Báo cáo khoa học: A novel metallocarboxypeptidase-like enzyme from the marine annelid Sabellastarte magnifica – a step into the invertebrate world of proteases pdf

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A novel metallocarboxypeptidase-like enzyme from themarine annelid Sabellastarte magnifica a step into theinvertebrate world of proteasesMaday Alonso-del-Rivero1, Sebastian A. Trejo3,Mo´nica Rodrı´guez de la Vega3, Yamile Gonza´lez1,Silvia Bronsoms3, Francesc Canals2, Julieta Delfı´n1, Joaquin Diaz1, Francesc X. Aviles3and Marı´aA. Cha´vez11 Centro de Estudio de Proteı´nas, Facultad de Biologı´a, Universidad de la Habana, Cuba2 Institut de Recerca Hospital Vall d’Hebron, Barcelona, Spain3 Institut de Biotecnologı´a i Biomedicina and Departament de Bioquı´mica i Biologı´a Molecular, Universitat Autonoma de Barcelona, SpainIntroductionNatural evolution has frequently generated a largeadaptative variety of forms among protein functionalfamilies, and metallocarboxypeptidases (MCPs) havealso followed this trend. Such enzymes are exopeptid-Keywordsenzyme specificity; marine annelid;metallocarboxypeptidases; metalloproteins;Sabellastarte magnificaCorrespondenceF. X. Aviles, Institut de Biotecnologı´aiBiomedicina (IBB) and Departament deBioquı´mica i Biologia Molecular, UniversitatAutonoma de Barcelona, 08193 Bellaterra(Barcelona), SpainFax: +34 93 581 2011Tel: +34 93 581 1231E-mail: francescxavier.aviles@uab.es(Received 16 March 2009, revised 16 June2009, accepted 30 June 2009)doi:10.1111/j.1742-4658.2009.07187.xAfter screening 25 marine invertebrates, a novel metallocarboxypeptidase(SmCP) has been identified by activity and MS analytical approaches, andisolated from the marine annelid Sabellastarte magnifica. The enzyme,which is a minor component of the molecularly complex animal body, asshown by 2D gel electrophoresis, has been purified from crude extracts tohomogeneity by affinity chromatography on potato carboxypeptidase inhib-itor and by ion exchange chromatography. SmCP is a protease of33792 Da, displaying N-terminal and internal sequence homologies withM14 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 Ca2+and is drastically inhibited by the metal chelator1,10-phenanthroline, as well as by excess Zn2+or Cu2+, but moderately soby EDTA. SmCP is also strongly inhibited by specific inhibitors of metallo-carboxypeptidases, such as benzylsuccinic acid and the protein inhibitorsfound in potato and leech (i.e. recombinant forms, both at nanomolarlevels). The enzyme displays high peptidase efficiency towards pancreaticcarboxypeptidase-A synthetic substrates, such as those with hydrophobicresidues 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 inthe present study indicate that SmCP is a novel member of the M14 metal-locarboxypeptidases family (assignable to the M14A or pancreatic-likesubfamily) with a wider specificity that has not been described previously.AbbreviationsAAFP, 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 4875ases that catalyze the hydrolysis of peptide bonds atthe C-terminus of peptides and proteins. They belongto the catalytic classes of either metalloproteases (clanMC, family M14) or serine proteases (clan SC, familyS10) [1] and their action causes strong effects in thebiological activity of their peptide and protein sub-strates [2]. M14 MCPs, including those from animals,plants and bacteria, have been divided into three mainsubfamilies based on structural similarity and sequencehomology. The first one, which includes the digestiveenzymes carboxypeptidase (CP) A (CPA) 1, CPA2,carboxypeptidase B (CPB) 1 and mast cell CPA3, aswell 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 thebioactive 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]. Veryrecently, a novel subfamily composed of enzymes oflarger size and apparently with a predominant cyto-solic location, termed M14D, Nna-like or CCPs, hasbeen proposed [4]. Furthermore, three main classesmay 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 differentsources [3,5,6], mainly from vertebrates, but a few ofthem have come from