Purification and cDNA cloning of the ovigerous-hair stripping substance (OHSS) contained in the hatch water of an estuarine crab Sesarma haematocheir
1 Laboratory of Animal Behavior and Evolution, Graduate School of Natural
Science and Technology, Okayama University, Tsushima 3-1-1, Okayama 700-8530,
Japan
2 Developmental Mechanisms Laboratory, Developmental Biology Department,
National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba 305-8634,
Japan
3 Laboratory for Evolutionary Regeneration Biology, Center for Developmental
Biology, RIKEN Kobe, Minatojima-minamimachi 2-2-3, Chuo-ku, Kobe 650-0047,
Japan
4 Department of Bioscience and Biotechnology, Faculty of Engineering,
Okayama University, Tsushima 3-1-1, Okayama 700-8530, Japan
* Author for correspondence (e-mail: saigusa{at}cc.okayama-u.ac.jp)
Accepted 10 November 2003
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Summary |
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Key words: crab, Sesarma (or Chiromantes) haematocheir, ovigerous hair, embryo attachment system, investment coat, stripping, ovigerous-hair stripping substance (OHSS), serine protease
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Introduction |
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Hatching in most crustaceans differs greatly from that of other animals. It
is characterized not by dissolution of the egg case, but by its sudden rupture
(Davis, 1981). Ultrastructural
studies on the egg case of an estuarine crab Sesarma haematocheir
indicated that no morphological changes occur in the thick outer layers
(E1+E2; 1.5 µm in total) upon hatching. Only the innermost thin layer (E3;
0.2 µm) is markedly digested (Saigusa
and Terajima, 2000
). At least two kinds of active factors are
contained in the hatch water (i.e. the filtered medium in which zoea larvae
are released by the female), caseinolytic proteases and OHSS (ovigerous-hair
stripping substance) (Saigusa,
1996
). OHSS plays a role in the stripping of the embryo attachment
system from the maternal ovigerous hairs just after hatching, in preparation
for the next clutch of embryos (Saigusa,
1995
). OHSS is clearly secreted by the embryo and not by the
female (Saigusa, 1995
).
However, physiological mechanisms by which the stripping of ovigerous hairs is
caused by OHSS are not known.
Embryos of a number of animals, including the sea urchin Paracentrotus
lividus (Lepage and Gache,
1990), the ascidian Ciona intestinalis
(D'Aniello et al., 1997
),
teleosteans Oryzias latipes
(Yamagami, 1988
) and
Hippoglossus hippoglossus (Helvik
et al., 1991
), release proteases upon hatching, and help to break
down the fertilization envelope. They are called `hatching enzymes'. The
hatching enzyme of Oryzias latipes is in fact two distinct enzymes,
each of which differs in its action against the egg case (Yasumasu et al.,
1989
,
1992
), whereas the hatching
enzyme of the sea urchin is a single protease
(Lepage and Gache, 1990
). The
hatching enzyme would be contained in the water in which embryos have hatched.
In Sesarma haematocheir, caseinolytic proteases might digest the
innermost thin layer, but ultrastructural analysis did not reveal evidence
that OHSS plays a role in digestion of the egg case
(Saigusa et al., 2002
).
To investigate its properties, OHSS has been partially purified by three
steps of chromatography, and the molecular mass eluted on the molecular sieve
chromatography was roughly estimated to be 30 and 32 kDa
(Saigusa and Iwasaki, 1999).
Furthermore, polyclonal antibodies raised against purified OHSS detected a 55
kDa protein. However, further investigation of the properties and functions of
this substance require a more elaborate purification and cDNA cloning.
In the present study, we have purified OHSS from hatch water using a reverse phase high-performance liquid chromatography (RP-HPLC), and cloned the OHSS cDNA and its gene. The deduced amino acid sequence matched with partially determined N-terminus and internal amino acid sequences, and the cloned cDNA was identified as that of OHSS. The primary structure of OHSS indicates that it belongs to the family of trypsin-like serine proteases. We confirmed that OHSS is expressed in the embryos. Furthermore, this paper provides evidence of recycling of the maternal ovigerous hairs by the action of OHSS.
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Materials and methods |
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Ovigerous females captured on the road were first disinfected in ice-cold
3070% ethanol for a few minutes, then washed with a large quantity of
distilled water (DW), and finally placed individually into plastic containers
(10 cm in diameter, 15 cm in height), but without water. These containers were
transferred to the laboratory, where each crab was immediately placed in a
small, covered plastic cup (5 cm in diameter, 6 or 8 cm in height) containing
10 ml of DW. As soon as zoea larvae were released, the zoeas were removed by
filtration through nylon mesh, and the remaining water was then passed again
through a filter paper. The resulting hatch water was pooled in a 50 ml
plastic bottle and immediately stored at 40°C until used. Most
females incubate their next clutch of embryos a few days after larval release
(Saigusa et al., 2002). The
females were therefore kept in the laboratory for about 2 months, and hatch
water was obtained from their second larval release (for further details, see
Saigusa 1995
,
1996
).
