Directory UMM :Data Elmu:jurnal:I:Insect Biochemistry and Molecular Biology:Vol30.Issue10.Oct2000:

(1)

www.elsevier.com/locate/ibmb

Purification, characterization and molecular cloning of

prophenoloxidases from

Sarcophaga bullata

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 enzyme is involved in three physiologically important processes viz., cuticular hardening, defense reactions and wound healing in insects. It was isolated from the larval hemolymph of Sarcophaga bullata and purified by employing ammonium sulfate precipitation, Phenyl Sepharose chromatography, DEAE–Sepharose chromatography, and Sephacryl S-200 column chromatography. The purified enzyme exhibited two closely moving bands on 7.5% SDS–PAGE under denaturing conditions. From the estimates of molecular weight on Sephacryl S-100, TSK-3000 HPLC column and SDS–PAGE, which ranged from 90,000 to 100,000, it was inferred that the enzyme is made up of a single polypeptide chain. Activation of PPO (Ka=40µM) was achieved by the cationic detergent, cetyl

pyridinium chloride below its critical micellar concentration (0.8 mM) indicating that the detergent molecules are binding specifically to the PPO and causing the activation. Neither anionic, nor nonionic (or zwitterionic) detergents activated the PPO. The active enzyme exhibited wide substrate specificity and marked thermal unstability. Using primers designed to conserved amino acid sequences from known PPOs, we PCR amplified and cloned two PPO genes from the sarcophagid larvae. The clones encoded polypeptides 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 sites and 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 a bifunctional enzyme possessing both monophenol monooxygenase activity (E.C. 1.14.18.1. tyrosine, dihy-droxyphenylalanine, oxygen, oxidoreductase) and o -diphenoloxidase activity (E.C. 1.10.3.1.o-diphenol, oxy-gen, oxidoreductase). It is responsible for initiating the biosynthesis of widely distributed melanin pigment in nature (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 the} GenBank 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).

cle used for color and camouflage, PO is also uniquely associated with three different physiologically important biochemical processes in insects and other arthropods. These are (a) sclerotization of insect cuticle (Andersen et al., 1996; Sugumaran, 1998), (b) encapsulation and melanization of foreign organisms observed as defense reaction (Ashida and Brey, 1995; Gillespie et al., 1997; So¨derha¨ll et al., 1990; Sugumaran, 1996) and (c) wound healing (Lai-Fook, 1966; Sugumaran, 1996). In the first process, PO generated 4-alkylquinones serve as sclerot-izing agents for quinone tanning reactions—one of the mechanisms by which the insect cuticle is hardened to protect the soft bodies of animals (Andersen et al., 1996; Sugumaran, 1998). Quinones are converted by quinone isomerase to quinone methides (Saul and Sugumaran 1988a, 1990; Ricketts and Sugumaran, 1994) which are reactive intermediates for the second mechanism of tan-ning called quinone methide sclerotization. Some of the quinone methides are converted to 1,2-dehydro-N-acyl-dopamines by the action of quinone methide isomerase

(2)

(Saul and Sugumaran, 1989a,b; Ricketts and Sugumaran, 1994). The resultant dehydro-N-acyldopamines are further oxidized by PO to their corresponding quinones which rapidly isomerize nonenzymatically to form quin-one methide imine amides necessary for α,β -sclerotiz-ation (Sugumaran et al., 1992; Ricketts and Sugumaran, 1994). The reactions of quinones, quinone methides and quinone methide imine amides with cuticular structural proteins 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 and pathogens which are too large to be phagocytosed are found to be usually encapsulated and melanized in insect blood by the action of phenoloxidase (Ashida and Brey, 1995; Gillespie et al., 1997; So¨derha¨ll et al., 1990; Sugu-maran, 1996; Sugumaran and Kanost, 1993). This pro-cess not only limits the growth and development of the foreign object, but also prevents the damage it can cause to host by creating a physical barrier. Finally during wounding, continuous loss of hemolymph is prevented by the rapid deposition melanin polymer at the wounding site (Lai-Fook, 1966; Sugumaran, 1996). Apart from stopping 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 physiology and biochemistry certainly demands a serious study on this enzyme. But, numerous problems such as instability and rapid loss of activity during purification, ‘stickiness,’ (=insolubilization on biotic and abiotic matters, various gels and glassware used for the purification of the enzyme) and self inactivation have prevented the detailed characterization of insect POs in the past (Sugumaran and Kanost, 1993). By taking advantage of the fact that PO is present in the inactive proenzyme form, some scientists have successfully purified and characterized the PPO. Thus PPOs from Bombyx 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),Galleria mellonella (Kopa´cek et al., 1995), Holotrichia diomph-alia(Kwon et al., 1997),Calliphora erythrocephala(Pau and Eagles, 1975), Musca domestica(Hara et al., 1993; Tsukamoto et al., 1986), Drosophila melanogaster

(Fujimoto et al., 1993), Blaberus discoidalis(Durrant et al., 1993), Tenebrio molitor (Heyneman, 1965), and

Locusta 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 encoding Manduca sexta PPO (Hall et al., 1995), several investigators have also characterized different insect PPO genes. These include one from Dro-sophila melanogaster (Fujimoto et al., 1995), two from

Bombyx mori (Kawabata et al., 1995) a second from

Manduca sexta (Jiang et al., 1997a), two from

Hyphantria cunea(Park et al., 1997) six fromAnopheles gambiae(Jiang et al., 1997b; Lee et al., 1998; Muller et al., 1999), one fromTenebrio molitor(Lee et al., 1999), and one from Armigeres 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 we report the purification, characterization and molecular cloning of PPO from the larval hemolymph of Sarco-phaga bullata.

2. Materials and methods

2.1. Animals

Larvae of Sarcophaga bullata were obtained from Carolina Biological Supplies Co., NC and maintained on a 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 from Pharmacia Fine Chemicals, Nutley, NJ. Molecular weight markers for molecular weight determination, sil-ver staining kit, and Coomassie blue protein assay kit were obtained from Bio Rad Laboratories, Hercules, CA.