marine invertebrate organisms:the digestive crayfish carboxypeptidase (CPB) [7], thecarboxypeptidase E-like enzyme from the sea hareAplysia 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 andone 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 areinvertebrates [14]. The ecological services provided byinvertebrates are immeasurable; life as we know itwould be quite different or decline without them (seeCenter for Applied Biodiversity Science; http://sci-ence.conservation.org). Overall, our knowledge aboutMCPs in invertebrates is very limited given the tremen-dous variety of such organisms and compared to themuch larger number of characterized CP from verte-brates [6]. In the present study, we screened for thepresence of CP activity in marine invertebrates belong-ing to the Phyla Cnidaria, Annelida, Mollusca, Echi-nodermata, Arthropoda and Chordata, amongstothers, collected on the coasts of Havana, Cuba. Thestudy has been based on the use of N-(4-meth-oxyphenylazoformyl)-l-phenylalanine (AAFP), a sensi-tive, specific and known colorimetric substrate forCPA enzymes. One of the highest activity levels wasdetected in extracts from the marine annelid S. magni-fica. This marine invertebrate, also termed ‘magnificentfeather duster’, was obtained from coral reefs. Itbelongs to the Phylum Annelida, Class Polychaeta,which shows a clear delimitation between its tentaclecrown and its body (Fig. 1) [15]. Some studies per-formed on another annelid, belonging to the Sabellidaefamily, have only detected proteolytic activity assign-able to serine proteases, which appeared to be involvedin reproduction [16] despite their digestive origin.The presence of a carboxypeptidase-like enzyme inAnnelida marine invertebrates has not been describedso far.The present study describes the enzymatic activityand 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 thencharacterized by size, metal content, location, basicinteractions, sequence analysis of different regions ofthe 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 M14MCP (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 theM14 family of proteolytic enzymes, which is now quitediverse and contains more than 25 different variantsFig. 1. S. magnifica Phylum Annelida, Class Polychaeta, SubclassPalpata, Order Canalipalpata, Suborder Sabellida, Family Sabellidae,Genus Sabellastarte [14] The ‘tentacle crown’ and the ‘body’ partsof 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 frominvertebrates have been characterized until now.ResultsDetection of MCP activities in marine organismsTwenty-five marine species belonging to differentinvertebrate Phyla were screened for CPA activityusing 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 occidentalisand Pyura vittata); 11 species of Cnidaria (Bartholo-mea annulata, Budonosoma granulifera, Cassiopeaxamachana, Condylactys gigantea, Gorgonia ventalina,Lebrunia danae, Palythoa caribaeorum, Physalia phy-salis, Plexaura homomalla, Stichodactyla helianthus andZoanthus pulchellus); two species of Annelida (Sabellas-tarte magnifica and Hermodice carunculata); two speciesof 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 positiveresults, with specific activity values of 56.0, 1.6 and1.8 UÆ100 mg)1extract, respectively. In these threecases, we found a linear relationship between CP-likeactivity and the quantity of extract used in the assay.Given that the material of the annelid S. magnificashowed by far the highest specific activity, it wasselected for further characterization studies. In thiscase, 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 MSOnce we focused our attention on S. magnifica bodyextracts, we found there direct evidence of at least oneMCP enzyme, of approximately 35 kDa by ‘intensityfading’ MALDI-TOF MS [17]. In the present study,the added ‘binder’ was the recombinant form of potatocarboxypeptidase 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 thebody extract. The control spectra, as well as the ‘per-turbed’ one (by rPCI addition, followed by removal ofthe captured targets by sedimentation of the beads),are