Purification of OHSS
OHSS was partially purified through three steps of chromatography
(hydrophobic chromatography, ion-exchange chromatography and molecular sieve
chromatography; Saigusa and Iwasaki,
1999). The procedures were all performed with a fast protein
liquid chromatography system (FPLC; Amersham-Pharmacia, Piscataway, NJ, USA)
in an experimental chamber with the temperature controlled at 4°C. The
pooled active fractions (1 ml/fraction) eluted by gel filtration were
collected, concentrated to about 50 µl by ultrafiltration (Centricon YM-10;
Millipore, Bedford, USA), and fractionated by reverse phase high-performance
liquid chromatography (RP-HPLC) (YMC-Pack ODS-A reverse-phase HPLC column; 150
mmx 6 mm; YMC Co., Ltd., Kyoto, Japan). The proteins were eluted using a
linear gradient of 852% acetonitrile containing 0.1% HCl over 80 min.
The flow rate was at 0.6 ml min1. The procedure was
performed using a Waters 626 LC system (Millipore) equipped with a model 600E
controller and a model 486 ultraviolet light (Millipore). The eluate was
monitored at 278 nm. Each fraction was tested for biological activity of
OHSS.
Egg attachment system and bioassay of OHSS
Embryos of crabs attached to ovigerous hairs arranged on the four pairs of
the ovigerous seta of the female (Fig.
1A,B). The egg attachment system consists of an outermost envelope
(E1) originating from the vitelline membrane (envelope of the ovum)
(Saigusa et al., 2002). The
adhesion and plasticity of this envelope changes just after egg-laying, and
kneading of the eggs by the ovigerous setae forms the investment coat on the
ovigerous hair (Fig. 1C).
|
After hatching, the larvae are released into the water by a vigorous
fanning movement of the abdomen (Saigusa,
1982), but the egg attachment system (broken egg envelope,
funiculus, and investment coat) remains on the hairs
(Fig. 1D). The egg attachment
system is finally removed from the hairs by the actions of OHSS
(Fig. 1E). If the embryos
attached to the female are gently pulled with forceps, the ovigerous hairs are
broken (Fig. 1F,G), whereas the
embryo clusters treated with an OHSS solution easily slip off the hairs
without damage (Fig. 1H).
The biological assay of OHSS is based on the ability of living or
chemically fixed ovigerous setae to respond to the OHSS solution. In brief, an
ovigerous seta with its attached embryos, all in the early stages of
development, was excised from a female, fixed in 70% ethanol, and then stored
at 4°C until used. Shortly before the bioassay, the fixed ovigerous setae
were suspended in DW to wash out the ethanol, and then placed in a glass dish
with DW. The ovigerous seta was subdivided into four segments under a
stereomicroscope (for further details of biological assay, see
Saigusa, 1995).
The subdivided segments with their attached embryos were placed in the well
of a plastic culture dish, with medium (300 µl) in which 50 µl of each
fraction eluted by RP-HPLC was diluted with 250 µl of PBS
(phosphate-buffered saline; pH 7.4). The culture dish was shaken on a
mechanical shaker at constant temperature (25±1°C). After
incubation for 1 and 1.5 h, each segment with its attached embryos was again
placed in a glass dish with DW. The embryos were gently pulled away from the
ovigerous hairs using fine forceps. The percentage of ovigerous hairs that
were stripped clean but were still undamaged was calculated under the
stereomicroscope (for further details, see
Saigusa, 1995).
SDS-polyacrylamide gel electrophoresis
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according
to Laemmli (1970) in a 15%
polyacrylamide gel. Prior to SDS-PAGE, the aqueous phase of each fraction
obtained by RP-HPLC was evaporated on a centrifugal evaporator (CVE-1000;
EYELA, Tokyo, Japan) equipped with a cold trap (EYELA, UT-2000). The amount of
protein in each fraction was calculated using a Protein Assay Kit (BioRad,
Hercules, USA). 200 ng of precipitated protein was dissolved in lysis buffer
(0.0625 mol l1 Tris, 2.5% SDS, 2.5% ß-mercaptoethanol,
4 mol l1 urea, 0.025 mol l1 EDTA, 2.5%
sucrose and 0.0025% Bromophenol Blue) and then denaturated at 95°C for 3
min. Electrophoresis was performed for 3 h at 30 mA in Tray buffer (0.025 mol
l1 Tris, pH 8.3, 9.6 mol l1 glycine and
0.1% SDS), according to the method of Ikeuchi and Inoue
(1988
). The molecular mass
marker employed was a Rainbow colored protein molecular mass marker
(Amersham-Pharmacia). The gels were stained with Coomassie Brilliant Blue
(CBB) R-250.
