Báo cáo khoa học: Purification and characterization of zebrafish hatching enzyme – an evolutionary aspect of the mechanism of egg envelope digestion pot

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

enzyme; molecular evolution
Correspondence
S. Yasumasu, Department of Materials and
Life Sciences, Faculty of Science and
Technology, Sophia University, 7-1 Kioi-cho,
Chiyoda-ku, Tokyo 102-8554, Japan
Fax / Tel: +81 3 3238 3393
E-mail:
(Received 17 June 2008, revised
22 September 2008, accepted 2
October 2008)
doi:10.1111/j.1742-4658.2008.06722.x
There are two hatching enzyme homologues in the zebrafish genome:
zebrafish hatching enzyme ZHE1 and ZHE2. Northern blot and RT-PCR
analysis revealed that ZHE1 was mainly expressed in pre-hatching
embryos, whereas ZHE2 was rarely expressed. This was consistent with the
results obtained in an experiment conducted at the protein level, which
demonstrated that one kind of hatching enzyme, ZHE1, was able to be
purified from the hatching liquid. Therefore, the hatching of zebrafish
embryo is performed by a single enzyme, different from the finding that the
medaka hatching enzyme is an enzyme system composed of two enzymes,
medaka high choriolytic enzyme (MHCE) and medaka low chorio-
lytic enzyme (MLCE), which cooperatively digest the egg envelope. The six
ZHE1-cleaving sites were located in the N-terminal regions of egg envelope
subunit proteins, ZP2 and ZP3, but not in the internal regions, such as the
ZP domains. The digestion manner of ZHE1 appears to be highly analo-
gous to that of MHCE, which partially digests the egg envelope and swells
the envelope. The cross-species digestion using enzymes and substrates of
zebrafish and medaka revealed that both ZHE1 and MHCE cleaved the
same sites of the egg envelope proteins of two species, suggesting that the

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

euteleostean fishes, such as fugu Takifugu rubripes,
spotted green pufferfish Tetraodon nigroviridis and ayu
Plecoglossus altivelis altivelis [10]. Thus, the hatching
of euteleostean fishes can be performed by the HCE-
LCE system.
According to the phylogenetic tree based on the
mitochondorial DNA of Teleostei, Osteoglossomorpha
first branched off from an ancestor, followed by
Elopomorpha, and then branched paraphyletically to
Otocephala and Euteleostei [11–14]. The cDNA
cloning analysis using Japanese eel Anguilla japonica
belonging to Elopomorpha revealed that several hatch-
ing enzyme cDNAs were present, their nucleotide
sequences were similar to each other, and all formed a
monophyletic clade in the phylogenetic tree of fish
hatching enzymes [15]. These results suggest that the
hatching of eel embryos is performed by a single type
of enzyme. Therefore, HCE and LCE are considered
to have been produced by a gene duplication event
after Elopomorpha had diverged [16].
At present, and in contrast with such genetic
information, information at the protein level is
restricted to euteleostean fishes and is not available
for fishes belonging to Elopomorpha and Otocepha-
la. In the present study, we purified hatching enzyme
from embryos of zebrafish Danio rerio belonging to
Cypriniformes in Otocephala, analyzed the mecha-
nism of its egg envelope digestion and compared it
with that of medaka hatching enzyme. Finally, the
evolution of the mechanism of egg envelope diges-

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

observed at the 28th cycle (Fig. 1B). The result
suggests that the amount of ZHE1 transcript is quite
different from that of ZHE2. The amount of ZHE2
transcript is considered to be much lower than that of
ZHE1. Taken together with the results of the northern
blot analysis, the ZHE2 gene in developing embryos is
considered to be expressed to a very small extent.
Finally, the expression of ZHE genes was observed
by whole mount in situ hybridization. ZHE1 gene
expression was observed in the hatching gland cells
located on the yolk sac of 24 h embryos (Fig. 1C).
However, no positive signal for ZHE2 was observed in
the same staining condition. Several more days of
incubation with a substrate solution showed only a
weak ZHE2 signal in the cells (Fig. 1C). Thus, the
ZHE1 gene, and not the ZHE2 gene, is predominantly
expressed in zebrafish embryos.
Purification of ZHE
The hatching liquid (i.e. culture medium after embryos
hatched out) was used to purify ZHE. First, we
applied the concentrated hatching liquid onto a Super-
dex 75 10 ⁄ 300 GL column in the HPLC system
(Fig. 2A). Most of the protein was eluted just after the
K. Sano et al. Hatching enzyme of zebrafish
FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS 5935
void volume, and a single peak of caseinolytic activity
was eluted near the bed volume. After dialysis against
a25mm Tris–HCl buffer (pH 7.5), the fraction having
caseinolytic activity was applied onto a Source 15S
column in the HPLC system and eluted with a linear