shown in Fig. 2A,B. It is apparent that some ofthe ion signals of the spectra were faded when theextract was treated with immobilized PCI. Subse-quently, MS analysis of the protein eluted from thebeads (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-likeenzyme activity found in S. magnifica body extract.The experiment indicates not only the occurrence inthe extract of the strong ligand (the enzyme SmCP) forthe added protease inhibitor, but also that this ligandis probably functional in the very complex extract (i.e.not in the zymogen state). It is worth noting that theapparent simplicity of the MALDI-TOF spectrum ofthe extract shown in Fig. 2C is most likely caused notonly by the low expansion scale used, but also by10001500Control MS (body extract)AIntens. (a.u.)Intens. (a.u.)Intens. (a.u.)05002000+PCI 050010001500100150Elution05010 000 15 000 20 000 25 000 30 000 35 000m/zBCFig. 2. MALDI-TOF MS of the ‘intensityfading’ experiment (A) Mass spectra of theS. magnifica body extract (control sample)before rPCI-agarose addition (B) Unboundproteins 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 arrowindicates the ‘perturbed’ signal by rPCI-aga-rose addition.M. Alonso-del-Rivero et al. A novel metallocarboxypeptidase from S. magnificaFEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS 4877‘signal suppression effects’; such phenomena usuallyaffect 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 bodyextract by 2D-PAGEThe 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). Agreat number of visible protein bands [as revealedeither by staining with silver or using difference gelelectrophoresis (DIGE)] appeared in the analysis ofboth parts of the animal, with a major presence ofbands in the body (upper part) versus the crown (lowerpart). 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 labeledwith fluorescent dyes using the DIGE approach. Thatis, the different materials (i.e. crown and body extracts,purified enzyme) were pre-labeled independently withDIGE 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 wasperformed not only to allow the differential trackingof their components, but also to deal with the veryhigh content of dyes and interfering materials from thecrown, which required a harsh cleaning (and denatur-ing) procedure. Such interfering materials strongly per-turbed the electrophoretic separation, and also gaverise to severe band strikes and decreased resolution.Only after testing several pre-cleaning and stainingprocedures (not shown), and selecting an adequateone, were we able to unveil the real band complexityof the extracts (see Experimental procedures). We hopethat this experience might be useful for the analysis ofother invertebrates with a high content in dyes andother similar problems.Overall, more than 200 protein species are detectedby this procedure, among which those in the17–37 kDa range are the most prominent. To facilitateidentification, we repeated the 2D-PAGE with threedifferent initial samples from the body, after passingthem 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 gelfor each case (see Experimental procedures). The anal-ysis of the ‘captured’ spots allowed us to potentiallyABCFig. 3. 2D gel electrophoresis of pre-labeled protein extracts from S. magnifica The gel contained 30 lg of total protein, separated by IEFusing 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 DIGEapproach (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 correspondingposition of SmCP enzyme is shown when it was run in an individual 2D-PAGE (and visualized by immunostaining) The spots labeled withnumbers correspond to molecular species affected by affinity capture on the immobilized inhibitors cystatin C (3, 4, 5, 6, 7 and 14) andsoybean 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 FEBSidentify at least 14 proteins captured differentially forthe first two microcolumns, which are labeled withnumbers in Fig. 3B (1 and 2 by both; 3, 4, 5, 6, 7 and14 by the cystatin one; and 8, 9, 10, 11, 12 and 13 bythe SBTI one). An initial validation of these assign-ments as proteolytic