From the Biochemistry Laboratory, The Institute of Low Temperature Science, Hokkaido University, Sapporo 060-0819, Japan
Received for publication, September 14, 2000, and in revised form, December 14, 2000
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ABSTRACT |
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Pro-phenoloxidase (proPO) in insects is activated
through the action of a protease cascade triggered by minute amounts of microbial cell wall components. It is an important molecule for the
defense against invading microorganisms and for the repair of wounds.
In the accompanying paper (Asano, T., and Ashida, M. (2001)
J. Biol. Chem. 276, 11100-11112), a proPO isoform,
proPO-HS, in the hemolymph of the silkworm, Bombyx mori, is
reported to be transported to the cuticle. The transported proPO
isoform was recovered from the cuticle and named proPO-CS. The elution
profiles of proPO-CS and proPO-HS in reversed-phase high performance
liquid chromatography (HPLC) were found to be different, giving a basis to the inference that proPO-CS is a modified form of proPO-HS. In the
present study, we investigated the nature of the modifications occurring in proPO-CS, in which proteolytically and chemically cleaved
fragments originating from the subunits of proPO-CS and proPO-HS were
analyzed by reversed-phase HPLC, amino acid sequencing, and mass
spectrometry. A subunit of the heterodimeric proPO-CS was found to
contain five or six methionine sulfoxides, and another subunit was
found to contain one methionine residue oxidized to the sulfoxide. All
of the oxidized methionyl residues were identified. Other than
oxidation of the methionyl residues, no additional modification of
proPO-CS was found. In the model structure of each subunit of proPO-CS
constructed by protein modeling with the known structures of the
horseshoe crab, Limulus polyphemus, hemocyanin type II
subunit as templates, the methionine residues identified as methionine
sulfoxide had high degrees of accessibility to the solvent. The
implication of the oxidation at the methionine residues is discussed in
relation to the mechanism of transepithelial transport of proPO from
the hemolymph to the cuticle.
The chitinous exoskeleton of the insect is a nonliving matrix of
carbohydrate and protein secreted from a monolayer of epidermal cells
that covers the entire surface of the insect, including respiratory
tracheae, the anterior and posterior portions of the digestive tract,
and reproductive ducts (1). The cuticle is a structure featuring the
body plan of the insect and displays various functions (2): a physical
protective barrier between the internal tissues and the external
environment, a structural support of the body that enables the insect
to fly and walk, and a stage for the chemical defense against microbes
by relaying the signal generated by the presence of microorganisms to
epidermal cells underneath and by directing the cells to secrete
anti-microbial peptides into the matrix (3). The proteins and chitin
fibers secreted by epidermal cells form the complexes that make up a lamellate structure. Proteins secreted by the epidermal cells play
important roles in cuticular functions. Although all of the proteins in
the chitinous exoskeleton of the insect are thought to be secreted by
epidermal cells, their origins have been shown to be different. Some of
cuticular proteins are thought to be synthesized in hemocytes and the
fat body (an insect organ equivalent to the vertebrate liver) and to be
transepithelially transported to the cuticle via the plasma fraction of
the hemolymph. Recently, some hemolymph proteins have been shown to be
transported from the hemolymph to the cuticle and vice versa in
lepidopteran insects (4, 5). However, there has been little
investigation on the mechanisms for the transepithelial transport of
proteins in insects, in contrast to studies on transepithelial and
transendothelial protein transport in mammals in which the mechanisms
of transcytosis of proteins such as polymeric immunoglobulin A and
transferrin have been investigated in detail (6-10).
In our laboratory, we have been studying the activation mechanism of
pro-phenoloxidase (zymogen of phenoloxidase
(proPO))1 (3). Our study has
recently demonstrated that the silkworm cuticle contains proPO and the
protease cascade for its activation. Furthermore, the epidermal cells
underneath the cuticle were found not to contain the transcripts of the
genes encoding proPO subunits, suggesting that cuticular proPO is
transepithelially transported from the hemolymph (11).
In the silkworm, Bombyx mori, two proPO isoforms have been
purified from both the hemolymph and cuticle (12). The isoforms from
the hemolymph have been named proPO-HF and proPO-HS, and the isoforms
from the cuticle have been named proPO-CF and proPO-CS. The isoforms
proPO-HF and proPO-CF have the same mobilities in polyacrylamide gel
electrophoresis under nondenaturing conditions (native PAGE), and they
are collectively referred to as F-type proPO. Similarly,
proPO-HS and proPO-CS migrated to the same position in native PAGE and
are referred to as S-type proPO. F-type proPOs have slightly greater
mobility than do S-type proPOs in native PAGE. ProPOs purified from the
hemolymph and cuticle have been shown to be heterodimeric proteins
(12). One of the two subunits of proPO-HS and proPO-HF is common, but
the other subunits are different, and five amino acid substitutions
have been detected between them (12).
In the accompanying paper (12), proPO-HS is reported to be transported
from the hemolymph to the cuticle and to be recovered as proPO-CS from
the cuticle. ProPO-HF is thought to be transported similarly, although
the direct evidence of such transportation has not yet been obtained.
The isoforms proPO-CS and proPO-HS did not exhibit any appreciable
difference in their molecular mass, as determined by gel permeation
chromatography, or in their enzymatic properties after activation by a
specific endogenous protease, but their elution profiles in
reversed-phase high performance liquid chromatography (RP-HPLC) on an
octadecyl (ODS) column were different. These results indicate that
modifications occur in cuticular proPO. We observed that little
proPO-CS purified from cuticles was transported from the hemolymph to
the cuticle even if it was injected into the hemocoel of the larval
silkworm (12). It seems that the sites susceptible to modification play
an important role in the transport of hemolymph proPO to the cuticle.
We considered elucidation of the nature of the modification to be the
first step in advancing our understanding of the mechanism of
transepithelial protein transport in insects.
Here, we report that the modification in proPO-CS is oxidation of
methionine residues. All of the oxidized methionine residues are
identified. This is the first report on the structural characterization of transepithelially transported protein in insects.
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.
Purification of Cuticular and Hemolymph ProPO
Isoforms--
Purification of proPO isoforms from cuticle and
hemolymph were carried out as described in the accompanying paper
(12).
Separation of Subunits of Each ProPO Isoform by
RP-HPLC--
Subunits of each proPO isoform were separated on an ODS
column (YMC-Pack ODS-AP; pore size, 300 Å; column size, 4.6 × 250 mm) as described previously (Fig. 4 in Ref. 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
in 0.1% trifluoroacetic acid. Adsorbed polypeptides were eluted with
two consecutive acetonitrile gradients (5-30%/5 min and 30-65%/65
min) in 0.1% trifluoroacetic acid at a flow rate of 0.8 ml/min.
