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Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804

Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804
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  Hemocyanin-derived phenoloxidase activity in the spiny lobster   Panulirus argus  (Latreille, 1804) Rolando Perdomo-Morales  a, ⁎ , Vivian Montero-Alejo  a  , Erick Perera  b ,Zenia Pardo-Ruiz  a  , Esther Alonso-Jiménez  a  a   Biochemistry Department, Center for Pharmaceuticals Research and Development, Ave. 26 No. 1605 e/ Ave 51 y Boyeros, Plaza, CP 10600, Havana, Cuba  b Center for Marine Research, University of Havana, Calle 16 No. 114, e/1ra y 3ra, Miramar, Playa, CP 10300, Havana, Cuba Received 27 May 2007; received in revised form 31 December 2007; accepted 3 January 2008Available online 12 January 2008 Abstract Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. Inthis study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster   Panulirus argus  shows phenoloxidase activity in vitro after treatmentwithtrypsin,chymotrypsinandSDS(0.1%optimalconcentration),butitisnotactivatedbysodiumperchlorateorisopropanol.Theoptimal pHsoftheSDS-activatedhemocyaninwere5.5and7.0.Hemocyaninfromspinylobsterbehavesasacatecholoxidase.Kineticcharacterizationusingdopamine,L-DOPAandcatechol showsthatdopamine isthemostspecificsubstrate. Catechol and dopamineproducedsubstrateinhibition above16and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin withtrypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.© 2008 Elsevier B.V. All rights reserved.  Keywords:  Hemocyanin; Phenoloxidase; Tyrosinase; Hemolymph; Spiny lobster  1. Introduction Hemocyanins are abundant and large proteins found in thehemolymph of mollusks and arthropods [1]. They are membersof a protein superfamily that includes the arthropod phenolox-idases, crustacean non-respiratory pseudohemocyanins (crypto-cyanins), insect storage hexamerins and dipteran hexamerinreceptors [2]. Although the primary function of hemocyanins isthe transport of oxygen in the body fluid [1], ithas been reportedthat hemocyanins from several species of arthropods and mol-lusks exhibit tyrosinase activity under certain artificial condi-tions such as limited proteolysis, SDS and denaturing agents[3,4]. Endogenous components of the hemolymph can lead tothe activation of hemocyanin of horseshoe crabs [5,6] andKuruma prawns [7], suggesting a possible participation of thehemocyanin-derived phenoloxidase activity in the immuneresponse.Tyrosinases (EC are enzymes able to catalyze the o -hydroxylation of monophenols (monophenolase or cresolaseactivity) and the oxidation of   o -diphenols ( o -diphenolase or catecholase activity) to quinones, initiating the synthesis of melanin. Catecholoxidase enzymes (EC catalyze onlythe oxidation step. In arthropods, both tyrosinase and cate-choloxidase are usually referred to as phenoloxidase. Phe-noloxidases from arthropods are crucial enzymes of the prophenoloxidase activating system, which is triggered by pathogen associated molecular patterns and results in melanin production, cell adhesion, encapsulation, and phagocytosis [8].Recently,thepresenceofphenoloxidaseactivityintheplasmafraction of the hemolymph of the spiny lobster   Panulirus argus has been described [9]. In the present study, it is demonstrated  Available online at Biochimica et Biophysica Acta 1780 (2008) 652 – ⁎ Corresponding author. Tel.: +537 881 1424; fax: +537 335556.  E-mail address: (R. Perdomo-Morales).0304-4165/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.bbagen.2008.01.001  that hemocyanin is the source of phenoloxidase activity in suchfraction. This activity is characterized and compared with other hemocyanins previously studied. 2. Materials and methods 2.1. Animals and experimental conditions Adult spiny lobsters were captured, maintained and handled as describedearlier  [9]. 