Advertisement

Planta

, Volume 236, Issue 1, pp 283–296 | Cite as

Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii

  • Carla Malaquias Almeida
  • Cláudia Pereira
  • Diana Soares da Costa
  • Susana Pereira
  • José Pissarra
  • Isaura Simões
  • Carlos FaroEmail author
Original Article

Abstract

Aspartic proteinases have been extensively characterized in land plants but up to now no evidences for their presence in green algae group have yet been reported in literature. Here we report on the identification of the first (and only) typical aspartic proteinase from Chlamydomonas reinhardtii. This enzyme, named chlapsin, was shown to maintain the primary structure organization of typical plant aspartic proteinases but comprising distinct features, such as similar catalytic motifs DTG/DTG resembling those from animal and microbial counterparts, and an unprecedentedly longer plant specific insert domain with an extra segment of 80 amino acids, rich in alanine residues. Our results also demonstrated that chlapsin accumulates in Chlamydomonas chloroplast bringing this new enzyme to a level of uniqueness among typical plant aspartic proteinases. Chlapsin was successfully expressed in Escherichia coli and it displayed the characteristic enzymatic properties of typical aspartic proteinases, like optimum activity at acidic pH and complete inhibition by pepstatin A. Another difference to plant aspartic proteinases emerged as chlapsin was produced in an active form without its putative prosegment domain. Moreover, recombinant chlapsin showed a restricted enzymatic specificity and a proteolytic activity influenced by the presence of redox agents and nucleotides, further differentiating it from typical plant aspartic proteinases and anticipating a more specialized/regulated function for this Chlamydomonas enzyme. Taken together, our results revealed a pattern of complexity for typical plant aspartic proteinases in what concerns sequence features, localization and biochemical properties, raising new questions on the evolution and function of this vast group of plant enzymes.

