| The unicellular eukaryotic green alga Dunaliella salina(D.salina) grows over a wide range of salt concentrations from 0.05 to 5.0 M and is one of the most halotolerant organisms. D.salina has received considerable attention in studies of halotolerance mechanisms and some salt-induced proteins have been isolated and identified. Although the genome of D. salina is not completely available, proteome analysis is an efficient technique to detect and characterize proteins from D. salina.Many glycometabolic enzymes have been identified being involved in responding to salt stress and meanwhile, they have been proved to be related to the flagella/cilia of D.salina, the model organism Chlamydomonas reinhardti and Drosophila et al. Flagella/cilia are specialized and widespread cell organelles. More and more evidences suggest that flagella/cilia serve diverse roles in motility, sensory signaling and development of many eukaryots and implicate cilia in polycystic kidney disease (PKD) and developmental disorders, even in cancer.Being one of the evidences for evolution theory, glucose-6-phosphate isomerase (GPI) is universally distributed among eukaryotes, bacteria and some archaea and catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate, which is an essential reaction involved in both catabolic glycolysis and anabolic gluconeogenesis. GPIs also act as an atuocrine motility factor (AMF) that can promote tumor cells metastasis and invasion by binding to their receptors in animals. Although GPI is related to flagella/cilia in D.salina, whether it has the cytokine function is still unknown.In this study, we analyzed changes of proteins between algal cells cultured at 1.5 or 3.5 M NaCl using proteomic method and obtained the differential expression maps. Twenty one proteins increased by more than 3-fold in 3.5 M NaCl than that in 1.5 M NaCl medium were isolated and analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS), making twelve proteins were identified including GPI. In order to verify GPI is involved in responding to salt stress at gene level, the full-length cDNA of GPI was obtained using rapid amplification of cDNA end technique and heterologous expressed to. Real-time quantitative RT-PCR was used to detect its expression changes in response to light and salinity. Bioinformatics analysis revealed that the GPI from D.salina(DsGPI) have the similar structure and key active sites with that from mammalian cells and hence, it was used to transfect esophageal squamous cell carcinoma cells to check whether it has the potential ability like human GPI to promote invasion of tumor cells. The results showed that DsGPI is involved in responding to salt stress not only at the gene level but also at the protein level and that DsGPI has the same cellular localization with human GPI that mainly localized in the nucleus and also distributed in cytoplasm in esophageal squamous cell carcinoma cell (ESCC) line EC9706 cells. In addition, western blots confirmed that DsGPI was stable expressed in EC9706 and able to enhance the invasion ability of EC9706, suggesting that DsGPI may has the cytokine functions like human GPI.Methods1 Proteomics analysis of salt-induced proteins from D.salina 1.1 Identification of D. salina strainContaminated algal cells containing not only one species were spread on semisolid medium and cultured for 10 to 14 days and then, single cells were picked out and continue cultured in test tube, respectively. Seven to 10 days later, algal cells were observed under microscope and samples with the larger or the smaller uniform size were cultured to logarithmic phase and harvested for DNA extraction. A pair of primers was designed to amplify the internal transcribed space and the PCR results were blasted to determine the pure D.salina species.1.2 Two-dimensional gel electrophoresis (2-DE)Proteins of algal cells cultured in 1.5M and 3.5M NaCl medium were precipitated by Trichloroacetic acid-actone and dissolved in a buffer, respectively. Protein concentrations were determined using the Bradford method. A total of 400μg soluble proteins were applied to isoelectric focusing (IEF) on IPGphor using 24 cm ImmobilineTM DryStrips (pH 3-10, linear; GE Healthcare) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).1.3 Analysis of the 2-DE gels and identification of salt-induced proteins by MS analysisThe 2-DE gels stained with Coomassie Brilliant Blue (CBB) R-250 or AgNO3 were scanned using Image-Scanner (GE Healthcare). Protein spots from the 3.5 M NaCl medium were compared to those from the 1.5 M NaCl medium (control) using ImageMaster 2D Platinum software After images were adjusted, protein spots were detected and matched, and protein intensities were measured, the differential expressed proteins were determined combined with eye observation. Each sample was replicated at least three times, and protein spots for comparative analyses were detected on all of the gels.Among the matched proteins, proteins up-regulated in by more than three times from the 2-DE gels were selected and digested by trypsin, the resulting peptides were extracted and analyzed using MALDI-TOF/TOF MS (Applied Biosystems). MS data were collected and protein identification was searched against the database of plants in NCBI using the MASCOT search engine. An identified protein was matched with the known protein(s) in the database only when its