From the Department of Entomology and Plant
Pathology, Oklahoma State University, Stillwater, Oklahoma 74078 and the ¶ Department of Biochemistry, Kansas State University,
Manhattan, Oklahoma 66506
Received for publication, June 10, 2002, and in revised form, October 18, 2002
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ABSTRACT |
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Proteolytic activation of prophenoloxidase in
insects is a component of the host defense system against invading
pathogens and parasites. We have purified from hemolymph of the tobacco hornworm, Manduca sexta, a new serine proteinase that
cleaves prophenoloxidase. This enzyme, designated
prophenoloxidase-activating proteinase-2 (PAP-2), differs from another
PAP, previously isolated from integuments of the same insect (PAP-1).
PAP-2 contains two clip domains at its amino terminus and a catalytic
domain at its carboxyl terminus, whereas PAP-1 has only one clip
domain. Purified PAP-2 cleaved prophenoloxidase at Arg51
but yielded a product that has little phenoloxidase activity. However,
in the presence of two serine proteinase homologs, active phenoloxidase
was generated at a much higher level, and it formed covalently linked,
high molecular weight oligomers. The serine proteinase homologs
associate with a bacteria-binding lectin in M. sexta
hemolymph, indicating that they may be important for ensuring that the
activation of prophenoloxidase occurs only in the vicinity of invading
microorganisms. PAP-2 mRNA was not detected in naive larval fat
body or hemocytes, but it became abundant in these tissues after the
insects were injected with bacteria.
Phenoloxidase (PO)1 is
implicated in several defense mechanisms in insects, including cuticle
sclerotization and melanotic encapsulation (1-3). Quinones produced by
phenoloxidase may also participate in wound healing and killing of
sequestered parasites and pathogens (4). To minimize detrimental
effects of the reactive intermediates to host tissues and cells,
arthropod phenoloxidases known so far are all produced as inactive
proenzymes and require specific proteinases for proteolytic activation.
Activation of proPO in insects is probably mediated by a serine
proteinase cascade, analogous to the coagulation pathway and complement
system in human plasma (1, 2, 5, 6). Components of this proteinase
system in insects may be already present in circulating hemolymph or
released from hemocytes or fat body when pathogens or parasites are
encountered. Recognition of invading microorganisms or aberrant host
tissues may trigger the autoactivation of the first proteinase in the
pathway, leading to the sequential activation of other components in
the system through limited proteolysis. Prophenoloxidase-activating proteinase (PAP) (also known as
prophenoloxidase-activating enzyme (PPAE)) is the terminal enzyme
that directly converts proPO to PO. The reactions catalyzed by
phenoloxidase can then result in melanization of foreign organisms
trapped in capsules or hemocyte nodules (7). Presumably,
protein-protein interactions ensure that the defense response occurs
near the site of infection. Inhibitors of serine proteinases and
phenoloxidases may further reduce unwanted damage caused by the active
enzymes (8).
The molecular mechanisms of proPO activation have been investigated in
several insect species. We have isolated a PAP from a cuticular extract
of the tobacco hornworm, Manduca sexta (9). This serine
proteinase, now renamed PAP-1, hydrolyzes a synthetic peptidyl-p-nitroanilide substrate but requires another
protein factor for generating active PO. A cDNA clone for PAP-1 was
obtained from an M. sexta hemocyte library. Sequence
comparison indicated that the protein belongs to a family of arthropod
serine proteinases containing a clip domain (10, 11).
In this paper, we report the purification and characterization of a
second serine proteinase from M. sexta hemolymph that activates proPO. To distinguish it from PAP-1 isolated previously from
cuticles (9), we designate this new proteinase PAP-2. Molecular cloning
of PAP-2 indicates that it is most similar to the silkworm PPAE and has
two clip domains at its amino terminus (12). The PAP-2 zymogen is
present at a higher level in hemolymph of M. sexta larvae
that were challenged with killed bacteria. PAP-2 requires serine
proteinase homologs from M. sexta plasma (13) as cofactors
for proPO activation. Since the serine proteinase homologs associate
with immulectin-2, a C-type lectin isolated from Manduca
hemolymph (13-15), they may serve as anchoring/auxiliary factors for
PAP-2 so that proPO activation only occurs as a local defense response
against nonself.
Insects and Hemolymph Collection--
M. sexta eggs
were originally purchased from Carolina Biological Supply, and larvae
were reared on an artificial diet (16). Pharate pupae with metathoracic
brown bars were chilled and dissected for hemolymph collection. When
all tissues were cautiously removed from the integument with a spatula,
hemolymph was pooled and collected carefully with a 1-ml pipetter,
avoiding contaminating tissue fragments. This method yields
significantly greater amounts of hemolymph than cutting a proleg of
pharate pupae, because their hemolymph volume is quite low. Individual
hemolymph samples (0.8-1 ml/insect) were immediately mixed with 100%
saturated ammonium sulfate (pH 7.0) to prevent the rapid melanization
of hemolymph, which occurs at this developmental stage. The ammonium
sulfate was adjusted to 50% saturation, and the suspension was stored at Measurement of proPO Activation and Amidase Activity--
proPO
was purified from M. sexta larval hemolymph as described
previously (17). Column fractions (10 µl) were mixed with 0.5 µg of
proPO and 20 mM Tris-HCl, 5 mM
CaCl2, pH 7.5, to a final volume of 20 µl. The reaction
mixtures were incubated in microplate wells on ice for 60 min. PO
activity was measured by adding 200 µl of 2 mM dopamine
in 50 mM sodium phosphate, pH 6.5, to each sample well.
Absorbance at 470 nm was then monitored continuously using a microplate
reader (Molecular Devices). One unit of PAP activity is defined as the
amount of enzyme yielding PO that produces an increase of 0.001 absorbance units/min.
Amidase activity was assayed by using
acetyl-Ile-Glu-Ala-Arg-p-nitroanilide (A0180; Sigma) as a
chromogenic substrate. Samples of column fractions (10-20 µl) were
mixed with 200 µl of 50 µM substrate in 0.1 M Tris-HCl, 0.1 M NaCl, 5 mM
CaCl2, pH 7.8. One unit of activity is defined as
Initial Isolation of PAP-2--
Since a detailed, optimized
purification scheme is presented below, we briefly describe here how
PAP-2 was first isolated from M. sexta hemolymph. Unless
otherwise specified, conditions for separation remained the same in the
first and second PAP-2 purification. The plasma (42.5 ml) from bar
stage prepupae was first fractionated with 15-40% saturation of
ammonium sulfate and then dialyzed before separation on a
hydroxylapatite column. Fractions were assayed for PO and
proPO-activating activities. The fractions that activated proPO were
pooled, concentrated in a Centriplus-30 (Millipore Corp.), and applied
to a Sephacryl S100-HR column. Since proPO-activating activity was low
in this and the following steps,
acetyl-Ile-Glu-Ala-Arg-p-nitroanilide hydrolysis (Sigma) was
used to monitor an amidase activity (IEARase) that activates proPO only
in the presence of other protein factors. Combined fractions of IEARase
were passed through concanavalin A (ConA)-Sepharose 4B (5 ml; Amersham
Biosciences) and Jacalin-agarose columns (5 ml; Vector Laboratories) to
remove minor proteins that are difficult to separate in the final step.
