The Trypsin Inhibitor Panulirin Regulates the Prophenoloxidase-activating System in the Spiny...

The Trypsin Inhibitor Panulirin Regulates theProphenoloxidase-activating System in the SpinyLobster Panulirus argusReceived for publication, February 27, 2013, and in revised form, September 17, 2013 Published, JBC Papers in Press, September 18, 2013, DOI 10.1074/jbc.M113.464297

Rolando Perdomo-Morales‡1, Vivian Montero-Alejo‡, Gerardo Corzo§, Vladimir Besada¶, Yamile Vega-Hurtado‡,Yamile González-González�, Erick Perera**, and Marlene Porto-Verdecia‡

From the ‡Biochemistry Department, Center for Pharmaceuticals Research and Development, Ave. 26 No. 1605 e/ Ave. 51 yBoyeros, Plaza, CP 10400, Havana, Cuba, the §Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología,Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos 62250, México, the ¶Department ofProteomics, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, CP 10600 Havana, Cuba, the �BiochemistryDepartment, Federal University of Sao Paulo, Rua 3 de Maio 100, CEP 04044-020 Sao Paulo, Brazil, and the **Center for MarineResearch, University of Havana, Calle 16 No. 114 e/ 1ra y 3ra, Miramar, Playa, CP 11300 Havana, Cuba

Background: The melanization reaction is an essential immune response in arthropods that should be tightly regulated.Results: A novel competitive and tight-binding trypsin inhibitor, panulirin, that inhibits the melanization response to lipopo-lysaccharides was found in lobster.Conclusion: Panulirin regulates serine peptidases in the pathway toward the activation of the prophenoloxidase enzyme.Significance: Serine peptidase inhibitors play a key role in controlling the immune response in arthropods.

The melanization reaction promoted by the prophenoloxi-dase-activating system is an essential defense response in inver-tebrates subjected to regulatory mechanisms that are still notfully understood. We report here the finding and characteriza-tion of a novel trypsin inhibitor, named panulirin, isolated fromthe hemocytes of the spiny lobster Panulirus argus with regula-tory functions on the melanization cascade. Panulirin is a cat-ionic peptide (pI 9.5) composed of 48 amino acid residues (5.3kDa), with six cysteine residues forming disulfide bridges. Itsprimary sequence was determined by combining Edman degra-dation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similaritywith known proteins indicates that it represents a new family ofpeptidase inhibitors. Panulirin is a competitive and reversibletight-binding inhibitor of trypsin (Ki � 8.6 nM) with a notablespecificity because it does not inhibit serine peptidases such assubtilisin, elastase, chymotrypsin, thrombin, and plasmin. Theremoval of panulirin from the lobster hemocyte lysate leads toan increase in phenoloxidase response to LPS. Likewise, theaddition of increasing concentrations of panulirin to a lobsterhemocyte lysate, previously depleted of trypsin-inhibitory activ-ity, decreased the phenoloxidase response to LPS in a concen-tration-dependent fashion. These results indicate that panulirinis implicated in the regulation of the melanization cascade inP. argus by inhibiting peptidase(s) in the pathway toward theactivation of the prophenoloxidase enzyme.

Invertebrates lack adaptive immunity, and their protectionagainst pathogens relies mainly on innate immunity mecha-nisms, which could be divided into closely related humoral andcellular defense responses (1, 2). The prophenoloxidase(proPO)2-activating system is a major humoral defense mech-anism in arthropods involved in a series of responses, such asmelanization, encapsulation, cytotoxic reactions, and phagocy-tosis (for reviews, see Refs. 1–4). In general, it could be com-posed by pattern recognition proteins, trypsin-like serinepeptidases, serine peptidase homologues, and the prophenol-oxidase (4). Minute amounts of pathogen-associated molec-ular patterns, such as lipopolysaccharides (LPS), peptidogly-can, and �-1,3-glucans, activate the proPO cascade, leadingultimately to the activation of the prophenoloxidase-activat-ing enzyme (ppA), a trypsin-like serine peptidase that con-verts the proPO zymogen into active phenoloxidase (PO) (4).Phenoloxidase (EC 1.14.18.1) catalyzes the hydroxylation ofmonophenols to o-diphenols (monophenolase or cresolaseactivity) and the oxidation of o-diphenols to o-quinones (o-di-phenolase or catecholase activity), therein initiating the synthe-sis of melanin (5).Several cytotoxic molecules are produced during melano-

genesis, which have recently been demonstrated to be effectiveto combat infections (4, 6). These intermediates can be alsohighly deleterious to the host if they are produced uncontrolla-bly (5, 7). Therefore, arthropods have developed several meansto regulate the melanization reaction, both spatially and tem-porally, to avoid damage to the host.Location of the PO response seems to be an important regu-

latory mechanism for insect defense against infections (8). InThe nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/

EBI Data Bank with accession number(s) KC154047 and AGE44005.The protein sequence data of panulirin reported in this paper appear in the Uni-

Prot Knowledgebase under accession number B3EWX6.1 To whom correspondence should be addressed. Fax: 537-833-5556; E-mail:

[email protected].

2 The abbreviations used are: proPO, prophenoloxidase; BAPNA, N-benzoyl-DL-arginyl-p-nitroanilide; pNA, p-nitroanilide; LHL, lobster hemocyte lysate;AC, anticoagulant solution; ppA, prophenoloxidase-activating enzyme(s);PO, phenoloxidase; BPTI, bovine pancreatic trypsin inhibitor; ESI, electro-spray ionization.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 44, pp. 31867–31879, November 1, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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this sense, components of the proPO-activating system mayassociate to form a large noncovalent complex, which localizesthe melanization to the surface of invading microorganisms orat the injury site (8–10). This complex ensures a high localconcentration of quinone products where necessary, whereas itavoids their dissemination. In addition, the stickiness of acti-vated phenoloxidase promotes its deposition on pathogen oranomalous surfaces, where it assists localized melanization (5).The melanin formation can be controlled even at late stages

of the melanogenesis. For example, the presence of melaniza-tion inhibition proteins has been described in plasma fromcrustaceans (11) and insects (12). These proteins hamper thesynthesis of melanin from quinones but have no effect on POenzyme activity. Interestingly, melanization inhibition proteinsfromcrustaceans and insects share a similarmolecularmass (43kDa) but are remarkably different in their primary structure(11).The most straightforward solution for controlling the

proPO-activating system could be that enzymatic components,such as PO and peptidases, exist inactive as zymogens (3, 4).However, it is reasonable to expect the occurrence of mecha-nisms to control their activities once they become active. Sev-eral phenoloxidase inhibitors have been characterized ininsects (13–15). Also in insects, genetic evidence suggests thatserpin-type peptidase inhibitors are involved in regulating themelanization cascade (16). However, the peptidases inhibitedby serpins have only been identified in the beetleTenebriomoli-tor (17) and the tobacco hornworm Manduca sexta (16). Ser-pins regulate the proPO system in insects by inhibiting both theppA (18–20) and peptidases upstream of the ppA in the cas-cade (10, 16, 21).Until the present report, pacifastin from the crayfish Paci-

fastacus leniusculus (22, 23) was the only known peptidaseinhibitor regulating the proPO system in crustaceans, despitethe fact that this system has been investigated in a variety ofcrustaceans for decades. Pacifastin regulates the activity of theppA. It is a heterodimeric protein (155 kDa) composed of twocovalently linked subunits, each encoded by two differentmRNAs. The light chain (44 kDa) contains the inhibitorydomains, whereas the heavy chain (105 kDa) is instead relatedto transferrins (24).In our first study on the proPO-activating system in spiny

lobster, we indicated the presence of trypsin-inhibitory activityin the hemocyte lysate (25). Here, we describe the purificationand somemolecular and biological properties of a novel trypsininhibitor that we have named panulirin. The low similarity toother protein inhibitors found through amino acid sequencecomparison suggests the finding of a new class of peptidaseinhibitor. Panulirin is a 5.3-kDa basic peptide (pI 9.5) composedof 48 amino acid residues, which contains six cysteine residuesengaged in disulfide bridges. It is a competitive, reversible andtight-binding inhibitor of trypsin. Experimental evidence indi-cated that panulirin is implicated in the regulation of theproPO-activating system in the spiny lobster.