Keywords

Aspartic proteinase Chlamydomonas Chloroplast Plant specific insert 

Abbreviations

AP

Aspartic proteinase

PSI

Plant specific insert

CDR1

Constitutive disease resistance 1

EST

Expressed sequence tag

EDANS

5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid

DABCYL

4-(Dimethylaminoazo)benzene-4-carboxylic acid

E-64

l-trans-Epoxysuccinylleucylamide-(4-guanidino)butane

IPTG

Isopropyl β-d-1-hiogalactopyranoside

MCA

(7-Methoxycoumarin-4-yl)acetyl

DNP

2,4-dinitrophenyl

rchlapsin

Recombinant chlapsin

References

  1. Asakura T, Watanabe H, Abe K, Arai S (1995) Rice aspartic proteinase, oryzasin, expressed during seed ripening and germination, has a gene organization distinct from those of animal and microbial aspartic proteinases. Eur J Biochem 232:77–83PubMedCrossRefGoogle Scholar
  2. Balsera M, Soll J, Buchanan BB (2010) Redox extends its regulatory reach to chloroplast protein import. Trends Plant Sci 15:515–521PubMedCrossRefGoogle Scholar
  3. Blanc G, Duncan G, Agarkova I, Borodovsky M, Gurnon J, Kuo A, Lindquist E, Lucas S, Pangilinan J, Polle J, Salamov A, Terry A, Yamada T, Dunigan DD, Grigoriev IV, Claverie JM, Van Etten JL (2010) The Chlorella variabilis NC64A genome reveals adaptation to photosymbiosis, coevolution with viruses, and cryptic sex. Plant Cell 22:2943–2955PubMedCrossRefGoogle Scholar
  4. Bleukx W, Delcour JA (1999) A second aspartic proteinase associated with wheat gluten. J Cereal Sci 32:31–42CrossRefGoogle Scholar
  5. Castanheira P, Samyn B, Sergeant K, Clemente JC, Dunn BM, Pires E, Van Beeumen J, Faro C (2005) Activation, proteolytic processing, and peptide specificity of recombinant cardosin A. J Biol Chem 280:13047–13054PubMedCrossRefGoogle Scholar
  6. Chen X, Pfeil JE, Gal S (2002) The three typical aspartic proteinase genes of Arabidopsis thaliana are differentially expressed. Eur J Biochem 269:4675–4684PubMedCrossRefGoogle Scholar
  7. Chen J, Ouyang Y, Wang L, Xie W, Zhang Q (2009) Aspartic proteases gene family in rice: Gene structure and expression, predicted protein features and phylogenetic relation. Gene 442:108–118PubMedCrossRefGoogle Scholar
  8. Consortium TU (2011) Ongoing and future developments at the Universal Protein Resource. Nucleic Acids Res 39:D214–D219CrossRefGoogle Scholar
  9. da Costa DS, Pereira S, Moore I, Pissarra J (2010) Dissecting cardosin B trafficking pathways in heterologous systems. Planta 232:1517–1530PubMedCrossRefGoogle Scholar
  10. Davies D (1990) The structure and function of the aspartic proteinases. Annu Rev Biophys Biophys Chem 19:189–215PubMedCrossRefGoogle Scholar
  11. D’Hondt K (1993) An aspartic proteinase present in seeds cleaves Arabidopsis 2 S albumin precursors in vitro. J Biol Chem 268:20884–20891PubMedGoogle Scholar
  12. Duarte P, Pissarra J, Moore I (2008) Processing and trafficking of a single isoform of the aspartic proteinase cardosin A on the vacuolar pathway. Planta 227:1255–1268PubMedCrossRefGoogle Scholar
  13. Dunn B (1997) Splitting image. Nat Struct Mol Biol 4:969–972CrossRefGoogle Scholar
  14. Dunn BM (2002) Structure and mechanism of the pepsin-like family of aspartic peptidases. Chem Rev 102:4431–4458PubMedCrossRefGoogle Scholar
  15. Eberhard S, Finazzi G, Wollman FA (2008) The dynamics of photosynthesis. Annu Rev Genet 42:463–515PubMedCrossRefGoogle Scholar
  16. Egas C, Lavoura N, Resende R, Brito RMM, Pires E, de Lima MCP, Faro C (2000) The saposin-like domain of the plant aspartic proteinase precursor is a potent inducer of vesicle leakage. J Biol Chem 275:38190–38196PubMedCrossRefGoogle Scholar
  17. Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971PubMedCrossRefGoogle Scholar
  18. Faro C, Gal S (2005) Aspartic proteinase content of the Arabidopsis genome. Curr Protein Pept Sci 6:493–500PubMedCrossRefGoogle Scholar