The unbound fraction from the lectin affinity columns was concentrated
in a Centriplus-30 concentrator, and buffer exchange was carried out in
the same centrifugal filter device. The sample was separated by HPLC on an MA7Q anion exchange column (Bio-Rad). Fractions containing the
IEARase activity were individually concentrated in a Microcon-30 concentrator (Millipore Corp.).
Amino Acid Sequence Determination--
The partially purified
IEARase (PAP-2) was treated with SDS-sample buffer containing
2-mercaptoethanol and separated by electrophoresis on a 12%
polyacrylamide gel (18). The protein was then transferred to a
polyvinylidene difluoride membrane and stained with Amido Black
(Sigma). The 35-kDa polypeptide, which corresponded to a [3H]DFP-labeled band detected as described by Jiang
et al. (9), was subjected to automated Edman degradation. To
determine the amino-terminal sequence of its 25-kDa light chain,
purified PAP-2 (2 µg) was mixed with 1 µg of pyroglutamate
aminopeptidase (Roche Molecular Biochemicals) in 100 µl of 100 mM sodium phosphate (pH 8.0) containing 10 mM
EDTA, 5 mM dithiothreitol, 5% glycerol (v/v), and 1 mM p-aminobenzamidine. The reaction was carried
out at 4 °C for 18 h followed by 25 °C for 4 h. The
proteins in the mixture were precipitated by trichloroacetic acid at a
final concentration of 10%. After centrifugation, the pellet was
washed with cold anhydrous acetone and dissolved in 20 µl of the SDS
sample buffer at 95 °C for 5 min. After gel separation and transfer,
the first six residues of the 25-kDa band were determined as described above.
Molecular Cloning and Sequence Analysis of PAP-2--
Northern and Western Blot Analyses of PAP-2 mRNA and Protein
Levels--
Fat body and hemocyte total RNA samples were prepared
according to Wang et al. (20), separated by electrophoresis
in agarose gels containing formamide, transferred to nitrocellulose,
and hybridized with 32P-labeled PAP-2 cDNA. A duplicate
blot was hybridized with a cDNA for M. sexta ribosomal
protein S3 (21) as a loading control. To detect a possible change of
proPAP-2 in larval hemolymph upon bacterial challenge,
Manduca larvae were injected with buffer, Escherichia
coli, or M. luteus. Cell-free hemolymph samples
collected at 24 h after injection were resolved by electrophoresis
on an SDS-polyacrylamide gel (18). Immunoblot analysis was performed using the polyclonal antiserum to proPAP-2 as the first
antibody.2
Optimized Purification of PAP-2--
All procedures for
purification of PAP-2 were carried out at 4 °C. A frozen bar stage
hemolymph sample (40 ml) was thawed, and the protein precipitate was
collected by centrifugation at 12,000 × g for 25 min.
The pellet was resuspended in 80 ml of HT buffer (pH 6.8, 10 mM potassium phosphate, 0.5 M NaCl),
supplemented with 0.001% 1-phenyl-2-thiourea and 0.5 mM
p-aminobenzamidine. To remove the
The protein solution was diluted with equal volume of H2O
and applied to a hydroxylapatite column (2.5-cm inner diameter × 7 cm; Bio-Rad) equilibrated with 0.5 × HT buffer. Following a washing step with 60 ml of 0.5× HT and 50 ml of 1× HT buffers, bound
proteins were eluted at 0.5 ml/min for 4 h with a linear gradient
of 20-150 mM potassium phosphate (pH 6.8), 0.5 M NaCl. Fractions were analyzed by immunoblotting and an
IEARpNa amidase assay. Active fractions containing PAP-2
were combined and precipitated with ammonium sulfate (60% saturation).
The precipitate was collected by centrifugation at 15,000 × g for 30 min, dissolved in 3.0 ml, 20 mM
Tris-HCl, 0.5 M NaCl, pH 7.5 (S100 buffer), and then
immediately applied to a Sephacryl S100-HR column (2.5-cm inner
diameter × 100 cm; Amersham Biosciences) equilibrated with the
same buffer. The column was eluted with S100 buffer at a flow rate of
0.7 ml/min, and fractions were collected at 2.5 ml/tube after the first
100 ml.
Fractions containing PAP-2 were pooled and supplemented with
CaCl2 and MgCl2 at a final concentration of 1 mM each. The sample was loaded onto a ConA-Sepharose 4B
column (5.0 ml) equilibrated with 20 mM Tris-HCl, 0.5 M NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.4 and washed with the same
buffer until A280 was lower than 0.05.
The flow-through fraction was dialyzed against Q buffer (25 mM Tris-HCl, pH 7.5), supplemented with 0.5 mM
p-aminobenzamidine (2 liters each time for 8 h,
twice). After passing through a syringe filter, the dialyzed sample (45 ml) was applied to a UNO-Q6 column (6 ml; Bio-Rad) equilibrated by Q
buffer. Following a washing step, the bound proteins were eluted with a
gradient of 0-0.2 M NaCl in Q buffer at a flow rate of 1.0 ml/min for
30 min. This step was performed in a cold chamber using a Biologic
Duo-Flow Protein Purification System (Bio-Rad). Fractions (0.5 ml/tube) were collected and analyzed by electrophoresis on a SDS-polyacrylamide gel (10%) followed by silver staining (23) or immunoblot analysis. Affinity labeling with [3H]DFP and fluorographic
detection of PAP-2 were carried out according to Skinner and
Griswold (24). Purified PAP-2 was stored at MALDI Mass Spectrometry of PAP-2 and Cleaved proPO--
The
purified PAP-2 was desalted using a C18 zip tip (Millipore) and eluted
with 70% acetonitrile, 0.1% trichloroacetic acid. The sample was
mixed with an equal volume of saturated sinapinic acid matrix on a
MALDI plate, air-dried, and subjected to mass determination on a
Voyager Elite mass spectrometer (PerkinElmer Life Sciences) with
delayed extraction. The spectra were calibrated using bovine serum
albumin as an external standard.
