A novel metallocarboxypeptidase-like enzyme from the marine annelid Sabellastarte magnifica – a step into the invertebrate world of proteases Maday Alonso-del-Rivero 1 , Sebastian A. Trejo 3 ,Mo ´ nica Rodrı ´ guez de la Vega 3 , Yamile Gonza ´ lez 1 , Silvia Bronsoms 3 , Francesc Canals 2 , Julieta Delfı ´ n 1 , Joaquin Diaz 1 , Francesc X. Aviles 3 and Marı ´ a A. Cha ´ vez 1 1 Centro de Estudio de Proteı ´ nas, Facultad de Biologı ´ a, Universidad de la Habana, Cuba 2 Institut de Recerca Hospital Vall d’Hebron, Barcelona, Spain 3 Institut de Biotecnologı ´ a i Biomedicina and Departament de Bioquı ´ mica i Biologı ´ a Molecular, Universitat Autonoma de Barcelona, Spain Introduction Natural evolution has frequently generated a large adaptative variety of forms among protein functional families, and metallocarboxypeptidases (MCPs) have also followed this trend. Such enzymes are exopeptid- Keywords enzyme specificity; marine annelid; metallocarboxypeptidases; metalloproteins; Sabellastarte magnifica Correspondence F. X. Aviles, Institut de Biotecnologı ´ ai Biomedicina (IBB) and Departament de Bioquı ´ mica i Biologia Molecular, Universitat Autonoma de Barcelona, 08193 Bellaterra (Barcelona), Spain Fax: +34 93 581 2011 Tel: +34 93 581 1231 E-mail: francescxavier.aviles@uab.es (Received 16 March 2009, revised 16 June 2009, accepted 30 June 2009) doi:10.1111/j.1742-4658.2009.07187.x After screening 25 marine invertebrates, a novel metallocarboxypeptidase (SmCP) has been identified by activity and MS analytical approaches, and isolated from the marine annelid Sabellastarte magnifica. The enzyme, which is a minor component of the molecularly complex animal body, as shown by 2D gel electrophoresis, has been purified from crude extracts to homogeneity by affinity chromatography on potato carboxypeptidase inhib- itor and by ion exchange chromatography. SmCP is a protease of 33792 Da, displaying N-terminal and internal sequence homologies with M14 metallocarboxypeptidase-like enzymes, as determined by MS and auto- mated Edman degradation. The enzyme contains one atom of Zn per mole- cule, is activated by Ca 2+ and is drastically inhibited by the metal chelator 1,10-phenanthroline, as well as by excess Zn 2+ or Cu 2+ , but moderately so by EDTA. SmCP is also strongly inhibited by specific inhibitors of metallo- carboxypeptidases, such as benzylsuccinic acid and the protein inhibitors found in potato and leech (i.e. recombinant forms, both at nanomolar levels). The enzyme displays high peptidase efficiency towards pancreatic carboxypeptidase-A synthetic substrates, such as those with hydrophobic residues at the C-terminus but, remarkably, also towards the acidic ones. This property, previously described as for carboxypeptidase O-like activity, has been shown on long peptide substrates by MS. The results obtained in the present study indicate that SmCP is a novel member of the M14 metal- locarboxypeptidases family (assignable to the M14A or pancreatic-like subfamily) with a wider specificity that has not been described previously. Abbreviations AAFP, N-(4-methoxyphenylazoformyl)- L-phenyl-alanine; AAFR, N-(4-methoxyphenylazoformyl)-L-Arg; ACTH fragment (18–39), adrenocorticotropic hormone (RPVKVYPNGAEDESAEAFPLEF); BAEE, benzoyl arginyl ethyl ester; BTEE, benzoyl tyrosine ethyl ester; CP, carboxypeptidase; CPA, carboxypeptidase A; CPB, carboxypeptidase B; CPO, carboxypeptidase O; DIGE, difference gel electrophoresis; E-64, L-carboxy-trans-2,3-epoxypropyl-leycylamido (4-guanidino) butane; FAAK, [3-(2-furyl)acryloyl]-L-alanyl-L-lysine; FAPP, N-(3-[2- furyl]acryloyl)-Phe-Phe; Hippuryl-Phe, N-benzoyl-Gly-Phe; MCP, metallocarboxypeptidase; rLCI, recombinant leech carboxypeptidase inhibitor; rPCI, recombinant potato carboxypeptidase inhibitor; V15E, synthetic substrate [VKKKARKAAGC(Amc)AWE]. FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS 4875 ases that catalyze the hydrolysis of peptide bonds at the C-terminus of peptides and proteins. They belong to the catalytic classes of either metalloproteases (clan MC, family M14) or serine proteases (clan SC, family S10) [1] and their action causes strong effects in the biological activity of their peptide and protein sub- strates [2]. M14 MCPs, including those from animals, plants and bacteria, have been divided into three main subfamilies based on structural similarity and sequence homology. The first one, which includes the digestive enzymes carboxypeptidase (CP) A (CPA) 1, CPA2, carboxypeptidase B (CPB) 1 and mast cell CPA3, as well as CPA4, CPA5 CPA6 and carboxypeptidase O (CPO) (known at the gene level), has been termed sub- family M14A or A ⁄ B; the second one, including the bioactive peptide-processing or regulatory enzymes (e.g. carboxypeptidases N, E, M and D, amongst oth- ers) has been termed subfamily M14B or N ⁄ E [3]. Very recently, a novel subfamily composed of enzymes of larger size and apparently with a predominant cyto- solic location, termed M14D, Nna-like or CCPs, has been proposed [4]. Furthermore, three main classes may be distinguished according to their substrate spec- ificity: (a) for aromatic ⁄ hydrophobic residues (A-like), (b) for basic residues (B-like) and (c) for acidic resi- dues (O-like) [3,5]. MCP enzymes have been isolated from different sources [3,5,6], mainly from vertebrates, but a few of them have come from marine invertebrate organisms: the digestive crayfish carboxypeptidase (CPB) [7], the carboxypeptidase E-like enzyme from the sea hare Aplysia californica, with important regulatory func- tions in this organism [8], two CPs (A and B types) from the hepatopancreas of the crab Paralithodes cam- tschatica [9], the CPA-like protease from squid hepato- pancreas of Illex illecebrosus [10], and CPs (two A and one B type) isolated