ProPO-CS was eluted in three peaks. Polypeptides contained in the peaks
were designated proPO-CS-pI, proPO-CS-pI*, and proPO-CS-pII in
the order of the elution (12). On the other hand, proPO-HS was eluted
in two peaks. The polypeptides contained in the peaks were named
proPO-HS-pI and proPO-HS-pII in the order of the elution (12).
Separated proPO subunits were lyophilized and stored at S-Pyridylethylation of ProPO Subunits and Peptide Mapping of the
S-Pyridylethylated ProPO Subunits--
Experiments on the
S-pyridylethylation of proPO was carried out in the same way
as described in the accompanying paper (12). 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% acetonitrile/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
Peptide Digestion with Trypsin and CNBr--
Some Lys-C-digested
fragments were subsequently digested with trypsin as follows. Peptides
(1-2 µg) were separately dissolved in 0.1 ml of 0.2 M
Tris-HCl buffer, pH 7.5, containing 8 M urea. The solutions
were diluted 2-fold with 0.1 ml of 0.2 M Tris-HCl buffer,
pH 7.5, followed by the addition of 5 µl of trypsin (Sigma) solution
(10 µg trypsin/ml of 0.001 N HCl) and incubation at
37 °C for 6 h. The trypsin-treated peptides were stored at
CNBr cleavage was carried out by using the method of Gross (14). 1-2
µg of the isolated Lys-C-digested fragment was dissolved in 70%
formic acid containing 1% CNBr. After incubation at room temperature
for 4 h, the solution was dried under a nitrogen atmosphere. The
dried materials were dissolved in a small volume of 70% formic acid
again and dried with a centrifuge evaporator. The resulting materials
were stored at Mass Spectrometry--
Electrospray ionization mass spectra and
matrix-assisted laser desorption ionization (MALDI) mass spectra were
obtained using a JEOL JMS-SX102A mass spectrometer (JEOL Co., Ltd.) and
a Kompact MALDI IV (Shimadzu Corp.), respectively, as described in the
accompanying paper (12). Post-source decay (PSD) spectra were obtained
using Kompact MALDI IV operated in the reflectron mode with an ion gate.
Protein Modeling--
Molecular models of the three-dimensional
structures of the subunits of proPO-HS (proPO-HS-pI and proPO-HS-pII)
were generated using the knowledge-based protein modeling method that
is implemented in the Swiss-Model server (15, 16). Both of the
molecular models for proPO-HS-pI and proPO-HS-pII were built using
known structures of Limulus hemocyanin type II subunit
(Protein Data Bank codes: 1LLA, 1NOL, and 1OXY) as template structures. The portions of putative amino acid sequences deduced from the cDNAs, pPO-5 (Leu66-Asp675) and pPO-23
(Pro60-Gln678) (12, 13), were submitted as
target sequences via the First Approach Mode. N-terminal and C-terminal
regions other than the submitted sequences were rejected by the program
because of low similarity between the target sequences and template
sequences. The surface accessibility of amino acid residues in the
models was calculated by submitting predicted models to a GETAREA
server (17), and the accessibility of the residues is expressed as the
value relative to that of Xaa in the tripeptide Gly-Xaa-Gly (18).
Amino Acid Sequence Analysis--
Peptides were sequenced
according to the method of Edman and Begg (19) using an automated
protein sequencer PPSQ-10 (Shimadzu Corp.).
Determination of Protein--
Protein was determined according
to the method of Bradford (20) with a Bio-Rad Protein Assay using
bovine serum albumin fraction V as a standard.
Amino Acid Sequencing and Molecular Mass Analyses of the Peptides
in the Digests of Pyridylethylated ProPO Subunits--
As has been
reported in the accompanying paper (12), both proPO-HS and proPO-CS
migrated in SDS-PAGE to the position corresponding to that of 71-kDa
proteins. No appreciable difference between the isoforms was observed
in the electrophoresis. On the other hand, proPO-CS was eluted in three
peaks in RP-HPLC on an ODS column, whereas proPO-HS was eluted in only
two peaks (Figs. 2 and 4 in Ref. 12). Polypeptides eluted in those
peaks were designated as described under "Experimental Procedures."
The polypeptides obtained in the RP-HPLC of proPO-CS and proPO-HS were
pyridylethylated and digested by Lys-C. The resulting digests were
subjected to RP-HPLC on an ODS column (Fig.
1). The elution profiles of the digests
of proPO-CS-pI, proPO-CS-pI*, and proPO-HS-pI (Fig. 1A) were
very similar except at the peaks indicated by arrows.
Peptides in the indicated peaks were named as shown in the figure. In
Fig. 1A, it can be seen that peptides (HSPIa, HSPIb, HSPIc,
and HSPId) originating from proPO-HS-pI were eluted at longer retention
times than were the peptides originating from proPO-CS-pI and
proPO-CS-pI* with the corresponding Roman letters at the end of their
names. Peptides in each of the peptide groups, "CSPIa and CSPI*a,"
"CSPIb and CSPI*b," and "CSPIc and CSPI*c," were eluted at the
same retention times. CSPId and CSPI*d, however, were not eluted at the
same retention times; the former was eluted earlier than CSPI*d. The
elution profiles in RP-HPLC of the digests of proPO-CS-pII and
proPO-HS-pII were also very similar except at the peaks indicated by
arrows (Fig. 1B). The peptides in the peaks that appeared at
different retention times in panels d and e were
named CSPIIe and HSPIIe, respectively. CSPIIe appeared at a slightly
shorter retention time than did HSPIIe.
Among the digests obtained from the pyridylethylated proPO-CS-pI,
proPO-CS-pI*, and proPO-HS-pI, peptides in the peaks that appeared at
the same retention times in the chromatography (Fig. 1A)
were determined to have the same molecular masses (data not shown).
Some of the peptide pairs with the same retention times were chosen at
random, and their N-terminal sequences were analyzed. The peptides of
each pair had the same N-terminal sequences without exception (data not
shown). Similarly, among the digests obtained from the pyridylethylated
proPO-CS-pII and proPO-HS-pII, the peptides in the peaks that appeared
at the same retention times in the chromatography (Fig. 1B)
were determined to have the same molecular masses and N-terminal amino
acid sequences (data not shown).
The N-terminal amino acid sequences and molecular masses of peptides in
the peaks indicated by arrows in Fig. 1 were also analyzed.
