Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata

Insect Biochemistry and Molecular Biology 30 (2000) 953–967www.elsevier.com/locate/ibmb

Purification, characterization and molecular cloning ofprophenoloxidases fromSarcophaga bullataq

Michael R. Chase, Kiran Raina, James Bruno, Manickam Sugumaran*

Department of Biology, University of Massachusetts–Boston, Boston, MA 02125, USA

Received 6 December 1999; received in revised form 8 March 2000; accepted 9 March 2000

Abstract

Prophenoloxidase (PPO) is a key enzyme associated with both melanin biosynthesis and sclerotization in insects. This enzymeis involved in three physiologically important processes viz., cuticular hardening, defense reactions and wound healing in insects.It was isolated from the larval hemolymph ofSarcophaga bullataand purified by employing ammonium sulfate precipitation,Phenyl Sepharose chromatography, DEAE–Sepharose chromatography, and Sephacryl S-200 column chromatography. The purifiedenzyme exhibited two closely moving bands on 7.5% SDS–PAGE under denaturing conditions. From the estimates of molecularweight on Sephacryl S-100, TSK-3000 HPLC column and SDS–PAGE, which ranged from 90,000 to 100,000, it was inferred thatthe enzyme is made up of a single polypeptide chain. Activation of PPO (Ka=40 µM) was achieved by the cationic detergent, cetylpyridinium chloride below its critical micellar concentration (0.8 mM) indicating that the detergent molecules are binding specificallyto the PPO and causing the activation. Neither anionic, nor nonionic (or zwitterionic) detergents activated the PPO. The activeenzyme exhibited wide substrate specificity and marked thermal unstability. Using primers designed to conserved amino acidsequences from known PPOs, we PCR amplified and cloned two PPO genes from the sarcophagid larvae. The clones encodedpolypeptides of 685 and 691 amino acids. They contained two distinct copper binding regions and lacked the signal peptide sequence.They showed a high degree of homology to dipteran PPOs. Both contained putative thiol ester site, two proteolytic activation sitesand a conserved C-terminal region common to all known PPOs. 2000 Elsevier Science Ltd. All rights reserved.

Keywords:Prophenoloxidase; Melanin biosynthesis; Sclerotization; Insect immunity; Wound healing

1. Introduction

Phenoloxidase (PO)* also known as tyrosinase is abifunctional enzyme possessing both monophenolmonooxygenase activity (E.C. 1.14.18.1. tyrosine, dihy-droxyphenylalanine, oxygen, oxidoreductase) ando-diphenoloxidase activity (E.C. 1.10.3.1.o-diphenol, oxy-gen, oxidoreductase). It is responsible for initiating thebiosynthesis of widely distributed melanin pigment innature (Prota, 1992). In addition to melanization of cuti-

Abbreviations:Bp, base pairs; CPC, Cetyl pyridinium chloride; PCR,polymerase chain reaction; PO, Phenoloxidase; PPO, Prophenoloxi-dase; RT, Reverse transcription.

q The sequence reported in this paper has been deposited in theGenBank Database (accession numbers: SbPPO1 AF 161260;SbPPO2 AF161261)

* Corresponding author. Tel.:+1-617-287-6598; fax:+1-617-287-6650.

E-mail address: [email protected] (M.Sugumaran).

0965-1748/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved.PII: S0965-1748 (00)00068-0

cle used for color and camouflage, PO is also uniquelyassociated with three different physiologically importantbiochemical processes in insects and other arthropods.These are (a) sclerotization of insect cuticle (Andersenet al., 1996; Sugumaran, 1998), (b) encapsulation andmelanization of foreign organisms observed as defensereaction (Ashida and Brey, 1995; Gillespie et al., 1997;Soderhall et al., 1990; Sugumaran, 1996) and (c) woundhealing (Lai-Fook, 1966; Sugumaran, 1996). In the firstprocess, PO generated 4-alkylquinones serve as sclerot-izing agents for quinone tanning reactions—one of themechanisms by which the insect cuticle is hardened toprotect the soft bodies of animals (Andersen et al., 1996;Sugumaran, 1998). Quinones are converted by quinoneisomerase to quinone methides (Saul and Sugumaran1988a, 1990; Ricketts and Sugumaran, 1994) which arereactive intermediates for the second mechanism of tan-ning called quinone methide sclerotization. Some of thequinone methides are converted to 1,2-dehydro-N-acyl-dopamines by the action of quinone methide isomerase

954 M.R. Chase et al. / Insect Biochemistry and Molecular Biology 30 (2000) 953–967

(Saul and Sugumaran, 1989a,b; Ricketts and Sugumaran,1994). The resultant dehydro-N-acyldopamines arefurther oxidized by PO to their corresponding quinoneswhich rapidly isomerize nonenzymatically to form quin-one methide imine amides necessary forα,β-sclerotiz-ation (Sugumaran et al., 1992; Ricketts and Sugumaran,1994). The reactions of quinones, quinone methides andquinone methide imine amides with cuticular structuralproteins and chitin result in the hardening of the cuticle(Sugumaran, 1998).