  19. Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer EL, Eddy SR, Bateman A (2010) The Pfam protein families database. Nucleic Acids Res 38:D211–D222PubMedCrossRefGoogle Scholar
  20. Francis SE, Gluzman Y, Oksman A, Knickerbocker A, Mueller R, Bryant ML, Sherman DR, Russell DG, Goldberg DE (1994) Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase. EMBO J 1:306–317Google Scholar
  21. Glathe S, Kervinen J, Nimtz M, Li GH, Tobin GJ, Copeland TD, Ashford DA, Wlodawer A, Costa J (1998) Transport and activation of the vacuolar aspartic proteinase phytepsin in barley (Hordeum vulgare L.). J Biol Chem 273:31230–31236PubMedCrossRefGoogle Scholar
  22. Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, Lopez R (2010) A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res 38:W695–W699PubMedCrossRefGoogle Scholar
  23. Grossman AR (2000) Chlamydomonas reinhardtii and photosynthesis: genetics to genomics. Curr Opin Plant Biol 3:132–137PubMedCrossRefGoogle Scholar
  24. Guruprasad K, Törmäkangas K, Kervinen J, Blundell TL (1994) Comparative modelling of barley-grain aspartic proteinase: A structural rationale for observed hydrolytic specificity. FEBS Lett 352:131–136PubMedCrossRefGoogle Scholar
  25. Harris EH (2009) The Chlamydomonas sourcebook, 2nd edn. Elsevier-Academic Press, OxfordGoogle Scholar
  26. Horimoto Y, Dee DR, Yada RY (2009) Multifunctional aspartic peptidase prosegments. N Biotechnol 25:318–324PubMedCrossRefGoogle Scholar
  27. Hortensteiner S (2006) Chlorophyll degradation during senescence. Annu Rev Plant Biol 57:55–77PubMedCrossRefGoogle Scholar
  28. Jain E, Bairoch A, Duvaud S, Phan I, Redaschi N, Suzek BE, Martin MJ, McGarvey P, Gasteiger E (2009) Infrastructure for the life sciences: design and implementation of the UniProt website. BMC Bioinformatics 10:136PubMedCrossRefGoogle Scholar
  29. Kato Y, Murakami S, Yamamoto Y, Chatani H, Kondo Y, Nakano T, Yokota A, Sato F (2004) The DNA-binding protease, CND41, and the degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase in senescent leaves of tobacco. Planta 220:97–104PubMedCrossRefGoogle Scholar
  30. Kato Y, Yamamoto Y, Murakami S, Sato F (2005) Post-translational regulation of CND41 protease activity in senescent tobacco leaves. Planta 222:643–651PubMedCrossRefGoogle Scholar
  31. Kervinen J, Sarkkinen P, Kalkkinen N, Mikola L, Saarma M (1993) Hydrolytic specificity of the barley grain aspartic proteinase. Phytochemistry 32:799–803PubMedCrossRefGoogle Scholar
  32. Kervinen J, Tobin GJ, Costa J, Waugh DS, Wlodawer A, Zdanov A (1999) Crystal structure of plant aspartic proteinase prophytepsin: inactivation and vacuolar targeting. EMBO J 18:3947–3955PubMedCrossRefGoogle Scholar
  33. Koelsch G, Mares M, Metcalf P, Fusek M (1994) Multiple functions of pro-parts of aspartic proteinase zymogens. FEBS Lett 343:6–10PubMedCrossRefGoogle Scholar
  34. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948PubMedCrossRefGoogle Scholar
  35. Levitan A, Trebitsh T, Kiss V, Pereg Y, Dangoor I, Danon A (2005) Dual targeting of the protein disulfide isomerase RB60 to the chloroplast and the endoplasmic reticulum. Proc Natl Acad Sci USA 102:6225–6230PubMedCrossRefGoogle Scholar
  36. Liepinsh E, Andersson M, Ruysschaert JM, Otting G (1997) Saposin fold revealed by the NMR structure of NK-lysin. Nat Struct Mol Biol 4:793–795CrossRefGoogle Scholar
  37. Lin X, Tang J, Koelsch G, Monod M, Foundling S (1993) Recombinant canditropsin, an extracellular aspartic protease from yeast Candida tropicalis. Escherichia coli expression, purification, zymogen activation, and enzymic properties. J Biol Chem 268:20143–20147PubMedGoogle Scholar