To determine the site at which PAP-2 cleaves proPO, 1.0 µg of proPO
was incubated with 0.2 µg of PAP-2 on ice for 60 min, and the
reaction mixture was desalted and eluted as described above. Molecular
masses of peaks that were not present in the control spectra of proPO
and PAP-2 were compared with calculated values of the amino-terminal
peptides to determine the cleavage site in proPO.
Activation of proPO by PAP-2 in the Presence of Serine Proteinase
Homologs (SPHs)--
The SPH-1 and -2 were isolated from induced
larval hemolymph according to Yu et al. (13). The protein
preparation (10 µl) was incubated with 40 µl of 5 mM
phenylmethylsulfonyl fluoride (Sigma) in 5 mM
CaCl2, 25 mM Tris-HCl (pH 7.5) at 4 °C for
16 h to inactivate serine proteinases that might be associated
with the SPHs. The treated sample was dialyzed against the same buffer without phenylmethylsulfonyl fluoride (100 ml/8 h each time,
twice). To test the effect of M. sexta SPH-1 and SPH-2 on
the activation of proPO, purified proPO was incubated with buffer,
PAP-2, SPH-1 and -2, or a mixture of PAP-2 and the SPHs at 4 °C for
60 min. PO activity in the reaction mixtures was assayed, using
dopamine as a substrate. As described in the legend to Fig. 6, the
reaction mixtures were also subjected to electrophoresis followed by
silver staining or immunoblot analysis using antiserum against
Manduca proPO (diluted 1:2000) as the first antibody. To
study whether PO activity is involved in the formation of SDS-stable PO
polymers, we included 0.001% 1-phenyl-2-thiourea (PTU), a PO
inhibitor, in some of the reaction mixtures and analyzed the samples similarly.
Partial Purification of PAP-2 and Its Requirement for a
Cofactor--
Preliminary experiments indicated that active PO and
proteinases are already present in the hemolymph of bar stage prepupal M. sexta larvae in the absence of microbial challenge. As a
step toward understanding the prophenoloxidase-activating system in M. sexta hemolymph, we attempted to purify a PAP from plasma
of bar stage insects. To preserve the enzyme activities and minimize protein cross-linking caused by active PO, we kept the plasma and
column fractions at high salt concentrations in the presence of
inhibitors of phenoloxidase and proteases. Based on our previous experience with the cuticular PAP (PAP-1) (9), we employed three
different assays to follow each purification step: proPO activation,
[3H]DFP labeling, and an amidase assay using
acetyl-Ile-Glu-Ala-Arg-p-nitroanilide as a substrate. Among
the commercially available peptidyl-p-nitroanilide substrates, IEARpNa is most similar to the amino-terminal
side of the putative activation site in M. sexta proPO-p1
(Leu-Ser-Asn-Arg51) and proPO-p2
(Leu-Asn-Asn-Arg51) (17). When the 15-40% ammonium
sulfate fraction of the cell-free hemolymph was separated on a
hydroxylapatite column, the proPO-activating activity roughly coincided
with one of the IEARase peaks (data not shown). These fractions were
pooled, concentrated, and then resolved by gel filtration
chromatography on a Sephacryl S100-HR column (Fig.
1A). We detected a major
IEARase activity peak in fractions 30-34 as well as low levels of
IEARpNa hydrolysis in some other column fractions. This
amidase peak did not correspond to the proPO-activating activity found
in fractions 10-16 (data not shown). The proPO-activating activity in
all of the fractions accounted for only 5-10% of the total activity
loaded onto the column. One possibility is that, like PAP-1 (9), proPO
activation is mediated by a complex of a proteinase and a protein
cofactor, and separation of this complex led to the low activity of
PAP-2.
To test this hypothesis, we incubated aliquots of fraction 32 with a
small amount of other column fractions for 1 h along with purified
proPO. Fraction 32 did not contain PO activity, and it generated little
active PO when incubated with proPO (Fig. 1B). After
fraction 32 was incubated with proPO in the presence of fraction
10-16, which contained only low levels of PO and proPO-activating activities, a large increase in PO activity was observed. This result
supported the hypothesis that proPO activation requires PAP and a
cofactor. Because high amidase activity was detected in fraction 32 but
not in fraction 14, we concluded that the PAP is probably present in
fraction 32 and that the cofactor is in fraction 14. Their molecular
masses are estimated to be about 50 and 200-500 kDa, respectively,
based on their elution volume from the Sephacryl S-100 column. The
pooled fractions 30-34 from the gel filtration column were passed
through a ConA-Sepharose column and a Jacalin-agarose column to remove
some plasma glycoproteins. The flow-through fractions from the lectin
affinity columns were concentrated and separated on an anion exchange
HPLC column. A single small IEARase peak was present in fractions
26-28, and a [3H]DFP labeling experiment indicated that
these fractions contained a single radioactive band at 35 kDa (data not
shown). As shown on an SDS-polyacrylamide gel stained with Coomassie
Blue, this band was prominent and well separated from other proteins
(Fig. 1C). This 35-kDa band and a 25-kDa band were shifted
to a 48-kDa position under nonreducing conditions (see Fig. 5), which
suggests that the proteinase, named PAP-2, is composed of a catalytic
heavy chain (35 kDa) and a regulatory light chain (25 kDa) linked by a
disulfide bond. The first 29 residues of the 35-kDa polypeptide were
determined to be
Ile-Leu-Gly-Gly-Glu-Ala-Thr-Ala-Ile-Asp-Gln-Tyr-Pro-Trp-Leu-Ala-Leu-Ile-Glu-Tyr-His-Lys-Leu-Ala-Glu-Ile-Lys-Leu-Met (Fig. 2). This sequence is similar to a
region at the beginning of the catalytic domain in PPAE from the
silkworm Bombyx mori (12). The amino terminus of the 25-kDa
light chain starts with a pyro-Glu residue, since, after deblocking
with pyroglutamate aminopeptidase, the newly exposed sequence was
determined to be Ala-(Cys)-Thr-Leu-Pro-Asn (Fig. 2).
