59 N. Parkinson et al. Insect Biochemistry and Molecular Biology 31 2001 57–63
Fig. 1. Deduced amino acid sequences of Pimpla POI, POII and POIII and alignment with Manduca PPO and Limulus haemocyanin. The PPO
sequences used in the alignment are listed in Fig. 2, though only the moth sequence M.S.PPO Hall et al., 1995 is shown. Residues highlighted with arrowheads are conserved in all PPO sequences and in Limulus haemocyanin L.P.Hcn but not in POs I–III. The RF residues indicated
by a single overline constitute the proteolytic cleavage site in insect PPOs. Conserved histidine-containing motifs involved in copper binding are underlined.
2.4. PO purification, NH
2
-terminal sequencing, PAGE analysis and determination of enzyme specific activity
Venom was size-fractionated as described previously Parkinson and Weaver, 1999. Fractions 17–20, which
contained maximum PO activity, were analysed by SDS–PAGE Laemmli, 1970 without the addition of
reducing agent and compared to the peptide profile from non-fractionated venom. These fractions were also used
for determination of the NH
2
-terminal PO sequence, by Edman degradation, as well as for determination of
enzyme specific activity. The latter was done spectro- photometrically using a wavelength of 492 nm with 15
mM L-DOPA as substrate in 10 mM cacodylate, 10 mM calcium chloride buffer pH 6.9.
3. Results
3.1. Cloning, cDNA sequence analysis and gene expression
Approximately 2 of clones in the cDNA library hybridized to the PCR-generated probe. Three clones
POI, POII and POIII were selected, according to insert size and restriction profile, and sequenced. The POI open
reading frame encoded a polypeptide of 699 amino acids M
r
79,499, POII a polypeptide of 690 amino acids M
r
79,020, and POIII a polypeptide of 708 amino acids M
r
79,333. POI shares 77 amino acid identity with POII and
60 amino acid identity with POIII. Fig. 1 shows deduced amino acid sequences of POI–POIII, and their
alignment with a PPO and a haemocyanin cloned from the moth Manduca sexta and the horseshoe crab Limulus
polyphemus
, respectively Hall et al., 1995; Nakashima et al., 1986. The alignment was made using seven PPO
genes Hall et al., 1995; Kawabata et al., 1995; Fujimoto et al., 1995; Aspa´n et al., 1995; Jiang et al., 1997a,b;
Park et al., 1997, though only the Manduca sequence is shown. Conserved residues encoded by all the PPOs
used to produce the alignment are highlighted on the Manduca
sequence in Fig. 1, as are eight conserved resi- dues arrowheads which are absent in the deduced
sequences of POI, POII or POIII. A search of the Gen- Bank database using BLASTP indicated that polypep-
tides encoded by POI, POII and POIII were most similar to PPOs approximately 48 identity, with lower simi-
larity to haemocyanins up to 40 identity.
Interestingly, POs I–III each encoded a similar NH
2
- terminal amino acid sequence which contained a high
proportion of hydrophobic residues and conformed to a putative signal peptide secretory signal Leader, 1979.
The NH
2
-terminal amino acid sequences were analysed
60 N. Parkinson et al. Insect Biochemistry and Molecular Biology 31 2001 57–63
using the SignalP program http:www.cbs.dtu.dk servicessignalP, which predicted signal sequences for
all three enzymes with potential cleavage sites located between the conserved glycine and aspartate residues
positions 19 and 20, Fig. 1.
A phylogram indicating the degree of relatedness of POI, POII and POIII to previously reported PPOs and
to Limulus haemocyanin is shown in Fig. 2. PPOs from flies and moths clustered into two distinct groups, the
moth group being divided into two subgroups each con- taining one of two PPO genes cloned from each species.
A third major grouping comprised POs I–III and, as a subgroup, the crayfish PPO and Limulus haemocyanin.
Northern hybridization Fig. 3 using the POI-specific oligonucleotide indicated a transcript length for this gene
of approximately 2.4 kb, similar to the size of the POI cDNA clone 2.2 kb. POI was expressed at a similar
level from the time of adult emergence through to the third sampling time at 6 days post-emergence, thereafter
declining to relatively low levels by day 9.
3.2. SDS–PAGE analysis of purified venom PO, specific activity and determination of NH
2
-terminal amino acid sequence
SDS–PAGE analysis of fractionated venom separated by size exclusion chromatography and containing
Fig. 2. Phylogenetic analysis of PO encoded by POI, POII and POIII
and relationship to PPOs from the lepidopterans Hyphantria cunea H.C. PPO 1, 2, Manduca sexta M.S. PPO 1, 2 and Bombyx mori
B.M. PPO 1, 2; the dipterans Drosophila melanogaster D.M. PPO A1 and Anopheles gambiae A.G. PPO 1, 2; the crayfish Pacifasticus
leniusculus P.L. PPO; and to a haemocyanin from Limulus poly-
phemus L.P. Hcn Hall et al., 1995; Kawabata et al., 1995; Fujimoto
et al., 1995; Aspa´n et al., 1995; Jiang et al., 1997a,b; Park et al., 1997; Nakashima et al., 1986.
Fig. 3. POI gene expression analysis. Total RNA 4
µ g isolated from
venom-producing tissues dissected from insects at days 0, 1, 3, 6, and 9 post-emergence was hybridized using a POI-specific oligonucleotide
as described in the text. The inset black background shows the same RNA samples prior to blotting, stained with ethidium bromide to vis-
ualize rRNA and indicate RNA loading. The positions of RNA size standards Life Technologies are indicated.
maximum PO activity is shown in Fig. 4. Two polypep- tides of similar electrophoretic mobility were detected
with M
r
s of approximately 80,000, in good agreement with the M
r
s deduced from the cDNA coding region
Fig. 4. Purification of venom PO. Venom was size-fractionated as
described previously Parkinson and Weaver, 1999 and fractions 17– 20, containing maximum activity, were pooled and analysed using a
7 polyacrylamide gel lane P. The polypeptide profile of whole venom is shown in the adjacent lane W. Arrows indicate molecular
mass standards kDa.
61 N. Parkinson et al. Insect Biochemistry and Molecular Biology 31 2001 57–63
sequences of POs I–III. These polypeptides co-migrated with two major constituents of whole venom Fig. 4, lane
W, indicating that PO is abundant in the Pimpla venom- producing gland. The specific activity of purified PO,
assayed
using 15
mM L-DOPA,
was DA
490
8.4minmg protein. The NH
2
-terminal amino acid sequence of PO purified from
Pimpla venom
was determined
to be
DEXDRDINQEILDQ, which, apart from the ambiguous third residue, is identical to residues 20–33 of POI and
POII Fig. 1. This sequence occurs immediately after the predicted signal peptide sequence, which is thus con-
firmed as authentic. Purified venom PO submitted for sequencing contained two polypeptides in approximately
equal proportions Fig. 4, lane P. Apart from a trace sequence, attributed to small quantities of a contaminat-
ing peptide, no other sequence was detectable, indicating that both polypeptides in the purified venom shared an
identical NH
2
-terminal amino acid sequence which is present in both POI and POII. In contrast, the polypep-
tide encoded by POIII, which has a similar but distinct sequence at the putative mature NH
2
terminus, was not present in detectable quantities in the venom sample
used for sequence analysis.
4. Discussion