Amino acid sequencing
The N terminus of OHSS eluted on RP-HPLC was determined with the amino acid
sequencer (Applied Biosystems, Foster, USA). The N terminus of OHSS was
determined using fraction no. 6. Furthermore, the OHSS containined in fraction
no. 6 (Fig. 2C) was digested by
lysylendopeptidase, eluted by RP-HPLC, and amino acid sequences of the
peptides were determined by the amino acid sequencer.
|
Extraction of total RNA
Total RNA was extracted from the embryos, zoea larvae, and tissues of adult
females. Female crabs including ovigerous individuals were maintained under 15
h:9 h light:dark cycle (LD15:9), similar to that in the field in summer
(lights-on at 05.00 h and lights-off at 20.00 h), and at constant temperature
(25±1°C). Muscles, ovigerous hair, brains and hepatopancreas were
excised from the adult female. Embryos at different stages of development were
removed from ovigerous females. Just after the larval release, zoeas were
transferred to an aquarium containing clean seawater with very weak aeration.
Zoeas were collected on the day of hatching and 3 days after hatching. All
these samples were individually frozen in liquid nitrogen and stored at
80°C until used.
Total RNA was extracted and purified with an RNeasy Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. To prevent possible DNA contamination in the RNA, samples were subjected to DNase treatment using DNA-free Kit (Ambion, Austin, TX, USA). The obtained total RNA was dissolved in nuclease-free DW and stored at 80°C until used.
Construction of the cDNA library of embryos
The poly(A)+RNA (9 µg) was purified from total RNA (400
µg) extracted from the embryos using the QuickPrep Micro mRNA purification
kit (Amersham-Pharmacia). The embryonic cDNA library was constructed using a
Marathon cDNA amplification kit (BD Biosciences, Palo Alto, CA, USA). In
brief, first strand cDNA synthesis was carried out using 2 µg of
poly(A)+RNA, the modified lock-docking oligo(dT) primer provided
with the Marathon cDNA amplification kit (BD Biosciences), and the Superscript
II reverse transcriptase (BD Biosciences). Second strand synthesis was
achieved using the Marathon cDNA amplification kit following the
manufacturer's instructions.
cDNA cloning and DNA sequencing of OHSS
The following two degenerate primers were used:
5'-GA(A/G)TGGCCATGGGC(C/T)GT(C/T)GT(C/T)GT(C/T)-3' (D1 in
Fig. 3) and
5'-(C/T)ACCAA(A/G)AC(G/T)CC(C/T)AC(G/T)TC-3' (D2 in
Fig. 3) corresponding to the
amino acid sequences EWPWAVVV and DVGVLV, respectively. The reactions were
carried out in a total volume of 20 µl of solution containing 1x PCR
reaction buffer, 150 µmol l1 dNTP mix, 0.5 U of Taq DNA
polymerase, 2 mmol l1 MgCl2, each of the primers
at 0.60.8 µmol l1, and 50 ng of cDNA. The
amplification was performed in a GeneAmp PCR System 9700 (Applied Biosystems)
programmed for 35 cycles of 94°C (1 min), 57°C (1 min) and 72°C (1
min).
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The PCR products were separated on a 2% agarose gel, and the DNA fragment (about 270 bp) was cut out from the gel and purified using a QIAEX II Gel Extraction kit (Qiagen). The yields were cloned into pCR2.1-TOPO cloning vector using a TOPO TA Cloning kit (Invitrogen, Carlsbad, USA) according to the supplier's protocol.
The cDNA clones were cycle-sequenced using a Thermo Sequenase Cycle Sequencing kit (Amersham-Pharmacia) with M13 forward (20) and M13 reverse primers specific to the flanking regions of a multi-cloning site in the pCR2.1-TOPO cloning vector according to the manufacturer's direction, and analyzed with an automatic DNA sequencer, the DSQ-2000L (Shimadzu, Kyoto, Japan).
Rapid amplification of cDNA ends (RACE)-PCR
To clone full-length cDNA encoding the entire open reading frame (ORF) of
OHSS, RACE-PCR was performed with embryonic cDNA library as template, using a
Marathon cDNA Amplification kit (BD Biosciences). The OHSS-sequence-specific
primer: 5'-AGGACAAGAACGACGTCCAC-3' (corresponding to nucleotides
954-973 in Fig. 3) and the AP1
primer provided in the kit (BD Biosciences) were used for 3'RACE. For
5'RACE, we used the OHSS-sequence-specific primer
5'-GTCGTTGTTTTTTGGGGTGG-3' (complementary to nucleotides
11641183 in Fig. 3) and
the AP1 (BD Biosciences). The RACE-PCR conditions were: 94°C for 30 s
followed by 25 cycles of 94°C for 5 s and 70°C for 2 min. The PCR
products were separated on 1% agarose gel and DNA fragments were cut out from
the gels, purified, cloned into pCR2.1-TOPO vector (Invitrogen), and
sequenced.
