Purification and characterization of zebrafish hatching
enzyme – an evolutionary aspect of the mechanism of
egg envelope digestion
Kaori Sano
1
, Keiji Inohaya
2
, Mari Kawaguchi
3
, Norio Yoshizaki
4
, Ichiro Iuchi
5
and
Shigeki Yasumasu
5
1 Graduate Program of Biological Science, Graduate School of Science and Technology, Sophia University, Tokyo, Japan
2 Department of Biological Information, Tokyo Institute of Technology, Yokohama, Japan
3 Ocean Reseach Institute, The University of Tokyo, Japan
4 Department of Biological Diversity, Faculty of Agriculture, Gifu University, Japan
5 Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan
Hatching enzyme is an enzyme that digests an egg
envelope at the time of embryo hatching. Fish hatch-
ing enzymes have been purified from several fish
species [1–5]. Among them, the hatching enzyme of
medaka Oryzias latipes has been extensively studied,
and its study field was extended not only to character-
ization of the enzyme itself, but also to the mechanism
of its egg envelope digestion [6,7]. The hatching of
Keywords
astacin family; egg envelope; hatching

substrate specificity of ZHE1 is quite similar to that of MHCE. However,
MLCE did not show such similarity. Because HCE and LCE are the result
of gene duplication in the evolutionary pathway of Teleostei, the present
study suggests that ZHE1 and MHCE maintain the character of an
ancestral hatching enzyme, and that MLCE acquires a new function, such
as promoting the complete digestion of the egg envelope swollen by
MHCE.
Abbreviations
MCA, 7-amino-4-methylcoumarin; MHCE, medaka high choriolytic enzyme; MLCE, medaka low choriolytic enzyme; ZHE, zebrafish hatching
enzyme; ZPD, ZP domain.
5934 FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS
medaka embryos is performed by two enzymes, high
choriolytic enzyme, choriolysin H (HCE; EC
3.4.24.67) and low choriolytic enzyme, choriolysin L
(LCE; EC 3.4.24.66), which cooperatively digest egg
envelope. Two enzymes have been separately purified
from hatching liquid [4,5]. HCE swells the egg enve-
lope by its limited proteolytic action, whereas LCE
efficiently digests the HCE-swollen envelope and solu-
bilizes it completely. We have named this digesting
system the ‘HCE-LCE system’. cDNA cloning analysis
revealed that both enzymes belong to the astacin fam-
ily and comprise 200 amino acid residues in mature
enzyme portions with 55% identity in amino acid
sequence [8]. In addition, two hatching enzymes have
been purified from killifish Fundulus heteroclitus
embryos, and hatching has been demonstrated to be
performed by the HCE-LCE system [9]. Two types of
enzymes homologous to medaka HCE (MHCE) and
medaka LCE (MLCE) were cloned from other

tion is discussed on the basis of the manner of the
reciprocal or cross-species egg envelope digestion
using enzymes and substrates of both species: zebra-
fish and medaka.
Results
Expression of zebrafish hatching enzyme ZHE1
and ZHE2 genes
It has been reported that two cDNAs, ZHE1 and
ZHE2, are cloned from the RNA of prehatching
embryos [17]. According to the zebrafish genome pro-
ject, three orthologues, ZHE1a, ZHE1b and ZHE2,
were clustered in the genome [16]. The amino acid
sequence of ZHE1a is 99% identical to that of ZHE1b,
and 60.8% identical to that of ZHE2. Therefore, we
considered that two types of hatching enzyme genes
are present in the zebrafish genome.
First, we observed the expression of ZHE1 and
ZHE2 genes by northern blot analysis (Fig. 1A). The
expression of ZHE1 was detected in embryos at
11.5 h, and a strong signal was observed at 24 h. After
hatching, no expression was observed. The size of the
band ( 1 kbp) was in agreement with that deduced
from ZHE1 cDNA (800 bp). By contrast, no signal for
ZHE2 gene expression was detected at any of the
developmental stages (Fig. 1A).
Next, RT-PCR, a method more sensitive than nor-
thern blot analysis, was used to detect expression using
RNA of 24 h embryos. An amplified band for ZHE1
transcript became visible at the 19th cycle of PCR,
whereas only a faint band for the ZHE2 transcript was

