From the Department of Cell Biology, National
Institute for Basic Biology, Okazaki 444, Japan and the
§ Department of Molecular Biomechanics, School of Life
Science, The Graduate University for Advanced Studies,
Okazaki 444, Japan
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ABSTRACT |
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Precursor-accumulating vesicles mediate transport
of the precursors of seed proteins to protein storage vacuoles in
maturing pumpkin seeds. We isolated the precursor-accumulating vesicles and characterized a 100-kDa component (PV100) of the vesicles. Isolated
cDNA for PV100 encoded a 97,310-Da protein that was composed of a
hydrophobic signal peptide and the following three domains: an 11-kDa
Cys-rich domain with four CXXXC motifs, a 34-kDa
Arg/Glu-rich domain composed of six homologous repeats, and a 50-kDa
vicilin-like domain. Both immunocytochemistry and immunoblots with
anti-PV100 antibodies showed that <10-kDa proteins and the 50-kDa
vicilin-like protein were accumulated in the vacuoles. To identify the
mature proteins derived from PV100, soluble proteins of the vacuoles were separated, and their molecular structures were determined. Mass
spectrometry and peptide sequencing showed that two Cys-rich peptides,
three Arg/Glu-rich peptides, and the vicilin-like protein were produced
by cleaving Asn-Gln bonds of PV100 and that all of these proteins had a
pyroglutamate at their NH2 termini. To clarify the cleavage
mechanism, in vitro processing of PV100 was performed with
purified vacuolar processing enzyme (VPE). Taken together, these
results suggested that VPE was responsible for cleaving Asn-Gln bonds
of a single precursor, PV100, to produce multiple seed proteins. It is
likely that the Asn-Gln stretches not only provide cleavage sites for
VPE but also produce aminopeptidase-resistant proteins. We also found
that the Cys-rich peptide functions as a trypsin inhibitor. Our
findings suggested that PV100 is converted into different functional
proteins, such as a proteinase inhibitor and a storage protein, in the
vacuoles of seed cells.
In higher plants, proprotein precursors of most seed proteins are
synthesized on the rough endoplasmic reticulum and are then transported
to protein storage vacuoles in maturing seed cells (1-3). We have
shown that the vesicles with a density of 1.24 g/cm3
mediate the delivery of proprotein precursors of seed proteins to the
vacuoles (4-6). We have succeeded in isolation of the vesicles from
maturing pumpkin seeds and have shown that they contained a large
amount of precursors of various seed proteins, including 11S globulin
and 2S albumin (7). Thus, these vesicles were designated
precursor-accumulating (PAC)1
vesicles. Recently, we have found that the PAC vesicles contain a type
I integral membrane protein with epidermal growth factor-like motifs
and have shown that the membrane protein binds to peptides derived from
the 2S albumin precursor (8). The membrane protein of the PAC vesicles
might function as a sorting receptor for seed protein precursors to the vacuoles.
Just after arriving at the vacuoles, the precursor proteins are
converted into their respective mature forms by proteolytic cleavages
(6, 9). The posttranslational cleavages occur at the carbonyl sides of
Asn residues in precursors of various seed proteins of different
plants, including storage proteins, lectins, and toxins, as reviewed by
Hara-Nishimura et al. (10). We have found an enzyme
responsible for maturation of these seed proteins and have designated
it vacuolar processing enzyme (VPE) (11, 12). VPE recognizes exposed
Asn residues on the molecular surface of the precursor proteins and
then cleaves the peptide bonds at the carbonyl sides of the Asn
residues (6). The VPE-mediated processing system plays a crucial role
in maturation of various seed proteins in protein storage vacuoles.
Our previous study showed that VPE homologs can be separated into two
subfamilies: one specific to seeds and the other specific to vegetative
organs (13, 14). This is consistent with the fact that the plant
vacuoles are classified into two types, protein storage vacuoles in
seeds and lytic vacuoles in vegetative organs. A VPE-mediated
processing system similar to that in protein storage vacuoles is
involved in maturation of vacuolar proteins in lytic vacuoles (15).
Vegetative VPE might be responsible for conversion of inactive
precursor into their mature active proteins, such as proteinase
inhibitors and hydrolytic enzymes (15). Similarly, it is likely that
seed VPE also plays a role in the activation of some functional
proteins in seeds.
Each of most precursor proteins is composed of a functional domain and
an NH2- and/or COOH-terminal propeptide(s), except for a
precursor protein of proteinase inhibitors of tobacco that is processed
into five homologous inhibitors and an NH2-terminal propeptide (16). On the other hand, it is not known whether multiple
vacuolar proteins with distinct functions are derived from a single
precursor. In this study, we demonstrated that a 100-kDa component of
the PAC vesicles (PV100) is converted into multiple proteins with a
pyroglutamate at their NH2 termini by cleaving Asn-Gln
bonds of PV100 by the action of VPE, after arrival at protein storage
vacuoles. We show here a unique mechanism for vacuolar processing at
Asn-Gln cassettes in the precursor sequence to produce
aminopeptidase-resistant proteins in the plant vacuoles.
Plant Materials--
Pumpkin (Cucurbita maxima cv.
Kurokawa Amakuri Nankin) seeds were purchased from Aisan Shubyo Seed
Co. (Nagoya, Japan). For isolation of PAC vesicles and
immunocytochemical analysis, pumpkin seeds were planted in the farm of
Nagoya University during the summer season, and cotyledons of the
maturing seeds, freshly harvested 22-28 days after pollination, were collected.