In the second process, PO serves as a terminal compo-nent of an elaborate defense mechanism. Parasites andpathogens which are too large to be phagocytosed arefound to be usually encapsulated and melanized in insectblood by the action of phenoloxidase (Ashida and Brey,1995; Gillespie et al., 1997; So¨derhall et al., 1990; Sugu-maran, 1996; Sugumaran and Kanost, 1993). This pro-cess not only limits the growth and development of theforeign object, but also prevents the damage it can causeto host by creating a physical barrier. Finally duringwounding, continuous loss of hemolymph is preventedby the rapid deposition melanin polymer at the woundingsite (Lai-Fook, 1966; Sugumaran, 1996). Apart fromstopping the blood loss, phenoloxidase might also pro-vide cytotoxic quinonoid compounds to kill the oppor-tunistically invading microorganism at the wound site(Sugumaran, 1996; Nappi and Sugumaran, 1993).

The unique roles played by PO in insect physiologyand biochemistry certainly demands a serious study onthis enzyme. But, numerous problems such as instabilityand rapid loss of activity during purification, ‘stickiness,’(=insolubilization on biotic and abiotic matters, variousgels and glassware used for the purification of theenzyme) and self inactivation have prevented thedetailed characterization of insect POs in the past(Sugumaran and Kanost, 1993). By taking advantage ofthe fact that PO is present in the inactive proenzymeform, some scientists have successfully purified andcharacterized the PPO. Thus PPOs fromBombyx mori(Ashida, 1971; Yasuhara et al., 1995),Manduca sexta(Aso et al., 1985; Hall et al., 1995; Jiang et al., 1997a),Hyalophora cecropia(Andersson et al., 1989),Galleriamellonella(Kopacek et al., 1995),Holotrichia diomph-alia (Kwon et al., 1997),Calliphora erythrocephala(Pauand Eagles, 1975),Musca domestica(Hara et al., 1993;Tsukamoto et al., 1986),Drosophila melanogaster(Fujimoto et al., 1993),Blaberus discoidalis(Durrant etal., 1993), Tenebrio molitor (Heyneman, 1965), andLocusta migratoria(Cherqui et al., 1996) have been pur-ified and several of their properties have been charac-terized. Following the initial report on the characteriz-ation of cDNA encodingManduca sextaPPO (Hall etal., 1995), several investigators have also characterizeddifferent insect PPO genes. These include one fromDro-sophila melanogaster(Fujimoto et al., 1995), two fromBombyx mori(Kawabata et al., 1995) a second from

Manduca sexta (Jiang et al., 1997a), two fromHyphantria cunea(Park et al., 1997) six fromAnophelesgambiae(Jiang et al., 1997b; Lee et al., 1998; Muller etal., 1999), one fromTenebrio molitor(Lee et al., 1999),and one fromArmigeres subalbatus(Cho et al., 1998).We have been usingSarcophaga bullatalarvae success-fully for unraveling the molecular mechanisms of cuticu-lar sclerotization for over two decades. In this paper wereport the purification, characterization and molecularcloning of PPO from the larval hemolymph ofSarco-phaga bullata.

2. Materials and methods

2.1. Animals

Larvae of Sarcophaga bullatawere obtained fromCarolina Biological Supplies Co., NC and maintained ona dog food diet.

2.2. Chemicals

L-3,4-dihydroxyphenylalanine (L-dopa), and dopam-ine were procured from Sigma Chemical Co., St Louis,MO. Sephacryl S-100, Sephacryl S-200, DEAE–Sepharose, and Phenyl Sepharose were purchased fromPharmacia Fine Chemicals, Nutley, NJ. Molecularweight markers for molecular weight determination, sil-ver staining kit, and Coomassie blue protein assay kitwere obtained from Bio Rad Laboratories, Hercules, CA.

2.3. Enzyme purification

All operations were carried out at 0–5°C unless statedotherwise. Last instar sarcophagid larvae were anesthet-ized on ice and cold 50 mM sodium phosphate buffer,pH 6.0 (buffer A) was injected into the animals. Thehemolymph was directly collected into a flask chilled ondry ice. The frozen mass was stored at280°C for threeto four weeks before use, or processed immediately. Thisprocedure prevented the activation of PPO as well asdarkening of hemolymph only inSarcophaga. Use inother insects such asManducacaused rapid activationof PPO and darkening of the hemolymph. Use of deco-agulation buffer outlined under ‘RNA extraction’ is rec-ommended for these organisms.

