Porto-Verdecia Marlene Gonzalez-Gonzalez, Erick ...

Immunology: The trypsin inhibitor panulirin regulates the prophenoloxidase activating system in the spiny lobster Panulirus argus Rolando Perdomo-Morales, Vivian Montero-Alejo, Gerardo Corzo, Vladimir Besada, Yamile Vega-Hurtado, Yamile Gonzalez-Gonzalez, Erick Perera and Marlene Porto-Verdecia J. Biol. Chem. published online September 18, 2013

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JBC Papers in Press. Published on September 18, 2013 as Manuscript M113.464297 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M113.464297 Novel trypsin inhibitor regulates the immune response in lobsters

The trypsin inhibitor panulirin regulates the prophenoloxidase activating system in the spiny lobster Panulirus argus* Rolando Perdomo-Morales1, Vivian Montero-Alejo1, Gerardo Corzo2, Vladimir Besada3, Yamile Vega-Hurtado1, Yamile González-González4, Erick Perera5, Marlene Porto-Verdecia1 1

From the Biochemistry Department, Center for Pharmaceuticals Research and Development, Ave. 26 No. 1605 e/ Ave 51 y Boyeros, Plaza, CP 10400, La Habana, Cuba

2

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos 62250, México. 3

Department of Proteomics, Centre for Genetic Engineering and Biotechnology, P.O. Box 6162, La Habana, Cuba.

4

Biochemistry Department, Federal University of Sao Paulo, rua 3 de maio 100, CEP 04044-020, Sao Paulo, Brazil.

5

Center for Marine Research, University of Havana, Calle 16 No. 114 e/ 1ra y 3ra, Miramar, Playa, CP 11300, La Habana, Cuba *Running title: Novel trypsin inhibitor regulates the immune response in lobsters

To whom correspondence should be addressed: Rolando Perdomo-Morales. Ave. 26 No. 1605 Esq. Puentes grandes. Plaza. CP 10400. La Habana. Cuba. Fax: 537-833-5556; E-mail: [email protected] and [email protected] Keywords: prophenoloxidase activating system; melanization; protease inhibitor; serine proteases; crustacean; innate immunity; phenoloxidase 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, was found in lobster that inhibits the melanization response to lipopolysaccharides. Conclusion: Panulirin regulates serino-peptidases in the pathway towards the activation of the prophenoloxidase enzyme. Significance: Serine peptidase inhibitors play a key role controlling the immune response in arthropods.

finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined combining Edman degradation N-terminal sequencing and ESI-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (Ki = 8.6 nM) with a notable specificity as it doesn’t inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin and plasmin. The removal of panulirin from the lobster hemocyte lysate (LHL) leads to an increase in phenoloxidase

ABSTRACT The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms which are still not fully understood. We report here the 1

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Novel trypsin inhibitor regulates the immune response in lobsters a large noncovalent complex, which localizes the melanization to the surface of invading microorganisms or at the injury site (8-10) . This complex assures local high concentration of quinone products where necessary, whereas avoids their dissemination. In addition, the stickiness of activated phenoloxidase promotes its deposition on pathogen or anomalous surfaces where it assist localized melanization (5). The melanin formation can be controlled even at late stages of the melanogenesis. For example, the presence of melanization inhibition proteins (MIPs) has been described in plasma from crustaceans (11) and insects (12). These proteins hamper the synthesis of melanin from quinones, but have no effect on PO enzyme activity. Interesting, MIPs from crustacean and insects share similar molecular weight (43 kDa) 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 mechanisms to control their activities once they become active. Several phenoloxidase inhibitors (POI) have been characterized in insects (13-15). Also in insects, genetic evidences support that serpins-type peptidase inhibitors are involved in regulating the melanization cascade (16). Though, the peptidases inhibited by serpins have been only identified in the beetle Tenebrio molitor (17) and the tobacco hornworm Manduca sexta (16). Serpins regulate the proPO system in insects by inhibiting both the ppA (18-20), as well as peptidases upstream the ppA in the cascade (10,16,21). Until the present report, pacifastin from the crayfish Pacifastacus leniusculus (22,23) was the only known peptidase inhibitor regulating the proPO system in crustacean, despite that this system has been investigated in a variety of crustaceans for decades. Pacifastin regulates the activity of the ppA. It is a heterodimeric protein (155 kDa) composed of two covalently linked subunits each encoded by two different mRNAs. The light chain (44 kDa) contains the inhibitory domains, while the heavy chain (105 kDa) is rather related to transferrins (24).

response to LPS. Likewise, the addition of increasing concentration of panulirin to a LHL, previously depleted of trypsin inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme. Invertebrates lack adaptive immunity and their protection against pathogens relies mainly on innate immunity mechanisms, which could be divided into closely-related humoral and cellular defense responses (1,2). The prophenoloxidase (proPO)-activating system is a major humoral defense mechanism in arthropods involved in a series of responses such as melanization, encapsulation, cytotoxic reactions and phagocytosis (for reviews see (1-4)). In general, it could be composed by pattern recognition proteins, trypsin-like serine peptidases, serine peptidase homologues, and the prophenoloxidase (4). Minute amounts of pathogen-associated molecular patterns such as lipopolysaccharides (LPS), peptidoglycan and β1,3-glucans activates the proPO cascade leading ultimately to the activation of the prophenoloxidase activating enzyme (ppA), a trypsin-like serine peptidase that converts the phenoloxidase zymogen (proPO) into active phenoloxidase (PO) (4). Phenoloxidase (EC 1.14.18.1) catalyzes the hydroxylation of monophenols to o-diphenols (monophenolase or cresolase activity), and the oxidation of odiphenols to o-quinones (o-diphenolase or catecholase activity), therein initiating the synthesis of melanin (5). Several cytotoxic molecules are produced during melanogenesis, which have recently been demonstrated effective to combat infections (4,6). These intermediates can be also highly deleterious to the host if are produced uncontrollably (5,7). Therefore, arthropods have developed several means to regulate the melanization reaction, both spatially and temporally, to avoid damage to the host. Location of the PO response seems to be an important regulatory mechanism for insect defense against infections (8). In this sense, components of the proPO-activating system may associate to form 2