from the pyloric ceca of the starf- ishes Asterias amurensis [11,12] and Asterina pectinifera [13]. More than 95% of the Earth’s animal species are invertebrates [14]. The ecological services provided by invertebrates are immeasurable; life as we know it would be quite different or decline without them (see Center for Applied Biodiversity Science; http://sci- ence.conservation.org). Overall, our knowledge about MCPs in invertebrates is very limited given the tremen- dous variety of such organisms and compared to the much larger number of characterized CP from verte- brates [6]. In the present study, we screened for the presence of CP activity in marine invertebrates belong- ing to the Phyla Cnidaria, Annelida, Mollusca, Echi- nodermata, Arthropoda and Chordata, amongst others, collected on the coasts of Havana, Cuba. The study has been based on the use of N-(4-meth- oxyphenylazoformyl)-l-phenylalanine (AAFP), a sensi- tive, specific and known colorimetric substrate for CPA enzymes. One of the highest activity levels was detected in extracts from the marine annelid S. magni- fica. This marine invertebrate, also termed ‘magnificent feather duster’, was obtained from coral reefs. It belongs to the Phylum Annelida, Class Polychaeta, which shows a clear delimitation between its tentacle crown and its body (Fig. 1) [15]. Some studies per- formed on another annelid, belonging to the Sabellidae family, have only detected proteolytic activity assign- able to serine proteases, which appeared to be involved in reproduction [16] despite their digestive origin. The presence of a carboxypeptidase-like enzyme in Annelida marine invertebrates has not been described so far. The present study describes the enzymatic activity and MS detection of a novel MCP (termed SmCP) from S. magnifica, and its occurrence as a minor com- ponent within the animal body extracts by 2D- PAGE. The enzyme has been isolated and purified, and then characterized by size, metal content, location, basic interactions, sequence analysis of different regions of the enzyme, and by a description of the main parame- ters related to enzyme kinetics, specificity and inhibi- tion ranges, as well as other basic molecular features. From this, it is apparent that SmCP is a novel M14 MCP (belonging to the pancreatic-like subfamily), showing simultaneous CPA- and CPO-like activities, which is an unusual feature. The present study com- prises an attempt to expand the growing field of the M14 family of proteolytic enzymes, which is now quite diverse and contains more than 25 different variants Fig. 1. S. magnifica Phylum Annelida, Class Polychaeta, Subclass Palpata, Order Canalipalpata, Suborder Sabellida, Family Sabellidae, Genus Sabellastarte [14] The ‘tentacle crown’ and the ‘body’ parts of the animal are clearly visible. A novel metallocarboxypeptidase from S. magnifica M. Alonso-del-Rivero et al. 4876 FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS [4–6], but for which only very few members from invertebrates have been characterized until now. Results Detection of MCP activities in marine organisms Twenty-five marine species belonging to different invertebrate Phyla were screened for CPA activity using AAFP as a substrate: four species of Mollusca (Aplysia dactylomela, Aplysia juliana, Isognomun radia- tus and Lima scabra); four species of Chordata (Pallu- sia nigra, Microcosmus gamus, Molgula occidentalis and Pyura vittata); 11 species of Cnidaria (Bartholo- mea annulata, Budonosoma granulifera, Cassiopea xamachana, Condylactys gigantea, Gorgonia ventalina, Lebrunia danae, Palythoa caribaeorum, Physalia phy- salis, Plexaura homomalla, Stichodactyla helianthus and Zoanthus pulchellus); two species of Annelida (Sabellas- tarte magnifica and Hermodice carunculata); two species of Echinodermata (Holothuria mexicana and Isostisch- opus badionotus); and two species of Arthropoda (Lito- peaeus schmitti and Litopenaeus vannamei). Among them, only the three species S. magnifica (Phyllum Annelida), B. granulifera (Phyllum Cnidaria) and P. vittata (Phyllum Chordata) gave rise to positive results, with specific activity values of 56.0, 1.6 and 1.8 UÆ100 mg )1 extract, respectively. In these three cases, we found a linear relationship between CP-like activity and the quantity of extract used in the assay. Given that the material of the annelid S. magnifica showed by far the highest specific activity, it was selected for further characterization studies. In this case, it was also found that extracts from the ‘body’ showed CP activity, whereas the feather-like ‘crown’ was devoid of it. ‘Intensity fading’ MALDI-TOF MS Once we focused our attention on S. magnifica body extracts, we found there direct evidence of at least one MCP enzyme, of approximately 35 kDa by ‘intensity fading’ MALDI-TOF MS [17]. In the present study, the added ‘binder’ was the recombinant form of potato carboxypeptidase inhibitor (rPCI) (4.5 kDa), immobi- lized on agarose beads, with the aim of both perturb- ing the MS spectrum and capturing the MCP in the body extract. The control spectra, as well as the ‘per- turbed’ one (by rPCI addition, followed by removal of the captured targets by sedimentation of the beads), are shown in Fig. 2A,B. It is apparent that some of the ion signals of the spectra were faded when the extract was treated with immobilized PCI. Subse- quently, MS analysis of the protein eluted from the beads (Fig. 2C) detected a molecular ion of 34 kDa. This molecular species, which is able to strongly inter- act with PCI, presumably represents the CP-like enzyme activity found in S. magnifica body extract. The experiment indicates not only the occurrence in the extract of the strong ligand (the enzyme SmCP) for the added protease inhibitor, but also that this