The results are presented in Table I. The
peptides HSPIa, HSPIb, HSPIc, and HSPId, each of which originated from proPO-HS, had N-terminal sequences and molecular masses corresponding to Val159-Lys227,
Tyr40-Lys87,
Leu419-Lys523, and
Leu275-Lys416, respectively, of the amino acid
sequence of proPO-HS-pI deduced from the base sequence of a cDNA
clone, pPO-5. The N-terminal amino acid sequence and molecular mass of
the peptide HSPIIe corresponded to those of the peptide
Lys639-Val692, which is the C-terminal region
of the amino acid sequence of proPO-HS-pII deduced from the base
sequence of a cDNA clone, pPO-23 (Table I and Fig.
2). On the other hand, among the Lys-C
fragments that originated from proPO-CS, each of those indicated by
arrows in Fig. 1 was found to be larger by about 16 or 32 Da
than the fragment from proPO-HS with the corresponding N-terminal
sequence (Table I). These results indicate that proPO-CS-pI and
proPO-CS-pI* are modified forms of proPO-HS-pI and that proPO-CS-pII is
a modified form of proPO-HS-pII.
Considering that proPO-HS, composed of two subunits (proPO-HS-pI and
proPO-HS-pII), was found to be transported from the hemolymph to the
cuticle and to become proPO-CS and that no appreciable difference
between the molecular masses of native proPO-CS and proPO-HS was
detected when their molecular masses were determined from the elution
volume in gel permeation chromatography (12), the results so far
obtained in the present study indicate the following possibility:
ProPO-CS is a mixture of two molecular species. One of them is composed
of proPO-CS-pI and proPO-CS-pII, and the other is composed of
proPO-CS-pI* and proPO-CS-pII. After proPO-HS is modified to proPO-CS,
the overall change in their molecular masses is not large enough to be
detected by their behaviors in gel permeation chromatography, as is
reported in the accompanying paper, but the reaction that brought about
the modification results in an increase in molecular mass of about 16 or 32 at the site to be subjected to the modification.
In the above-described peptide mapping of the subunits derived from
proPO-CS and proPO-HS, the peptides corresponding to
His88-Arg89-Lys90 (439.6 Da) and
Tyr417-Lys418 (309.4 Da) of the sequence
deduced from pPO-5 and
Ile59-Pro60- Leu-61-Lys62
(469.7 Da),
Val144-Arg145-Val146-Lys147
(500.7 Da), and Asp637-Lys638 (261.3 Da) of the
sequence deduced from pPO-23 could not be recovered in RP-HPLC as shown
in Fig. 1. However, over 98% of the sequences deduced from pPO-5 and
pPO-23 could be assigned to the sequences of the peptides recovered in
the chromatography of the Lys-C digests of pyridylethylated subunits of
proPO-HS and proPO-CS.
Only N-acetylation of the N-terminal amino acids of the
nascent polypeptides of the silkworm proPO has so far been reported as
a post-translational modification (12, 13). The molecular masses of the
subunits of proPO-CS observed in MALDI-MS, which are presented in the
accompanying paper (12), were in good agreement with the theoretical
molecular masses in which acetylation and modification of the subunits
identified in the present study (as will be described below) were taken
into account. Therefore, the above results seem to imply that all of
the modifications that have occurred in proPO-CS are in peptides with
names CSPIa, CSPIb, CSPIc, CSPId, CSPI*a, CSPI*b, CSPI*c, CSPI*d, and
CSPIIe indicated in Fig. 1 and listed in Table I. In the sequences of
the Lys-C peptides that were not recovered in RP-HPLC, there is no
consensus sequence for glycosylation, sulfation, and phosphorylation.
Silkworm proPO in hemolymph has not been shown to be a glycoprotein by chemical analysis (21).
To make it easier to understand the experimental design and results
described in the sections below, the deduced amino acid sequences of
proPO-HS-pI and proPO-HS-pII and sequence of horse shoe crab
(Limulus) hemocyanin type II subunit are aligned (Fig. 2).
In Fig. 2, Lys-C fragments containing the modified amino acid residues
in the subunits of proPO-CS and the amino acid residues that will be
identified as modified amino acids are also indicated.
Identification of the Modification Occurring in Cuticular
proPO--
The Lys-C fragments listed in Table I were digested with
trypsin, and the resulting digests were subjected directly to MALDI-MS. In the spectrum of the digest of HSPIa, all of the fragments that were
expected to be produced were detected except for
Ala223-Lys227 (m/z
544.68) (Fig. 3A, panel
c, and Table II). The absence of this fragment may have been caused by the suppression of ionization, which often occurs in the analysis of a peptide mixture (22). In the
case of the tryptic digests of peptides, CSPIa and CSPI*a, originating
from proPO-CS-pI and proPO-CS-pI*, respectively, spectra similar to the
spectrum of the digest of HSPIa were observed, the only major
difference being that the ion at m/z 2149.4, which corresponds to the fragment
Met176-Arg194, was not detected. Also instead
of the peak at m/z 2149.4, another peak appeared
at m/z 2165.3-2166.3 in each spectrum of the
digests of CSPIa and CSPI*a (panels a and b in
Fig. 3A). The PSD spectrum of the ion at
m/z 2165.39 derived from the digest of CSPIa is shown in panel d of Fig. 3B. A predominant
fragment 64 Da smaller than the precursor ion was detected at
m/z 2102.3 in addition to minor peaks at
m/z 2149.3, m/z 2073.5, and
m/z 2016.5. This fragmentation is characteristic
of a peptide with a methionine sulfoxide (Met(O)) at the N terminus as
is depicted in panel e of Fig. 3B (23, 24). The
64-Da smaller size is the result of the release of methanesulfenic acid
from the side chain of the oxidized methionine. Between
Met176 and Arg194 of the polypeptide sequence
deduced from pPO-5 (Table II), there was no methionine residue other
than the terminal methionine. Therefore, Met176 of CSPIa
was concluded to be an oxidized methionine residue, Met(O).
The same experiments as those performed on CSPIa were performed on
CSPI*a. The results indicated that Met176 of CSPI*a was a
residue oxidized to Met(O) (data not shown). Tryptic fragments
corresponding to Met176-Arg194 were isolated
from all of the three digests of CSPIa, CSPI*a, and HSPIa in RP-HPLC.
The isolated fragments derived from CSPIa and CSPI*a were eluted in
chromatography with shorter retention times than that for the fragment
from HSPIa (data not shown). The isolated fragments, however, were
confirmed to have the same amino acid sequence (MIPIVSNYTASDTEPEQR) in
Edman degradation. Met(O) was not detected in the sequencing,
suggesting that Met(O) was reduced to methionine under the conditions
of Edman degradation as reported by Märki et al.
(25).
All of the results presented above are consistent with the inference
that the observed difference (16 Da) between CSPIa and HSPIa and
between CSPI*a and HSPIa was caused by the presence of one Met(O) in
CSPIa and CSPI*a and that the residue is Met176 in the
amino acid sequence deduced from pPO-5.