Novel trypsin inhibitor regulates the immune response in lobsters obtained from the fourth walking leg coxa using sterile and precooled modified Alsever anticoagulant solution (AC) containing 27 mM sodium citrate, 115 mM glucose, 336 mM NaCl, 9 mM EDTA, pH 7 (26). The hemolymph was centrifuged immediately after collection at 700 g for 10 min at 4 °C and the supernatant discarded. The hemocyte pellet was washed twice with cold AC, suspended in the corresponding lysis buffer (see particular experiments below), and disrupted by sonication 3 times at 40 W for 10 s each. The clarified lobster hemocyte lysate (LHL) was obtained by centrifuging the homogenate at 4,000 g for 30 min at 4 °C. Determination of trypsin inhibitory activity− Trypsin activity was determined using 0.9 mM BAPNA (1 x Km) as substrate (27). Nominal trypsin concentration was determined at 280 nm using an extinction coefficient (E1%280) of 14.4 (28), and 23.3 kDa molecular weight. Stock solution of bovine trypsin was prepared at 10 mg/ml in 1 mM HCl, 20 mM CaCl2, pH 3, while stock solution of BAPNA (125 mM) was in DMSO. For the assay, 20 µl of trypsin were mixed with 180 µl of assay buffer (0.1 M Tris-HCl, pH 8, 150 mM NaCl, and 20 mM CaCl2) in a well of 96well plate. The reaction was started by the addition of 50 µl of 4.5 mM BAPNA in the assay buffer. The pNA released was measured kinetically at 405 nm for 10 min at 37 °C in a microplate reader ELx808 IU (BioTek Instruments, Winooski, VT). Initial velocities were obtained using the kinetic application of the program KC4 v. 3.4 (BioTek Instruments, Winooski, VT, USA). One unit of trypsin activity was defined as the amount of trypsin causing the release of 1 μmol of pNA per minute. The extinction coefficient of pNitroanilide at 405 nm for a volume of 250 µl was 6.8 mM-1 as determined empirically. Trypsin inhibitory activity was calculated at dilutions where the inhibitory percent fell between 25 and 70%. One unit of inhibitory activity (IU) was defined as the amount of inhibitor producing 50% inhibition of two units of the trypsin (29). Specific activity was defined as the inhibitory activity per mg of protein. Where indicated, the fractional activity (Vi/Vo) was determined as the ratio between the initial velocity in presence (Vi) and absence (Vo) of inhibitor. Protein concentration determination−The protein concentration was determined by the

In our first study on proPO-activating system in spiny lobster, we indicated the presence of trypsin inhibitory activity in the hemocyte lysate (25). Here, we describe the purification, and some molecular and biological properties of a novel trypsin inhibitor we have named panulirin. The low similarity to other protein inhibitors found through amino acid sequence comparison suggests the finding of a new class of peptidase inhibitor. Panulirin is a 5.3 kDa basic peptide (pI 9.5) composed of 48 amino acid residues, which contains six cysteine residues engaged in disulfide bridges. It is a competitive, reversible and tightbinding inhibitor of trypsin. Experimental evidences indicated that panulirin is implicated in the regulation of the proPO-activating system in the spiny lobster. EXPERIMENTAL PROCEDURES Materials−Bovine pancreatic trypsin (EC 3.4.21.4), elastase from porcine pancreas type IV (EC 3.4.21.36), subtilisin A from Bacillus licheniformis (EC 3.4.21.62), bovine pancreatic chymotrypsin (EC 3.4.21.1), plasmin from human plasma (EC 3.4.21.7), thrombin from bovine plasma (EC 3.4.21.5), HEPES, N-benzoylArginine-p-Nitroanilide (Bz-Arg-pNA; BAPNA), LPS from E. Coli O55:B5, N-Succinyl-Ala-ProPhe-p-Nitroanilide and dopamine were purchased from Sigma (St. Louis, MO). 4-nitrophenyl 4´guanidinobenzoate (NPGB) was from ICN Biomedicals Inc. (Aurora, OH). DMSO, EDTA, NaCl, Tris, DTT, Acetonitrile LiChrosolv® (hypergrade for LC/MS), TFA (for protein sequence analysis grade), papain from Carica papaya (EC 3.4.22.2), Triton X-100, protamine sulfate, glucose, sodium citrate and calcium chloride were all obtained from Merck (Darmstadt, Germany). Sephadex G-50 Superfine was from Amersham Biosciences (Buckinghamshire, UK). HiTrap SP HP column, low molecular weight calibration kit for SDS electrophoresis, and low molecular weight gel filtration calibration kit were from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). The substrates S-2251 (Val-Leu-Lys-p-Nitroanilide), S-2238 (Phe-Pip-Arg-p-Nitroanilide) and S-2586 (MeO-Suc-Arg-Pro-Tyr-p-Nitroanilide) were from Chromogenix AB (Mondal, Sweden). Preparation of hemocyte lysate supernatant−The spiny lobster hemolymph was 3

Novel trypsin inhibitor regulates the immune response in lobsters performance liquid chromatography (RP-HPLC) in a Knauer Smartline HPLC system (Germany), using a Discovery BIO Wide Pore C5 column (4.6 x 250 mm, 5 µm, Supelco) equilibrated with 0.1% (v/v) TFA in water (Solvent A). The elution system comprised Solvent A and 0.07% TFA in 70% acetonitrile (Solvent B). Separation was performed with a linear gradient of acetonitrile from 5% to 70% over 45 min at a flow rate of 1 ml/min. The absorbance was monitored at 214 nm. Determination of the equilibrium dissociation constant (Ki)−The concentration of active trypsin was determined by active-site titration with NPGB (33). The time to reach the equilibrium of trypsininhibitor complex was determined by incubating fixed concentrations of trypsin (48 µg/ml) and inhibitor (0.9 µg/ml) at room temperature for 0, 5, 10, 30 and 60 min before substrate addition. The active concentration of inhibitor was determined by titration against fixed concentration of activesite-titrated trypsin (1.2 µM) 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 2.0 Km) to demonstrate substrate induced dissociation. To determine the apparent dissociation constant (Kiapp), trypsin (80 nM) was mixed with increasing inhibitor concentrations (2.4-288 nM) and the residual trypsin activity determined. The Kiapp value was calculated by fitting the experimental data to the quadratic Morrison equation for tightbinding inhibitors (35), using GraphPad Prism version 5 for Windows (GraphPad Software, San Diego, CA). Determination of inhibitor specificity−The inhibitory activity was evaluated against peptidases belonging to three mechanistic classes: metallo, cysteine and serine. The enzymatic activities were assessed at 37 ºC in a mixture composed by 20 µl of each enzyme, 180 µl of assay buffer and 50 µl of the corresponding substrate as follows: 50 nM chymotrypsin, 50 mM Tris-HCl, pH 8.0, 10 mM CaCl2, 0.05 mM S-2586; 5 nM subtilisin, 0.1 M Tris-HCl, pH 8, 0.15 mM Suc-Ala-Pro-Phe-pNa; 10 nM elastase, 0.1 M TrisHCl, pH 8, 1.2 mM N-Suc-Ala-Ala-Ala-pNa; 0.9 mM papain, 200 mM sodium acetate, pH 6.0, containing 8 mM DTT and 4 mM sodium EDTA, 1 mM BAPNA; 29 nM thrombin, 0.1 M Tris-HCl, pH 8, 0.15 M NaCl, 0.3 µM S-2238; 48 nM