Hemolymph was subjected to 40% ammonium sulfatesaturation and the proteins precipitated within 20 minwere discarded after centrifugation at 13,000g for 15min. The supernatant was brought to 60% saturationwith respect to ammonium sulfate and the proteins pre-cipitated within 20 min were collected by centrifugationat 10,000g for 15 min. The pellet was dissolved in mini-mum amount of buffer A containing 10% ammoniumsulfate and chromatographed on a phenyl Sepharose col-

955M.R. Chase et al. / Insect Biochemistry and Molecular Biology 30 (2000) 953–967

umn (13.5×2 cm) equilibrated with the same buffer.After loading and washing the column to removeunbound proteins, bound PPO was eluted with water. Aflow rate of 4 ml/min was maintained throughout. Frac-tions containing PPO were pooled and chromatographedon a DEAE Sepharose column (13.5×3 cm) equilibratedwith 10 mM buffer A. The column was washed exten-sively with this buffer and bound proteins were elutedwith step gradients of (a) 50 mM buffer A and (b) 100mM buffer A at a flow rate of 3 ml/min. PPO activityeluting with 100 mM buffer A was pooled and concen-trated by 65% ammonium sulfate precipitation. The pre-cipitate obtained after 30 min was collected by centrifug-ation at 10,000g for 15 min. It was dissolved inminimum amount of 10 mM buffer A and chromato-graphed on a Sephacryl S-200 column (100×3.5 cm)equilibrated with the same buffer. A flow rate of 0.4ml/min was maintained and fractions of 4 ml were col-lected. The PPO containing fractions were pooled andused as the pure proenzyme.

2.4. Enzyme assay

Since PPO was devoid of any activity, it needed to beactivated before detecting PO activity. For this purpose areaction mixture (1 ml) containing 2 mM dopamine, 50mM sodium phosphate buffer, pH 6.0 and enzyme pro-tein (5–10µg) was incubated at room temperature andthe increase in absorbance at 475 nm associated with theproduction of dopaminechrome was continuously moni-tored after activating the PPO by the addition of 10µlof 10% CPC. One unit was defined as 0.1 absorbanceincrease at 475 nm per min. For some assays, oxygenuptake was monitored using the same reaction at 30°C.

2.5. Molecular weight estimation

The purity of the PPO was determined by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis(SDS–PAGE) followed by silver staining. SDS–PAGEwas performed following the method of Laemmli (1970).About 10 µg of PPO was dissolved in 30µl of 0.5 MTris HCl buffer, pH 6.8 containing 10% SDS, 10% gly-cerol, 5% β-mercaptoethanol and 0.05% bromophenolblue and loaded on a 7.5% polyacrylamide gel. Afterextensive washing, protein bands on the gel were vis-ualized by silver staining.

The approximate molecular weight of PPO wasdetermined by three different techniques. The firstmethod employed the above mentioned SDS–PAGE.The second method utilized the gel filtration HPLC ona TSK 3000 (30 cm×7.5 mm) column coupled with aBeckman pre-column (4 cm×7.5 mm). The standards andPPO were separately chromatographed using isocraticelution with 100 mM buffer A at a flow rate of 0.7ml/min. The elution time of PPO was determined by

measuring the PO activity in various fractions after acti-vation. By comparing its retention time with those ofmolecular weight markers, the approximate molecularweight of the PPO was determined. The molecularweight of the native PPO was also determined on aSephacryl S-100 column (55×2 cm) equilibrated with 10mM buffer A containing 0.2 M NaCl. A flow rate of 12ml/h was maintained. The column was calibrated withdifferent molecular weight markers.

2.6. RNA extraction

RNA was isolated from the hemocytes of last instarS. bullata larval hemolymph. About 300 larvae wereinjected with decoagulation buffer (100 mM glucose, 15mM NaCl, 10 mM disodium ethylene diamine tetraacet-ate, 30 mM trisodium citrate and 26 mM citric acid, pH4.6) and a drop of fluid from each animal was collectedinto a 15 ml tube placed on ice. Extract was allowed tosettle, then the upper layer was transferred to a freshtube and centrifuged at 1000g to pellet the cells. Thesupernatant was discarded and the cells were stored at280°C until needed. Hemocytes were removed from the280°C freezer, mixed with 10 ml of Trizol Reagent(Gibco/Brl) and vortexed for 30 sec. The remaining pro-tocol follows the manufacturer’s recommendations. TheRNA pellet was dissolved in 200µl of diethyl pyrocar-bonate treated water, divided into two aliquots and pur-ified with an RNeasy kit (Qiagen).

2.7. Isolation of prophenoloxidase cDNAs from S.bullata

We employed a reverse transcription–polymerasechain reaction (RT–PCR) strategy to isolate the PPOcDNAs from S. bullata by amplifying reverse tran-scribed (RT) total RNA extracted from larval hemocytewith degenerate PCR primers designed to conservedamino acid motifs among all insect PPO sequences avail-able from the GenBank. Multiple PCR fragments weresequenced, compared to previously known PPO genesand used to design gene specific primers to obtain theremainder of the molecule with a 39 and 59 RACE strat-egy. Thus isolating each PPO cDNA, in three inde-pendent overlapping fragments. To ensure integrity ofeach PPO cDNA contig, primers were designed withinthe 59 and 39 untranslated regions to amplify the entirecoding domain. The entire coding domain was clonedand resequenced.