2.3. Enzyme purification

All operations were carried out at 0–5°C unless stated otherwise. 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. The hemolymph was directly collected into a flask chilled on dry ice. The frozen mass was stored at280°C for three to four weeks before use, or processed immediately. This procedure prevented the activation of PPO as well as darkening of hemolymph only in Sarcophaga. Use in other insects such as Manduca caused rapid activation of 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 sulfate saturation and the proteins precipitated within 20 min were discarded after centrifugation at 13,000g for 15 min. The supernatant was brought to 60% saturation with respect to ammonium sulfate and the proteins pre-cipitated within 20 min were collected by centrifugation at 10,000gfor 15 min. The pellet was dissolved in mini-mum amount of buffer A containing 10% ammonium sulfate and chromatographed on a phenyl Sepharose

(3)

col-umn (13.5×2 cm) equilibrated with the same buffer. After loading and washing the column to remove unbound proteins, bound PPO was eluted with water. A flow rate of 4 ml/min was maintained throughout. Frac-tions containing PPO were pooled and chromatographed on a DEAE Sepharose column (13.5×3 cm) equilibrated with 10 mM buffer A. The column was washed exten-sively with this buffer and bound proteins were eluted with step gradients of (a) 50 mM buffer A and (b) 100 mM buffer A at a flow rate of 3 ml/min. PPO activity eluting 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 in minimum 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 0.4 ml/min was maintained and fractions of 4 ml were col-lected. The PPO containing fractions were pooled and used as the pure proenzyme.

2.4. Enzyme assay

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

2.5. Molecular weight estimation

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

The approximate molecular weight of PPO was determined by three different techniques. The first method employed the above mentioned SDS–PAGE. The second method utilized the gel filtration HPLC on a TSK 3000 (30 cm×7.5 mm) column coupled with a Beckman pre-column (4 cm×7.5 mm). The standards and PPO were separately chromatographed using isocratic elution with 100 mM buffer A at a flow rate of 0.7 ml/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 of molecular weight markers, the approximate molecular weight of the PPO was determined. The molecular weight of the native PPO was also determined on a Sephacryl S-100 column (55×2 cm) equilibrated with 10 mM buffer A containing 0.2 M NaCl. A flow rate of 12 ml/h was maintained. The column was calibrated with different molecular weight markers.

2.6. RNA extraction

RNA was isolated from the hemocytes of last instar

S. bullata larval hemolymph. About 300 larvae were injected with decoagulation buffer (100 mM glucose, 15 mM NaCl, 10 mM disodium ethylene diamine tetraacet-ate, 30 mM trisodium citrate and 26 mM citric acid, pH 4.6) and a drop of fluid from each animal was collected into a 15 ml tube placed on ice. Extract was allowed to settle, then the upper layer was transferred to a fresh tube and centrifuged at 1000g to pellet the cells. The supernatant was discarded and the cells were stored at

280°C until needed. Hemocytes were removed from the

280°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. The RNA 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–polymerase chain reaction (RT–PCR) strategy to isolate the PPO cDNAs from S. bullata by amplifying reverse tran-scribed (RT) total RNA extracted from larval hemocyte with degenerate PCR primers designed to conserved amino acid motifs among all insect PPO sequences avail-able from the GenBank. Multiple PCR fragments were sequenced, compared to previously known PPO genes and used to design gene specific primers to obtain the remainder of the molecule with a 39and 59RACE strat-egy. Thus isolating each PPO cDNA, in three inde-pendent overlapping fragments. To ensure integrity of each PPO cDNA contig, primers were designed within the 59 and 39 untranslated regions to amplify the entire coding domain. The entire coding domain was cloned and resequenced.

2.8. Degenerate primer design

Insect PPO amino acid sequences were downloaded in FASTA format from GENBANK and aligned with CLUSTAL W (Thompson et al., 1994), using default parameters. Conserved domains were identified and

(4)

reverse translated. The forward primer, PPO-FS (5₉ -CAY -CAY TKB -CAY TGG -CAY YTN GT-3₉) targets HH (W/Y) HWHLVY (Copper binding domain) and the reverse primer PPO-RAS (59-CKR TCR AAN GGR WAN CCC AT-39), MGYPFDR (conserved carboxy domain).

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

Independent RT reactions (Superscript II Gibco BRL) with 5µl of 1/5 or 1/10 dilutions ofS. bullatatotal RNA eluted from the RNeasy column and primed with RT-1 (59-CTC TGG GCC CAA GCT TTT TTT TTT TTT TTV-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 5 µl of 1/5, 1/10 and 1/50 dilutions of the RT reaction with PPO-FS and PPO-RAS. The reactions were set up as follows: 5 µl template, 5

µl 10 PCR buffer (Promega) 2.5 mM MgCl2, 200 µM

each 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, manually adding the 1 unit of TAQ to each reaction and cycled at 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 allowed to grow overnight and screened by contaminating a PCR reaction containing M13 forward and reverse primers with a single colony. Replicate clones were plated and grown for 5 h at 37°C. Selected colonies (based on PCR results) 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 other insect PPOs were then used to design nested primers of about 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, using template from the initial RT reaction primed with RT-1.

2.11. 5₉ race

To obtain the 5₉ end of each DNA fragment, two nested anti-sense primers were designed 200–250 bp away from the 59end of the clone. Independent RT reac-tions were primed with the outer most gene specific primer 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 was changed as follows: The anneal temperature was

decreased to 48°C for five cycles, then increased to 60°C for the 30 remaining cycles. If the PCR product was heterogeneous, 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 coding domain

To ensure the integrity of each identified gene, we amplified the entire coding domain. Primers were designed to the 39 and 59 untranslated regions of each gene. PCR conditions were as previously described, but the anneal temperature was altered to accommodate each primer 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 data at 500 base intervals and three independent clones sequenced.

