue distribution of hTJ6 mRNA. Northern blot analysis of Hybond filters containing multiple human tissue samples was performed using a full-length hTJ6 cDNA probe that was nick translated in the presence of [32P]dCTP (A). Each lane contains 2.5 µg of poly A+ mRNA and the tissue source is indicated above the lane. The relative amount of hTJ6 mRNA in each lane was normalized by comparison to ß-actin mRNA (B), and presented in relative units (C). The data represent one of two separate experiments performed with freshly made probes on two different Hybond filters. Numbers on the left indicate the position of RNA size markers.

 
hTJ6 protein expression
To confirm the expression of a putative protein with a sequence and molecular mass that coincides with those of the predicted hTJ6 gene product, we first prepared a rabbit antiserum against a synthetic peptide (hTJ6-2) corresponding to residues 140–153 of hTJ6. Utilization of the anti-hTJ6-2 to immunoprecipitate and immunoblot Jurkat T cell lysates revealed one predominant immunoreactive protein band with an apparent molecular mass of ~100 kDa (as determined by SDS-PAGE), consistent with the predicted size of hTJ6. Immunoblot analysis of Jurkat whole cell lysates yielded an undetectable or very low response, suggesting either low level of expression of hTJ6 by Jurkat cells or low affinity and/or specificity of this antibody toward the denatured hTJ6 protein.

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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Fig. 5. Localization of hTJ6 in transfected Cos-7 cells. Cos-7 cells were transfected with the pEF-hTJ6-Myc expression vectors, cultured for 48 h, followed by fixation in PFA, permeabilization in saponin and staining with anti-Myc mAb and CyTM3-conjugated secondary antibody. Stained cells were analyzed by confocal laser microscopy. Panels (A) and (B) represent fluorescence and Nomarski imaging of one single field, respectively. Analysis of the relative distribution of hTJ6 in Jurkat T cells was performed by preparation of cytosolic and particulate fractions of cell lysates and their immunoblotting with anti-hTJ6 antiserum (C). The effect of PMA + ionomycin on the cellular distribution of hTJ6 was tested by treatment of the Jurkat cell with 100 ng ml–1 of PMA and 200 ng ml–1 of ionomycin for 20 min, prior to their lysis.

 
To further analyze the relative cellular distribution of hTJ6, we prepared cytoplasmic and membrane-rich particulate fractions from Jurkat T cells and immunoblotted them with anti-hTJ6 antiserum. Over 85% of the total cellular hTJ6 protein was found to reside in the particulate fraction (Fig. 5C). In addition, PMA + ionomycin treatment of cells, which affects the cellular distribution of some proteins (i.e. protein kinase C), had no effect on the subcellular distribution of hTJ6.

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{zeta} 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{zeta} by using ZAP-70-specific antibodies is that anti-TCR{zeta} antibodies precipitate very well the non-phosphorylated TCR{zeta}, but much less efficiently the tyrosine phosphorylated TCR{zeta} (Fig. 6E). On the other hand, the tyrosine phosphorylated TCR{zeta} 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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Fig. 6. Analysis of the capacity of hTJ6 to serve as an in vivo substrate for PTKs. Jurkat T cells were treated with hydrogen peroxide (10 mM, 5 min) or pervanadate (100 µM Na3VO4 + 0.01% H2O2, 30 min) at 37°C. Cell lysates were left untreated or subjected to immunoprecipitation with anti-hTJ6 (A and B) or anti-ZAP-70 (C–E) antibodies. Samples were fractionated by SDS-PAGE and sequentially immunoblotted with anti-phosphotyrosine (A) and anti-hTJ6 (B) antibodies or with anti-phosphotyrosine (C) and anti-ZAP-70 (D, upper part of the membrane) and anti-TCR{zeta} (E, lower part of the membrane) antibodies.