Isolation of PAC Vesicles--
PAC vesicles were isolated from
pumpkin cotyledons at the middle stage of seed maturation essentially
as described previously (7). The cotyledons (15 g) were homogenized in
a solution (7 ml/g fresh weight of cotyledons) of 20 mM
sodium pyrophosphate (pH 7.5), 1 mM EDTA, and 0.3 M mannitol with an ice-chilled mortar and pestle, and the
homogenate was filtered through cheesecloth. The filtrate was
centrifuged at 3000 × g for 15 min and the supernatant was centrifuged again at 8000 × g for 20 min at
4 °C. The pellet was suspended in 1 ml of 10 mM
Hepes-KOH (pH 7.2), 1 mM EDTA, and 0.3 M
mannitol. The suspension was layered on a solution of 28% Percoll
(Amersham Pharmacia Biotech) in 10 mM Hepes-KOH (pH 7.2), 1 mM EDTA, and 0.3 M mannitol on a cushion of 2 ml of 90% Percoll. Centrifugation was at 40,000 × g
for 30 min at 4 °C. The vesicle fraction was centrifuged again in a
self-generated Percoll gradient. The resulting vesicle fraction was
washed in the above-described Hepes-KOH buffer and used for
immunoelectron microscopy and immunoblot analysis.
Isolation of Protein Storage Vacuoles and Purification of
Proteins Derived from PV100--
Protein storage vacuoles (protein
bodies) were isolated from dry pumpkin seeds (50 g) by a nonaqueous
isolation method, as described previously (17). Isolated protein
storage vacuoles were burst in 100 ml of 10 mM Tris-MES (pH
6.5), 0.1 M sucrose, 1 mM EDTA; sonicated; and
then centrifuged at 100,000 × g for 1 h at
4 °C to remove insoluble proteins and membranes as a pellet, as
described previously (18). Ammonium sulfate was added to the
supernatant solution to a concentration of 30% saturation. The mixture
was incubated for 1 h at 5 °C and then centrifuged at
200,000 × g for 15 min. The ammonium sulfate
concentration of the supernatant was then increased to 100%
saturation, and the incubation and centrifugation steps were repeated.
The precipitate was suspended in 4 ml of a solution of 25 mM sodium acetate (pH 5.5) and 5 mM EDTA and
then applied to an Econo-PacI0 DG column (Bio-Rad) to remove
ammonium sulfate. The preparation was used as the matrix fraction of
the protein storage vacuoles.
The matrix fraction was found to contain a large amount of the 4-6-kDa
proteins and the 50-kDa protein that were derived from PV100 by
posttranslational cleavage. To purify the 4-6-kDa proteins, the matrix
fraction was applied to either a reverse phase column (µRPC C2/C18 PC
3.2/3) on a SMART system (Amersham Pharmacia Biotech) or a reverse
phase column (µRPC C2/C18 ST 4.6/100) on an ÄKTA system
(Amersham Pharmacia Biotech). Elution was carried out with a gradient
starting from 0.065% trifluoroacetic acid in distilled water to 0.05%
trifluoroacetic acid in acetonitrile, at a flow rate of 200 µl/min
for SMART and at a rate of 500 µl/min for ÄKTA. Each fraction
was subjected to immunoblot analysis, mass spectrometry, and digestion
by pyroglutamate aminopeptidase followed by automatic Edman
degradation, as described below, to determine the molecular structures
of these proteins. We also measured trypsin inhibitory activity in the
fractions to demonstrate the function of the PV100-derived small proteins.
Determination of NH2-terminal and Internal Amino Acid
Sequences--
The PAC vesicles and the matrix fraction of the
protein storage vacuoles were subjected to SDS-PAGE, and then the
separated proteins were transferred electrophoretically to an
Immobilon-P membrane (0.22 mm) (Nihon Millipore Ltd., Tokyo, Japan).
After staining of proteins on the blot with Coomassie Blue, the band corresponding to either PV100 or the 50-kDa protein was cut out from
the blot and subjected to automatic Edman degradation on a peptide
sequencer (model 492, Applied Biosystems Inc.).
To determine the internal sequence of PV100, the separated proteins of
the PAC vesicles were stained with Coomassie Blue, and the band
corresponding to PV100 on the SDS gels was cut out from the gel, as
described previously (19). The gel piece was incubated with 5 µg of
V8 protease (Sigma) by the method of Cleveland et al. (20).
After Tricine-SDS-PAGE (21), the separated peptides were transferred to
an Immobilon-P membrane and subjected to automatic Edman degradation.
Digestion with Pyroglutamate Aminopeptidase Followed by Edman
Degradation--
Each 3 µg of the purified 4-6-kDa proteins of the
protein storage vacuoles was digested with 0.3 µg of pyroglutamate
aminopeptidase (Boehringer Mannheim) in 20 µl of solution of 0.1 M sodium phosphate (pH 8.0), 5% glycerol, 5 mM
dithiothreitol, and 1 mM EDTA for 6 h at 50 °C. The
digests were directly subjected to automatic Edman degradation.
Purification of VPE--
Protein storage vacuoles (protein
bodies) were isolated from castor bean endosperm by a nonaqueous
isolation method, as described above. VPE was purified from the soluble
fraction of the protein storage vacuoles by using ammonium sulfate
precipitation and Con-A Sepharose and MonoS column chromatographies, as
described previously (11).
In Vitro Processing by VPE--
The PAC vesicles (60 µg of
proteins) were incubated with the purified VPE in a solution of 50 mM sodium acetate buffer (pH 5.5) and 50 mM
dithiothreitol for 15 h at 37 °C. The reaction was subjected to
SDS-PAGE and the separated proteins on the gels were transferred
electrophoretically to a polyvinylidene difluoride membrane (0.22 µm)
(Nihon Millipore Ltd.). The band corresponding to the 50-kDa protein
was cut out from the blot to determine the NH2-terminal
sequence. The membrane piece was incubated with 0.5% polyvinylpyrrolidone for 30 min at 37 °C, followed by digestion by
2.5 µg of pyroglutamate aminopeptidase (Boehringer Mannheim) in 40 µl of solution of 0.1 M sodium phosphate (pH 8.0), 5%
glycerol, 5 mM dithiothreitol, and 10 mM EDTA
at room temperature for 18 h. After the removal of a pyroglutamate
at the NH2 terminus, the 50-kDa protein on the membrane
piece was subjected to automatic Edman degradation on a peptide
sequencer (model 492, Applied Biosystems Inc.).
