Multiplicity of aspartic proteinases from Cynara cardunculus L.
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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.
KeywordsAspartic proteinases Mass spectrometry Protein characterisation Specificity
Matrix-assisted laser desorption/ionisation
Time of flight
This research work was financed by POCI/QUI/60791/2004. AC Sarmento, R Vitorino and CS Oliveira were supported by Fundação para a Ciência e Tecnologia (grant nos. BPD/38008/2007, BPD/14968/2004, BPD/26670/2006). B. Samyn is a postdoctoral fellow of the Fund for Scientific Research-Flanders (F.W.O.-Vlaanderen, Belgium).
- 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
- 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
- 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
- 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
- DeLano WL (2002) The PyMOL molecular graphics system. DeLano Scientific, San Carlos. Available at: http://www.pymol.org
- 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
- 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
- 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