Lowry method (30), using bovine serum albumin (BSA) as standard. Samples containing HEPES above the interfering level with the Lowry assay were firstly precipitated with deoxycholatetrichloroacetic acid (31). Influence of ionic strength on inhibitory activity−Hemocytes from four centrifuge tubes containing 50 ml of hemolymph: anticoagulant (1:1, v/v) were collected and washed as above, and then pooled to a final volume of 50 ml made up with AC. The homogeneous hemocyte suspension was divided into 6 ml aliquots and centrifuged. Finally, each pellet was suspended in 50 mM TrisHCl pH 7.5 containing various concentration of NaCl (from 0 to 650 mM). The hemocytes were lysed and clarified as described earlier, and the inhibitory activity of trypsin for each fraction determined. Where is indicated, nucleic acids were precipitated by adding 0.1% protamine sulfate (final concentration) to lysed hemocytes before the clarification step. Reversed zymography−Reversed zymography for detection of inhibitory activity in the hemocyte lysate was determined by SDS-PAGE in 15% polyacrylamide gel copolymerized with 0.1% gelatin (w/v) (32). Low molecular weight calibration kit composed by Phophorylase b (97 kDa), Albumin (66 kDa), Ovalbumin (45 kDa), Carbonic anhydrase (30 kDa), Trypsin inhibitor (20.1 kDa) and α-Lactalbumin (14.4 kDa) was used as standard. Purification of peptidase inhibitor−The LHL was obtained in lysis buffer containing 450 mM NaCl and treated with protamine sulfate as above. The supernatant (10 ml at 9.5 mg/ml) was fractionated by gel filtration chromatography in a Sephadex G-50 Superfine column (2.6 x 65 cm), equilibrated with 25 mM 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 collected for determining trypsin-inhibiting activity. Gel filtration column was calibrated with molecular weight standards (carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa) and aprotinin (6.5 kDa)). Pooled inhibitory fraction was applied to a 5 ml HiTrap SP HP column equilibrated with buffer A. The bound proteins were eluted with 135 ml of a lineal NaCl gradient (100-500 mM) in the same buffer at 0.5 ml/min. Protein elution was monitored at 280 nm. The inhibitory peak was further purified by reverse phase high4

Novel trypsin inhibitor regulates the immune response in lobsters (Micromass, UK) fitted with a Z-spray nanoflow electrospray ion source operated at 80 oC with a drying gas flow at 50 L/h. The analyzer was calibrated in a wide mass range (50 to 2,000 Da) using a reference mixture of sodium and cesium iodides. Intact protein and peptide digest samples were loaded into the borosilicate nanoflow tips and submitted to 900 and 35 V of capillary and cone voltage, respectively. To acquire the ESI-MS spectra, the first quadrupole was used to select the precursor ion within a window of 3 Th approximately. Argon gas was used in the collision chamber at ~3x10-2 Pa pressure and collision energies between 15 and 48 eV were set to fragment precursor ions. Data acquisition and processing were performed using MassLynx v3.5 (Micromass, UK). N-terminal sequence−Panulirin and a trypticderived peptide (1550.2 Da) were directly sequenced on a Shimadzu PPSQ-31A (Shimadzu, Kyoto, Japan) automated gas-phase sequencer. Samples were dissolved in 10 µl of a 37% CH3CN (v/v) solution and applied to TFA-treated glass fiber membranes, precycled with Polybrene (Aldrich, USA). Data were recorded using the Shimadzu PPSQ-31A software. Sequence analysis−The physical-chemical properties of panulirin were determined using ProtParam tool at http://us.expasy.org/tools/protparam.html. Influence of panulirin on PO response to LPS− PO activity was determined spectrophotometrically by recording the formation of dopaminechrome and derivatives from dopamine as substrate (36). In brief, 20 µl of 0.25 mg/ml LHL or pooled fraction eluted at the void volume from the gel filtration chromatography of the LHL (F1) at 0.025 mg/ml, was mixed in flatbottom microplate wells with 100 µl of 50 mM Tris-HCl buffer, pH 7.5, 50 mM CaCl2, and 50 µl of 0.1 mg/ml LPS. Control experiment was LPSfree water instead LPS. PO activity was assessed continuously at 490 nm and 37 ºC immediately after the addition of 50 µl of 0.6 mg/ml dopamine. In order to evaluate the influence of panulirin on the PO response to LPS, 20 µl of LHL fraction depleted of trypsin inhibitory activity (F1) at 0.025 mg/ml was incubated for 15 min with 50 µl of two-fold serial dilutions of purified panulirin (16.2 IU/ml starting inhibitory activity), and PO activity was assessed as described above.

plasmin, 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.3 mM S-2251. The pNA released was determined at 405 nm as described earlier for trypsin. In the case of carboxypeptidase A, the activity was determined kinetically at 254 nm for 3 min at room temperature in a reaction mixture composed by 85 µl of 25 mM Tris-HCl, pH 7.5, 0.5 M NaCl (reaction buffer), 1 ml of 1 mM Hippuryl-L-Phe, and 15 µl of the enzyme at 2.2 µM. All enzymatic activities were determined under initial velocity conditions. After 30 min of incubation of each enzyme with 100-fold molar concentrations of panulirin, the inhibitory activity was evaluated by determining the fractional activity using the assay conditions described above. Disulfide reduction and carbamidomethylation−Panulirin was mixed with 13.3 µl of a solution containing 0.75 M Tris, pH 8.0, 33.3 µl of 6 M guanidinium chloride and 4.5 µl of 1.3 M DTT. The reaction was incubated for 2 h at 37 °C. After incubation, 25 µl of 0.8 M iodoacetamide was added and allowed to react for 25 minutes at room temperature. The reaction was stopped with 10 µl of pure formic acid. This solution was passed through a ZipTip-C4 microcolumn (Millipore, USA) for desalting prior to enzymatic digestion. The protein was eluted with 2.5 µl of 60% acetonitrile (v/v). Enzymatic treatment−Samples of the reduced and carbamidomethylated panulirin were separately enzymatically digested with trypsin, chymotrypsin and endoproteinase Glu-C. The eluates from the Ziptip were dissolved in 60 µl of 0.19 M Tris, pH 8.0 and the enzyme was added at 50:1 (trypsin) or 25:1 (chymotrypsin) substrate: enzyme ratios, in each case. For the enzymatic treatment with Glu-C, the eluate was dissolved in 60 µl of 0.25 M ammonium hydrogen carbonate, pH 8.0, and the endoproteinase was added in a 50:1 ratio. Chemical treatment− Reduced and carbamidomethylated panulirin (10 µg) was desalted through Ziptip C4 (Millipore, USA), dried in speed-vac, and resuspended in 30 mM hydrochloric acid containing 6 M guanidine hydrochloride. The solution was transferred to a 1 ml vial (Pierce, USA) and kept under vacuum and 104 oC during 12 h. Mass spectrometry−ESI-MS and ESI-MS/MS spectra were acquired using a QTof-1TM 5