Mass Spectrometry--
To determine the exact molecular mass of
the 4-6-kDa proteins of the protein storage vacuoles, each fraction
that was separated on a SMART system as described above was applied to
an API 300 triple quadrupole mass spectrometer (PE SCIEX, Foster City,
CA) in positive ion detection mode, equipped with ion spray interface. Samples were dissolved in 0.1% formic acid and 50% acetonitrile and
then delivered at 3 µl/min. The sprayer was held at a potential of
4.5 kV. Orifice potential was maintained at 25 V.
Assay of Trypsin Inhibitory Activity--
Trypsin inhibitory
activity was assayed essentially as described by Cechova (22).
Isolation of cDNA for PV100 and Determination of Nucleotide
Sequence--
A cDNA library in pBluescript II SK+ (Stratagene, La
Jolla, CA) was constructed with the poly(A)+ RNA from
maturing pumpkin cotyledons, as described previously (23). Four
degenerate primers, 1F
(5'-GG(A/C/G/T)GC(A/C/G/T)GG(A/C/G/T)GT(A/C/G/T)GA(C/T)CA-3'), 2F
(5'-CA(C/T)GA(C/T)GG(A/C/G/T)TG(C/T)GT(A/C/G/T)-3'), 3R
(5'-GG(A/G/T)AT(A/C/G/T)GTCAT(A/C/G/T)AC(A/G)TC-3'), and 4R
(5'-TA(G/A)TC(T/C)TT(G/A)AA(T/C)TC(A/C/G/T)CC)-3'), were designed on
the basis of the NH2-terminal and internal amino acid sequences of PV100 and synthesized on a DNA synthesizer (model 394, Applied Biosystems Inc., Foster City, CA). Polymerase chain reaction
was performed using a set of the 1F and 4R primers and the cDNA
library as a template. A 1454-bp DNA was amplified. A second polymerase
chain reaction using a set of 2F and 3R primers and the 1454-bp DNA was
performed to amplify a 1340-bp DNA. The 1340-bp DNA was inserted into
the T-vector to confirm the nucleotide sequence. The 1340-bp DNA was
labeled with [
DNA sequencing was performed with a DNA sequencer (model 377, Applied
Biosystems Inc.) and Preparation of Specific Antisera--
The isolated PAC vesicles
were subjected to SDS-PAGE on a 12.5% polyacrylamide gel with
subsequent staining with Coomassie Blue. The band corresponding to the
PV100 protein with a molecular mass of 100 kDa was cut out from the gel
and gently shaken in phosphate-buffered saline for several hours. The
gel was emulsified with complete Freund's adjuvant and injected
subcutaneously into a rabbit. After 3 weeks, two booster injections
with incomplete adjuvant were given at 7-day intervals. One week after
the booster injections, blood was drawn, and the antiserum was prepared.
Immunoblot Analysis--
Both the PAC vesicles and protein
storage vacuoles were subjected to SDS-PAGE followed by either
Coomassie Blue staining or immunoblotting. The purified 4-6-kDa
proteins of the protein storage vacuoles were also subjected to
immunoblot analysis. The immunoblot was performed essentially as
described previously (18). The separated proteins on gels
were transferred electrophoretically to a polyvinylidene difluoride
membrane (0.22 µm) (Nihon Millipore Ltd., Tokyo, Japan). The membrane
blot was incubated overnight with anti-PV100 antibodies that were
diluted 2000-fold in a solution of 50 mM Tris-HCl (pH 7.5),
0.15 M NaCl, 0.05% (v/v) Tween 20, and 3% (w/v) skim
milk. Alkaline phosphatase-conjugated antibodies (Cappel, West Chester,
PA) and horseradish peroxidase-conjugated antibodies (Amersham
Pharmacia Biotech) that were raised in goat against rabbit IgG were
diluted 2000-fold and used as second antibodies.
Immunoelectron Microscopy--
Maturing pumpkin seeds were
freshly harvested. The cotyledons were vacuum-infiltrated for 1 h
with a fixative that consisted of 4% paraformaldehyde, 1%
glutaraldehyde, and 0.06 M sucrose in 0.05 M
cacodylate buffer (pH 7.4). The tissues were then cut into slices of
less than 1 mm in thickness with a razor blade and treated for another
2 h with freshly prepared fixative. The isolated PAC vesicles were
fixed in 4% paraformaldehyde, 1% glutaraldehyde, 0.3 M
mannitol, 1 mM EDTA, and 10 mM Hepes-KOH (pH
7.2) for 1 h at 4 °C. The samples were dehydrated in a graded
dimethylformamide series at PV100 Is a 100-kDa Protein Component of PAC Vesicles--
We have
shown that the PAC vesicles are responsible for the intracellular
transport of precursors of major seed proteins, including 11S globulin
and 2S albumin, to protein storage vacuoles in maturing pumpkin seeds
(4, 6-8). The PAC vesicles were highly purified from cotyledons of
maturing pumpkin seeds. Electron microscopy revealed that each PAC
vesicle contained an electron-dense core with a diameter of 300-500 nm
and that the isolated vesicles were barely contaminated by other
cellular components (Fig. 1B). Fig. 1A (lane 1) shows the protein components of
the vesicles that were separated on an SDS-gel with Coomassie Blue
staining. Three major proteins were found in the PAC vesicle fraction.
Two of them have been shown to correspond to proglobulin, a proprotein precursor of 11S globulin (4), and to pro2S albumin, a proprotein precursor of 2S albumin (6), as indicated by pG and
p2S in Fig. 1A (lane 1), respectively.
The third component of the PAC vesicles, with a molecular mass of 100 kDa, was designated PV100.
For immunochemical characterization of PV100, polyclonal antibodies
were raised against the PV100 protein. An immunoblot of the PAC
vesicles with the anti-PV100 antibodies showed that the antibodies
specifically recognized PV100 on the blot, as shown in Fig.