2.13. Sequence assembly and analysis

Contigs for each sequence was assembled and edited with the program Sequencher (Gene Codes Corporation). Sequencing primers were designed with the program Primer3 (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3Fwww.cgi). Each sequence was

com-pared to other sequences in GenBank with a Blastx search. Prophenoloxidase amino acid sequences were downloaded from GenBank in fasta format and aligned with Clustal W using default parameters (except for changing output format to Phylip). This file was imported to PAUP (Swofford, 1999 (4.0b2)) and used to calculate pairwise identities.

3. Results

Employing the protein purification scheme outlined in Table 1, PPO from the hemolymph was purified 221-fold with a recovery of 16%. The recovery of total activity was drastically reduced at the ammonium sulfate step due to the presence of proteins that interfere with quinonoid metabolism and high protein concentrations. However, during the succeeding steps, such interference is removed and the recovery of total activity units is restored. The finally purified PPO exhibited two closely moving bands on SDS–7% polyacrylamide gel (Fig. 1). Both bands when cut and incubated with dopamine and CPC, exhibited PO activity. Without CPC treatment, neither of them showed any detectable phenoloxidase activity, thereby indicating that they are isozymes of PPO.

(5)

Table 1

Purification 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 1

NH4SO4 2.8 1092 420 2.6 17.5 5

Phenyl sepharose 122 4148 152.5 27.2 66.4 5.2

DEAE-sepharose 30 2760 33 83.6 44.2 16.1

Sephacryl 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 were stained 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 Sephacryl S-100 (or Sephacryl S-200) gel filtration column, it eluted with an apparent molecular weight of 100,000 [Fig. 2(a)]. On HPLC, it exhibited a molecular weight of 85,000 [Fig. 2(b)]. Under denaturing conditions on SDS–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 chain with no apparent subunits.

3.2. Activation of PPO

Fig. 3 inset shows the activation of PPO caused by different detergents. As is evident only the cationic detergent, CPC activated the proenzyme specifically and neither anionic detergents such as SDS, sodium capry-late, sodium lauryl sarcosidate, deoxychocapry-late, nor non-ionic detergents such as octyl-β-glucopyranoside, Non-idet-P-40, Triton X-100, digitonin, Brij 58 and Tween 20 activated the proenzyme. Similarly, zwitterionic detergent, 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 gel filtration chromatography. A Sephacryl S-100 column (55×2 cm) equi-librated with 10 mM sodium phosphate buffer pH 6.0 containing 200 mM sodium chloride was used at a flow rate of 12 ml/h. Molecular weight 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 exclusion HPLC. A Beckmann TSK 3000 column (30 cm×7.5 mm) was used to estimated the approximate molecular weight of PPO. Proteins were separated on the column using isocratic elution with 100 mM sodium phosphate buffer pH 6.0 at a flow rate of 0.7 ml/min. Molecular weight markers used are: (A) carbonic anhydrase (29 kDa); (B) bovine serum albumin (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 and methods. Pre-stained molecular weight markers were used to calculate the approximate molecular weight of PPO. (A) carbonic anhydrase (29 kDa); (B) ovalbumin (43 kDa); (C) Phosphorylase b (96 kDa); and (D) alcohol dehydrogenase (150 kDa).

(6)

Fig. 2. (continued)

Fig. 3. Activation of PPO by different concentrations of CPC. PPO was assayed using standard assay conditions except for variation in the concentration 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; Nonidet P 40; Brij 35; Tween 20; CHAPS; sodium lauryl sarcosidate; sodium caprylate; Triton X-100; deoxycholate or digitonin.

critical micellar concentration of CPC. From the dose response curves shown in Fig. 3, theKafor the activation

of PPO by CPC was estimated to be 40µM. The critical micellar concentration of CPC under these conditions is 0.8 mM. Therefore, CPC must be binding to the proen-zyme specifically below its critical micellar concen-tration and activating the enzyme. This is in confirmation with our previous result on the activation of Manduca

PPO (Hall et al., 1995).

(7)

potent activators of PPO (Sugumaran and Nellaiappan, 1991; Sugumaran and Kanost, 1993). In order to check whether sarcophagid PPO can also activated by these reagents, the following studies were carried out. Various phospholipids and fatty acids have been incubated with PPO and the appearance of PO activity in the incubation mixture was assessed by standard PO assay. As shown in Fig. 4, fatty acids caused either mild or no activation at all. Among the different phospholipids tested, phos-phatidyl glycerol activated the PPO immediately, while phosphatidyl inositol activated the PPO after a lag.

3.3. pH optimum and thermal stability

The activated PO exhibited a typical bell shaped pH curve with optimum activity observed at about pH 7 (Fig. 5). Fig. 6 shows the comparative stability of pro-phenoloxidase and detergent activated pro-phenoloxidase. The activated enzyme rapidly lost its activity even at room temperature (25°C) within 15 min (curve C), while the raising the temperature to 55°C resulted in total loss of its activity within one min (curve D). In contrast, the proenzyme was much more stable towards heat treat-ment (Curves A and B, Fig. 6). Heating the proenzyme for 10 min at 60°C resulted in 50% loss of its activity and 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 is shown in Fig. 7. As is evident, N-acetyldopamine, cat-echol, 4-methyl catcat-echol, norepinephrine, dopamine, 3,4-dihydroxyphenylacetic acid, and N-β

-alanyldopam-Fig. 4. Activation of PPO by different phospholipids. PPO was assayed using standard assay conditions except for using different lip-ids in the place of CPC. The liplip-ids were dissolved in 100µl of ethanol and made up to 1 ml with water (concentration of lipids=5 mg/ml). An aliquot (6µl) of this solution, was used to test the activation of PPO. Compounds used are (A) phosphatidyl glycerol; (B) phosphatidyl inositol; (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 was determined at indicated pH values using the standard assay conditions. Sodium acetate buffer (50 mM) was used from pH 3 to 4.5 (No activity was detected from pH 3 to 4). Sodium phosphate buffer (50 mM) was used from pH 5 to 9.