  38. Malyan AN (2010) Nucleotide binding to noncatalytic sites is essential for ATP-dependent stimulation and ADP-dependent inactivation of the chloroplast ATP synthase. Photosynth Res 105:243–248PubMedCrossRefGoogle Scholar
  39. Mazorra-Manzano MA, Tanaka T, Dee DR, Yada RY (2010) Structure-function characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana. Phytochemistry 71:515–523PubMedCrossRefGoogle Scholar
  40. Mittag M, Kiaulehn S, Johnson CH (2005) The circadian clock in Chlamydomonas reinhardtii. What is it for? What is it similar to? Plant Physiol 137:399–409PubMedCrossRefGoogle Scholar
  41. Mourioux G, Douce R (1981) Slow passive diffusion of orthophosphate between intact isolated chloroplasts and suspending medium. Plant Physiol 67:470–473PubMedCrossRefGoogle Scholar
  42. Olinares PD, Kim J, van Wijk KJ (2011) The Clp protease system; a central component of the chloroplast protease network. Biochim Biophys Acta 1807:999–1011PubMedCrossRefGoogle Scholar
  43. Pereira CS, da Costa DS, Pereira S, Nogueira FM, Albuquerque PM, Teixeira J, Faro C, Pissarra J (2008) Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases. Protoplasma 232:203–213PubMedCrossRefGoogle Scholar
  44. Pissarra J, Pereira C, Costa DS, Figueiredo R, Duarte P, Teixeira J, Pereira S (2007) From flower to seed germination in Cynara cardunculus: a role for aspartic proteinases. Int J Plant Develop Biol 1:274–281Google Scholar
  45. Prochnik SE, Umen J, Nedelcu AM, Hallmann A, Miller SM, Nishii I, Ferris P, Kuo A, Mitros T, Fritz-Laylin LK, Hellsten U, Chapman J, Simakov O, Rensing SA, Terry A, Pangilinan J, Kapitonov V, Jurka J, Salamov A, Shapiro H, Schmutz J, Grimwood J, Lindquist E, Lucas S, Grigoriev IV, Schmitt R, Kirk D, Rokhsar DS (2010) Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri. Science 329:223–226PubMedCrossRefGoogle Scholar
  46. Radhamony RN, Theg SM (2006) Evidence for an ER to Golgi to chloroplast protein transport pathway. Trends Cell Biol 16:385–387PubMedCrossRefGoogle Scholar
  47. Ramalho-Santos M, Verissimo P, Cortes L, Samyn B, Van Beeumen J, Pires E, Faro C (1998) Identification and proteolytic processing of procardosin A. Eur J Biochem 255:133–138PubMedCrossRefGoogle Scholar
  48. Rawlings ND, Bateman AJ (2009) Pepsin homologues in bacteria. BMC Genomics 10:437PubMedCrossRefGoogle Scholar
  49. Rawlings ND, Barrett AJ, Bateman A (2010) MEROPS: the peptidase database. Nucleic Acids Res 38:D227–D233PubMedCrossRefGoogle Scholar
  50. Runeberg-Roos P, Kervinen J, Kovaleva V, Raikhel NV, Gal S (1994) The aspartic proteinase of barley is a vacuolar enzyme that processes probarley lectin in vitro. Plant Physiol 105:321–329PubMedCrossRefGoogle Scholar
  51. Sakamoto W (2006) Protein degradation machineries in plastids. Annu Rev Plant Biol 57:599–621PubMedCrossRefGoogle Scholar
  52. Schaaf A, Reski R, Decker EL (2004) A novel aspartic proteinase is targeted to the secretory pathway and to the vacuole in the moss Physcomitrella patens. Eur J Cell Biol 83:145–152PubMedCrossRefGoogle Scholar
  53. Sigrist CJ, Cerutti L, de Castro E, Langendijk-Genevaux PS, Bulliard V, Bairoch A, Hulo N (2010) PROSITE, a protein domain database for functional characterization and annotation. Nucleic Acids Res 38:D161–D166PubMedCrossRefGoogle Scholar
  54. Simoes I, Faro C (2004) Structure and function of plant aspartic proteinases. Eur J Biochem 271:2067–2075PubMedCrossRefGoogle Scholar
  55. Simoes I, Faro R, Bur D, Faro C (2007) Characterization of recombinant CDR1, an Arabidopsis aspartic proteinase involved in disease resistance. J Biol Chem 282:31358–31365PubMedCrossRefGoogle Scholar
  56. Tanaka R, Tanaka A (2011) Chlorophyll cycle regulates the construction and destruction of the light-harvesting complexes. Biochim Biophys Acta 1807:968–976PubMedCrossRefGoogle Scholar