Isolation of genomic DNAs
Genomic DNAs were prepared from individual frozen embryo clusters using the
method described by Blin and Stafford
(1976). The purified genomic
DNA was suspended in TE (10 mmol l1 Tris-HCl, 1 mmol
l1 EDTA, pH 8.0) and stored at 20°C until
used.
Reverse transcription PCR (RT-PCR)
2 µg each of total RNA obtained from embryos, zoeal larvae,
hepatopancreas, muscles and brains of adult female S. haematocheir
were subjected to reverse transcription using a First Strand cDNA Synthesis
kit (Roche Diagnostics, Basel, Switzerland). The PCRs were carried out in a
total volume of 20 µl of solution containing 1xPCR reaction buffer,
150 µmol l1 dNTP mix, 0.5 U of Taq DNA polymerase, 2 mmol
l1 MgCl2, each of the primers at 0.30.4
µmol l1 and 50 ng of cDNA. For a negative control, a PCR
using genomic DNA was performed with the same reaction mixture using 150 ng of
genomic DNA as template. The amplification was performed in a GeneAmp PCR
System 9700 (Applied Biosystems) programmed for 30 cycles of 94°C (1 min),
57°C (1 min) and 68°C (3 min). Samples were removed from each reaction
during the PCR every four cycles starting from 15th cycle (i.e. 15th, 19th,
23rd, 27th) and PCR products were separated on 2% agarose gel.
The primers 5'-GTCGGATGTAGCGGCCATCACTC-3' (F1 in Fig. 3, corresponding to nucleotides 1638) and 5'-GCTAAACACTCAGTATTTCGTC-3' (R1 in Fig. 3, complementary to nucleotides 16231644) were used.
PCR on genomic DNA
To isolate the OHSS gene, approximately 150 ng ofgenomic DNA were used in
20 µl PCR reactions that consisted of 1x PCR reaction buffer, 150
µmol l1 of each nucleotide, 0.5 units of Taq DNA
polymerase (Takara, Otsu, Japan), 2 mmol l1
MgCl2, and each of the primers at 0.30.4 µmol
l1. The primers 5'-GTCGGATGTAGCGGCCATCACTC-3'
(F1 in Fig. 3, corresponding to
nucleotides 1638) and 5'-GCTAAACACTCAGTATTTCGTC-3' (R1 in
Fig. 3, complementary to
nucleotides 16231644) were used.
The amplification was performed in a GeneAmp PCR System 9700 (Applied Biosystems) programmed for 35 cycles of 94°C (1 min), 57°C (1 min) and 68°C (4 min), followed by elongation for 10 min at 72°C. The PCR products were separated on 1% agarose gel and DNA fragments were cut out from the gels, purified, cloned into pCR2.1-TOPO vector (Invitrogen) and sequenced.
Sequence analyses
Multiple sequence alignment and comparisons were made using GeneDoc
Multiple Sequences Alignment Editor 2.6 computer software
(Nicholas et al.,
1997).
A homology search in a protein database was carried out using BLAST 2.0. Protein features were analyzed using ProtoScale software via the Internet (http://us.expasy.org). A putative signal peptide sequence was predicted with SignalP V1.1 software (http://www.cbs.dtu.dk/services/SignalP/). For the calculation of molecular mass and primer design and analysis, we used Vector NTI Suite 8 (InforMax) computer software.
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Results |
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The biological activity of OHSS was detected in fractions 36, if the bioassay was carried out immediately after the chromatography (Fig. 2B). But when active fractions were stored in a refrigerator (4°C, overnight) or in a deep freezer (30°C, overnight), the biological activity was lost.
Amino acid sequence of OHSS
Twenty residues of the N-terminal amino acid sequence of purified OHSS were
determined: IIGGLLASCGEWPWAVVVKD. The N-terminal amino acid sequences of three
polypeptides obtained by digestion of purified OHSS (fraction 6) with
lysylendopeptidase were: NNDVGVLVVQ (Pep-1), LWVIGWGATM (Pep-2) and LRDVEVTVLA
(Pep-3). The polypeptides are boxed in Fig.
3.
cDNA cloning and nucleotide sequencing
A PCR-based approach was employed for cloning a complete sequence of OHSS
cDNA. The degenerate primers designed to the sequences corresponding to the
determined sequence of the N terminus (D1 in
Fig. 3) and Pep-1(D2 in
Fig. 3) generated a 270 bp PCR
(data, not shown). The full-length cDNA amplified by 5' RACE and
3' RACE PCR was 1759 bp long, containing a single open reading frame
(ORF) encoding 492 amino acids (sequence submitted to GenBank, accession
number AY306010), a putative polyadenylation signal (AATAAA), and a
poly(A)+ tail (Fig.
