From the Biochemistry Laboratory, The Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan 060-0819
Received for publication, September 14, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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Pro-phenoloxidase (proPO) in insects is
implicated in the defense against microbes and wounding. The presence
of proPO in the cuticle was suggested more than 30 years ago, but it
has not been purified. The extract of cuticles of the silkworm,
Bombyx mori, was shown to contain two proPO isoforms
(F-type and S-type proPOs, which have slightly different mobilities in
polyacrylamide gel electrophoresis under nondenaturing conditions). The
two isoforms were purified to homogeneity. From hemolymph of the same
insect, two types of proPO with the same electrophoretic mobilities as those of cuticular isoforms were separated and were shown to be different at five amino acid residues in one of their subunits. The
isoforms in the hemolymph and cuticle were activated by a specific
activating enzyme. The resulting active phenoloxidases exhibited almost
the same substrate specificities and specific activities toward
o-diphenols. The substrate specificities and the
susceptibilities to inhibitors, including carbon monoxide, indicated
that the purified proPO isoforms were not zymogens of laccase-type
phenoloxidase. The proPO in hemolymph was shown to be transported to
the cuticle. This demonstration was corroborated by the failure to
detect proPO transcripts by Northern analysis of total RNA from
epidermal cells. In reversed-phase column chromatography, cuticular and
hemolymph proPOs gave distinct elution profiles, indicating that some
yet to be identified modification occurs in hemolymph proPO and results
in the formation of cuticular proPO. There was little transportation of
cuticular proPO to the cuticle when it was injected into the hemocoel.
The nature of the modification is described in the accompanying paper
(Asano, T., and Ashida, M. (2001) J. Biol. Chem. 276, 11113-11125).
The outer surface of arthropods, including the insect, is covered
by a protective armor, the cuticle. The cuticle is a nonliving matrix
of carbohydrates and proteins secreted by the underlying monolayer of
epithelial cells (1). Because of its strategic localization between the
environment and internal organs, the cuticle has been considered to be
the first line of defense that is indispensable to safeguard the
homeostasis against unfavorable environmental factors such as
infectious microbes and drought. At the same time, it has often been
evaluated only as a nonliving physical barrier. This view, however, is
changing dramatically because of the recent demonstration of active
participation of the cuticle in the defense reaction against microbial
invasion and in the storage of proteins (2, 3).
The cuticle has been shown to have a certain mechanism to sense the
presence of bacteria and fungi in an injured part and to relay signals
to the underlying epithelial cells to direct the synthesis of a
bacteriocidal peptide and its secretion to the cuticular matrix (3).
However, it is still not clear what kind of mechanism is involved in
the signaling in the cuticle. Interestingly, the existence of a
pro-phenoloxidase (proPO)1
cascade (proPO-activating system) in the cuticle of the silkworm, Bombyx mori, has been reported (4). In the 1980s, this
cascade has been shown to be present also in the hemolymph of this
insect and to be composed of recognition proteins with specific
affinity to microbial cell wall components, serine protease zymogens
and proPO (5). The cascade is triggered by very minute amounts of cell
wall components such as peptidoglycan, Epithelial cells underneath the cuticle secrete proteins. The secretion
is directed apically to the cuticular matrix or basolaterally to the
hemolymph or bidirectionally to the cuticle and hemolymph (8). In
addition to this secretion, the epithelial cells have been shown to be
capable of transcytosis of proteins, a process in which the epithelial
cells take up proteins from the hemolymph and secrete them apically to
the cuticle or vice versa. Thus, the cuticle has been shown to function
as a reservoir of proteins (2).
What are the underlying mechanisms by which these physiological
functions of the cuticle and the epithelial cells are exhibited? How
are the components playing roles in the cuticular functions organized
in the cuticular matrix? Almost nothing is known at present about these
mechanisms. Several years ago, one of the authors of this paper
reported that immunogold staining of proPO displays an orderly arrayed
pattern in the cuticular matrix of the silkworm, B. mori,
and that proPO may be transported from the hemolymph to the cuticle
through epithelial cell layer (4). To understand the function of the
cuticular proPO, the properties of the proenzyme and the enzymatic
properties of its active form must be determined. Furthermore, a clear
demonstration of transport of hemolymph proPO to the cuticle and a
comparison of the properties of cuticular and hemolymph proPOs should
provide clues to understand the mechanism by which hemolymph proteins
are transported to the cuticle.
The principal aim of the present paper and the accompanying paper (9)
is to get keys for solving the mechanism of transepithelial protein
transport in insects by using proPO as a probe. Our knowledge on
molecular properties of proPOs and their active forms has not reached
to the level compatible with the purpose. Neither homogeneous cuticular
proPO nor its active enzyme (PO) has been characterized (10), since the
original suggestion of the presence of proPO in insect cuticle by
Lai-Fook (11). With regard to the hemolymph proPO of lepidopteran
insects, the presence of two isoforms has been indicated (12, 13),
but they have yet to be isolated and characterized.
Here, we report the purification and characterization of isoforms of
cuticular and hemolymph proPOs and some enzymatic properties of the
active forms. We also present evidence of transport of hemolymph proPO
to the cuticle and of modification occurring in the cuticular
proenzymes. On the other hand, we found that cuticular proPO was not
transported when it was injected into the hemocoel, indicating that the
mechanism for recognition of subtle differences between cuticular and
hemolymph proPOs operates in the process for the transport.
Silkworm (B. mori)--
Silkworm larvae (Kinsyu × Showa)
were reared on the artificial diet Silkmate 2M (Kyodo Shiryo, Tokyo) at
25 °C under a 12-h photoperiod.
ProPO-activating Enzyme (PPAE)--
Crude PPAE preparation
referred to as PPAE (AS-salt) and purified PPAE preparation were
obtained from larval cuticles of silkworms as previously reported
(14).
Assay of PO Activity and Determination of the Amount of
ProPO--
The activity of PO was assayed by the spectrophotometric
method that had originally been reported by Pye (15) and has
subsequently been used in our laboratory with modifications (16).
Briefly, PO was incubated at 30 °C for 5 min in 1.25 ml of a
reaction mixture containing 80 mM potassium phosphate
buffer, pH 6.0, 8 mM 4-methylcatechol, and 8 mM
4-hydroxyproline ethylester. The increase in absorbance at 520 nm was
measured with a spectrophotometer (Shimadzu, model UV-240). One unit of
enzyme was defined as the amount causing an increase in absorbance of
0.01 under the above conditions. PO was assayed by this method
throughout the present study, unless otherwise specified. The amount of
proPO was quantified by assaying the PO activity after the conversion
of proPO to PO by PPAE (AS-salt) at pH 7.5 as described previously
(16). One unit of proPO was defined as the amount of the protein that
had 1 unit of PO activity upon activation.
Extraction of Cuticular Proteins--
Cuticular proteins were
extracted as described previously (4). Briefly, each cuticle of fifth
instar larvae reared to day 5-6 was subjected to extraction of
proteins on ice for 1 h by using 1 ml of 50 mM acetate
buffer, pH 5.2, containing 10 mM EDTA and 33.3 µM p-amidinophenyl-methanesulfonylfluoride
(Sigma). The extract was centrifuged at 15,000 × g for
10 min at 4 °C to remove the flocculent materials.
Purification of Cuticular ProPO Isoforms--
Purification,
except for column chromatography, was carried out at 4 °C. Column
chromatography was performed at room temperature (20-25 °C).
Cuticles from 70 larvae were subjected to extraction as described
above, and the combined extracts were applied to a CM-Toyopearl (Toso)
column (15 × 120 mm) equilibrated with 50 mM acetate
buffer, pH 5.2, containing 10 mM EDTA. After application to
the column, the nonadsorbed proteins were eluted with 60 ml of the same
buffer as that used for the equilibration. During the chromatography,
the flow rate was maintained at 2 ml/min. The flow-through fraction was
saved, and the pH was adjusted to around 7.5 with 7 ml of 1 M Tris-HCl buffer, pH 7.5, followed by dialysis against 10 mM Tris-HCl buffer, pH 7.5, containing 0.1 mM
EDTA. The dialyzed solution was stored at 4 °C and used as a
CM-Toyopearl fraction. The CM-Toyopearl fraction was applied to a Super
Q-Toyopearl (Toso) column (18 × 180 mm) equilibrated with10
mM Tris-HCl buffer, pH 7.5. After washing the column with 20 ml of the buffer, adsorbed proteins were eluted with two consecutive linear gradients of NaCl (0-100 mM/10 min and 100-250
mM/120 min) established in 10 mM Tris-HCl
buffer, pH 7.5, at a flow rate of 0.5 ml/min. 5-ml fractions were
collected. Fractions eluted between 120 and 150 mM NaCl
were pooled and dialyzed against 10 mM potassium phosphate
buffer, pH 6.5. The dialyzed solution was applied to a hydroxyapatite
(Wako Pure Chemical Industries) column (8.5 × 50 mm). Adsorbed
proteins were eluted with two consecutive linear gradients (10-65
mM potassium phosphate buffer, pH 6.5/110 min and 65-300
mM potassium phosphate buffer, pH 6.5/50 min) at a flow
rate of 0.3 ml/min. Fractions of 1.5 ml were collected. Fractions eluted between 30 and 50 mM potassium phosphate were
diluted 3-fold with 10 mM Tris-HCl buffer, pH 7.5, and
applied to a Mono Q column HR5/5 (Amersham Pharmacia Biotech)
equilibrated with the same buffer. Adsorbed proteins were eluted at a
flow rate of 0.25 ml/min with two consecutive linear NaCl gradients
(0-100 mM/20 min and 100-200 mM/120 min)
established in 10 mM Tris-HCl buffer, pH 7.5. Monitoring
the absorbance at 280 nm of the effluent, 0.5-1-ml fractions were
collected manually. Cuticular proPO was eluted from the column in two
fractions: fraction I between 160 and 166 mM NaCl and
fraction II between 169 and 175 mM NaCl. These two fractions were rechromatographed separately on the Mono Q column under
the same eluting conditions as those described above. In the
rechromatography, proPOs in fractions I and II were eluted between 163 and 167 mM NaCl and between 169 and 175 mM
NaCl, respectively. The purified proPOs originating from fractions I
and II were designated proPO-CS and proPO-CF, respectively.