 
hTJ6 function
The lack of tyrosine phosphorylation and the homology to the 116-kDa vacuolar H+ pump suggest that TJ6 may function as a subunit of the multisubunit cytoplasmic sector of the V-type pump that is expressed in all organelles, including the Golgi and the ER, to mediate their acidification. This possibility was tested directly by comparing the accumulation of the weak base AO in control cells and cells transiently transfected with hTJ6. Figure 7 shows that expression of hTJ6 increased AO uptake by ~30%. Parallel transient over-expression of GFP (data not shown) revealed transfection efficiency of ~60%, indicating that expression of hTJ6 increased acidification of intracellular organelles by as much as 50%. The specificity of the AO signal was verified by complete dissipation of the gradient with the H+/K+ exchanger ionophore nigericin and by the complete inhibition of the influx by the selective vacuolar H+ pump inhibitor bafilomycin. Hence, the results in Fig. 7 clearly show that hTJ6 function in H+ transport into intracellular organelles.



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Fig. 7. Effect of hTJ6 on organelle H+ pumping. Untransfected (A, control) and hTJ6 transfected (B and C) cells were added to a medium containing 10 µM AO and AO fluorescence was measured. The cells in (C) were pre-incubated with 0.75 µM bafilomycin (Baf) for 3 min at room temperature, followed by incubation in medium containing a similar concentration of Baf. Upright arrows indicate the addition of 5 µM nigericin (Nig) to dissipate the H+ gradient and unquench the AO. Panel (D) shows the summary of the results given as mean ± SEM, with significance values calculated using analysis of variance.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have cloned and partially characterized the human homolog of the mTJ6 gene, termed hTJ6, which encodes a transmembrane protein containing a conserved ITAM-like sequence. Analysis of the hTJ6 protein structure revealed a high degree of similarity to a large family of proteins that includes the 116-kDa V-type ATPase [vacuolar (H+)-ATPases] subunits and the V-type ATP synthase subunit i (2931). Members of the family of the V-type ATPases function as subunits of large complexes of proteins that form the cytoplasmic sector of the pumps that pump H+ from the cytoplasm to the lumen of different organelles (e.g. ER, Golgi, lysosomes and secretory granules), using the energy released by the hydrolysis of ATP. As such, these proteins function as integral parts of a cellular machinery that regulates the pH inside organelles (3032). In some cells, such as the kidney collecting duct and osteoclasts, the pump is expressed in the luminal membrane and participates in H+ secretion. However, in all cells, the main function of the pump is the control of organellar pH, which is essential for protein sorting (33). In addition, the pump also mediates acidification that is needed for the activation of hydrolytic enzymes in lysosomes (34), dissociation of receptor–ligand complexes (35) and storage of secretory products in granules of secretory cells. The 116-kDa subunit (subunit a) in the V-type ATPase is part of the V0 functional domain, which is responsible for proton transport and assembly of the V-type ATPase complex (36, 37). ‘Subunit a’ is a transmembrane glycoprotein with multiple putative transmembrane helices that possesses a hydrophilic amino terminus and a hydrophobic carboxy terminal region (36, 37). Thus, by controlling H+ pumping into organelles, hTJ6 is likely to have a prominent role in many cellular functions, including in lymphocytes.

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 (452–466 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 ({gamma}, {delta}, {varepsilon} and {zeta}), the BCR subunits ({alpha} and ß), the common FcR-{gamma} 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{varepsilon}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 ~18–20 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.


    Acknowledgements
 
We thank A. Elson, J. Takeda and L. Samelson for gifts of reagents. The work reported herein was supported, in part, by grants from the Israel Science Foundation, founded by the Israel Academy of Sciences and Humanities, the USA–Israel Binational Science Foundation, the German–Israeli Foundation (GIF) for Scientific Research and Development, the Chief Scientist's office, Israel Ministry of Health, the Israel Cancer Association, Yael Research Fund and the Israel Cancer Research Fund.


    Abbreviations
 
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

    Notes
 
Transmitting editor: I. Pecht

Received 3 May 2005, accepted 15 July 2005.


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 Discussion
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