gradient of 0–1 m NaCl (Fig. 2B). Most of the activity
was retained in the column and eluted at the concen-
tration of approximately 0.35 m NaCl as a sharp single
peak. SDS ⁄ PAGE of the active fraction gave a single
band, with an estimated molecular mass of 23 kDa
(Fig. 3). A partial amino acid sequence from the N-ter-
minus of the 23 kDa protein was NALIXE, which
matched with the sequence from the N-terminus of
mature protein portion deduced from ZHE1 cDNA,
but not from ZHE2. Thus, a single enzyme, ZHE1,
was contained in the hatching liquid. This is consistent
with the results of the gene expression analysis: ZHE1
10 20 300
0.01
0.2
0.1
Time (min)
0.02
0
1
NaCI (
M
)
Caseinolytic activity (ΔA
280
)
Caseinolytic activity (ΔA
280
)
0.5

. The dotted line indicates the caseinolytic
activity.
Fig. 3. SDS ⁄ PAGE patterns of rec. ZHE1 (lane 1) and purified
ZHE1 (lane 2). The gel was stained with silver. Numbers on the left
refer to the sizes of molecular markers.
A
B
C
Fig. 1. Expression analysis of ZHE1 and ZHE2 genes. (A) The result
of the northern blot analysis probed with ZHE1 and ZHE2 cDNAs.
Total RNAs were isolated from 11.5 h embryos (lane 1), 24 h
embryos (lane 2) or embryos after hatching (lane 3). Arrowheads
indicate the positions of 28S and 18S rRNA. (B) Expression of
ZHE1 and ZHE2 was analyzed by RT-PCR using RNA isolated from
24 h embryos. b-actin was used as a control. (C) The result of
whole mount in situ hybridization probed with ZHE1 and ZHE2
cDNAs. The color precipitation was developed for 2 h (ZHE1) or
several days (ZHE2). Arrowheads in (C) indicate positive signals
observed in hatching gland cells. Scale bars = 200 lm.
Hatching enzyme of zebrafish K. Sano et al.
5936 FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS
is mainly expressed in the developing embryo, but very
little ZHE2 is expressed.
Generation of recombinant ZHE1
Recombinant ZHE1 (rec. ZHE1) was generated by an
Escherichia coli expression system using pET3c as an
expression vector, and the active enzyme was obtained
by the astacin-refolding method with slight modifica-
tions [18]. The specific caseinolytic activity of rec. ZHE1
(900 min

)1
protein. The sub-
strate specificity of rec. ZHE1 was similar to that of
the protease contained in hatching liquid. This result
supported the findings of the purification indicating
that only a single enzyme, ZHE1, was contained in
hatching liquid.
Changes of fertilized egg envelopes treated with
recombinant ZHE1
Figure 4B shows an egg envelope after hatching. At
the natural hatching of zebrafish embryo, the egg enve-
lope was not completely solubilized, but was softened
and ruptured by the contractile movement of the
embryo. When isolated egg envelopes were incubated
with rec. ZHE1, no marked structural changes could
be observed under a binocular microscope (Fig. 4C).
Using electron microscopy, we observed changes of
the fine structure of envelope. Figure 4D shows the
structure of an intact egg envelope, which was composed
of a thick inner layer and a thin outer layer. The inner
layer comprised a lamellar structure with microvillous
Table 1. The specific activity of rec. ZHE1 examined by various
MCA substrates. The activity of hatching liquid was normalized by
caseinolytic activity per 1 lg of rec. ZHE1. ND, not detected.
MCA substrate
Specific activity
(nmolÆ30 min
)1
Ælg
)1