Molecular Cloning and Structural Features of PAP-2--
We
designed two degenerate primers encoding part of the amino-terminal
sequence of the 35-kDa polypeptide. Using these primers along with a
vector-specific primer (T7) and a degenerate primer based on a
conserved sequence around the active site serine, we obtained a 0.6-kb
cDNA fragment in a nested polymerase chain reaction and cloned it
into a pGem-T plasmid vector. Sequence analysis confirmed that the
cDNA encoded a sequence starting with
Leu-Ala-Leu-Ile-Glu-Tyr-His-Lys-Leu-Ala-Glu-Ile-Lys-Leu-Met. The rest
of the sequence is typical of a serine proteinase from the S1 family
(25). Using the 32P-labeled cDNA fragment as a probe,
we screened a bacteria-induced M. sexta larval fat body
cDNA library. Approximately 1.0 × 103 positives
were found in the first round screening of 6.0 × 105
plaques, indicating that PAP-2 mRNA is abundant at 24 h after immune challenge. Plaque purification and in vivo excision
of phagemids were carried out with 16 of the putative positive clones. Sequence analysis of their 3' termini indicated that these clones are
all identical except for clone 4. Therefore, we determined the complete
nucleotide sequences of clone 2 (the longest) and clone 4.
Clone 2 encompasses a 2299-nucleotide cDNA, which includes a
complete open reading frame spanning nucleotides 34-1359 (Fig. 2). The
same coding region was found in clone 4 with a few nucleotide substitutions, most of which do not alter the amino acid sequence. A
significant difference occurs in the 3'-untranslated regions of the two
sequences; clone 2 contains 915 nucleotides between the stop codon and
poly(A) tail, but clone 4 has only six. Apparently, in clone 4 the
AATAAA sequence near the end of the coding region was recognized as a
signal for polyadenylation. Northern blot analysis (Fig.
3) indicated that this 1.4-kb form may
represent a small but significant portion of the total PAP-2 mRNA
population.
The deduced amino acid sequence of PAP-2 is 441 residues long,
including a predicted 19-residue secretion signal peptide, which is
consistent with the experimentally determined N-terminal sequence of
the mature PAP-2: pyro-Glu-Ala-Cys-Thr-Leu-Pro-Asn. The molecular mass
of mature proPAP-2 (deduced from clone 2) is 45,780 Da. Potential
N-linked and O-linked glycosylation sites are
identified in the sequence as Asn219, Asn349,
and Ser133. The calculated isoelectric point of proPAP-2 is
6.0. Based on N-terminal sequence of the proteinase catalytic domain,
which perfectly matches that deduced from the cDNAs, the activation cleavage site is located between Lys153 and
Ile154. The light chain (residues 20-172; 16,429 Da) of
PAP-2 contains 25 acidic and 18 basic residues, whereas the heavy chain
(residues 173-441, 29,373 Da) has 26 acidic and 36 basic residues. The
calculated pI values are 4.7 for the N-terminal regulatory domain and
8.9 for the C-terminal catalytic domain. The catalytic domain is
similar in sequence to serine proteinases of the chymotrypsin family, including the conserved catalytic triad consisting of
His198, Asp265, and Ser372. Based
on the results from a multiple sequence alignment (data not shown), the
primary substrate specificity pocket is composed of Asp366,
Gly393, and Gly404, suggesting that PAP-2 has a
specificity for cleavage after basic residues.
Similar to the silkworm PPAE (12), the mature proPAP-2 contains 24 cysteine residues, which may form 12 disulfide bonds (Fig. 2). The
protein contains two clip domains at the amino-terminal end between
Ala2 and Pro56 and between Ser62
and Gly114. The connection between the clip domains
(Glu-Ser-Asp-Thr-Leu) is longer than that of B. mori PPAE,
in which Cys-6 of clip domain-1 and Cys-1 of clip domain-2 are
separated by a single Pro residue (12).
Induced Expression of proPAP-2 Gene--
To test
whether transcription of the proPAP-2 gene is up-regulated upon immune
challenge, we carried out a Northern blot analysis of RNA from
hemocytes and fat body from M. sexta larvae injected with
water or killed E. coli (Fig. 3A). PAP-2 mRNA
was not detected in the water-injected control or induced hemocyte
samples. In induced fat body, a major 2.3-kb RNA band was observed
along with a minor 1.4-kb band, which probably correspond to PAP-2
mRNAs represented by cDNA clones 2 and 4.
We constructed a recombinant plasmid (proPAP-2/H6pQE60), which encodes
the mature proPAP-2 fused with a hexahistidine tag at its amino
terminus, purified the fusion protein by Ni2+ affinity
chromatography, and prepared a polyclonal antiserum against
proPAP-2.2 Immunoblot analysis did not detect any proPAP-2
in larval hemolymph from naive M. sexta (Fig.
3B), but we observed an inducible 55-kDa band in plasma from
insects challenged with E. coli or M. luteus, consistent with the result from Northern blot analysis. The mobility of
this band is the same as that of the recombinant proPAP-2 from a
baculovirus/insect cell expression system.2 Slightly below
the 55-kDa band was another inducible protein at around 52 kDa, which
cross-reacts with the proPAP-2 antiserum.
Purification of M. sexta PAP-2 from Bar Stage Hemolymph--
With
the polyclonal antiserum available, we detected proPAP-2 in the
hemolymph of naive M. sexta larvae in the days just before pupation. At day 5 of the wandering stage, ~10% of the zymogen was
converted to active enzyme (PAP-2). Preliminary experiments indicated
that almost all PAP-2 was present in the 0-35% ammonium sulfate
fraction of the plasma sample and that a
First, we incubated the 0-50% ammonium sulfate fraction of the plasma
with curdlan in the presence of 0.5 M NaCl and 0.001% 1-phenyl-2-thiourea. Phenoloxidase activity was greatly reduced under
these conditions, and
After small amounts of contaminating proteins were removed from the
combined fractions by lectin chromatography on ConA-Sepharose, the
PAP-2 sample was dialyzed and separated by ion exchange chromatography on an HPLC Q column (Fig. 4C). A major protein peak eluted
from the column at about 150 mM NaCl. Most impurities from
the loaded protein sample were removed in this step, and the specific
activity of PAP-2 increased about 90-fold (Table I). Although PAP-2 is present at a low concentration in bar stage hemolymph of M. sexta, we successfully purified the enzyme with an overall yield
of 73% and an increase in specific activity of 6 × 104.
Properties of Purified M. sexta PAP-2--
As indicated by
SDS-PAGE (Fig. 5A), purified
PAP-2 is near homogeneity with an apparent molecular mass of 48 kDa
under nonreducing conditions. In the presence of 0.1 M
dithiothreitol, the heavy and light chains of PAP-2 were separated as
35- and 25-kDa bands. Due to possible allelic variations and/or
glycosylation differences, microheterogeneity was observed in both
polypeptides. Immunoblot analysis (Fig. 5B) indicated that
both polypeptide chains were recognized by the polyclonal antibodies,
although the polyclonal antiserum appears to recognize the light chain
better than the heavy chain. Affinity labeling of the active site
serine residue showed that the catalytic domain of M. sexta
PAP-2 is located in the 35-kDa chain, which is attached to the
unlabeled light chain by a disulfide bond (Fig. 5C). The
apparent molecular masses of both chains are greater than the masses
calculated from sequence, suggesting that both of the polypeptides
are glycosylated (Fig. 2).