Purification of ProPO Isoforms from Hemolymph--
The
collection of hemolymph from silkworm larvae and the first ammonium
sulfate fractionation of the hemolymph proteins were performed
according to the method of Ashida (17). Column chromatography was
carried out at room temperature (20-25 °C), and other purification procedures were performed at 4 °C, unless otherwise specified. Hemolymph (275 ml) was subjected to ammonium sulfate fractionation during which pH of the hemolymph was maintained at pH 6.5 by the addition of 0.1% ammonia water. The precipitate that appeared between
0.4 and 0.5 saturation of ammonium sulfate was dissolved in 30 ml of
0.2 M potassium phosphate buffer, pH 6.5, which contained 20% (v/v) of saturated ammonium sulfate solution. The volume of the
resulting slurry was adjusted to 80 ml by adding saturated ammonium
sulfate solution. After being stirred for 1 h, the slurry was
centrifuged at 8000 × g for 30 min. The supernatant
was added to 0.5 ml of 33.3 mM of
p-amidinophenyl-methanesulfonylfluoride and dialyzed for
20 h against 3 liters of 10 mM Tris-HCl buffer, pH
7.5, containing 0.5 mM EDTA and 150 mM NaCl.
71% of proPO precipitated between 0.4 and 0.5 saturation in the first
ammonium sulfate fractionation was recovered in the dialyzed solution
(data not shown). The dialyzed solution was incubated at 50 °C for 5 min and centrifuged at 20,000 × g for 30 min. The
supernatant was dialyzed against 3 liters of 10 mM Tris-HCl
buffer, pH 7.5, containing 0.5 mM EDTA. ProPO in one-third
of the dialyzed solution was purified by chromatography on a Super
Q-Toyopearl column, a hydroxyapatite column, and first and second
chromatography on a Mono Q column in the same way as the cuticular
proPO isoforms were purified. In the first Mono Q column
chromatography, proPO was eluted in two fractions, fractions III and
IV, at NaCl concentrations between 164 and 170 mM and between 173 and 180 mM, respectively. Fractions III and IV
were separately subjected to the second Mono Q column chromatography. The proPOs purified from fractions III and IV were designated proPO-HS
and proPO-HF, respectively.
Assay of PO Used in the Study of Their Enzymatic Properties and
the Preparation of POs from Zymogens--
10 µg of each proPO
isoform in 0.1 ml of 10 mM Tris-HCl buffer, pH 7.5, was
added to 1 µg of purified PPAE in 10 µl of 3.5 mM
N,N'-bis(2-hydroxyethyl)-glycine buffer, pH 7.5, containing 35 mM KCl. The mixtures were incubated on ice
for 30 min to complete the conversion of proPOs to POs. The active POs
thus obtained from proPO-HS, proPO-HF, proPO-CS, and proPO-CF were
designated PO-HS, PO-HF, PO-CS, and PO-CF, respectively. Substrate
specificity of the POs was assessed at a fixed concentration (0.33 mM) of substrate as described by Andersen (18). Briefly,
100-500 units of POs were incubated at 25 °C in 2 ml of 100 mM potassium phosphate buffer, pH 6.0, containing 0.33 mM of substrate and 7.5 × 10
Michaeris-Menten (Km) constants of POs for NADA, DA,
and L-DOPA were examined as follows: 70-400 units of POs
were incubated at 25 °C in 2 ml of a reaction mixture containing 100 mM potassium phosphate buffer, pH 6.0, and 0.0375-15
mM of each substrate. The rates of the initial absorbance
changes were monitored at 390, 430, and 475 nm for NADA, DA, and
L-DOPA, respectively (19). The results were graphically
analyzed according to the method of Lineweaver and Burk (20).
To examine the effect of carbon monoxide on the PO activity, the
reaction mixture (0.1 M potassium phosphate buffer, pH 6.0, containing 0.5 mM of L-DOPA) for the enzyme
assay was equilibrated with a gas of which the composition was 80% CO
and 20% O2. After equilibration, PO was added to the
reaction mixture, and the activity was assayed by measuring the rate of
the initial absorbance change at 475 nm. As a control, a gas with 80%
N2 instead of CO was used.
Separation of Subunits of Each ProPO Isoform by Reversed-phase
High Performance Liquid Chromatography (RP-HPLC)--
Subunits of each
proPO isoform were separated on an octadecyl (ODS) column (YMC-Pac
ODS-AP; pore size, 300 Å; column size, 4.6 × 250 mm) as
described previously (12). Briefly, purified proPO isoforms were
diluted 5-fold with 0.1% trifluoroacetic acid and applied separately
to the ODS column equilibrated with 5% acetonitrile (AcCN) in 0.1%
trifluoroacetic acid. Adsorbed polypeptides were eluted with two
consecutive AcCN gradients (5-30%/5 min and 30-65%/65 min) in 0.1%
trifluoroacetic acid at a flow rate of 0.8 ml/min. Separated proPO
subunits were lyophilized and stored at S-Pyridylethylation of ProPO Subunits--
100-300 µg of each
proPO subunit was dissolved in 200 µl of 0.1 M Tris-HCl
buffer, pH 8.2, containing 5 M guanidine-HCl and 5 mM EDTA. 10 µl of 10 mg/ml dithiothreitol was added to
the solution, and the solution was incubated at room temperature for
2 h. Then 4 µl of 4-vinylpyridine was added to the solution, and
the solution was incubated at room temperature for 6 h. After the
addition of 20 µl of formic acid, the solution was desalted by ODS
column chromatography and then subjected to lyophilization. The
lyophilized S-pyridylethylated proPO subunits were stored at
Peptide Mapping of the S-Pyridylethylated ProPO
Subunits--
Each of the S-pyridylethylated proPO subunits
was dissolved in 0.15 ml of 0.2 M Tris-HCl buffer, pH 7.5, containing 8 M urea. The solution was diluted 2-fold with
0.2 M Tris-HCl buffer, pH 7.5, and incubated with 1-3 µg
of lysylendopeptidase (Lys-C) (Wako Pure Chemical Industries) at
37 °C for 6 h. The incubated solution was applied to the same
ODS column as that used for the separation of proPO subunits. The
adsorbed peptides were eluted with a linear gradient (5-65% AcCN/120
min) in 0.1% trifluoroacetic acid at a flow rate of 0.6 ml/min.
Peptides eluted in well separated peaks were lyophilized and stored at
Electrophoresis--
SDS-PAGE was performed in a 1-mm-thick slab
gel according to the method of Laemmli (21), with 14% acrylamide in
the separating gel. Proteins were stained with Coomassie Brilliant Blue
R-250 (CBB). Phosphorylase a (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin
inhibitor (21.5 kDa), and lysozyme (14.4 kDa) were used as molecular
mass standards.
PAGE under nondenaturing conditions was performed by adopting the
buffer system of Davis (22) in a 1-mm-thick slab gel with 4.5%
acrylamide in the separating gel. In the case of crude proPO samples,
proPO was converted to PO in the gel after the electrophoresis and
localized by the method of Asada et al. (23).
Determination of the Molecular Mass of Cuticular ProPO under
Nondenaturing Conditions--
The molecular masses of native
cuticular proPO isoforms were determined using a Superose 12 HR 10/30
column (Amersham Pharmacia Biotech) as previously reported (12).
Briefly, 50 µg of proPO-CS or proPO-CF was applied to the column
equilibrated with 10 mM Tris-HCl buffer, pH 7.5, containing
150 mM KCl. The column was calibrated by the chromatography
of a mixture of molecular mass standards (Combithek Calibration
Proteins II, Roche Diagnostics) consisting of ferritin (450 kDa),
catalase (240 kDa), aldolase (158 kDa), bovine serum albumin (68 kDa),
ovalbumin (45 kDa), Mass Spectrometry--
Electrospray ionization mass spectra of
peptides were obtained using a JEOL JMS-SX102A mass spectrometer (JEOL
Co., Ltd.). The peptides were subjected to analyses at the
concentration of 1 pmol/µl in a solvent containing 1% acetic acid
and 50% AcCN. The spectrometer was operated at the source voltage of
65 V and source temperature of 100 °C. Samples were scanned over a
mass range of 700-1600 Da. The instrument was calibrated using bovine insulin (5733.5 Da).
Matrix-assisted laser desorption ionization mass spectrometric analyses
were performed using a Kompact MALDI IV (Shimadzu Corp.).
Sinapinic acid, Amino Acid Sequence Analysis--
Peptides were sequenced
according to the method of Edman and Begg (24) using an automated
protein sequencer PPSQ-10 (Shimadzu Corp.).