1A (lane 2). Immunoelectron microscopy of the
isolated PAC vesicles with the anti-PV100 antibodies shows that gold
particles are inside the PAC vesicles (Fig. 1B). These
results indicate that PV100 is localized in the PAC vesicles together
with proproteins of the major seed proteins, 11S globulin and 2S albumin.
PV100 Is Composed of Three Domains--
The next issue to be
solved was the molecular structure of PV100 to clarify the manner of
posttranslational cleavage. We determined the amino acid sequences of
the NH2 terminus and two internal fragments of PV100 and
then isolated a cDNA with a 4.3-kb insert from the library of
maturing pumpkin seeds based on the amino acid sequences. The cDNA
encoded a 97,310-Da protein of 810 amino acids, as shown in Fig.
2. The determined
NH2-terminal amino acid sequence,
DKGESLSSGAGVDHDGCVNRCEELKGXNVDEFAA (X, not
determined), and the two internal sequences,
XYNVESGDVMTIPAGTTLYLANQEN and DLQIVKLVQPVNNPGEFKDY, were
found in the deduced sequence (Fig. 2, double-underlined
sequences), indicating that this clone was a cDNA for PV100. The
deduced primary structure of the PV100 protein was composed of a
hydrophobic signal peptide followed by the PV100 sequence. The
NH2-terminal sequence of PV100 revealed that the signal
peptide is cleaved off co-translationally on the carbonyl side of
Gly27, as indicated in Fig. 2 (open triangle).
The cleavage site was consistent with that predicted by application of
the rules of Von Heijne (27).
The PV100 sequence was composed of three domains: an 11-kDa Cys-rich
domain, a 34-kDa Arg/Glu-rich domain, and a 50-kDa vicilin-like domain,
as shown in Fig. 2. The Cys-rich domain contained four CXXXC
motifs (Fig. 2, boxes) of two Cys residues separated by three other amino acids. The 50-kDa domain (Fig. 2, light
shading) exhibited a 30-35% identity in amino acids to the
vicilin homologs, pea vicilin (28), soybean
The Arg/Glu-rich domain of PV100 is composed of 37 mol % Arg and 27 mol % Glu, as shown in Fig. 2 (large box). It should be noted that this domain is unique to pumpkin PV100. A homology plot of
PV100 shows that six homologous repeats are found in the Arg/Glu-rich
domain, as shown in Fig. 3A.
An alignment revealed that the six homologous repeats were separated by
Asn-Gln (Glu) sequences (Fig. 3B). The six repeats rich in
Arg and Glu were designated RE1-RE6 in order from the NH2
terminus. They show a sequence homology to pumpkin basic peptide of 36 amino acids that was isolated from pumpkin (C. maxima cv.
Mexican-papitas) seeds (33). In particular, the 36-amino acid form of
RE3 that was found in the seeds could be identical to the pumpkin
basic peptide (Fig. 3C, discussed below).
Multiple Seed Proteins That Are Derived from PV100 Are Accumulated
in Protein Storage Vacuoles--
Our previous works have shown that
both proglobulin and pro2S albumin are transported from the PAC
vesicles to the protein storage vacuoles and then are converted into
their respective mature forms (4, 6). This raises the question of
whether PV100 is also incorporated into the protein storage vacuoles. Immunocytochemistry of the maturing pumpkin seeds with the anti-PV100 antibodies answered this question. Electron-dense PAC vesicles with
diameters of 300-500 nm and protein storage vacuoles composed of
crystalloids of 11S globulin and the matrix were observed in the cells,
as shown in Fig. 4A. Gold
particles can be seen distributed in the vacuolar matrix region, the
PAC vesicles, and the rough endoplasmic reticulum. In contrast, none of
the vacuolar crystalloid, the lipid bodies, the mitochondria, or the
cell wall was labeled with gold particles. These results suggested that
PV100, which is synthesized on rough endoplasmic reticulum, is
transported to PAC vesicles and then to protein storage vacuoles.
The molecular structure of PV100 implies that multiple seed proteins
are derived from PV100. The next question is whether PV100 is processed
to make such seed proteins in the protein storage vacuole. To answer
this question, the protein storage vacuoles were isolated from dry
pumpkin seeds and subjected to SDS-PAGE followed by immunoblot
analysis, as shown in Fig. 4B. The vacuoles contained a
large amount of 11S globulin (Fig. 4B, lane 1). On the
immunoblot with anti-PV100 antibodies, two bands, corresponding to a
50-kDa protein and a <10-kDa small protein(s), were detected (Fig.
4B, lane 2). The NH2-terminal sequence of the
50-kDa protein was IRRTEQEQSNNPYYFQ, which corresponds to a sequence
starting from the fourth amino acid of the vicilin-like domain, as
indicated in Fig. 2, dotted line (discussed below). These
results suggested that the 50-kDa vicilin-like protein and <10-kDa
small protein(s) were produced from PV100 and were accumulated in the
protein storage vacuoles.
To identify the <10-kDa small protein(s), soluble proteins of the
protein storage vacuoles were separated by HPLC, as shown in Fig.
5A. Each peak fraction of the
HPLC was subjected to both mass spectrometry and automatic Edman
degradation (Fig. 6). Fractions 37 and 41 contained the known C. maxima trypsin inhibitor (34), which
is not related to PV100. The NH2 termini of all proteins of
fractions 12, 14, 17, 43, and 45 were blocked. When digested by
pyroglutamate aminopeptidase, each protein gave an
NH2-terminal amino acid sequence that was consistent with
the sequence starting from the second residues of the respective small
protein derived from PV100, as indicated in Figs. 2 and 6B
(arrow). Fractions 12, 14, and 17 corresponded to RE4, RE3,
and RE5 of the Arg/Glu-rich domain, respectively, and fractions 43 and
45 corresponded to the latter half (C2) of the Cys-rich domain (Fig.