Novel trypsin inhibitor regulates the immune response in lobsters Isolation of panulirin−Panulirin was purified by gel filtration on Sephadex G-50 followed by a cation-exchange chromatography on HiTrap SPSepharose HP column (Table 1). Reverse zymography revealed a band with trypsin inhibitory activity of around 16 kDa (Fig. 2A). Hence, it was decided to fractionate the LHL first by gel filtration chromatography in Sephadex G50 (30 kDa exclusion limit), presuming that the possible targets of inhibitors in the LHL are endogenous peptidases over 30 kDa that would elute at the void volume along with other components of the proPO-activating system. A single peak of around 5 kDa showing trypsin inhibitory activity eluted from the gel filtration between two major fractions leading to a high degree of fractionation (Fig. 2B). We also found that precipitation of nucleic acids with protamine sulfate improved the resolution between the inhibitory peak and the fraction eluting at the void volume (not shown), which allowed to fractionate higher volumes of LHL per chromatographic step. The inhibitory fraction (205-242 ml) was pooled and applied to a cation-exchange chromatography. Two trypsin-inhibiting peaks were identified (Fig. 2C). The second and major peak with strongest trypsin inhibitory activity (66-74 ml) was pooled and used throughout this study. RP-HPLC analysis on a Supelco C5 column (Fig. 2D), showed that this fraction, hereafter referred as panulirin, was around 95% pure. Isolated panulirin from RPHPLC was used for N-terminal, MS and MS/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 experiments showed concave inhibition curves in the plot between the fractional activity (Vi/Vo) vs. increasing concentration of panulirin at constant trypsin and substrate concentrations, which could mean that the incubation time for association of trypsin and panulirin was incomplete or that association was completed but the inhibition was reversible with [Eo]/Ki≤ 10 (34). Therefore, the time dependence to reach the association between trypsin and panulirin was determined. The inhibitory activity remained constant since no significant differences (p>0.05) were observed in the fractional activities among each incubation time evaluated (Fig. 3A), suggesting that completed association between

RESULTS Influence of ionic strength on trypsin-inhibitory activity of the LHL−Preliminary observations lead us to suspect that the extent of trypsin inhibitory activity in the lysate could be related with the ionic strength in the lysis buffer used to prepare the LHL. Therefore, we evaluated the inhibitory activity of LHL obtained in lysis buffer containing different concentrations of NaCl (0-650 mM). A direct relationship between trypsin inhibitory activity in the LHL and NaCl concentration was found. This effect occurs up to concentrations as high as 450 mM. Further increase in NaCl concentration did not affect the inhibitory activity (Fig. 1). When an aliquot of LHL prepared in the lysis buffer without NaCl was treated with 0.1% protamine sulfate (w/v), a well-known precipitant of nucleic acids, the trypsin inhibitory activity increased from 7.4 IU/ml to 38.60 IU/ml. In a different experiment, no significant differences (p > 0.05) were found between the inhibitory 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 that protamine sulfate did not impair the inhibitory activity under the experimental condition used. Taking together, these results might suggest that trypsin inhibitors in the LHL are bound to nucleic acids, probably electrostatically, and that this binding is abrogated by high ionic strength. To confirm whether panulirin binds to nucleic acids, we isolated DNA from the hemocytes using TRI reagent according to the manufacturer instructions. Panulirin (25 µl at 8 µM) was incubated with 25 µl of 0.3 µg/µl DNA for one hour at room temperature using buffers lacking NaCl. The control experiment was also incubated but with reaction buffer instead of DNA. Thereafter, the trypsin activity was determined as described above in a mixture containing 80 nM trypsin, and 80 nM panulirin diluted from the above incubations. The inhibitory activity in the control experiment was 78%, and it dropped to 9.5% in the sample previously incubated with DNA. The corresponding DNA concentration in absence of panulirin had no influence on trypsin activity (data not shown). These results suggest that panulirin is able to bind to nucleic acids at low ionic strength. 6

Novel trypsin inhibitor regulates the immune response in lobsters chymotrypsin, elastase, subtilisin, thrombin and plasmin. In all cases the inhibitory activities after 30 min incubation of 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 enzymes assayed. Determination of the primary structure of Panulirin−Molecular weight determination by MS of untreated inhibitor revealed the presence of a main protein with a molecular mass of 5,367.1 Da. The first 19 cycles with no contaminating residues of the N-terminal Edman degradation were clearly SYKARSXTAYGYFXMIPPR, where X represents the cysteine residues. Furthermore, panulirin when treated with DTT at pH 8.0 showed a shift of mass signal of 6 mass units, which suggested the existence of three disulfide bridges and six cysteines. This was later confirmed by peptide alkylation of the cysteines with carbamidomethyl groups, which added 6 times (342 Da) to the molecular mass of panulirin. Enzymatic digestions, after carbamidomethyl alkylation of the cysteines with trypsin or chymotrypsin, were performed. The enzymatic cleavages were mainly found in the residues Lys and Arg for trypsin, and in the residues Trp, Phe, Ile/Leu and with good frequency in Arg for chymotrypsin. The manual interpretation of such tryptic and chymotryptic MS/MS spectra revealed the amino acid sequences of the ion species [680.30, 3+], [600.30, 2+], [636.30, 3+], [861.89, 2+], [505.95, 3+] and [840.40, 2+] for trypsin, and [489.25, 4+], [363.85, 3+], [690.80, 2+] and [481.70, 4+] for chymotrypsin (Fig. 4A). Additionally, the enzymatic digestion with endoproteinase Glu-C was incomplete suggesting the absence of glutamic acid in the primary structure. Furthermore, the interpretation of the partial acid hydrolysis MS/MS spectra disclosed the amino acid sequences of ion species [769.35, 4+] and [625.26, 3+] (Fig. 4A). Finally, the Nterminal sequence of a tryptic fragment with a molecular mass of 1,550.2 Da gave the amino acid sequence of the first 10 residues (ARGHIXXSSP) that help to reveal the presence of an Ile residue and to confirm the primary structure of panulirin (Fig. 4A). The complete amino acid sequence of panulirin has been deposited in the Swiss Protein data base Uniprot with accession number B3EWX6.

trypsin and panulirin was reached upon mixing, and therefore, the concave inhibition curves observed probably describe a reversible interaction. It is worth mentioning that all progress curves in this experiment were linear (data not shown). On the other hand, we found that the fractional activity increased with substrate concentration at constant trypsin and inhibitor concentrations (Fig. 3B), indicating substratedependent inhibition and thus confirming that the interaction is reversible and competitive (34). The active concentration of panulirin was determined by titration with a constant concentration of active-site-titrated trypsin (1.5 µM). At this trypsin concentration, the plot between the residual trypsin activity and inhibitor concentration track linear behavior (Fig. 3C), allowing determining an active inhibitor concentration of 22.5 µM, which corresponded to 97% of nominal concentration of purified panulirin. Having the active concentration of trypsin and inhibitor, we proceed to determine the inhibitor dissociation constant. For this purpose, constant concentrations of trypsin at 80 nM were mixed with increasing concentration of inhibitor and the residual trypsin activity determined. The concave inhibition curve obtained (Fig. 3D) indicated experimental conditions of [Eo]/Kiapp between 110 (34), which further demonstrated the reversible interaction between panulirin and trypsin. The Kiapp value obtained by fitting the data to the Morrison equation (Fig. 3C), was converted to true [S] app Ki value by the equation 𝐾i = 𝐾i �1 + � 𝐾m

taking into account the competitive nature of the inhibitor (35). The real Ki was 8.6 ± 0.81 nM indicating that panulirin binds trypsin with relatively high affinity. Inhibitory specificity− It is widely accepted that most inhibitors are specific for one of the four mechanistic classes of peptidases (37,38), although a few inhibitors have shown a broader specificity, for instance, against serine and cysteine peptidases, or against serine and metallo peptidases (37-39). Therefore, we assayed the inhibitory activity of panulirin against papain and carboxypeptidase A as representative of cysteine and metallo peptidases, respectively. Thereafter, the selectivity of panulirin was evaluated against a wider panel of serine peptidases, which included 7