ligand is probably functional in the very complex extract (i.e. not in the zymogen state). It is worth noting that the apparent simplicity of the MALDI-TOF spectrum of the extract shown in Fig. 2C is most likely caused not only by the low expansion scale used, but also by 1000 1500 Control MS (body extract) A Intens. (a.u.)Intens. (a.u.)Intens. (a.u.) 0 500 2000 +PCI 0 500 1000 1500 100 150 Elution 0 50 10 000 15 000 20 000 25 000 30 000 35 000 m/z B C Fig. 2. MALDI-TOF MS of the ‘intensity fading’ experiment (A) Mass spectra of the S. magnifica body extract (control sample) before rPCI-agarose addition (B) Unbound proteins mass spectra obtained after rPCI- agarose addition (C) MS spectra of recov- ered m ⁄ z signal after elution of the sample, corresponding to CP-like enzyme The arrow indicates the ‘perturbed’ signal by rPCI-aga- rose addition. M. Alonso-del-Rivero et al. A novel metallocarboxypeptidase from S. magnifica FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS 4877 ‘signal suppression effects’; such phenomena usually affect visualization of signals in media crowded in mol- ecules [17–19], as will be reported and discussed subse- quently. Molecular complexity of the S. magnifica body extract by 2D-PAGE The molecular complexity of the S. magnifica extracts (both from the body and from the crown, or mixed) was demonstrated by 2D-PAGE analysis (Fig. 3). A great number of visible protein bands [as revealed either by staining with silver or using difference gel electrophoresis (DIGE)] appeared in the analysis of both parts of the animal, with a major presence of bands in the body (upper part) versus the crown (lower part). In Fig. 3, we show, in the uncombined (Fig. 3A,B) or in the combined way (Fig. 3C), the pro- tein components of both parts of the animal labeled with fluorescent dyes using the DIGE approach. That is, the different materials (i.e. crown and body extracts, purified enzyme) were pre-labeled independently with DIGE reagents before they were mixed and run simul- taneously in a single 2D-PAGE separation. The inde- pendent labeling of the crown and body extracts was performed not only to allow the differential tracking of their components, but also to deal with the very high content of dyes and interfering materials from the crown, which required a harsh cleaning (and denatur- ing) procedure. Such interfering materials strongly per- turbed the electrophoretic separation, and also gave rise to severe band strikes and decreased resolution. Only after testing several pre-cleaning and staining procedures (not shown), and selecting an adequate one, were we able to unveil the real band complexity of the extracts (see Experimental procedures). We hope that this experience might be useful for the analysis of other invertebrates with a high content in dyes and other similar problems. Overall, more than 200 protein species are detected by this procedure, among which those in the 17–37 kDa range are the most prominent. To facilitate identification, we repeated the 2D-PAGE with three different initial samples from the body, after passing them through microcolumns with immobilized protein- aceous inhibitors of serine (soya bean protease inhibi- tor, SBTI), cysteine (chicken cystatin) and aspartic (pepstatin) proteases. The intact, flow-through (depleted) and captured (released) materials were deriv- atized with DIGE and run in the same 2D-PAGE gel for each case (see Experimental procedures). The anal- ysis of the ‘captured’ spots allowed us to potentially A B C Fig. 3. 2D gel electrophoresis of pre-labeled protein extracts from S. magnifica The gel contained 30 lg of total protein, separated by IEF using a pH 3–10 IPG strip in the first dimension and 15% SDS ⁄ PAGE in the second dimension The gel was first stained with the DIGE approach (see Experimental procedures), and subsequently checked by silver staining (A) Labeling with Cy5 fluorofor for the tentacle crown (B) Labeling with Cy2 fluorofor for the body (C) Body and tentacle crown alltogether (overlapped images) In the light box, the corresponding position of SmCP enzyme is shown when it was run in an individual 2D-PAGE (and visualized by immunostaining) The spots labeled with numbers correspond to molecular species affected by affinity capture on the immobilized inhibitors cystatin C (3, 4, 5, 6, 7 and 14) and soybean trypsin inhibitor (8, 9, 10, 11, 12 and 13), or on both (1 and 2). A novel metallocarboxypeptidase from S. magnifica M. Alonso-del-Rivero et al. 4878 FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS identify at least 14 proteins captured differentially for the first two microcolumns, which are labeled with numbers in Fig. 3B (1 and 2 by both; 3, 4, 5, 6, 7 and 14 by the cystatin one; and 8, 9, 10, 11, 12 and 13 by the SBTI one). An initial validation of these assign- ments as proteolytic enzymes (awating MS ⁄ MS analy- sis) was made by ‘intensity fading’ MALDI-TOF MS using the mentioned set of immobilized inhibitors, employing a strategy similar to the one for PCI described above. It is important to note that the band corresponding to the SmCP enzyme, the target of the present study, did not appear at around 34 kDa, which is the mass assigned to it as a potential MCP (see MALDI-TOF MS analysis and below), when the extracts (either from the body or body + crown) were analyzed. However, such a band is clearly visible when the enzyme is puri- fied, concentrated and subsequently applied to the 2D- PAGE (Fig. 3, encircled region). We assume that such a difference is a result of the very low abundance of SmCP in the animal. Also, it is relevant that the use of an antibody raised against the sequence around Asn144-Arg145, preserved in CPs [4], gave rise to a spot in the same location by immunostaining (not