Construction of a Hemocyte cDNA
Library--
Poly(A)+ mRNAs were obtained from
silkworm hemocytes as described previously (25). A hemocyte cDNA
library was constructed using a Screening of the cDNAs Encoding a Subunit, ProPO-HS-pII of
ProPO-HS--
A 1.9-kbp EcoRI fragment of a cDNA clone,
pPO-17, which was described in our previous paper (25), was labeled
with [
The PCR products were all 2.1 kbp, corresponding to the size of the
entire open reading frame of pPO-17. The amino acid sequence Asn12-Pro13-Gly14 found in the
peptide fragment, 12c from proPO-HF-pII (see Table II), was judged to
correspond to the nucleotide sequence of 5'-AACCCGGGC-3' in pPO-17. On
the other hand, in the peptide fragment, 12d from proPO-HS-pII, the Pro
residue in the sequence of
Asn12-Pro13-Gly14 was displaced by
Gln (see Table II). The cDNA sequence encoding the sequence
Asn-Gln-Gly was thought to have mutated at least in one of two ways, to
5'-AACCAGGGC-3' or 5'-AACAAGGGC-3'. The nucleotide sequence
5'-AACCCGGGC-3' in the sequence of pPO-17 contains the sequence
(5'-CCCGGG-3') recognized by the restriction endonuclease
SmaI, but the 5'-AACCAGGGC-3' and 5'-AACAAGGGC-3' sequences
do not. Based on these observations and reasoning, the PCR products
were treated with SmaI. Five of eight PCR products were
cleaved by SmaI to give two fragments, the 1.3-kbp fragment and the 0.8-kbp fragment. The other three PCR products were not cleaved
by the enzyme. The cDNA clones that had been the templates of the
three PCR products were subcloned into pBbluescipt SK. Three clones
(pPO-21, pPO-22, and pPO-23) with 2.3-kbp inserts were obtained. The
inserts were sequenced and found to have identical sequences, although
the lengths of their noncoding regions on the 5' side varied. One of
them (pPO-23; accession number AB048761) was used as a clone containing
the cDNA encoding proPO-HS-pII in the present study.
Preparation of Crude Hemolymph ProPO Fractions and Crude
Cuticular ProPO Fractions for Electrophoretic Analyses of the Presence
of ProPO Isoforms--
Fifth-instar larvae were bled by cutting the
abdominal legs, and hemolymph was collected individually from each
larva. To each 0.1-ml aliquot of the hemolymph samples, 2 µl of 33.3 mM p-APMSF in a mixture of 1 volume of AcCN and
9 volumes of dimethylformamide and 5 µl of 0.2 M EDTA, pH
5.5, were added. After incubation at 50 °C for 3 min, the mixtures
were centrifuged at 10,000 × g for 10 min. The
supernatants were used as crude hemolymph proPO fractions. Cuticular
proteins were individually extracted from cuticles, and chromatography
of each extract was performed using a CM-Toyopearl column as described
in the above section except that a column (4 × 3 mm) in an
Ultrafree centrifuge tube UFC30 GV00 (Millipore) was used. The
flow-through fractions were dialyzed against 10 mM Tris-HCl
buffer, pH 7.5, and the dialyzed solutions were concentrated to make
their protein concentrations 1 mg/ml. The concentrated solutions were
used as crude cuticular proPO fractions.
Tissue Collection for RNA Isolation--
Epidermal cells were
collected and pooled, as previously described (4), from larvae on the
second day of the fifth instar. Hemocytes were collected as described
previously (26). Tissues were stored in liquid N2 until use.
RNA Extraction and Northern Blot Hybridization--
Total RNA
was isolated from epidermal cells and hemocytes using Isogen
(Nippongene). About 20-40-µg aliquots of the RNA preparations were
separated by electrophoresis in 1% agarose gel with formaldehyde, transferred to a Hybond-N+ (Amersham Pharmacia Biotech)
membrane, and hybridized with probes. The proPO hybridization probe
consisted of an [ Injection of ProPO-HS or ProPO-CS into Larvae with Only One ProPO
Isoform, ProPO-HF--
Silkworm larvae at day 4 of the fourth instar
were bled by cutting one of the abdominal legs. About 30 µl of
hemolymph from each larva was analyzed individually for the presence of
proPO isoforms by electrophoresis under nondenaturing conditions in essentially the same way as that described in the above section. As has
been reported previously (12), there were larvae with only proPO-HF in
their hemolymph (referred to as F-type larvae) and larvae with
proPO-HF and proPO-HS (referred to as FS-type larvae) in the strain
used in the present study. The larvae that had been bled molted to the
fifth instar and grew without any appreciable abnormalities.
Purified proPO-HS was concentrated to about 5 mg/ml with a centrifuge
evaporator Speed-Vac (Savant) and dialyzed against 10 mM
potassium phosphate buffer, pH 6.5, containing 10 mM KCl.
100-150 µg of the proPO-HS was injected twice into the hemocoel of
an F-type larva at a 24-h interval with a fine capillary inserted through footpads of the abdominal legs. The first injection was given
on day 0 of the fifth instar. 200 µg of proPO is the amount contained
in the plasma fraction of hemolymph of a larva on day 2 of the fifth
instar.2
Purification of ProPO from the Cuticles of F-type Larvae to Which
ProPO-HS Had Been Injected--
The 12 F-type larvae to which proPO-HS
had been injected as described above were sacrificed at 96 h after
the first injection to obtain their cuticles. Extraction of cuticular
proteins and purification of proPO isoforms in the cuticular extract
were carried out in essentially the same way as that described above.
Briefly, the dialyzed CM-Toyopearl fraction was applied to a Super
Q-Toyopearl column (4 × 40 mm) equilibrated with 10 mM Tris-HCl buffer, pH 7.5. Adsorbed proteins were eluted
with two consecutive linear gradients of NaCl (0-100 mM/20
min and 100-200 mM/80 min) in 10 mM Tris-HCl
buffer, pH 7.5, at a flow rate of 0.2 ml/min. Fractions of 0.6 ml were
collected. Fractions eluted between 115 and 130 mM NaCl
were pooled and dialyzed against 10 mM potassium phosphate buffer, pH 6.5. The dialyzed solution was applied to a prepacked hydroxyapatite column (4.6 × 30 mm) (Koken) equilibrated with 10 mM potassium phosphate buffer, pH 6.5. Adsorbed proteins
were eluted with two consecutive gradients of potassium phosphate
buffer, pH 6.5 (10-65 mM/85 min and 65-400
mM/80 min) at a flow rate of 0.2 ml/min. Fractions of 0.6 ml were collected. Fractions eluted between 18.5 and 24 mM
of potassium phosphate were diluted 3-fold with 10 mM
Tris-HCl buffer, pH 7.5, and applied to a Mono Q HR5/5 column
equilibrated with the buffer. Adsorbed proteins were eluted with two
consecutive linear gradients of NaCl (0-70 mM/20 min and
70-200 mM/100 min) at a flow rate of 0.25 min. ProPO was
eluted in two peaks, which appeared at the same NaCl concentrations as proPO-CS and proPO-CF eluted in the Mono Q column chromatography described in the above section. The proPO fractions in the first and
second peaks were rechromatographed on the Mono Q column under the same
conditions as those used in the first Mono Q column chromatography. The
purified proPO preparations were analyzed by electrophoresis under
nondenaturing conditions and under denaturing conditions. Fractions
giving a single band in electrophoresis were pooled and used for
subsequent analyses.
Determination of Protein--
Protein was determined according
to the method of Bradford (27) with a Bio-Rad Protein Assay using
bovine serum albumin fraction V as a standard.
Purification of Cuticular ProPO Isoforms--
ProPO isoforms
present in the cuticles of silkworm larvae at day 5 of the fifth instar
were extracted and purified by column chromatography on CM-Toyopearl,
Super Q-Toyopearl, hydroxyapatite, and Mono Q columns as described
under "Experimental Procedures." In the first Mono Q column
chromatography, they were eluted in two peaks at NaCl concentrations of
0.165 and 0.171 M (Fig.
1A). Each of the isoforms was
further purified by the second Mono Q column chromatography, in which
they were eluted in a single peak (Fig. 1, B and
C). The proPO isoforms eluted at lower and higher NaCl
concentrations were designated proPO-CS and proPO-CF, respectively. Both preparations of the purified proPO isoforms were homogeneous, as
judged from the results of PAGE under denaturing and nondenaturing conditions (Fig. 2). A summary of the
purification of proPO isoforms is presented in Table
I. From cuticles of 70 larvae, 0.282 and 0.374 mg of purified proPO-CS and proPO-CF, respectively, were obtained.
Isolation of ProPO Isoforms from Hemolymph--
ProPO isoforms
present in hemolymph were purified by ammonium sulfate fractionation,
heat treatment, and column chromatography on Super Q-Toyopearl,
hydroxyapatite, Mono Q columns as described under "Experimental
Procedures." In the first Mono Q column chromatography, the isoforms
were eluted in two peaks at NaCl concentrations of 0.168 and 0.175 M (Fig. 1D). The isoforms in the peaks were
further purified by Mono Q column chromatography, in which they were
eluted in a single peak (Fig. 1, E and F). The
proPO isoforms eluted at lower and higher NaCl concentrations were
designated proPO-HS and proPO-HF, respectively. Both preparations of
the purified proPO isoforms were homogeneous, as judged from the
results of PAGE under denaturing and nondenaturing conditions (Fig. 2).