2.2. Purification of hemocyanin The spiny lobster hemolymph was obtained from the fourth walking leg coxausing a sterile and precooled anticoagulant solution composed of 0.2 M  N  -ethylmaleimide(NEM)and0.45MNaCl[9].Becauseofthepresenceofphenoloxidaseactivity in the hemocytes of   P. argus  [9], microbial contamination and hemocytesinjury were avoided in order to prevent the liberation of phenoloxidase into the plasma fraction. The hemolymph was centrifuged immediately after collection at 600 ×  g   for 5 min at 4 °C and the cell pellet was discarded. The supernatant containinghemocyaninwasdialyzedagainst50mMTris – HCl,10mMCaCl 2 ,and10 mM MgCl 2  pH 7.5 at 4 °C to stabilize the protein [10]. Dialyzed plasma wascentrifuged at 10,000 ×  g   for 15 min to remove any insoluble material.Hemocyanin was purified by size exclusion chromatography in a SephacrylS-300 HR column (100×1.5 cm) previously equilibrated with the stabilization buffer. Fractions were assayed for absorbance at 280 nm for total protein, 340 nmfor copper containing protein and 492 nm for dopachrome formation due to phenoloxidase activity (see Fig. 1 for details). The column was calibrated with gelfiltrationmolecularweightmarker(Biorad)composedbythyroglobulin(670kDa), bovine  γ -globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin(17 kDa) and vitamin B-12 (1.35 kDa). Hemocyanin monomers were obtained bydialyzing the purified hemocyanin multimer against50 mM glycine/NaOH buffer,and 5 mM EDTA pH 9.5 (dissociating buffer) at 4 °C for 4 days [11]. 2.3. Protein concentration The total protein concentration was determined by the Bradford method[12], using bovine serum albumin (BSA) as standard. 2.4. Determination of phenoloxidase activity of hemocyanin Phenoloxidase activity was measured spectrophotometrically by recordingthe formation of dopachrome and its derivates from  L -3,4-dihydroxyphenyla-lanine (L-DOPA) [9]. Briefly, the activity induced by SDS was measured by theaddition of 50  μ l of 0.12% SDS to 10  μ l of hemocyanin in a 96-well plate,followed by the addition of 150 μ l of 50 mM Tris – HCl pH 7.0. Finally, 50 μ l of substrate solution (3 mg/ml L-DOPA in distilled water) was added.Immediately,the time course of the reaction was measured kinetically at 492 nm using amicroplate reader  [9]. Activation of hemocyanin was also evaluated usingtrypsin (0.1 mg/ml), chymotrypsin (0.1 mg/ml), isopropanol (25%) and sodium perchlorate (50 – 200 mM in 50 mM Tris – HCl pH 7). Initial rates were obtainedfrom the lineal portion of the absorbance vs. time (min) plot. Reaction velocitywas defined as the increment of absorbance per minute and specific activity asreaction velocity per milligram of proteins. 2.5. Detection of phenoloxidase activity by zymography Zymography of phenoloxidase was performed as described recently [9].Briefly, the hemocyanin was subjected to SDS-PAGE using the same buffer system as Laemmli [13] in 7% analytical polyacrylamide gels, but omitting boiling of the sample in order to preserve the enzymatic activity. Bands with phenoloxidase activity were identified using L-DOPA, followed by CoomassieBrilliant Blue R-250 staining for total protein, which allowed the determinationofphenoloxidasebandsamongtotalproteins[9].Highmolecularweightmarkers(HMWM, Amersham Bioscience) were used as standard. 2.6. Kinetic characterization of hemocyanin-derived phenoloxidaseactivity Kinetic characterization of SDS-activated hemocyanin (0.032 mg) was performed spectrophotometrically using different substrates: dopamine (0.075to 3 mM), L-DOPA (0.3 to 5.8 mM) and catechol (0.8 to 30 mM). Absorbancevalues were registered kinetically at 492 nm for dopamine and L-DOPA [9], andat 405 nm for catechol [14]. 3. Results We have recently shown the presence of phenoloxidaseactivity in the plasma fraction of the hemolymph obtained fromthe spiny lobster   P. argus  [9]. The phenoloxidase activity of  plasmasamplesstoredinanticoagulantsolution,atboth4°Cand Fig. 1. Chromatographic profile of hemocyanin purification on Sephacryl S-300HR. One milliliter of whole plasma at 32 mg/ml was applied onto the column.Flow of 0.3 ml/min. Fractions of 1 ml were collected for analysis. Solid line:280 nm for total protein, dash dot dot line: absorbance at 340 nm, short dot line:absorbance at 492 nm due to phenoloxidase activity induced by SDS in 10 μ l of each fraction as described in Materials and methods for the spectrophotometricassay. The absorbance was measured immediately after the addition of L-DOPA.Trypsin activation produced the same profile (data not shown).Fig. 2. Phenoloxidase activity of hemocyanin from the spiny lobster. Fortymicrograms of hemocyanin was incubated for 20 min at 25 °C with 200  μ l of 0.1mg/ml trypsinin 50mM Tris – HCl buffer pH7( □ );0.1 mg/mlchymotrypsinin 50 mM Tris – HCl buffer pH 7 ( ○ ); 0.1% SDS final concentration as describedin Materials and methods ( ■ ). Controls consisted in the addition of 200  μ l of 50 mM Tris – HCl buffer pH 7 (r). Absorbance values were