The Lys-C peptides other than CSPIa, CSPI*a, and HSPIa listed in Table
I were similarly examined as above by tryptic digestion and MALDI-MS
analyses. The data are summarized in Table II. In the examination of
the peptides HSPIb, HSPIc, HSPId, and HSPIIe, which originated from
proPO-HS, all the observed molecular masses of the fragments obtained
in their tryptic digestion were almost identical to one of the
molecular masses predicted from the sequences deduced from pPO-5 and
pPO-23 (Table II). On the other hand, several peaks in the MALDI-MS of
the tryptic digests of CSPIb, CSPI*b, CSPIc, CSPI*c, CSPId, CSPI*d, and
CSPIIe were observed at positions 16 or 32 Da larger than the expected
molecular mass. Gains in the observed molecular mass were detected only
with the tryptic peptides that were thought to contain one or two
methionine residues (Table II). The tryptic fragments with molecular
masses of 16 or 32 Da greater than the predicted molecular mass gave
PSD spectra characteristic of a peptide containing Met(O) (data not
shown). From these observations and the fact that
Met58-Arg62 and
Asn63-Lys87 of proPO-CS-pI and proPO-CS-pI*
and Asn687-Val692 of proPO-CS-pII contain one
methionine residue in their sequences (Table II and Fig. 2), it was
concluded that Met58 and Met73 of both
proPO-CS-pI and proPO-CS-pI* and Met690 of proPO-CS-pII are
residues oxidized to Met(O)s.
In tryptic digests of CSPIc and CSPI*c, peptides with sequence
corresponding to Phe475-Arg502 of proPO-CS-pI
and proPO-CS-pI* were expected to be produced. Two methionine residues,
Met498 and Met500, are present in
Phe475-Arg502. The molecular mass of
Phe475-Arg502 calculated from the deduced
amino acid sequence is 3246.61 Da (m/z 3247.62),
but the tryptic digests of CSPIc and CSPI*c gave peaks at
m/z 3263. 92 and m/z
3263.4, respectively, suggesting that one of the two methionine
residues was Met(O). The peptides CSPIc and CSPI*c were treated with
CNBr, and the resulting fragments were directly analyzed by MALDI-MS.
The fragmentation patterns of CSPIc and CSPI*c with CNBr were thought
to depend on the position of the Met(O) residue in the peptides, as
depicted in panels a and b of Fig.
4B, because CNBr does not
cleave the C-terminal side of Met(O) (22). In the spectrum of the CNBr
fragments of CSPIc, three major peaks were observed (panel a
in Fig. 4A). A peak at m/z 4831.4 corresponded to the calculated molecular mass (4831.4 Da) of
Leu419-Met463, of which the C terminus was
homoserine lactone, and another peak at m/z
2716.07 corresponded to the calculated molecular mass (2715.1 Da) of
Phe501-Lys523. The postulated CNBr fragment
spanning from Asp464 to Met500 with unoxidized
Met498 should have had the molecular mass of 4096.6 Da, but
instead one peak at m/z 4112.05 was observed in
the spectrum corresponding to the theoretical molecular mass
(m/z 4112.6) of the CNBr fragment Asp464-Met500 with Met(O) at the 498th
residue. Essentially the same results as above were obtained in the
case of the peptide CSPI*c (data not shown). Met498 of
proPO-CS-pI and proPO-CS-pI* was concluded to be the oxidized residue
Met(O).
The Lys-C peptides CSPId, CSPI*d, and HSPId were digested with trypsin.
The digests were subjected to MALDI-MS as above. The peptides
corresponding to Phe317-Arg332 of CSPId should
have appeared in the spectrum at m/z 1821.19, but
they were observed at m/z 1853.58, 32 Da larger
than the theoretical molecular mass of the peptide with the sequence of
Phe317-Arg332 of proPO-HS-pI. The PSD spectrum
of the precursor ion at m/z 1853.58 showed the
characteristic features of a peptide containing two Met(O)s (Fig.
5). Because two methionine residues are
present in Phe317-Arg332 of the sequence of
proPO-HS-pI deduced from pPO-5, CSPId was concluded to have two
oxidized methionines at Met324 and Met327.
The peptide CSPI*d appeared to have the same amino acid sequence as
that of CSPId, but its molecular mass was found to be larger by 16 Da
than the theoretical value (Table I), suggesting that one of the
methionine residues was oxidized to Met(O). The results of analyses of
the tryptic digest indicated that one of the two methionines,
Met324 and Met327, in the postulated tryptic
fragment of Phe317-Arg332 was the oxidized
residue (Table II). To identify the oxidized methionine residue, CSPI*d
was treated with CNBr, and the resulting fragments were directly
subjected to MALDI-MS (Fig. 4). The fragmentation pattern was thought
to differ depending on the position of the oxidized methionine, and two
possible patterns are depicted in panels e and f
of Fig. 4B. The spectrum of CNBr-treated CSPI*d indicated
the presence of a fragment with m/z 5803.83, which corresponded to the singly protonated
Leu275-Met324 because the C terminus was
expected to have been converted to homoserine, and the theoretical mass
of such a peptide was calculated to be 5802.1. This result indicates
that CSPI*d was cleaved at Met324, suggesting that
Met324 was not the residue oxidized to Met(O). Although a
fragment with a molecular mass corresponding to
Ser325-Met349, which might contain Met(O) at
the position of Met327, was not detected in the spectrum
shown in Fig. 5, Met327 was concluded to be Met(O).
Observations supporting this conclusion were as follows:
Phe317-Arg332, which was derived from CSPI*d
by tryptic digestion, had two methionine residues, Met324
and Met327; the peptide
Phe317-Arg332 was larger by 16.1 Da than the
theoretical molecular mass (Table II); and Met324 was not
shown to be the residue oxidized to Met(O).
Taking into account all the data obtained in the present study on the
modification of the subunits of the cuticular proPO isoform (proPO-CS),
proPO-CS-pI can be said to have the same amino acid sequence as that of
proPO-HS-pI except that it has six Met(O)s at Met58,
Met73, Met176, Met324,
Met327, and Met498. Similarly, proPO-CS-pI* has
the same amino acid sequence as that of proPO-CS-pI except that its
Met324 is not Met(O), and proPO-CS-pII has the same amino
acid sequence as that of proPO-HS-pII except that it has a Met(O) at
Met690. The theoretical molecular masses of proPO-CS-pI,
proPO-CS-pI*, and proPO-CS-pII, in which acetylation of their N termini
and modification at the methionyl residues were taken into account, were calculated to be 78,791, 78,775, and 80,107 Da, respectively. The
observed molecular masses in MALDI-MS of the same polypeptides, proPO-CS-pI, proPO-CS-pI*, and proPO-CS-pII were 78,887, 78,861, and
80,190 Da, respectively. The observed molecular masses and theoretical
masses are in good agreement within the experimental errors in the
MALDI-MS (22).
