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The use of immobilised metal affinity chromatography (IMAC) to compare expression of copper-binding proteins in control and copper-exposed marine microalgae

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Abstract

Toxicity of metals to aquatic organisms is dependent on both external factors, such as exposure concentration and water quality parameters, and intracellular processes including specific metal-binding sites and detoxification. Current models used to predict copper toxicity in microalgae do not adequately consider these intracellular processes. This study compared the copper-binding proteins from four species of marine microalgae, Dunaliella tertiolecta, Tetraselmis sp., Phaedactylum tricornutum and Ceratoneis closterium, in controls (no added copper) and following a 72-h exposure to copper (sufficient to inhibit growth by approximately 50 %). Cells were lysed by sonication, which was optimised to obtain 54–94 % cell rupture for the different algae. Cell lysates were processed by immobilised metal affinity chromatography (IMAC) using Cu2+ as the bound metal (i.e. Cu-IMAC). Bound proteins were subsequently analysed by SDS-PAGE, comparing proteins recovered from algae that were exposed to copper versus untreated control cells. Individual proteins for which copper exposure resulted in changes to proteins present were excised from gels and further analysed by nano LC ESI-MS/MS; proteins were identified using the Mascot database. Proteins identified in this way included heat-shock proteins, rubisco, α- and β-tubulins and ATP synthase (β subunit). The results established that Cu-IMAC is a useful approach to identify proteins involved in copper binding in algae. This study identified several proteins that may play an active role in responses to copper toxicity in marine microalgae.

Diagram representing the sample preparation steps from algal growth and copper exposure through to mass spectrometry (MS) analysis

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References

  1. Levy JL, Angel BM, Stauber JL, Poon WL, Simpson SL, Cheng SH, Jolley DF (2008) Uptake and comparison of copper by three marine microalgae: Comparison of copper-sensitive and copper-tolerant species. Aquat Toxicol 89:82–93

    Article  CAS  Google Scholar 

  2. Stauber JL, Davies CM (2000) Use and limitations of microbial bioassays for assessing copper availability in the aquatic environment. Environ Rev 8:255–301

    Article  CAS  Google Scholar 

  3. Jonsson CM, Aoyama H (2010) Effect of copper on the activation of the acid phosphatase from the green algae Pseudokirchneriella subcapitata. BioMetals 23(1):93–98

    Article  CAS  Google Scholar 

  4. Fernandes JC, Henriques FS (1991) Biochemical, physiological, and structural effects of excess copper in plants. Bot Rev 57(3):246–273

    Article  Google Scholar 

  5. Levy JL, Stauber JL, Jolley DF (2007) Sensitivity of marine microalgae to copper: The effect of biotic factors on copper adsorption and toxicity. Sci Total Environ 387:141–154

    Article  CAS  Google Scholar 

  6. Pagenkopf GK (1983) Gill surface interaction model for trace metal toxicity to fishes: role of complexation, pH and water hardness. Environ Sci Technol 17:342–347

    Article  CAS  Google Scholar 

  7. Morel FMM (1983) In: Principles of Aquatic Chemistry. Wiley-Interscience, New York, pp 301–308

    Google Scholar 

  8. Brown PL, Markich SJ (2000) Evaluation of the free ion activity model of metal-organism interaction: extension of the conceptual model. Aquat Toxicol 51(2):177–194

    Article  CAS  Google Scholar 

  9. Di Toro DM, Allen HE, Bergman HL, Meyer JS, Paquin PR, Santore RC (2001) A biotic ligand model of the acute toxicity of metals (I). Environ Toxicol Chem 20:2383–2396

    Article  Google Scholar 

  10. Santore RC, Di Toro DM, Paquin PR, Allen HE, Meyer JS (2001) A biotic ligand model of the acute toxicity of metals (II) Application to acute copper toxicity in freshwater fish and daphnia. Environ Toxicol Chem 20:2397–2402

    CAS  Google Scholar 

  11. Merchant SS, Allen MD, Kropat J, Moseley JL, Long JC, Tottey S, Terauchi AM (2006) Between a rock and a hard place: Trace element nutrition in Chlamydomonas. Biochimica et Biophysica Acta-Mol Cell Res 1763(7):578–594

    Article  CAS  Google Scholar 

  12. Wolfe-Simon F, Grzebyk D, Schofield O (2005) The role and evolution of superoxide dismutases in algae. J Phycol 41:453–465

    Article  CAS  Google Scholar 

  13. Yadav SK (2010) Heavy metal toxicity in plants: An overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants. S Afr J Bot 76:167–179

    Article  CAS  Google Scholar 

  14. Ahner BA, Wei L, Oleson JR, Ogura N (2002) Glutathione and other low molecular weight thiols in marine phytoplankton under metal stress. Mar Ecol Prog Ser 232:93–103

    Article  CAS  Google Scholar 

  15. Lavoie M, Faucheur S, Fortin C, Campbell PGC (2009) Cadmium detoxification strategies in two phytoplankton species: Metal binding by newly synthesized thiolated peptides and metal sequestration in granules. Aquat Toxicol 92:65–75