Novel trypsin inhibitor regulates the immune response in lobsters observed that the progress curve of the reaction showed a lag phase, which is likely due to the cascade mechanism of the proPO-system. The lag phase might represent the time required since the activation of the system until the conversion of proPO into active PO, the final component of the cascade. This behavior rules out the possibility of LHL contamination as possible cause of the small difference found 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). The PO response to LPS increased significantly in F1 fraction compared to LHL under similar experimental conditions (Fig. 5B), suggesting that panulirin might be involved regulating phenoloxidase response to LPS. In addition, the phenoloxidase activity found in F1confirmed our assumption that component of the proPO system responsible for recognizing microbial elicitors leading to melanization response in the LHL have all a molecular weight above 30 kDa. However, it is conceivable that factors regulating the PO response other than panulirin (such as peptidase inhibitors, MIP or POI), although currently unknown in P. argus, were also absent from F1 and help explaining the differences in PO activity. To ascertain whether the increment on PO response to LPS was due to the lack of panulirin, F1 was incubated with constant LPS concentrations, and two-fold dilutions of decreasing concentrations of panulirin for 15 min at room temperature before determining PO activity. It was found that PO response to LPS decreased in a dose-response fashion with increasing panulirin concentration, while controls without LPS remained similar for each inhibitor concentration (Fig. 6). This result suggests that panulirin is implicated in the regulation of peptidase(s) that are in the pathway toward the activation of proPO into PO, and therefore involved in the regulation of the proPO-activating system in the spiny lobster.

Sequence analysis−Sequence similarity search at NCBI databases using BLASTP with default parameters retrieved only three hits which corresponded to hypothetical proteins from Aspergillus niger, with bit score of 32 and Expect (E) values greater than 6. Thereafter, we searched at MEROPS database, which is devoted to peptidases and peptidase inhibitors (40). The hits retrieved showing closer sequence relationships with panulirin were unassigned peptidase inhibitor homologues belonging to the I63 family according to MEROPS classification (39). However, no significant relationship was found for the hits retrieved as the lower E value obtained was 0.67. According to MEROPS developers (41), to include a sequence in a family it must be related directly or indirectly (transitive relationship) to the type-example of the family in a statistically significant way (i.e. E value below 10-10) in the alignment using BLAST on MEROPS database. Therefore, these results suggest that panulirin represent a new family of peptidase inhibitors. Sequence analysis using ProtParam tool revealed that panulirin is a basic peptide with a theoretical pI of 9.5, aliphatic index of 42.7, and extinction coefficient of 11.8 mM-1cm-1 assuming the three pairs of Cys residues to form cystines. Later, several sequences showing high identities (> 60%) with panulirin were found at the EST database from the hemocytes of the spiny lobster Panulirus japonicus. An ORF identified in the PJ_EST01_03A01 mRNA coding for a putative P. japonicus trypsin inhibitor (PjTI1) was deposited as third party annotation in GenBank with accession number KC154047. The N terminus of the translated sequence showed properties attributable to a signal peptide as assessed by SignalP program, with a predicted cleavage site located between positions 22-23 (VHG-DP). The TBLASTN 2.2.25+ homology comparison between panulirin and PjTI1 showed 65.9 as maximal score value covering 93% of panulirin sequence (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 phenoloxidase activity increased slightly in presence of LPS (Fig. 5A). Ten-fold or lower concentrations of LPS produced negligible activity compared to the control (not shown). Also in Figure 5A can be

DISCUSSION Peptidases intervene in several immune response mechanisms of invertebrates such as coagulation, melanization, activation of the Toll receptor and complement-like reactions (42). As the presence of peptidases in biological systems 8

Novel trypsin inhibitor regulates the immune response in lobsters usually implies the occurrence of peptidase inhibitors to maintain homeostasis (43), peptidase inhibitors to control such processes are likely to occur, e.g. avoiding unnecessary activation of PO zymogen. Peptidase inhibitors from Kazal, serpin, Kunitz, α-macroglobulin and pacifastin families have been identified in arthropods so far, and some are thought to be involved in immunity (43-46). However, the exact physiological function of most of them remains unknown. We have previously reported the presence of trypsin inhibitory activity in the hemocytes of the spiny lobster P. argus (25). Recent evidences indicate the existence of genes encoding Kazal, Kunitz and serpin type inhibitors in the hemocytes of a closely related species, the spiny lobster Panulirus japonicus (47). Therefore, it is conceivable to expect the occurrence of these inhibitors also in P. argus. In the current study we present the finding and purification of a trypsin inhibitor from the hemocytes of P. argus, but surprisingly, it showed no sequence similarity with any known protein. Since it have been suggested that the finding of new peptidase inhibitor with no sequence homology to any existing inhibitor family will lead to build a new family (41), we propose that this protein, named here panulirin, represent a new family of peptidase inhibitors. In this sense, it worth to mention that the first systematic organization of peptidase inhibitors in families was accomplished for standard mechanism inhibitors of serine peptidases (48). It was mainly based on primary sequence homology, although the topology of disulfides bridges and their relationship to the reactive site were also considered (37,48,49). Currently, protein inhibitors of peptidases are organized in a hierarchical classification system that attempt overcome some disadvantages of previous classification approaches (39-41). The system is composed of three main levels, those of inhibitor unit, family and clan. A family consists of protein sequences that are homologous, while membership of a clan is determined by similarities in protein tertiary structures, and hence all the members of a clan will share a similar protein fold despite limited sequence similarity (39-41). This classification system is implemented at the MEROPS database (http://merops.sanger.ac.uk) and is widely used nowadays.