hatching (E) and the egg envelope digested by rec. ZHE1 (F).
Arrowheads indicate outer layers. Scale bars = 1 lm.
K. Sano et al. Hatching enzyme of zebrafish
FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS 5937
channels. After incubation with rec. ZHE1, the fibrous
structure of the inner layer became evident, and its
thickness was increased two-fold more than that of the
intact envelope. Figure 4E shows an egg envelope after
natural hatching. Its fine structure was similar to that of
the egg envelope incubated with rec. ZHE1 (Fig. 4F).
Taken together with the result of the purification, the
single enzyme, ZHE1, is suggested to act on the egg
envelope at the time of natural hatching.
Digestion of unfertilized egg envelope by ZHE1
It is well known that the egg envelope becomes hard-
ened after fertilization. The hardening of the envelope
is considered to be achieved by the polymerization of
egg envelope subunits. The polymerization is due to
the formation of e-(c-glutamyl) lysine isopeptide cross-
links by transglutaminase [19–21]. Such cross-links
make it difficult to clearly determine the sites of egg
envelope cleaved by ZHE1. Therefore, initially, an
unfertilized egg envelope was used as a substrate.
The zebrafish egg envelope is known to be mainly
constructed by two glycoproteins, ZP2 (44 kDa) and
ZP3 (49 kDa), which were visualized by the
SDS ⁄ PAGE analysis of unfertilized egg envelopes
(Fig. 5, lane 1). The isolated unfertilized egg envelopes
were digested by rec. ZHE1 and analyzed by
SDS ⁄ PAGE. After incubation for 2 min, bands with

by the sequence analysis. ZP domains and trifoiled domain are indi-
cated in light gray and dark gray boxes, respectively. Predicted
N-glycosylation site is underlined. Black and white triangles indicate
putative signal sequence cleaving sites and predicted C-terminal
processing sites, respectively.
Fig. 5. SDS ⁄ PAGE patterns of unfertilized egg envelopes digested
by rec. ZHE1. The envelopes isolated from unfertilized egg of
zebrafish (lane 1) were incubated with rec. ZHE1 for 2 min (lane 2),
10 min (lane 3) and 40 min (lane 4). Numbers on the right show
the molecular masses of the major bands.
Hatching enzyme of zebrafish K. Sano et al.
5938 FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS
site E). The molecular mass of the 43 kDa product
was somewhat larger than the molecular mass pre-
dicted from ZP3 cDNA (39 070.60; from Ala80 to
lle431; Fig. 6). Because the amino acid sequence of
ZP3 contains one of the consensus sequences for
N-glycosylation site, such a difference is considered to
be due to the existence of a sugar chain. The N-termi-
nal amino acid sequences of the 39 and 36.5 kDa prod-
ucts were DYLIKEIVQP and VEEVVVK, respectively,
and these matched with the sequences from Asp48 and
Val67 deduced from ZP2 cDNA, respectively. There-
fore, the cleaving sites are Ser47 ⁄ Asp48 and Arg66 ⁄
Val67 (Fig. 6, sites A and B). The molecular masses of
the 39 and 36.5 kDa products of ZP2 were consistent
with those calculated from ZP2 cDNA (39 107.06 from
Asp48 to Arg405; 36 902.50 from Val67 to Arg405).
Digestion of fertilized egg envelope by ZHE1
Next, a fertilized egg envelope was digested by