3). The calculated molecular mass of the deduced amino acids was
54.7 kDa.
N-terminal sequences of OHSS (IIGGLLASVGEWPWAVVVKD) corresponded to the residues 252271. The partially determined amino acid sequences of the digest of purified OHSS were in agreement with the deduced amino acid sequence: Pep-1, NNDVGVLVVQ corresponding to residues 344353; Pep-2, LWVIGVGATM corresponding to residues 380389; and Pep-3, LRAVEVTVLA corresponding to residues 398407 (Fig. 3). Only two residues were different: the sixth residue of Pep-2 was V (Valine) instead of W (Tryptophan); and the third residue of Pep-3 was A (Alanine) instead of D (Aspartic acid).
The first 23 N-terminal residues of the deduced amino acid sequence were highly hydrophobic, and were predicted to be a signal peptide. Three potential N-glycosylation sites were found at the 35th, 47th and 53th residues after the putative signal peptide (Fig. 3).
Serine protease domain
The amino acid sequence deduced from the cDNA
(Fig. 3) was compared with
other proteins using a BLAST homology search. The search showed that the
residues in the C-terminal region of the OHSS extending from positions
243492 had high similarities to trypsin-like serine protease domain. An
alignment of the homologous sequence of this domain of OHSS and other serine
proteases is shown in Fig. 4.
Homologies with these proteases ranged from 33% to 38%. Homology with prawn
Penaeus vanameii chymotrypsin
(Sellos and Van Wormhoudt,
1992) was 35%, that with crab Paralithodes camtschaticus
trypsin (Rudenskaya et al.,
1998
) was 38%, that with a proclotting enzyme of the horseshoe
crab Tachypleus tridentatus (Muta
et al., 1990
) was 34%, that with prophenoloxidase activating
enzyme (defensin) of the freshwater crayfish Pacifastacus leniusculus
(Wang et al., 2001
) was 37%,
that with human hepsin (Leytus et al.,
1988
) was 33%, and that with matriptase
(Lin et al., 1999
) was
33%.
|
The OHSS serine protease domain contained the invariant catalytic triad His-293, Asp-346 and Ser-441. The substrate specificity pocket (S1) of OHSS is likely to be composed of Asp-435, positioned at its bottom, with Gly-463 and Gly-473 at its neck, indicating that OHSS is a typical trypsin-like serine protease.
Genomic analysis
The primary structure of the OHSS gene was examined. A series of PCRs were
conducted using genomic DNA as a template with a set of two gene-specific
primers (F1 and R1 in Fig. 3).
The primers were designed to correspond to the 5' and 3' ends of
the OHSS cDNA. The PCR product was a single DNA fragment of 3.4 kb (lane G in
Fig. 5). Further cloning and
sequencing of the fragment revealed that it was the OHSS gene. Three introns
of 240 bp (Int-1), 316 bp (Int-2) and 842 bp (Int-3) were present within the
coding region of the serine protease domain of OHSS
(Fig. 3). All introns displayed
canonical GT-AG boundaries and were flanked by consensus matching exonic
acceptor and donor sequences (Table
1). So far we have not observed any PCR signals indicating the
existence of alternatively spliced transcripts of the OHSS gene.
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Expression of the OHSS gene
The expression patterns of the OHSS gene were examined by RT-PCR
(Fig. 5). 2 µg of total RNA
extracted from embryos in different stages of development, zoeas (larval
stage) and tissues of the adult female were reverse-transcribed and used as
template for RT-PCR. After 15 cycles, very weak visible products were
amplified only from embryos 1 day before hatching and zoeas just after
hatching (data not shown). After 27 cycles of PCR, amplified DNA fragments
that reflect the OHSS gene expression were found at all stages examined,
excluding zoeas 3 days after hatching, in the brain, but not in either muscle,
ovigerous setae or hepatopancreas of the female
(Fig. 5).
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Discussion |
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Putative process of conversion from the 54.7 kDa form to an active 25 kDa form
The OHSS cDNA clone of 1759 bp was found to encode a protein of 492 amino
acids whose molecular mass was estimated to be 54.7 kDa
(Fig. 3). A homology search
indicated that the C-terminal part of the deduced amino acid sequence was
composed of a trypsin-like serine protease domain
(Fig. 4). Serine proteases are
involved in many biological process including digestion, blood clotting,
proenzyme activation and complement activation (e.g.
Neurath, 1984;
Lanz et al., 1993
;
Rawlings and Barrett, 1994
;
Klein et al., 1996
;
Levine et al., 2001
). Numerous
serine proteases are synthesized as inactive zymogens. Zymogens prevent
premature physiological functioning of the active portion of the protease,
thus protecting host cells from enzymatic damage
(Neurath and Walsh, 1976
;
Rawlings and Barrett,
1994
).
