Planta

, Volume 230, Issue 2, pp 429–439 | Cite as

Multiplicity of aspartic proteinases from Cynara cardunculus L.

  • Ana Cristina Sarmento
  • Henrique Lopes
  • Cláudia S. Oliveira
  • Rui Vitorino
  • Bart Samyn
  • Kjell Sergeant
  • Griet Debyser
  • Jozef Van Beeumen
  • Pedro Domingues
  • Francisco Amado
  • Euclides Pires
  • M. Rosário M. Domingues
  • Marlene T. Barros
Original Article

Abstract

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC–MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO2)AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity.

Keywords

Aspartic proteinases Mass spectrometry Protein characterisation Specificity 

Abbreviations

AP

Aspartic proteinase

LC

Liquid chromatography

MALDI

Matrix-assisted laser desorption/ionisation

MS

Mass spectrometry

TOF

Time of flight

TFA

Trifluoroacetic acid

MQ

Ultra-pure water

DIEA

N,N-diisopropylethylamine

Supplementary material

425_2009_948_MOESM1_ESM.doc (180 kb)
Supplementary material 1 (DOC 180 kb)

References

  1. Beyer BM, Dunn BM (1998) Prime region subsite specificity characterization of human cathepsin D: the dominant role of position 128. Protein Sci 7:88–95PubMedGoogle Scholar
  2. Brodelius PE, Cordeiro MC, Pais MS (1995) Aspartic proteinases (cyprosins) from Cynara cardunculus spp flavescens cv cardoon: purification, characterisation, and tissue-specific expression. Aspartic Proteinases 362:255–266Google Scholar
  3. Brodelius M, Hiraiwa M, Marttila S, Al Karadaghi S, Picaud S, Brodelius PE (2005) Immunolocalization of the saposin-like insert of plant aspartic proteinases exhibiting saposin C activity. Expression in young flower tissues and in barley seeds. Physiol Plant 125:405–418Google Scholar
  4. 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
  5. Chow RB, Kassell B (1968) Bovine pepsinogen and pepsin I. Isolationm purification and some properties of pepsinogen. J Biol Chem 243:1718–1724PubMedGoogle Scholar
  6. Cordeiro M, Jakob E, Puhan Z, Pais MS, Brodelius PE (1992) Milk clotting and proteolytic activities of purified cynarases from Cynara cardunculus—a comparison to chymosin. Milchwiss Milk Sci Int 47:683–687Google Scholar
  7. Cordeiro MC, Xue ZT, Pietrzak M, Pais MS, Brodelius PE (1994) Isolation and characterisation of a cDNA from flowers of Cynara cardunculus encoding cyprosin (an aspartic proteinase) and its use to study the organ-specific expression of cyprosin. Plant Mol Biol 24:733–741PubMedCrossRefGoogle Scholar
  8. Costa J, Ashford DA, Nimtz M, Bento I, Frazao C, Esteves CL, Faro CJ, Kervinen J, Pires E, Verissimo P, Wlodawer A, Carrondo MA (1997) The glycosylation of the aspartic proteinases from barley (Hordeum vulgare L) and cardoon (Cynara cardunculus L). Eur J Biochem 243:695–700PubMedCrossRefGoogle Scholar
  9. DeLano WL (2002) The PyMOL molecular graphics system. DeLano Scientific, San Carlos. Available at: http://www.pymol.org
  10. Dingle JT, Leaback DH (1975) Secretion of enzymes into pericellular environment. Philos Trans R Soc Lond B Biol Sci 271:315–324PubMedCrossRefGoogle Scholar
  11. Dunn BM (2002) Structure and mechanism of the pepsin-like family of aspartic peptidases. Chem Rev 102:4431–4458PubMedCrossRefGoogle Scholar
  12. Dwek RA (1996) Glycobiology: toward understanding the function of sugars. Chem Rev 96:683–720PubMedCrossRefGoogle Scholar
  13. 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
  14. Faro C, Ramalho-Santos M, Verissimo P, Pissarra J, Frazao C, Costa J, Lin XL, Tang J, Pires E (1998) Structural and functional aspects of cardosins. Aspartic Proteinases 436:423–433Google Scholar