Fig. 6. Thermal stability of PPO and PO. The thermal stability of PPO and PO were determined by incubating the PPO and CPC acti-vated PO at indicated temperature for varying amounts of time. After cooling to room temperature, the residual activity was determined using 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 is routinely 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 towards hydroquinone, methyl hydroquinone and gentisic acid indicates that the enzyme is a typicalo-diphenoloxidase

(8)

Fig. 7. Substrate specificity of sarcophagid phenoloxidase. Standard assay conditions were employed except for variation of substrates instead of dopamine as indicated. Oxygen uptake assay was used. Sub-strate used are: (A) 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 to possess 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 of the enzyme drastically (Table 2). Other copper chelators also 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. This further emphasizes the superiority of dopamine over dopa as a substrate for insect PO.

3.6. Molecular cloning

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

Table 2

Inhibition of detergent activated Prophenoloxidase

Inhibitor Concentration (%) Inhibition

Phenylthiourea 10µM 100

1µM 93

0.3µM 90

0.1µM 74

Diethyldithiocarbamate 90µM 100

70µM 92

50µM 84

10µM 49

Sodium cyanide 0.5 mM 100

0.1 mM 64

Mimosine 5 mM 26

1 mM 20

Neocuprine 5 mM 74

1 mM 38

8-Hydroxyquinoline sulfonate 1 mM 100

0.1 mM 33

o-Phenanthroline 5 mM 0

α,α9-Dipyridyl 1 mM 14

Sodium fluoride 5 mM 0

Ethylenediamine tetra acetate 5 mM 23

1 mM 19

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

Comparison of deduced amino acid sequence of the PPO 1 and 2 with other arthropod PPOs in GenBank (Fig. 10) shows that the two copper binding sites are preserved in all these proteins (underlined sequences). Both copper binding sites show extensive homology. The six histidine residues, which ligate the two copper atoms, are present at the conserved sites in all the phe-noloxidases. The proteolytic cleavage site (RF, indicated by an arrow) is conserved in all dipteran PPOs. A second possible proteolytic cleavage site, (REE, also designated by an arrow) is conserved in all dipteran PPOs with the exception of sarcophagid PPO 2 which has RAE and

Drosophila PPOA1 which has RQE at this site. In addition, previously characterized putative thiol ester site CGCGWPQHML (double underlined) (Hall et al., 1995) is preserved in SbPPO1 in its entirety, but the SbPPO2 has E instead of Q at position 7. At the C-terminal region, there is a conserved region in all the PPOs (marked by asterisks). In particular, the motif MG(Y/F)PFDR is present in all known PPOs.

(9)

(10)

(11)

Fig. 10. Alignment of known dipteran PPO sequences. The two copper binding domains in the amino acid sequence are underlined. Arrows indicate 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 in Materials and methods, we have purified the PPO from the hemolymph of the Sarcophaga larvae to apparent homogeneity. The purified enzyme exhibited two closely moving bands on the polyacrylamide gel electrophoresis; both capable of oxidizing dopamine when activated with CPC. Thus the protein bands are due to the presence of two isozymic forms of the same enzyme. Two isoforms

of PPO have been characterized from Manduca sexta

(Hall et al., 1995; Jiang et al., 1997a), Bombyx mori

(Yasuhara et al., 1995), and Galleria mellonella

(Kopa´cek et al., 1995). However, only one isoform has been characterized fromBlaberus discoidalis(Durrant et al., 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 indicating the presence of six different isozymes.

(12)

(13)

monomerization and polymerization depending on the ionic strength of the medium (Jiang et al., 1997a; Yasu-hara et al., 1995; Kwon et al., 1997). However, the PPO from H. cecropia has been shown to be a monomeric protein (Andersson et al., 1989). The sarcophagid PPOs isolated in the present study also seems to be a mono-meric protein.

The phenoloxidase fromGallaria mellonellahas been shown to be a glycoprotein (Kopa´cek et al., 1995). But, both Manduca sexta (Jiang et al., 1997a) and Bombyx mori (Yasuhara et al., 1995) PPOs are devoid of any carbohydrates and are not glycoproteins. Sarcophagid PPOs have few N-glycosylation sites and do not stain for sugars with Schiff’s reagent (data not shown). They also do not exhibit any binding affinity for Concanavalin A Sepharose. Therefore, sarcophagid PPOs do not appear to be glycosylated.

The sarcophagid PPO can be activated either by endogenous proteases (Saul and Sugumaran, 1988b) or by detergents. Among the detergents tested only the cat-ionic detergent, CPC activated the proenzyme. The acti-vation caused by CPC (Ka=40µM) occurred well below

its critical micellar concentration (0.8 mM) thereby indi-cating that CPC is binding to the proenzyme specifically and causing its activation. Earlier, we reported a similar findings 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 were tested for their ability to activate the proenzyme. In gen-eral, fatty acids showed only marginal activation of sar-cophagid PPO. Phosphatidyl glycerol activated the PPO readily, while phosphatidyl inositol activated the enzyme after a lag (Fig. 4).

The substrate specificity of the sarcophagid PPO is similar to that of other insect enzymes and is distinctly different from that of the mammalian tyrosinase. Thus, N-acetyldopamine, N-β-alanyldopamine, and dopamine proved to be far better substrates for the enzyme, than dopa which is routinely used for the assay of PPOs. This distinct difference in substrate specificity between mam-malian and arthropod enzymes and the absence signifi-cant sequence identity between these two class of enzymes confirms that they are quite different in spite of the fact that they catalyze the same biochemical trans-formations.