EXPERIMENTAL PROCEDURES

Materials—Bovine pancreatic trypsin (EC 3.4.21.4), elastasefrom porcine pancreas type IV (EC 3.4.21.36), subtilisin A from

Bacillus licheniformis (EC 3.4.21.62), bovine pancreatic chymo-trypsin (EC 3.4.21.1), plasmin from human plasma (EC3.4.21.7), thrombin from bovine plasma (EC 3.4.21.5), HEPES,N-benzoyl-DL-arginyl-p-nitroanilide (BAPNA), LPS from Esch-erichia coli O55:B5, N-succinyl-Ala-Pro-Phe-p-nitroanilide,anddopaminewerepurchased fromSigma.4-Nitrophenyl4�-gua-nidinobenzoate was from ICN Biomedicals Inc. (Aurora, OH).DMSO, EDTA, NaCl, Tris, DTT, acetonitrile (LiChrosolv�,hypergrade for LC/MS), TFA (for protein sequence analysisgrade), papain fromCarica papaya (EC 3.4.22.2), TritonX-100,protamine sulfate, glucose, sodium citrate, and calcium chlo-ride were all obtained from Merck. Sephadex G-50 Superfinewas from Amersham Biosciences. The HiTrap SP HP column,the low molecular weight calibration kit for SDS electrophore-sis, and the low molecular weight gel filtration calibration kitwere fromGEHealthcare. The substrates S-2251 (Val-Leu-Lys-p-nitroanilide), S-2238 (Phe-Pip-Arg-p-nitroanilide), andS-2586 (MeO-Suc-Arg-Pro-Tyr-p-nitroanilide) were fromChromogenix AB (Mondal, Sweden).Preparation of Hemocyte Lysate Supernatant—The spiny

lobster hemolymph was obtained from the fourth walking legcoxa using sterile and precooled modified Alsever anticoagu-lant solution (AC) containing 27 mM sodium citrate, 115 mM

glucose, 336mMNaCl, 9mMEDTA, pH7 (26). The hemolymphwas centrifuged immediately after collection at 700 � g for 10min at 4 °C, and the supernatant was discarded. The hemocytepellet was washed twice with cold AC, suspended in the corre-sponding lysis buffer (see particular experiments below), anddisrupted by sonication three times at 40 watts for 10 s each.The clarified lobster hemocyte lysate (LHL) was obtained bycentrifuging the homogenate at 4,000 � g for 30 min at 4 °C.Determination of Trypsin-inhibitory Activity—Trypsin activ-

ity was determined using 0.9 mM BAPNA (1 � Km) as substrate(27). The nominal trypsin concentrationwas determined at 280nm using an extinction coefficient (E2801% ) of 14.4 kDa (28), and23.3 kDamolecularmass.A stock solution of bovine trypsinwasprepared at 10mg/ml in 1mMHCl, 20mMCaCl2, pH3,whereasthe stock solution of BAPNA (125 mM) was in DMSO. For theassay, 20 �l of trypsin were mixed with 180 �l of assay buffer(0.1 MTris-HCl, pH8, 150mMNaCl, and 20mMCaCl2) in awellof a 96-well plate. The reactionwas started by the addition of 50�l of 4.5 mM BAPNA in the assay buffer. The pNA released wasmeasured kinetically at 405 nm for 10min at 37 °C in an ELx808IUmicroplate reader (BioTek Instruments,Winooski, VT). Ini-tial velocities were obtained using the kinetic application of theprogram KC4 version 3.4 (BioTek Instruments). One unit oftrypsin activitywas defined as the amount of trypsin causing therelease of 1 �mol of pNA/min. The extinction coefficient ofp-nitroanilide at 405 nm for a volume of 250 �l was 6.8 mM�1,as determined empirically.Trypsin-inhibitory activity was calculated at dilutions where

the inhibitory percentage fell between 25 and 70%. One unit ofinhibitory activity (IU) was defined as the amount of inhibitorproducing 50% inhibition of 2 units of the trypsin (29). Specificactivity was defined as the inhibitory activity/mg of protein.Where indicated, the fractional activity (Vi/Vo) was determinedas the ratio between the initial velocity in the presence (Vi) andabsence (Vo) of inhibitor.

Novel Trypsin Inhibitor Regulates Immune Response in Lobsters

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Protein Concentration Determination—The protein concen-trationwas determined by the Lowrymethod (30), using bovineserumalbumin (BSA) as a standard. Samples containingHEPESabove the interfering level with the Lowry assay were first pre-cipitated with deoxycholate-trichloroacetic acid (31).Influence of Ionic Strength on InhibitoryActivity—Hemocytes

from four centrifuge tubes containing 50 ml of hemolymph/anticoagulant (1:1, v/v) were collected andwashed as above andthen pooled to a final volume of 50 ml made up with AC. Thehomogeneous hemocyte suspension was divided into 6-ml ali-quots and centrifuged. Finally, each pellet was suspended in 50mM Tris-HCl, pH 7.5, containing various concentrations ofNaCl (from 0 to 650 mM). The hemocytes were lysed and clar-ified as described earlier, and the inhibitory activity of trypsinfor each fraction was determined. Where indicated, nucleicacids were precipitated by adding 0.1% protamine sulfate (finalconcentration) to lysed hemocytes before the clarification step.Reversed Zymography—Reversed zymography for the detec-

tion of inhibitory activity in the hemocyte lysate was deter-mined by SDS-PAGE in 15% polyacrylamide gel copolymerizedwith 0.1% gelatin (w/v) (32). A lowmolecular weight calibrationkit composed of phosphorylase b (97 kDa), albumin (66 kDa),ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsininhibitor (20.1 kDa), and �-lactalbumin (14.4 kDa) was used asa standard.Purification of Peptidase Inhibitor—The LHL was obtained

in lysis buffer containing 450 mM NaCl and treated with prota-mine sulfate as above. The supernatant (10ml at 9.5mg/ml)wasfractionated by gel filtration chromatography in a SephadexG-50 Superfine column (2.6� 65 cm), equilibrated with 25mM

HEPES, pH 8.2, 100 mM NaCl, 0.01% Brij 35 (w/v) (buffer A).The flow rate was 0.8 ml/min, and fractions of 4 ml were col-lected for determining trypsin-inhibiting activity. The gel filtra-tion column was calibrated with molecular mass standards(carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa), andaprotinin (6.5 kDa)). The pooled inhibitory fractionwas appliedto a 5-mlHiTrap SPHP column equilibratedwith buffer A. Thebound proteins were eluted with 135 ml of a linear NaCl gradi-ent (100–500 mM) in the same buffer at 0.5 ml/min. Proteinelution was monitored at 280 nm. The inhibitory peak was fur-ther purified by reversed phase HPLC in a Knauer SmartlineHPLC system (Germany), using a Discovery BIOWide Pore C5column (4.6 � 250 mm, 5 �m; Supelco) equilibrated with 0.1%(v/v) TFA in water (solvent A). The elution system comprisedsolvent A and 0.07% TFA in 70% acetonitrile (solvent B). Sepa-ration was performed with a linear gradient of solvent B from 5to 80% over 55 min at a flow rate of 1 ml/min. The absorbancewas monitored at 214 nm.Determination of the Equilibrium Dissociation Constant (Ki)—