enzymes (awating MS ⁄ MS analy-sis) was made by ‘intensity fading’ MALDI-TOF MSusing the mentioned set of immobilized inhibitors,employing a strategy similar to the one for PCIdescribed above.It is important to note that the band correspondingto the SmCP enzyme, the target of the present study,did not appear at around 34 kDa, which is the massassigned to it as a potential MCP (see MALDI-TOFMS analysis and below), when the extracts (either fromthe 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 sucha difference is a result of the very low abundance ofSmCP in the animal. Also, it is relevant that the use ofan antibody raised against the sequence aroundAsn144-Arg145, preserved in CPs [4], gave rise to aspot in the same location by immunostaining (notshown), confirming its assignment.Purification and partial molecular characterizationof SmCPAfter detection of carboxypeptidase activity in theannelid worm (‘bodies’) of S. magnifica, SmCP wasfractionated to homogeneity using affinity chromatog-raphy on a PCI-Sepharose column as the first step ofpurification. The enzymatic activity was detected in theeluted fraction with a 79% yield and a 286-fold purifi-cation with respect to the crude extract (Table 1). Thesecond step of purification comprised anion exchangechromatography on a TSK-DEAE 5PW column(FPLC) (Tosoh Bioscience LLC, Montgomeryville,PA, USA) (Fig. 4A). SmCP eluted in a single fractionwith a specific activity of 322 UÆmg)1and 1150-foldpurification (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 permolecule.A single band with a molecular mass of 34 kDa wasdetected by SDS ⁄ PAGE (Fig. 4B). This result agreeswith the molecular mass of 33 792 Da that was obtainedwhen it was analyzed by MALDI-TOF MS (Fig. 4C).In addition, Edman degradation analysis revealed aunique N-terminal sequence, confirming the homogene-ity of SmCP at this end of the molecule. Despite therather limited size of the N-terminal region sequenced(19 residues: AFDLNDFNTLEDTYDQMNV), ablast search for this sequence revealed a consistentTable 1. Summary of a typical purification procedure for SmCPThe assays were carried out as described in the Experimentalprocedures. Substrate AAFP at 0.1 mM, pH 7.5, 25 °C.StepProtein(mg)Enzymaticactivity(U)Specificsctivity(UÆmg)1)Yield(%)Purification(n-fold)Extract 404 114 0.28 100 1Affinitychromatography1.12 90 80.3 79 286Ion exchangechromatography0.23 74 322 65 115014.2122834.15190120203IIIIII(mAU)20.0UV1/280 nmConcCP activity15.010.05.0–5.00 20 40 60 80 1000100200300Unit·m–1400500mL0.0020040060080015 000 20 000 25 000 30 000 35 000m/z40 000Intens. (a.u.)16 928.95633 792.855.ABCFig. 4. Purification of SmCP from the body extract of S. magnificaand its molecular weight (A) Ion exchange chromatography on aTSK-DEAE gel (7.5 · 7.5 cm) column Buffer A: 20 mM Tris–HCl (pH8.0); buffer B: 1M Tris–HCl (pH 8.0) (I) Equilibration: 0% B for45 min; (II) 60% B for 20 min; and (III) gradient 60% to 80% B for170 min; flow rate: 68 cmÆh)1–––, A280; ,EnzAct; –––, ConcNaCl (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 purifiedby PCI-Sepharose and anionic exchange chromatography (C) MSspectrum (MALDI-TOF) of SmCP.M. Alonso-del-Rivero et al. A novel metallocarboxypeptidase from S. magnificaFEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS 4879homology with other MCPs, such as porcine and bovinecarboxypeptidase A1 precursor, mosquito Aedes ae-gipty CPA, and the carboxypeptidase homolog fromBothrops jaraca, amongst others (Fig. 5). Subsequently,and as a result of SmCP trypsin digestion followed byLC-MS ⁄ MS analyses, we identified nine internal pep-tides (termed T1–T9), which showed identity to internalsequences of different CPs (Fig. 5). Some of theminclude important ‘canonical’ residues of the catalyticsite of these enzymes [3]. Thus, in peptides T2 and T6,respectively, His69 and His196 (using canonical num-bering) were found, which are tetrahedrally coordinatedto the catalytic zinc ion in all MCPs (i.e. the numberingsystem corresponds to bovine pancreatic CPA and isused 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, actsas