We determined by MALDI mass spectrometry that the molecular mass of
PAP-2 is 46,450 ± 30 Da (Fig. 6),
which is larger than the calculated molecular mass for PAP-2 (45,801 Da). The mass difference (~651 Da) may result from glycosylation at
Ser or Asn residues (Fig. 2). In agreement with the results of SDS-PAGE
analysis (Fig. 5), we have also detected a shoulder peak at both
MH+ and MH2+ positions, which may correspond to
a minor component in the PAP-2 preparation. Genetic variations and/or
posttranslational modifications could be responsible for the 176-Da
mass increase.
Activation of proPO by M. sexta PAP-2 and SPHs--
To test
whether purified PAP-2 can activate Manduca proPO, we
incubated PAP-2 with proPO for 60 min and detected a low level of PO
activity (Fig. 7A). Since the
control reaction of proPO alone does not have any PO activity,
Manduca PAP-2 must have converted a small amount of proPO to
PO. However, the activation reaction was much more efficient in the
presence of a cofactor.
We have isolated from M. sexta hemolymph two SPHs
that bind to a C-type lectin, M. sexta immulectin-2 (13,
14). cDNA cloning indicates that both SPHs lack a Ser residue at
the expected position of the active site. Neither protein, therefore,
is expected to have a catalytic activity. However, they are similar in
amino acid sequence to prophenoloxidase-activating factor (PPAF)-III from a coleopteran insect, Holotrichia diomphalia (27).
PPAF-III is a serine proteinase-like protein that assists PPAF-I in the conversion of proPO to PO. The M. sexta SPHs and H. diomphalia PPAF-III contain a clip domain at their amino termini.
Because of the sequence similarity, we tested whether the M. sexta SPHs can serve as cofactors of PAP-2. In the control
reactions, proPO and SPHs did not have PO activity (Fig.
7A). After proPO was incubated with PAP-2 and SPHs, however,
we detected phenoloxidase activity at a level much higher than the
control reactions of proPO with either PAP-2 or SPHs. These results
indicate that the SPHs are required for efficient activation of proPO
by PAP-2.
To explore the mechanism of proPO activation, we separated the reaction
mixtures by electrophoresis on an SDS-polyacrylamide gel followed by
either silver staining (Fig. 7B) or immunoblot analysis
(Fig. 7C). M. sexta proPO is composed of 79-kDa
proPO-p1 and 80-kDa proPO-p2 (17). After proPO had been incubated with PAP-2 for 1 h, a 74-kDa protein was generated, which was
recognized by antibodies against proPO-p2 (data not shown). Therefore,
we concluded that a significant amount of proPO-p2 was converted to a
74-kDa product, although a low level of PO activity was detected (Fig.
7A). In contrast, the cleavage of proPO-p1 was hardly
detected. When proPO was reacted with PAP-2 in the presence of the
SPHs, the 74-kDa band became more prominent. In addition, several other bands were detected: one at about 140 kDa and two at greater than 250 kDa (Fig. 7B), which were recognized by antibodies against Manduca proPO (Fig. 7C). These may be protein
complexes that contain PO or PO oligomers. The protein complexes appear
to be connected through covalent bonds, since they are present after
the reaction mixture was separated by SDS-polyacrylamide gel
electrophoresis under reducing conditions. To test whether active
phenoloxidase itself is required for the oligomer formation, we
included a PO inhibitor (PTU) in the activation mixture. While the
conversion of proPO-p2 to its 74-kDa form was not affected, we did not
detect any high molecular weight complex in the presence of PO
inhibitor (Fig. 7, B and C). Thus, active PO may
be involved in formation of covalent cross-links between proteins to
form the observed high molecular weight species.
Determination of Cleavage Site in proPO--
To further
characterize the proPO activation reaction, we used MALDI mass
spectrometry to determine the site at which PAP-2 cleaves proPO (Fig.
8). In the control samples of PAP-2 or
proPO only, we did not detect any significant mass peak within the
range from 2500 to 6500 Da (data not shown). However, after
Manduca proPO was incubated with PAP-2, we detected a major
peak at 5851 Da and a corresponding MH2+ peak of 2926 Da.
There was also a small peak of 6032 Da in the spectrum. These peaks
probably correspond to the propeptides released from proPO-p1 and
proPO-p2 as a result of proteolytic cleavage by PAP-2. Previous study
indicated that proPO-p1 and proPO-p2 are blocked at the amino terminus
(17). We calculated the molecular masses for the putative 50-residue
propeptides to be 5990 Da for proPO-p1 and 5808 Da for proPO-p2. Taking
into account the accuracy of MALDI time-of-flight mass spectrometry,
the mass differences between the theoretical and observed mass values
(42 Da for propeptide 1 and 43 Da for propeptide 2) are in good
agreement with N-acetylation of the amino-terminal residue.
The silkworm proPO-p1 and proPO-p2, cleaved by PPAE at
Arg51, were also found to be N-acetylated
(28).
As an integral part of the host defense system against invading
pathogens and parasites, prophenoloxidase activation has been investigated in various insects and crustaceans for more than 30 years
(3, 4). Even so, our knowledge of the prophenoloxidase-activating cascade is rather limited. A major breakthrough currently occurring in
this field is the isolation and molecular cloning of clip domain serine
proteinases that directly activate prophenoloxidase through limited
proteolysis (9, 12, 30, 31). We previously isolated PAP-1 from M. sexta and cloned a cDNA encoding the proPAP-1 zymogen (9).
PAP-1 contains a single amino-terminal clip domain. proPO-activating proteinases from the beetle H. diomphalia (30) and from a
crayfish Pacifastacus leniusculus (31) also contain one clip
domain. In this paper, we describe a second PAP from M. sexta, PAP-2. PAP-2 is most similar to the silkworm PPAE (12).
M. sexta PAP-2 and silkworm PPAE contain two amino-terminal
clip domains with 24 cysteine residues that are perfectly aligned. The
functions of clip domains in arthropod serine proteinases are not yet
well established. It seems likely that they are involved in regulating the enzymes' activity or localization. The clip domain in the crayfish
proteinase exhibits antibacterial activity toward Gram-positive bacteria (31). If clip domains interact with bacteria to exert an
antimicrobial function, they might also anchor the PAP-2 catalytic domain that converts proPO to PO onto a bacterial surface. Clip domains
linked to serine proteinases may have multiple functions in arthropod
immune responses (10).