Protein Modeling of the ProPO Subunits with the Sequences Deduced
from pPO-5 and pPO-23--
ProPO is a protein homologous to the
arthropod hemocyanin. Because the three-dimensional structure of proPO
has not been determined yet, model structures of the silkworm proPO
subunits were constructed by protein modeling in which the known
structures of the horse- shoe crab hemocyanin (Limulus) type
II subunit were employed as templates. When the entire sequences of
proPO-HS-pI and proPO-HS-pII were submitted for protein modeling, they
were rejected by the Swiss-Model Server (14, 15) because the sequences
of the N-terminal and C-terminal regions of the proPO subunits have low
identity to those of the horseshoe crab hemocyanin type II subunit.
Therefore, the Lue66-Asp675 deduced from pPO-5
and the Pro60-Gln678 deduced from pPO-23 were
submitted to the Server. The former and latter were calculated to have
sequence identities of 44 and 42%, respectively, to the sequence of
horseshoe crab hemocyanin type II subunit. The foldings of the models
shown in Fig. 6A were very
similar to those of the template structures (26-28). Two disulfide bridges, which had been observed in the template structures, were formed in each of the models (not shown). The models were used for
calculation of the surface accessibility of methionine side chains. The
results are summarized in Table
III and visually presented in Fig.
6B. Methionine residues exposed (surface accessibility > 50%) or partially exposed (surface accessibility > 20%) to
the surface are indicated in green in the figure. All of the
methionine residues detected as Met(O)s except for Met58 of
proPO-CS-pI and proPO-CS-pI* and Met690 of proPO-CS-pII are
located at the surface in the model with surface accessibility of more
than 20%. The surface accessibility of Met58 and that of
Met690 could not be determined in the present molecular
modeling.
Invertebrate epithelia have the septate junction that is
functionally equivalent to the tight junction of vertebrates. The septate junction is thought to limit the diffusion of molecules through
intercellular spaces in the epithelial layer (29, 30). The transcripts
of the proPO subunit genes have not been shown to be detected in
epidermal cells (11), but proPO has been detected immunocytochemically
in the epidermal cells of 5th instar silkworm larvae.2 Furthermore, proPO
has been shown to be transported from the hemolymph to the cuticle
(12). These results have indicated that hemolymph proPO is
transepithelially transported from the hemolymph to the cuticle.
The mechanisms of transepithelial or transendothelial transportation of
mammalian proteins such as polymeric immunoglobulin A and transferrin
have become clearer in recent years (6, 7, 8, 9, 10). Because of the
presence of the tight junction sealing extracellular space between the
epidermal cells, macromolecules such as proteins cannot pass freely
between mammalian epithelial cells. It is well documented that proteins
taken up into the epithelial cells by receptor-mediated endocytosis at
the basolateral surface are transported via cytoplasm to the apical
surface and secreted (6, 7). However, in insects, the mechanism by
which proteins are transepithelially transported has not been studied.
There is not even any concrete evidence that a protein is transported by transcytosis through insect epidermal cells. We considered the
silkworm proPO to be a molecule that would offer a rare opportunity to
study the mechanism of transepithelial protein transport in the insect.
Purified cuticular proPO had the same enzymatic properties as those of
hemolymph proPO when they were activated by a specific activating
protease, pro-phenoloxidase-activating enzyme (PPAE) (12). However,
subunits of cuticular proPO were not eluted at the same retention times
as those of hemolymph proPO subunits in RP-HPLC, indicating that
cuticular proPO is a modified form of hemolymph proPO. There are some
reports suggesting that hemolymph proteins are transepithelially
transported from hemolymph to the cuticle (4, 5). However, there have
been no detailed structural analyses of the transported proteins.
Elucidation of the nature of the modification of transepithelially
transported proteins would be the first step to understanding the
mechanisms of transepithelial transport of macromolecules from
hemolymph to the cuticle in insects.
In the hemolymph of the silkworm, B. mori, two proPO
isoforms (referred to as proPO-HS and proPO-HF) are present. Only
proPO-HS has been proved to be transported to the cuticle and become
proPO-CS, but it is almost certain that proPO-HF is also transported as proPO-HS (12). In the present study, modification occurring in proPO-CS
was analyzed. In RP-HPLC, proPO-CS was eluted in three peaks. The
polypeptides in the peaks were named proPO-CS-pI, proPO-CS-pI*, and
proPO-CS-pII. The polypeptides proPO-CS-pI and proPO-CS-pI* were
revealed to be modified forms of proPO-HS-pI (a subunit of proPO-HS),
and the former was revealed to have six methionine sulfoxides, Met(O)s,
at Met58, Met73, Met176,
Met324, Met327, and Met498, and the
latter was revealed to have five Met(O)s at Met58,
Met73, Met176, Met327, and
Met498. The polypeptide proPO-CS-pII was shown to be a
modified form of proPO-HS-pII (another subunit of proPO-HS) and to have
a Met(O) at Met690. Molecular mass data for the subunits of
proPO-CS, results of mass mapping of the peptides in protease digests
of the subunits, N-terminal amino acid sequences of the isolated
peptides in the digests, and the results of analyses of peptides
containing methionine sulfoxide(s) by PSD mass spectrometry were all
consistent with the contention that modification of the subunits of
proPO-CS involves only oxidation of the methionine residues detected in
the present study. In the case of F-type proPOs (proPO-HF and
proPO-CF), our preliminary analyses indicated that proPO-HF and
proPO-CF have the same amino acid sequences and that essentially the
same modifications as those observed in proPO-CS occur on
proPO-CF.3
Oxidation of methionyl residues in many proteins caused by atmospheric
oxygen or other reactive oxygen species has been reported (31, 32). It
was therefore speculated that the oxidized methionyl residues detected
in the cuticular proPO were artifactually introduced during the
purification process employed in the present study. However, the
purified or crude hemolymph proPO preparations could be stored for
several months without any appreciable change of their elution profiles
in RP-HPLC, indicating that the oxidation of methionyl residues
detected in cuticular proPO did not occur during storage. In another
experiment, an extraction buffer supplemented with hemolymph proPO was
used to extract cuticular proteins, and immune precipitate of proPOs in
the resulting extract was prepared. From the precipitate, a subunit
(proPO-HS-pI) of hemolymph proPO was recovered in a high
yield,3 indicating that the oxidation of methionine
residues of proPO does not take place during the extraction of
cuticular proteins. Considering these observations, cuticular proPO
isoforms are likely to exist in the oxidized form in situ
before extraction.