shown), confirming its assignment. Purification and partial molecular characterization of SmCP After detection of carboxypeptidase activity in the annelid worm (‘bodies’) of S. magnifica, SmCP was fractionated to homogeneity using affinity chromatog- raphy on a PCI-Sepharose column as the first step of purification. The enzymatic activity was detected in the eluted fraction with a 79% yield and a 286-fold purifi- cation with respect to the crude extract (Table 1). The second step of purification comprised anion exchange chromatography on a TSK-DEAE 5PW column (FPLC) (Tosoh Bioscience LLC, Montgomeryville, PA, USA) (Fig. 4A). SmCP eluted in a single fraction with a specific activity of 322 UÆmg )1 and 1150-fold purification (Table 1). The purified enzyme was submit- ted to metal analysis by inductive coupled plasma-MS, which indicated that it contains 0.96 atoms of Zn per molecule. A single band with a molecular mass of 34 kDa was detected by SDS ⁄ PAGE (Fig. 4B). This result agrees with the molecular mass of 33 792 Da that was obtained when it was analyzed by MALDI-TOF MS (Fig. 4C). In addition, Edman degradation analysis revealed a unique N-terminal sequence, confirming the homogene- ity of SmCP at this end of the molecule. Despite the rather limited size of the N-terminal region sequenced (19 residues: AFDLNDFNTLEDTYDQMNV), a blast search for this sequence revealed a consistent Table 1. Summary of a typical purification procedure for SmCP The assays were carried out as described in the Experimental procedures. Substrate AAFP at 0.1 m M, pH 7.5, 25 °C. Step Protein (mg) Enzymatic activity (U) Specific sctivity (UÆmg )1 ) Yield (%) Purification (n-fold) Extract 404 114 0.28 100 1 Affinity chromatography 1.12 90 80.3 79 286 Ion exchange chromatography 0.23 74 322 65 1150 14.2 12 28 34.1 51 90 120 203 I II III (mAU) 20.0 UV1/ 280 nm Conc CP activity 15.0 10.0 5.0 –5.0 0 20 40 60 80 100 0 100 200 300 Unit·m –1 400 500 mL 0.0 0 200 400 600 800 15 000 20 000 25 000 30 000 35 000 m/z 40 000 Intens. (a.u.) 16 928.956 33 792.855. A B C Fig. 4. Purification of SmCP from the body extract of S. magnifica and its molecular weight (A) Ion exchange chromatography on a TSK-DEAE gel (7.5 · 7.5 cm) column Buffer A: 20 m M Tris–HCl (pH 8.0); buffer B: 1 M Tris–HCl (pH 8.0) (I) Equilibration: 0% B for 45 min; (II) 60% B for 20 min; and (III) gradient 60% to 80% B for 170 min; flow rate: 68 cmÆh )1 –––, A 280 ; ,EnzAct; –––, Conc NaCl (B) SDS ⁄ PAGE gel (125%) of the purified enzyme Lane 1, Standard molecular weights [myosin (203 kDa), galactosidase (120 kDa), bovine serum albumin (90 kDa), ovoalbumin (51 kDa), carbonic anhydrase (34.1 kDa), soybean trypsin inhibitor (28 kDa) and lysosyme (14.2 kDa)] Lane 2: Fraction of S. magnifica purified by PCI-Sepharose and anionic exchange chromatography (C) MS spectrum (MALDI-TOF) of SmCP. M. Alonso-del-Rivero et al. A novel metallocarboxypeptidase from S. magnifica FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS 4879 homology with other MCPs, such as porcine and bovine carboxypeptidase A1 precursor, mosquito Aedes ae- gipty CPA, and the carboxypeptidase homolog from Bothrops jaraca, amongst others (Fig. 5). Subsequently, and as a result of SmCP trypsin digestion followed by LC-MS ⁄ MS analyses, we identified nine internal pep- tides (termed T1–T9), which showed identity to internal sequences of different CPs (Fig. 5). Some of them include important ‘canonical’ residues of the catalytic site of these enzymes [3]. Thus, in peptides T2 and T6, respectively, His69 and His196 (using canonical num- bering) were found, which are tetrahedrally coordinated to the catalytic zinc ion in all MCPs (i.e. the numbering system corresponds to bovine pancreatic CPA and is used throughout). The other three most important resi- dues found in the sequenced peptides are Glu270 (T9), Asn144 and Arg145 (T2). Glu270, in the S1 subsite, acts as a general base for catalysis, whereas Asn144 and Arg145, in the S1¢ subsite, bind the C-terminal carboxyl- ate group of the substrate. The peptide T6 appears to contain Tyr198, which usually belongs to the S2 CP sub- site. In addition, peptides T4 and T5 appear to contain two cysteine residues conserved in all members of MCP A ⁄ B subfamily, forming the disulfide bridge Cys138- Cys161 [1]. Any peptide assignable to the putative speci- ficity site [3] was found. Overall, these results indicate that SmCP represents a CP-like enzyme of the M14A subfamily [1,4]. Fig. 5. Alignment of the amino terminal and internal sequences of SmCP with the sequences of carboxypeptidases from other organisms SmCP sequences were derived after trypsin treatment of the purified enzyme followed by LC-MS ⁄ MS (de novo sequencing) and bioinfor- matics analyses (see Experimental procedures) Similar and identical residues are shown in light and dark grey, respectively ‘Canonical’ resi- dues of CP (based on bovine CPA1) that are present in the trypsin peptides of SmCP are labeled with an asterisk The sequences are CPA from Aedes aegypti (yellow fever mosquito) (Q9U9K2 AEDAE); Carboxypeptidase A1 precursor from Mus musculus (CBPA1 MOUSE); car- boxypeptidase A2 from Paralichthys olivaceus (Japanese flounder) (Q8QAXN5 PAROL); carboxypeptidase A1 precursor from Sus scrofa (CBPA1 PIG); carboxypeptidase A1 precursor from Bos taurus (CPBPA1 BOVIN); carboxypeptidase homolog from B. jaraca (Q9PUF2 BOT- JA); CPO from Homo sapiens (CBPO HUMAN); CPB from Astacus fluviatilis (broad-fingered crayfish) (CBPB ASTFL); CPA precursor from H. armigera (cotton bollworm) (097434_HELAM); carboxypeptidase precursor from H. armigera (cotton bollworm) (Q6H962_HELAM); MCP from Culicoides sonorensis (Q5QBL3_9DIPT); and carboxypeptidase A2 precursor from H. sapiens (CBPA2_HUMAN). A novel metallocarboxypeptidase