A putative trypsin inhibitor from P. japonicus (PjTI1; AC: KC154047) showing high homology with panulirin was identified by reverse searching in an expressed sequence tag (EST) database. The high value in identity found (64%) between panulirin and PjTI1 supports our primary sequence elucidation results, and strongly indicate the occurrence of genes encoding panulirin-like inhibitors in the Panulirus genus. Interestingly, both the signal peptide and the cleaving site predicted in PjTI1 are highly similar to those described in defensin-like peptides from P. japonicus (50) and P. argus (26). Panulirin is constitutively expressed in the hemocytes of P. argus at ca 3% of total protein. It is a non-glycosylated basic peptide composed of 48 amino acid residues containing six cysteine residues forming disulfide bridges. The small protein size resembles that of Kunitz (51) and pacifastin inhibitory domains (52), whereas the presence of six cysteine residues forming disulfide bridges is a typical feature found across most inhibitory units of serine peptidase inhibitors, regardless the family or the inhibitory mechanism (39,48,52,53). Three different types of natural protein inhibitors of serine peptidases can be distinguished based on their mechanism of action: standard mechanism canonical inhibitors, non-canonical inhibitors, and serpins (38,54). We found that panulirin is a reversible, competitive and tightbinding inhibitor of trypsin. Hence, it should be either a canonical or non-canonical inhibitor. The serpin possibility was ruled out as they are much larger proteins (350-500 amino acid residues), which interact with the cognate peptidase as irreversible suicide substrates through a trapping mechanism (38,55,56). It has been stated that standard mechanism inhibitors are by far the most prevalent of the three classes of inhibitors of serine peptidases (54). In 2004, the MEROPS database grouped 48 families of peptidase inhibitors from which 19 were serine peptidase inhibitors that obey the standard mechanism (39). Since then, around nine new families of inhibitors that probably act through such mechanism have been added (41). On the other hand, the non-canonical inhibitors are much less abundant and the few existing are solely known for thrombin and factor Xa (38,57). Taking the above together, it is reasonable to suggest that panulirin is a standard 9

Novel trypsin inhibitor regulates the immune response in lobsters reactive site are conserved in one or more families (38,48,60,61). Thus, we were encouraged to conduct a visual inspection of those conserved residues along panulirin sequence. Considering that panulirin is likely a canonical inhibitor, the Lys3 and Arg5 residues from the N-terminal, and the Lys47 from the C-terminus were discarded from the analysis because their location near the center of a loop is improbable. This first approach reduced the putative P1 position to Arg19, Arg21, Arg31 and Arg33 residues. The BPTI, antistasin, elafin, arrowhead, hirustasin, and chelonianin families present Cys at P2 (38,60), which occurs in the putative P1 Arg21 and Arg31 of panulirin. It is also known that Ala and Gly residues are very well conserved in P1' of sequences homologous to BPTI (62), being more likely Ala (61). Three of the putative P1 position (Arg21, Arg31 and Arg33) matched this sequential motif in panulirin sequence. Considering both observations, it was noticeable that putative P1 sites, Arg21 and Arg31, have P2 and P1’ amino acid residues which are conserved in the BPTI family. Surprisingly, we also found a marked similarity between the regions Cys-Lys-Ala-Arg (P2P1P1'P2') of BPTI with panulirin sequence fragment Cys30-Arg31-Ala32-Arg33, with a Lys→Arg31 isofunctional substitution. Considering the apparent similarity described above, and that residues P3-P3’ describe better the length and convex shape of the loop in canonical inhibitors (60), we selected for further analysis the Arg31 residue in panulirin sequence as putative P1, and thus the Trp-Cys-Arg-Ala-Arg-Gly fragment as putative P3-P3’ reactive site region. It was found that this fragment had not significant homology to any inhibitory unit as no hit were retrieved using Blast at MEROPS database, even at E values as high as 100. Similar results were obtained with larger sequence fragments (P7-P7'). This result could be explained by the hypervariability in primary sequence of the reactive site regions (59,63,64). Furthermore, this fragment lacks Pro at P3, which along with Cys at P2, is a distinctive combination found in the BPTI family (60). Therefore, the similarity found could be rather fortuitous, but might deserve future consideration. On the other hand, the reactive site region of BPTI is located towards the N-terminus, while the sequence fragment analyzed in panulirin tends more to the C-terminus.

mechanism canonical inhibitor. However, confirming this assumption will require further studies. Standard mechanism canonical inhibitors interact with the cognate enzyme through an exposed binding loop of convex shape having similar or canonical conformation, which is complementary to the concave active site of the enzyme (37,38,49,53,54). The loop is made up 611 contiguous amino acid residues (the reactive site region) (54). Its central part contains the most exposed P1-P1’ peptide bond (Schechter and Berger nomenclature (58)), called the reactive site, which is recognized by the peptidase in a substrate-like manner (37,38,55). Panulirin did not inhibit papain and carboxypeptidase A, which were studied as type examples of cysteine and metallo peptidases, respectively. It is widely accepted that inhibitors of serine peptidase rarely exceed this mechanistic class (34,37-39). On the other hand, selectivity of serine peptidases inhibitors against different serine peptidases is usually broader (37). In this sense, it was found that panulirin did not inhibit typical non-trypsin-like serine peptidases such as chymotrypsin, elastase and subtilisin. This result was not completely unexpected since the reactive site (P1 residue) of serine peptidases inhibitors is usually the primary specificity determinant, being Arg or Lys in most trypsin-like inhibitors (53). Yet, panulirin neither inhibited trypsin-like peptidases such as thrombin and plasmin, indicating a notable selectivity of panulirin among these closely related enzymes. In the case of canonical inhibitors, this ability could be explained by the negative influence of the inhibitor scaffolding, i.e. the remainder of the inhibitor molecule other than the reactive site region, which is known may influence in the inhibitory specificity toward related peptidases (54). Since panulirin sequence presents two Lys and five Arg residues, the reactive site (P1) position cannot be predicted with certainty without structural or experimental evidence. However, at this moment some speculation is tempting at least for suggesting the most likely candidates. Although it is known that the reactive site region is the most variable region in the primary sequence of serine peptidases inhibitors, even among those belonging to the same family (53,59), several amino acid residues at certain positions around the 10

Novel trypsin inhibitor regulates the immune response in lobsters side chains from Arg21 and Arg31 residues are solvent-exposed, while in Arg33 it is toward the protein core and hence 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 molecular weight found by gel filtration and ESI-MS (5 kDa), and reverse zymography (16 kDa). In other experiments using SDS-PAGE electrophoresis we found molecular weights of around 12 kDa (not shown). This difference could be due to the wellknown anomalous behavior showing basic proteins on SDS-PAGE based electrophoresis. In addition, such high positive charge seems to be responsible for panulirin interaction with nucleic acids in the LHL, causing the trypsin inhibitory activity go unnoticed or underestimated if the ionic strength in the lysis buffer is unsuitable. We have even found that the peak of inhibitor at 280 nm in the gel filtration chromatography (Fig. 2A), disappear in similar chromatographic conditions if the LHL is prepared in a buffer lacking NaCl (not shown). The stated above suggest that the electrostatic interaction between panulirin and the nucleic acids in the LHL could be eventually stronger than its affinity for trypsin, even when our results indicated that panulirin bound to trypsin with relatively high affinity. Previous studies have shown that nucleic acids bind to cationic peptides and that the salt concentration influences such interaction (67,68). However, in vitro panulirin-nucleic acids interaction observed in this work probably have no repercussion to exert its biological role. The major contributions to knowledge the proPO system of crustaceans have been made by the group of Söderhäll in the fresh water crayfish P. leniusculus, and therefore it has been widely used for building a general model of melanization cascade in crustaceans (2-4), where the regulatory role of pacifastin on the proPO system is depicted. Recently, cDNA encoding pacifastin-related peptides haven been described in the Chinese mitten crab Eriocheir sinensis (69), and in the swimming crab Portunus trituberculatus (70). They also seem to be implicated in the immune response because are up-regulated upon microbial challenge, although their role in the proPOactivating system remain to be established. On the