was located 12 amino acid residues upstream from the
cleaving site obtained from the 43 kDa product of ZP3
(site E). Therefore, the finding that the 36.5 kDa prod-
uct is a mixture of two peptides from ZP2 and ZP3
suggested that the 36.5 kDa product of ZP2 obtained
from unfertilized digest binds the 12 amino acid resi-
dues fragment (from site D to site E) of ZP3 via an
e-(c-glutamyl) lysine cross-link.
Further analysis revealed that 150 kDa product also
contained two amino acid sequences identical to those
of the 36.5 kDa product, VEEVVVKAGPVDK and
APLDLXE. The sequence APLDLXE is quite similar
to the sequence APLDLQE of ZP3 deduced from
cDNA. However, the sixth glutamine residue (Q of
APLDLQE; Fig. 6, circle) was not detected in sequenc-
ing of the 36.5 and 150 kDa product by Edman degra-
dation. There is evidence that Edman degradation did
not release amino acid residues at the e-(c-glutamyl)
lysine cross-linked position [24]. Although further
investigation is necessary, we conclude that Gln73 in
ZP3 is one of the glutamine acceptor sites for e-(c-glut-
amyl) lysine cross-link formation. The lysine donor site
presumed to exist in the ZP2 sequence of the 36.5 and
150 kDa product was not determined in the present
study. The 150 kDa proteins disappeared after further
digestion (90 min; Fig. 7, lane 3), and this analysis
identified a new cleaving site, Gln79 ⁄Ala80 peptide
bond (Fig. 6, site E) in ZP3, which is identical to the
site found in the 43 kDa product. Therefore, this
digestion probably resulted in further digestion of the

site F), suggesting that the 43 kDa product of ZP3 is
further digested and decreases its molecular mass to
approximately 36.5 kDa. The other was AGPVDK
(from Ala74 of ZP2; Fig. 6, site C), which was shifted
seven amino acid residues to the C-terminal from the
site B. Thus, the cleaving sites obtained from the
90 min incubation and the post-hatching egg envelopes
are considered to contain the sites that can be cleaved,
although inefficiently, by ZHE1. Considering that the
perivitelline space where hatching enzyme is secreted is
only a small area, a rather considerably high concen-
tration of ZHE1 appears to act on egg envelope, and
therefore the ZHE1-cleaving sites at natural hatching
are suggested to include not only its preferred sites,
but also inefficient cleaving sites for ZHE1.
Specific activity of ZHE1 judged by synthetic
peptide substrates
The cleaving efficiency of ZHE1 was quantitatively
estimated with synthetic peptide substrates that were
designed from the determined ZHE1-cleaving sites.
The specific activities of rec. ZHE1 toward five pep-
tides (Fig. 6, sites A, B, C, D and E) were determined.
The most efficient substrate was site A peptide and the
second most efficient was site E peptide (Table 2). Sites
A and E corresponded to the N-termini of the 39 kDa
product of ZP2 and the 43 kDa product of ZP3
observed in the 2 min ZHE1 digestion of unfertilized
egg envelopes, respectively. By contrast, the specific
activities toward site B and D peptides were much
lower than those toward the former two (5.86% and

a 120 min incubation (Fig. 8A, lanes 3–5). These corre-
sponded to three bands obtained from the ZHE1
digest after a 10 min incubation (Fig. 8A, lane 2). The
N-terminal sequence analyses of three digests revealed
that each of the MHCE-cleaving sites on zebrafish egg
envelope was the same as the three ZHE1-cleaving
Table 2. The specific activity of ZHE1, MHCE and MLCE examined
by synthetic peptide substrates. The cleaving site of each peptide
is indicated by an arrow. ND, not detected.
Peptide
name Peptide sequence
Specific activity
(nmolÆ30 min
)1
Ælg
)1
enzyme)
ZHE1 MHCE MLCE
Site A TVQQSflDYLIK 85.5 74.1 3.6
Site B PLPVRflVEEVV 6.2 5.4 ND
Site C EVVVKflAGPVD ND – –
Site D GKPVQflAPLDL 2.1 – –
Site E KLMLKflAPEPF 32.4 39.0 2.4
Pro-X-Y-1 NPSYPQflNPSYPQ 27.3 41.7 0.12
Pro-X-Y-2 NPQVPQflYPSKPQ 14.4 32.1 1.5
ZPD-center EVQPPDflSPLSI 0.27 0.06 49.8
Hatching enzyme of zebrafish K. Sano et al.
5940 FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS
sites (Fig. 6, sites A, E and B). The results suggest that
ZHE1 and MHCE have the same substrate specificity