from S. magnifica M. Alonso-del-Rivero et al. 4880 FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS Kinetic characterization of SmCP Kinetic analyses for isolated SmCP was performed using different types of standard synthetic substrates for carb- oxypeptidases that were clearly cleaved by the enzyme. The K m , k cat and k cat ⁄ K m determined for the enzyme against AAFP, N -benzoyl-Gly-Phe (Hippuryl-Phe) and N-(3-[2-furyl]acryloyl)-Phe-Phe (FAPP) as substrates are shown in Table 2. Such kinetic parameters indicate that SmCP is highly efficient against the three CPA type substrates used. On the other hand, we found that SmCP is unable to cleave CPB type substrates such as [3-(2-furyl)acryloyl]-L-alanyl-l-lysine (FAAK) or N-(4- methoxyphenylazoformyl)-l-Arg (AAFR). Therefore, SmCP appears to be more related to the A-type than to the B-type MCPs [1–4]. The influence of pH on SmCP activity was also ana- lyzed using the AAFP substrate, and indicated an opti- mum pH value in the range 7.0–7.5. The effect of various protease inhibitors on the SmCP enzymatic activity is shown in Table 3. Inhibitors of cysteine proteases (l-carboxy-trans-2,3-epoxypropyl-leycylami- do (4-guanidino) butane, E-64; cystatin), aspartic pro- teases (pepstatin) and serine proteases (Pefabloc, soybean trypsin–chymotrypsin inhibitor, soybean tryp- sin inhibitor, aprotinin) did not have noticeable effects on SmCP activity. The enzyme was drastically inhib- ited by the chelating agent 1,10-phenanthroline at 1mm. However, EDTA at 10 mm, which might act by metal chelation, did not produce any inhibition at sim- ilar concentrations and inhibitor ⁄ enzyme (I o ⁄ E o ) rela- tionships (3 · 10 5 m). Nevertheless, EDTA partial inhibitory effects were observed when preincubation times were increased. By contrast, benzylsuccinic acid, a well-known organic inhibitor of A-type carboxypep- tidases, fully cancelled the enzyme activity, at 1 mm. Furthermore, the addition of the protein inhibitor of carboxypeptidases PCI (in fact rPCI, a recombinant form, reactive towards CPA and CPB type enzyme) at 0.4 lm produced a 70% inhibition of SmCP activity. The apparent K i value for this inhibitor towards SmCP was 7.37 · 10 )8 m; however, the adjusted value considering the substrate-induced dissociation was 2.45 · 10 )8 m. Another protein inhibitor from leech (rLCI, also recombinant) at 13.5 lm produced a 70% inhibition of SmCP activity. The estimated K i value for rLCI was 2.95 · 10 )8 m, and its adjusted value considering the substrate induced dissociation was 1.45 · 10 )8 m (Table 4). Preincubation of the inhibi- tors with the enzymes for various periods of time did not affect its inhibitory activity, suggesting that rLCI and rPCI are fast tight binding inhibitors. Table 2. Kinetic parameters for substrate hydrolysis catalyzed by SmCP in comparison with data reported for bovine pancreatic CPA (bCPA) The assays were carried out under the same conditions as those described for AAFP Substrate concentrations in the range 0.11–1.2 m M (3.29 nM of the enzyme in assay), 0.1–2 mM (24 lM of the enzyme in assay) and 0.02–0.25 nM (3.29 nM of the enzyme in assay) were used for AAFP, Hippuryl-Phe and FAPP, respectively. Enzyme AAFP Hippuryl-Phe FAPP K m (mM) k cat s )1 k cat ⁄ K m M )1 Æs )1 K m (mM) k cat s )1 k cat ⁄ K m M )1 Æs )1 K m (mM) k cat s )1 k cat ⁄ K m M )1 Æs )1 SmCP 0.05 ± 0.01 42.5 79 · 10 5 0.36 ± 0.03 145 3.8 · 10 5 0.14 ± 0.01 15 1.7 · 10 5 bCPA 0.11 ± 0.01 a 44.0 41 · 10 5 0.88 ± 0.05 b 60 6.8 · 10 4 0.05 ± 0.01 b 340 6.8 · 10 6 a Mock et al [23]. b Cho et al [24] Table 3. Effect of protease inhibitors on the relative activity of SmCP SmCP: 3.29 n M; AAFP: 0.1 mM; pH 7.5, 25 °C The enzyme was preincubated with the inhibitors for 10 min at 25 °C. Inhibitor Concentration % Enzymatic activity I o ⁄ E o E-64 0.1 mM 100 3.0 · 10 4 M Pefabloc 10 mM 100 3.03 · 10 6 M Pepstatin A 50 lM 94 1.51 · 10 4 M Trypsin-chymotrysin inhibitor (soybean) 3mM 100 9.1 · 10 5 M 1,10-Phenanthroline 1 mM 21 3.03 · 10 5 M Benzylsuccinic acid 1 mM < 1 3.03 · 10 5 M EDTA 10 mM 117 3.03 · 10 5 M PCI 0.4 lM 28.5 1.21 · 10 2 M LCI 13.5 lM 30 4.1 · 10 2 M Aprotinin 3 mM 100 9.1 · 10 5 M Trypsin inhibitor (soybean) 2mM 100 6.0 · 10 5 M Table 4. K i values of rPCI and rLCI against SmCP compared to pre- vious data obtained for bovine pancreatic CPA (bCPA) SmCP: 3.29 n M; AAFP: 0.1 mM; pH 7.5, 25 °C The enzyme was preincu- bated with the inhibitors for 10 min at 25 °C. Carboxypeptidase K i (nM) rPCI rLCI SmCP 24.5 ± 03 14.5 ± 05 bCPA 1.5 ± 02 a 1.6 ± 01 b a Ryan et al [25]. b Reverter et al [27]. M. Alonso-del-Rivero et al. A novel metallocarboxypeptidase from S. magnifica FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS 4881 On the other hand, we evaluated the effect on SmCP of metal ions after overnight dialysis against EDTA at 10 mm (followed by the removal of excess EDTA by dialysis against metal-free buffers; see Experimental procedures). After this, SmCP only retains 40% of its initial activity. This apoform subsequently was used as a control for the studies with metals. We observed that 1mm Ca 2+ ,Mn 2+ or Mg 2+ enhanced the enzyme activity of apoSmCP above 100% of the control activ- ity, whereas the addition of Cd 2+ at 1 mm or Co 2+ at 1mm or 10 mm did not affect the enzymatic activity of the control (Fig. 6). However, Cu at 1 mm and 10 mm reduced the apoenzyme activity to 11% and 15% of its residual activity. Noteworthy, under our conditions, the addition of Zn 2+ at 1 mm or 10 mm brought the activity to 100% (full rescue) and to 70%, respectively, with the latter assignable to inhibition by this metal. Specificity of cleavage Two different long peptides were used as substrate models to analyze the ability of SmCP to cleave differ- ent kinds