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

Table 3

Percent similarity of known PPOsa

PPO GenBank A. no. SbPPO1 SbPPO2 SbPPO1 AF161260

SbPPO2 AF161261 52

DmPPOA1 D45835 52 62

AgPPO1 L76038 73 52

AgPPO2 AF004915 53 54

AgPPO3 AF004916 55 54

AgPPO4 AG010193 51 49

AgPPO5 AG010194 49 46

AgPPO6 AG010195 53 52

AsPPO AF062034 54 52

TmPPO AB020738 65 50

MsPPO1 AF003253 59 46

MsPPO2 L42556 51 44

BmPPO1 D49370 60 48

BmPPO2 D49371 53 45

HcPPO1 U86875 56 47

HcPPO2 AF020391 51 45

PlPPO 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 binding regions are highly conserved among all dipteran PPOs. The putative proteolytic cleavage site 1 in Sarcophaga

PPO1, RFG (marked with an arrow in Fig. 10) resembles lepidopteran PPO better than the dipteran PPO sequences. This site is the same as that found in all lepi-dopterans. Sarcophaga PPO2 has the sequence, RFS similar to Drosophila A1, and anopheline PPO2 and anopheline PPO6 at this site. The second cleavage site on Sarcophaga PPO1, REE (also marked by an arrow at amino acid 164) is conserved at all the mosquito sequences. SarcophagaPPO2 has RAE and Drosophila

has RQE at this site.

Inα2-macroglobulin and complement proteins C3 and C4, the site GCGEQNM is responsible for binding to other macromolecules (for review see, Dodds and Day, 1996). The thiol group of cysteine displaces the amide group on glutamine at this site in the native proteins. Upon proteolytic cleavage, this site is exposed. The exposed thiol ester is susceptible to attack by nucleo-philes such as amines and hydroxyl groups resulting in the formation of amides and esters. In general, such reac-tion leads to the immobilizareac-tion of these proteins on other macromolecules. In an earlier work, we used a primer that is specific for this sequence to isolate the cDNA encoding the Manduca PPO (Hall et al., 1995). Subsequently, the corresponding putative thiol ester sequence, CGWPQH, was identified in a number of insect PPOs. However, chemical modification studies (unpublished results) indicate that Manduca enzyme is not sensitive to methylamine, a typical reaction exhibited

(14)

by thiolesters (Dodds and Day, 1996). In addition, a close scrutiny of this site in different PPOs reveals that glutamine occurs only sporadically in different sequences, but the nearby histidine seems to be always conserved. 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 acid at this site suggests that perhaps, histidine might be involved in adduct formation reaction with cysteine. In fungal tyrosinase, a thiol histidine adduct has been characterized 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 found in PPO forms a similar adduct or not remains to be determined. Also it is not clear at present, whether this site 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 and Scheller, 1996).

Acknowledgements

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

References

Andersen, S.O., Peter, M.G., Roepstorff, P., 1996. Cuticular sclerotiz-ation in insects. Comparative Biochemistry and Physiology 113B, 689–705.

Andersson, K., Sun, S.C., Boman, H.G., Steiner, H., 1989. Purification of the prophenoloxidase fromHyalophora cecropiaand four pro-teins involved in its activation. Insect Biochemistry 19, 629–637. Ashida, M., 1971. Purification and characterization of prephenoloxi-dase from hemolymph of the silkwormBombyx mori. Archives of Biochemistry and Biophysics 144, 749–762.

Ashida, M., Brey, P., 1995. Role of the integument in insect defense: prophenoloxidase cascade in the cuticular matrix. Proceedings of the National Academy of Science USA 92, 10698–10702. Aso, Y., Kramer, K.J., Hopkins, T.L., Lookhart, G.L., 1985.

Charac-terization of hemolymph protyrosinase and a cuticular activator fromManduca sexta(L). Insect Biochemistry 15, 9–17.

Barrett, F.M., 1991. Phenoloxidases andthe integument. In: Binning-ton, K., Retnakaran, A. (Eds.), The Physiology of Insect Epidermis. CSIRO Publications, Victoria, Australia, pp. 195–212.

Brumester, T., Scheller, K., 1996. Common origin of arthropod tyro-sinase, arthropod hemocyanin, insect hexamerin and dipteran aryl-phorin receptors. Journal of Molecular Evolution 42, 713–728. Cherqui, A., Duvic, B., Brehelin, M., 1996. Purification and

charac-terization of prophenoloxidase from the hemolymph of Locusta migratoria. Archives of Insect Biochemistry and Physiology 32, 225–235.

Cho, W.L., Liu, H.S., Lee, C.H., Kuo, C.C., Chang, T.Y., Liu, C.T., Chen, C.C., 1998. Molecular cloning, characterization and tissue expression of prophenoloxidase cDNA from the mosquito

Armigeres subalbatusinoculated withDirofilaria immitis microfil-ariae. Insect Molecular Biology 7, 31–40.

Dodds, A.W., Day, A.J., 1996. Complement related proteins in invert-ebrates. In: So¨derha¨ll, K., Iwanaga, K., Vastha, G.R. (Eds.), New Directions in Invertebrate Immunology. SOS Publications, Fair Haven, NJ, pp. 303–341.

Durrant, H.J., Ratcliffe, N.A., Hipkin, C.R., Aspan, A., So¨derha¨ll, K., 1993. Purification of the prophenoloxidase enzyme from hemocytes of the cockroachBlaberus discoidalis. Biochemistry Journal 289, 87–91.

Fujimoto, K., Masuda, K., Asada, N., Ohnishi, E., 1993. Purification and characterization of prophenoloxidase from the pupae of Droso-phila melanogaster. Journal of Biochem (Tokyo) 113, 285–291. Fujimoto, K., Okino, N., Kawabata, S.I., Iwanaga, S., Ohnishi, E.,

1995. Nucleotide sequence of the cDNA encoding the proenzyme of phenoloxidase A1ofDrosophila melanogaster. Proceedings of the National Academy of Science USA 92, 7769–7773.