The two PAPs we have identified in M. sexta have some
similarities and some differences in their patterns of expression.
M. sexta PAP-1 was initially isolated from a prepupal
cuticular extract, and its mRNA is present in prepupal
epidermis.3 PAP-1 mRNA
was also detected in fat body of naive (no microbial injection) feeding
stage larvae, and proPAP-1 is present in hemolymph of naive larvae
(32). Upon bacterial challenge, the concentration of proPAP-1 protein
increases in hemolymph, as transcription of the PAP-1 gene is
up-regulated in fat body and hemocytes during immune responses (9, 32).
However, neither proPAP-2 nor its mRNA was detected in naive
feeding stage larvae (Fig. 3). During the larval stage, PAP-2
expression was only observed after microbial challenge. However, we did
detect proPAP-2 and active PAP-2 in hemolymph of naive prepupal
insects, but PAP-1 was not present in hemolymph at this
stage.4 These results suggest
that the transcription of the PAP genes might be regulated by
developmental and immune pathways. Hormonal signals (presumably
ecdysteroids) prior to pupation may stimulate expression of PAP-1 in
epidermis and PAP-2 in fat body, whereas both genes are expressed in
fat body in response to microbial infection. The existence of more than
one PAP in M. sexta indicates that the proPO-activating
cascade in this species is more complex than previously expected.
Purified M. sexta PAP-1 and PAP-2 require the presence of a
nonproteolytic protein cofactor for proPO-activating function, although
they can hydrolyze a small peptide substrate in the absence of the
cofactor (Fig. 1) (9). The cofactors responsible for this modulation of
PAP activity appear to be SPHs, which have a structure similar to the
PAPs themselves. These SPHs contain an amino-terminal clip domain and a
carboxyl-terminal proteinase-like domain, but their active site serine
residue is replaced with glycine (13). The SPHs isolated from M. sexta plasma are a mixture of two related proteins, which we have
not yet been able to separate. We are pursuing experiments with
recombinant SPHs to determine whether one or both of the SPHs interact
with PAP-1 and PAP-2.
Similar to M. sexta PAP-1 and PAP-2, PPAF-I from a beetle,
H. diomphalia, was also reported to require a proteinaceous
factor, PPAF-II or PPAF-III, to activate proPO (27, 30). cDNA
cloning revealed that PPAF-III is a serine proteinase homolog
containing a clip domain. H. diomphalia PPAF-I cuts
proPO at Arg51 to generate an inactive intermediate. When a
40-kDa PPAF-II was also present, proPO was cleaved at an additional
site (Arg at around position 165) to yield active PO. Under our
experimental conditions, PAP-2 cleaved proPO at Arg51 in
the absence of SPH, and we did not observe a secondary cleavage site in
proPO after incubation with PAP-2 and SPHs (Fig. 7). In contrast to the
proteinases from M. sexta and H. diomphalia, the silkworm PPAE, which contains two clip domains like PAP-2, does not
appear to require any protein cofactor for the activation (12). The
reason for this difference is not clear. Perhaps in vivo at
a low enzyme concentration, the silkworm PPAE may also interact with
SPH or other protein cofactors.
Nonself recognition, an essential first step of the insect immune
responses against microorganisms, is mediated by hemolymph proteins
that specifically bind to molecules present on the surface of invading
pathogens, such as lipopolysaccharide and peptidoglycan from bacteria or glucans and mannans from fungi (33, 34). Lectins
involved in proPO activation (14, 35) and some cellular defense
responses (36) have been identified in several insect species. M. sexta immulectin-2, a C-type lectin, binds to bacterial lipopolysaccharide, interacts with the SPHs, and stimulates proPO activation in response to lipopolysaccharide (14, 15).
It can be envisioned that bacterial infection results in formation of a
protein complex, containing immulectin-2 bound to the bacterial surface
and interacting with SPHs, which bring PAP and proPO together in a
suitable orientation for activation of proPO by PAP-1 or PAP-2 (13).
Such a complex serves the purpose of activating proPO and localizing
the active enzyme at a microbial surface. We observed that activation
of proPO by PAP-2 in the presence of SPHs resulted in the formation of
high molecular weight species that contained PO (Fig. 7) but not PAP-2
or SPH.4 Therefore, we interpret these bands, detected in
SDS-PAGE and immunoblot analysis, to represent covalently linked
oligomers of PO or PO associated with other proteins, because they were not dissociated by 1% SDS. Formation of the oligomers did not occur in
the presence of a PO inhibitor, indicating that the PO enzymatic
activity is required to cross-link the proteins, perhaps through
oxidation of tyrosine residues. Beck et al. (26) observed a
high molecular weight protein complex, including proPO and PO, that
formed in M. sexta plasma only when the hemolymph was
collected under nonsterile conditions, suggesting that it was a
response to the presence of microorganisms. Formation of a protein
complex at a surface is a common feature of serine proteinase cascade pathways such as the vertebrate blood coagulation and complement systems. A similar process may occur in arthropod proPO activation cascades, serving to activate proteinases in discrete locations near
the surface of pathogens. We are planning to express the PAPs, SPHs,
and their individual clip and proteinase domains as recombinant
proteins for further studies to understand the interactions among the
protein factors involved in proPO activation in M. sexta.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. Hemolymph collected this way contains many active
proteinases and is stable for at least 2 years. For the bacterial
induction experiment, fifth instar M. sexta larvae (day 2)
were injected with Micrococcus luteus (10 µl/larva, 10 µg/µl; Sigma), formalin-killed Escherichia coli XL1-blue
(108 cells/larva, 10 µl), or H2O (10 µl) as
a negative control.
A405/min = 0.001.
phage
DNA (0.1 µg) isolated from a bacteria-induced fat body cDNA
library was used as a template in a PCR to amplify a PAP-2 cDNA
fragment. Two degenerate primers were designed based on the amino-terminal sequence of PAP-2 catalytic domain: 660 (5'-ACA GCC ATC
GAY CAR TAY CCN TGG-3') and 661 (5'-G CTG GCG CTG ATH GAR TAY CAY
AA-3'), which encode TAIDQYPW and LALIEYHK, respectively. Primer 625 (5'-CAT GAG SGG RCC RCC SGA RTC NCC-3') is the reverse complement of
the sequence encoding GDSGGPLM, a highly conserved sequence around the
active site serine residue in the chymotrypsin family of serine
proteinases. In a first PCR, primer 660 was used with primer T7, which
anneals with sequence in the cloning vector near the 3'-end of the
inserted cDNA, under the following conditions: 94 °C, 30 s;
53 °C, 40 s; and 72 °C, 80 s for 30 cycles. The
reaction product (1 µl) was used directly as a template for a second,
nested PCR by using primers 661 and 625 under the same cycling
conditions. After electrophoresis of the resulting products on a 1%
agarose gel, a DNA band of the expected size (about 0.6 kb) was
recovered and cloned into the pGem-T vector (Promega). After this clone was confirmed by DNA sequence analysis to encode PAP-2, the PCR-derived cDNA fragment was labeled by 32P and used as a probe to
screen the induced M. sexta fat body cDNA library in
ZAP II (Stratagene) (19). Positive clones were purified to
homogeneity and subcloned by in vivo excision of pBluescript phagemids. Nucleotide sequence analysis was carried out using the
BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems).