of residues at the C-terminus, in comparison Fig. 6. Effect of divalent metals on SmCP activity The concentrations used in the assays were 329 n M for the enzyme SmCP and 0.1 m M for the substrate AAFP, at pH 7.5 and 25 °C The enzyme, after EDTA treatment and dialysis against metal-free buffer (see Experimental procedures), was preincubated with the different ion metal salts at 1 m M, for 10 min at 25 °C The assays were also performed, under the same conditions, at 10 m M for Zn 2+ ,Co 2+ and Cu 2+ . SmCP vs ACTH A B SmCP vs V15E E F E A W E A W E E F E F F F F 2188 2317 2466 1427 1529 1541 1563 1587 1619 1693 1716 1748 ACTH control 60 min bCPA vs ACTH 2466 2317 ACTH control 60 min bCPA vs V15E 1793 1716 1748 V15E control 60 min V15E control 60 min SmCP + PCI 60 min 15 min 30 min 60 min bCPA + PCI 60 min 15 min 30 min 60 min Fig. 7. Determination of SmP specificity for C-terminal substrate residues. Comparative analysis by MALDI-TOF MS of the degrada- tion of two synthetic substrates by SmCP and bovine pancreatic CPA (bCPA). The assays were performed in 10 m M Tris–HCl buffer (pH 8.0) with 1 l M of peptides and 2.19 n M of SmCP or 1 nM of bCPA in 10 lL of final volume for 60 min. (A) represents the enzymatic activity of SmCP against the ACTH fragment and V15E peptide, whereas (B) represents the enzymatic activity of bCPA against the same substrate. Sequence of the ACTH fragment (residues 18–39): RPVKVYPNGAEDESAEAFPLEF, MW: 2466 Da; ACTHdes-F, MW: 2317 Da; ACTHdes-EF, MW: 2188 Da; V15E peptide sequence, VKKKARKAAGC(Amc)AWE: MW 1716 Da; V15Edes-E, MW: 1587 Da; V15Edes-WE, MW: 1400 Da; V15Edes-AWE, MW: 1329 Da. A novel metallocarboxypeptidase from S. magnifica M. Alonso-del-Rivero et al. 4882 FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS with bovine pancreatic CPA (a reference enzyme in the field). After 15 min of incubation of SmCP with the adrenocorticotropic hormone (ACTH) fragment used as substrate (residues 18–39, 2466 Da), the enzyme was able to release phenylalanine (ACTHdes-F, 2317 Da) and glutamic acid (ACTHdes-EF, 2188 Da) residues from the substrate C-terminus (Fig. 7A). No further amino acids were released after a 30-min incubation period. Under the same conditions, bovine pancreatic CPA was only able to hydrolyze the C-terminal phen- ylalanine residue from ACTH to obtain the ACTHdes- F (2317 Da). The addition of the protein inhibitor rPCI prevented cleavage in all cases. To confirm the capability of SmCP to hydrolyze acidic residues from the C-terminus of peptides, the specificity of SmCP against synthetic substrate [VKKKARKAAGC(Amc)AWE] (V15E peptide) (resi- due 15, 1716 Da) was evaluated (Fig. 7B). After 15 min of incubation, the release of glutamic acid from the peptide was observed and, after 60 min, the new C-terminus residues formed and tryptophan and ala- nine were further released, as shown by the trimming scale: 1716, 1587 and 1329 Da. However, bovine pan- creatic CPA was unable to hydrolyze the first of such C-terminal residues, glutamic acid, even after 60 min of incubation. Again, the addition of rPCI prevented any kind of hydrolysis by the enzyme. The release of a glutamic acid residue from the C-terminus of peptides is a very unusual capability of a CPA-like enzyme and is reminiscent of the so-called CPO forms [3,5]. Discussion The growing application of genomics and related tech- nologies is facilitating an expanding view of the pres- ent enzymatic families, including proteases [20] and CPs in particular [4]. However, such an advance is lim- ited in the invertebrate world because of the great diversity of organisms within it, which complicates the study, but has the potential to generate enzyme vari- ants of great biological and biotechnological values. To gain insight into the field of MCPs, one of the most unknown among proteases in invertebrates, we have used a mix of both modern and more classical approaches to identify and characterize them, estab- lishing comparisons with the vertebrate species (i.e. the reference ones). The present study started with a sys- tematic screening in extracts from 25 invertebrates from marine Caribbean species, using a specific and sensitive enzymatic assay; this allowed us to detect the presence of CPA-like activity in the body extract of the marine annelid S. magnifica. Given that we previ- ously reported the successful use of MALDI-TOF MS for the initial detection of CPs and carboxypeptidase inhibitors in other crude biological extracts [17–19], we have applied such approaches to the S. magnifica case. The use of affinity capture on microbeads or microcol- umns derivatized with a recombinant carboxypeptidase inhibitor from potatoes, specific for such class of enzymes, and the use of MALDI-TOF MS signal anal- ysis approaches, allowed us to quickly identify in this annelid a 35-kDa species as a potential MCP, which we named SmCP. Different fractionation methods have been per- formed to purify SmCP from the body extract of S. magnifica. In initial attempts, using anion exchange and gel filtration chromatographies, we found a frac- tion with clear carboxypeptidase activity, which, inter- estingly, conveyed two additional activities against typical substrates for trypsin-like (benzoyl arginyl ethyl ester; BAEE) and chymotrypsin-like (benzoyl tyrosine ethyl ester; BTEE) serine proteases (data not shown). This suggests that, in the fractionation, SmCP could co-elute with serine proteases, perhaps establishing bin- ary or ternary complexes with such enzymes, as shown in other organisms [21,22]. Nevertheless, the substitu- tive use of affinity chromatography on rPCI-agarose, in subsequent experiments, allowed the selective cap- ture of SmCP and contributed to its separation from the other enzymes. Potentially, rPCI could