6). Interestingly, all of these small proteins had a pyroglutamate at
their NH2 termini. It should be noted that an Asn residue
always preceded all Gln residues to be converted into a pyroglutamate,
as indicated in Fig. 2 (boldfaced NQ in the PV100 sequence)
(discussed below).
The observed molecular masses of fractions 12, 14, and 17 showed good
agreement with the theoretical masses of sequence d of RE4, sequence c
of RE3, and sequence e of RE5, respectively, each of which has a
pyroglutamate at the NH2 terminus and an Asp residue at the
COOH terminus (Fig. 6). Thus, two steps of processing might be
required to produce the mature forms of RE peptides from PV100: the
first is cleavage at Asn-Gln bonds of PV100, and the second is trimming
2 or 5 amino acids off at the COOH termini of RE intermediates. All of
the mature peptides of the Arg/Glu-rich RE3, RE4, and RE5 found in
seeds are basic ones with estimated pIs of 11.90, 11.54, and 10.20, respectively. This is in contrast to the neutral pIs of RE
intermediates before trimming their COOH-terminal few amino acids.
The observed molecular masses of fractions 43 and 45 also showed good
agreement with the theoretical masses of sequences a and b of C2 from
the Cys-rich domain, respectively, indicating that each sequence has a
pyroglutamate at the NH2 terminus and two intramolecular
disulfide bonds (Fig. 6). The disulfide bridges are deduced from the
data of buckwheat trypsin inhibitor, an allergenic protein, that
exhibits a similar characteristic to the C2 peptide and has two
CXXXC motifs and two disulfide bridges (35).
To explore function of the C2 peptide, we examined trypsin inhibitory
activity of the C2 peptide using BAPA as a substrate of trypsin. The C2
peptide was highly purified. Mass spectrometry showed that the final
preparation of the C2 peptide used for the assay was not contaminated
by C. maxima trypsin inhibitor. We found that the C2 peptide
had an inhibitory activity against trypsin, as shown in Fig.
7. Ten µg of trypsin was completely
inhibited by 1.2 nmol of C2 peptide. The C2 peptide of 49 amino acids
exhibits an 18% identity in amino acids to buckwheat trypsin inhibitor of 51 amino acids. In contrast to the low identity between the two
sequences, they have a similar characteristic in the presence of two
CXXXC motifs in their sequences. The reactive site of
buckwheat trypsin inhibitor for trypsin was reported to be
Arg19, between the two CXXXC motifs (35). The C2
peptide conserves Arg21, between the two CXXXC
motifs (Fig. 6B), and the residue might be the reactive site
for trypsin (discussed below).
None of RE1, RE2, RE6, or the former half (C1) of the Cys-rich domain
was detected in protein storage vacuoles. They might be degraded in the
vacuoles during seed maturation (discussed below). These results
indicated that ~6-kDa C2, ~5-kDa RE3, ~4-kDa RE4, and ~5-kDa
RE5 are accumulated in the protein storage vacuoles. Fig. 5B
shows an immunoblot of RE3 (fraction 14), RE4 (fraction 12), RE5
(fraction 17), and C2 (fractions 43 and 45) with anti-PV100 antibodies.
Surprisingly, the polyclonal antibodies recognized C2 peptide
efficiently, but no RE peptides at all appeared on the blot. These
results suggested that the antigenicity of the Cys-rich peptides was
much higher than that of the extremely hydrophilic Arg/Glu-rich
peptides. On the immunoblot of protein storage vacuoles, the signal
corresponding to <10 kDa might be caused by C2 peptide (Fig.
4B). The intensity of the signal was much higher than that of the 50-kDa vicilin-like protein. It seems likely that such CXXXC motifs cause allergy to animals as buckwheat trypsin
inhibitor does (35).
VPE Mediates the Conversion of PV100 into Multiple Seed
Proteins--
We previously showed that VPE is involved in maturation
of various seed proteins in the protein storage vacuoles by cleaving a
peptide bond on the carbonyl side of Asn residues (6, 10, 15). This
raised the question of whether VPE mediates the proteolytic processing
of PV100. To answer this question, we performed an in vitro
processing of PV100 by the purified VPE from castor bean seeds. We used
proproteins in the isolated PAC vesicles as substrates, including
PV100, proglobulin, and pro2S albumin, as shown in Fig. 1A
(lane 1). After incubation of these proteins with the
purified VPE, the amount of PV100 decreased in association with the
increase of the amount of a 50-kDa protein and <10-kDa proteins, as
shown in Fig. 8. The <10-kDa proteins
contained not only PV100-derived small proteins but also 2S albumin,
composed of 3.8- and 8.0-kDa subunits, which was produced from pro2S
albumin. This indicated that VPE was involved in the conversion of
PV100 into the 50-kDa protein and the <10-kDa proteins.
The 50-kDa protein was subjected to automatic Edman degradation after
digestion by pyroglutamate aminopeptidase (Fig. 8). The determined
NH2-terminal sequence, <QVAIRRTEQEQSNNPY, was found in the
sequence of PV100, as in Fig. 2 (arrow). The
NH2-terminal sequence determined after in vitro
processing was consistent with that of the 50-kDa protein accumulated
in maturing seeds (data not shown). This suggests that processing
similar to the in vitro processing of PV100 occurs by
endogenous VPE during seed maturation. The result indicated that VPE
mediated the production of the 50-kDa vicilin-like protein by cleaving
an Asn375-Gln376 bond of PV100. The cleavage
was consistent with the substrate specificity of VPE toward Asn
residues. Further degradation to remove NH2-terminal three
amino acids must occur at the later stage of seed maturation and
produce a mature 50-kDa vicilin-like protein with the
NH2-terminal sequence, IRRTEQEQSNNPYYFQ (Figs. 2 and
4B). We previously reported that protein storage vacuoles accumulated not only VPE but also aspartic proteinase (36). It seems
likely that such aspartic proteinase might be involved in the
proteolytic trimming.