From 91.6 ml of hemolymph from the silkworm larvae at day 5 of the fifth instar, 0.472 and 0.882 mg of purified proPO-HS and proPO-HF, respectively, were obtained. Their specific activities were 31.3 × 104 units/mg protein for proPO-HS and 33.7 × 104 units/mg protein for proPO-HF.
Comparison of Some Molecular and Enzymatic Properties of Native
ProPO Isoforms from the Hemolymph and Cuticle--
In PAGE under
nondenaturing conditions (Fig. 2B), mobilities of proPO-HS
(lane a) and proPO-CS (lane c) were about the
same and slightly slower than those of proPO-HF (lane b) and
proPO-CF (lane d), which also migrated to about the same
positions. Hereafter, the pair of proPO-CS and proPO-HS will be
collectively referred to as S-type proPO, and the pair of proPO-CF and
proPO-HF will be collectively referred to as F-type proPO (S-type and
F-type meaning slower and faster migrating, respectively). Despite the difference in mobilities in the electrophoresis under nondenaturing conditions, all of the proPO isoforms migrated as a single band and did
not exhibit any appreciable difference in their mobilities in SDS-PAGE.
They migrated to the position corresponding to that of a 71-kDa protein
(Fig. 2A).
Both proPO-CS and proPO-CF were determined to be 142-kDa proteins based
on their retention times in the Superose 12 column chromatography (data
not shown). ProPO-CS and proPO-CF each were eluted in symmetrical peaks
at the same elution volumes. The elution volumes of proPO-CS and
proPO-CF were the same as those of proPO-HS and proPO-HF (data not shown).
The S-type proPO isoforms, proPO-CS and proPO-HS, were activated by
purified PPAE, and the time courses of the activation were followed by
SDS-PAGE. Both isoforms were converted to smaller polypeptides, each
with a molecular mass of 66 kDa, and the time courses of the conversion
appeared to be the same (Fig.
3A). Both proPO-CS and
proPO-HS were converted to active POs within 30 min under the
experimental conditions used in this study. ProPO-CS and proPO-HS were
activated for 30 min under the same conditions as those used in the
experiment for which the results are shown in Fig. 3A, and
the resulting POs were examined for their abilities to oxidize DA,
NADA, and L-DOPA at various concentrations. In Lineweaver and Burk plots, PO-CS and PO-CF exhibited very similar kinetic parameters with each of the three substrates. The plots indicate that the substrates have inhibitory effects on the enzymes at
high concentrations. Km values of the POs for DA, NADA, and L-DOPA were estimated to be about 0.53, 0.8, and
1.9 mM, respectively (Fig. 3B).
Substrate specificities of the POs were examined at a fixed
concentration of several o-diphenols and
p-diphenols (Fig. 3C). The phenols were found to
serve as better substrates in the following order: DA, NADA,
L-DOPA, methylhydroquinone, and hydroquinone. The
p-diphenols were barely oxidized under the experimental
conditions used in this study. PO-CF and PO-HF exhibited slightly
higher activities than did PO-CS and PO-HS toward the
o-diphenols. The difference in specific activities was
10-15% in the oxidation of DA. This tendency appears to hold in the
oxidation of 4-methylcatechol judging from the data presented in Table
I and in the above section, "Isolation of ProPO Isoforms from Hemolymph."
Activities of the POs were inhibited almost completely by
phenylthiourea at a concentration of 1 µM in the
oxidation of any of the substrates used in the present study. The
effect of CO on the PO activity was also investigated as described
under "Experimental Procedures." Activities of the four POs in air
composed of 20% O2 and 80% CO were about 75% less than
those assayed in air composed of 20% O2 and 80%
N2 (data not shown).
Separation and Analyses of the Subunits of ProPO Isoforms--
The
four purified proPO isoforms were separated into their subunits by
RP-HPLC. Both proPO-CS and proPO-CF were eluted in three peaks (Fig.
4, A and B). On the
other hand, proPO-HS and proPO-HF were eluted in only two peaks. In
SDS-PAGE, polypeptides contained in the peaks all migrated to a
position corresponding to a 71-kDa protein (data not shown).
Polypeptides in the peaks originating from proPO-CS were proPO-CS-pI,
proPO-CS-pI*, and proPO-CS-pII, in the order of the elution. Similarly,
polypeptides of proPO-CF were proPO-CF-pI, proPO-CF-pI*, and
proPO-CF-pII. Polypeptides in the peaks of the chromatogram of proPO-HS
were proPO-HS-pI and proPO-HS-pII, in the order of the elution, and, similarly, those of proPO-HF were proPO-HF-pI and proPO-HF-pII (Fig. 4,
C and D). The molecular masses of all of the
polypeptides were analyzed by matrix-assisted laser desorption
ionization mass spectrometry. The molecular masses of proPO-CS-pI,
proPO-CS-pI*, proPO-CS-pII, proPO-CF-pI, proPO-CF-pI*, and proPO-CF-pII
were determined to be 78,887, 78,861, 80,190, 78,867, 78,857, and
80,080 Da, respectively, and the molecular masses of proPO-HS-pI,
proPO-HS-pII, proPO-HF-pI, and proPO-HF-pII were determined to be
78,657, 80,110, 78,681, and 80,050 Da, respectively.
Considering the molecular masses of these subunits and the molecular
masses of native proPO isoforms (proPO-HS, proPO-HF, proPO-CS, and
proPO-CF), each of which was determined to be 142 kDa as stated in the
above section, all the proPO isoforms appear to be composed of two
subunits. One of the present authors has reported the similar
conclusion that hemolymph proPO is composed of two distinct subunits
(12, 25). The conclusion, however, was drawn from the experiment using
a mixture of proPO-HS and proPO-HF and, therefore, has yet to be
confirmed for each of the hemolymph proPO isoforms. The exact
compositions of subunits of each cuticular proPO and the reason why
each cuticular proPO isoform was eluted in three peaks in RP-HPLC are
described in the accompanying paper (9).
Determination of the Difference between ProPO-HS and
ProPO-HF--
ProPO-HS-pI, proPO-HS-pII, proPO-HF-pI, and proPO-HF-pII
were S-pyridylethylated and digested with Lys-C. The
resulting digests were subjected to RP-HPLC. The digests of proPO-HS-pI
and proPO-HF-pI gave very similar elution profiles (Fig.
5, A and B).
Peptides 1a-16a and 1b-16b in Fig. 5 (A and B)
were analyzed by mass spectrometry. The results indicated that peptides
with the corresponding Arabic numerals in the two elution profiles have
the same molecular masses. Several peptides obtained in the RP-HPLC
were chosen at random and were sequenced to the 10th residue from their
N termini. Observed sequences matched without exception to those in the
amino acid sequence deduced from the base sequence of the cDNA,
pPO-5 (24) (data not shown). We calculated theoretical molecular mass
of the predicted Lys-C fragments that could be produced from the polypeptide with the deduced sequence. All of the observed molecular masses of peptides 1a-16a and 1b-16b matched the expected molecular masses except for those of peptides 1a and 1b, which were 42 Da larger
than expected, the difference corresponding to the increase in
molecular mass caused by acetylation of the amino group of amino acid
at the N terminus (data not shown) (25). Although peptides
corresponding to His88-Lys90 (439.6 Da) and
Tyr417-Lys418 (309.4 Da) in the deduced
sequence were not identified in RP-HPLC of the digests, these results
seem to demonstrate that proPO-HS-pI and proPO-HF-pI have the same
primary structures and that their sequences are the same as that
encoded by pPO-5.
To study the difference between the structures of proPO-HS-pII and
proPO-HF-pII, cDNA (pPO-23) encoding proPO-HS-pII was newly cloned,
and the same methodology as that described above was used. In the
RP-HPLC of the Lys-C digests of proPO-HS-pII and proPO-HF-pII, two
peaks in an elution profile did not have corresponding peaks in another
elution profile, as indicated by arrows in Fig. 5
(C and D). The other parts of the elution
profiles of the digests were almost the same. The N-terminal sequences
and molecular masses of the peptides in the peaks (12c, 12d, 14c, and
14d) were analyzed, and the results are shown in Table
II. The N-terminal sequence of 12c seems
to correspond to that of Glu417-Gln435 of the
polypeptide deduced from cDNA, pPO-17, which has been shown to
encode one of the proPO subunits before (25). Peptide 12d had a
sequence very similar to that of 12c. However, Pro at the 13th residues
of 12c was found to be displaced with Gln in 12d. The same amino acid
displacement was found between the putative amino acid sequences
deduced from pPO-17 and pPO-23 (Fig. 6). Sequencing of the N-terminal regions of 14c and 14d did not reveal any
difference between them (Table II), although the observed sequences
were shown to correspond to Met526-Pro556 of
the sequences deduced from pPO-17 and pPO-23 (Fig. 6).