The molecular mass of purified native proPO-CS was estimated to be 142 kDa by permeation gel chromatography, and the same value was also
obtained for the molecular mass of proPO-HS. Because proPO-HS has been
shown to be composed of two different subunits, the molecular mass data
seem to indicate that proPO-CS is also a heterodimeric protein. Thus,
the purified proPO-CS was concluded to be a mixture of proPO-CS
variants, one of which is composed of proPO-CS-pI and proPO-CS-pII and
the other of which is composed of proPO-CS-pI* and proPO-CS-pII. We
have not succeeded in separating the variants by any purification
procedure under nondenaturing conditions.
The silkworm proPO is a protein homologous to arthropod hemocyanins.
Among the two types of arthropod hemocyanins of which the
crystallographic three-dimensional structures have been determined (26,
27, 28, 33), cheliceratan (Limulus) hemocyanin is closer to
the silkworm proPO in molecular phylogeny than is crustacean (Panulirus) hemocyanin (34). Three structures of
Limulus hemocyanin type II subunits have been reported. Two
of them were obtained from protein crystallized in buffer containing
0.5 M NaCl, and the other one was obtained from protein
crystallized in the presence of nitrate (26, 27, 28). They have been
shown to have essentially the same structures (27, 28). Protein
modeling of proPO subunits was carried out with these three
crystallographic structures as templates. The resulting model
structures were very similar to those of the templates (Fig. 6). From
the model structures, surface accessibilities of methionine residues
were calculated (Table III). Methionine residues identified to be
Met(O)s in cuticular proPO subunits were shown to have high surface
accessibilities in the model, with Met73 bearing the lowest
value, 32.5%, and Met176 bearing the highest value,
87.2%. Both Met73 and Met176 are the residues
of proPO-HS-pI. The surface accessibility of Met58 of
proPO-HS-pI and Met690 of proPO-HS-pII, which were both
identified as methionine sulfoxides in proPO-CS, could not be assessed
in the modeling because the regions containing these methionines were
not included in the sequences submitted as targets in the present
protein modeling. Although Met295 and Met456 of
proPO-HS-pII were shown to have relatively high surface accessibilities (40.1 and 48.4%, respectively) in the model, they were not detected as
oxidized methionine residues in proPO-CS-pII. This observation seems to
be explainable if it is assumed that the methionyl residues are located
at the surfaces where subunits of proPO interact and are therefore not
exposed to the solvent surrounding the proPO molecule. Methionine
residues in proteins are classified into three groups: exposed,
partially exposed, and buried (31). Oxidation of methionine is thought
to be restricted to the exposed or partially exposed residues (31).
Considering the previous report, the calculated high surface
accessibilities of the methionine residues detected as methionine
sulfoxides in proPO-CS suggest that the model structures obtained in
the present protein modeling reflect rather faithfully the real
structures of proPO subunits, especially that of proPO-HS-pI.
The oxidation of methionine residues of proteins sometimes induces
conformational change and causes loss of their activity (31, 32). In
the case of the silkworm proPO, such oxidative inactivation did not
seem to occur. We have observed (12) that PPAE purified from the
silkworm cuticle converted hemolymph proPO and cuticular proPO to
phenoloxidases (monophenol,
L-3,4-dihydroxyphenylalanine:oxygen oxidoreductase,
EC1.14.18.1) of which the enzymatic properties were very similar and
that the conversion catalyzed by PPAE did not appear to be influenced
by the oxidation of methionyl residues. The only conspicuous effect of
oxidation detected was on the transport of proPO from hemolymph to the
cuticle; cuticular proPO-CS injected into the hemocoel was not
transported to the cuticle, whereas the injected purified proPO-HS was
transported to the cuticle (12). The most plausible model for proPO
transport may be receptor-mediated transcellular transport as has been
observed in mammals (35). It is possible that the putative receptor for
proPO does not have appreciable affinity to the oxidized proPO isolated
from cuticles. Methionyl residues of some proteins have been shown to
participate in molecular interaction (36-44). Oxidation of methionyl
residues has been observed to have a crucial effect on interaction
between molecules without causing large conformational changes of the proteins (42-44). The reduced hydrophobicity at the oxidized methionyl residues of cuticular proPO may lower the binding ability of molecules to the putative proPO receptors on epidermal cells. Such lowered affinity of the cuticular proPO for the putative receptor may imply
that epidermal cells employ oxidation of the methionine residues to
facilitate the secretion of proPO, which is destined to be
transepithelially transported from the hemolymph to the cuticle. It is
also possible that the lowered affinity plays a role in preventing the
cuticular proPO from being taken up from the cuticle into epidermal
cells. To the best of our knowledge, no structural studies have been
carried out on insect proteins that have been shown to be transported
from the hemolymph to the cuticle. Accordingly, it is not known whether
methionyl residues of other transepithelially transported proteins are
also oxidized.
Since the first cDNA cloning of insect proPOs in 1995 (11, 45, 46),
the number of reports on cloned proPO cDNAs in literature has been
increasing. A phylogenetic tree of proPOs of insects was constructed
from the reported sequences (Fig.
7A). Insect proPOs were
grouped in two clusters. The subunits proPO-HS-I and proPO-HS-II
separately belong to each cluster. ProPO subunits belonging to the
cluster in which proPO-HS-I is located have often been referred to as
type I proPO subunits, and those belonging to the cluster in which
proPO-HS-pII is located have often been referred to as type II proPO
subunits. In the alignments of sequences around oxidized methionines of
Bombyx proPO subunits, the methionine residue corresponding
to Met73 of proPO-HS-pI was found in all type I subunits
except for a coleopteran proPO subunit. Next to Met73,
Met176 is well conserved among the type I proPO subunits
with the exceptions of the subunits of coleopteran proPO and
Drosophila proPO-1b (Fig. 7). With regard to
Met690 of proPO-HS-pII, none of the type II proPO subunits
of other insects were found to have a methionine residue at the
position corresponding to Met690. These alignment data seem
to raise the possibility that the methionine residue corresponding to
Met73 or Met176 of proPO-HS-pI plays a role in
the transport of proPOs from hemolymph to the cuticle in insects. The
coleopteran proPO subunit, which does not have a methionine residue
corresponding to Met73 or Met176 of
proPO-HS-pI, cannot rule out this possibility because all of the proPOs
so far characterized at protein level have been shown to be composed of
more than two subunits. There has been no report of a partner of the
coleopteran proPO subunit to form a complete proPO molecule, and it is
not known whether coleopteran proPO containing the subunit is
transported to the cuticle. In a dipteran species, Anopheles
gambiae, as many as six genes coding for proPO subunits are known
to be present, but there has been no examination of whether the gene
products are transported to the cuticle. Further investigation is
needed to determine whether particular proPOs in other insects are
transported from hemolymph to the cuticle and whether the transported
proPO subunints have conserved methionine at the position corresponding
to Met73 or Met176 of proPO-HS-pI. More direct
evidence of the roles of these two methionines of proPO-HS-pI in the
transport of proPO subunits could be obtained if mutagenized proPO
molecules at the methionine residues were made. However, we have not so
far succeeded in the synthesis of such mutagenized molecules.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C
until use. In the accompanying paper (12), the amino acid sequences of
proPO-HS-pI and proPO-HS-pII deduced from the base sequences of
cDNA clones, pPO-5 (13) and pPO-23, respectively, are presented.
