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 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 from Anopheles gambiae
Jiang et al., 1997b; Lee et al., 1998; Muller et al., 1999, one from Tenebrio molitor Lee et al., 1999,
and one from Armigeres subalbatus Cho et al., 1998. We have been using Sarcophaga bullata larvae 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 at 280
° 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 10,000g for 15 min. The pellet was dissolved in mini- mum amount of buffer A containing 10 ammonium
sulfate and chromatographed on a phenyl Sepharose col-
955 M.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 remove
unbound proteins, bound PPO was eluted with water. A flow rate of 4 mlmin 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 mlmin. 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 mlmin 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
µ g 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 µ
l 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 µ
g of PPO was dissolved in 30 µ
l 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
mlmin. 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 mlh 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 GibcoBrl and vortexed for 30 sec. The remaining pro-
tocol follows the manufacturer’s recommendations. The RNA pellet was dissolved in 200
µ l 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 39 and 59 RACE 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
956 M.R. Chase et al. Insect Biochemistry and Molecular Biology 30 2000 953–967
reverse translated. The forward primer, PPO-FS 59- CAY CAY TKB CAY TGG CAY YTN GT-39 targets
HH WY 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 5
µ l of 15 or 110 dilutions of S. bullata total 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 µ
l of 15, 110 and 150 dilutions of the RT reaction with PPO-FS and PPO-RAS.
The reactions were set up as follows: 5 µ
l template, 5 µ
l 10 PCR buffer Promega 2.5 mM MgCl
2
, 200 µ
M each dNTP, 10–20 pM each primer, 1 unit Taq Poly-
merase Promega in a total volume of 50 µ
l. 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. 3 9 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. 5 9 race
To obtain the 59 end of each DNA fragment, two nested anti-sense primers were designed 200–250 bp
away from the 59 end 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 GIBCOBRL 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.educgi-
binprimerprimer3 Fwww.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
