Cite this paper:
HAN Xiaolin, LIU Ping, GAO Baoquan, WANG Haofeng, DUAN Yafei, XU Wenfei, CHEN Ping. Na+/K+-ATPase α-subunit in swimming crab Portunus trituberculatus : molecular cloning, characterization, and expression under low salinity stress[J]. Journal of Oceanology and Limnology, 2015, 33(4): 828-837

Na+/K+-ATPase α-subunit in swimming crab Portunus trituberculatus : molecular cloning, characterization, and expression under low salinity stress

HAN Xiaolin1,2, LIU Ping1, GAO Baoquan1, WANG Haofeng1, DUAN Yafei1, XU Wenfei1,2, CHEN Ping1
1 Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;
2 Dalian Ocean University, Dalian 116023, China
Na+/K+-ATPases are membrane-associated enzymes responsible for the active transport of Na+ and K+ ions across cell membranes, generating chemical and electrical gradients. These enzymes' α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na+/K+-ATPase α-subunit cDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end methods. Analysis of the nucleotide sequence revealed that the cDNA had a full-length of 3 833 base pairs (bp), with an open reading frame of 3 120 bp, 5' untranslated region (UTR) of 317 bp, and 3' UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na+/K+-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of amino acid sequences showed that the P. trituberculatus α-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit's transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab's Na+/K+-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.
Key words:    cloning|expression|Na+/K+-ATPase|α-subunit|Portunus trituberculatus|salinity   
Received: 2014-02-20   Revised: 2014-11-24
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Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. 1990. Basic local alignment search tool. J. Mol. Biol., 215 (3): 403-410.
Axelsen K B, Palmgren M G. 1998. Evolution of substrate specificities in the P-type ATPase superfamily. J. Mol. Evol., 46 (1): 84-101.
Bachère E, Chagot D, Grizel H. 1988. Separation of Crassostrea gigas hemocytes by density gradient centrifugation and counterflow centrifugal elutriation. Dev. Comp. Immunol., 12 (3): 549-559.
Baxter-Lowe L A, Guo J Z, Bergstrom E E, Hokin L E. 1989. Molecular cloning of the Na, K-ATPase α-subunit in developing brine shrimp and sequence comparison with higher organisms. FEBS Lett., 257 (1): 181-187.
Béguin P, Wang X Y, Firsov D, Puoti A, Claeys D, Horisberger J D, Geering K. 1997. The γ subunit is a specific component of the Na, K-ATPase and modulates its transport function. Embo J., 16 (14): 4 250-4 260.
Choi C Y, An K W. 2008. Cloning and expression of Na+/K+- ATPase and osmotic stress transcription factor 1 mRNA in black porgy, Acanthopagrus schlegeli during osmotic stress. Comp. Biochem. Physiol. Part B : Biochem. Mol. Biol., 149 (1): 91-100.
Cooper A R, Morris S. 1997. Osmotic and ionic regulation by Leptograpsus variegatus during hyposaline exposure and in response to emersion. J. Exp. Mar. Biol. Ecol., 214 (1- 2): 263-282.
Corotto F S, Holliday C W. 1996. Branchial Na, K-ATPase and osmoregulation in the purple shore crab Hemigrapsus nudus (Dana). Comp. Biochem. Physiol., 113A (4): 361- 368.
Cutler C P, Sanders I L, Hazon N, Cramb G. 1995. Primary sequence, tissue specificity and expression of the Na+, K+- ATPase α 1 subunit in the European eel (Anguilla anguilla). Comp. Biochem. Physiol. B : Biochem. Mol. Biol., 111 (4): 567-573.
Deane E E, Woo N Y S. 2005. Cloning and characterization of sea bream Na+-K+-ATPase α and β subunit genes: in vitro effects of hormones on transcriptional and translational expression. Biochem. Biophys. Res. Commun., 331 (4): 1 229-1 238.