score is greater than 65 (P<0.05)2 Cloning, heterologous expression and functional analysis of the DsGPI gene2.1 Cloning of full-length cDNA of DsGPI geneA pair of degenerated PCR primers "GARTTYGGNATHGAYCC" (sense) and "NACNCCCCAYTGRTCRAA"(antisense) corresponding to the highly conserved amino acid residues "EFGIDP" and "FDQWGV" was used to amplify a cDNA fragment of the DsGPI, and then 5'-and 3'-RACEs were performed. The putative 5'-and 3'-RACE cDNAs and the partial sequences cloned using the degenarate primers were organized to form a cDNA contigue that was applied to bioinformatics analysis.2.2 Heterologous expression of DsGPIThe DsGPI cDNA was subcloned into the prokaryotic protein expression vector pET28a(+) (Novagen) with NdeI andoRI sites and recombinant plasmid was screened and identified by sequencing. Recombinant plasmids were expressed in E. coli BL21 and IPTG was added in to produce recombinant fusion DsGPI with a 6×His-tag that was subjected to SDS-PAGE.2.3 Analysis of DsGPI gene expressionTo confirm whether DsGPI responds to salt stress, algal cells were collected at 0,1,2,4,8,12,16,20 and 24 h after being transferred from 1.5 to 3.5 M NaCl medium, and thecells transferred to fresh 1.5 M NaCl medium were served as controls. Besides, to investigate whether DsGPI is lightrelated, algal cells having been cultured in the dark for 48 h were exposed to light and then collected at 1,2,4,8, 10 and 12 h, and cells without exposure to light were used as controls. Real time quantitative RT-PCR was performed for the expression change of DsGPI. 3 Effects of DsGPI gene on the invasion ability of EC9706 cells3.1 Preparation of DsGPI polyclonal antibodyThe DsGPI fusion proteins expressed in E. coli cells were purified using Ni-NTA His Bind column and subjected to immunize albino rabbit for the preparation of polyclonal antibody. The antibody titer and specificity were determined by ELISIA and Western blots, respectively.3.2 Localization of DsGPI in MDCK cellsThe complete cDNA sequences of DsGPI was cloned into the plasmid pEGFP-C1 to create a recombinant plasmid pEGFP-C1-DsGPI by using BamH I and EcoR I sites and identified. The same method was used to construct recombinant plasmid pEGFP-C1-GPI with complete cDNA sequences of human GPI.Both recombinant plasmids transfected in MDCK cells with blank plasmid pEGFP-C1 and cells without transfection were used as control. Localization the EGFP fusion proteins were examined by fluorescence microscopy.3.3 Invasion ability Changes of EC9706 cellsRecombinant plasmids pcDNA3.1(+)-DsGPI and pcDNA3.1(+)-GPI were constructed and transfected in EC9706 cells, respectively. Cells transfected with blank plasmid pcDNA3.1(+) and cells without transfection were used as control. Protein expressions were examined by Western blots and cell invasion assay was performed using transwell chambers.4 Statistical analysisResults of Western blotting were analyzed by TotalLab2.0 software and all experiments were replicated at least three times. The data was performed by one-way analysis of variance using SPSS version13.0 (SPSS, Chicago, USA). Summary statistics were expressed as the means±the standard deviations, except as otherwise stated. In all statistical analyses, a P value of< 0.05 was considered statistically significant, and all P values were two-sided. Results1 Proteomics analysis of salt-induced proteins from D.salina1.1 Identification of D.salinaThe contaminated algal cells were purified as two groups, one with the larger and the other with the smaller cell volume. The internal transcribed spaces (ITS) cloned by PCR and sequenced of the two species showed that the larger one resulted a 680 bp while the smaller one resulted a 1042 bp fragment. Analysis of the ITS sequences indicated that one (the larger) was Dunaliella salina UTEX 1644 and the other (the smaller) was Dunaliella viridis.1.2 Two-dimensional gel electrophoresis (2-DE)Pigments such as chlorophyll were removed and the protein precipitation was easy to dissolve in the IEF buffer using the TCA-actone method for the extraction of total proteins; the voltage gradually increased up to the final 8000 v during the process of IEF suggesting that the sample was properly treated.1.3 Analysis of the 2-DE gels and identification of salt-induced proteins by MS analysisEight hundreds and three protein spots were reproducibly detected in each CBB-stained gel and 739 spots were matched with a matching rate of 92.1%. Intensity quantification of protein spots revealed that intensities of a total of 21 protein spots were increased by over threefold at 3.5 M NaCl as compared to control gels and these protein spots were selected for MS analysis.The selected 21 protein spots digested by trypsin were applied to MALDI-TOF/TOF MS. According to the search results by MASCOT, there was not a good match (score<65) in nine spots while 12 spots had a good match (score>65). Of the 12 spots, one protein spot was identified as DsGPI, two spots were unknown proteins, and the nine proteins have been identified as follows:Fe superoxide dismutase, ATP synthase, heat shock protein 70B,30S ribosomal protein S4, retrotransposon protein, dynein 1-alpha heavy chain (flagellar inner arm), G-protein, calcium-dependent protein kinase, and alcohol dehydrogenase.2 Cloning, heterologous expression and functional analysis of the DsGPI gene2.1 Cloning of full-length cDNA