  15. Fernandez-Salguero J, Prados F, Calixto F, Vioque M, Sampaio P, Tejada L (2003) Use of recombinant cyprosin in the manufacture of ewe’s milk cheese. J Agric Food Chem 51:7426–7430PubMedCrossRefGoogle Scholar
  16. Foltmann B (1992) Chymosin—a short review on fetal and neonatal gastric proteases. Scand J Clin Lab Invest 52:65–79CrossRefGoogle Scholar
  17. Frazao C, Bento I, Costa J, Soares CM, Verissimo P, Faro C, Pires E, Cooper J, Carrondo MA (1999) Crystal structure of cardosin A, a glycosylated and Arg-Gly-Asp-containing aspartic proteinase from the flowers of Cynara cardunculus L. J Biol Chem 274:27694–27701PubMedCrossRefGoogle Scholar
  18. Fruton JS (2002) A history of pepsin and related enzymes. Q Rev Biol 77:127–147PubMedCrossRefGoogle Scholar
  19. Guruprasad K, Tormakangas K, Kervinen J, Blundell TL (1994) Comparative modeling of barley-grain aspartic proteinase—a structural rationale for observed hydrolytic specificity. FEBS Lett 352:131–136PubMedCrossRefGoogle Scholar
  20. Heimgartner U, Pietrzak M, Geertsen R, Brodelius P, Figueiredo ACD, Pais MSS (1990) Purification and partial characterization of milk clotting proteases from flowers of Cynara cardunculus. Phytochemistry 29:1405–1410CrossRefGoogle Scholar
  21. Huff JR (1991) Hiv protease—a novel chemotherapeutic target for AIDS. J Med Chem 34:2305–2314PubMedCrossRefGoogle Scholar
  22. Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  23. Lapolla A, Fedele D, Traldi P (2000) The role of mass spectrometry in the study of non-enzymatic protein glycation in diabetes. Mass Spectrom Rev 19:279–304PubMedCrossRefGoogle Scholar
  24. Mutlu A, Gal S (1999) Plant aspartic proteinases: enzymes on the way to a function. Physiol Plant 105:569–576CrossRefGoogle Scholar
  25. Oconnell KL, Stults JT (1996) Identification of mouse liver proteins on two-dimensional electrophoresis gels by matrix-assisted laser desorption ionization mass spectrometry of in situ enzymatic digests. 2nd Siena 2D electrophoresis—from genome to proteome. Vch Publishers Inc, Siena, Italy, pp 349–359Google Scholar
  26. Pereira CS, da Costa DS, Pereira S, Nogueira FD, 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
  27. Picon A, Fernandez J, Gaya P, Medina M, Nunez M (1999) Short communication: stability of chymosin and cyprosins under milk-coagulation and cheese-ripening conditions. J Dairy Sci 82:2331–2333CrossRefGoogle Scholar
  28. Pimentel C, Van der Straeten D, Pires E, Faro C, Rodrigues-Pousada C (2007) Characterization and expression analysis of the aspartic protease gene family of Cynara cardunculus L. FEBS J 274:2523–2539PubMedCrossRefGoogle Scholar
  29. Pina DG, Oliveira CS, Sarmento AC, Barros M, Pires E, Zhadan GG, Villar E, Gavilanes F, Shnyrov VL (2003) Thermostability of cardosin A from Cynara cardunculus L. Thermochimica Acta 402:123–134Google Scholar
  30. Prados F, Pino A, Fernandez-Salguero J (2007) Effect of a powdered vegetable coagulant from cardoon Cynara cardunculus in the accelerated ripening of Manchego cheese. Int J Food Sci Technol 42:556–561CrossRefGoogle Scholar
  31. Ramalho-Santos M, Pissarra J, Verissimo P, Pereira S, Salema R, Pires E, Faro CJ (1997) Cardosin A, an abundant aspartic proteinase, accumulates in protein storage vacuoles in the stigmatic papillae of Cynara cardunculus L. Planta 203:204–212PubMedCrossRefGoogle Scholar
  32. 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
  33. Samyn B, Sergeant K, Castanheira P, Faro C, Van Beeumen J (2005) A new method for C-terminal sequence analysis in the proteomic era. Nat Methods 2:193–200PubMedCrossRefGoogle Scholar
  34. Sandra K, Devreese B, Van Beeumen J, Stals I, Claeyssens M (2004) The Q-trap mass spectrometer, a novel tool in the study of protein glycosylation. J Am Soc Mass Spectrom 15:413–423PubMedCrossRefGoogle Scholar