The protein encoded in the OHSS cDNA would be a zymogen of OHSS, which is likely to be proteolytically activated. The Arg-Ile-Ile-Gly-Gly motif (Fig. 3) clearly corresponds to the typical Arg-Ile-(Ile or Val)-Gly-Gly motif in other serine proteases (Fig. 4), indicating that Arg251-Ile252 is a putative proteolytic activation site of OHSS.
The six conserved cysteines required to form three intramolecular disulfide
bonds that stabilize the catalytic pocket were demonstrated in trypsin-like
serine proteases (e.g. Lin et al.,
1999). The most likely cysteine pairings for OHSS are:
Cys278-Cys294, Cys411-Cys426, and Cys437-Cys466. Furthermore, an additional
cysteine (Cys366) is also contained in the OHSS serine protease domain. While
this cysteine is not present in a single chain protease such as trypsin
(Rudenskaya et al., 1998
) and
chymotrypsin (Sellos and Van Wormhoudt,
1992
), it is found in two-chain proteases, e.g. hepsin
(Leytus et al., 1988
),
prophenoloxidase activating enzyme (defensin)
(Wang et al., 2001
) and
matriptase (Lin et al., 1999
).
The active form of the two-chain protease, representing the majority of plasma
serine proteases, consists of two polypeptides held together by a disulfide
bond, a highly conserved catalytic chain derived from the C-terminal region of
the precursor polypeptide, and a unique noncatalytic chain derived from the
N-terminal region of the polypeptide chain. The presence of noncatalytic
chain(s) distinguishes the plasma serine proteases from digestive proteases
(Neurath and Walsh, 1976
).
Noncatalytic chain(s) mediate interaction with other proteins, affecting the
action of proteases on their selected substrates
(Leytus et al., 1988
).
Comparative sequence analysis (Fig.
4) suggests that OHSS is originally synthesized as a single-chain
zymogen, and then proteolytically activated to take the two-chain form. The
conserved intramolecular disulfide bond in OHSS is likely to be formed at
Cys246 and Cys366 (Fig. 4). If
this is the case, the majority of OHSS molecules in developing embryos would
be present in the zymogen form. This suggestion is consistent with
immunoblotting data obtained before. In our previous study, a polyclonal
antiserum was raised against the active fractions (corresponding to the
mixture of 25 kDa and 22 kDa proteins in
Fig. 2C) eluted by
molecular-sieve chromatography (Saigusa
and Iwasaki, 1999). Antibodies purified from this antiserum
(anti-OHSS antibody) recognized not only both proteins but also a band at
about 55 kDa. It is highly probable that the 55 kDa protein detected by the
anti-OHSS antibody is a zymogen form of OHSS. The estimated molecular mass of
the polypeptides encoded in the OHSS cDNA was 54.7 kDa, and agreed well with
the results of immunoblotting. While the 55 kDa protein was clearly detected
from 2 weeks to 2 days before hatching, the 25 kDa proteins (active form)
appeared from 4 days before hatching to the day of hatching. OHSS biological
activity appeared only 1 day before hatching
(Saigusa and Iwasaki, 1999
).
It is plausible that OHSS activity would be suppressed until 1 day before
hatching, and that OHSS is activated by some (unidentified) factor(s) before
hatching.
Action of OHSS on the egg attachment system
Just after laying her eggs, the female kneads them by moving the ovigerous
setae. By this action the layer investing the embryo (E1) is stretched, and
wraps around the ovigerous hair (Saigusa
et al., 2002). The wrapping of and adherence to the ovigerous hair
(enclosed by open rectangle in Fig.
6A) has been speculated to occur without any adhesive substance
(Cheung, 1966
; Goudeau and
Lachaise, 1980
,
1983
;
Goudeau et al., 1987
). However,
we found a electron-dense, slender structure arranged at intervals of
130160 nm around the hair (Fig.
6B). The stretched embryonic envelope (E1) would attach to the
ovigerous hair on this structure, finally forming the investment coat
(Fig. 6A). An adhesive
substance, which is possibly secreted from the maternal ovigerous seta, would
appear at this structure (i.e., egg attachment site) upon egg-laying, making
the bond with the investment coat (Fig.
6C).
|
Several hours after hatching, the egg attachment system of S.
haematocheir slips off the ovigerous hairs due to actions of OHSS
(Saigusa, 1995,
1996
;
Saigusa et al., 2002
).
Ultrastructural analysis indicated that the stripping is due to separation of
the attachment sites from the ovigerous hair
(Fig. 6D, left). OHSS might act
specifically at the attachment sites of the investment coat, lysing the bond
with the coat (Fig. 6C,D,
right), thus disposing of the embryo attachment system in preparation for the
next clutch of embryos.