A few other specific residues conserved in other families were also found. For instance, the putative P1 position Arg21 has Pro in P3, which is conserved among soybean trypsin inhibitors (Kunitz) (38,60). Or the presence of Ser at P1' of the same Arg21 that along with Pro at P3', absent in Arg21, are conserved residues in the first and second domains of Bowman-Birk inhibitors (60,61). However, these minor similarities are divergent 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 classify standard mechanism inhibitors within known families (54). We found that it cannot be suggested with acceptable confidence the P1 site through comparison with other families and thus establish similarities with them, or vice versa. The main reason could be that panulirin represents a newborn family which is still partially characterized. Once the P1 site become determined experimentally the analysis of the reactive region will allow proper identification of conserved residues, if any, shared with other inhibitor families. In addition, the expected appearance of new protein sequences encoding panulirin-like inhibitors will help identify conserved residues within this family. Finally, a panulirin model was constructed to assess its putative structure (Figure 7). The core of 34 residues (the inner length between the cysteines at the ends plus the two adjacent serine residues, see underlined sequence of panulirin in figure 4B) of panulirin was modeled using the ESyPred3D web server based on the solved structure of the bovine neutrophil beta-defensin 12 (PDB 1bnb). This template shares 30.8% identities with panulirin using the ALIGN program (65). The model was refined with the program SSBond (http://hazeslab.med.ualberta.ca/forms/ssbond.htm l) (66) to predict the disulfide bonds, which were Cys7-Cys37, Cys14-30 and Cys20-Cys38. This disulfide pattern has been observed in beta_defensins and other defensin-like peptides (i.e. antimicrobial peptide tachystatin A from horseshoe crab, defensin-like peptides from Platypus, and beta-defensins 2 and 3 from human), which are part of the innate defensive system of animals. Panulirin probably folds in a core of 31 residues leaving two tails of 6 and 10 residues at the N- and C-terminus. The model showed that 11

Novel trypsin inhibitor regulates the immune response in lobsters other hand, members of pacifastin family are widely found among insects (45,71), but interestingly there are not involved in the regulation of the melanization cascade (72). Our knowledge about molecular components of the humoral immune response in the spiny lobster is still rather limited. In the current study, we have found that PO response to LPS is lower than that reported in other crustaceans like shrimps (73), black tiger prawn (74) and crayfish (75). The substantial increase in sensitivity of PO response to LPS in LHL fraction depleted of trypsin inhibitory activity and other molecules below 30 kDa obtained by gel filtration chromatography (F1), indicated that LPS considerably activate the proPO system of P. argus, but such response is tightly regulated. Our results also demonstrated that panulirin is much involved in the regulation of PO response (see Fig. 6), although it cannot be rule out the presence of other regulatory factors acting on phenoloxidase, peptidases or the melanization reaction. The in vivo significance of such a tight regulation of melanization cascade in the LHL of spiny lobster is yet unknown and prompt for further studies, mostly if we consider the wellknown importance of melanization response to host defense. One conceivable cause could be related with hemocyanin-derived phenoloxidase activity. It is known that hemocyanin from P. argus exerts phenoloxidase activity when it is partially hydrolyzed by trypsin (76). Furthermore, it has even been suggested that hemocyaninderived phenoloxidase activity may be involved in host defense (77). Considering the aforementioned, it is conceivable that ppA or any other serine peptidase released from the hemocytes to the plasma in response to a microbial stimulus, are capable of activating hemocyanin into phenoloxidase, thus contributing the overall melanization reaction. Under this probable scenario, tighter control of peptidase activity may be required to avoid excessive melanization 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 that panulirin may inhibit this enzyme. However, further studies are required to ascertain whether panulirin inhibit the calciumdependent ppA or other serine peptidase, if any, upstream the cascade. It worth to mention that conversely to insect, serine peptidase upstream ppA in the ProPO cascade has not yet been identified in crustacean, although their presence have been inferred in P. leniusculus (4). Once purified ppA or any other serine peptidase involved in the melanization response in P. argus become available, it will be possible to evaluate the relative affinity with panulirin and to identify where and how panulirin regulates the proPO activation pathway. As ppA in crustacean are trypsin-like serine peptidases, commercial trypsin from mammals has been widely used to activate proPO in vitro in several investigations pursuing the isolation and characterization of proPO (78-81). We have previously found that higher amounts of trypsin are required to activate prophenoloxidase in P. argus (25). Considering the significant presence of panulirin in the LHL, it is likely that this behavior was mostly due to the inhibitory activity of panulirin, which impose higher concentration of trypsin to accomplish complete proPO activation. Since the discovery of pacifastin more than 20 years ago (23,24) and up to the present report, the regulatory role of peptidase inhibitors in the proPO-activating system of crustaceans has not been proven for inhibitor other than pacifastin, though serpins has been recently suggested to also regulate proPO cascade in crustaceans (82). Yet, up until know the role of serpins in regulating melanization cascade has been truly demonstrated just in insects (16). We described here for first time that panulirin, a novel peptidase inhibitor, is involved in the regulation of proPO cascade in lobster. The results of this study, together with the recent discovery of novel genes encoding defensin-like antimicrobial peptides in lobsters (26,50), could indicate that Panulirus can be an attractive genus for deepening understanding in the immune system in crustaceans.

12

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16


Somnuk, S., Tassanakajon, A., and Rimphanitchayakit, V. (2012) Gene expression and characterization of a serine proteinase inhibitor PmSERPIN8 from the black tiger shrimp Penaeus monodon. Fish Shellfish Immunol. 33, 332-341