  35. Sarmento AC, Silvestre L, Barros M, Pires E (1998) Cardosins A and B, two new enzymes available for peptide synthesis. J Mol Catal B Enzym 5:327–330CrossRefGoogle Scholar
  36. Sarmento AC, Oliveira C, Pires E, Amado F, Barros M (2004a) Reverse hydrolysis by cardosin A: specificity considerations. J Mol Catal B Enzym 28:33–37CrossRefGoogle Scholar
  37. Sarmento AC, Oliveira CS, Pires EM, Halling PJ, Barros MT (2004b) Evaluation of cardosin A as a proteolytic probe in the presence of organic solvents. J Mol Catal B Enzym 31:137–141CrossRefGoogle Scholar
  38. Sarmento AC, Oliveira CS, Duarte AS, Pires E, Barros MT (2006) Evaluation of cardosin A as a probe for limited proteolysis in non-aqueous environments—complex substrates hydrolysis. Enzyme Microb Technol 38:415–421CrossRefGoogle Scholar
  39. Sarmento AC, Oliveira CS, Pereira A, Esteves VI, Moir AJG, Saraiva J, Pires E, Barros M (2009) Unfolding of cardosin A in organic solvents and detection of intermediaries. J Mol Catal B Enzym 57:115–122CrossRefGoogle Scholar
  40. Sergeant K, Samyn B, Debyser G, Van Beeumen J (2005) De novo sequence analysis of N-terminal sulfonated peptides after in-gel guanidination. Proteomics 5:2369–2380PubMedCrossRefGoogle Scholar
  41. Simoes I, Faro C (2004) Structure and function of plant aspartic proteinases. Eur J Biochem 271:2067–2075PubMedCrossRefGoogle Scholar
  42. Simoes I, Mueller EC, Otto A, Bur D, Cheung AY, Faro C, Pires E (2005) Molecular analysis of the interaction between cardosin A and phospholipase D alpha—identification of RGD/KGE sequences as binding motifs for C2 domains. FEBS J 272:5786–5798PubMedCrossRefGoogle Scholar
  43. 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
  44. Takahashi K, Niwa H, Yokota N, Kubota K, Inoue H (2008) Widespread tissue expression of nepenthesin-like aspartic protease genes in Arabidopsis thaliana. Plant Physiol Biochem 46:724–729PubMedCrossRefGoogle Scholar
  45. Tejada L, Vioque M, Gomez R, Fernandez-Salguero J (2008) Effect of lyophilisation, refrigerated storage and frozen storage on the coagulant activity and microbiological quality of Cynara cardunculus L. extracts. J Sci Food Agric 88:1301–1306CrossRefGoogle Scholar
  46. Vassar R (2004) BACE1—the beta-secretase enzyme in Alzheimer’s disease. J Mol Neurosci 23:105–113PubMedCrossRefGoogle Scholar
  47. Verissimo P, Faro C, Moir AJG, Lin YZ, 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
  48. White PC, Cordeiro MC, Arnold D, Brodelius PE, Kay J (1999) Processing, activity, and inhibition of recombinant cyprosin, an aspartic proteinase from cardoon (Cynara cardunculus). J Biol Chem 274:16685–16693PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Ana Cristina Sarmento
    • 1
  • Henrique Lopes
    • 2
  • Cláudia S. Oliveira
    • 1
  • Rui Vitorino
    • 2
  • Bart Samyn
    • 3
  • Kjell Sergeant
    • 3
    • 4
  • Griet Debyser
    • 3
  • Jozef Van Beeumen
    • 3
  • Pedro Domingues
    • 2
  • Francisco Amado
    • 2
  • Euclides Pires
    • 5
  • M. Rosário M. Domingues
    • 2
  • Marlene T. Barros
    • 5
    • 6
  1. 1.CESAM, Department of BiologyUniversity of AveiroAveiroPortugal
  2. 2.Department of ChemistryUniversity of AveiroAveiroPortugal
  3. 3.Department of Biochemistry, Physiology and Microbiology, Laboratory of Protein Biochemistry and Protein EngineeringGhent UniversityGhentBelgium
  4. 4.Department of Environment and AgrobiotechnologiesCRP-GLBelvauxLuxembourg
  5. 5.Center for Neuroscience and Cell BiologyIBILICoimbraPortugal
  6. 6.Instituto de Ciências da SaúdeUniversidade Católica PortuguesaViseuPortugal

Personalised recommendations