-1,3-glucan recognition
proteins in the hemolymph (22), curdlan (0.2 g) was incubated with the
protein solution with gentle agitation for 10 min on ice. The reaction
mixture was centrifuged at 15,000 × g for 30 min to
remove the flocculent materials including curdlan. Saturated ammonium
sulfate solution was slowly added to the supernatant to a final
saturation of 35%. After centrifugation at 15,000 × g
for 30 min, the pellet was collected and dissolved in 20 ml of HT
buffer. The fractionated plasma sample was dialyzed against the same
buffer (1.0 liter for 8 h, twice), and the resulting particulate
substances were removed by passing the sample through a syringe filter
(0.45 µm, low protein binding; Fisher).
70 °C for
characterization and activity assays.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Partial purification of M. sexta
PAP-2 and requirement of a cofactor for proPO activation.
A, gel filtration chromatography of partially purified PAP-2
from hemolymph of bar stage insects. As described under "Experimental
Procedures," a 15-40% ammonium sulfate (AS) fraction of
the plasma sample was separated by hydroxylapatite chromatography.
Concentrated fractions that activated proPO and hydrolyzed
IEARpNa were combined and resolved on a Sephacryl S100-HR
column (2.5-cm inner diameter × 120 cm) with 20 mM
Tris-HCl, 0.5 M NaCl, pH 7.5. , absorbance at 280 nm;
, amidase activity (units) in 10 µl of the fraction
measured with IEARpNa. B, activation of proPO by
S-100 column fractions. M. sexta proPO (0.2 µg) was
incubated with a sample from the high molecular weight peak (fraction
14, 2 µl), a sample from the IEARase activity peak (fraction 32, 2 µl), or a mixture of these two samples (14 + 32, 2 µl + 2 µl) in
pH 7.5, 20 mM Tris-HCl, 5 mM CaCl2
on ice for 60 min. Phenoloxidase activity in the reaction mixtures and
the controls of fraction 14 (2 µl), fraction 32 (2 µl), proPO (0.2 µg), and a mixture of fractions 14 and 32 (2 µl + 2 µl) were
measured as described under "Experimental Procedures."
C, analysis of MA7Q column fractions by SDS-polyacrylamide
gel electrophoresis. After removal of some glycoproteins by the lectin
affinity columns, the IEARase activity peak was further separated by
HPLC on an anion exchange column. Concentrated active fractions 26-28
(equivalent to 120 µl of the unconcentrated sample) were subjected to
SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue
staining. C and R, catalytic and regulatory
domains of PAP-2. M, protein molecular mass standards.
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Fig. 2.
Nucleotide and deduced amino acid
sequence of M. sexta PAP-2. Amino acid
residues, shown in one-letter codes, are aligned with the
first nucleotide of each codon. The secretion signal peptide is
underlined. The double underlined
sequence of the mature protein was confirmed by deblocking and
sequencing of the PAP-2 light chain (Fig. 1C). The
proteolytic activation site is indicated (
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Fig. 3.
Changes in PAP-2 mRNA and protein levels
in bacteria-challenged M. sexta larvae.
A, total RNA samples (10 µg) from hemocytes of control
insects (CH), hemocytes collected 24 h after injection
of E. coli (IH), fat body of control insects
(CF), or fat body collected 24 h after injection of
E. coli (IF) were separated by electrophoresis on
a 1% agarose gel containing formaldehyde. Northern blot analysis was
performed using 32P-labeled M. sexta PAP-2
cDNA as a probe (upper panel). The positions
of RNA standards are shown on the right side, and
the PAP-2 mRNA is marked with an arrow. Approximately
equal loading of each lane was confirmed by hybridizing a duplicate
blot with 32P-labeled cDNA for M. sexta
ribosomal protein S3 (lower panel) (21).
B, cell-free hemolymph samples (1 µl) collected 24 h
after fifth instar day 2 larvae had been injected with water, killed
E. coli cells, or M. luteus; were treated with
SDS sample buffer containing -mercaptoethanol; and were separated by
SDS-polyacrylamide gel electrophoresis on a 10% gel. Immunoblot
analysis was carried out using antiserum against proPAP-2 as the first
antibody. The proPAP-2 band is indicated with an arrow.
Molecular masses of the protein standards are marked on the
right.
-1,3-glucan recognition
protein was one of the major contaminating proteins in several
chromatographic steps in the original purification scheme. Based on
these results, an efficient method was developed to purify PAP-2 from
the bar stage hemolymph.
-1,3-glucan recognition protein bound to the
insoluble curdlan. After curdlan and cell debris were removed by
centrifugation, ammonium sulfate saturation of the supernatant was
slowly adjusted to 35%. The protein precipitate was collected by
centrifugation and dissolved in and dialyzed against the
hydroxylapatite HT buffer. Compared with the starting material, over
90% of total proteins were removed in this simple step (Table
I). However, PAP-2 represented only a
small portion of the total IEARpNa-hydrolyzing activity in
the sample. PAP-2 (fractions 41-46) was hidden under a broad IEARase
peak in fractions 38-52 (Fig.
4A). Guided by results from
immunoblot analysis, we pooled fractions 41-46, which contained 90%
of PAP-2 loaded. The specific activity was increased ~9.4-fold by
this step. The proteins in the combined fractions were collected by
ammonium sulfate precipitation and then separated by gel filtration chromatography on a Sephacryl S100-HR column (Fig. 4B). Most
of the PAP-2 eluted in fractions 37-44, whereas hydrolysis of
IEARpNa was also detected in many other fractions. The last
IEARase peak (fractions 52-60) may represent a proteinase that
interacts with the column matrix, since its apparent molecular mass is
only about 10 kDa, a value much smaller than the size of a typical
serine proteinase without a regulatory domain (25 kDa).
Purification of M. sexta PAP-2 from hemolymph of bar
stage insects
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Fig. 4.