MHCE and MLCE judged by synthetic peptide
substrates
Cleaving efficiencies of ZHE1, MHCE and MLCE
were quantitatively estimated using synthetic peptide
substrates (Fig. 9). For the zebrafish egg envelope, the
peptides designed from sites A, B and E (Fig. 6) were
employed. As mentioned earlier, the best substrate for
ZHE1 was a site A peptide and the second best was a
site E peptide, and the specific activity toward the site
B peptide is lower than one tenth of that toward the
site A peptide. In respective peptide substrates, the
values of the specific activity of MHCE were similar
to those of ZHE1.
By contrast, the specific activity of MLCE was much
lower than those of ZHE1 and MHCE. As was true in
the egg envelope digestion experiment, the cleaving
sites of site A peptides of MLCE did not coincide with
those of ZHE1 and MHCE. However, the ratios of the
specific activities of MLCE toward the three substrates
were similar to that of ZHE1 and MHCE. In
summary, the substrate specificity of MHCE toward
peptides for zebrafish egg envelopes is quite similar to
that of ZHE1, whereas that of MLCE is similar to a
certain extent.
The medaka egg envelope is known to consist of the
subunits proteins having a ZP domain (i.e. ZI-1,2 and
ZI-3) that are homologous to zebrafish ZP2 and ZP3,
respectively [25,26]. One of the obvious differences of
the subunit protein between medaka and zebrafish is
AB

was consistent with the results obtained in the diges-
tion experiment using unfertilized egg envelopes. It is
interesting to note that ZHE1 cleaves Pro-X-Y
sequences that are not present in the subunit proteins
of zebrafish egg envelope.
Around the cleaving sites of ZHE1 and MHCE, we
were unable to find a common or consensus amino acid
sequence between zebrafish and medaka. This is sup-
ported by the finding that ZHE1 has broad substrate
specificity, as judged by the MCA substrate experiment.
Discussion
Gene expression analyses revealed that ZHE1, one of
two zebrafish hatching enzyme genes, was mainly
expressed, whereas ZHE2 was scarcely expressed. This
was supported by the result that only a single enzyme,
ZHE1, was purified from hatching liquid. In addition,
the fine morphology of fertilized egg envelope digested
by rec. ZHE1 was similar to that after natural hatch-
ing. Thus, only one enzyme, ZHE1, is suggested to be
essential for hatching of zebrafish embryo, and ZHE2
does not contribute to the hatching.
We have suggested that the ZHE1 and ZHE2 genes
were produced by gene duplication and subsequent
diversification during the evolutionary process to
zebrafish [16]. The whole mount in situ hybridization
revealed that ZHE2 transcript was expressed specifi-
cally, but weakly, in the hatching gland cells. At an
earlier period of evolution, ZHE2 is inferred to have
worked as a hatching enzyme and to have lost its abil-
ity of egg envelope digestion during its further evolu-

–1
·µg
–1
enzymen mole
–1
·30 min
–1
·µg
–1
enzyme n mole
–1
·30 min
–1
·µg
–1
enzyme
n mole
–1
·30 min
–1
·µg
–1
enzymen mole
–1
·30 min
–1
·µg
–1
enzyme n mole
–1