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

10
20
30
40 500
Time (min)
0.1
0.2
0.3
0.2
0.4
0.6
0.8
B
A
A
280
A
280
Fig. 2. Purification of the hatching enzyme of zebrafish. (A) Super-
dex 75 10 ⁄ 300 GL column chromatogram of hatching liquid. The
solid line indicates A
280
and the dotted line shows caseinolytic
activity. (B) Source-15S column chromatogram of the caseinolytic
active fractions obtained from the Superdex 75 10 ⁄ 300 GL column
chromatograqphy. The sample was eluted with a line gradient from
0–1
M NaCl (broken line). The solid line indicates the protein
amount measured at A
280

. 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
Æmg
)1
protein) was higher than that of
purified medaka hatching enzymes (800 min
)1
Æmg
)1
protein for MHCE, 540 min
)1
Æmg
)1
protein for MLCE)
[4,5]. The result suggests that almost all rec. ZHE1
molecules were correctly refolded and had activity. By
contrast, rec. ZHE2 failed in the refolding.
rec. ZHE1 was completely inhibited by 1 mm
EDTA, but not by 10 mm diisopropylfluorophosphate
or 10 mm iodoacetic acid, consistent with the fact that
fish hatching enzymes generally belong to the metallo-
protease family. The substrate specificity of rec. ZHE1
was determined using various 7-amino-4-methylcou-
marin (MCA) peptides (Table 1). ZHE1 cleaved the
peptide bonds at the C-terminal side of Arg, Tyr, Asn,
Trp, Ala, Asp, Phe and Gly, suggesting that ZHE1 has
broad substrate specificity. One of the most suitable
substrates was Z-Phe-Arg-MCA, and the specific activ-
ity was 27.02 nmolÆ30 min
)1
Æmg

)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

enzyme)
rec. ZHE1
Hatching
liquid
Z-Phe-Arg-MCA 27.02 32.26
Suc-Leu-Leu-Val-Tyr-MCA 11.09 8.36
Boc-Phe-Ser-Arg-MCA 2.57 3.91
Z-Ala-Ala-Asn-MCA 2.33 1.26
Suc-Ile-Ile-Trp-MCA 1.55 2.01
Boc-Val-Pro-Arg-MCA 1.00 2.34
Suc-Ala-Pro-Ala-MCA 0.48 0.14
Ac-Asp-Glu-Val-Asp-MCA 0.40 0.28
Bz-Arg-MCA 0.34 0.47
Suc-Ala-Ala-Pro-Phe-MCA 0.22 0.32
Z-Leu-Arg-Gly-Gly-MCA 0.19 0.23
Suc-Ala-Glu-MCA ND ND
Z-Val-Lys-Met-MCA ND ND
Suc-GLy-Pro-Leu-Gly-Pro-MCA ND 0.39
A
D
B
E
C
F
Fig. 4. Morphological changes of egg envelope. The isolated egg
envelope of zebrafish before hatching (A), after hatching (B) and
digested by rec. ZHE1 (C) were observed using a binocular micro-
scope. Scale bars = 200 lm. (D–F) Electron microscopic observa-
tion of zebrafish egg envelopes. Sections of the egg envelope
isolated from pre-hatching embryo (D), the egg envelope after

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

molecular masses of 43 and 39 kDa were observed in
addition to undigested bands of ZP2 and ZP3. After
incubation for 10 min, three major bands with molecu-
lar masses of 43, 39 and 36.5 kDa were observed
(Fig. 5, lane 2). After further incubation (60 min), the
39 kDa band disappeared, and only two bands with
the same mobility as the 43 and 36.5 kDa products
were detected (Fig. 5, lane 3). These results indicate
that the 43 and 39 kDa products appear first, and the
39 kDa product is then further digested and shifted to
the 36.5 kDa product.
To determine rec. ZHE1-cleaving sites in egg enve-
lope subunits, we analyzed the sequence of each prod-
uct from its N-terminus. The sequences were compared
with those deduced from ZP2 and ZP3 cDNA [22,23].
It is well known that ZP2 is composed of an
N-terminal region (95 amino acids), an internal trefoil
domain and a C-terminal ZP domain ( 260 amino
acids) including eight consensus Cys residues (Fig. 6A).
ZP3 is composed of an N-terminal region (45 amino
acids) and a C-terminal ZP domain (Fig. 6B).
The detected N-terminal amino acid sequence of the
43 kDa product was APEPFT, which matched with
the sequence from Ala80 of ZP3, and we therefore
deduced that the cleaving site is Gln79 ⁄ Ala80 (Fig. 6,
Fig. 6. Amino acid sequences of ZP2 and ZP3 deduced from their
cDNAs. The arrows and capital letters (sites A to F) indicate the
cleaving sites of ZHE1 determined from the N-terminal amino acid
sequences of the ZHE1 digests. Circled Q indicates a glutamine
residue that is presumed to form a e-(c-glutamyl) lysine cross-link

