Phytochemistry 81 (2012) 7–18 Contents lists available at SciVerse ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Molecular cloning and characterization of procirsin, an active aspartic protease precursor from Cirsium vulgare (Asteraceae) Daniela Lufrano a, Rosário Faro b, Pedro Castanheira c, Gustavo Parisi d, Paula Veríssimo b,e, ⇑ Sandra Vairo-Cavalli a, Isaura Simões b,c, , Carlos Faro b,c,e a Laboratorio de Investigación de Proteínas Vegetales (LIPROVE), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, C.C. 711, 1900 La Plata, Argentina b CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal c Biocant, Biotechnology Innovation Center, 3060-197 Cantanhede, Portugal d Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, Bernal, Buenos Aires, B1876BXD, Argentina e Department of Life Sciences, University of Coimbra, 3004-517 Coimbra, Portugal article info abstract Article history: Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well- Received 16 February 2012 known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on Received in revised form 23 May 2012 other Astereaceae plant species for identification of new vegetable rennets. Here we report on the clon- Available online 21 June 2012 ing, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, Keywords: with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form Cirsium vulgare of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its Asteraceae pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described Bull thistle Cirsin for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical Procirsin proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage Aspartic proteinase between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Proc- Pepsin-like protease irsin also displayed a high specificity towards j-casein and milk-clotting activity, suggesting it might be Milk-clotting activity an effective vegetable rennet. The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction different clans and families according to MEROPS database (Raw- lings et al., 2010) and appear to be the second-largest class of pro- Aspartic proteinases (APs, EC. 3.4.23) belong to one of the seven teases in these organisms (van der Hoorn, 2008). Members of the catalytic types of proteolytic enzymes and are widely distributed A1 family are by far the best characterized and constitute the in nature in a variety of organisms such as viruses, bacteria, yeast, largest group of plant APs. They are found in a variety of tissues fungi, plants and animals (Dunn, 2002; Rawlings and Bateman, (Simões and Faro, 2004; Feijoo-Siota and Villa, 2011; González- 2009; Simões et al., 2011). In plants, APs are distributed among Rábade et al., 2011) and their diversity triggered a redefinition of the classification of A1 plant APs into typical, nucellin-like and atypical proteases (Faro and Gal, 2005). Abbreviations: AP, aspartic proteinase; DABCYL, [4-((4-(dimethylamino) Although plant typical APs display differences in activity and phenyl)azo)benzoic acid]; E-64, trans -epoxysucciny-L-leucyl-amido (4-guanidino) butane; EDANS, 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid; EDTA, ethyl- specificity, they share several common features with their non- enediaminetetraacetic acid; EPNP, 1,2-epoxy-3-p-nitrophenoxy propane; MCA, (7- plant homologues. Typical plant APs are characterized by the pres- methoxycoumarin-4-yl) acetyl; PSI, plant-specific insert; SAPLIPs, saposin-like ence of two aspartic acid residues organized in two conserved Asp- proteins. Thr/Ser-Gly (DT/SG) motifs responsible for catalytic activity, are ⇑ Corresponding author. Address: Biocant, Parque Tecnológico de Cantanhede, Núcleo 4-Lote 3, 3060-197 Cantanhede, Portugal. Tel.: +351 231410890; fax: +351 most active at acidic pH, have a preference for cleavage between 231 410 899. hydrophobic amino acids and are sensitive to pepstatin A. The E-mail addresses: [email protected] (D. Lufrano), [email protected] (R. Faro), overall three-dimensional structure is also conserved, consisting [email protected] (P. Castanheira), [email protected] (G. Parisi), of two b-barrel-like domains that create a deep cleft where the [email protected] (P. Veríssimo), [email protected] (S. Vairo-Cavalli), isi- active site is buried (Simões and Faro, 2004). Typical plant APs, [email protected] (I. Simões), [email protected] (C. Faro). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.05.028 8 D. Lufrano et al. / Phytochemistry 81 (2012) 7–18 however, contain a distinctive additional segment of about 100 countries. In an attempt to identify new milk coagulants, several residues called plant-specific insert (PSI). This insert is folded as species of the Asteraceae family found in Argentina were screened an independent domain, shares high topological similarity with for their ability to clot milk. Cirsium vulgare flower extracts were the saposin-like proteins (SAPLIPs) and is suggested to be involved shown to be positive in this screening, thereby suggesting the pres- in membrane interactions and in the vacuolar sorting of several ence of APs which are known to be responsible for this activity APs (Kervinen et al., 1999; Egas et al., 2000; Bryksa et al., 2011). among Asteraceae family members. Typical APs are synthesized as single-chain preproenzymes and To identify and further characterize bull thistle’s AP(s), cDNA subsequently processed to single- or two-chain mature forms. In was synthesized using total RNA isolated from young flower buds general, the PSI domain is totally or partially removed from the and used as PCR template with primers designed for conserved re- precursor, yet some variations exist on the mechanism and order gions of the ORF 50 and 30 ends of plant APs. The amplified product by which this insert and the pro-segment are excised during con- of 1530 bp was cloned and several clones were sequenced. This version towards the active form (Simões and Faro, 2004). Process- analysis resulted in the identification of at least four different de- ing of the precursor forms is suggested to involve sequential duced amino acid sequences exhibiting high levels of sequence proteolytic cleavages along the secretory pathway that ultimately identity among themselves and with other plant typical APs avail- generate a complex mixture of heterogeneous mature aspartic pro- able in public databases, clearly suggesting the presence of several teinases within plant tissues (Duarte et al., 2008). Although no par- APs in C. vulgare flowers. One of these cDNA sequences was se- ticular enzyme has been identified as a trigger in these processing lected for further characterization and its nucleotide and deduced events, evidence from in vitro processing experiments suggests amino acid sequences are shown in Fig. 1 (GenBank accession that complete maturation of proteases may require the action of number: JN703462). This protein product (named cirsin) encoded other(s) proteinase(s), in addition to autoproteolytic processing a preproenzyme of 509 residues with a putative hydrophobic sig- (White et al., 1999; Domingos et al., 2000; Castanheira et al., nal peptide of 24 amino acids predicted by SignalP 4.0 (Petersen 2005; Sampaio et al., 2008). Diversity among plant typical APs et al., 2011), a pro-segment of 44 residues, and a 441 amino may, therefore, be caused by the occurrence of different proteolytic acid-long polypeptide interrupted by 105 residues corresponding cleavage sites on the same precursor and also by the expression of to the PSI domain. The preproenzyme has a predicted molecular several genes encoding closely related enzymes, as shown with mass of 55 kDa with a theoretical pI of 5.3. The deduced amino acid Cynara cardunculus typical APs multigene family (Pimentel et al., sequence displays the characteristic primary structure of typical 2007). Although well documented in the literature, the correlation plant APs in addition to two conserved catalytic triads (DTG 103– between diversity patterns of plant typical APs and their biological 105 and DTG 290–292) and a tyrosine residue in position 147 (cor- functions is still far from being deciphered. responding to DTG 32–34, DTG 215–217 and Y75, respectively, Interestingly, typical plant APs have been extensively investi- using pepsin numbering). Furthermore, two putative N-glycosyla- gated over the years for their commercial potential as coagulants tion sites conserved in several typical plant APs (Domingos et al., for cheesemaking. Aqueous extracts from flowers of species of the 2000; Vieira et al., 2001) were also predicted by NetNGlyc (Blom Cardueae tribe (Asteraceae family) like Cynara cardunculus, Cynara et al., 2004) at residues N139 (NGT motif) and N400 (NET