promote the dissociation of SmCP from ‘complexes with serine proteases’ that it might establish in the crude extracts. This is an issue that merits further research. The 2D-PAGE analysis of the crude extracts indi- cates that they are very complex in protein species, and that a stainable band at around 35 kDa, attribut- able to SmCP, is not directly visible with such approach unless high sensitivity approaches (i.e immu- nostaining) are employed. This is probably a result of the low representation of this enzyme in the animal extracts, in agreement with its subsequent analysis and visualization in the purified form. Additionally, we obtained evidence by affinity cap- ture on three different kinds of immobilized proteina- ceous inhibitors (soybean trypsin inhibitor, cystatin, pepstatin), indicating that different main 2D-PAGE protein bands around 20–55 kDa correspond to cyste- ine and serine protease enzymes present in the S. mag- nifica body extract. At least 14 species that gave stainable and clearly visible bands were detected by this approach. They were provisionally validated by ‘intensity fading’ MALDI-TOF MS perturbation stud- ies carried out by the addition of such protein inhibi- tors on the extracts. Full validation would require either direct isolation or MS ⁄ MS analyses. The later type of study is under way in our laboratory, but is M. Alonso-del-Rivero et al. A novel metallocarboxypeptidase from S. magnifica FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS 4883 proving more difficult than expected because of the very low homologies shown by S. magnifica proteases with respect to equivalent ones found in databases. Given the poor representation of invertebrate proteases in databases, this is not an unexpected problem when carrying out identification proteomics. It is worth noting that the preliminary detection of serine and cysteine proteases species in the body extracts correlates with the measure of their activities by enzymatic analysis of the crude samples. Interest- ingly, neither approach revealed evidence of the occu- rence of aspartic proteases. Overall, although the presence of pigments and other interfering products initially constituted a very serious problem, once this was technically solved, the feasibility and data genera- tion capability of both the 2D-PAGE and ‘intensity fading’ MALDI-TOF MS of this annelid indicated that such proteomic-like approaches (and probably related ones) are very promising for the analysis of proteolytic enzymes in marine invertebrates. A central question in the analysis of novel MCPs from biological sources is whether they occur in their precursor or mature forms [2–5]. In the present study, using direct extracts from S. magnifica, we found only a monomeric and activated form of SmCP, as shown by its enzymatic activity, molecular mass, derived N-terminal sequence and homology analysis. Procarb- oxypeptidases are usually activated by proteolytic removal of their activation segment by serine prote- ases, mostly trypsin. Studies on procarboxypeptidases from several species have indicated that its activation is dependent of the environmental ionic conditions and, sometimes, the influence of quaternary structure [2,5]. Under our experimental conditions, quick activa- tion of SmCP by autologous serine-like proteases, which appeared to be present in large quantities in the extract, could be favored. On the other hand, the coincidence between the N-terminal sequences of SmCP and those from several other MCPs included in alignments (Fig. 5) also suggests that SmCP has been purified in the active mature form. In addition, we found that the sequences of a number of SmCP inter- nal peptides included important residues that belong to catalytic site and domain of this enzyme family, confirming our interpretation. All the experimental data reported in the present study indicate that SmCP belongs to the M14A sub- family of metalloproteases [6], the so-called pancreatic- like forms (or A ⁄ B), favoring its potential digestive function in the marine annelid. Its molecular weight (33.7 kDa), N-terminal sequence and behavior towards a panel of substrates and inhibitors are similar to those of mammalian pancreatic CP (i.e. the best known). These types of enzymes have molecular masses close to 35 kDa after the removal of the propeptide, whereas the regulatory CPs (or N ⁄ E) display higher mass val- ues as a result of the presence of other domains in addition to the CP domain [2,3]. On the other hand, SmCP shows sequence homology with some CPs iso- lated from different vertebrates and invertebrates, belonging to the A ⁄ B subfamily with CPA substrate preferences. Only a few CPs have been isolated from marine invertebrates, and in not one case have the whole or extended sequences been disclosed. This would be the case for the two CPAs and CPBs isolated from the hepatopancreas of the crab P. camtschatica [9] and the CPA-like enzyme from the squid hepato- pancreas of I. illecebrosus [10]. SmCP is able to cleave different types of CPA sub- strates such as AAFP, Hippuryl-Phe and FAPP, with an overall efficiency similar to bovine pancreatic CPA, but with some significant differences in k cat , K m and k cat ⁄ K m for certain substrates [23,24]. In addition, SmCP has a maximum activity at pH 7.5, in agreement with the optimum pH activity of almost all M14A CP- like forms, including marine enzymes [7–13], which lie in the neutral range (pH 6.5–8.5), and is consistent with the pH at their sites of biological action [1,2]. As previously shown for mammalian CPs [25–27], potato and leech proteinaceous inhibitors efficiently inhibit SmCP, displaying similar K i values. In addition, two smaller organic molecules (benzylsuccinic acid and 1,10-phenantroline) known to act