The embryo has a special developmental program for hatching (hatching
program) for 2 nights (4849.5 h), during which ecdysis occurs twice
(Saigusa and Terajima, 2000).
OHSS biological activity begins to appear 1 day before hatching
(Saigusa and Iwasaki, 1999
).
OHSS may cause separation of the embryonic exuviae from the zoeal cuticle
before hatching. Furthermore, a PCR based analysis of mRNA expression showed
that OHSS is highly expressed in the embryos just before hatching. However,
less intensive expression of the gene also can be detected in the earlier
stage embryos. The OHSS gene was also expressed not only in the brain of the
female (Fig. 5), but also the
eyestalk ganglia of the female (O. Gusev, H. Ikeda and M. Saigusa, unpublished
data), and further studies are needed to elucidate the effects of OHSS gene
expression in the brain of females in addition to the expression in the
earlier embryonic stages.
Yasumasu et al. (1989)
reported that the hatching enzyme of the fish Oryzias latipes
consists of two kinds of proteases that act together on the egg envelope; one
of them (HCE: high choriolytic enzyme) has two isomers (HCE-1 and HCE-2), as
demonstrated by cation-exchange chromatography. Two distinct cDNAs were
obtained and the nucleotide sequences had 92.8% similarity
(Yasumasu et al., 1992
). At
present, we do not have any evidence that OHSS takes part directly in
hatching, having obtained only one sequence of OHSS cDNA. However, we found a
slight discrepancy in the deduced amino acid sequence with that of the
purified protein and some minor variation in nucleotide sequences of the PCR
products (O. Gusev, H. Ikeda and M. Saigusa, unpublished data). Thus, OHSS
might consist of multiple isomers as well as one of the medaka hatching
enzymes (HCE). This possibility remains to be explored.
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Blin, N. and Stafford, D. W. (1976). A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3,2303 -2308.[Abstract]
Cheung, T. S. (1966). The development of egg-membranes and egg attachment in the shore crab, Carcinus maenas, and some other related decapods. J. Mar. Biol. Assn. UK 46,373 -400.
Davis, C. C. (1981). Mechanisms of hatching in aquatic invertebrates eggs. Oceanogr. Mar. Biol. Annu. Rev. 19,95 -123.
D'Aniello, A., de Vincentiis, M., Di Fiore, M. M. and Scippa, S. (1997). Hatching enzyme from the sea-squirt Ciona intestinalis: purification and properties. Biochim. Biophys. Acta 1339,101 -112.[Medline]
Goudeau, M. and Lachaise, F. (1980). Fine structure and secretion of the capsule enclosing the embryo in a crab (Carcinus maenas (L.)). Tissue Cell 12,287 -308.[Medline]
Goudeau, M. and Lachaise, F. (1983). Structure of the egg funiculus and deposition of embryonic envelopes in a crab. Tissue Cell 15,47 -62.[CrossRef][Medline]
Goudeau, M., Talbot, P. and Harper, R. (1987). Mechanism of egg attachment stalk formation in the lobster, Homarus.Gamete Res. 18,279 -289.[Medline]
Helvik, J. V., Oppen-Berntsen, D. O. and Walther, B. T. (1991). The hatching mechanism in Atlantic halibut (Hippoglossus hippoglossus). Int. J. Dev. Biol. 35,9 -16.[Medline]
Herrick, F. H. (1895). The American lobster: a study of its habits and development. Bull. US Fish. Comm. 1895,1 -252.
Ikeuchi, M. and Inoue, Y. (1988). A new 4.8-kDa polypeptide intrinsic to the PS II reaction center, as revealed by modified SDS-PAGE with improved resolution of low-molecular-weight proteins. Plant Cell Physiol. 29,1233 -1239.
Klein, B., Le Moullac, G., Sellos, D. and Van Wormhoudt, A. (1996). Molecular cloning and sequencing of trypsin cDNAs from Penaeus vanamei (Crustacea, Decapoda): use in assessing gene expression during the moult cycle. Int. J. Biochem. Cell Biol. 28,551 -563.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Lanz, H., Hernandez, S., Garrido-Guerrero, E., Tsutsumi, V. and Arechiga, H. (1993). Prophenoloxidase system activation in the crayfish Procambarus clarkii. Dev. Comp. Immunol. 17,399 -406.[CrossRef][Medline]
Lepage, T. and Gache, C. (1990). Early expression of a collagenase-like hatching enzyme gene in the sea urchin embryo. EMBO J. 9,3003 -3012.[Abstract]
Levine, M. Z., Harrison, P. J. H., Walthall, W. W., Tai, P. C. and Derby, C. D. (2001). A CUB-serine protease in the olfactory organ of the spiny lobster Panulirus argus. J. Neurobiol. 49,277 -302.[CrossRef][Medline]
Leytus, S. P., Loeb, K. R., Hagen, F. S., Kurachi, K. and Davie, E. W. (1988). A novel trypsin-like serine protease (hepsin) with a putative transmembrane domain expressed by human liver and hepatoma cells. Biochemistry 27,1067 -1074.[Medline]
Lin, C. Y., Anders, J., Johnson, M., Sang, Q. A. and Dickson, R.