Gillespie, J.P., Kanost, M.R., Trenczek, T., 1997. Biological mediators of insect immunity. Annual Reviews in Entomology 42, 611–643. Hall, M., Scott, M., Sugumaran, M., So¨derha¨ll, K., Law, J.H., 1995. Proenzyme of Manduca sexta phenoloxidase: purification, acti-vation, substrate specificity of the active enzyme and molecular cloning. Proceedings of the National Academy of Science USA 92, 7764–7768.

Hara, T., Miyoshi, T., Funatsu, M., 1993. Comparative studies on lar-val and pupal phenoloxidases of the housefly Musca domestica. Comparative Biochemistry and Physiology 106B, 287–292. Heyneman, R.A., 1965. Final purification of a latent phenoloxidase

with mono- and diphenoloxidase fromTenebrio molitor. Biochem-istry and Biophysics Research Communications 21, 162–169. Jiang, H., Wang, Y., Ma, C., Kanost, M.R., 1997a. Subunit

compo-sition of prophenoloxidase fromManduca sexta: molecular cloning of subunit ProPo-P1. Insect Biochemistry and Molecular Biology 27, 835–850.

Jiang, H., Wang, Y., Korochkina, S.E., Benes, H., Kanost, M.R., 1997b. Molecular cloning of cDNAs for two prophenoloxidases subunits from the Malaria vectorAnopheles gambiae. Insect Bio-chemistry and Molecular Biology 27, 693–699.

Kawabata, T., Yasuhara, Y., Ochiai, M., Matsuura, S., Ashida, M., 1995. Molecular cloning of insect prophenoloxidase: a copper con-taining protein homologous to arthropod hemocyanin. Proceedings of the National Academy of Science USA 92, 7774–7778. Kopa´cek, P., Weise, C., Gotz, P., 1995. The prophenoloxidase from

the wax mothGalleria mellonella: purification and characterization of proenzyme. Insect Biochemistry and Molecular Biology 25, 1081–1091.

Kwon, T.H., Lee, S.Y., Lee, J.H., Choi, J.S., Kawabata, S.I., Iwanaga, S., Lee, B.L., 1997. Purification and characterization of prophe-noloxidase from the hemolymph of coleopteran insectHolotrichia diomphlia. Molecules and Cells 7, 90–97.

Lai-Fook, J., 1966. The repair of wounds in the integument of insects. Journal of Insect Physiology 12, 195–226.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage, T4. Nature 227, 680–685. Lerch, K., 1982. Primary structure of tyrosinase from Neurospora

crassa. Journal of Biological Chemistry 257, 6414–6419. Lee, W.J., Ahmed, A., Torre, A.D., Kobayashi, A., Ashida, M., Brey,

P.T., 1998. Molecular cloning and chromosomal localization of a prophenoloxidase cDNA from the malaria vectorAnapheles gam-biae. Insect Molecular Biology 7, 41–50.

Lee, H.S., Cho, S.Y., Lee, K.M., Kwon, T.H., Homma, K., Natori, S., Lee, B.L., 1999. The pro-phenoloxidase of coleopteran insect,

Tenebrio molitor, larva was activated during cell clump/cell adhesion of insect cellular defense reactions. FEBS Letters 444, 255–259.

Muller, H.M., Dimopoulos, G., Blass, C., Kafatos, F.C., 1999. A hemo-cyte-like cell line established from the malaria vector Anopheles

(15)

gambiae expresses six prophenoloxidase genes. Journal of Biologi-cal Chemistry 274, 11727–11735.

Nappi, A.J., Sugumaran, M., 1993. Some biochemical aspects of eume-lanin in insect immunity. In: Pathak, J.P.N. (Ed.), Insect Immunity. Oxford and IBH Publishing Co, New Delhi, pp. 131–148. Park, D.S., Shin, W., Kim, M.G., Park, S.S., Lee, W.J., Brey, P.T.,

Park, H.Y., 1997. Isolation and characterization of the cDNA enco-ding the prophenoloxidase of Fall webworm Hyphantria cunea. Insect Biochemistry and Molecular Biology 27, 983–992. Pau, R.N., Eagles, P.A.M., 1975. The isolation of o-diphenoloxidase

from the third instar larvae of blowfly Calliphora erythocephala. Biochemistry Journal 149, 707–712.

Prota, G., 1992. Melanins and Melanogenesis. Academic Press, San Diego.

Ricketts, D., Sugumaran, M., 1994. 1,2-dehydro-N-β-alanyldopamine as a new intermediate in insect cuticular sclerotization. Journal of Biological Chemistry 269, 22217–22221.

Saul, S.J., Sugumaran, M., 1988a. A novel quinone: quinone methide isomerase generates quinone methides in insect cuticle. FEBS Let-ters 237, 155–158.

Saul, S.J., Sugumaran, M., 1988b. Prophenoloxidase activation in the hemolymph ofSarcophaga bullatalarvae. Archives of Insect Bio-chemistry and Physiology 7, 91–103.

Saul, S.J., Sugumaran, M., 1989a. Characterization of a new enzyme system that desaturates the side chain of N-acetyldopamine. FEBS Letters 251, 69–73.

Saul, S.J., Sugumaran, M., 1989b. N-acetyldopamine quinone methide /1,2-dehydro-N-acetyldopamine tautomerase—a new enzyme involved in sclerotization of insect cuticle. FEBS Letters 255, 340–344.

Saul, S.J., Sugumaran, M., 1990. 4-alkyl-o-quinone/2-hydroxy-p -quin-one methide isomerase from the larvae hemolymph ofSarcophaga bullata. I. purification and characterization of enzyme catalyzed reaction. Journal of Biological Chemistry 265, 16992–16999. So¨derha¨ll, K., Aspa´n, A., Duvic, B., 1990. The ProPO system and

associated proteins. Role in cellular communication in arthropods. Research in Immunology 141, 896–907.