20 °C until use.
20 °C until use.
20 °C until use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Peptide mapping of the Lys-C digests of the
subunits of proPO isoforms from the cuticle and hemolymph. After,
S-pyridylethylation, each subunit was digested with Lys-C.
The digests were analyzed using the RP-HPLC with an ODS column.
A, panels a, b, and c show
elution profiles of the digests of proPO-CS-pI, proPO-CS-pI* and
proPO-HS-pI, respectively. B, panels d, and
e show elution profiles of the digests of proPO-CS-pII and
proPO-HS-pII, respectively. Details of the experiments are given under
"Experimental Procedures."
N-terminal sequences and molecular masses of some fragments in the
lysylendopeptidase digests of proPO subunits
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Fig. 2.
Assignments of Lys-C-digested fragments with
oxidized methionine to the amino acid sequences of the subunits of a
silkworm proPO isoform (proPO-CS), and comparison of amino acid
sequences of cheliceratan hemocyanin and the subunits of the
isoform. Sequences of whole proteins were aligned by the Clustal W
method (47). As is stated in the text, proPO-HS-pI, proPO-CS-pI, and
proPO-CS-pI* have the same amino acid sequences, and proPO-HS-pII and
proPO-CS-pII also have the same sequences. The aligned sequences except
for that of Limulus hemocyanin were obtained from the DNA
Data Bank of Japan. The sequence of Limulus
hemocyanin type II subunit was obtained from Protein Data Bank. Their
accession numbers are as follows: Limulus hemocyanin type II
subunit (Limulus type II), 1LLA; proPO-HS-pI, D49370;
proPO-HS-pII, AB048761. Residues identical to those of
Limulus hemocyanin in the aligned sequences are
shaded. Four conserved cysteines are boxed.
Disulfide bonds indicated by dashed lines are those found in
Limulus hemocyanin. Gaps are indicated by
hyphens. The vertical arrow indicates the
cleavage site in the activation of the silkworm proPO by PPAE.
Asterisks show histidine residues that have been shown to
form copper binding sites in Limulus hemocyanin type II
subunit. Of the Lys-C-digested fragments of proPO-HS-pI, proPO-HS-pII,
proPO-CS-pI, proPO-CS-pI*, and proPO-CS-pII, the sequences of the
fragments listed in Table I are indicated with highlighted
letters, and the names of the fragments are indicated above the
aligned sequences. Among the tryptic fragments listed in Table II,
those with the sequences containing the methionine residue(s) to be
oxidized to methionine sulfoxide(s) in cuticular proPO-CS are also
indicated by horizontal bars with an arrowhead at
each end above the aligned sequences. Open triangles with
Roman numerals indicate methionine residues and residue
numbers that were determined to be oxidized methionine residues in
proPO-CS-pI. In proPO-CS-pI*, the same methionine residues as those in
proPO-CS-pI were detected as oxidized methionine residues except for
Met327. The closed triangle with a Roman
numeral indicates Met 690 that was detected as a
residue oxidized to methionine sulfoxide in the sequence of
proPO-CS-pII.
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Fig. 3.
MALDI mass spectra of tryptic digests of
CSPIa, CSPI*a, and HSPIa (A) and PSD mass spectrum of
a fragment in the tryptic digest of CSPIa (B).
A, CSPIa, CSPI*a, and HSPIa were obtained as described in
the legend of Fig. 1A and were secondly digested with
trypsin. The tryptic digests were subjected to MALDI- MS in a linear
mode. The spectra of the digests derived from CSPIa, CSPI*a, and HSPIa
are shown in panels a, b, and c,
respectively. B, a precursor ion at
m/z 2165.39 indicated by an arrow in panel
a in A was analyzed by the PSD method, and the
resultant PSD spectrum is shown in panel d. In panel
e, the possible fragmentation of the Met(O) at the N terminus of
Met176-Arg194 in the mass spectrometry is
depicted together with the molecular mass of the resultant
fragments.
Summary of peptide mass mapping analysis of tryptic digests
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Fig. 4.
MALDI mass spectra of CNBr fragments of the
Lys-C fragments, CSPIc (Leu419-Lys523) and
CSPI*d (Leu275-Lys416), from proPO-CS-pI and
proPO-CS-pI*. A, Lys-C fragments, CSPIc and CSPI*d,
obtained as described in the legend of Fig. 1A were treated
with CNBr and subjected to MALDI-MS in a linear mode. Panel
a, spectrum of the digest of CSPIc. Panel b, spectrum
of the digest of CSPI*d. B, schematic representation of
postulated CNBr fragments to be produced from CSPIc or CSPI*d. The
cleavage with CNBr is expected to be blocked at Met(O)s. Thus, the
fragmentation pattern by CNBr depends on the site of the oxidized Met
in the fragments. Panel c, fragmentation of CSPI*c with
Met(O) 498. Panel d, fragmentation of CSPIc with
Met(O) 500. Panel e, fragmentation of CSPI*d
with Met(O) 324. Panel f, fragmentation of
CSPI*d with Met(O) 327. The equilibrium between homoserine
and homoserine lactone shifts depending on the acidity of the medium.
Therefore, the methionine residue that had been located at the C
terminus of the peptide bond cleaved by CNBr should be detected as
homoserine or homoserinelactone under the present experimental
conditions. The molecular masses of the postulated fragments whose C
termini are homoserine, homoserine lactone, and other amino acids are
indicated in parentheses, brackets, and
angle brackets, respectively, in the schemes.
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Fig. 5.