The concentration of active trypsin was determined by activesite titration with 4-nitrophenyl 4�-guanidinobenzoate (33).The time to reach the equilibrium of trypsin-inhibitor complexwas determined by incubating fixed concentrations of trypsin(48�g/ml) and inhibitor (0.9�g/ml) at room temperature for 0,5, 10, 30, and 60min before the addition of substrate. The activeconcentration of inhibitorwas determined by titration against afixed concentration of active site-titrated trypsin (1.5 �M)assuming an equimolar binding between the enzyme and the

inhibitor and E0/Kiapp � 100 (34). In addition, inhibitory activity

was determined at different substrate concentrations (0.5, 1.0,1.5, and 2.0Km) to demonstrate substrate-induced dissociation.To determine the apparent dissociation constant (Ki

app), trypsin(80 nM) was mixed with increasing inhibitor concentrations(2.4–288 nM), and the residual trypsin activity was determined.The Ki

app value was calculated by fitting the experimental datato the quadraticMorrison equation for tight-binding inhibitors(35), using GraphPad Prism version 5 for Windows (GraphPadSoftware, San Diego, CA).Determination of Inhibitor Specificity—The inhibitory activ-

ity was evaluated against peptidases belonging to three mecha-nistic classes: metallo, cysteine, and serine. The enzymaticactivities were assessed at 37 °C in amixture composed of 20 �lof each enzyme, 180 �l of assay buffer, and 50 �l of the corre-sponding substrate as follows: 50 nM chymotrypsin, 50 mM

Tris-HCl, pH8.0, 10mMCaCl2, 0.05mMS-2586; 5 nM subtilisin,0.1 M Tris-HCl, pH 8, 0.15 mM Suc-Ala-Pro-Phe-pNA; 10 nMelastase, 0.1 M Tris-HCl, pH 8, 1.2 mM N-Suc-Ala-Ala-Ala-pNA; 0.9 mM papain, 200 mM sodium acetate, pH 6.0, contain-ing 8 mM DTT and 4 mM sodium EDTA, 1 mM BAPNA; 29 nMthrombin, 0.1 M Tris-HCl, pH 8, 0.15 MNaCl, 0.3�M S-2238; 48nM plasmin, 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.3 mM

S-2251. The pNA released was determined at 405 nm, asdescribed earlier for trypsin. In the case of carboxypeptidase A,the activity was determined kinetically at 254 nm for 3 min atroom temperature in a reaction mixture composed of 85 �l of25 mM Tris-HCl, pH 7.5, 0.5 M NaCl (reaction buffer), 1 ml of 1mM hippuryl-L-Phe, and 15 �l of the enzyme at 2.2 �M. Allenzymatic activities were determined under initial velocityconditions. After 30 min of incubation of each enzyme with100-fold molar concentrations of panulirin, the inhibitoryactivity was evaluated by determining the fractional activityusing the assay conditions described above.Disulfide Reduction and Carbamidomethylation—Panulirin

was mixed with 13.3 �l of a solution containing 0.75 M Tris, pH8.0, 33.3 �l of 6 M guanidinium chloride, and 4.5 �l of 1.3 M

DTT. The reaction was incubated for 2 h at 37 °C. After incu-bation, 25 �l of 0.8 M iodoacetamide was added and allowed toreact for 25 min at room temperature. The reaction wasstoppedwith 10�l of pure formic acid. This solutionwas passedthrough a ZipTip-C4 microcolumn (Millipore) for desaltingprior to enzymatic digestion. The proteinwas elutedwith 2.5�lof 60% acetonitrile (v/v).Enzymatic Treatment—Samples of the reduced and carbam-

idomethylated panulirin were separately enzymatically digestedwith trypsin, chymotrypsin, and endoproteinase Glu-C. The elu-ates from the ZipTip were dissolved in 60 �l of 0.19 M Tris, pH8.0, and the enzymewas added at 50:1 (trypsin) or 25:1 (chymo-trypsin) substrate/enzyme ratios in each case. For the enzy-matic treatmentwithGlu-C, the eluate was dissolved in 60�l of0.25 M ammonium hydrogen carbonate, pH 8.0, and the endo-proteinase was added in a 50:1 ratio.Chemical Treatment—Reduced and carbamidomethylated

panulirin (10 �g) was desalted through ZipTip-C4 (Millipore),dried in a SpeedVac, and resuspended in 30 mM hydrochloricacid containing 6 M guanidine hydrochloride. The solution was




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transferred to a 1-ml vial (Pierce) and kept under vacuumand at104 °C for 12 h.Mass Spectrometry—ESI-MS and ESI-MS/MS spectra were

acquired using a QTof-1TM mass spectrometer (Micromass)fittedwith a Z-spray nanoflow electrospray ion source operatedat 80 °C with a drying gas flow at 50 liters/h. The analyzer wascalibrated in awidemass range (50–2,000Da) using a referencemixture of sodium and cesium iodides. Intact protein and pep-tide digest samples were loaded into the borosilicate nanoflowtips and submitted to 900 and 35 V of capillary and cone volt-age, respectively. To acquire the ESI-MS spectra, the first qua-drupole was used to select the precursor ionwithin a window ofapproximately 3 Thomson. Argon gas was used in the collisionchamber at �3 � 10�2 pascal pressure, and collision energiesbetween 15 and 48 eVwere set to fragment precursor ions. Dataacquisition and processing were performed using MassLynxversion 3.5 (Micromass).N-terminal Sequence—Panulirin and a tryptic digestion-de-

rived peptide (1550.2 Da) were directly sequenced on a Shi-madzu PPSQ-31A (Shimadzu, Kyoto, Japan) automated gasphase sequencer. Samples were dissolved in 10 �l of a 37%CH3CN (v/v) solution and applied to TFA-treated glass fibermembranes, precycled with Polybrene (Aldrich). Data wererecorded using the Shimadzu PPSQ-31A software.Sequence Analysis—The physical-chemical properties of

panulirin were determined using the ProtParam tool (availableon the ExPASy Web site).Influence of Panulirin on PO Response to LPS—PO activity

was determined spectrophotometrically by recording the for-mation of dopaminechrome and derivatives from dopamine assubstrate (36). In brief, 20 �l of 0.25 mg/ml LHL or pooledfraction eluted at the void volume from the gel filtration chro-matography of the LHL (F1) at 0.025 mg/ml was mixed in flatbottommicroplate wells with 100 �l of 50 mM Tris-HCl buffer,pH 7.5, 50 mM CaCl2, and 50 �l of 0.1 mg/ml LPS. The controlexperiment was LPS-free water instead of LPS. PO activity wasassessed continuously at 490 nm and 37 °C immediately afterthe addition of 50 �l of 0.6 mg/ml dopamine.In order to evaluate the influence of panulirin on the PO

response to LPS, 20 �l of LHL fraction depleted of trypsin-inhibitory activity (F1) at 0.025mg/mlwas incubated for 15minwith 50 �l of 2-fold serial dilutions of purified panulirin (16.2IU/ml starting inhibitory activity), and PO activity was assessedas described above.