2.8. Degenerate primer design

Insect PPO amino acid sequences were downloadedin FASTA format from GENBANK and aligned withCLUSTAL W (Thompson et al., 1994), using defaultparameters. Conserved domains were identified and


reverse translated. The forward primer, PPO-FS (59-CAY CAY TKB CAY TGG CAY YTN GT-3 9) targetsHH (W/Y) HWHLVY (Copper binding domain) and thereverse primer PPO-RAS (59-CKR TCR AAN GGRWAN CCC AT-39), MGYPFDR (conserved carboxydomain).

2.9. Reverse transcription–polymerase chain reaction(RT–PCR)

Independent RT reactions (Superscript II Gibco BRL)with 5 µl of 1/5 or 1/10 dilutions ofS. bullatatotal RNAeluted from the RNeasy column and primed with RT-1(59-CTC TGG GCC CAA GCT TTT TTT TTT TTTTTV-59) were set up following the manufacturer’s proto-col. All RT reactions were incubated at 42°C. PCR reac-tions were set up using 5µl of 1/5, 1/10 and 1/50dilutions of the RT reaction with PPO-FS and PPO-RAS.The reactions were set up as follows: 5µl template, 5µl 10 PCR buffer (Promega) 2.5 mM MgCl2, 200 µMeach dNTP, 10–20 pM each primer, 1 unit Taq Poly-merase (Promega) in a total volume of 50µl. The reac-tions were then heated to 94°C for 2 min, manuallyadding the 1 unit of TAQ to each reaction and cycledat 94°C 1 min, 54°C 1 min, 72°C 1 min, 35 times.

A DNA fragments was cloned into a Pgem T-easy(Promega) and transformed into XL-1 Blue MRF9 elec-trocompetent cells (Stratagene). Colonies were allowedto grow overnight and screened by contaminating a PCRreaction containing M13 forward and reverse primerswith a single colony. Replicate clones were plated andgrown for 5 h at 37°C. Selected colonies (based on PCRresults) were grown overnight in 10 ml of LB media,purified and sequenced on an ABI model 377 sequencer,using a dye terminator kit (Perkin Elmer Applied Biosys-tems, Foster City, CA).

2.10. 39 race

Sequence data from clones with homology to otherinsect PPOs were then used to design nested primers ofabout 250–300 bases from the 39 end of the initial frag-ment. PCR reactions were set up as described above,but pairing with a gene specific primer and RT-1, usingtemplate from the initial RT reaction primed with RT-1.

2.11. 59 race

To obtain the 59 end of each DNA fragment, twonested anti-sense primers were designed 200–250 bpaway from the 59 end of the clone. Independent RT reac-tions were primed with the outer most gene specificprimer and dCTP tailed with terminal transferase sup-plied in the GIBCO/BRL 59RACE kit. The PCR con-ditions are the same as above, but the cycling profile waschanged as follows: The anneal temperature was

decreased to 48°C for five cycles, then increased to 60°Cfor the 30 remaining cycles. If the PCR product washeterogeneous, another round of amplification was car-ried out with the second nested primer. The DNA frag-ments were gel purified with a Gel Purification kit(QIAGEN) and cloned into a T-vector and sequenced.

2.12. Amplification and sequencing of entire codingdomain

To ensure the integrity of each identified gene, weamplified the entire coding domain. Primers weredesigned to the 39 and 59 untranslated regions of eachgene. PCR conditions were as previously described, butthe anneal temperature was altered to accommodate eachprimer pair and the extension time was increased to 90 s.PCR products were purified and ligated into a T-vector.Primers were designed from the original sequence dataat 500 base intervals and three independent clonessequenced.

2.13. Sequence assembly and analysis

Contigs for each sequence was assembled and editedwith the program Sequencher (Gene Codes Corporation).Sequencing primers were designed with the programPrimer3 (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3Fwww.cgi). Each sequence was com-pared to other sequences in GenBank with a Blastxsearch. Prophenoloxidase amino acid sequences weredownloaded from GenBank in fasta format and alignedwith Clustal W using default parameters (except forchanging output format to Phylip). This file wasimported to PAUP (Swofford, 1999 (4.0b2)) and usedto calculate pairwise identities.

3. Results

Employing the protein purification scheme outlined inTable 1, PPO from the hemolymph was purified 221-fold with a recovery of 16%. The recovery of totalactivity was drastically reduced at the ammonium sulfatestep due to the presence of proteins that interfere withquinonoid metabolism and high protein concentrations.However, during the succeeding steps, such interferenceis removed and the recovery of total activity units isrestored. The finally purified PPO exhibited two closelymoving bands on SDS–7% polyacrylamide gel (Fig. 1).Both bands when cut and incubated with dopamine andCPC, exhibited PO activity. Without CPC treatment,neither of them showed any detectable phenoloxidaseactivity, thereby indicating that they are isozymes ofPPO.