Purification of M. sexta
PAP-2 from hemolymph of bar-stage insects. As described
under "Experimental Procedures," 0-50% AS-fraction of hemolymph
(40 ml) was incubated with curdlan in HT buffer containing
phenyl-2-thiourea and p-aminobenzamidine. After
refractionation with 0-35% saturated AS and dialysis against the HT
buffer, the treated sample was separated on a hydroxylapatite column
(A). Fractions containing PAP-2, indicated by the
horizontal bar, were pooled and precipitated with
60% saturated AS. The dissolved sample (3.0 ml) was applied to a
Sephacryl S100-HR column (B) and eluted as described under
"Experimental Procedures." Based on results of immunoblot analysis
and amidase activity measurement, fractions containing PAP-2, indicated
by a horizontal bar, were combined and subjected
to lectin affinity chromatography on a ConA-Sepharose column (5.0 ml). , absorbance at 280 nm;
, amidase activity (units)
in 5 µl of the fraction measured with IEARpNa.
C, UNO-Q6 anion-exchange chromatography. After dialysis, the
ConA flow-through fraction containing PAP-2 was loaded onto an HPLC
UNO-Q6 column equilibrated in Q buffer. A linear gradient of 0-0.2
M NaCl was applied for 30 min at a flow rate of 1.0 ml/min.
The elution profile was monitored by measurement of absorbance at 280 nm, and 0.5-ml fractions were collected. The PAP-2 peak is denoted by a
horizontal bar.
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Fig. 5.
SDS-polyacrylamide gel electrophoretic
analysis of purified M. sexta PAP-2. Purified
PAP-2 (50 ng/lane) was treated with SDS sample buffer in the absence or
presence of 0.1 M dithiothreitol and separated by
electrophoresis on a 12% polyacrylamide gel (18). A, silver
staining. B, immunoblot analysis was performed as described
in the legend to Fig. 3. C, to affinity-label the active
site serine residue of PAP-2, 18 µl of purified PAP-2 (12 µg/µl)
was incubated with [3H]DFP (2 µl, 0.1 mM,
10 mCi/mmol) at 37 °C for 1 h. After treatment with SDS sample
buffer, the labeling mixture was resolved by electrophoresis on a 12%
gel and subjected to fluorography according to Skinner and Griswold
(24). Positions of the 14C-labeled molecular mass standards
are marked on the right.
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Fig. 6.
MALDI time-of-flight mass spectrometry of
M. sexta PAP-2. A representative strong single
accumulation spectrum for PAP-2 is presented. The spectrum was
calibrated with an external bovine serum albumin standard and subjected
to noise removal. The mass values are indicated above the
MH+ and MH2+ peaks, which are slightly
different from the average reported under "Results" (the mean ± S.D., n = 4). Experimental details for sample
preparation and mass spectrometry are described under "Experimental
Procedures."
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Fig. 7.
Effect of M. sexta SPH-1 and
SPH-2 on the activation of prophenoloxidase by PAP-2.
A, PO activity. Buffer, SPHs (5 ng), PAP-2 (50 ng), or a
mixture of PAP-2 and SPHs (50 ng + 5 ng) was incubated with 0.1 µg of
purified proPO in 20 mM Tris-HCl, 5 mM
CaCl2, pH 7.5, at 4 °C for 60 min. Activated
phenoloxidase was assayed using dopamine as a substrate. B,
SDS-polyacrylamide gel electrophoretic analysis. Purified proPO (0.125 µg) was reacted with buffer (lanes 1 and
4), 20 ng of PAP-2 (lanes 2 and
5), or a mixture of 20 ng of PAP-2 and 7.5 ng of SPHs
(lanes 3 and 6) in the absence
(lanes 1-3) or presence (lanes
4-6) of 0.001% PTU. The reaction mixtures were treated
with SDS-sample buffer containing -mercaptoethanol, separated by
electrophoresis on a 6% polyacrylamide gel, and visualized by silver
staining. C, immunoblot analysis. Purified proPO (0.1 µg)
was incubated with buffer (lane 1) or a mixture
of 50 ng of PAP-2 and 30 ng of SPHs in the absence (lane
2) or presence (lane 3) of PTU. The
activation mixtures were subjected to SDS-polyacrylamide gel
electrophoresis under reducing conditions. Immunoblot analysis was
performed using 1:2000 diluted antiserum against proPO as the first
antibody. Positions of the prestained molecular weight standards are
indicated on the right.
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Fig. 8.
MALDI time-of-flight mass spectrometry of
propeptides released from proPO after PAP-2 cleavage. The reaction
mixture of proPO and PAP-2 was desalted and eluted from a C18 zip tip.
A representative strong single accumulation spectrum is presented. The
spectrum was subjected to noise removal and calibrated with external
standards of bovine insulin, E. coli thioredoxin, and horse
apomyoglobulin. The mass values are indicated above the
MH+ and MH2+ peaks. Other experimental details
are described under "Experimental Procedures."
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Gary Radke at the KSU Microchemical Laboratory for amino acid sequence analysis and Dr. Steve Hartson at OSU Recombinant DNA/Protein Resource Facility for MALDI mass determination. We also thank Drs. Dilwith, Sauer, Gorman, and Kramer for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants GM58634 (to H. J.) and GM41247 (to M. K.). This article was approved for publication by the Director of the Oklahoma Agricultural Experimental Station and supported in part under project OKLO2450. This is contribution 02-448-J from the Kansas Agricultural Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY077643.
§ To whom correspondence should be addressed: Dept. of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078. Tel.: 405-744-9400; Fax: 405-744-6039; E-mail: haobo@okstate.edu.
Published, JBC Papers in Press, November 26, 2002, DOI 10.1074/jbc.M205743200
2 C. Ji, Y. Wang, J. Ross, and H. Jiang, manuscript in preparation.
3 Y. Wang and H. Jiang, unpublished results.
4 Y. Wang and H. Jiang, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PO, phenoloxidase; PAP, prophenoloxidase-activating proteinase; proPAP, precursor of proprophenoloxidase-activating proteinase; PPAE, prophenoloxidase-activating enzyme; PPAF, prophenoloxidase-activating factor; SPH, serine proteinase homolog; proPO, prophenoloxidase; DFP, diisopropyl fluorophosphate; IEARpNa, acetyl-Ile-Glu-Ala-Arg-p-nitroanilide; PTU, 1-phenyl-2-thiourea; MALDI, matrix-assisted laser desorption ionization; ConA, concanavalin A; HPLC, high performance liquid chromatography; IEARase, IEARpNa-hydrolyzing enzyme.
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