on MCPs are also able to inhibit the enzyme. By contrast, EDTA, which chelates metal ions, at 10 mm, failed to inhibit SmCP activity significantly after 10 min of preincubation, which is in agreement with the reported properties of other invertebrate MCPs isolated from the gut of Tion- ela bisselliella [28] and from Helicoverpa armigera larvae [29] for which EDTA effects are also time dependent. The capability of divalent metal ions to substitute the essential active site Zn 2+ of MCPs [30,31], or bind a second atom nearby [32], interfering with the cata- lytic mechanism, is well known. We also observed diverse effects by the addition of such metals to SmCP. After its dialysis against EDTA at 10 mm, SmCP reduced its activity to 40% of initial activity. Starting from this state, the capacity of different metal ions to regenerate SmCP activity demonstrated that, in certain cases [Mn, Mg and Ca], there is an enhancement of activity of the enzyme; in others [Cd and Co], no changes are observed; and, in a third case [Cu], a clear inhibition is produced. Such results are quite congru- ent with the well-known properties of mammalian CPs [33]. In the case of Zn, an enhancement of SmCP activity was observed when added at 1 mm, whereas, A novel metallocarboxypeptidase from S. magnifica M. Alonso-del-Rivero et al. 4884 FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... by Bachem (Weil am Rhein, Germany) Preparation of extracts The marine organisms belonging to the kingdom Methazoa (Phyla: Annelida, Urochordata, Echinodermata, Cnidaria, Mollusca, Artropoda) were collected in the north coast of Havana and classified by Cuban specialists at the National Institute of Oceanology (Havana, Cuba) The organisms were homogenized in their own sea water liquid (1 : 2, w ⁄ v) The. .. magnifica annelid) Before an ample characterization of other proteolytic enzymes present in this invertebrate is achieved (several other proteases, such as serine proteases, appear to be there by 2D-PAGE and MS analyses; not shown), such requirements can only be a matter of guesswork We are still far from a consistent characterization of the ‘degradomes’ of invertebrates (i.e the genomically and proteomically... the hepatopancreas of the crab Paralithodes camtschatica Mar Biotechnol (NY) 2, 25 9–2 66 10 Raksakulthai R & Haard NF (2001) Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus) J Agric Food Chem 49, 501 9–5 030 11 Kishimura H & Hayashi K (2002) Isolation and characterization of carboxypeptidase B from the pyloric ceca FEBS Journal 276 (2009) 487 5–4 890 ª... Referencia en Biotecnologia (XeRBa, Generalitat de 4888 Catalunya) M .A. C acknowledges a Visitor Grant from AGAUR (Generalitat de Catalunya) Professor Magnus Abrahamson and colleagues (Lund, Sweden) are acknowledged for kindly providing immobilized cystatin The authors are grateful for technical support provided by Dagmara Diaz and Rachel Lopez, as well as ´ from ProteoRed-Instituto Nacional de Proteomica, and... v) The homogenates were centrifuged at 10 000 g for 30 min at 4 °C In the case of the marine invertebrate S magnifica, belonging to the Phylum Annelida, the animals were separated into two parts, tentacle crowns and bodies, which were homogeneized as described above Carboxypeptidase assays The general assay for CPA-like activity was carried out using AAFP as substrate [23] It was prepared at 10 mm in... Characteristics of carboxypeptidase B from pyloric ceca of the starfish Asterina pectinifera Food Chem, 95, 26 4–2 69 Brusca RC & Brusca GJ (2003) Invertebrates 2 edn Sinauer Associates Inc., Sunderland, Massashusetts Knight-Jones P & Mackie ASY (2003) A revision of Sabellastarte (Polychaeta: Sabellidae) J Nat Hist 37, no 19, 226 9–2 301 Peaucellier G (1983) Purification and characterization of proteases from. .. kinetic characterization Kinetic parameters The Km and Vmax values for the purified enzyme were evaluated using different CPA substrates such as AAFP [23], Hippuryl-Phe [39] and FAPP [40] in accordance with the experimental conditions described above for the CP assays Kinetic parameters were graphically calculated by adjusting the experimental data to the rectangular hyperbola curve, using origin software... acquired in the linear positive ion mode, using 25 kV acceleration voltage The analysis of proteins or peptide fragments A novel metallocarboxypeptidase from S magnifica was carried out using 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) and a- cyano-4-hydroxicinnamic acid as matrices Samples were prepared by mixing them with equal volumes of a saturated solution of the matrices From this mixture,...M Alonso-del-Rivero et al at 10 mm, little recovery of the initial activity occurred The sense and intensity of the changes in the enzymatic parameters show different degrees of fitting with what has been described for other invertebrate CPs, such as the sea hare A californica [8], the squid I illecebrosus [10] and the larvae Helicoverpa armiguera [29], as well as for other mammalian CPs [2,34]... 8.5 and 9.0) All other experimental conditions were as described for the CP assay using AAFP as substrate [23] Effect of inhibitors and metal cations Inhibition studies of SmCP by proteinaceous inhibitors was evaluated against pepstatin A, rPCI, rLCI, aprotinin, FEBS Journal 276 (2009) 487 5–4 890 ª 2009 The Authors Journal compilation ª 2009 FEBS 4887 A novel metallocarboxypeptidase from S magnifica M Alonso-del-Rivero . A novel metallocarboxypeptidase-like enzyme from the marine annelid Sabellastarte magnifica – a step into the invertebrate world of proteases Maday Alonso-del-Rivero 1 ,. the Phyla Cnidaria, Annelida, Mollusca, Echi- nodermata, Arthropoda and Chordata, amongst others, collected on the coasts of Havana, Cuba. The study has