registered kineticallyimmediately after the addition of 50  μ l of L-DOPA (3 mg/ml).653  R. Perdomo-Morales et al. / Biochimica et Biophysica Acta 1780 (2008) 652  –  658  − 80 °C, decays to undetectable levels in around a week. Thisdeclinewasfasterat  − 80°C.Ontheotherhand,plasmadialyzedagainststabilizationbuffercanbeconservedforseveralweeksat 4 °C without significant decay in its phenoloxidase activity.Size exclusion chromatography of plasma samples showsthat hemocyanin eluted as a major leading peak (Fig. 1), with anapparent molecular weight of around 450 kDa. The 340 nm/ 280 nm ratio for the whole plasma fraction before the chroma-tographic step was 0.17, while it was 0.21 for the purifiedhemocyanin.Spectrophotometric assays for phenoloxidase activity of the purified hemocyanin using L-DOPA as substrate showed that SDS, trypsin and chymotrypsin induced phenoloxidase activity(Fig. 2), with specific activities ( Δ Abs 492 nm min − 1 mg − 1 ;mean±SD) of 1.44±0.086, 1.70±0.040 and 1.63±0.018 res- pectively. Activation by SDS occurred instantaneously, withan optimal final concentration of 0.1% (Fig. 3). High con-centrations of SDS such as 6% can still produce phenoloxidaseactivity (Fig. 3). No decline in the enzymatic activity wasobserved after 1 h of incubation for the hemocyanin – 0.1% SDSmixture. On the other hand, no activity was observed in the presence of isopropanol or sodium perchlorate at any of theconcentrations tested.Biochemical studies were undertaken in order to describesome features of the phenoloxidase activity derived from hemo-cyanin. Such studies were performed at hemocyanin concentra-tions where the tyrosinase reaction was kinetically controlled bytheamountofhemocyanin.ThereweretwopeaksofoptimalpH,at pH 5.5 and pH 7.0. Phenoloxidase activity was not observedwhen tyramine and tyrosine (monophenols) were used assubstrates, but it was evident with all the  o -diphenols assayed.The enzyme reaction was subject to substrate inhibition withreaction rates starting to decline at substrate concentrationsgreater than 2 and 16 mM for dopamine and catechol respec-tively (Fig. 4). Hence, to determine the kinetic parameters  K  m and  V  max  by the Henri – Michaelis – Menten equation, substrate-inhibiting concentrations werenotconsidered. Resultsofkineticcharacterization of hemocyanin using selected  o -diphenols are presented in Table 1, which shows that dopamine is the most specific substrate. An interesting behaviour was observed whenanalyzing the progress curves obtained with different hemocya-nin concentrations. The velocity of product formation decreasedwith time for each of the hemocyanin concentrations tested untila plateau is reached where no further increment in absorbance is Fig. 3. Phenoloxidase activity of hemocyanin at several concentrations of SDS.Purifiedhemocyanin(40 μ g) was mixedwith50 μ l ofdifferent concentrations of SDS and 150  μ l of 50 mM Tris – HCl pH 7. The absorbance was recorded after 5 min of incubation of the assay mixture with 50  μ l L-DOPA (3 mg/ml).Fig. 4. Lineweaver  – Burk double-reciprocal plot for substrate concentrations ranged between 0.075 – 3 mM and 0.8 – 30 mM for dopamine (left panel) and catechol(right panel) respectively.Table 1Kinetic characterization of SDS-activated hemocyanin (0.032 mg) usingdopamine (0.075 – 2 mM), L-DOPA (0.3 – 5.8 mM) and catechol (0.8 – 16 mM) V  max  ( Δ Abs min − 1 )  K  m  (mM)  V  max /   K  m Catechol 0.161±0.0052 7.174±0.4873 0.022L-DOPA 0.112±0.0023 2.565±0.1159 0.044Dopamine 0.143±0.0029 0.181±0.0011 0.79  K  m  and  V  max  values were obtained using the Henri – Michaelis – Mentenequation.654  R. Perdomo-Morales et al. / Biochimica et Biophysica Acta 1780 (2008) 652  –  658  evident. In Fig. 5 (left panel), the assay using L-DOPA is pre-sented. The other substrates showed similar patterns (data not shown). In addition, differences in the duration of the linealresponse among different substrates were also observed (Fig. 5,right panel). Of all substrates tested, L-DOPA shows the highest output of absorbance values, which suggests higher levels of  product formation.Zymographic analysis was used to study whether the pheno-loxidase activity of the hemocyanin from  P. argus  is produced by the multimeric form, the monomers, or both. Because of the presence of SDS in the electrophoresis sample buffer that activatestheprotein[9],nofurthertreatmentofthesampleorthegelwasneededinordertotransformthenativehemocyaninprior