It should be noted that most processing occurs at Asn-Gln bonds in the
hydrophilic region of PV100, and all of the mature proteins, the 50-kDa
protein, and C2 and RE peptides have a pyroglutamate at their
NH2 termini, as shown in Fig.
9. Similar VPE-mediated processing of
PV100 might occur to produce the multiple seed proteins (discussed
below).
PV100 Is a Unique Precursor to Multiple Functional
Proteins--
The present study demonstrates that PV100 is not only a
precursor of vicilin storage protein but also a precursor of the
Arg/Glu-rich RE peptides and a precursor of the Cys-rich C2 peptide
that acts as a trypsin inhibitor. PV100 is a unique precursor for
multiple seed proteins with different functions.
The C2 peptide was shown to have trypsin inhibitory activity. However,
the sequence of the C2 peptide has no homology to known trypsin
inhibitors, including members of squash trypsin inhibitor family (34),
except for buckwheat trypsin inhibitor, showing a 18% identity to the
C2 peptide. Interestingly, despite such low homology in primary
structure, the higher structure of the C2 peptide might be analogous to
that of buckwheat inhibitor. It has been shown that the buckwheat
trypsin inhibitor forms a hairpin structure, in which two
CXXXC motifs are linked by two disulfide bonds, and that
Arg19, between the two CXXXC motifs, is the
reactive site for trypsin (35). Similarly, all four Cys residues of the
C2 peptide formed two disulfide bonds, and Arg21 is found
between the two CXXXC motifs (Fig. 6B). The
result suggests that both the C2 peptide and buckwheat inhibitor belong
to a novel family of trypsin inhibitors. These inhibitors might play a
role in protecting the seeds from animals.
Among the Arg/Glu-rich RE peptides, the mature RE3 with the highest pI
value (pI 11.90) shows the highest content in pumpkin seeds. We
compared the RE3 composed of 36 amino acids with the pumpkin basic
peptide that has been shown to be toxic to mouse B-16 cells (33) (Fig.
3C). The cytotoxic basic peptide was composed of 36 amino
acids, and the probable amino acid sequence of the peptide was reported
by Naisbitt et al. (33). Both sequences are identical to
each other, except for two residues. It is likely that the mature RE3
accumulated in the vacuoles of pumpkin seeds might be identical to the
cytotoxic basic peptide that was characterized by Naisbitt et
al. (33). This suggests that the mature RE3 might function as a
toxin to prevent animals from eating the seeds.
Most vacuolar proteins are synthesized as a proprotein precursor on the
rough endoplasmic reticulum and are then transported to vacuoles. The
vacuolar targeting signals have been shown to be present in the
propeptides of some vacuolar proteins, including barley aleurain (37),
barley lectin (38), sweet potato sporamin (39), and tobacco chitinase
(40). It has been thought that the propeptides are cleaved off and
degraded after arrival of the proproteins at the vacuoles. However, the
possibility cannot be excluded that the propeptides exhibit some
functions in the vacuoles after being removed from the precursor
proteins, as the 4-6-kDa RE and C2 peptides are accumulated to act as
functional proteins in the vacuoles.
VPE-mediated Cleavage at Asn-Gln Bonds of PV100 to Produce Multiple
Seed Proteins with a Pyroglutamate at Their NH2
Termini--
Fig. 9 shows the hydrophobicity plot of PV100 and a
hypothetical mechanism for vacuolar processing of PV100 to produce C2 peptide, RE peptides, and a vicilin-like protein. PV100 contains nine
Asn-Gln bonds in the sequence. All six Asn-Gln bonds to be cleaved are
located in the hydrophilic region of the PV100 sequence, whereas the
other three noncleavable Asn-Gln bonds are found in the hydrophobic
region of the vicilin-like domain. The result is consistent with our
previous data showing that VPE recognizes Asn residues that are located
in the hydrophilic region and are exposed on the surface of precursor
molecules (6).
We previously reported that one subunit of pumpkin 11S globulin has a
pyroglutamate at the NH2 terminus (41).
NH2-terminal sequencing of the proglobulin in the isolated
PAC vesicles revealed that a cleavage of Asn-Gln bond by VPE produced a
pyroglutamate at the NH2 terminus of the 11S globulin
subunits (11). VPE cleavage of an Asn-Gln bond gives a new
NH2-terminal Gln residue, which might be spontaneously
converted into a pyroglutamate under the acidic condition in the
vacuoles. Proteins with an NH2 terminus blocked by
pyroglutamate are resistant to aminopeptidases that are localized in
the vacuoles. These results suggested that the Asn-Gln sequences not
only provide sites that can be cleaved by VPE but also produce
aminopeptidase-resistant functional proteins in the vacuoles.
In contrast to the accumulation of RE3, RE4, RE5, and C2 peptide in the
vacuoles, none of RE1, RE2, RE6, or C1 peptides were detected in the
vacuoles (Fig. 6). They might be sensitive to proteinases in the
vacuoles. The C1 peptide has two Asn residues inside the sequence (Fig.
2) and can be attacked by VPE to be degraded. It should be noted that
the RE3, RE4, RE5, and C2 peptides have no Asn residue inside their
sequences, indicating that they are resistant to VPE. They are also
resistant to aminopeptidase because of a pyroglutamate at their
NH2 termini. However, both RE1 and RE2, with a Glu residue
at each NH2 terminus, could be sensitive to
aminopeptidases, if they were produced by cleaving an
Asp118-Glu119 bond and an
Asn161-Glu162 bond by VPE, respectively.