B. (1999). Molecular cloning of cDNA for matriptase, a
matrix-degrading serine protease with trypsin-like activity. J.
Biol. Chem. 274,18231
-18236.
Muta, T., Hashimoto, R., Miyata, T., Nishimura H., Toh, Y. and
Iwanaga S. (1990). Proclotting enzyme from horseshoe crab
hematocytes. cDNA cloning, disulfide locations, and subcellular localization.
J. Biol. Chem. 265,22426
-22433.
Neurath, H. (1984). Evolution of proteolytic enzymes. Science 224,350 -357.[Medline]
Neurath, H. and Walsh, K. A. (1976). Role of proteolytic enzymes in biological regulation (a review). Proc. Natl. Acad. Sci. USA 73,3825 -3832.[Abstract]
Nicholas, K. B., Nicholas H. B., Jr and Deerfield, D. W. (1997). GeneDoc: Analysis and Visualization of Genetic Variation. Embnew. News 4,14 .
Rawlings, N. D. and Barrett, A. J. (1994). Families of serine peptidases. Methods Enzymol. 244, 19-61.[Medline]
Rudenskaya, G. N., Isaev V. A., Kalebina, T. S., Stepanov, V. M., Mal'tsev, K., Shvets, S. V., Luk'yanova, N. A., Kislitsin, Yu. A. and Miroshnikov, A. I. (1998). Isolation and properties of trypsin PC from the King crab Paralithodes camtschaticus. Russian J. Bio-organic Chem. 24,98 -105 (in Russian).
Saigusa, M. (1982). Larval release rhythm coinciding with solar day and tidal cycles in the terrestrial crab Sesarma-harmony with the semilunar timing and its adaptive significance. Biol. Bull. 162,371 -386.
Saigusa, M. (1995). Bioassay and preliminary
characterization of ovigerous-hair stripping substance (OHSS) in hatch water
of crab larvae. Biol. Bull.
189,175
-184.
Saigusa, M. (1996). Two kinds of active factor
in crab hatch water: ovigerous-hair stripping substance (OHSS) and a
proteinase. Biol. Bull.
191,234
-240.
Saigusa, M. and Iwasaki, H. (1999).
Ovigerous-hair stripping substance (OHSS) in an estuarine crab: purification,
preliminary characterization, and appearance of the activity in the developing
embryos. Biol. Bull.
197,174
-187.
Saigusa, M. and Terajima, M. (2000). Hatching of an estuarine crab, Sesarma haematocheir: from disappearance of the inner (E3) layer to rupture of the egg case. J. Exp. Zool. 287,510 -523.[CrossRef][Medline]
Saigusa, M., Terajima, M. and Yamamoto, M.
(2002). Structure, formation, mechanical properties, and disposal
of the embryo attachment system of an estuarine crab, Sesarma
haematocheir. Biol. Bull.
203,289
-306.
Sellos, D. and Van Wormhoudt, A. (1992). Molecular cloning of a cDNA that encodes a serine protease with chymotryptic and collagenolytic activities in the hepatopancreas of the shrimp Penaeus vanameii (Crustacea, Decapoda). FEBS Lett. 309,219 -224.[CrossRef][Medline]
Wang, R., Lee, S. Y., Cerenius, L. and Soderhall, K.
(2001). Properties of the prophenoloxidase activating enzyme of
the freshwater crayfish, Pacifastacus leniusculus. Eur. J.
Biochem. 268,895
-902.
Yamagami, K. (1988). Mechanisms of hatching in fish. Fish Physiology XIA,447 -499.
Yasumasu, S., Iuchi, I. and Yamagami, K. (1989). Purification and partial characterization of high choriolytic enzyme (HCE), a component of the hatching enzyme of the teleost, Oryzias latipes. J. Biochem. 105,204 -211.[Abstract]
Yasumasu, S., Yamada, K., Akasaka, K., Mitsunaga, K., Iuchi, I., Shimada, H. and Yamagami, K. (1992). Isolation of cDNAs for LCE and HCE, two constituent proteases of the hatching enzyme of Oryzias latipes, and concurrent expression of their mRNAs during development. Dev. Biol. 153,250 -258.[Medline]
Yonge, C. M. (1937). The nature and significance of the membranes surrounding the developing eggs of Homarus vulgaris and other Decapoda. Proc. Zool. Soc. Lond. (Ser. A) 107,499 -517.
Yonge, C. M. (1946). Permeability and properties of the membranes surrounding the developing egg of Homarus vulgaris. J. Mar. Biol. Assn. UK 26,432 -438.
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