Table 1Purification chart for prophenoloxidase

Step Total volume (ml) Total units Total protein (mg) Specific activity Recovery (%) Fold purification(units/mg)

Crude 25 6250 1200 5.2 100 1NH4SO4 2.8 1092 420 2.6 17.5 5Phenyl sepharose 122 4148 152.5 27.2 66.4 5.2DEAE-sepharose 30 2760 33 83.6 44.2 16.1Sephacryl S-200 22 1012 0.88 1150 16.2 221

Fig. 1. SDS–PAGE of PPO fromS. bullata. SDS–PAGE was perfor-med on 7.5% gel as outlined in Materials and methods. Proteins werestained with silver staining.

3.1. Molecular weight

Fig. 2 shows the calibration curves used for the esti-mation of the molecular weight of PPO. On SephacrylS-100 (or Sephacryl S-200) gel filtration column, iteluted with an apparent molecular weight of 100,000[Fig. 2(a)]. On HPLC, it exhibited a molecular weightof 85,000 [Fig. 2(b)]. Under denaturing conditions onSDS–PAGE, it migrated as a single band with a molecu-lar weight of 90,000 [Fig. 2(c)]. Thus the native proen-zyme seems to be made up of a single polypeptide chainwith no apparent subunits.

3.2. Activation of PPO

Fig. 3 inset shows the activation of PPO caused bydifferent detergents. As is evident only the cationicdetergent, CPC activated the proenzyme specifically andneither anionic detergents such as SDS, sodium capry-late, sodium lauryl sarcosidate, deoxycholate, nor non-ionic detergents such as octyl-β-glucopyranoside, Non-idet-P-40, Triton X-100, digitonin, Brij 58 and Tween20 activated the proenzyme. Similarly, zwitterionicdetergent, CHAPS also failed to activate the enzyme.The activation caused by CPC occurred well below the

Fig. 2. Molecular weight estimation of sarcophagid PPO. (a) By gelfiltration chromatography. A Sephacryl S-100 column (55×2 cm) equi-librated with 10 mM sodium phosphate buffer pH 6.0 containing 200mM sodium chloride was used at a flow rate of 12 ml/h. Molecularweight markers used to calibrate the column are: (A) carbonic anhyd-rase (29 kDa); (B) ovalbumin (43 kDa); (C) Phosphorylase b (96 kDa);and (D) alcohol dehydrogenase (150 kDa). (b) By size exclusionHPLC. A Beckmann TSK 3000 column (30 cm×7.5 mm) was used toestimated the approximate molecular weight of PPO. Proteins wereseparated on the column using isocratic elution with 100 mM sodiumphosphate buffer pH 6.0 at a flow rate of 0.7 ml/min. Molecular weightmarkers used are: (A) carbonic anhydrase (29 kDa); (B) bovine serumalbumin (66 kDa); (C) alcohol dehydrogenase (150 kDa); (D) Myosin(200 kDa); (E) apoferritin (443 kDa) and thyroglobulin (669 kDa). (c)By SDS–PAGE on 7.5% gel. Conditions are outlined in Materials andmethods. Pre-stained molecular weight markers were used to calculatethe approximate molecular weight of PPO. (A) carbonic anhydrase (29kDa); (B) ovalbumin (43 kDa); (C) Phosphorylase b (96 kDa); and(D) alcohol dehydrogenase (150 kDa).


Fig. 2. (continued)

Fig. 3. Activation of PPO by different concentrations of CPC. PPOwas assayed using standard assay conditions except for variation in theconcentration of CPC. Inset: activation of PPO by different detergents.Standard assay conditions were used except for varying the detergents.Detergents used are: (a) CPC; (b) SDS; octylglucopyranoside; NonidetP 40; Brij 35; Tween 20; CHAPS; sodium lauryl sarcosidate; sodiumcaprylate; Triton X-100; deoxycholate or digitonin.

critical micellar concentration of CPC. From the doseresponse curves shown in Fig. 3, theKa for the activationof PPO by CPC was estimated to be 40µM. The criticalmicellar concentration of CPC under these conditions is0.8 mM. Therefore, CPC must be binding to the proen-zyme specifically below its critical micellar concen-tration and activating the enzyme. This is in confirmationwith our previous result on the activation ofManducaPPO (Hall et al., 1995).

Phospholipids and fatty acids have been shown to be


potent activators of PPO (Sugumaran and Nellaiappan,1991; Sugumaran and Kanost, 1993). In order to checkwhether sarcophagid PPO can also activated by thesereagents, the following studies were carried out. Variousphospholipids and fatty acids have been incubated withPPO and the appearance of PO activity in the incubationmixture was assessed by standard PO assay. As shownin Fig. 4, fatty acids caused either mild or no activationat all. Among the different phospholipids tested, phos-phatidyl glycerol activated the PPO immediately, whilephosphatidyl inositol activated the PPO after a lag.