Mass spectrometric analysis was performed to further characterize the
peptides 12c, 12d, 14c, and 14d. Peptides 12c and 12d were found to
have molecular masses of 10,754.8 and 10,802.1 Da, respectively (Table
II). These values are very close to the theoretical masses, 10,755.1 and 10,802.1 Da, which were calculated for the peptide fragments
spanning from Glu417 to Lys510 of the amino
acid sequences deduced from pPO-17 and pPO-23, respectively. Peptides
14c and 14d were found to have molecular masses of 8196.1 and 8208.3 Da, respectively (Table II). These values also match the theoretical
masses, 8195.4 and 8210.4 Da, that were calculated for the peptide
fragments spanning from Met526 to Lys594 of the
sequences deduced from pPO-17 and pPO-23, respectively. These data
suggest that the differences in the masses of 12c and 12d and those of
14c and 14d are due to amino acid displacements rather than some kind
of modification, although only one amino acid displacement was actually
detected by N-terminal sequencing (Table II). As to peptides other than
12c, 12d, 14c, and 14d, the peptides in the corresponding peaks in the
two elution profiles in Fig. 5 (C and D) have the
same molecular masses. All of the observed molecular masses of the
peptides 1c-20c and 1d-20d matched the calculated values of the
predicted Lys-C fragments that could be produced from polypeptides with
sequences deduced from pPO-17 and pPO-23, respectively, except for
molecular masses of 20c and 20d, which were 42 Da larger than the
expected masses (data not shown). The difference corresponds to the
increase of molecular mass by the acetylation of N-terminal amino group
(25). The peptides corresponding to
Ile59-Lys62 (469.7 Da),
Val144-Lys147 (500.7 Da), and
Asp637-Lys638 (261.3 Da) in the sequences
deduced from pPO-17 and pPO-23 were not identified in chromatograms of
the Lys-C digests of proPO-HS-pII and proPO-HF-pII. From the above
data, we concluded that pPO-17 encodes proPO-HF-pII and that pPO-23
encodes proPO-HS-pII. All of the data presented above indicate that
nascent polypeptides encoded by pPO-5, pPO-17, and pPO-23 are not
subjected to post-translational modification except for the acetylation
of the amino acids at the N termini. Thus, the difference in the
properties between proPO-HS and proPO-HF appear to be caused by the
different primary structures of proPO-HS-pII and proPO-HF-pII.
With the analysis of amino acid sequences of cuticular proPO subunits,
it has been observed that the amino acid sequences of proPO-CS and
proPO-CF are the same as those of proPO-HS and proPO-HF, respectively,
except that same methionine residues are oxidized to methionine
sulfoxides (9).
Demonstration of Transport of ProPO from the Hemolymph to the
Cuticle--
Previously, proPO in the cuticle was suggested to be
synthesized in hemocytes (4, 26). In experiments to prove the
transportation of proPO from the hemolymph to the cuticle, we utilized
the existence of two types of larvae, one with only F-type proPO in the
hemolymph and the other with both F- and S-type proPO in the hemolymph
(12). Larvae were examined individually for the presence of isoforms in
their hemolymph and cuticles. Crude hemolymph proPO fractions and crude
cuticular proPO fractions were analyzed by PAGE under nondenaturing
conditions. It was found that larvae with both proPO-HS and proPO-HF in
their hemolymph had both proPO-CS and proPO-CF in their cuticles.
However, crude cuticular fractions obtained from larvae with only
proPO-HF in their hemolymph contained proPO-CF but not proPO-CS
(Fig. 7A).
Purified proPO-HS was injected into the hemocoels of each F-type larva
at day 0 of the fifth instar as described under "Experimental Procedures." The larvae that received the proPO isoform were
sacrificed at 24 h after the second injection to prepare crude
cuticular proPO fractions. ProPO isoforms in the fractions were
analyzed by PAGE under nondenaturing conditions. A proPO band with the same mobility as that of proPO-CS was detected in the crude cuticular fractions (Fig. 7B, lane 6). The intensity of the
band seemed to be similar to that observed with crude cuticular proPO
fractions obtained from cuticles of FS-type larvae. This clearly shows
that the proPO-HS injected into the hemocoel of F-type larva was
transported to the cuticle of this larva. ProPO was purified from the
cuticles of 12 F-type larvae to which proPO-HS had been injected. The
total amount of the purified proPO was about 136 µg. About 53 µg of the total purified proPO exhibited the same electrophoretic mobility under nondenaturing conditions as that of the proPO-CS (Fig.
8A, inset) and was
eluted in three peaks in RP-HPLC (Fig. 8B). The retention
times of the peaks were the same as those of peaks observed in RP-HPLC
of the purified proPO-CS as shown in Fig. 4A. These results
indicate that the injected proPO-HS was transported from the hemolymph
to the cuticle and that the proPO-HS had been modified to become
proPO-CS during the process of transportation or in the cuticle after
transportation. We examined the specific activities of proPO isoforms
purified from 12 cuticles of F-type larvae to which the purified
proPO-HS had been injected. The relative specific activity of the
transported proPO-CS was 0.90 if that of proPO-CF copurified in the
present experiment is taken as 1.0. There was no apparent significant
loss of activity of the injected proPO-HS during the entire process of
the experiment.
The modification of some methionyl residues of cuticular proPO to
methionine sulfoxides is described in the accompanying paper (9).
Interestingly, little of the proPO-CS injected into the hemocoel was
transported to the cuticle (Fig. 7B, lane 9).
Northern Blot Analysis of ProPO Transcripts--
In a previous
study (4), proPO mRNA was not detected by Northern blot analysis in
total RNA extracted from epidermal cells. Only cDNA of pPO-17,
which encodes proPO-HF-pII, was used as a probe in that study. In the
present study, however, pPO-5 was used as a probe in Northern blot
analysis, because it was shown to encode proPO-HF-pI and proPO-HS-pI.
The results indicated the presence of abundant proPO mRNA in total
hemocyte RNA but not in total RNA from the epidermis (Fig.
9). The amount of proPO in the body wall
cuticle was shown to increase from days 0 to 5 of the fifth
instar.3 This observation
supports our contention that transcripts for proPO must be detected if
proPO is synthesized in the epidermal cells.
In this study, the properties and the molecular identity of
cuticular PO and its zymogen (proPO) of the silkworm, B. mori, were investigated. The cuticular PO is often referred to as
the injury PO in the literature. In addition to the injury PO, two other types of PO have been shown to be present in the insect cuticle:
granular PO and laccase-type PO (10, 28). Different physiological
functions have been proposed for these three POs. Granular PO is
thought to be responsible for making the body color pattern by
synthesizing melanin in the course of normal development, and
laccase-type PO is thought to be involved in sclerotization of a newly
ecdysed cuticle. The injury PO appears to work in an injured part of
the cuticle, possibly by synthesizing cytotoxic quinones and by sealing
off the injury. All of these POs have been suggested to be present as
inactive precursors, although none of them have been purified (10, 28).
The active forms of granular PO and laccase-type PO have been purified
from the tobacco hornworm, Manduca sexta (29), and the
silkworm, B. mori (30), respectively. Several attempts to
purify the injury PO from orthopteran, dipteran, lepidopteran, and
hymenopteran insects have been reported in the literature. All of
the attempts, however, failed to give a homogeneous preparation (19,
31-35). The reason for the failure seems to be the adhesive property
of the injury PO. As is often encountered with PO from hemolymph, the
injury PO appears to form aggregates of itself with progressively
higher degrees of association or complexes with other proteins. Thus, the injury PO becomes heterogeneous molecules and has not been able to
be purified by conventional techniques.
Insect hemolymph contains PO that is present as a zymogen (proPO) under
normal physiological conditions. The precursor forms have been purified
from several insect species (12, 17, 28, 36-38). Because the finding
of homology between arthropod hemocyanin and proPO in insect hemolymph
(25, 38, 39), intensive studies have been carried out, resulting in a
rapid accumulation of our molecular biological knowledge on the
precursor. Multiplicity of as many as six of the genes encoding proPO
subunits in the genome of the mosquito Anopheles gambiae has
been demonstrated (40), and an upstream region of an A. gambiae proPO subunit gene has been shown to have an
ecdysone-responsive cis-element and an NF- Lai-Fook suggested the presence of a zymogen of PO (injury PO) in
1966 (11). Thirty years later, a new method for the extraction of cuticular proteins from cuticles of the silkworm, B. mori, was developed, and the existence of the zymogen and the
cascade for activation of the zymogen was clearly demonstrated for the first time (4). In the present study, we used this method for extraction of the zymogen, and we isolated two homogeneous isoforms of
proPO (referred to as proPO-CF and proPO-CS) from the extract (Figs. 1
and 2 and Table I). Previously, silkworm hemolymph was shown to contain
two isoforms of proPO (12). We also purified them (referred to as
proPO-HF and proPO-HS) for the first time to homogeneity, as judged by
the results of SDS-PAGE and PAGE under nondenaturing conditions (Figs.
1 and 2). ProPO-HF and proPO-CF were found to have the same
electrophoretic mobilities under nondenaturing conditions, and they are
collectively referred to as F-type proPO. ProPO-HS and proPO-CS are
collectively referred to as S-type proPO. They were found to have the
same mobilities, which are slightly smaller than those of F-type
proPOs (Fig. 2B).