Sugumaran, M., 1996. Role of insect cuticle in immunity. In: So¨der-ha¨ll, K., Iwanaga, S., Vastha, G. (Eds.), New Directions in Invert-ebrate Immunology. SOS Publications, Fair Haven, NJ, pp. 355– 374.

Sugumaran, M., 1998. Unified mechanism for sclerotization of insect cuticle. Advances in Insect Physiology 27, 229–334.

Sugumaran, M., Kanost, M., 1993. Regulation of insect hemolymph phenoloxidases. In: Beckage, N.E., Thompson, S.N., Frederick, B.A. (Eds.). Parasites and Pathogens, 1. Academic Press, San Diego, pp. 317–342.

Sugumaran, M., Nellaiappan, K., 1991. Lysolecithin—a potent acti-vator of prophenoloxidase from the hemolymph of the lobster

Homarus americanus. Biochemistry and Biophysics Research Communications 176, 1371–1376.

Sugumaran, M., Semensi, V., Kalyanaraman, B., Bruce, J.M., Land, E.J., 1992. Evidence for the formation of a quinone methide during the oxidation of the insect cuticular sclerotizing precursor, 1.2-dehydro-N-acetyldopamine. Journal of Biological Chemistry 267, 10355–10361.

Swofford, D.L., 1999. PAUP*: Phylogenetic Analysis Using Parsi-mony, (*And Other Methods) Version 4. Sinauer Associates Inc Publishers, Sunderland, MA.

Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence align-ment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acid Research 22, 4673–4680. Tsukamoto, T., Ishiguro, M., Funatsu, M., 1986. Isolation of a latent phenoloxidase from prepupae of the house fly Musca domestica. Insect Biochemistry 16, 573–581.

Yasuhara, Y., Koizumi, Y., Katagiri, C., Ashida, M., 1995. Reexamin-ation of properties of prophenoloxidase isolated from larval hemo-lymph of the silkworm,Bombyx mori. Archives of Insect Biochem-istry and Physiology 32, 14–23.

(1)

(2)

4. Discussion

Using the protein purification protocols outlined in

Materials and methods, we have purified the PPO from

the hemolymph of the

Sarcophaga

larvae to apparent

homogeneity. The purified enzyme exhibited two closely

moving bands on the polyacrylamide gel electrophoresis;

both capable of oxidizing dopamine when activated with

CPC. Thus the protein bands are due to the presence of

two isozymic forms of the same enzyme. Two isoforms

of PPO have been characterized from

Manduca sexta

(Hall et al., 1995; Jiang et al., 1997a),

Bombyx mori

(Yasuhara et al., 1995), and

Galleria mellonella

(Kopa´cek et al., 1995). However, only one isoform has

been characterized from

Blaberus discoidalis

(Durrant et

al., 1993) and

Locusta migratoria

(Cherqui et al., 1996).

In anopheline cell line, Muller et al. (1999) have

charac-terized as many as six different PPO genes indicating

the presence of six different isozymes.

(3)

(4)

monomerization and polymerization depending on the

ionic strength of the medium (Jiang et al., 1997a;

Yasu-hara et al., 1995; Kwon et al., 1997). However, the PPO

from

H. cecropia

has been shown to be a monomeric

protein (Andersson et al., 1989). The sarcophagid PPOs

isolated in the present study also seems to be a

mono-meric protein.

The phenoloxidase from

Gallaria mellonella

has been

shown to be a glycoprotein (Kopa´cek et al., 1995). But,

both

Manduca sexta

(Jiang et al., 1997a) and

Bombyx

mori

(Yasuhara et al., 1995) PPOs are devoid of any

carbohydrates and are not glycoproteins. Sarcophagid

PPOs have few N-glycosylation sites and do not stain

for sugars with Schiff’s reagent (data not shown). They

also do not exhibit any binding affinity for Concanavalin

A Sepharose. Therefore, sarcophagid PPOs do not

appear to be glycosylated.

The sarcophagid PPO can be activated either by

endogenous proteases (Saul and Sugumaran, 1988b) or

by detergents. Among the detergents tested only the

cat-ionic detergent, CPC activated the proenzyme. The

acti-vation caused by CPC (

K

=40

µM) occurred well below

its critical micellar concentration (0.8 mM) thereby

indi-cating that CPC is binding to the proenzyme specifically

and causing its activation. Earlier, we reported a similar

findings 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 were

tested for their ability to activate the proenzyme. In

gen-eral, fatty acids showed only marginal activation of

sar-cophagid PPO. Phosphatidyl glycerol activated the PPO

readily, while phosphatidyl inositol activated the enzyme

after a lag (Fig. 4).

The substrate specificity of the sarcophagid PPO is

similar to that of other insect enzymes and is distinctly

different from that of the mammalian tyrosinase. Thus,

N-acetyldopamine, N-β-alanyldopamine, and dopamine

proved to be far better substrates for the enzyme, than

dopa which is routinely used for the assay of PPOs. This

distinct difference in substrate specificity between

mam-malian and arthropod enzymes and the absence

signifi-cant sequence identity between these two class of

enzymes confirms that they are quite different in spite

of the fact that they catalyze the same biochemical

trans-formations.

The sequence identities of the sarcophagid PPOs with

other reported PPO sequences, range from 73 to 38%

(Table 3). SbPPO1 and SbPPO2 share 52% homology

between them. SbPPO1 shows 52% homology with

Dro-sophila

PPO A1; while SbPPO2 shows 62% homology.

SbPPO1 is quite similar to mosquito AgPPO1 (73%).

Our finding of two divergent PPO lineages within a

species is consistent with what others have found (Jiang

et al., 1997a; Muller et al., 1999). These results clearly

associate the PPO genes we have isolated with other

dip-teran PPOs.