Further proteolysis for COOH-terminal trimming at the Asp residues must
occur to make the final mature forms of the RE3, RE4, and RE5 peptides
(Fig. 6). Recently, we have found that VPE also cleaves a peptide bond
at carbonyl side of Asp, although the activity toward Asp is less than
that toward Asn (data not shown). The finding is consistent with the
report that the VPE homolog of vetch has a substrate specificity toward
both Asn and Asp residues (42). Therefore, it seems likely that the
COOH-terminal trimming of RE3, RE4, and RE5 peptides is also mediated
by VPE.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-N-Benzoyl-DL-arginine-p-nitroanilide HCl (BAPA) was used as a substrate of trypsin. One of the PV100-derived small proteins, C2 peptide, purified by a reverse phase chromatography, was dissolved in a solution of 0.1 M Tris-HCl (pH 8.0) and
25 mM CaCl2. After preincubation of the C2
peptide (0-2.4 nmol) with 10 µg of trypsin (Sigma) in a 676 µl of
0.1 M Tris-HCl (pH 8.0) and 25 mM
CaCl2 at room temperature for 30 min, 333 µl of a
substrate BAPA solution (1 mg/ml) was added to the mixture to start the reaction. After incubation of the mixture at room temperature for 30 min, 100 µl of acetic acid was added to stop the reaction. The
residual enzyme activity was measured at 405 nm. The amount of the C2
peptide was estimated from the absorbance at 280 nm, and the molar
absorption coefficient at 280 nm of the C2 peptide was computed.
-32P]dCTP and Megaprime DNA labeling
systems (Amersham Pharmacia Biotech). The cDNA library was screened
by colony hybridization using the 32P-labeled DNA as a
probe. The isolated cDNA lacked an initiation codon. Subsequently,
we amplified DNAs covering the 5' region of PV100 cDNA using a
5'-Full RACE Core Set (Takara, Tokyo, Japan). Two identical clones were
amplified, and the nucleotide sequences were overlapped with the
isolated cDNA sequence that lacked an initiation codon.
21M13 forward and M13 reverse fluorescent
primers in accordance with the manufacturer's directions. The
nucleotide and the deduced amino acid sequences were analyzed with DNA
analytical software (Gene Works, IntelliGenetics, Mountain View, CA).
The hydrophobicity profile of the amino acid sequences was computed by
application of the algorithm of Kyte and Doolittle (24), with a window
size of 10 residues. A homology plot was computed with the PAM-250
algorithm (25).
20 °C and embedded in LR white resin
(London Resin Co. Ltd., Basingstoke, Hampshire, UK). Immunogold
labeling procedures were essentially the same as those described
previously (26), except for the use of the anti-PV100 antibodies that
were diluted 1000-fold in blocking solution (1% bovine serum albumin
in phosphate-buffered saline). Protein A-gold (15 nm) (Amersham
Pharmacia Biotech) was diluted 40-fold and used. The ultrathin sections
were examined with a transmission microscope (model 1200EX) (JEOL,
Tokyo, Japan) at 80 kV.
RESULTS
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Fig. 1.
PV100 is one of the major proteins in the PAC
vesicles from maturing pumpkin seeds. A, isolated PAC
vesicles were subjected to SDS-PAGE and subsequent staining with
Coomassie Blue (lane 1) or immunoblot with anti-PV100
antibodies (lane 2). pG and p2S
represent proprotein precursors of 11S globulin and 2S albumin,
respectively. The molecular mass of each marker protein is given on the
left in kDa. PAC vesicles were isolated from the cotyledons
at the middle stage of seed maturation of pumpkin. B,
immunogold labeling of the isolated PAC vesicles with anti-PV100
antibodies. Bar, 500 nm.
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Fig. 2.
Deduced amino acid sequence from a cDNA
that encodes PV100. Isolated cDNA encodes a 97,310-Da protein
of 810 amino acids, which consists of a hydrophobic signal peptide
followed by the PV100 sequence. The NH2-terminal sequence
and two internal sequences of PV100 that were determined are indicated
by double underlining. An open triangle indicates
a cleavage site of a signal peptide. The PV100 sequence was divided
into three domains: an 11-kDa Cys-rich domain (indicated by dark
shading) with four CXXXC motifs (enclosed in the
small boxes), a 34-kDa Arg/Glu-rich domain (enclosed in the
large box), and a 50-kDa vicilin-like domain (indicated by
light shading). The arrows indicate the
determined NH2-terminal sequences of PV100-derived mature
proteins that had been digested by pyroglutamate aminopeptidase (see
Figs. 6 and 8), and a dotted line indicates the
NH2-terminal sequence of the vicilin-like protein from dry
seeds. Boldfaced NQ (Asn-Gln) stretches, marked with a
closed triangle, represent posttranslational processing
sites to produce multiple seed proteins, each with a pyroglutamate at
its NH2 terminus. The nucleotide sequence has been
submitted to the DNA Data Bank of Japan and GenBankTM with
the accession number AB019195.
-conglycinin (29), and
jack bean canavalin (30). Precursors of vicilin homologs of pea,
soybean, and jack bean are composed of a signal peptide followed by a
vicilin domain, whereas the precursors of cacao vicilin (31) and upland cotton
-globulin-A (32) have six and four CXXXC motifs,
respectively, preceding a vicilin domain. However, the amino acid
sequences around the CXXXC motifs of pumpkin, cacao, and
cotton exhibited a very low similarity to each other.
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Fig. 3.
Homology plot of PV100 and an amino acid
alignment of the six homologous repeats in the Arg/Glu-rich
domain. A, a homology plot was performed with the
PAM-250 algorithm (25) with a window of 10 residues. Each pair of
windows that exhibited more than 35% identity in amino acids is
indicated by a dot in the matrix. Six homologous repeats
were found in the Arg/Glu-rich domain. B, Asn-Gln/Glu bonds
separate the Arg/Glu-rich domain into six repeats (see Fig. 9). The six
Arg/Glu-rich repeats that were designated RE1 to RE6 in order from the
NH2 terminus were aligned. Numbers on the right
side of each sequence refer to the positions of the amino acids
starting from the initiation Met. C, the mature RE3 peptide
(see Fig. 6B) was aligned with the sequence of pumpkin basic
peptide, where the second and the third possible amino acids are also
shown, as reported by Naisbitt et al. (33). Both peptides
are composed of 36 amino acids, as indicated on the right
side of each sequence. Boxes enclose identical amino acids,
and shading indicates homologous amino acids.
