on-line at: www.actabp.pl Identification of a novel protein encoded by third conserved gene within RAG locus

Recently, a third evolutionarily conserved gene, NWC, was discovered within the recombination activating gene (RAG) locus, known to contain the RAG1 and RAG2 genes. Here, we identify and characterize the murine endogenous NWC protein which has no homology to any known protein and is ubiquitously expressed. In the cell, the NWC protein which has been suggested to function as a transcriptional repressor, is found in the cytoplasm as well as in the nucleus.


INtRoduCtIoN
Recombination activating gene (RAG)1 and 2, two closely linked, evolutionarily conserved lymphocyte specific genes, encode transposase, an essential component of the V(D)J recombinase which is responsible for the generation of diversity of antigen receptors on T and B lymphocytes (Schatz & Baltimore, 2004).It is believed that the acquisition of RAG genes represented a turning point in evolution of vertebrates, allowing for the development of lymphocytes and adaptive immunity (Thompson, 1995).NWC is a third evolutionarily conserved gene, recently identified within the RAG locus (Cebrat et al., 2005).In contrast to RAG genes, NWC is ubiquitously expressed but in lymphocytes, unlike in non-lymphocytes, it is subject to RAG1-dependent, lymphocyte specific regulation (Cebrat et al., 2005).In non-lymphocytes, it is controlled by its own, RAG2 intragenic promoter, which in lymphocytes is inactivated due to methylation (Cebrat et al., 2008).The predicted amino-acid sequence of NWC protein has no significant homology to any known protein and contains three highly conserved regions with identical sequence motifs that had not been previously described (Cebrat et al., 2005).
The above-mentioned observations suggest that NWC could play an important role in evolution and in development of lymphocytes (Kisielow et al., 2008).Unfortunately, the function of the NWC is not yet known and until now the endogenous protein product of NWC gene has not been identified.Very recently, a protein encoded by cDNA representing a splice variant of the human ortholog of the NWC gene was identified and preliminary findings suggest that it has properties of a transcriptional repressor, but attempts to demonstrate expression of this protein from the endogenous gene have failed (Hong et al., 2008).
Here we identify endogenous NWC protein in the mouse and show that it has a very wide tissue distribution.In the cell it can be found both in the cytoplasm and in the nucleus.