RESULTS

Influence of Ionic Strength on Trypsin-inhibitory Activity ofthe LHL—Preliminary observations led us to suspect that theextent of trypsin-inhibitory activity in the lysate could berelated to the ionic strength in the lysis buffer used to preparethe LHL. Therefore, we evaluated the inhibitory activity of LHLobtained in lysis buffer containing different concentrations ofNaCl (0–650 mM). A direct relationship between trypsin-in-hibitory activity in the LHL andNaCl concentration was found.This effect occurs for concentrations as high as 450 mM. Fur-ther increase in NaCl concentration did not affect the inhibi-tory activity (Fig. 1).

When an aliquot of LHL prepared in the lysis buffer withoutNaCl was treated with 0.1% protamine sulfate (w/v), a wellknown precipitant of nucleic acids, the trypsin-inhibitory activ-ity increased from 7.4 to 38.60 IU/ml. In a different experiment,no significant differences (p � 0.05) were found between theinhibitory activity in protamine-treated (55.4 � 1.11 IU/ml;mean � S.E., n � 5) and untreated (54.0 � 0.31 IU/ml; mean �S.E., n � 5) LHL prepared in 450 mM NaCl, indicating thatprotamine sulfate did not impair the inhibitory activity underthe experimental conditions used. Taken together, these resultsmight suggest that trypsin inhibitors in the LHL are bound tonucleic acids, probably electrostatically, and that this binding isabrogated by high ionic strength. To confirmwhether panulirinbinds to nucleic acids, we isolated DNA from the hemocytesusing TRI reagent according to the manufacturer’s instruc-tions. Panulirin (25 �l at 8 �M) was incubated with 25 �l of 0.3�g/�l DNA for 1 h at room temperature using buffers lackingNaCl. The control experiment was also incubated but withreaction buffer instead of DNA. Thereafter, the trypsin activitywas determined as described above in a mixture containing 80nM trypsin and 80 nM panulirin diluted from the above incuba-tions. The inhibitory activity in the control experiment was78%, and it dropped to 9.5% in the sample previously incubatedwith DNA. The corresponding DNA concentration in theabsence of panulirin had no influence on trypsin activity (datanot shown). These results suggest that panulirin is able to bindto nucleic acids at low ionic strength.Isolation of Panulirin—Panulirin was purified by gel filtra-

tion on Sephadex G-50 followed by cation exchange chroma-tography on a HiTrap SP-Sepharose HP column (Table 1).Reversed zymography revealed a band with trypsin-inhibitoryactivity of around 16 kDa (Fig. 2A). Hence, it was decided tofractionate the LHL first by gel filtration chromatography inSephadex G-50 (30 kDa exclusion limit), presuming that thepossible targets of inhibitors in the LHL are endogenous pepti-dases over 30 kDa that would elute at the void volume alongwith other components of the proPO-activating system.

FIGURE 1. Influence of ionic strength on trypsin-inhibitory activity in lob-ster hemocyte lysate. A homogenous suspension of washed hemocytes wasevenly aliquoted, and each aliquot was lysed in 50 mM Tris-HCl, pH 7.5, con-taining various concentrations of NaCl (0 – 650 mM). Trypsin-inhibitory activ-ities were determined at dilutions producing 25–70% inhibition of 48 �g/mltrypsin in the assay using 0.9 mM BAPNA as substrate. One unit of inhibitoryactivity (IU) was defined as the amount of inhibitor-containing sample pro-ducing 50% inhibition of 2 units of trypsin. Values represent the mean of atleast three replicates plus S.D. (error bars).




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A single peak of around 5 kDa showing trypsin-inhibitoryactivity eluted from the gel filtration between two major frac-tions, leading to a high degree of fractionation (Fig. 2B).We alsofound that precipitation of nucleic acids with protamine sulfateimproved the resolution between the inhibitory peak and thefraction eluting at the void volume (not shown), which allowedus to fractionate higher volumes of LHL per chromatographicstep. The inhibitory fraction (205–242 ml) was pooled andapplied to cation exchange chromatography. Two trypsin-in-hibiting peaks were identified (Fig. 2C). The secondmajor peakwith the strongest trypsin-inhibitory activity (66–74 ml) waspooled and used throughout this study. Reversed phase HPLCanalysis on a Supelco C5 column (Fig. 2D) showed that thisfraction, hereafter referred as panulirin, was around 95% pure.Isolated panulirin from reversed phase HPLC was used forN-terminal and MS analysis.Characterization of Panulirin-Trypsin Interaction—The

study was partially based on the strategies described by Bieth

(34), using the steady state approach. Preliminary experimentsshowed concave inhibition curves in the plot between the frac-tional activity (Vi/Vo) versus increasing concentration of panu-lirin at constant trypsin and substrate concentrations, whichcould mean that the incubation time for association of trypsinand panulirin was incomplete or that association was com-pleted but the inhibition was reversible with [Eo]/Ki � 10 (34).Therefore, the time dependence to reach the associationbetween trypsin and panulirin was determined. The inhibitoryactivity remained constant because no significant differences(p� 0.05) were observed in the fractional activities among eachincubation time evaluated (Fig. 3A), suggesting that completedassociation between trypsin and panulirin was reached uponmixing, and therefore, the concave inhibition curves observedprobably describe a reversible interaction. It is worth mention-ing that all progress curves in this experiment were linear (datanot shown). On the other hand, we found that the fractionalactivity increased with substrate concentration at constant

TABLE 1Panulirin purification

Stage Volume ProteinTotal inhibitory

activityaSpecificactivity Purification Yield

ml mg IU IU/mg -fold %Lobster hemocyte lysate 9.5 95 361 3.8 1 100Sephadex-G50 40 3.6 222 61.6 16.2 61.5Cation exchange 8 0.8 171 213 56.2 47.3

a Inhibitory activity: 1 unit of inhibitory activity (IU) was defined as the amount of protein needed to inhibit 2 units of trypsin activity. One unit of trypsin activity was definedas the enzyme activity that produces 1 �mol of pNA/min under specified conditions.

FIGURE 2. Purification of panulirin. A, reversed zymography of LHL. Lane 1, molecular weight marker; lane 2, 0.08 �g of LHL loaded per well. The gel wasstained with Coomassie Brilliant Blue R-250. B, lobster hemocyte lysate was fractionated by gel filtration chromatography on a Sephadex G-50 superfinecolumn (2.6 � 65 cm); 9.5 ml of LHL (10 mg/ml) treated with 0.1% protamine sulfate (w/v) was loaded onto the column previously equilibrated with 25 mM

HEPES, 100 mM NaCl, pH 8.2, 0.01% Brij 35 (w/v) (buffer A) and eluted with the same buffer at 0.8 ml/min. Fractions of 4 ml were collected, and 10 �l of each wasassayed for inhibitory activity against 48 �g/ml trypsin using 0.9 mM BAPNA. C, fractions from the gel filtration containing trypsin-inhibitory activity werecombined and applied to a 5-ml HiTrap SP-Sepharose HP column equilibrated in buffer A. Bound proteins were eluted with 135 ml of a linear NaCl gradient(100 –500 mM) in buffer A. Fractions of 1 ml were collected during gradient elution and assayed for trypsin inhibition as above. D, the inhibitory peak from cationexchange was finally purified by reversed phase C5 column (4.6 � 250 mm) equilibrated with 0.1% (v/v) TFA in water. Bound samples were eluted with a lineargradient of acetonitrile from 3.5 to 56% over 55 min at 1 ml/min. The absorbance was monitored at 214 nm.