17

Novel trypsin inhibitor regulates the immune response in lobsters Acknowledgments– We thanks Leandro Rodríguez Viera and Lazaro Macías for support in capture and maintenance of lobster in captivity. Also, we acknowledge Timoteo Olamendi-Portugal for assistant during the N-terminal degradation of panulirin. FOOTNOTES 1 To whom correspondence should be addressed: Rolando Perdomo-Morales. Ave. 26 No. 1605 e/ Ave. 51 y Boyeros. Plaza. CP 10400. La Habana. Cuba. Fax: 537-833-5556; E-mail: [email protected] and [email protected] 5 Abbreviations: BAPNA, N-benzoyl-DL-arginyl-p-nitroanilide; NPGB, 4-nitrophenyl 4´guanidinobenzoate; LHL, lobster hemocyte lysate; AC, anticoagulant solution; ppA, prophenoloxidase activating enzyme; proPO, prophenoloxidase; PO, phenoloxidase; MIP, melanization inhibition proteins; LPS, lipopolysaccharide; Phenoloxidase inhibitors (POI); BPTI, bovine pancreatic trypsin inhibitor The protein sequence data of panulirin reported in this paper appear in the UniProt Knowledgebase under accession number B3EWX6. The nucleotide and protein sequences of panulirin-like peptide from P. japonicus (PjTI1) described here appear in GenBank with accession numbers KC154047 and AGE44005, respectively. FIGURE LEGENDS FIGURE 1. Influence of ionic strength on trypsin inhibitory activity in lobster hemocyte lysate. A homogenous suspension of washed hemocyte was evenly aliquoted and each was lysed in 50 mM TrisHCl, pH 7.5 containing various concentrations of NaCl (0-650 mM). Trypsin inhibitory activities were determined at dilutions producing 25-70% inhibition of 48 µg/ml trypsin in the assay using 0.9 mM BAPNA as substrate. One unit of inhibitory activity (IU) was defined as the amount of inhibitor containing sample producing 50% inhibition of two units of trypsin. Values represent the mean of at least three replicates plus S.D. FIGURE 2. Purification of panulirin. A, Reverse zymography of LHL. Lane 1: Molecular weight marker; Lane 2: 0.08 µg of LHL loaded per well. The gel was stained with Coomassie brilliant blue R-250. B, Lobster hemocyte lysate was fractionated by gel filtration chromatography on a Sephadex G-50 superfine column (2.6 x 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 µl of each was assayed for inhibitory activity against 48 µg/ml trypsin using 0.9 mM BAPNA. C, Fractions from the gel filtration containing trypsin inhibitory activity were combined 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 cation exchange was finally purified by reversed-phase C5 column (4.6 x 250 mm) equilibrated with 0.1% (v/v) TFA in water. Bound samples were eluted with a linear gradient of acetonitrile from 5% to 70% over 45 min at 1 ml/min. The absorbance was monitored at 214 nm. FIGURE 3. Characterization of panulirin interaction with trypsin. A, Time dependence of panulirintrypsin association. Trypsin (48 µg/ml) was incubated with 0.9 µg/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 fractional activity (Vi/Vo) calculated. B, Substrate dependence of inhibition. Fractional activity at constant concentrations of trypsin (80 nM) and panulirin (40 nM) were obtained 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 µM) was mixed with increasing volumes (10 -100 µl) of panulirin at 22.5 µM and the residual trypsin activity determined after adding 0.9 mM BAPNA. The lineal portion of the plot 18

Novel trypsin inhibitor regulates the immune response in lobsters between fractional activities vs. inhibitor volume was fitted to a line. The active inhibitor concentration was obtained from the intercept with the x-axis, which represents the equivalence point where the concentration of inhibitor equals the enzyme. The graphs include the means ± S.D. of at least four replicates. D, Determination of inhibitor dissociation constant (Ki). Constant concentration of trypsin (80 nM) were mixed with increasing concentration of panulirin (2.4-288 nM), and trypsin activity determined after the addition of 0.9 mM BAPNA. Connecting line represent the best fit to the quadratic Morrison´s equation for tight binding inhibitors. The Kiapp calculated from the fitting was 16.3 ± 1.43 nM (± S.E.). Vi/Vo represents the fractional activity in presence (Vi) and in absence of inhibitor (Vo). FIGURE 4. Elucidation of the primary structure of Panulirin. A, Amino acid sequence of panulirin elucidated by N-terminal Edman degradation and by MS/MS sequencing of enzymatic and chemical cleavages. B, Sequence similarity of panulirin and the putative mature peptide of PjTI 1 from the complete sequence mRNA (GenBank Accession number KC154047) from Panulirus japonicus hemocytes. FIGURE 5. Phenoloxidase activity in lobster hemocyte lysate. A, Phenoloxidase response to LPS of lobster hemocyte lysate (LHL) B, Phenoloxidase response to LPS of fraction eluted at the void volume from gel filtration chromatography of LHL (F1), which lack panulirin and other molecules below 30 kDa. For each assays, LHL (250 µg/ml) and F1 (25 µg/ml) were mixed with 150 µl of 50 mM Tris-HCl, pH 7.5, 50 mM CaCl2 and 50 µl of 0.1 mg/ml LPS or LPS-free water as control. The phenoloxidase activity was measured kinetically at 490 nm and 37 ºC using dopamine as substrate. The error bars represent the S.D. of the mean (n=3). FIGURE 6. Influence of panulirin on phenoloxidase response to LPS. Constant concentrations of F1 fraction (25 µg/ml) were incubated with 150 µl of 50 mM Tris-HCl, pH 7.5, 50 mM CaCl2 and decreasing concentration of purified panulirin for 15 min. LPS (0.1 mg/ml) or LPS-free water (Control) were added and the phenoloxidase activity was measured continuously at 490 nm and 37 ºC immediately after the addition of dopamine. Values represent the average of three replicates plus S.D. FIGURE 7. Schematic three-dimensional representation of panulirin. The structure backbone was achieved by homology modeling. The core of 34 residues (the inner length between the cysteines at the ends) show two β strands stabilized by three disulfide bridges in the arrangement Cys1-Cys5, Cys2-Cys4 and Cys3-Cys6. The residues Arg21, Arg31 and Arg33 are shown. The model structure was obtained using ESyPred3D and drawn with PyMol. TABLE 1. Panulirin purification table. Stage

Lobster Hemocyte Lysate SephadexG50 Cationexchange

Volume

Protein

Specific Activity

mg

Total Inhibitory Activitya IU

ml

9.5

95

361

3.8

1

100

40

3.6

222

61.6

16.2

61.5

8

0.8

171

213

56.2

47.3

IU/mg

19

Purification (x)

-fold

Yield

%

Novel trypsin inhibitor regulates the immune response in lobsters a

Inhibitory activity: one unit of inhibitory activity (IU) was defined as the amount of protein needed to inhibit two units of trypsin activity. One unit of trypsin activity was defined as the enzyme activity that produce 1 µmol of pNA per min under specified conditions. Figure 1

Figure 2

20

Novel trypsin inhibitor regulates the immune response in lobsters Figure 3

Figure 4

a

PjTI 1 (Panulirus japonicus trypsin inhibitor 1) corresponds to the putative mature peptide from panulirin precursor with GenBank protein ID AGE44005, translated from the complete sequence mRNA (GenBank Accession number KC154047) from Panulirus japonicus hemocytes. Panulirin and PjTI 1 are in alignment format that represent identical residue, conserved substitution, and semi-conserved substitution, respectively. b The primary structures were obtained based on the experimental masses from the MS/MS spectrometric analysis and the N-terminal Edman degradation. The positions of the cysteines are represented in bold and gray shadow while basic amino acids are in bold and light-gray shadow. The underlined sequence in 4B was used to construct the panulirin model. c,d Theoretical and experimental molecular masses, respectively.

21


Figure 6

22


23