Aso et al. (36) reported that POs from the hemolymph and
cuticle of the tobacco hornworm had different Km
values for each of several o-diphenols, including NADA, DA,
and L-DOPA. They, however, used only partially purified
cuticular PO in their experiments, and it was therefore possible that
the PO complexed with contaminated proteins and was rendered to have
different enzymatic properties from those of the homogeneous
preparation. They also purified the active cuticular PO in the
cuticular extract, implying that the conditions in which cuticular
proPO is activated could not be specified. One of the authors of the
present report found that the specific activity and the
Km value of PO obtained by activation of hemolymph
proPO by purified PPAE differed depending on the pH value at which
proPO was converted into PO (43). Therefore, the conditions under which
proPOs are activated should be specified to compare enzymatic
properties of POs from different sources. In the present study, the
homogeneous cuticular and hemolymph proPO isoforms were activated under
strictly controlled conditions with homogeneous PPAE purified from
cuticles of the silkworm, B. mori. The resulting active
enzymes, PO-HF, PO-HS, PO-CF, and PO-CS, obtained from proPO-HF,
proPO-HS, proPO-CF, and proPO-CS, respectively, were examined for their
enzymatic properties. It was found that PO-HS and PO-CS had almost the
same Km and Vmax values for
each of the o-diphenols such as NADA, DA, and
L-DOPA (Fig. 3). Among the three o-diphenols,
the Km value was lowest for DA (0.53 mM), and they were 0.80 and 1.9 mM for NADA and
L-DOPA, respectively.
All of the POs examined in the present study were inhibited by 75%
when their activities were assayed with L-DOPA as a
substrate under air composed of 80% CO and 20% O2. The
activities of the enzymes were also completely inhibited with 1 µM of phenylthiourea and minimally oxidized
p-diphenols (Fig. 3C). Considering their ability
to oxidize tyrosine, all of the POs examined in the present study
appear to be tyrosinase-type phenoloxidases, not laccase-type phenoloxidases, according to the classification of Keilin and Mann
(44).
The transportation of hemolymph proPO to the cuticle has been suggested
before in a paper from our laboratory (4). To directly demonstrate this
transportation and to elucidate the mechanism of the transportation, we
thought that it was important to characterize the molecular properties
of proPO isoforms to be transported to the cuticle. Both proPO-HF and
proPO-HS each gave two polypeptides in RP-HPLC on an ODS column (Fig.
4). Subunits of proPO-HF were named proPO-HF-pI and proPO-HF-pII, and
those of proPO-HS were named proPO-HS-pI and proPO-HS-pII. The peptides
in the Lys-C digests of proPO-HF-pI and proPO-HS-pI were analyzed by
peptide mapping, electrospray ionization mass spectrometry, and Edman degradation. All of the data obtained from these analyses indicated that proPO-HF-pI and proPO-HS-pI were identical polypeptides. Both
subunits were proved to have the same amino acid sequence as that
deduced from a cDNA clone, pPO-5, reported previously (25).
ProPO-HF-pII and proPO-HS-pII were shown to have the same amino acid
sequence except at five amino acid residues where amino acid
displacements were detected (Fig. 5, C and D, and
Fig. 6 and Table II). ProPO-HF-pII was shown to be encoded by a
previously known cDNA clone, pPO-17 (25). A cDNA clone, pPO-23,
encoding proPO-HS-pII was isolated from a cDNA library of the
silkworm hemocytes in the present study. All of the data obtained
regarding the four proPO subunits of hemolymph proPO isoforms indicated that the subunits are simple polypeptides without modifications such as
glycosylation and phosphorylation. This knowledge of the structure made
it easier to determine that modification occurred in cuticular proPO,
as described in the accompanying paper (9).
Yasuhara et al. (12) reported that the silkworm proPO in the
hemolymph is a heterodimer composed of subunits referred to as proPO
polypeptides I and II. The proPO preparation in that study, however,
was a mixture of proPO-HF and proPO-HS. Therefore, proPO-HF-pI and
proPO-HS-pI in the present study correspond to the proPO polypeptide I,
and a mixture of proPO-HF-pII and proPO-HS-pII corresponds to the proPO
polypeptide II. In a previous study (12), silkworm proPO in the
hemolymph was found to migrate as a doublet in SDS-PAGE. As shown in
Figs. 2A and 3A, each proPO isoform migrated as a
single band in SDS-PAGE. These results appear to be inconsistent. In
reality, however, they are not contradictory, if we consider our
finding that proPO gave a doublet in the SDS-PAGE with a separating gel
polymerized with a cross-linker, AcrylAide (olefinic derivative of
agarose that is no longer commercially available) (12), but gave a
single band with separating gels polymerized with bis-acrylamide. In
the present study, the separating gel was polymerized with bis-acrylamide.
ProPO transcripts were concluded to be undetectable in total RNA
extracted from epidermis of the silkworm larvae at day 2 of the fifth
instar, on the basis of the results of Northern blot analyses with a
random primed pPO-17 probe (4) and pPO-5 probe (Fig. 9). This was the
basis for the speculation that the proPO is transported from the
hemolymph to the cuticle. If proPO is labeled with a certain tag and
injected into the hemocoel, the recovery of proPO from the cuticle
would be direct evidence of transportation. Fluoresceinisothiocyanate
or 125I2 have often been used to label
proteins. It is possible, however, for these tags to cause
conformational changes to the proteins and to introduce artifacts into
the experimental results. We took advantage of the presence of two
kinds of larvae in the silkworm strain used in the present study. One
of them has only F-type proPO in the hemolymph and cuticle, whereas the
other has both F-type and S-type proPO isoforms in the hemolymph and
cuticle. The larvae with only an F-type isoform are referred to as
F-type larvae, and those with F-type and S-type proPO isoforms are
referred to as FS-type larvae. To the hemocoel of F-type larvae at the beginning of the fifth instar, purified proPO-HS was injected two times
with a 24-h interval, and the larvae were sacrificed to extract
proteins from the cuticles at 24 h after the last injection. The
extract was analyzed for the presence of S-type proPO by PAGE under
nondenaturing conditions. The proPO-HS that had been injected into the
hemocoel of the F-type larvae was recovered in their cuticle extracts
and showed the same electrophoretic mobility as that of proPO-CS (Fig.
7B, lane 6). This result clearly demonstrates that proPO was transported to the cuticle. Transported S-type proPO was
recovered from the F-type larvae and purified to homogeneity (Fig.
7B, lane 6, and Fig. 8A,
inset, lane a). No apparent loss of activity
occurred during the transportation. Moreover, its elution profile in
RP-HPLC was the same as that of proPO-CS as shown in Fig. 4. These
results seem to support the speculation that the proPO-HS artificially
introduced into the hemocoel is transported to the cuticle by the same
process as that by which endogenous hemolymph proPO is transported to
the cuticle under normal physiological conditions. Interestingly,
little of the proPO-CS injected into the hemocoel of F-type larvae was
transported to the cuticles (Fig. 7B, lane 7).
The transport of proPO-HF to the cuticle was not studied because
silkworm larva with only S-type proPO isoform in the hemolymph and
cuticle was not found in the silkworm strain used in the present study.
However, the molecular properties of proPO-HS and proPO-HF were found
to be the same except for amino acid displacements at five residues
between proPO-HF-pII and proPO-HS-pII, and transcripts of proPO-HF
subunits were not detected in total RNA prepared from integumental
epidermis as stated above. It therefore seems reasonable to assume that
proPO-HF is also transported to the cuticle.
What is the physiological meaning of the presence of proPO isoforms in
hemolymph and cuticle? We do not have any definite answer to this
question at present. Silkworms with only F-type proPO develop normally,
indicating that S-type proPO is not essential molecule for the
silkworms as long as they have F-type proPO. Silkworms with only S-type
proPO have not been found in the silkworm strain we used in the present
report. Therefore, it cannot be said with certainty that silkworms with
S-type proPO live normally in the absence of F-type proPO, even though
molecular properties of F-type and S-type proPO and enzymatic
properties of their active forms were determined to be almost the same.
In insect species other than the silkworm, B. mori, the
presence of at least two kinds of proPO polypeptides has been detected,
for example, two in the tobacco hornworm, M. sexta (13), six
in the mosquito, A. gambiae (40), and three in the fruit
fly, Drosophila melanogaster (FlyBase). The physiological
significance of the presence of multiple proPO polypeptides in these
organisms also remains to be studied.
The epidermis of the body wall integument has been examined
histochemically for the presence of the activity to oxidize DA in a few
insect species. The oxidation of DA to melanin was thought to be
catalyzed by PO and has been used to detect the enzyme histochemically. Binnington and Barrett (45) used this technique and reported PO
activity in the granules of epithelial cells underlying the cuticle of
the sheep blowfly, Lucilia cuprina. Locke and Kirshnan (46)
observed PO activity in the secretary granules and cis-gorge apparatus of the epidermis of Calpodes ethlius, and they
suggested that PO is synthesized in the epidermis and secreted into the cuticle. Because the histochemical techniques used in these studies were not so specific to enable detection of a particular kind of PO, it
is possible that laccase-type PO and granular PO might have been
detected instead of injury PO. Furthermore, the results should be
assessed bearing in mind that the precursor form of PO cannot be
detected by the histochemical techniques unless it is converted to the
active form during the process of preparing the specimens for the
histochemical study. Antibodies raised against granular PO and
laccase-type PO have been reported not to cross-react to injury PO (28,
29). Employment of a specific probe such as a monospecific antibody and
cDNA would give clear proof of the presence or the synthesis of a
particular PO in insect epidermal cells by histochemical techniques.