a general base for catalysis, whereas Asn144 andArg145, in the S1¢ subsite, bind the C-terminal carboxyl-ate group of the substrate. The peptide T6 appears tocontain Tyr198, which usually belongs to the S2 CP sub-site. In addition, peptides T4 and T5 appear to containtwo cysteine residues conserved in all members of MCPA ⁄ B subfamily, forming the disulfide bridge Cys138-Cys161 [1]. Any peptide assignable to the putative speci-ficity site [3] was found. Overall, these results indicatethat SmCP represents a CP-like enzyme of the M14Asubfamily [1,4].Fig. 5. Alignment of the amino terminal and internal sequences of SmCP with the sequences of carboxypeptidases from other organismsSmCP 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 CPAfrom 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 fromH. armigera (cotton bollworm) (097434_HELAM); carboxypeptidase precursor from H. armigera (cotton bollworm) (Q6H962_HELAM); MCPfrom 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 FEBSKinetic characterization of SmCPKinetic analyses for isolated SmCP was performed usingdifferent types of standard synthetic substrates for carb-oxypeptidases that were clearly cleaved by the enzyme.The Km, kcatand kcat⁄ Kmdetermined for the enzymeagainst AAFP, N -benzoyl-Gly-Phe (Hippuryl-Phe) andN-(3-[2-furyl]acryloyl)-Phe-Phe (FAPP) as substratesare shown in Table 2. Such kinetic parameters indicatethat SmCP is highly efficient against the three CPA typesubstrates used. On the other hand, we found thatSmCP 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 tothe 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 ofvarious protease inhibitors on the SmCP enzymaticactivity is shown in Table 3. Inhibitors of cysteineproteases (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 effectson SmCP activity. The enzyme was drastically inhib-ited by the chelating agent 1,10-phenanthroline at1mm. However, EDTA at 10 mm, which might act bymetal chelation, did not produce any inhibition at sim-ilar concentrations and inhibitor ⁄ enzyme (Io⁄ Eo) rela-tionships (3 · 105m). Nevertheless, EDTA partialinhibitory effects were observed when preincubationtimes 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 ofcarboxypeptidases PCI (in fact rPCI, a recombinantform, reactive towards CPA and CPB type enzyme) at0.4 lm produced a 70% inhibition of SmCP activity.The apparent Kivalue for this inhibitor towards SmCPwas 7.37 · 10)8m; however, the adjusted valueconsidering the substrate-induced dissociation was2.45 · 10)8m. Another protein inhibitor from leech(rLCI, also recombinant) at 13.5 lm produced a 70%inhibition of SmCP activity. The estimated Kivaluefor rLCI was 2.95 · 10)8m, and its adjusted valueconsidering the substrate induced dissociation was1.45 · 10)8m (Table 4). Preincubation of the inhibi-tors with the enzymes for various periods of time didnot affect its inhibitory activity, suggesting that rLCIand 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 mM(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 usedfor AAFP, Hippuryl-Phe and FAPP, respectively.EnzymeAAFP Hippuryl-Phe FAPPKm(mM) kcats)1kcat⁄ KmM)1Æs)1Km(mM) kcats)1kcat⁄ KmM)1Æs)1Km(mM) kcats)1kcat⁄ KmM)1Æs)1SmCP 0.05 ± 0.01 42.5 79 · 1050.36 ± 0.03 145 3.8 · 1050.14 ± 0.01 15 1.7 · 105bCPA 0.11 ± 0.01a44.0 41 · 1050.88 ± 0.05b60 6.8 · 1040.05 ± 0.01b340 6.8 · 106aMock et al [23].bCho et al [24]Table 3. Effect of protease inhibitors on the relative activity ofSmCP SmCP: 3.29 nM; AAFP: 0.1 mM; pH 7.5, 25 °C The enzymewas preincubated with the inhibitors for 10 min at 25 °C.Inhibitor Concentration% Enzymaticactivity Io⁄ EoE-64 0.1 mM 100 3.0 · 104MPefabloc 10 mM 100 3.03 · 106MPepstatin A 50 lM 94 1.51 · 104MTrypsin-chymotrysininhibitor (soybean)3mM 100 9.1 · 105M1,10-Phenanthroline 1 mM 21 3.03 · 105MBenzylsuccinic acid 1 mM < 1 3.03 · 105MEDTA 10 mM 117 3.03 · 105MPCI 0.4 lM 28.5 1.21 · 102MLCI 13.5 lM 30 4.1 · 102MAprotinin 3 mM 100 9.1 · 105MTrypsin inhibitor(soybean)2mM 100 6.0 · 105MTable 4. Kivalues of rPCI and rLCI against SmCP compared to pre-vious data obtained for bovine pancreatic