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Fig. 4.
PV100-derived proteins are localized in
protein storage vacuoles in pumpkin seeds. A,
immunoelectron micrograph of maturating pumpkin seeds after staining
with anti-PV100 antibodies. Gold particles were distributed in the PAC
vesicles (PV), the matrix region (VM) of protein
storage vacuoles and ER. VC, vacuolar crystalloid composed
of 11S globulin; Mt, mitochondrion; LB, lipid
body; CW, cell wall. Bar, 1 µm. B,
isolated protein storage vacuoles (protein bodies) from dry pumpkin
seeds were subjected to SDS-PAGE and subsequent staining with Coomassie
Blue (lane 1) or immunoblot with anti-PV100 antibodies
(lane 2). PV100-derived proteins, the 50-kDa vicilin-like
protein (V) and a ~6-kDa C2 peptide (C) were
detected on the blot. The determined NH2-terminal sequence
of the 50-kDa vicilin-like protein is shown. G represents
the 11S globulin. The molecular mass of each marker protein is given on
the left in kDa.
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Fig. 5.
An HPLC profile of PV100-derived peptides
from the protein storage vacuoles. A, soluble fraction
of the protein storage vacuoles that contained the PV100-derived
peptides was applied to a reverse phase column. Elution was carried out
with a gradient starting from 0.065% trifluoroacetic acid in distilled
water to 0.05% trifluoroacetic acid in acetonitrile. Chromatography
was monitored in terms of absorbance at 214 nm. B,
immunoblot analysis of each peak fraction with anti-PV100
antibodies.
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Fig. 6.
Molecular structures of PV100-derived
peptides from the protein storage vacuoles. A,
molecular masses of the peptides in each HPLC fraction, as shown in
Fig. 5, were determined by mass spectrometry. Theoretical molecular
masses of RE3, RE4, and RE5 that had a pyroglutamate (<Q) at their
NH2 termini are consistent with the observed values. The
number of disulfide bonds in the C2 peptide was determined to be 2. B, primary structures of peptide components of fractions 12, 14, 17, 43, and 45 were determined to be sequence d (RE4),
sequence c (RE3), sequence e (RE5), and
sequences a and b (C2), respectively. The
determined NH2-terminal sequences after digestion by
pyroglutamate aminopeptidase are indicated by arrows below
the respective sequences. Numbers on the right side of each
sequence refer to the positions of the amino acids starting from the
initiation Met of PV100. The disulfide bridges were deduced from the
data of buckwheat trypsin inhibitor that exhibits a characteristic
similar to the C2 peptide and has two CXXXC motifs and two
disulfide bridges (35).
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Fig. 7.
PV100-derived C2 peptide functions as a
trypsin inhibitor. The C2 peptide was highly purified from soluble
fraction of the protein storage vacuoles of pumpkin seeds by HPLC. The
reaction mixture contained 0-2.4 nmol of the C2 peptide, 10 µg of
trypsin and 333 µg of BAPA in a 0.9-ml solution of 0.1 M
Tris-HCl (pH 8.0) and 25 mM CaCl2 (see under
"Experimental Procedures"). The residual enzyme activity was
monitored with absorbance at 405 nm.
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Fig. 8.
In vitro processing of PV100 by
purified VPE produced the vicilin-like protein. PAC vesicles that
contained PV100 were incubated with the purified VPE and then subjected
to SDS-PAGE followed by staining with Coomassie Blue. The resultant
band corresponding to 50-kDa vicilin-like protein (V) that
had been blotted to a polyvinylidene difluoride membrane was incubated
with pyroglutamate aminopeptidase and then was subjected to automatic
Edman degradation. The determined NH2-terminal sequence
corresponds to the sequence in PV100, as indicated by an
arrow in Fig. 2. pG and G represent
proglobulin and 11S globulin, respectively. The <10-kDa band
(M) contained a mixture of the PV100-derived peptides and 2S
albumin subunits that had been produced from pro2S albumin by VPE in
the reaction.
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Fig. 9.
Hydropathy profile of PV100 and a
hypothetical mechanism for the VPE-mediated cleavage at Asn-Gln bonds
to produce multiple seed proteins. The mean hydrophobicity index
was computed according to the algorithm of Kyte and Doolittle (24) with
a window of 10 residues. VPE is responsible for maturation of multiple
seed proteins by cleaving Asn-Gln bonds that are found in the
hydrophilic region of the PV100. Gln at the new NH2 termini
of the mature proteins might be spontaneously converted into
pyroglutamate (<Q) under the acidic condition in the
vacuoles. The cysteine-rich C2 peptide, the Arg/Glu-rich RE3, RE4, and
RE5 peptides, and the vicilin-like protein are produced. SP
represents a signal peptide.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Prof. Shigekata Yoshida of Nagoya University Experimental Farm for growing the pumpkin plants. We are grateful to L. M. Melgarejo of the National University of Colombia for help with cDNA cloning, to Y. Koumoto and J. Morita for their skillful technique on column chromatography, to Y. Makino for helpful support on mass spectrometry and peptide sequencing, and to C. Nanba for growing pumpkin and castor plants.
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FOOTNOTES |
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* This work was supported by Grants-in-Aid 10440244, 10163242, and 09267241 for the Research for the Future Program (JSPS-RFTF96L00407) from the Japan Society for the Promotion of Science and for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB019195.
¶ To whom correspondence should be addressed. Tel.: 81-564-55-7500; Fax: 81-564-55-7505; E-mail: ihnishi{at}nibb.ac.jp.
The abbreviations used are:
PAC, precursor-accumulating; PV100, a 100-kDa component of PAC vesicles; VPE, vacuolar processing enzyme; BAPA, -N-benzoyl-DL-arginine-p-nitroanilide
HCl; bp, base pair; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.
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REFERENCES |
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