Arthropods, including insects, have septate junctions that are
considered to be functional equivalents to tight junctions of
vertebrates (47). The tight junction has been shown to be a barrier
that prevents materials from going through the intercellular space and
maintains cell polarity. Insect epithelial cells are highly polarized,
and receptor-mediated transport of proteins across cells (transcytotic
transport), such as that observed in vertebrates, has been postulated
(48). Recently, several of the hemolymph proteins of the tobacco
hornworm, M. sexta, have been reported to be transported to
the cuticle (2, 8). The transportation was studied by
immunocytochemical and immunochemical techniques. However, other than
their molecular masses, which have been estimated by SDS-PAGE, the
molecular properties or the functions of the transported proteins have
not been determined. It is not known whether the tobacco hornworm
proteins are subjected to any kind of modification during the
transportation. As is described above, proPO in the silkworm hemolymph
seems to move to the cuticle by transcytotic transportation. By using
mono-specific rabbit anti-silkworm proPO/IgG and anti-rabbit goat IgG
conjugated to gold colloid, we have detected proPO in the granules of
epidermal cells underlying the body wall cuticle of the
silkworm.4 There is no doubt
that transcytotic transport of proteins takes place in insect at
various epithelial cell layers. Thus, the transportation must be
important for the physiology of an insect, but nothing is known about
the mechanisms of the transportation. In the present study, proPOs from
the hemolymph and cuticle, which are the starting point and
destination, respectively, of the transcytotic transportation, were
purified to homogeneity and characterized. Modification occurring during the transportation could be inferred from the difference between
the chromatographic behaviors of cuticular and hemolymph proPO. It has
been proved that modification does not take place during the extraction
of proPO from cuticles and subsequent purification.3 The
mechanisms of the transcytotic transportation of macromolecules in
insects have yet to be studied. Elucidation of the modification that
occurs in the cuticle proPO and how it takes place would be the first
important step for advancing our understanding of the mechanism of
transcytotic transportation of proteins in insects. In the accompanying
paper (9), we report that modification of cuticular proPO involves
oxidation of some methionine residues to methionine sulfoxides.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1,3-glucan and possibly
lipopolysaccaride. It is considered to be one of the recognition
systems for bacteria and fungi (5). Once the proPO cascade is
triggered, one of the results is activation of proPO. Active
phenoloxidase (PO) is a key enzyme for oxidation of phenolic substances
such as tyrosine, DOPA, and dopamine to melanin (6). During the
oxidation, quinones are formed in the melanized cell layers surrounding
the encapsulated microbes, which are too big to be phagocytosed (7).
These quinones are thought to be cytotoxic and facilitate the killing
of encapsulated microbes. It is possible that the cascade in the
cuticle also plays a similar role (4).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4% (w/v)
ascorbate. The substrates used were N-acetyldopamine (NADA), dopamine (DA), L-DOPA, methylhydroquinone, and
hydroquinone. The decrease in absorbance at 265 nm was monitored. The
intervals of the monitoring were from 5 to 30 s depending on the
kind of substrate and the amount of the enzyme used for the particular assay. From the change in absorbance decreasing linearly with time, the
oxidation rates of substrates were determined.
20 °C until use. The
isolated subunits of each isoform were named by adding suffixes
such as pI, pI*, or pII, where the p stands for polypeptide and Roman
numerals, including that with an asterisk, indicate the lengths of
retention times of subunits of the proPO isoform in RP-HPLC, being
shorter in the order of I, I*, and II. The following proPO subunits
were isolated from proPO isoforms: proPO-HS-pI and proPO-HS-pII from
proPO-HS; proPO-HF-pI and proPO-HF-pII from proPO-HF; proPO-CS-pI,
proPO-CS-pI*, and proPO-CS-pII from proPO-CS; and proPO-CF-pI,
proPO-CF-pI*, and proPO-CF-pII from proPO-CF.
20 °C until use.
20 °C until use.
-chymotrypsin (25 kDa), and cytochrome
c (12.5 kDa).
-cyano-4-hydroxycinnamic acid, or gentisic acid was
used as a matrix. The saturated matrix solution was prepared in 0.1%
trifluoroacetic acid containing 50% AcCN. 100-300 pmol of each sample
was dissolved in 0.1 ml of 0.1% trifluoroacetic acid containing 50%
AcCN. After 1 µl of the matrix solution had dried and the matrix
crystals had formed on a sample slide, 1 µl of the sample solution
was applied on the matrix crystals and dried. Forty laser shots were
applied to each sample, and a spectrum was obtained as a sum of 40 spectra observed after each laser shot. Substance P (1347.6 Da), bovine
insulin (5733.5 Da), horse heart myoglobin (16,950.9 Da), and bovine
serum albumin (66,430 Da) were used as mass number standards for
calibration of the instrument.
Zap II cDNA Synthesis kit
(Stratagene) and Gigapack III Goldpacking Extract (Stratagene)
according to the manufacturer's instructions.
-32P]dCTP using a Ready-To-Go-DNA Labeling kit
(Amersham Pharmacia Biotech). About 5000 clones of the cDNA library
were screened at high stringency using the labeled 1.9-kbp fragment as
a probe. Eight positive clones were detected. They were investigated
for the presence of variants of pPO-17 with PCR and the susceptibility of the PCR products to the restriction enzyme, SmaI.
Primers, 5'-ATGGCTGACGTTTTTGAAAGCCTCGAGTTG-3' and
5'-CATGGGAGGGTTCCGTGGGTTAGGTTCGG-3', were synthesized to obtain a PCR
product that covers the entire open reading frame of the cDNA of
pPO-17.
-32P]-labeled, random-primed 2.1-kbp
EcoRI-KpnI fragment of hemocyte proPO cDNA
(pPO-5) (25). An [
-32P]-labeled, random-primed
-tubulin probe was used as a constitutively expressed internal
standard. Hybridization was performed for 12 h at 42 °C in 50%
formamide, 4 × SSPE (40 mM sodium phosphate buffer,
pH 7.4, containing 1.2 M NaCl and 4 mM EDTA),
5× Denhardt's (0.1% polyvinylpyrrolidone, 0.1% bovine serum
albumin, and 0.1% Ficoll), 0.1% SDS, and 100 µg/ml denatured salmon
sperm DNA. The membrane was washed once in 2× SSPE containing 0.1%
SDS and subsequently twice in 1× SSPE containing 0.1% SDS at 50 °C
for 10 min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification of proPO isoforms present in
cuticles and hemolymph. A, Mono Q column chromatography
of cuticular proPO isoforms. Hydroxyapatite fraction of the proPO was
subjected to Mono Q column chromatography. The parts of effluent
indicated by horizontal bars with the Roman numerals
I and II were pooled separately and named
fractions I and II, respectively. B, chromatography of
fraction I in A on a Mono Q column. The purified proPO
isoform was designated proPO-CS. C, chromatography of
fraction II in A on Mono Q column. The purified proPO
isoform was designated proPO-CF. D, Mono Q column
chromatography of proPO isoforms in hemolymph. Hydroxyapatite fraction
of hemolymph proPO isoforms was subjected to Mono Q column
chromatography. The parts of effluent indicated by horizontal
bars with Roman numerals III and IV were
pooled separately and named fractions III and IV, respectively.
E, chromatography of fraction III in D on a Mono Q column.
The purified proPO isoform was designated proPO-HS. F,
chromatography of fraction IV in D on a Mono Q column. The
purified proPO isoform was designated proPO-HF. The column
chromatography was performed using a fast protein liquid chromatography
system of Amersham Pharmacia Biotech. , amount of proPO; solid
line, absorbance at 280 nm; dashed line, NaCl
concentration. Other experimental conditions are described under
"Experimental, Procedures."
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Fig. 2.
Polyacrylamide gel electrophoresis of
purified proPO isoforms. A, 1 µg of each isoform was
subjected to SDS-PAGE under reducing conditions and stained with CBB.
ProPO isoforms applied to lanes a-d were as follows:
lane a, proPO-HS; lane b, proPO-HF; lane
c, proPO-CS; lane d, proPO-CF. To lane M,
marker proteins were applied. The molecular masses of the marker
proteins in kDa are indicated at the left. B,
PAGE of proPO isoforms under nondenaturing conditions in 4.5%
separating gel. Samples and the amounts of protein applied to
lanes a-d were the same as those in A. S and F at the left of the figure indicate the
positions where S-type proPO isoforms and F-type proPO isoforms
migrated. Proteins were stained with CBB.
Summary of purification of cuticular proPO
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Fig. 3.
Comparison of the POs obtained by activating
proPO isoforms from the cuticle and hemolymph. A,
demonstration by SDS-PAGE of the conversion of proPO to PO catalyzed by
the purified PPAE. The reaction mixtures for the activation of proPO-CS
and proPO-HS were prepared as described under "Experimental
Procedures." They were incubated on ice, and at intervals an aliquot
(10 µl) of each mixture was subjected to SDS-PAGE. Proteins were
stained with CBB. Arabic numerals at the top of
the figure indicate the incubation time (min) when the mixtures were
sampled for the SDS-PAGE. The left and right
panels show the results with proPO-CS and proPO-HS, respectively.
To lane M, the same marker proteins as those shown in Fig.
2A were applied. B, kinetic analysis of POs.