1
MHCE
MLCE
Fig. 9. Specific activity of ZHE1, MHCE and MLCE examined by
synthetic peptide substrates. Names of the synthetic peptides are
indicated at the bottom of the figures. Sites A, B and C indicate
the ZHE1-cleaving sites on the zebrafish egg envelope. Pro XY-1
and Pro XY-2 indicate MHCE-cleaving sites, whereas ZPD-center is
the MLCE-cleaving site on the medaka egg envelope.
Hatching enzyme of zebrafish K. Sano et al.
5942 FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS
According to molecular phylogenetic analysis of fish
hatching enzyme genes, hatching enzyme originally
consisted of a single type of enzyme, and HCE and
LCE were produced by duplication and diversification
of the gene [16]. As comparing the egg envelope diges-
tion mechanism between zebrafish and medaka, we will
discuss the evolution of hatching enzyme function.
In medaka egg envelope digestion, it has been
reported that MHCE mainly cleaves Pro-X-Y repeat
sequences located at the N-terminal region of ZI1,2
and releases small peptides containing most of
the e-(c -glutamyl) lysine isopeptide cross-links [7]. The
present study revealed that ZHE1 also cleaved the
N-terminal regions of egg envelope subunits where
most of cross-links are located, and swelled the egg
envelope. Therefore, the manner of egg envelope diges-
tion is analogous between ZHE1 and MHCE.
The cross-species digestion experiments and the
experiments using synthetic peptide substrates revealed

identity of the ZP domains was approximately 60%
(ZP3 ⁄ ZI3 = 55%; ZP2 ⁄ ZI1,2 = 65%); however, there
was no similarity in their N-terminal regions in which
the cleaving sites for ZHE1 or MHCE are located.
Hatching enzyme recognition sites on the egg envelope
are suggested to have changed with a relatively higher
substitution rate during evolution. By contrast, one of
the present studies using MCA substrates showed that
ZHE1 had broad substrate specificity. MHCE also had
broad substrate specificity [4]. In addition, some stud-
ies report that astacin and meprin A, members of the
same astacin family as hatching enzyme, have broad
substrate specificity [28,29], suggesting that the sub-
strate specificity of proteases belonging to this family
is not so strict. Therefore, due to such a character
common to the astacin family proteases, fish hatching
enzymes could flexibly adapt the changes in amino
acid residues around the cleaving sites on the N-termi-
nal regions that had a relatively higher substitution
rate, and the manner of egg envelope digestion was
conserved between ZHE1 and MHCE.
During evolution, mutations would be independently
generated in the genes of egg envelope and hatching
enzyme. Some mutations of the two genes would be
selected and accumulated under a common pressure
with respect to egg envelope digestion. Such evolution
of an enzyme and substrate is one typical of the phe-
nomena called ‘molecular co-evolution’. Therefore, the
cleaving site recognition of both enzymes would be
established under a rule that makes it possible to

USA) using ZHE1 and ZHE2 cDNA as templates. After
prehybridization was performed in DIG Easy Hyb (Roche)
at 37 °C for 1 h, the total RNA on the membrane was
hybridized with DNA probe in DIG Easy Hyb at 37 °C
overnight. The membrane was washed twice with 2·
NaCl ⁄ Cit ⁄ 0.1% SDS for 5 min at room temperature, once
with 1· NaCl ⁄ Cit ⁄ 0.1% SDS for 15 min at 60 °C and twice
with 0.2· NaCl ⁄ Cit ⁄ 0.1% SDS for 15 min at 60 °C. The
membrane was incubated with a 0.2% blocking reagent in
NaCl ⁄ P
i
-Tween for 30 min at room temperature and with
1 : 5000-diluted alkaline phosphatase-conjugated antibody
to digoxigenin in the same buffer for 1 h. After being
washed three times with the NaCl ⁄ P
i
-Tween for 5 min, the
membrane was incubated in a substrate solution comprising
1% CSPD (Roche), 0.1% diethanolamine and 1 mm MgCl
2
for 5 min, and was exposed to scientific imaging film
(Kodak, Rochester, NY, USA) in the dark.
RT-PCR
RT-PCR was performed using OneStep RT-PCR Kit (Qia-
gen, Valencia, CA, USA) according to the manufacturer’s
instructions under the PCR cycle of at 94 °C for 30 s,
55 °C for 30 s, and 72 °C for 1 min. The primers specific
for ZHE1 and ZHE2 were: ZHE1, forward: 5¢-CTGAACT
TCTCTACACACTGAGG-3¢, reverse: 5¢-CCTTATCACC
ATCACCTCACTTC-3¢; ZHE2 forward: 5¢-CTCCACACA