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trypsin and inhibitor concentrations (Fig. 3B), indicating sub-strate-dependent inhibition and thus confirming that the inter-action is reversible and competitive (34).The active concentration of panulirin was determined by

titration with a constant concentration of active site-titratedtrypsin (1.5�M).At this trypsin concentration, the plot betweenthe residual trypsin activity and inhibitor concentration trackslinear behavior (Fig. 3C), allowing the determination of anactive inhibitor concentration of 22.5 �M, which correspondedto 97% of the nominal concentration of purified panulirin.Having the active concentration of trypsin and inhibitor, we

proceeded to determine the inhibitor dissociation constant. Forthis purpose, constant concentrations of trypsin at 80 nM weremixed with increasing concentrations of inhibitor, and theresidual trypsin activity was determined. The concave inhibi-tion curve obtained (Fig. 3D) indicated experimental condi-tions of [Eo]/Ki

app between 1 and 10 (34), which further demon-strated the reversible interaction between panulirin andtrypsin. The Ki

app value obtained by fitting the data to the Mor-rison equation (Fig. 3C), was converted to true Ki value by theequation, Ki

app � Ki(1 � [S]/Km), taking into account the com-petitive nature of the inhibitor (35). The real Ki was 8.6 � 0.81nM, indicating that panulirin binds trypsin with relatively highaffinity.Inhibitory Specificity—It is widely accepted that most inhib-

itors are specific for one of the four mechanistic classes of pep-tidases (37, 38), although a few inhibitors have shown a broader

specificity, for instance, against serine and cysteine peptidasesor against serine andmetallopeptidases (37–39). Therefore, weassayed the inhibitory activity of panulirin against papain andcarboxypeptidase A as representative of cysteine and metallo-peptidases, respectively. Thereafter, the selectivity of panulirinwas evaluated against a wider panel of serine peptidases, whichincluded chymotrypsin, elastase, subtilisin, thrombin, and plas-min. In all cases, the inhibitory activities after a 30-min incuba-tion of an at least 100-fold molar excess (active concentration)of panulirin over each peptidase was below 10% (not shown),thus indicating that panulirin did not inhibit the enzymesassayed.Determination of the Primary Structure of Panulirin—Mo-

lecular weight determination by MS of untreated inhibitorrevealed the presence of a main protein with a molecular massof 5,367.1 Da. The first 19 cycles with no contaminating resi-dues of the N-terminal Edman degradation were clearlySYKARSXTAYGYFXMIPPR, where X represents the cysteineresidues. Furthermore, panulirin, when treatedwithDTTat pH8.0, showed a shift of mass signal of 6 mass units, which sug-gested the existence of three disulfide bridges and six cysteines.This was later confirmed by peptide alkylation of the cysteineswith carbamidomethyl groups, which added 6 times (342Da) tothe molecular mass of panulirin. Enzymatic digestions, aftercarbamidomethyl alkylation of the cysteines with trypsin orchymotrypsin, were performed. The enzymatic cleavages weremainly found in the residues Lys and Arg for trypsin and in the

FIGURE 3. Characterization of panulirin interaction with trypsin. A, time dependence of panulirin-trypsin association. Trypsin (48 �g/ml) was incubatedwith 0.9 �g/ml panulirin for 0, 5, 10, 30, and 60 min at room temperature. Trypsin activity was determined after the addition of 0.9 mM BAPNA, and the fractionalactivity (Vi/Vo) was calculated. B, substrate dependence of inhibition. Fractional activity at constant concentrations of trypsin (80 nM) and panulirin (40 nM) wasobtained at substrate concentrations corresponding to 0.5, 1.0, 1.5, and 2.0 Km. C, determination of panulirin active site concentration. Trypsin (1.5 �M) wasmixed with increasing volumes (10 –100 �l) of panulirin at 22.5 �M, and the residual trypsin activity was determined after adding 0.9 mM BAPNA. The linearportion of the plot of fractional activities versus inhibitor volume was fitted to a line. The active inhibitor concentration was obtained from the intercept withthe x axis, which represents the equivalence point where the concentration of inhibitor equals the enzyme. The graphs include the means � S.D. (error bars) ofat least four replicates. D, determination of the inhibitor dissociation constant (Ki). A constant concentration of trypsin (80 nM) was mixed with increasingconcentrations of panulirin (2.4 –288 nM), and trypsin activity was determined after the addition of 0.9 mM BAPNA. The connecting line represents the best fit tothe quadratic Morrison equation for tight binding inhibitors. The Ki

app calculated from the fit was 16.3 � 1.43 nM (S.E.). Vi/Vo represents the fractional activity inthe presence (Vi) and in the absence of inhibitor (Vo).




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residues Trp, Phe, Ile/Leu, and with good frequency in Arg forchymotrypsin. The manual interpretation of such tryptic andchymotryptic MS/MS spectra revealed the amino acidsequences of the ion species [680.30, 3�], [600.30, 2�], [636.30,3�], [861.89, 2�], [505.95, 3�], and [840.40, 2�] for trypsinand [489.25, 4�], [363.85, 3�], [690.80, 2�] and [481.70, 4�]for chymotrypsin (Fig. 4A). Additionally, the enzymatic diges-tionwith endoproteinaseGlu-Cwas incomplete, suggesting theabsence of glutamic acid in the primary structure. Furthermore,the interpretation of the partial acid hydrolysis MS/MS spectradisclosed the amino acid sequences of ion species [769.35, 4�]and [625.26, 3�] (Fig. 4A). Finally, the N-terminal sequence ofa tryptic fragment with amolecular mass of 1,550.2 Da gave theamino acid sequence of the first 10 residues (ARGHIXXSSP)that help to reveal the presence of an Ile residue and to con-firm the primary structure of panulirin (Fig. 4A). The com-plete amino acid sequence of panulirin has been deposited inthe Swiss Protein database Uniprot with accession numberB3EWX6.Sequence Analysis—Sequence similarity search at NCBI

databases using BLASTP with default parameters retrievedonly three hits, which corresponded to hypothetical proteinsfromAspergillus niger, with bit score of 32 andExpect (E) valuesgreater than 6. Thereafter, we searched at the MEROPS data-base, which is devoted to peptidases and peptidase inhibitors(40). The hits retrieved showing closer sequence relationshipswith panulirin were unassigned peptidase inhibitor homo-logues belonging to the I63 family according to the MEROPSclassification (39). However, no significant relationship wasfound for the hits retrieved because the lower E value obtainedwas 0.67. According to theMEROPS developers (41), to includea sequence in a family it must be related directly or indirectly

(transitive relationship) to the type-example of the family in astatistically significant way (i.e. E value below 10�10) in thealignment using BLAST on the MEROPS database. Therefore,these results suggest that panulirin represents a new family ofpeptidase inhibitors.Sequence analysis using the ProtParam tool revealed that

panulirin is a basic peptide with a theoretical pI of 9.5, aliphaticindex of 42.7, and extinction coefficient of 11.8 mM�1 cm�1,assuming that the three pairs of Cys residues form cystines.Later, several sequences showing high identities (�60%)with

panulirin were found at the expressed sequence tag databasefrom the hemocytes of the spiny lobster Panulirus japonicus.An ORF identified in the PJ_EST01_03A01mRNA coding for aputative P. japonicus trypsin inhibitor (PjTI1) was deposited asa third party annotation in GenBankTMwith accession numberKC154047. The N terminus of the translated sequence showedproperties attributable to a signal peptide, as assessed by theSignalP program, with a predicted cleavage site locatedbetween positions 22 and 23 (VHG-DP). The TBLASTN2.2.25� homology comparison between panulirin and PjTI1showed 65.9 as amaximal score value covering 93% of panulirinsequence (Fig. 4B).Influence of Panulirin on PO Response—We studied first the