Reaction mixtures containing NADA (panel a), DA (panel
b), and L-DOPA (panel c) at various
concentrations and 0.1 M potassium phosphate buffer, pH
6.0, were prepared. Measurements of oxidation of the substrates were
carried out as described under "Experimental Procedures." The
results are presented in Lineweaver-Burk plots. Open circles
and solid lines represent the results obtained with PO-CS,
and closed circles and dashed lines represent
those obtained with PO-HS. Fitted lines were obtained by the
least squares method. The velocity (v) in the figure is
expressed in Abs/min observed at the enzyme concentration
of 1 unit/ml in the reaction mixture. C, substrate
specificity of POs. The oxidation of some diphenols by POs was measured
at a fixed concentration (0.33 mM) by using ascorbate as
described under "Experimental Procedures." From the observed rates
of the decrease of the ascorbate, the rates of oxidation of the
substrates were calculated. When, DA was used as a substrate, the rates
(nmol of oxidized substrate/min/mg enzyme) obtained with PO-CS, PO-HS,
PO-CF, and PO-HF were 302.6, 300.9, 335.1, and 348.6, respectively. In
the figure, the rate of oxidation of DA by PO-HF was taken as 100. The
observed oxidation rate with other substrates or by other POs are
indicated as the values relative to that for DA by PO-HF.
HQ, hydroquinone; MHQ, methylhydroquinone.
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Fig. 4.
Elution profiles of proPO isoforms in RP-HPLC
on an ODS column. Each panel shows an elution profile of a proPO
isoform as follows: A, proPO-CS; B, proPO-CF;
C, proPO-HS; D, proPO-HF. About 100 µg of a
proPO isoform was subjected to each run of chromatography. Solid
lines, absorbance at 214 nm; dashed line, concentration
of AcCN in the effluent. Polypeptides eluted in the peaks in the
chromatograms were named as indicated at the corresponding peaks in the
figure. Other conditions for the chromatography are described under
"Experimental Procedures."
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Fig. 5.
Peptide mapping of the Lys-C digests of the
subunits of proPO isoforms from hemolymph. The subunits of
proPO-HS and proPO-HF were obtained as described in the legend of Fig.
4. After, S-pyridylethylation of the subunits, each subunit
was digested with Lys-C. The digests were analyzed in RP-HPLC on an ODS
column. Panels showing each chromatogram of the digests and the proPO
subunits used for preparing the digests are: A, proPO-HF-pI;
B, proPO-HS-pI; C, proPO-HF-pII; D,
proPO-HS-pII. Arabic numerals and small letters
at the peaks in the figures are to assign the name to the peptide
contained in each major peak. Arrowheads indicate the peaks
where N-acetylated peptides were detected. Inset, portions
(80-120 min) of the chromatograms in C and D are
expanded. Details of the experiments are described under
"Experimental Procedures."
N-terminal sequences and molecular masses of some lysylendopeptidase
fragments of proPO subunits, proPO-HS-pII and proPO-HF-pII
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Fig. 6.
The deduced amino acid sequence of the
subunit proPO-HS-pII of proPO-HS. The amino acid sequence deduced
from the cDNA of the clone pPO-23 (upper row) is
presented with the sequence deduced from the cDNA of pPO-17
(lower row). cDNAs of pPO-23 and pPO-17 have been
concluded to encode proPO-HS-pII and proPO-HF-pII, respectively (see
text). Lysine residues are boxed, and the residues where
amino acid displacements were detected are shaded. Among the
peptides that are predicted to be produced by Lys-C digestion of the
polypeptides with the deduced sequences, there are peptides with the
same N-terminal sequences and different molecular masses as those
observed with the peptides 12c, 12d, 14c, and 14d, which are listed in
Table II. The sequences corresponding to the peptides are indicated by
horizontal solid lines with arrows at each end
together with the calculated molecular masses (the masses with
pyridylethylated cysteines are given in parentheses). Amino
acid displacements were detected only among the four predicted Lys-C
peptides corresponding to 12c, 12d, 14c, and 14d. Open
triangles show the cleavage sites by PPAE.
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Fig. 7.
Identification of proPO isoforms in cuticles
of F-type and FS-type larvae (A) and demonstration of
the transport of proPO from the hemolymph to the cuticle
(B). The types of proPO isoforms were examined by
the electrophoresis under nondenaturing conditions. A, crude
proPO fractions were prepared individually from larvae. The sources of
crude proPO fractions applied were as follows: lane 1,
hemolymph of FS-type larva; lane 2, hemolymph of F-type
larva; lane 3, cuticle of FS-type larva; lane 4,
cuticle of F-type larva. B, purified proPO-HS was injected
twice into the hemocoel of an F-type larva, each injection with
100-150 µg of the protein. At 24 h after the second injection,
the larva was sacrificed and the crude cuticular proPO fraction was
prepared. In control experiments, F-type or FS-type larvae were
injected as described above except that only the medium for proPO-HS or
proPO-CS was injected. Crude cuticular proPO fractions applied were
obtained from larvae as follows: lane 5, FS-type larva
injected with medium; lane 6, F-type larva injected with
purified proPO-HS; lane 7, F-type larva injected with
medium; lane 8, F-type larva injected with proPO-HS;
lane 9, F-type larva injected with proPO-CS. The above
experiments were repeated with several larvae, and the results were
essentially the same as those in the figure presented here. Positions
where S-type proPO isoforms and F-type proPO isoforms migrated are
indicated by the capital letters S and F,
respectively. Other details are described under "Experimental
Procedures."
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Fig. 8.
Isolation of the transported proPO from
cuticles of F-type larvae to which proPO-HS had been injected
(A) and RP-HPLC of the transported proPO
(B). A, proPO-HS was injected into 12 F-type larvae as described in the legend to Fig. 7B. The
larvae were sacrificed at 72 h after the second injection. ProPO
in the cuticles was extracted and purified as described under
"Experimental Procedures." In the purification, a proPO peak in
addition to that of proPO-CF was observed in the first Mono Q column
chromatography. ProPO in the additional peak and proPO-CF were further
purified by rechromatography on a Mono Q column. The elution profile of
the proPO in the additional peak is shown in the figure. The effluent
indicated by a horizontal bar was pooled and used as the
transported proPO. , concentration of proPO; solid line,
absorbance at 280 nm; dashed line, concentration of NaCl.
Inset, 1 µg each of the transported proPO and the purified
proPO-CF were subjected to electrophoresis under nondenaturing
conditions. Proteins were stained with CBB. ProPOs applied: lane
a, transported proPO; lane b, purified proPO-CF.
B, the transported proPO obtained in the chromatography
shown in A was subjected to RP-HPLC on an ODS column. The
adsorbed proteins were eluted from the column as described in the
legend to Fig. 4. Trace c, chromatogram of the transported
proPO. Trace d, chromatogram of the purified proPO-HS. Other
details are described under "Experimental Procedures."
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Fig. 9.
Northern blot analysis of pro-phenoloxidase
mRNA in cuticular epidermal cells and hemocytes. Total RNA
from epidermal cells and hemocytes was analyzed by Northern
hybridization as described under "Experimental Procedures." The
blot was probed simultaneously with the 32P-labeled
random-primed 1.9-kbp EcoRI-KpnI fragment of
pro-phenoloxidase (pPO-5) cDNA and a tubulin cDNA. Lane
1 received 35 µg of epidermal cell total RNA, and lane
2 received 18 µg of hemocyte total RNA. From separate
experiments, pro-phenoloxidase mRNA and tubulin mRNA were
proved to migrate to the positions corresponding to 3.7 and 1.7 kilobases, respectively.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-responsive
element. The transcription of the gene was actually shown to be
up-regulated by ecdysone (41). Furthermore, the transcript of the gene
encoding a protein that possesses two copper binding sites, as observed
in proPO or hemocyanin, was detected in the hemocytes of early locust
embryos (42). Nascent polypeptide of the locust hemocyanin-like protein
has a putative signal sequence for secretion in the deduced sequence,
although none of the cDNAs of proPOs that have been isolated
contain such signal sequences. These recent observations, however, have
not directly contributed to advancing our understanding of cuticular POs, because of the lack of knowledge of the primary structures of
cuticular POs. Isolation of cuticular PO and elucidation of the
relationship between hemolymph PO and cuticular PO had not been
achieved in previous studies. The importance of such studies for insect
physiology was pointed out by Barrett nearly a decade ago (10).
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ACKNOWLEDGEMENT |
---|
We are grateful to Kenji Watanabe for analysis of molecular masses of the peptides by electrospray ionization mass spectrometer.
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FOOTNOTES |
---|
* This work was supported in part by Grants 09265201 and 06454023 from the Japan Ministry of Education, Science, Sports, and Culture.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.
To whom correspondence should be addressed. Tel.:
81-11-706-6877; Fax: 81-11-706-7142; E-mail:
ashida@pop.lowtem.hokudai.ac.jp.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M008426200
2 R. Iwama and M. Ashida, unpublished observation.
3 T. Asano and M. Ashida, unpublished observation.
4 M. Sass, T. Asano, and M. Ashida, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: proPO, pro-phenoloxidase; PO, phenoloxidase; proPO-CS, S-type cuticular proPO; proPO-CF, F-type cuticular proPO; proPO-HS, S-type hemolymph proPO; proPO-HF, F-type hemolymph proPO; PPAE, pro-phenoloxidase-activating enzyme; PAGE, polyacrylamide gel electrophoresis; RP, reversed-phase; HPLC, high performance liquid chromatography; ODS, octadecyl; AcCN, acetonitrile; Lys-C, lysylendopeptidase; CBB, Coomassie Brilliant Blue R- 250; DOPA, L-3,4-dihydroxyphenylalanine; DA, dopamine; NADA, N-acetyldopamine; PCR, polymerase chain reaction; kbp, kilobase pair(s).
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REFERENCES |
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