3.3. pH optimum and thermal stability

The activated PO exhibited a typical bell shaped pHcurve with optimum activity observed at about pH 7(Fig. 5). Fig. 6 shows the comparative stability of pro-phenoloxidase and detergent activated phenoloxidase.The activated enzyme rapidly lost its activity even atroom temperature (25°C) within 15 min (curve C), whilethe raising the temperature to 55°C resulted in total lossof its activity within one min (curve D). In contrast, theproenzyme was much more stable towards heat treat-ment (Curves A and B, Fig. 6). Heating the proenzymefor 10 min at 60°C resulted in 50% loss of its activityand heating at 70°C for the same time resulted in 100%loss of its activity (data not shown).

3.4. Substrate specificity

The substrate specificity of activated phenoloxidase isshown in Fig. 7. As is evident, N-acetyldopamine, cat-echol, 4-methyl catechol, norepinephrine, dopamine,3,4-dihydroxyphenylacetic acid, and N-β-alanyldopam-

Fig. 4. Activation of PPO by different phospholipids. PPO wasassayed using standard assay conditions except for using different lip-ids in the place of CPC. The lipids were dissolved in 100µl of ethanoland made up to 1 ml with water (concentration of lipids=5 mg/ml).An aliquot (6 µl) of this solution, was used to test the activation ofPPO. Compounds used are (A) phosphatidyl glycerol; (B) phosphatidylinositol; (C) phosphatidyl ethanolamine; (D) nondecanoic acid; (E) pal-mitic acid; (F) lauric acid; (G) stearic acid; (H) linoleic/oleic acid and(I) phosphatidyl choline.

Fig. 5. pH optimum of sarcophagid PPO. The activity of PO wasdetermined at indicated pH values using the standard assay conditions.Sodium acetate buffer (50 mM) was used from pH 3 to 4.5 (No activitywas detected from pH 3 to 4). Sodium phosphate buffer (50 mM) wasused from pH 5 to 9.

Fig. 6. Thermal stability of PPO and PO. The thermal stability ofPPO and PO were determined by incubating the PPO and CPC acti-vated PO at indicated temperature for varying amounts of time. Aftercooling to room temperature, the residual activity was determinedusing standard assay conditions. (A) stability of PPO at room tempera-ture; (B) stability of PPO at 55°C; (C) stability of PO at room tempera-ture; (D) stability of PO at 55°C.

ine proved to be better substrates than dopa, which isroutinely used for the assay of various phenoloxidases.In general, unlike mammalian tyrosinases, insect phenol-oxidases prefer dopamine better than dopa (Barrett,1991; Sugumaran, 1998). The marginal activity towardshydroquinone, methyl hydroquinone and gentisic acidindicates that the enzyme is a typicalo-diphenoloxidase


Fig. 7. Substrate specificity of sarcophagid phenoloxidase. Standardassay conditions were employed except for variation of substratesinstead of dopamine as indicated. Oxygen uptake assay was used. Sub-strate used are: (A) N-acetyldopamine; (B) 4-methylcatechol; (C) N-β-alanyldopamine; (D) dopamine; (E) 3,4-dihydroxyphenylacetic acid;(F) catechol; (G) norepinephrine; (H) 3,4-dihydroxymandelic acid; (I)dopa; (J) tyrosine methyl ester; (K) 3,4-dihydroxybenzoic acid; (L)hydroquinone; (M) gentisic acid; and base line=methylhydroquinone.

and not a laccase type enzyme. The enzyme seems topossess monophenol monooxygenase activity as evi-denced by its ability to attack tyrosine methyl ester.

3.5. Inhibition studies

Typical phenoloxidase inhibitors such as phenylthio-urea and diethyldithiocarbamate inhibited the activity ofthe enzyme drastically (Table 2). Other copper chelatorsalso showed marked inhibition of phenoloxidase activity.Iron chelators such aso-phenanthroline andα,α9-dipyri-dyl failed to inhibit the enzyme. Interestingly, mimosine,which is a structural analog of dopa, and a typical inhibi-tor of tyrosinase, showed marginal inhibition only. Thisfurther emphasizes the superiority of dopamine overdopa as a substrate for insect PO.

3.6. Molecular cloning

Using PCR amplification with degenerate primers, wehave identified two distinct PPO genes in the hemocytes

Table 2Inhibition of detergent activated Prophenoloxidase

Inhibitor Concentration (%) Inhibition

Phenylthiourea 10µM 1001 µM 930.3 µM 900.1 µM 74

Diethyldithiocarbamate 90µM 10070 µM 9250 µM 8410 µM 49

Sodium cyanide 0.5 mM 1000.1 mM 64

Mimosine 5 mM 261 mM 20

Neocuprine 5 mM 741 mM 38

8-Hydroxyquinoline sulfonate 1 mM 1000.1 mM 33

o-Phenanthroline 5 mM 0α,α9-Dipyridyl 1 mM 14Sodium fluoride 5 mM 0Ethylenediamine tetra acetate 5 mM 23

1 mM 19

of S. bullata larvae. The sequence of cDNA encodingsarcophagid PPO 1 and 2 are shown in Figs. 8 and 9,along with their deduced amino acid sequence. ThecDNA for PPO-1 is 2268 bp while that for 2 is 2246bp. The PPO cDNA-1 coded for a protein of 685 aminoacids with a molecular weight of 79,088, while 2 codesfor a protein of 691 amino acids with a molecular weightof 79,797. There is no evidence for the presence of thehydrophobic sorting signal sequence for the endoplasmicrecticulum in any of the PPOs.