PO response to LPS in the LHL. Surprisingly, it was found that,conversely to that described in other arthropods, the phe-noloxidase activity increased slightly in the presence of LPS(Fig. 5A). 10-Fold or lower concentrations of LPS producednegligible activity compared with the control (not shown). Alsoin Fig. 5A, it can be observed that the progress curve of thereaction showed a lag phase, which is probably due to the cas-cade mechanism of the proPO system. The lag phase mightrepresent the time required since the activation of the system

FIGURE 4. Elucidation of the primary structure of Panulirin. A, amino acid sequence of panulirin elucidated by N-terminal Edman degradation and by MS/MSsequencing of enzymatic and chemical cleavages. B, sequence similarity of panulirin and the putative mature peptide of PjTI1 from the complete sequencemRNA (GenBankTM accession number KC154047) from P. japonicus hemocytes. a, PjTI1 (P. japonicus trypsin inhibitor 1) corresponds to the putative maturepeptide from panulirin precursor with GenBankTM protein ID AGE44005, translated from the complete sequence mRNA (GenBankTM accession numberKC154047) from P. japonicus hemocytes. Panulirin and PjTI1 are in an alignment format that shows identical residues, conserved substitutions, and semicon-served substitutions, respectively. b, the primary structures were obtained based on the experimental masses from the MS/MS spectrometric analysis and theN-terminal Edman degradation. The positions of the cysteines are represented in boldface type and gray shading, whereas the basic amino acids are in boldfacetype and light gray shading. The underlined sequence in B was used to construct the panulirin model. c and d, theoretical and experimental molecular masses,respectively.




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until the conversion of proPO into active PO, the final compo-nent of the cascade. This behavior rules out the possibility ofLHL contamination as a possible cause of the small differencefound between the response to LPS and LPS-free water.We also tested the PO response to LPS in the F1 fraction,

which is devoid of trypsin-inhibitory activity (see Fig. 2B). ThePO response to LPS increased significantly in the F1 fractioncompared with LHL under similar experimental conditions(Fig. 5B), suggesting that panulirin might be involved in regu-lating the phenoloxidase response to LPS. In addition, the phe-noloxidase activity found in F1 confirmed our assumption thatcomponents of the proPO system responsible for recognizingmicrobial elicitors leading tomelanization response in the LHLall have a molecular weight mass above 30 kDa.However, it is conceivable that factors regulating the PO

response other than panulirin (such as peptidase inhibitors,melanization inhibition proteins, or phenoloxidase inhibitors),although currently unknown in Panulirus argus, were alsoabsent from F1, helping to explain the differences in PO activ-ity. To ascertain whether the increment on PO response to LPSwas due to the lack of panulirin, F1was incubatedwith constantLPS concentrations and 2-fold dilutions of decreasing concen-trations of panulirin for 15 min at room temperature beforedetermining PO activity. It was found that PO response to LPS

decreased in a dose-response fashion with increasing panulirinconcentration, whereas controls without LPS remained similarfor each inhibitor concentration (Fig. 6). This result suggeststhat panulirin is implicated in the regulation of peptidase(s)that are in the pathway toward the activation of proPO into POand therefore involved in the regulation of the proPO-activat-ing system in the spiny lobster.

DISCUSSION

Peptidases intervene in several immune response mecha-nisms of invertebrates, such as coagulation, melanization, acti-vation of theToll receptor, and complement-like reactions (42).Because the presence of peptidases in biological systems usuallyimplies the occurrence of peptidase inhibitors to maintainhomeostasis (43), peptidase inhibitors to control such pro-cesses are likely to occur (e.g. avoiding unnecessary activation ofPO zymogen).Peptidase inhibitors from the Kazal, serpin, Kunitz, �-mac-

roglobulin, and pacifastin families have been identified inarthropods so far, and some are thought to be involved inimmunity (43–46). However, the exact physiological functionof most of them remains unknown.We have previously reported the presence of trypsin-inhibi-

tory activity in the hemocytes of the spiny lobster P. argus (25).Recent evidence indicates the existence of genes encodingKazal, Kunitz, and serpin type inhibitors in the hemocytes of aclosely related species, the spiny lobster Panulirus japonicus(47). Therefore, it is conceivable to expect the occurrence ofthese inhibitors also inP. argus. In the current study,we presentthe finding and purification of a trypsin inhibitor from thehemocytes of P. argus, but surprisingly, it showed no sequencesimilarity with any known protein. Because it has been sug-gested that the finding of a new peptidase inhibitor with nosequence homology to any existing inhibitor family will lead tothe building of a new family (41), we propose that this protein,named here panulirin, represents a new family of peptidaseinhibitors.In this sense, it is worth mentioning that the first systematic

organization of peptidase inhibitors in families was accom-

FIGURE 5. Phenoloxidase activity in lobster hemocyte lysate. A, phenoloxi-dase response to LPS of LHL. B, phenoloxidase response to LPS of the fractioneluted at the void volume from gel filtration chromatography of LHL (F1),which lacks panulirin and other molecules below 30 kDa. For each assay, LHL(250 �g/ml) and F1 (25 �g/ml) were mixed with 150 �l of 50 mM Tris-HCl, pH7.5, 50 mM CaCl2, and 50 �l of 0.1 mg/ml LPS or LPS-free water as a control. Thephenoloxidase activity was measured kinetically at 490 nm and 37 °C usingdopamine as substrate.

FIGURE 6. Influence of panulirin on phenoloxidase response to LPS. Con-stant concentrations of the F1 fraction (25 �g/ml) were incubated with 150 �lof 50 mM Tris-HCl, pH 7.5, 50 mM CaCl2, and decreasing concentrations ofpurified panulirin for 15 min. LPS (0.1 mg/ml) or LPS-free water (Control) wasadded, and the phenoloxidase activity was measured continuously at 490 nmand 37 °C immediately after the addition of dopamine. Values represent theaverage of three replicates plus S.D. (error bars).




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plished for standard mechanism inhibitors of serine peptidases(48). This was mainly based on primary sequence homology,although the topology of disulfide bridges and their relation-ship to the reactive site were also considered (37, 48, 49). Cur-rently, protein inhibitors of peptidases are organized in a hier-archical classification system that attempts to overcome somedisadvantages of previous classification approaches (39–41).The system is composed of three main levels: inhibitor unit,family, and clan. A family consists of protein sequences that arehomologous, whereas membership of a clan is determined bysimilarities in protein tertiary structures, and hence all of themembers of a clan will share a similar protein fold despite lim-ited sequence similarity (39–41). This classification system isimplemented at the MEROPS database, which is now widelyused.A putative trypsin inhibitor from P. japonicus (PjTI1;

GenBankTM accession number KC154047) showing highhomology with panulirin was identified by reverse searching inan expressed sequence tag database. The high value in identityfound (64%) between panulirin and PjTI1 supports our primarysequence elucidation results and strongly indicates the occur-rence of genes encoding panulirin-like inhibitors in the Panu-lirus genus. Interestingly, both the signal peptide and the cleav-ing site predicted in PjTI1 are highly similar to those describedin defensin-like peptides from P. japonicus (50) and P. argus(26).Panulirin is constitutively expressed in the hemocytes of