For the construction of expression vector, the mature
enzyme portion of ZHE1 was amplified by PCR using a
sense primer, 5¢-CATATGAATGCTCTCATCTG CGAGG
ACA-3¢, containing a 5¢ NdeI site and start methionine resi-
due, and an antisense primer, 5¢-GGATCCTAGTGATG
GTGATGGTGGCATCCATACAGCTTATTGATCC-3¢,
which was added to a tail encoding five histidine residues
and a BamHI restriction site to the 3¢-end. After digestion
of NdeI and BamHI, the fragment was transferred into the
expression vector, pET3c. The pET3c-ZHE1 thus obtained
was transformed into E. coli strain BL21 (DE3) pLysE
(Invitrogen Corp., Carlsbad, CA, USA). BL21(DE3)
pLysE ⁄ pET3c-ZHE1 were grown in 20 mL of a LB culture
solution with 50 lgÆmL
)1
of carbenicillin and 34 lgÆmL
)1
of chloramphenicol at 37 °C in a shaking incubator for 4 h.
This culture was added to 250 mL of a prewarmed LB cul-
ture solution containing carbenicillin and chloramphenicol,
and the mixture was incubated at 37 °C. When A
600
of 0.6
was reached, a final concentration of 1 mm of isopropyl
thio-b-d-galactoside was added. After 4 h, cells were
harvested by centrifugation (5800 g for 10 min). Harvested
cell paste was re-suspended in 10 mL of a 50 mm Tris–HCl
buffer (pH 8.0) containing 1 mm EDTA and frozen at
)20 °C. The frozen sample was melted at 37 °C and
disrupted by sonication. The mixture was extracted three

mixture was subjected to SDS ⁄ PAGE.
Fertilized egg envelopes were manually isolated from
24 h embryos with sharp tweezers. Approximately 10 enve-
lopes were incubated in 50 mm Tris–HCl (pH 7.5) contain-
ing 0.4 lg of rec. ZHE1 at 30 °C and subjected to
SDS ⁄ PAGE.
Analysis of cleaving sites of hatching enzymes
using synthetic peptides
Eight synthetic peptides consisting of 10–12 amino acid resi-
dues were used in the analysis. The synthetic sequences were
designed from ZHE1-, MHCE- and MLCE-cleaving sites
that were determined from the egg envelope digests. A
100 lL reaction mixture was made comprising 100 nm of the
peptide and an appropriate amount of enzyme in 50 mm
Tris–HCl (pH 7.5). After incubation at 30 °C for 30 min, the
reaction was stopped by addition of 10 lL of 0.1 m EDTA.
Such final mixtures were applied onto a C18 column (YMC
Co., Ltd, Tokyo, Japan) on the HPLC system equilibrated
with 0.1% trifluoroacetic acid and eluted with a linear gra-
dient of 0–36% MeCN in 0.1% trifluoroacetic acid. The
activity was calculated from the ratio of peaks areas of
digested and undigested peptides. The cleaving sites of
peptides were determined by amino acid sequencing.
Determination of N-terminal amino acid
sequences
Egg envelopes were analyzed by SDS ⁄ PAGE and electri-
cally blotted onto poly(vinylidene difluoride) membrane
(Hybond-P; GE Healthcare). After staining with Coomassie
Brilliant Blue, the band portion was cut out and subjected
to a protein sequencer (Procise 491HT; Applied Biosystems,

Acknowledgements
We express our cordial thanks to Professor F. S.
Howell (Department of Materials and Life Sciences,
Faculty of Science and Technology, Sophia University,
Tokyo) for reading the manuscript and to Dr
K. Yamagami (former Professor of Developmental
Biology, Life Science Institute, Sophia University,
Tokyo) for providing valuable advice and reading the
manuscript. The present study was supported in part
by Grants-in-Aid for Scientific Research (C) from
JSPS to I. I. (No. 17570189) and to S. Y. (No.
15570102).
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