2009
M. Kasztura and others
expression vectors.To obtain a recombinant NWC-His tag protein-expressing vector (pQE-60-NWC) PCR amplified cDNA fragment containing NWC open reading frame was cloned into the NcoI/ BamHI sites of pQE-60 vector (Qiagen).To obtain a mammalian NWC expression vector, (pCI-Neo-NWC), PCR-amplified NWC cDNA was cloned into EcoRI/SmaI sites of pCI-Neo vector (Promega).After cloning, the NWC cDNA sequence and the in frame insertions were verified by sequencing.
Monoclonal anti-NWC antibody (MoM12).Wistar rats were immunized subcutaneously three times with recombinant His-tagged NWC protein (200 µg per injection), in incomplete Freund's adjuvant.Two days after last injection, spleen cells were mixed with SP2/0 myeloma at a 5 : 1 ratio and fused with PEG 1500 (Sigma).Hybridomas were grown and selected as described by Koehler and Milstein (1975).Supernatants from growing hybridoma were screened for the presence of antibodies recognizing NWC-His using ELISA.Hybridomas secreting such antibodies were subcloned two times by limiting dilution.
Fractionation of cell lysates.Cells and tissues (tissues were first mechanically disrupted using a Polytron homogenizer (Glen Mills Inc.)) were lysed on ice in 1% Nonidet P-40 in buffer (50 mM Hepes, pH 7.2, 150 nm NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1 × Complete protease inhibitors, Roche) for 30 min followed by sonication.Insoluble fragments were removed by centrifugation.For analysis by 2D electrophoresis protein lysates were fractionated with increasing concentration of ammonium sulfate.The fraction collected between 30 and 40% of salt saturation was centrifuged, dissolved in 50 mM acetate buffer pH 5.5 and dialyzed.Precipitated proteins were removed by centrifugation and supernatant was purified on a DE23 column.The fraction eluted with 300 mM NaCl was enriched in a protein recognized by the MoM12 antibody.This fraction was used for further characterization by 2D electrophoresis.To obtain the cytoplasmic protein fraction, cells were lysed in buffer A (25 mM Hepes, pH 7.4, 10 mM KCl, 150 mM NaCl, 0.2 mM EDTA, 1% Triton X-100, 1 × Complete protease inhibitors (Roche)) for 30 min and nuclei were removed by centrifugation at 600 × g for 10 min.To obtain the nuclear protein fraction, the pellet was washed 3 times with buffer A and sonicated in buffer B (25 mM Hepes, pH 7.4, 10 mM KCl, 150 mM NaCl, 0.2 mM EDTA, 20% glycerol 1 × Complete protease inhibitors).Protein concentration was determined using the BCA assay (Pierce).Possible cross-contamination of separated cytoplasmic and nuclear protein fractions was monitored by Western blotting using Raf-1-specific antibody (C-20, Santa Cruz -a marker of cytoplasmic fraction) and histone H3-specific antibody (Abcam -a marker of nuclear fraction).
Real-time Rt-PCR.Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions.Three micrograms of RNA was reverse transcribed with Omniscript reverse transcriptase (Qiagen) at 37 o C for 1 h with 100 ng of random hexamers (Amersham Pharmacia).Real-time RT-PCR was performed with the aid of a DNA Engine Opticon 2 apparatus (MJ Research) using the Quantitect SYBR Green PCR Kit (Qiagen).The cycling conditions were: 30 s at 94 o C, 30 s at 52 o C and 1 min at 72 o C. Standard curves generated by amplification of 5-fold serial dilutions of cDNA template were plotted for Nwc and Hprt transcripts.The Nwc expression values were normalized to Hprt.The following primer pairs were used: Nwc: 5'-GGGAGTTCCTTTCAAGGTGATATGACC-3' (forward), 5'-GGATCATAAGATGGAACGCTGGTGC-3' (reverse); Hprt: 5'-GCTGGTGAAAAGGACCTCT-3' (forward), 5'-CACAGGACTAGAACACCTGC-3' (reverse).

Monoclonal antibody against recombinant NWC protein identifies endogenous mouse protein
Long non-coding RNAs are now recognized as being four times as abundant as protein-coding genes (Kapranov et al., 2007).It was therefore important to establish whether NWC, a third evolutionarily conserved gene identified within the RAG locus (Cebrat et al., 2005), encodes a protein.
The recombinant His-tagged NWC protein was expressed in E. coli and purified using Ni-NTA column.Two proteins, one corresponding to a fulllength NWC protein and the second to its N-terminally truncated form (as established by MS sequencing; not shown) were obtained which under conditions of SDS/PAGE electrophoresis migrated as 37 kDa and 25 kDa species, respectively (not shown).The discrepancy between the predicted (26 kDa) and observed (37 kDa) approximate molecular mass of the full-length NWC protein is probably due to its high negative charge which can cause retardation of migration in denaturing polyacrylamide gel electrophoresis (SDS/PAGE) as is the case, for example, of LAT protein (Zhang et al., 1998).A rat monoclonal antibody (MoM12) against full-length recombinant NWC protein was obtained and used to identify endogenous NWC protein in mouse tissues.As shown in Fig. 1, MoM12 detects an endogenous mouse protein with a similar migration pattern in 2D gel electrophoresis to that of recombinant NWC protein.
Both proteins recognized by MoM12 are acidic proteins of 37 kDa. Figure 2A shows that transfection of mouse lymphoid cell line EL4 with NWC cDNA results in a very strong up-regulation of the level of the protein recognized by MoM12.The level of the MoM12-reactive protein in non-transfected EL4 cells  is very low, in many experiments hardly detectable.Thus, the above results demonstrate that NWC encodes a protein in eukaryotic cells and confirms the specificity of MoM12 for the endogenous NWC protein.The close correlation between the level of NWC RNA and the amount of the endogenous protein detected by MoM12 (Fig. 2B) provides further support for the conclusion that the identified protein is indeed encoded by NWC.