Fagan M J, Saier M H Jr. 1994. P-type ATPases of eukaryotes and bacteria: sequence analyses and construction of phylogenetic trees. J. Mol. Evol., 38 (1): 57-99.
Feng S Y, Zhao F, Zhuang P, Zhang L Z. 2012. Preliminary studies on molecular mechanism of salinity regulation of Na+, K+-ATPase α-subunit in gills of juvenile Chinese sturgeon (Acipenser sinensis). J. Fish. Chin., 36 (9): 1 386-1 391. (in Chinese with English abstract)
Geering K. 1990. Subunit assembly and functional maturation of Na, K-ATPase. J. Membr. Biol., 115 (2): 109-121.
Geering K, Beggah A, Good P, Girardet S, Roy S, Schaer D, Jaunin P. 1996. Oligomerization and maturation of Na, K-ATPase: functional interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit. J. Cell Biol., 133 (6): 1 193-1 204.
Harasywych M G, Adamkewicz S L, Blake J A, Suadek D, Spriggs T, Bolt C J. 1997. Phylogeny and relationships of pleurotomariid gastropods (Mollusca: Gastropoda): an assessment based on partial 18S rDNA and cytochrome c oxidase I sequences. Mol. Mar. Biol. Biotechnol., 6 (1): 1-20.
Horisberger J D, Lemas V, Kraehenbuhl J P, Rossier B C. 1991. Structure-function relationship of Na, K-ATPase. Annu. Rev. Physiol., 53 (1): 565-584.
Huong D T T, Jasmani S, Jayasankar V, Wilder M. 2010. Na/KATPase activity and osmo-ionic regulation in adult whiteleg shrimp Litopenaeus vannamei exposed to low salinities. Aquaculture, 304 (1-4): 88-94.
Jayasundara N, Towle D W, Weihrauch D, Spanings-Pierrot C. 2007. Gill-specific transcriptional regulation of Na+/K+- ATPase α-subunit in the euryhaline shore crab Pachygrapsus marmoratus : sequence variants and promoter structure. J. Exp. Biol., 210 (Pt 12): 2 070-2 081.
Jiang S, Xu Q H. 2011. Influence of salinity stress on the activity of gill Na+/K+-ATPase in swimming crab (Portunus trituberculatus). J. Fish. Chin., 35 (10): 1 475- 1 480. (in Chinese with English abstract)
Jørgensen P L, Andersen J P. 1988. Structural basis for E1 -E2 conformational transitions in Na, K-pump and Ca-pump proteins. J. Membr. Biol., 103 (2): 95-120.
Kinne O. 1971. Salinity: Animal invertebrates. In : Kinne O ed. Marine Ecology Vol. 1: Environmental Factors. Wiley Interscience, London, UK. p.821-995.
Lingrel J B, Kuntzweiler T. 1994. Na+/K+-ATPase. J. Biol. Chem., 269 (31): 19 659-19 662.
Liu Z S, Liu Z. 2008. China Fisheries Yearbook 2008. Beijing: Chinese Agriculture Express. (in Chinese)
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 -ΔΔC T method. Methods, 25 (4): 402-408.
Lu Y L, Wang F, Zhao Z Y, Dong S L, Ma S. 2012. Effects of salinity on growth, molt and energy utilization of juvenile swimming crab Portunus trituberculatus. J. Fish. Sci. Chin., 19 (2): 237-245. (in Chinese)
Lucu Č, Towle D W. 2003. Na++K+-ATPase in gills of aquatic crustacean. Comp. Biochem. Physiol. A : Mol. Integr. Physiol., 135 (2): 195-214.
Mercer R W. 1993. Structure of the Na, K-ATPase. Int. Rev. Cytol., 137 C : 139-168.
Mitsunaga-Nakatsubo K, Yamazaki K, Hatoh-Okazaki M, Kawashita H, Okamura C, Akasaka K, Shimada H, Yasumasu I. 1996. cDNA cloning of NA+, K+-ATPase α-subunit from embryos of the sea urchin, Hemicentrous pulcherrimus. Zool Sci., 13 (6): 833-841.