the SDS-PAGE. Phenoloxidase activity was observed in themultimeric hemocyanin both under reducing (not shown) andnon-reducing conditions (Fig. 6A, lane 1). Incubation of nativehemocyanin with trypsin and chymotrypsin produced a single band of 170 kDa and 190 kDa respectively with intense pheno-loxidase activities (Fig. 6). Protein Coomassie staining showsthat all bands with molecular weight above 190 kDa were hy-drolyzedbytheproteases(Fig.6B).Abandofaround190kDais present in all lanes which lacks phenoloxidase activity (Fig. 6). Fig.5.Leftpanel: Progress curves ofphenoloxidase activity induced by SDS at different finalconcentrationsof hemocyanin: 0.2mg/ml ( 5 );0.15mg/ml ( ● );0.08mg/ml( ○ ) and 0.04 mg/ml ( □ ). L-DOPAwas used as substrate as described in Materials and methods. Right panel: Progress curves of SDS-activated hemocyanin (0.12 mg/ml)obtained with 7 mM catechol ( ● ); 0.2 mM dopamine ( 4 ) and 2.9 mM L-DOPA ( ○ ).Fig.6.Zymographyforthedeterminationofphenoloxidaseactivityofhemocyaninin 7% acrylamide gel. Panel A: Phenoloxidase activity observed by L-DOPAstaining: 9.6  μ g per well of purified hemocyanin (lane 1); 1 ml of purifiedhemocyanin(3mg/ml)wereincubatedfor20minatroomtemperaturewith1mlof 2.5 mg/ml trypsin, 100 μ l of this mixture was mixed with 25 μ l of 5× SDS sample buffer. Finally, 20  μ l was applied per well, corresponding to 24  μ g of hydrolyzedhemocyanin (lane 2); purified hemocyanin previously incubated with chymo-trypsin using the same procedure described for trypsin, 24  μ g of hydrolyzedhemocyanin per well (lane 3). Panel B: Coomassie Brilliant Blue staining of thesame gel. Lanes 1, 2 and 3 are the same as (A), while lane 4 corresponded tomolecular weight markers.Fig. 7. SDS-PAGE analysis in 7% polyacrylamide gel using the same buffer system as Laemmli [14] of hemocyanin monomers obtained with dissociating buffer. Molecular weight marker (lane 1); 4.8  μ g of dissociated hemocyaninreduced but not boiled (lane 2). Protein bands were stained with CoomassieBrilliant Blue R-250. The same gel did not show phenoloxidase activity when it was previously soaked in 3 mg/ml L-DOPA solution.655  R. Perdomo-Morales et al. / Biochimica et Biophysica Acta 1780 (2008) 652  –  658  This protein band was resistant to proteolitic degradation in thecondition used in the assay.The same zymogram showed that monomers arediffusedandin low amount (see Coomassiestaining in Fig. 6,80-kDa range). No phenoloxidase activity induced by SDS was observed in theregion of the gel where the monomers are present (Fig. 6). Tofurther analyze the presence or not of phenoloxidase activity inthe monomeric forms, the purified hemocyanin was dialyzedagainst dissociating buffer. The activity of the monomers wasassayed by both zymography and spectrometry. The Coomassiestaining of monomers is depicted in Fig. 7. Two major poly- peptides of hemocyanin monomers with an apparent molecular weight of around 80 and 89 kDa were obtained. A third minor  band of around 77 kDa was also present. L-DOPA staining of this gel did not show detectable activity. In addition, dissociatedhemocyanin monomers didn't show phenoloxidase activity insolution when using trypsin or SDS as potential activators. 4. Discussion Asithas beenshowninthe caseofthe spinylobster   Palinuruselephas  [15], hemocyaninisvery abundantand can be purifiedina single step of size exclusion chromatography. Similarly, thechromatographic profile obtained during purification of hemo-cyanin from the spiny lobster   P. argus  showed a major peak which coincides with the elution profile at 340 nm, suggestingthatthisfractioniscomposedmainlybyhemocyanin.Inaddition,these profiles matched with those obtained by measuring pheno-loxidase activity (Fig. 1). The relation between optical density at 340and280nmhasbeenwidelyemployedasapuritycriterionof hemocyanins. In  P. argus , the ratio 340/280 nm for purifiedhemocyanin was 0.21, identical to that reported in  Penaeus japonicus  [16],  Carcinus maenas  and  Homarus americanus  [17].The possibility that phenoloxidase activity described in the pre-sent study was produced by phenoloxidases from hemocytes isexcluded due to the reasons given below.The band profile on SDS-PAGE of the active phenoloxidasefrom hemocytes of   P. argus  is spread. Both L-DOPA andCoomassie staining showed a high background along the geltrack, which is due to the stickiness of the activated protein [9].Conversely, trypsin-activated hemocyanin