    Article  CAS  Google Scholar 

  16. Porath J, Carlsson JAN, Olsson I, Belfrage G (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258(5536):598–599

    Article  CAS  Google Scholar 

  17. Hage DS, Anguizola JA, Bi C, Li R, Matsuda R, Papastavros E, Pfaunmiller E, Vargas J, Zheng X (2012) Pharmaceutical and biomedical applications of affinity chromatography: Recent trends and developments. J Pharm Biomed Anal 69:93–105

    Article  CAS  Google Scholar 

  18. Pearson RG (1968) Hard and soft acids and bases HSAB, part I fundamental principles. J Chem Educ 45(9):581–587

    Article  CAS  Google Scholar 

  19. Housecroft CE, Constable EC (2010) Chemistry 4th Edition. Pearson Education Ltd, England

    Google Scholar 

  20. Katz AK, Shimoni-Livny L, Navon O, Navon N, Bock CW, Glusker JP (2003) Copper-binding motifs: Structural and Theoretical Aspects. Helvetica Chimica Acta 86:1320–1338

    Article  CAS  Google Scholar 

  21. Gaberc-Porekar V, Menart V (2001) Perspectives of immobilized-metal affinity chromatography. J Biochem Biophys Methods 49(1–3):335–360

    Article  CAS  Google Scholar 

  22. Hage DS (ed) (2006) Handbook of Affinity Chromatography, vol 92. Taylor & Francis, Boca Raton, Florida

    Google Scholar 

  23. Gordon AS (1992) Isolation of compounds with affinity for copper from seawater using immobilized copper-ion affinity-chromatography. Mar Chem 38(1–2):1–12

    CAS  Google Scholar 

  24. Ross ARS, Ikonomou MG, Orians KJ (2003) Characterization of copper-complexing ligands in seawater using immobilized copper(Il)-ion affinity chromatography and electrospray ionization mass spectrometry. Mar Chem 83(1–2):47–58

    Article  CAS  Google Scholar 

  25. Gordon AS, Donat JR, Kango RA, Dyer BJ, Stuart LM (2000) Dissolved copper-complexing ligands in cultures of marine bacteria and estuarine water. Mar Chem 70(1–3):149–160

    Article  CAS  Google Scholar 

  26. Smith SD, She Y-M, Roberts EA, Sarkar B (2004) Using immobilized metal affinity chromatography, two-dimensional electrophoresis and mass spectrometry to identify hepatocellular proteins with copper-binding ability. J Proteome Res 3:834–840

    Article  CAS  Google Scholar 

  27. Mestek O, Polák J, Koplík R, Šantrůček J, Kodíček M (2008) Isolation of ligands of trace metals from plant samples by immobilized metal affinity chromatography. Anal Lett 41:1459–1467

    Article  CAS  Google Scholar 

  28. Mestek O, Komínková J, Koplík R, Šantrůček J, Polák J (2010) Trace elements distribution and species fractionation in the wheat (Triticum aestivum) plant. Chem Speciat Bioavailab 22(1):61–70

    Article  CAS  Google Scholar 

  29. Barnett JP, Scanlan DJ, Blindauer CA (2012) Fractionation and identification of metalloproteins from a marine cyanobacterium. Anal Bioanal Chem 402(3371–3377):3371–3377

    Article  CAS  Google Scholar 

  30. Johnson HL, Stauber JL, Adams MS, Jolley DF (2007) Copper and zinc tolerance of two tropical microalgae after copper acclimation. Environ Toxicol 22(3):234–244

    Article  CAS  Google Scholar 

  31. Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. Can J Microbiol 8:229–239

    Article  CAS  Google Scholar 

  32. Franklin NM, Stauber JL, Adams MS (2005) Improved methods of conducting microalgal bioassays using flow cytometry. In: Ostrander GK (ed) Techniques in Aquatic Toxicology. Taylor and Francis, Boca Raton, pp 735–756

    Google Scholar 

  33. Graham JM, Rickwood D (1997) Subcellular fractionation: A practical approach. Oxford University Press

  34. Storrie B, Madden EA (1990) Isolation of sub-cellular organelles. Methods Enzymol 182:203–225

    CAS  Google Scholar 

  35. Bairoch A, Apweiler R (2000) The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acids Res 28:45–48

    Article  CAS  Google Scholar 

  36. Wang W, Vinocur B, Shoseyov O, Altman A (2004) Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci 9(5):244–252

    Article  CAS  Google Scholar 

  37. De Maio A (1999) Heat shock proteins: Facts, thoughts and dreams. Shock 11(1):1–12

    Article  Google Scholar 

  38. Kiang J, Tsokos GC (1998) Heat shock protein 70 kDa: Molecular biology, biochemistry, and physiology. Pharmacol Ther 80(2):183–201