MALDI mass spectrum of the tryptic digest of
CSPId (Leu275-Lys416) and PSD spectrum of the
fragment (Phe317-Arg332, in the tryptic
digest. Inset, CSPId isolated as described in the
legend of Fig. 1A was digested with trypsin and subjected to
MALDI-MS in a linear mode. The molecular mass
(m/z 1853.58) of the peak indicated by an
arrow corresponds to a doubly oxygenated form of
Phe317-Arg332 of which the theoretical
molecular mass is 1820.2 Da. Other than the peak indicated by the
arrow, all the major peaks in the figure are assigned to one
of the peptides to be produced by tryptic digestion of the polypeptide
with the sequence of CSPId. The PSD spectrum of the peptide in the peak
(indicated by an arrow in the inset) at
m/z 1853.58 was examined. In the spectrum, the
fragments about 64 and 128 Da smaller than the precursor ion are
detected at m/z 1789.24 and 1725.71, indicating
the presence of two Met(O)s in the precursor ion.
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Fig. 6.
Protein modeling of proPO subunits. In
the modeling, Limulus hemocyanin type II subunit was used as
a template. Models of the proPO subunits with the sequences of
proPO-HS-pI and proPO-HS-pII were obtained by Swiss-Model Server as
described under "Experimental Procedures." A, ribbon
representation of a model of proPO-HS-pI (panel a) and a
model of proPO-HS-pII (panel b). Helixes and
-strands are represented in red and in blue,
respectively. B, space filling representation of the models
in A. Panels c and e, models of
proPO-HS-pI. Panels d and f, models of
proPO-HS-pII. Panels c and d, models viewed from
the same directions as the corresponding models are viewed in
A. Panels e and f, models viewed from
the opposite directions in which panels c and e,
respectively, are viewed. Methionine residues exposed or
partially exposed to the surfaces of the models (with surface
accessibility of greater than 20%) are colored in green,
and their residue numbers are given in black. Visualization
was performed by using WebLab Viewer Lite, version 3.20 (Molecular
Simulations, Inc., San, Diego, CA).
Surface accessibilities of methionyl residues in model structures of
proPO subunits
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
A phylogenetic tree of proPO subunits
of insects (A) and alignment of the sequences of
fragments with oxidized methionine residue(s) in the silkworm cuticular
proPO with the corresponding sequences of other insect proPOs
(B). A, all the sequences of proPO
subunits presently available in data bases were analyzed by the
Nearest-Joining method (48) to construct the phylogenetic tree.
ProPO-HS-pI and proPO-HS-pII are indicated by underlining.
B, sequences around methionines of proPO-HS-pI to be
oxidized to Met(O) in proPO-CS-pI were aligned with the corresponding
regions of other insect proPOs that belong to the cluster in the
phylogenetic tree. The methionine residue (Met690) of
proPO-HS-pII that is oxidized to Met(O) in proPO-CS-pII was found not
to be conserved at all in other insect proPOs. Therefore, the alignment
of the sequence around Met690 is not presented here. The
methionines identified as Met(O) in proPO-CS-pI are indicated with the
residue numbers. The conserved methionine residues among the proPO
subunits are presented with white letters on a black
background. The sequences extracted from data bases and their
accession numbers are as follows (in cases in which a data base other
than DNA Data Bank of Japan is used, the name of the data
base is given in parentheses): Drosophila melanogaster proPO
1a, CG2952 (fly base); Sarcophaga bullata proPO, AF161260;
A. gambiae proPO 1, AF031626; D. melanogaster
proPO 1b, CG8193 (fly base); Tenebrio molitor proPO,
AB020738; B. mori proPO-HS-pI, D49370; Manduca
sexta proPO 1, AF003253; Hyphantia cunea proPO
1, U86875; A. gambiae proPO 4, AJ010193; A. gambiae proPO 6, AJ010195; Anopheles stephensi proPO 1, AF062034; A. gambiae proPO 2, AF004915; A. gambiae proPO 3, AF004916; Armigeres subalbatus,
AF260567; A. gambiae proPO 5, AJ010194; D. melanogaster proPO A1, D45835; S. bullata proPO 2, AF161261; B. mori proPO-HS-pII, AB048761; M. sexta proPO 2, L42556; and H. cunea proPO 2, AF020391.
As stated above, the oxidized methionyl residues in cuticular proPO do not seem to have been artifactually introduced during the process of extraction and purification of the protein. Elucidation of the site where the oxidation takes place should contribute greatly to an understanding of the mechanisms of the transepithelial transport of hemolymph proPO. There are two possible sites for oxidation: cuticle and epithelial cells. We are currently trying to raise an antibody cross-reactive to hemolymph proPO but not to cuticular proPO by using peptides with sequences containing methionines identified as Met(O) in cuticular proPO. The use of such an antibody and an immunocytochemical technique would enable identification of the site where methionine residues are oxidized. All of the methionyl residues exposed to the surface of proPO appear to be oxidized. Therefore, there does not seem to be a mechanism by which specific methionyl residues are oxidized. It is certain that hemolymph proPO is subjected to an oxidative environment during the translocation from hemolymph to the cuticle.
To the best of our knowledge, this is the first report of a high
content of methionine sulfoxide in cuticular proteins. This may be
partly because methionyl residues oxidized to sulfoxide are detected as
methionines in Edman degradation (25). Although the general
physiological significance of the methionine residues oxidized to
Met(O) in cuticular proteins remains to be determined, the implication
of oxidation in the transepithelial transport of hemolymph proPO to the
cuticle is worthy of further study.
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ACKNOWLEDGEMENTS |
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We are grateful to Takashi Ozawa for analyses of the amino acid sequences of cuticular proPO isoforms at the beginning of the present study and to Kenji Watanabe for analysis of molecular masses of the peptides by electrospray ionization mass spectrometer.
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FOOTNOTES |
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* 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 14, 2000, DOI 10.1074/jbc.M008425200
2 M. Sass, T. Asano, and M. Ashida, unpublished observation.
3 T. Asano and M. Ashida, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: proPO, pro-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; Met(O), methionine sulfoxide; PPAE, pro-phenoloxidase-activating enzyme; Lys-C, lysylendopeptidase; PAGE, polyacrylamide gel electrophoresis; RP, reversed-phase; HPLC, high performance liquid chromatography; ODS, octadecyl; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; PSD, post-source decay.
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REFERENCES |
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1. | Wigglesworth, V. B. (1972) The Principles of Insect Physiology , 7th Ed. , pp. 25-53, Chapman and Hall, London |
2. | Andersen, S. O., Hojrup, P., and Roepstorff, P. (1995) Insect Biochem. Mol. Biol. 25, 153-176[CrossRef][Medline] [Order article via Infotrieve] |
3. | Ashida, M., and Brey, P. (1997) in Molecular Mechanisms of Immune Responses in Insects (Brey, P. T. , and Hultmark, D., eds) , pp. 135-172, Chapman & Hall, London |
4. | Sass, M., Kiss, A., and Locke, M. (1994) J. Insect Physiol. 40, 407-421 |
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Csikós, G.,
Molnár, K.,
Borhegyi, N. H.,
Talián, G. C.,
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