P. argus at �3% of total protein. It is a non-glycosylated basicpeptide composed of 48 amino acid residues containing six cys-teine residues forming disulfide bridges. The small protein sizeresembles that ofKunitz (51) andpacifastin-inhibitory domains(52), whereas the presence of six cysteine residues formingdisulfide bridges is a typical feature found across most inhibi-tory units of serine peptidase inhibitors, regardless of the familyor the inhibitory mechanism (39, 48, 52, 53).Three different types of natural protein inhibitors of serine

peptidases can be distinguished based on their mechanism ofaction: standard mechanism canonical inhibitors, non-canoni-cal inhibitors, and serpins (38, 54).We found that panulirin is areversible, competitive, and tight-binding inhibitor of trypsin.Hence, it should be either a canonical or non-canonical inhib-itor. The serpin possibility was ruled out because they aremuchlarger proteins (350–500 amino acid residues), which interactwith the cognate peptidase as irreversible suicide substratesthrough a trapping mechanism (38, 55, 56). It has been statedthat standard mechanism inhibitors are by far the most preva-lent of the three classes of inhibitors of serine peptidases (54). In2004, the MEROPS database grouped 48 families of peptidaseinhibitors, of which 19 were serine peptidase inhibitors thatobey the standard mechanism (39). Since then, around ninenew families of inhibitors that probably act through such amechanism have been added (41). On the other hand, the non-canonical inhibitors are much less abundant, and the few exist-ing are solely known for thrombin and factor Xa (38, 57). Tak-ing the above together, it is reasonable to suggest that panulirinis a standard mechanism canonical inhibitor. However, con-firming this assumption will require further studies. Standardmechanism canonical inhibitors interact with the cognate

enzyme through an exposed binding loop of convex shape hav-ing similar or canonical conformation,which is complementaryto the concave active site of the enzyme (37, 38, 49, 53, 54). Theloop is made up of 6–11 contiguous amino acid residues (thereactive site region) (54). Its central part contains the mostexposed P1–P1� peptide bond (Schechter and Berger nomen-clature (58)), called the reactive site, which is recognized by thepeptidase in a substrate-like manner (37, 38, 55).Panulirin did not inhibit papain and carboxypeptidase A,

which were studied as type examples of cysteine peptidases andmetallopeptidases, respectively. It is widely accepted that inhib-itors of serine peptidase rarely exceed this mechanistic class(34, 37–39). On the other hand, selectivity of serine peptidaseinhibitors against different serine peptidases is usually broader(37). In this sense, it was found that panulirin did not inhibittypical non-trypsin-like serine peptidases such as chymotryp-sin, elastase, and subtilisin. This result was not completelyunexpected because the reactive site (P1 residue) of serine pep-tidases inhibitors is usually the primary specificity determinant,being Arg or Lys in most trypsin-like inhibitors (53). However,panulirin did not inhibit trypsin-like peptidases, such as throm-bin and plasmin, indicating a notable selectivity of panulirinamong these closely related enzymes. In the case of canonicalinhibitors, this ability could be explained by the negative influ-ence of the inhibitor scaffolding (i.e. the remainder of the inhib-itor molecule other than the reactive site region, which isknown may influence the inhibitory specificity toward relatedpeptidases) (54).Because the panulirin sequence presents twoLys and fiveArg

residues, the reactive site (P1) position cannot be predictedwith certainty without structural or experimental evidence.However, at this moment, some speculation is tempting at leastfor suggesting the most likely candidates. Although it is knownthat the reactive site region is the most variable region in theprimary sequence of serine peptidase inhibitors, even amongthose belonging to the same family (53, 59), several amino acidresidues at certain positions around the reactive site are con-served in one or more families (38, 48, 60, 61). Thus, we wereencouraged to conduct a visual inspection of those conservedresidues along the panulirin sequence. Considering that panu-lirin is probably a canonical inhibitor, the Lys3 and Arg5 resi-dues from the N terminus and Lys47 from the C terminus werediscarded from the analysis because their location near the cen-ter of a loop is improbable. This first approach reduced theputative P1 position to Arg19, Arg21, Arg31, and Arg33 residues.The BPTI, antistasin, elafin, arrowhead, hirustasin, and che-

lonianin families present Cys at P2 (38, 60), which occurs in theputative P1 Arg21 and Arg31 of panulirin. It is also known thatAla andGly residues are verywell conserved in P1� of sequenceshomologous to BPTI (62), being more likely Ala (61). Three ofthe putative P1 position residues (Arg21, Arg31, and Arg33)matched this sequential motif in the panulirin sequence. Con-sidering both observations, it was noticeable that putative P1sites, Arg21 andArg31, have P2 and P1� amino acid residues thatare conserved in the BPTI family. Surprisingly, we also found amarked similarity between the regions Cys-Lys-Ala-Arg(P2P1P1�P2�) of BPTI and the panulirin sequence fragment




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Cys30-Arg31-Ala32-Arg33, with a Lys 3 Arg31 isofunctionalsubstitution.Considering the apparent similarity described above and that

residues P3–P3� better describe the length and convex shape ofthe loop in canonical inhibitors (60), we selected for furtheranalysis the Arg31 residue in panulirin sequence as the putativeP1 and thus the Trp-Cys-Arg-Ala-Arg-Gly fragment as theputative P3–P3� reactive site region. It was found that this frag-ment had no significant homology to any inhibitory unitbecause no hits were retrieved using BLAST at the MEROPSdatabase, even at E values as high as 100. Similar results wereobtained with larger sequence fragments (P7–P7�). This resultcould be explained by the hypervariability in primary sequenceof the reactive site regions (59, 63, 64). Furthermore, this frag-ment lacks Pro at P3, which, alongwithCys at P2, is a distinctivecombination found in the BPTI family (60). Therefore, the sim-ilarity found could be rather fortuitous butmight deserve futureconsideration. On the other hand, the reactive site region ofBPTI is located toward the N terminus, whereas the sequencefragment analyzed in panulirin tends more to the C terminus.A few other specific residues conserved in other families

were also found. For instance, the putative P1 position Arg21has Pro in P3, which is conserved among soybean trypsin inhib-itors (Kunitz) (38, 60), or the presence of Ser at P1� of the sameArg21 that, alongwith Pro at P3�, absent in Arg21, are conservedresidues in the first and second domains of Bowman-Birkinhibitors (60, 61). However, these minor similarities are diver-gent and less relevant at this moment.The presence of conserved residues in the reactive region has

usually served as a family signature and has helped to classifystandardmechanism inhibitors within known families (54).Wefound that the P1 site could not be deduced with acceptableconfidence through comparisonwith other families, thus estab-lishing similarities with them, or vice versa. The main reasoncould be that panulirin represents a new family that is still onlypartially characterized. Once the P1 site becomes determinedexperimentally, the analysis of the reactive region will allowproper identification of the conserved residues, if any, sharedwith other inhibitor families. In addition, the expected appear-ance of new protein sequences encoding panulirin-like inhibi-tors will help to identify conserved residues within this family.Finally, a panulirin model was constructed to assess its puta-