Tissue distribution of NWC protein
Figure 3A shows that NWC protein is expressed in all examined tissues.However, the detectability in some tissues (e.g.brain) varies strongly between experiments and the reason for such variablity is not clear.Testis and thymus are the only tissues in which the NWC protein is consistently detected at a high level.Figure 3B shows that NWC protein can be also detected in purified mature lymphocytes.In the context of our initial observations concerning the level of NWC transcription (Cebrat et al., 2005), the finding that the NWC protein is easily and consistently detectable in mature lymphocytes was surprising.Originally, the very low level of NWC transcript observed in RAG-negative lymphocytes was interpreted as resulting from contaminating cells (Cebrat et al., 2005).The present results, using highly purified populations of peripheral T cells, strongly suggest that NWC can be transcribed and translated in mature lymphocytes, albeit at a relatively low level.This is consistent with our recent observation (M.Cebrat, unpublished) that in a small fraction of mature lymphocytes the NWC promoter is not methylated, which could be responsible for the observed protein expression level.

Intracellular distribution of NWC protein
In order to determine the intracellular localization of NWC protein, the nuclear and cytoplasmic fractions were separated and Western blotting using MoM12 was performed.
As shown in Fig. 4, NWC protein can be detected in both fractions.The extent of possible contamination of the nuclear fraction by cytoplasmic proteins and vice versa was monitored by the presence of Raf-1 proteins and histone H3, respectively.The very week signal produced by the anti-Raf-1 antibody in the nuclear fraction indicates that some contamination with cytoplasmic proteins has occurred but this cannot explain the presence of NWC in this fraction because the strength of the NWC to Raf-1 signal (quantified by dilution) shows a reverse correlation to that observed in the cytoplasmic fraction.We conclude that the intracellular localization of NWC protein is not restricted to the nucleus or to the cytoplasm.Considering the possibility that NWC protein functions as a transcriptional regulator (Hong et al., 2008), this last observation suggests that it may belong to the family of proteins controlling gene expression at the transcriptional and post-transcriptional levels (Visa et al. 1996;Ladomery, 1997).

Figure 1 .
Figure 1.Identification of endogenous murine protein by MoM12 monoclonal antibody specific for recombinant NWC protein.Recombinant NWC protein (A) or partially purified protein lysate of HB10 murine cell line (B) was subjected to 2D gel electrophoresis and analysed by silver staining and Western blotting using MoM12 antibody.

Figure 2 .
Figure 2. endogenous murine protein identified by MoM12 antibody is encoded by NWC. A. Murine T cell lymphoma EL4 was transfected with pCI-Neo-NWC (cDNA-NWC) or empty pCI-Neo vector and cell lysates were analysed by Western blotting using MoM12.B. The levels of NWC transcript and of the protein identified by MoM12 antibody in indicated cell lines were assessed using real-time PCR and Western blotting, respectively.Ponceau staining of the PVDF membrane is shown as a control of the protein load in Western blotting experiment.

Figure 3 .
Figure 3. expression of NWC protein in different tissues, including mature lymphocytes.Protein extracts of indicated tissues (A) or indicated amounts of total cellular proteins from sorted CD4 and CD8 splenic T cells and CD4 + 8 + thymocytes (>99% pure) (B) were analysed by Western blotting using MoM12 antibody.Indicated tissues were analysed in 3 to 6 independent experiments.

Figure 4 .
Figure 4. Intracellular distribution of NWC protein.Protein extracts of cytoplasmic and nuclear fractions of STO cells were analysed by Western blotting using indicated antibodies.Possible contamination of cytoplasmic fraction by nuclear proteins and vice versa was monitored by anti-H3 and anti-Raf-1 antibodies, respectively.Inverse correlation between the strength of NWC to Raf-1 signal (quantified by protein dilution) indicates that the detection of NWC protein in nuclear fraction is not due to the contamination with cytoplasmic fraction.