CPA (bCPA) SmCP:3.29 nM; AAFP: 0.1 mM; pH 7.5, 25 °C The enzyme was preincu-bated with the inhibitors for 10 min at 25 °C.CarboxypeptidaseKi(nM)rPCI rLCISmCP 24.5 ± 03 14.5 ± 05bCPA 1.5 ± 02a1.6 ± 01baRyan et al [25].bReverter et al [27].M. Alonso-del-Rivero et al. A novel metallocarboxypeptidase from S. magnificaFEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS 4881On the other hand, we evaluated the effect on SmCPof metal ions after overnight dialysis against EDTA at10 mm (followed by the removal of excess EDTA bydialysis against metal-free buffers; see Experimentalprocedures). After this, SmCP only retains 40% of itsinitial activity. This apoform subsequently was used asa control for the studies with metals. We observed that1mm Ca2+,Mn2+or Mg2+enhanced the enzymeactivity of apoSmCP above 100% of the control activ-ity, whereas the addition of Cd2+at 1 mm or Co2+at1mm or 10 mm did not affect the enzymatic activityof the control (Fig. 6). However, Cu at 1 mm and10 mm reduced the apoenzyme activity to 11% and15% of its residual activity. Noteworthy, under ourconditions, the addition of Zn2+at 1 mm or 10 mmbrought the activity to 100% (full rescue) and to 70%,respectively, with the latter assignable to inhibition bythis metal.Specificity of cleavageTwo different long peptides were used as substratemodels to analyze the ability of SmCP to cleave differ-ent kinds of residues at the C-terminus, in comparisonFig. 6. Effect of divalent metals on SmCPactivity The concentrations used in theassays were 329 nM for the enzyme SmCPand 0.1 mM for the substrate AAFP, at pH7.5 and 25 °C The enzyme, after EDTAtreatment and dialysis against metal-freebuffer (see Experimental procedures), waspreincubated with the different ion metalsalts at 1 mM, for 10 min at 25 °C Theassays were also performed, under thesame conditions, at 10 mM for Zn2+,Co2+and Cu2+.SmCP vs ACTH ABSmCP 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 ACTH2466 2317 ACTH control 60 min bCPA vs V15E1793 1716 1748 V15E control 60 min V15E control 60 min SmCP + PCI 60 min 15 min 30 min 60 min bCPA + PCI 60 min15 min 30 min 60 min Fig. 7. Determination of SmP specificity forC-terminal substrate residues. Comparativeanalysis by MALDI-TOF MS of the degrada-tion of two synthetic substrates by SmCPand bovine pancreatic CPA (bCPA). Theassays were performed in 10 mM Tris–HClbuffer (pH 8.0) with 1 lM of peptides and2.19 nM of SmCP or 1 nM of bCPA in 10 lLof final volume for 60 min. (A) representsthe enzymatic activity of SmCP against theACTH fragment and V15E peptide, whereas(B) represents the enzymatic activity ofbCPA against the same substrate.Sequence of the ACTH fragment (residues18–39): RPVKVYPNGAEDESAEAFPLEF,MW: 2466 Da; ACTHdes-F, MW: 2317 Da;ACTHdes-EF, MW: 2188 Da; V15E peptidesequence, VKKKARKAAGC(Amc)AWE: MW1716 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 FEBSwith bovine pancreatic CPA (a reference enzyme in thefield). After 15 min of incubation of SmCP with theadrenocorticotropic hormone (ACTH) fragment usedas substrate (residues 18–39, 2466 Da), the enzyme wasable to release phenylalanine (ACTHdes-F, 2317 Da)and glutamic acid (ACTHdes-EF, 2188 Da) residuesfrom the substrate C-terminus (Fig. 7A). No furtheramino acids were released after a 30-min incubationperiod. Under the same conditions, bovine pancreaticCPA 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 inhibitorrPCI prevented cleavage in all cases.To confirm the capability of SmCP to hydrolyzeacidic residues from the C-terminus of peptides, thespecificity of SmCP against synthetic substrate[VKKKARKAAGC(Amc)AWE] (V15E peptide) (resi-due 15, 1716 Da) was evaluated (Fig. 7B). After15 min of incubation, the release of glutamic acid fromthe peptide was observed and, after 60 min, the newC-terminus residues formed and tryptophan and ala-nine were further released, as shown by the trimmingscale: 1716, 1587 and 1329 Da. However, bovine pan-creatic CPA was unable to hydrolyze the first of suchC-terminal residues, glutamic acid, even after 60 minof incubation. Again, the addition of rPCI preventedany kind