tive structure (Fig. 7). The core of 34 residues (the inner lengthbetween the cysteines at the ends plus the two adjacent serineresidues; see underlined sequence of panulirin in Fig. 4B) ofpanulirin was modeled using the ESyPred3DWeb server basedon the solved structure of the bovine neutrophil �-defensin 12(Protein Data Bank code 1bnb). This template shares 30.8%identities with panulirin using the ALIGN program (65). Themodel was refined with the program SSBond (66) to predict thedisulfide bonds, which were Cys7–Cys37, Cys14–Cys30, andCys20–Cys38. This disulfide pattern has been observed in �-de-fensins and other defensin-like peptides (i.e. antimicrobial pep-tide tachystatin A from horseshoe crab, defensin-like peptidesfrom platypus, and �-defensins 2 and 3 from humans), whichare part of the innate defensive system of animals. Panulirinprobably folds in a core of 31 residues, leaving two tails of 6 and10 residues at theN andC termini. Themodel showed that side

chains from Arg21 and Arg31 residues are solvent-exposed,whereas in Arg33, the side chain is toward the protein core andhence probably not accessible to the protease reactive site.On the other hand, the strong positive charge of panulirin

could explain several experimental findings of this study. First,it could be the cause of the discrepancy between the molecularmass found by gel filtration and ESI-MS (5 kDa) and that foundby reversed zymography (16 kDa). In other experiments usingSDS-PAGE, we found molecular masses of around 12 kDa (notshown). This difference could be due to the well known anom-alous behavior showing basic proteins on SDS-PAGE-basedelectrophoresis.In addition, such a high positive charge seems to be respon-

sible for panulirin interaction with nucleic acids in the LHL,causing the trypsin-inhibitory activity to go unnoticed or beunderestimated if the ionic strength in the lysis buffer is unsuit-able.Wehave even found that the peak of inhibitor at 280 nm inthe gel filtration chromatography (Fig. 2A) disappears in similarchromatographic conditions if the LHL is prepared in a bufferlacking NaCl (not shown). The above findings suggest that theelectrostatic interaction between panulirin and the nucleicacids in the LHL could be eventually stronger than its affinityfor trypsin, even when our results indicated that panulirinbound to trypsin with relatively high affinity. Previous studieshave shown that nucleic acids bind to cationic peptides and thatthe salt concentration influences such interaction (67, 68).However, the in vitro panulirin-nucleic acid interactionobserved in this work probably has no repercussions for thebiological role of panulirin.The major contributions to knowledge of the proPO system

of crustaceans have been made by the group of Söderhäll in the

FIGURE 7. Schematic three-dimensional representation of panulirin. Thestructure backbone was achieved by homology modeling. The core of 34residues (the inner length between the cysteines at the ends) shows two�-strands stabilized by three disulfide bridges in the arrangement Cys1-Cys5,Cys2-Cys4, and Cys3-Cys6. The residues Arg21, Arg31, and Arg33 are shown. Themodel structure was obtained using ESyPred3D and drawn with PyMOL.




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fresh water crayfish P. leniusculus, and therefore it has beenwidely used for building a general model of melanization cas-cade in crustaceans (2–4), where the regulatory role of pacifas-tin on the proPO system is depicted. Recently, cDNAs encodingpacifastin-related peptides have been described in the Chinesemitten crab Eriocheir sinensis (69) and in the swimming crabPortunus trituberculatus (70). They also seem to be implicatedin the immune response because they are up-regulated uponmicrobial challenge, although their role in the proPO-activat-ing system remains to be established. On the other hand, mem-bers of the pacifastin family arewidely found among insects (45,71), but interestingly, they are not involved in the regulation ofthe melanization cascade (72).Our knowledge aboutmolecular components of the humoral

immune response in the spiny lobster is still rather limited. Inthe current study, we have found that PO response to LPS islower than that reported in other crustaceans like shrimps (73),black tiger prawns (74), and crayfish (75). The substantialincrease in sensitivity of PO response to LPS in the LHL fractiondepleted of trypsin-inhibitory activity and other moleculesbelow 30 kDa obtained by gel filtration chromatography (F1)indicated that LPS considerably activate the proPO system ofP. argus, but such a response is tightly regulated. Our resultsalso demonstrated that panulirin is very involved in the regula-tion of PO response (see Fig. 6), althoughwe cannot rule out thepresence of other regulatory factors acting on phenoloxidase,peptidases, or the melanization reaction.The in vivo significance of such a tight regulation of the

melanization cascade in the LHL of spiny lobster is yetunknown and suggests the need for further studies, especially ifwe consider the well known importance of melanizationresponse to host defense. One conceivable cause could berelated to hemocyanin-derived phenoloxidase activity. It isknown that hemocyanin from P. argus exerts phenoloxidaseactivity when it is partially hydrolyzed by trypsin (76). Further-more, it has even been suggested that hemocyanin-derived phe-noloxidase activity may be involved in host defense (77). Con-sidering the aforementioned, it is conceivable that ppA or anyother serine peptidase released from the hemocytes to theplasma in response to amicrobial stimulus is capable of activat-ing hemocyanin into phenoloxidase, thus contributing to theoverall melanization reaction. In this probable scenario, tightercontrol of peptidase activity may be required to avoid excessivemelanization and its potential deleterious effects to the host.The ppA in P. argus seems to be a calcium ion-dependent

trypsin-like serine peptidase (25). At this time, we suggest thatpanulirinmay inhibit this enzyme.However, further studies arerequired to ascertain whether panulirin inhibits the calcium-dependent ppA or other serine peptidase, if any, upstream ofthe cascade. It isworthmentioning that, unlike in insects, serinepeptidase upstream of ppA in the ProPO cascade has not yetbeen identified in crustaceans, although its presence has beeninferred in P. leniusculus (4). Once purified ppA or any otherserine peptidase involved in the melanization response inP. argus becomes available, it will be possible to evaluate therelative affinity with panulirin and to identify where and howpanulirin regulates the proPO activation pathway.

Because ppA in crustaceans are trypsin-like serine pepti-dases, commercial trypsin frommammals has beenwidely usedto activate proPO in vitro in several investigations pursuing theisolation and characterization of proPO (78–81). We havefound previously that higher amounts of trypsin are required toactivate prophenoloxidase in P. argus (25). Considering the sig-nificant presence of panulirin in the LHL, it is likely that thisbehavior was mostly due to the inhibitory activity of panulirin,which impose a higher concentration of trypsin to accomplishcomplete proPO activation.Since the discovery of pacifastin more than 20 years ago (23,

24) andup to the present report, the regulatory role of peptidaseinhibitors in the proPO-activating system of crustaceans hasnot been proven for inhibitors other than pacifastin, althoughserpins have been recently suggested to also regulate the proPOcascade in crustaceans (82). However, until now, the role ofserpins in regulating melanization cascade has been truly dem-onstrated only in insects (16). We have described here for firsttime how panulirin, a novel peptidase inhibitor, is involved inthe regulation of the proPO cascade in lobsters. The results ofthis study, together with the recent discovery of novel genesencoding defensin-like antimicrobial peptides in lobsters(26, 50), could indicate that Panulirus can be an attractivegenus for deepening understanding of the immune system incrustaceans.

Acknowledgments—We thank Leandro Rodríguez Viera and LazaroMacías for support in the capture and maintenance of lobster in cap-tivity. Also, we acknowledge Timoteo Olamendi-Portugal for assist-ance during the N-terminal degradation of panulirin.

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Porto-VerdeciaYamile Vega-Hurtado, Yamile González-González, Erick Perera and Marlene

Rolando Perdomo-Morales, Vivian Montero-Alejo, Gerardo Corzo, Vladimir Besada,Panulirus argusin the Spiny Lobster

The Trypsin Inhibitor Panulirin Regulates the Prophenoloxidase-activating System

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