Nilsen T O, Ebbesson L O E, Madsen S S, McCormick S D, Andersson E, Björnsson B T, Prunet P, Stefansson S O. 2007. Differential expression of gill Na+, K+-ATPase α- and β-subunits, Na+, K+, 2Cl- cotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar. J. Exp. Biol., 210 (16): 2 885-2 896.
Palmgren M G, Axelsen K B. 1998. Evolution of P-type ATPases. Biochim. Biophys. Acta, 1365 (1-2): 37-45.
Pan L Q, Yue F, Miao J J, Zhang L, Li J. 2010. Molecular cloning and characterization of a novel c-type lysozyme gene in swimming crab Portunus trituberculatus. Fish. Shellfish Immunol., 29 (2): 286-292.
Péqueux A. 1995. Osmotic regulation in crustaceans. J. Crust. Biol., 15 (1): 1-60.
Semple J W, Green H J, Schulte P M. 2002. Molecular cloning and characterization of two Na/K-ATPase isoforms in Fundulus heteroclitus. Marine Biotechnology, 4 (5): 512- 519.
Serrano R. 1988. Structure and function of proton translocating ATPase in plasma membranes of plants and fungi. Biochim. Biophys. Acta, 947 (1): l-28.
Shull G E, Lane L K, Lingrel J B. 1986. Amino-acid sequence of the β -subunit of the (Na++K+)ATPase deduced from a cDNA. Nature, 321 (6068): 429-431.
Shull G E, Schwartz A, Lingrel J B. 1985. Amino-acid sequence of the catalytic subunit of the (Na++K+)ATPase deduced from a complementary DNA. Nature, 316 (6030): 691-695.
Sun H Y, Zhang L P, Ren C H, Chen C, Fan S G, Xia J J, Lin H J, Hu C Q. 2011. The expression of Na, K-ATPase in Litopenaeus vannamei under salinity stress. Mar. Biol. Res., 7 (6): 623-628.
Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol., 24 (8): 1 596-1 599.
Tang C H, Chang I C, Chen C H, Lee T H, Hwang P P. 2008. Phenotypic changes in mitochondrion-rich cells and responses of Na+/K+-ATPase in gills of tilapia exposed to deionized water. Zool Sci., 25 (2): 205-211.
Thompson J D, Higgins D G, Gibson T J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res., 22 (22): 4 673-4 680.
Towle D W. 1981. Role of Na+-K+-ATPase in ionic regulation by marine and estuarine animals. Mar. Biol. Lett., 2 : 107- 122.
Towle D W. 1990. Sodium transport systems in gills. In : Kinne R K H ed. Comparative Aspects of Sodium Cotransport Systems. 1st edn. Karger Publishing, Basel, USA. p.241- 263.
Towle D W. 1993. Ion transport systems in membrane vesicles isolated from crustacean tissues. J. Exp. Zool., 265 (4): 387-396.
Towle D W. 1997. Molecular approaches to understanding salinity adaptation of estuarine animals. Am. Zool., 37 (6): 575-584.
Towle D W, Paulsen R S, Weihrauch D, Kordylewski M, Salvador C, Lignot J H and Spanings-Pierrot C. 2001. Na++K+-ATPase in gills of the blue crab Callinectes sapidus : cDNA sequencing and salinity-related expression of α-subunit mRNA and protein. J. Exp. Biol., 204 : 4 005- 4 012.
Willms K, Shoemaker C B, Skelly P J, Landa A. 2004. Cloning and expression of a Na+, K+-ATPase α-subunit from Taenia solium (TNaK1α). Mol. Biochem. Parasitol., 138 (1): 79-82.
Wong M, Medrano J. 2005. Real-time PCR for mRNA quantitation. Biotechniques, 39 (1): 75-85.
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