    Article  CAS  Google Scholar 

  39. Ellis RJ (1979) The most abundant protein in the world. Trends Biochem Sci 4(11):241–244

    Article  CAS  Google Scholar 

  40. Taylor TC, Backlund A, Bjorhall K, Spreitzer RJ, Andersson I (2001) First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J Biol Chem 276(51):48159–48164

    CAS  Google Scholar 

  41. Hallegraeff GM, Bolch CJS, Hill DRA, Jameson I, Leroi J-M, McMinn A, Murray S, De Salas MF, Saunders K (2010) Phytoplankton of temperate coastal waters. Algae of Australia. Australian Biological Resources Study/CSIRO publishing, Canberra

    Google Scholar 

  42. Hajduch M, Rakwal R, Agrawal GK, Yonekura M, Pretova A (2001) High-resolution two-dimensional electrophoresis separation of proteins from metal-stressed rice (Oryza sativa L.) leaves: Drastic reductions/fragmentation of ribulose-1,5-bisphosphate carboxylase/oxygenase and induction of stress-related proteins. Electrophoresis 22(2824–2831)

    Google Scholar 

  43. Lindon FC, Henriques FS (1991) Limiting step on photosynthesis of rice plants treated with varying copper levels. J Plant Physiol 138(115–118)

    Google Scholar 

  44. Demirevska-Kepova K, Simova-Stoilova L, Stoyanova Z, Hölzer R, Feller U (2004) Biochemical changes in barley plants after excessive supply of copper and manganese. Environ Exp Bot 52:253–266

    Article  CAS  Google Scholar 

  45. Stephens RE (1978) Primary structural differences among tubulin subunits from flagella, cilia, and the cytoplasm. Biochem (Washington) 17(14):2882–2891

    Article  CAS  Google Scholar 

  46. Dutcher SK (2001) The tubulin fraternity: alpha to eta. Curr Opin Cell Biol 13:49–54

    Article  CAS  Google Scholar 

  47. Önfelt A (1983) Spindle disturbances in mammalian cells. I. changes in the quantity of free sulfhydryl groups in relation to survival and c-mitosis in V79 Chinese hamster cells after treatment with colemid, diamide, carbaryl and methyl mercury. Chem Biol Interact 46:201–217

    Article  Google Scholar 

  48. Runswick MJ, Walker JE (1983) The amino acid sequence of the β-subunit of ATP synthase from bovine heart mitochondria. J Biol Chem 258(5):3081–3089

    CAS  Google Scholar 

  49. Walker JE, Saraste M, Runswick MJ, Gay NJ (1982) Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO (Eur Mol Biol Org) J 1(8):945–951

    CAS  Google Scholar 

  50. Cid A, Herrero C, Torres E, Abalde J (1995) Copper toxicity on the marine microalga Phaeodactylum tricornutum: effects on photosynthesis and related parameters. Aquat Toxicol 31:165–174

    Article  CAS  Google Scholar 

  51. Steinebach OM, Wolterbeek HT (1994) Role of cytosolic copper, metallothionein and glutahtione in copper toxicity in rat hepatoma tissue culture cells. Toxicology 92:75–90

    Article  CAS  Google Scholar 

  52. Rodríguez-Celma J, Rellán-Álvarez R, Abadía A, Abadía J, López-Millán A-F (2010) Changes induced by two levels of cadmium toxicity in the 2-DE protein profile of tomato roots. J Proteome 73:1694–1706

    Article  Google Scholar 

  53. Ritter A, Ubertini M, Romac S, Gaillard F, Delage L, Mann A, Cock JM, Tonon T, Correa JA, Potin P (2010) Copper stress proteomics highlights local adaptation of two strains of the model brown alga Ectocarpus siliculosus. Proteomics 10:2074–2088

    Article  CAS  Google Scholar 

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Acknowledgements

The authors would like to thank Rebecca Ronchin for assistance in optimising the sonication parameters for cell lysis. C. Smith was awarded an Australian Postgraduate Award scholarship for the duration of this work. The protein identification work was undertaken at APAF, the infrastructure provided by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS).

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Authors and Affiliations

  1. School of Chemistry, University of Wollongong, Wollongong, NSW, 2522, Australia

    Cassandra L. Smith & Dianne F. Jolley

  2. CSIRO Land and Water, Locked Bag 2007, Kirrawee, NSW, 2232, Australia

    Jennifer L. Stauber

  3. School of Biological Sciences, Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, 2522, Australia

    Cassandra L. Smith & Mark R. Wilson

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  1. Cassandra L. Smith
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  2. Jennifer L. Stauber
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  4. Dianne F. Jolley
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Correspondence to Cassandra L. Smith or Dianne F. Jolley.

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Smith, C.L., Stauber, J.L., Wilson, M.R. et al. The use of immobilised metal affinity chromatography (IMAC) to compare expression of copper-binding proteins in control and copper-exposed marine microalgae. Anal Bioanal Chem 406, 305–315 (2014). https://doi.org/10.1007/s00216-013-7452-6

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  • DOI: https://doi.org/10.1007/s00216-013-7452-6

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