from  P. argus  showsabandprofileonSDS-PAGEthatismarkedlydifferentfromthat of phenoloxidase derived from hemocytes [9]. Difference instickiness among other properties between hemocyanin and phenoloxidase has been recently described [18]. On the other hand, most hemocyanins showed only catecholase activity [4],whereas phenoloxidases are capable to perform both cresolaseand catecholase activity. We found that phenoloxidase from  P. argus  is able to perform both steps (unpublished result), whilein the current study only catecholase activity has been dem-onstrated. Finally,microbialcontaminationthatcouldleadtotherelease of phenoloxidase into the plasma was avoided duringhemolymph handling. Considering all the above stated, it is possible to sustain that the hemocyanin from  P. argus  (as it occurs in other species [4]), is the source of phenoloxidase or tyrosinase activity presented in the plasma fraction of thehemolymph.Hemocyanin from  P. argus  can be activated artificially into phenoloxidase-like enzyme by SDS (Figs. 1 and 2). Thisactivation is thought to be due to a distortion or slight unfoldingof the protein. The resulting conformational change should thenallow substrates to reach the active site [3,4]. However, there areexceptions such as the hemocyanin from crayfish, which doesnot show phenoloxidase activity after treatment with different concentrations of SDS [19]. It seems that activation does not occur at the same rate. In the crab  Cancer magister  , hemocyaninshows phenoloxidase activity after extended incubation withSDS [20], while hemocyanin activation observed in the current study occurs instantaneously. Tyrosinase activity produced bySDS has been described as well in tyrosinases from plants[21,22] and crustacean [9,19]. Another particular feature of the SDS-activated hemocyanin from  P. argus  is that phenoloxidaseactivity can still be produced even at high concentrations of SDS (Fig. 3), probably due to a distinct resistance to completedenaturalization by SDS.The SDS-induced phenoloxidase activity shows two valuesof optimal pH, 5.5 and 7. This behaviour also differentiates theactivityproducedbyhemocyaninfromthatofthehemocytes[9].The presence of two major peaks in the pH profile of thehemocyanin-derived phenoloxidase activity has been documen-ted in  P. japonicus , with optimal pH of 4.9 and 8.3 [16]. Theactivation of hemocyanins to phenoloxidase by limited proteo-lysis also seems to be species-specific. The hemocyanins fromthe primitive crustacean  Bathynomus giganteus  [23] and the prawn  P. japonicus  [16] are not activated by proteolytic cleav-age. Trypsin and chymotrypsin are able to activate the hemo-cyanin from the spiny lobster   P. argus  into phenoloxidase-likeenzyme. The presence of trypsin and chymotrypsin cleavagesitesinhemocyaninofseveralspecies,someoftheminvolvedinthe activation of hemocyanin into phenoloxidase, has beendescribed [10,19]. Decker and Rimke [10] have explained acti- vating hemocyanin by proteolytic cleavage in tarantula. On theotherhand,perchlorate,asaltoftheHofmeisterseries,iscapableto induce a change in the hemocyanin conformation with conco-mitantincreaseofcatecholoxidaseactivityinhemocyaninsfrom C. maenas  and  H. americanus  [17].As for the hemocyanin from  Eurypelma californicum  [10], this treatment does not induce phenoloxidase activity in the hemocyanin from spiny lobster,even after prolonged incubation times. Whereas isopropanolactivates the hemocyanin from  P. japonicus  [16], it does not induce activity in  P. argus .SDS-induced phenoloxidase activity of the purified hemo-cyanin resides in its aggregated structure. It was also evidencedthat activation by proteolytic attack occurs upon native hemo-cyanin, producing single bands of 170 kDa and 190 kDa for trypsin and chymotrypsin respectively, which showed signifi-cant tyrosinase activity (Fig. 6). These multimers, which arelikely dimers of activated hemocyanin, do not occur naturally inthe plasma of   P. argus  (see the lack of these bands in lane 1,Fig. 6 Panels A and B). Our results suggest that due to pro-teolysis, hemocyanin hexamers are converted into a stabledimer, in which an opened active site is reachable by phenolicsubstrates. Whether these active dimers could be produced bythe action of an endogenous serine protease remains unknown 656  R. Perdomo-Morales et al. / Biochimica et Biophysica Acta 1780 (2008) 652  –  658
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