of hydrolysis by the enzyme. The release of aglutamic acid residue from the C-terminus of peptidesis a very unusual capability of a CPA-like enzyme andis reminiscent of the so-called CPO forms [3,5].DiscussionThe growing application of genomics and related tech-nologies is facilitating an expanding view of the pres-ent enzymatic families, including proteases [20] andCPs in particular [4]. However, such an advance is lim-ited in the invertebrate world because of the greatdiversity of organisms within it, which complicates thestudy, 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 themost unknown among proteases in invertebrates, wehave used a mix of both modern and more classicalapproaches to identify and characterize them, estab-lishing comparisons with the vertebrate species (i.e. thereference ones). The present study started with a sys-tematic screening in extracts from 25 invertebratesfrom marine Caribbean species, using a specific andsensitive enzymatic assay; this allowed us to detect thepresence of CPA-like activity in the body extract ofthe marine annelid S. magnifica. Given that we previ-ously reported the successful use of MALDI-TOF MSfor the initial detection of CPs and carboxypeptidaseinhibitors in other crude biological extracts [17–19], wehave applied such approaches to the S. magnifica case.The use of affinity capture on microbeads or microcol-umns derivatized with a recombinant carboxypeptidaseinhibitor from potatoes, specific for such class ofenzymes, and the use of MALDI-TOF MS signal anal-ysis approaches, allowed us to quickly identify in thisannelid a 35-kDa species as a potential MCP, whichwe named SmCP.Different fractionation methods have been per-formed to purify SmCP from the body extract ofS. magnifica. In initial attempts, using anion exchangeand gel filtration chromatographies, we found a frac-tion with clear carboxypeptidase activity, which, inter-estingly, conveyed two additional activities againsttypical substrates for trypsin-like (benzoyl arginyl ethylester; BAEE) and chymotrypsin-like (benzoyl tyrosineethyl ester; BTEE) serine proteases (data not shown).This suggests that, in the fractionation, SmCP couldco-elute with serine proteases, perhaps establishing bin-ary or ternary complexes with such enzymes, as shownin 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 fromthe other enzymes. Potentially, rPCI could promotethe dissociation of SmCP from ‘complexes with serineproteases’ 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 suchapproach unless high sensitivity approaches (i.e immu-nostaining) are employed. This is probably a result ofthe low representation of this enzyme in the animalextracts, in agreement with its subsequent analysis andvisualization 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-PAGEprotein 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 gavestainable and clearly visible bands were detected bythis 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 requireeither direct isolation or MS ⁄ MS analyses. The latertype of study is under way in our laboratory, but isM. Alonso-del-Rivero et al. A novel metallocarboxypeptidase from S. magnificaFEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS 4883proving more difficult than expected because of thevery low homologies shown by S. magnifica proteaseswith respect to equivalent ones found in databases.Given the poor representation of invertebrate proteasesin databases, this is not an unexpected problem whencarrying out identification proteomics.It is worth noting that the preliminary detection ofserine and cysteine proteases species in the bodyextracts correlates with the measure of their activitiesby enzymatic analysis of the crude samples. Interest-ingly, neither approach revealed evidence of the occu-rence of aspartic proteases. Overall, although thepresence of pigments and other interfering productsinitially constituted a very serious problem, once thiswas technically solved, the feasibility and data genera-tion capability of both the 2D-PAGE and ‘intensityfading’ MALDI-TOF MS of this annelid indicatedthat