of DsGPI genePCR amplification with degenerated primers resulted in a specific PCR product of 771 bp long, A 1,206 bp and a 687 bp of products were obtained by 5'and 3' RACE inner PCR, respectively. Based on fact that 123 bp of the 1206 bp and 137 bp of the 687 bp were overlapped with the 771 bp of PCR product, respectively, a full-length sequence of 2338 bp (GenBank accession number FJ210719), in which an ORF of 1,980 bp was included, was deduced. Bioinformatics analysis showed that DsGPI amino acids shared high identities with the known GPIs from other species and potential active site residues that have been proved to be extremely conserved in the GPIs from other organisms.2.2 Heterologous expression of DsGPIAfter competent cells of E. coli BL21 transformed with the pET28a(+)-DsGPI were induced by IPTG, the predicted. abundant fusion proteins with a molecular weight of-75 kDa were identified by SDS-PAGE, while null or trace of the protein band appeared in control E. coli BL21 cells that were untransformed or transformed but without induction.2.3 Analysis of DsGPI gene expressionReal time quantitative RT-PCR demonstrated that when the algal cells were transferred to higher NaCl (2.5 and 3.5 M) from lower (1.5 M) concentrations, DsGPI expressions were abruptly reduced at the first 2 h but then rapidly increased by up to eightfold or 14-fold in 2.5 or 3.5 M NaCl, respectively, suggesting that DsGPI is highly induced by salt and may play an important role in adaptation to salt stress. Furthermore, after algal cells that had been cultured in the dark for 48 h were exposed to light, expression of the DsGPI gene was reduced quickly in the first 2 h and then maintained at a relatively stable level.3 Effects of DsGPI gene on the invasion ability of EC9706 cells3.1 Preparation of DsGPI polyclonal antibodyThe fusion proteins purified using Ni-NTA spin column according to the affinity of His-tag to Ni2+ resulted in a purity reaching to 96.3%;The titer of anti-serum was determined as 1:256K-1:2512K by ELISA and Western blotting showed that the anti-serum from the immunized albino rabbit could specially bind with DsGPI protein in the total soluble proteins of BL21.3.2 Localization of DsGPI in MDCK cellsAs shown by fluorescence microscopy, EGFP fusion proteins were mainly localized in the nucleus area in MDCK cells transfected with plamids of pEGFP-Cl-DsGPI or pEGFP-Cl-GPI while uniform distributed with blank plasmid of EGFP-C1 and without fluorescence in untreated cells, suggesting that DsGPI has the similar cellular localization with that of human GPI (AMF).3.3 Invasion ability Changes of EC9706 cellsResults of Western blots revealed that expression of DsGPI was incresed by 2.2±0.06 fold (P< 0.05) and 1.9±0.13 fold (P< 0.05) in EC9706 cells transfected with pcDNA3.1(+)-DsGPI compared with the expression of AMF in EC9706 cells that transfected with blank pcDNA3.1(+) or without transfection; and that expression of AMF was incresed by 3.3±0.11 fold (P< 0.05) and 3.0±0.19 fold (P< 0.05)in EC9706 cells transfected with pcDNA3.1(+)-AMF compared with that with blank pcDNA3.1(+) or without transfection. In cell invasion assay, the values of OD of cells transfected with and without blank pcDNA3.1 (+) were 2.64±0.06 and 2.47±0.10, but increased to 3.86±0.14(P< 0.05) and 4.18±0.11 (P< 0.05) in the cells transfected with pcDNA3.1-DsGPI and pcDNA3.1-AMF (P< 0.05), respectively, suggesting that DsGPI can acts as the similar roles in promoting cell invasion. Conclusions1. D. salina was successfully isolated, purified and identified based on nuclear ITS rDNA sequences; two-dimensional gel electrophoresis maps of proteins from D.salina cells cultured in 1.5 M or 3.5 M NaCl medium were constructed and salt-induced proteins were isolated; twenty-one protein spots whose intensities were elevated 3-to 13-fold at 3.5 M NaCl as compared to 1.5 M NaCl were analyzed by MALDI-TOF MS and 12 proteins had a good match with databases providing useful peptides information for further studies on functions of these proteins and corresponding genes. The identified proteins are as follows: glucose-6-phosphate isomerase, Fe superoxide dismutase, ATP synthase, heat shock protein 70B,30S ribosomal protein S4, kinetochore protein, dynein 1-alpha heavy chain (flagellar inner arm), G-protein, calcium-dependent protein kinase, alcohol dehydrogenase and two unknown proteins.2. A full-length cDNA of GPI gene of 2338 bp in length has firstly been obtained from D. salina containing a complete open reading frame 1980 bp that has been verified by bioinformatics analysis and heterologous expression; expression of DsGPI gene is related to light induction and involves in responding to salt stress; polyclonal antibody of DsGPI has been prepared with specificity and a titer of 1:250K.3. Eukaryotic expression plasmids pEGFP-Cl-DsGPI and pcDNA 3.1-DsGPI containing full-length cDNA of DsGPI the are successfully constructed; DsGPI mainly localizes in the nucleus in MDCK cells as that of human GPI(AMF); DsGPI can enhance the invasion ability of EC9706 cells function as an cytokine. The cytokine function of DsGPI that promotes invasion of tumor cells and its correlation to flagella/cilia provides the possibility to study the functions of DsGPI in flagella/cilia and the relationship between flagella/cilia and tumorigenesis.
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