Comparison of deduced amino acid sequence of thePPO 1 and 2 with other arthropod PPOs in GenBank(Fig. 10) shows that the two copper binding sites arepreserved in all these proteins (underlined sequences).Both copper binding sites show extensive homology.The six histidine residues, which ligate the two copperatoms, are present at the conserved sites in all the phe-noloxidases. The proteolytic cleavage site (RF, indicatedby an arrow) is conserved in all dipteran PPOs. A secondpossible proteolytic cleavage site, (REE, also designatedby an arrow) is conserved in all dipteran PPOs with theexception of sarcophagid PPO 2 which has RAE andDrosophila PPOA1 which has RQE at this site. Inaddition, previously characterized putative thiol ester siteCGCGWPQHML (double underlined) (Hall et al., 1995)is preserved in SbPPO1 in its entirety, but the SbPPO2has E instead of Q at position 7. At the C-terminalregion, there is a conserved region in all the PPOs(marked by asterisks). In particular, the motifMG(Y/F)PFDR is present in all known PPOs.


Fig. 8. Nucleotide and the deduced amino acid sequences of SbPPO 1 fromS. bullata. (GenBank accession No. AF 161260).


Fig. 9. Nucleotide and the deduced amino acid sequences of SbPPO 2 fromS. bullata. (GenBank accession No. AF 161261).


Fig. 10. Alignment of known dipteran PPO sequences. The two copper binding domains in the amino acid sequence are underlined. Arrowsindicate the two putative proteolytic cleavage sites. The putative thiolester sites are double underlined. *Indicate the conserved C-terminal site.GenBank accession numbers are listed in Table 3.

4. Discussion

Using the protein purification protocols outlined inMaterials and methods, we have purified the PPO fromthe hemolymph of theSarcophagalarvae to apparenthomogeneity. The purified enzyme exhibited two closelymoving bands on the polyacrylamide gel electrophoresis;both capable of oxidizing dopamine when activated withCPC. Thus the protein bands are due to the presence oftwo isozymic forms of the same enzyme. Two isoforms

of PPO have been characterized fromManduca sexta(Hall et al., 1995; Jiang et al., 1997a),Bombyx mori(Yasuhara et al., 1995), andGalleria mellonella(Kopacek et al., 1995). However, only one isoform hasbeen characterized fromBlaberus discoidalis(Durrant etal., 1993) andLocusta migratoria(Cherqui et al., 1996).In anopheline cell line, Muller et al. (1999) have charac-terized as many as six different PPO genes indicatingthe presence of six different isozymes.

In general, PPO’s are dimers capable of undergoing


Fig. 10. (continued)


monomerization and polymerization depending on theionic strength of the medium (Jiang et al., 1997a; Yasu-hara et al., 1995; Kwon et al., 1997). However, the PPOfrom H. cecropiahas been shown to be a monomericprotein (Andersson et al., 1989). The sarcophagid PPOsisolated in the present study also seems to be a mono-meric protein.

The phenoloxidase fromGallaria mellonellahas beenshown to be a glycoprotein (Kopa´cek et al., 1995). But,both Manduca sexta(Jiang et al., 1997a) andBombyxmori (Yasuhara et al., 1995) PPOs are devoid of anycarbohydrates and are not glycoproteins. SarcophagidPPOs have few N-glycosylation sites and do not stainfor sugars with Schiff’s reagent (data not shown). Theyalso do not exhibit any binding affinity for ConcanavalinA Sepharose. Therefore, sarcophagid PPOs do notappear to be glycosylated.

The sarcophagid PPO can be activated either byendogenous proteases (Saul and Sugumaran, 1988b) orby detergents. Among the detergents tested only the cat-ionic detergent, CPC activated the proenzyme. The acti-vation caused by CPC (Ka=40 µM) occurred well belowits critical micellar concentration (0.8 mM) thereby indi-cating that CPC is binding to the proenzyme specificallyand causing its activation. Earlier, we reported a similarfindings with Manduca PPO also (Hall et al., 1995).Since lipids are likely to be the endogenous activator(Sugumaran and Kanost, 1993), some of the lipids weretested for their ability to activate the proenzyme. In gen-eral, fatty acids showed only marginal activation of sar-cophagid PPO. Phosphatidyl glycerol activated the PPOreadily, while phosphatidyl inositol activated the enzymeafter a lag (Fig. 4).