  57. Terauchi K, Asakura T, Ueda H, Tamura T, Tamura K, Matsumoto I, Misaka T, Hara-Nishimura I, Abe K (2006) Plant-specific insertions in the soybean aspartic proteinases, soyAP1 and soyAP2, perform different functions of vacuolar targeting. J Plant Physiol 163:856–862PubMedCrossRefGoogle Scholar
  58. Timotijevic GS, Milisavljevic MD, Radovic SR, Konstantinovic MM, Maksimovic VR (2010) Ubiquitous aspartic proteinase as an actor in the stress response in buckwheat. J Plant Physiol 167:61–68PubMedCrossRefGoogle Scholar
  59. Tormakangas K, Hadlington JL, Pimpl P, Hillmer S, Brandizzi F, Teeri TH, Denecke J (2001) A vacuolar sorting domain may also influence the way in which proteins leave the endoplasmic reticulum. Plant Cell 13:2021–2032PubMedGoogle Scholar
  60. Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, Luo Y, Fisher S, Fuller J, Edenson S, Lile J, Jarosinski MA, Biere AL, Curran E, Burgess T, Louis JC, Collins F, Treanor J, Rogers G, Citron M (1999) Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286:735–741PubMedCrossRefGoogle Scholar
  61. Veríssimo P, Faro C, Pires C (1995) The vegetable rennet of Cynara cardunculus L. contains two proteinases with chymosin and pepsin-like specificities. Biotechnol Lett 17:621–626CrossRefGoogle Scholar
  62. Veríssimo P, Faro C, Moir AJ, Lin Y, Tang J, Pires E (1996) Purification, characterization and partial amino acid sequencing of two new aspartic proteinases from fresh flowers of Cynara cardunculus L. Eur J Biochem 235:762–768PubMedCrossRefGoogle Scholar
  63. Vieira M, Pissarra J, Veríssimo P, Castanheira P, Costa Y, Pires E, Faro C (2001) Molecular cloning and characterization of cDNA encoding cardosin B, an aspartic proteinase accumulating extracellularly in the transmitting tissue of Cynara cardunculus L. Plant Mol Biol 45:529–539PubMedCrossRefGoogle Scholar
  64. Villarejo A, Buren S, Larsson S, Dejardin A, Monne M, Rudhe C, Karlsson J, Jansson S, Lerouge P, Rolland N, von Heijne G, Grebe M, Bako L, Samuelsson G (2005) Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast. Nat Cell Biol 7:1224–1231PubMedCrossRefGoogle Scholar
  65. Wilson NF, Iyer JK, Buchheim JA, Meek W (2008) Regulation of flagellar length in Chlamydomonas. Semin Cell Dev Biol 19:494–501PubMedCrossRefGoogle Scholar
  66. Worden AZ, Lee JH, Mock T, Rouze P, Simmons MP, Aerts AL, Allen AE, Cuvelier ML, Derelle E, Everett MV, Foulon E, Grimwood J, Gundlach H, Henrissat B, Napoli C, McDonald SM, Parker MS, Rombauts S, Salamov A, Von Dassow P, Badger JH, Coutinho PM, Demir E, Dubchak I, Gentemann C, Eikrem W, Gready JE, John U, Lanier W, Lindquist EA, Lucas S, Mayer KF, Moreau H, Not F, Otillar R, Panaud O, Pangilinan J, Paulsen I, Piegu B, Poliakov A, Robbens S, Schmutz J, Toulza E, Wyss T, Zelensky A, Zhou K, Armbrust EV, Bhattacharya D, Goodenough UW, Van de Peer Y, Grigoriev IV (2009) Green evolution and dynamic adaptations revealed by genomes of the marine picoeukaryotes Micromonas. Science 324:268–272PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Carla Malaquias Almeida
    • 1
  • Cláudia Pereira
    • 2
  • Diana Soares da Costa
    • 3
  • Susana Pereira
    • 2
  • José Pissarra
    • 2
  • Isaura Simões
    • 1
    • 4
  • Carlos Faro
    • 1
    • 4
    Email author
  1. 1.Biocant, Biotechnology Innovation Center, Molecular Biotechnology Unit, Parque Tecnológico de CantanhedeCantanhedePortugal
  2. 2.Departamento de Biologia, Faculdade de CiênciasBioFig-Centre for Biodiversity, Functional and Integrative Genomics, Universidade Do PortoPortoPortugal
  3. 3.Biomaterials, Biodegradables and Biomimetics Research Group, Department of Polymer EngineeringUniversidade do MinhoCaldas das TaipasPortugal
  4. 4.CNC-Center for Neuroscience and Cell Biology of Coimbra, University of CoimbraCoimbraPortugal

Personalised recommendations