Table 3

Percent similarity of known PPOsa

PPO GenBank A. no. SbPPO1 SbPPO2

SbPPO1 AF161260

SbPPO2 AF161261 52

DmPPOA1 D45835 52 62

AgPPO1 L76038 73 52

AgPPO2 AF004915 53 54

AgPPO3 AF004916 55 54

AgPPO4 AG010193 51 49

AgPPO5 AG010194 49 46

AgPPO6 AG010195 53 52

AsPPO AF062034 54 52

TmPPO AB020738 65 50

MsPPO1 AF003253 59 46

MsPPO2 L42556 51 44

BmPPO1 D49370 60 48

BmPPO2 D49371 53 45

HcPPO1 U86875 56 47

HcPPO2 AF020391 51 45

PlPPO 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 binding

regions are highly conserved among all dipteran PPOs.

The putative proteolytic cleavage site 1 in

Sarcophaga

PPO1, RFG (marked with an arrow in Fig. 10) resembles

lepidopteran

PPO better

than

the

dipteran

PPO

sequences. This site is the same as that found in all

lepi-dopterans.

Sarcophaga

PPO2 has the sequence, RFS

similar to

Drosophila

A1, and anopheline PPO2 and

anopheline PPO6 at this site. The second cleavage site

on

Sarcophaga

PPO1, REE (also marked by an arrow

at amino acid 164) is conserved at all the mosquito

sequences.

Sarcophaga

PPO2 has RAE and

Drosophila

has RQE at this site.

In

α2-macroglobulin and complement proteins C3 and

C4, the site GCGEQNM is responsible for binding to

other macromolecules (for review see, Dodds and Day,

1996). The thiol group of cysteine displaces the amide

group on glutamine at this site in the native proteins.

Upon proteolytic cleavage, this site is exposed. The

exposed thiol ester is susceptible to attack by

nucleo-philes such as amines and hydroxyl groups resulting in

the formation of amides and esters. In general, such

reac-tion leads to the immobilizareac-tion of these proteins on

other macromolecules. In an earlier work, we used a

primer that is specific for this sequence to isolate the

cDNA encoding the

Manduca

PPO (Hall et al., 1995).

Subsequently, the corresponding putative thiol ester

sequence, CGWPQH, was identified in a number of

insect PPOs. However, chemical modification studies

(unpublished results) indicate that

Manduca

enzyme is

not sensitive to methylamine, a typical reaction exhibited

(5)

by thiolesters (Dodds and Day, 1996). In addition, a

close scrutiny of this site in different PPOs reveals that

glutamine

occurs

only

sporadically

in

different

sequences, but the nearby histidine seems to be always

conserved. 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 acid

at this site suggests that perhaps, histidine might be

involved in adduct formation reaction with cysteine. In

fungal tyrosinase, a thiol histidine adduct has been

characterized 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 found

in PPO forms a similar adduct or not remains to be

determined. Also it is not clear at present, whether this

site 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 and

Scheller, 1996).

Acknowledgements

We thank Dr Kaliappanadar Nellaiappan, Mark

Zervas and Tim Scott for their help and assistance. This

research was supported in part by grants from U.

Mass/Boston and N.I.H. (grant # AI-14753).

References

Andersen, S.O., Peter, M.G., Roepstorff, P., 1996. Cuticular sclerotiz-ation in insects. Comparative Biochemistry and Physiology 113B, 689–705.

Charac-terization of hemolymph protyrosinase and a cuticular activator fromManduca sexta(L). Insect Biochemistry 15, 9–17.

Barrett, F.M., 1991. Phenoloxidases andthe integument. In: Binning-ton, K., Retnakaran, A. (Eds.), The Physiology of Insect Epidermis. CSIRO Publications, Victoria, Australia, pp. 195–212.

charac-terization of prophenoloxidase from the hemolymph of Locusta migratoria. Archives of Insect Biochemistry and Physiology 32, 225–235.

Cho, W.L., Liu, H.S., Lee, C.H., Kuo, C.C., Chang, T.Y., Liu, C.T., Chen, C.C., 1998. Molecular cloning, characterization and tissue expression of prophenoloxidase cDNA from the mosquito

Armigeres subalbatusinoculated withDirofilaria immitis microfil-ariae. Insect Molecular Biology 7, 31–40.

1995. Nucleotide sequence of the cDNA encoding the proenzyme of phenoloxidase A1ofDrosophila melanogaster. Proceedings of

the National Academy of Science USA 92, 7769–7773.

with mono- and diphenoloxidase fromTenebrio molitor. Biochem-istry and Biophysics Research Communications 21, 162–169. Jiang, H., Wang, Y., Ma, C., Kanost, M.R., 1997a. Subunit

compo-sition of prophenoloxidase fromManduca sexta: molecular cloning of subunit ProPo-P1. Insect Biochemistry and Molecular Biology 27, 835–850.

the wax mothGalleria mellonella: purification and characterization of proenzyme. Insect Biochemistry and Molecular Biology 25, 1081–1091.

Lai-Fook, J., 1966. The repair of wounds in the integument of insects. Journal of Insect Physiology 12, 195–226.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage, T4. Nature 227, 680–685. Lerch, K., 1982. Primary structure of tyrosinase from Neurospora

crassa. Journal of Biological Chemistry 257, 6414–6419. Lee, W.J., Ahmed, A., Torre, A.D., Kobayashi, A., Ashida, M., Brey,

P.T., 1998. Molecular cloning and chromosomal localization of a prophenoloxidase cDNA from the malaria vectorAnapheles gam-biae. Insect Molecular Biology 7, 41–50.

Lee, H.S., Cho, S.Y., Lee, K.M., Kwon, T.H., Homma, K., Natori, S., Lee, B.L., 1999. The pro-phenoloxidase of coleopteran insect, Tenebrio molitor, larva was activated during cell clump/cell adhesion of insect cellular defense reactions. FEBS Letters 444, 255–259.