The substrate specificity of the sarcophagid PPO issimilar to that of other insect enzymes and is distinctlydifferent from that of the mammalian tyrosinase. Thus,N-acetyldopamine, N-β-alanyldopamine, and dopamineproved to be far better substrates for the enzyme, thandopa which is routinely used for the assay of PPOs. Thisdistinct difference in substrate specificity between mam-malian and arthropod enzymes and the absence signifi-cant sequence identity between these two class ofenzymes confirms that they are quite different in spiteof the fact that they catalyze the same biochemical trans-formations.

The sequence identities of the sarcophagid PPOs withother reported PPO sequences, range from 73 to 38%(Table 3). SbPPO1 and SbPPO2 share 52% homologybetween them. SbPPO1 shows 52% homology withDro-sophilaPPO A1; while SbPPO2 shows 62% homology.SbPPO1 is quite similar to mosquito AgPPO1 (73%).Our finding of two divergent PPO lineages within aspecies is consistent with what others have found (Jianget al., 1997a; Muller et al., 1999). These results clearlyassociate the PPO genes we have isolated with other dip-teran PPOs.

Table 3Percent similarity of known PPOsa

PPO GenBank A. no. SbPPO1 SbPPO2

SbPPO1 AF161260SbPPO2 AF161261 52DmPPOA1 D45835 52 62AgPPO1 L76038 73 52AgPPO2 AF004915 53 54AgPPO3 AF004916 55 54AgPPO4 AG010193 51 49AgPPO5 AG010194 49 46AgPPO6 AG010195 53 52AsPPO AF062034 54 52TmPPO AB020738 65 50MsPPO1 AF003253 59 46MsPPO2 L42556 51 44BmPPO1 D49370 60 48BmPPO2 D49371 53 45HcPPO1 U86875 56 47HcPPO2 AF020391 51 45PlPPO X83494 42 38

a Sb=Sarcophaga bullata; Dm=Drosophila melanogaster; Ag=Ano-pheles gambiae; As=Armigeres subalbatus; Tm=Tenebrio molitor;Ms=Manduca sexta; Bm=Bombyx mori; Hc=Hyphantria cunea; Pl=Pa-cifastacus leniusculus.

Examination of Fig. 10 reveals that the copper bindingregions are highly conserved among all dipteran PPOs.The putative proteolytic cleavage site 1 inSarcophagaPPO1, RFG (marked with an arrow in Fig. 10) resembleslepidopteran PPO better than the dipteran PPOsequences. This site is the same as that found in all lepi-dopterans.SarcophagaPPO2 has the sequence, RFSsimilar to Drosophila A1, and anopheline PPO2 andanopheline PPO6 at this site. The second cleavage siteon SarcophagaPPO1, REE (also marked by an arrowat amino acid 164) is conserved at all the mosquitosequences.SarcophagaPPO2 has RAE andDrosophilahas RQE at this site.

In α2-macroglobulin and complement proteins C3 andC4, the site GCGEQNM is responsible for binding toother macromolecules (for review see, Dodds and Day,1996). The thiol group of cysteine displaces the amidegroup on glutamine at this site in the native proteins.Upon proteolytic cleavage, this site is exposed. Theexposed thiol ester is susceptible to attack by nucleo-philes such as amines and hydroxyl groups resulting inthe formation of amides and esters. In general, such reac-tion leads to the immobilization of these proteins onother macromolecules. In an earlier work, we used aprimer that is specific for this sequence to isolate thecDNA encoding theManducaPPO (Hall et al., 1995).Subsequently, the corresponding putative thiol estersequence, CGWPQH, was identified in a number ofinsect PPOs. However, chemical modification studies(unpublished results) indicate thatManducaenzyme isnot sensitive to methylamine, a typical reaction exhibited


by thiolesters (Dodds and Day, 1996). In addition, aclose scrutiny of this site in different PPOs reveals thatglutamine occurs only sporadically in differentsequences, but the nearby histidine seems to be alwaysconserved. Thus most insect PPOs have the motif,C(G/N)CGWP(2)H(M/L)L at this location. The remark-able conservation of histidine and lack of glutamic acidat this site suggests that perhaps, histidine might beinvolved in adduct formation reaction with cysteine. Infungal tyrosinase, a thiol histidine adduct has beencharacterized as a structural component (Lerch, 1982),but the two amino acids are only separated by a thre-onine in this protein. Whether the thiol ester motif foundin PPO forms a similar adduct or not remains to bedetermined. Also it is not clear at present, whether thissite is responsible for the ‘stickiness’ of PPO or not.Similarly the role of the conserved C-terminal site(marked by asterisks in Fig. 10) is unknown at this time.A similar site is present in arylphorins (Brumester andScheller, 1996).

Acknowledgements

We thank Dr Kaliappanadar Nellaiappan, MarkZervas and Tim Scott for their help and assistance. Thisresearch was supported in part by grants from U.Mass/Boston and N.I.H. (grant # AI-14753).

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Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata

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