such proteomic-like approaches (and probablyrelated ones) are very promising for the analysis ofproteolytic enzymes in marine invertebrates.A central question in the analysis of novel MCPsfrom biological sources is whether they occur in theirprecursor or mature forms [2–5]. In the present study,using direct extracts from S. magnifica, we found onlya monomeric and activated form of SmCP, as shownby its enzymatic activity, molecular mass, derivedN-terminal sequence and homology analysis. Procarb-oxypeptidases are usually activated by proteolyticremoval of their activation segment by serine prote-ases, mostly trypsin. Studies on procarboxypeptidasesfrom several species have indicated that its activationis dependent of the environmental ionic conditionsand, 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 theextract, could be favored. On the other hand, thecoincidence between the N-terminal sequences ofSmCP and those from several other MCPs included inalignments (Fig. 5) also suggests that SmCP has beenpurified in the active mature form. In addition, wefound that the sequences of a number of SmCP inter-nal peptides included important residues that belong tocatalytic site and domain of this enzyme family,confirming our interpretation.All the experimental data reported in the presentstudy indicate that SmCP belongs to the M14A sub-family of metalloproteases [6], the so-called pancreatic-like forms (or A ⁄ B), favoring its potential digestivefunction in the marine annelid. Its molecular weight(33.7 kDa), N-terminal sequence and behavior towardsa panel of substrates and inhibitors are similar to thoseof mammalian pancreatic CP (i.e. the best known).These types of enzymes have molecular masses close to35 kDa after the removal of the propeptide, whereasthe regulatory CPs (or N ⁄ E) display higher mass val-ues as a result of the presence of other domains inaddition 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 substratepreferences. Only a few CPs have been isolated frommarine invertebrates, and in not one case have thewhole or extended sequences been disclosed. Thiswould be the case for the two CPAs and CPBs isolatedfrom 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, withan overall efficiency similar to bovine pancreatic CPA,but with some significant differences in kcat, Kmandkcat⁄ Kmfor certain substrates [23,24]. In addition,SmCP has a maximum activity at pH 7.5, in agreementwith the optimum pH activity of almost all M14A CP-like forms, including marine enzymes [7–13], which liein the neutral range (pH 6.5–8.5), and is consistentwith the pH at their sites of biological action [1,2].As previously shown for mammalian CPs [25–27],potato and leech proteinaceous inhibitors efficientlyinhibit SmCP, displaying similar Kivalues. In addition,two smaller organic molecules (benzylsuccinic acid and1,10-phenantroline) known to act on MCPs are alsoable to inhibit the enzyme. By contrast, EDTA, whichchelates metal ions, at 10 mm, failed to inhibit SmCPactivity significantly after 10 min of preincubation,which is in agreement with the reported properties ofother 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 substitutethe essential active site Zn2+of MCPs [30,31], or binda second atom nearby [32], interfering with the cata-lytic mechanism, is well known. We also observeddiverse effects by the addition of such metals to SmCP.After its dialysis against EDTA at 10 mm, SmCPreduced its activity to 40% of initial activity. Startingfrom this state, the capacity of different metal ions toregenerate SmCP activity demonstrated that, in certaincases [Mn, Mg and Ca], there is an enhancement ofactivity of the enzyme; in others [Cd and Co], nochanges are observed; and, in a third case [Cu], a clearinhibition 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 SmCPactivity 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-Rivero1,. the Phyla Cnidaria, Annelida, Mollusca, Echi-nodermata, Arthropoda and Chordata, amongstothers, collected on the coasts of Havana, Cuba. The study has
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