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

rec. ZHE1. As a control, the SDS extract of intact egg
envelopes was analyzed by SDS ⁄ PAGE. Several bands
with a mobility that did not correspond to that of ZP2
and ZP3 were observed (Fig. 7, lane 1). These bands
are considered to be proteins that are secreted from
cortical alveoli and adhere to the envelope at fertiliza-
tion. The hardened, fertilized egg envelopes are not
considered to be solubilized by SDS.
The egg envelope is not soluble but became swollen
by rec. ZHE1. This swollen envelope was dissolved
into SDS and analyzed by SDS ⁄ PAGE. SDS ⁄ PAGE
of the fertilized egg envelope digested by rec. ZHE1
for 50 min gave three bands (150, 43 and 36.5 kDa),
which were not found in the control (Fig. 7, lane 2).
The N-terminal amino acid sequence of the 43 kDa
product (APEPFT) was identical to that of the 43 kDa
product obtained in the unfertilized egg envelope
digestion, suggesting that rec. ZHE1 cleaves the com-
mon sites of unfertilized and fertilized egg envelope
(Fig. 6, site E).
Amino acid sequence analysis of the 36.5 kDa
product revealed that this sequence was a mixture of
two peptide sequences. One of them was VEEV-
VVKAGPVDK and matched that of the 36.5 kDa
product from ZP2 in unfertilized egg envelope digests
(Fig. 6, site B). The other sequence, APLDLXE, did
not correspond to any cleaving site obtained in the
unfertilized egg envelope digestion. However, this
sequence was found in the sequence from Ala68 of
ZP3 (Gln67 ⁄ Ala68; Fig. 6, site D). This cleaving site

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

150 kDa products into the 36.5 and 43 kDa product.
All the results suggest that rec. ZHE1 cleaves the
N-terminal portions of ZP2 and ZP3 and eliminates
Fig. 7. SDS ⁄ PAGE patterns of fertilized egg envelopes. Lane 1,
SDS extract of intact fertilized egg envelopes; lanes 2 and 3, SDS
extract of egg envelopes digested by rec. ZHE1 for 50 and 90 min,
respectively; lane 4, SDS extract of egg envelopes after hatching.
Numbers on the left show the molecular masses of the major
bands.
K. Sano et al. Hatching enzyme of zebrafish
FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS 5939
the tight bindings between subunits by cleaving out the
small portion of peptides including e-(c-glutamyl)
lysine isopeptide cross-links.
We compared the cleaving sites determined from egg
envelopes after natural hatching (post-hatching) with
those artificially digested by rec. ZHE1. As shown in
Fig. 7 (lane 4), the SDS ⁄ PAGE pattern of the proteins
of the post-hatching egg envelopes was similar to that
digested by rec. ZHE1 for 90 min. The 43 kDa band
became weaker than that of the 50 min incubation,
suggesting further digestion of the 43 kDa product.
Furthermore, their 36.5 kDa bands became broader
than that for the 50 min incubation. The sequence
analyses of the 36.5 kDa bands revealed that two
sequences of further digested products were detected,
in addition to those of 36.5 kDa product obtained
from the 50 min incubation. One was a minor
sequence, QPASPG, which was found to locate at
Q106 of ZP3; the cleaving site is K105 ⁄ Q106 (Fig. 6,

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

2.47% of site A peptide, respectively). Therefore, these
values well reflected the results of the egg envelope
digestion experiment. According to the fertilized egg
envelope digestion experiment, the e-(c-glutamyl) lysine
isopeptide cross-links formed between ZP2 and ZP3
subunits are considered to be eliminated by cleaving of
site E. Thus, site E is conjectured to be a key cleaving
site leading to a conformational change that results in
swelling of the egg envelope. Therefore, it is reasonable
to consider that site E is one of the efficient cleaving
sites for ZHE1. The cleaving activity at site C, which
was detected in the 90 min digestion of the fertilized
egg envelope and considered as an inefficient cleaving
site, was not easily detected in this condition.
Species specificity of digestion by hatching
enzyme
As described earlier, zebrafish hatching is performed
by a single enzyme, ZHE1. Different from zebrafish,
hatching of medaka is performed by a two enzyme sys-
tem. To obtain an evolutionary aspect of the mecha-
nism of egg envelope digestion by hatching enzyme, we
changed the substrate–enzyme combination between
zebrafish and medaka and performed the cross-species
digestion experiment using unfertilized egg envelopes
as substrate. First, the unfertilized egg envelopes of
zebrafish were digested either by ZHE1, MHCE or
MLCE, and their SDS ⁄ PAGE patterns were com-
pared. SDS ⁄ PAGE of the MHCE digest after a 10 and
40 min incubation gave two bands (43 and 39 kDa),
and an additional band (36.5 kDa) was observed after

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

toward zebrafish unfertilized egg envelopes, although
the digestion efficiency of MHCE appears to be some-
what lower than that of ZHE1. By contrast, MLCE
digested the zebrafish egg envelopes and produced two
bands, the mobilities of which corresponded to 43 and
39 kDa ZHE1 digests (Fig. 8A, lanes 6–8). However,
the cleaving efficiency of MLCE appears to be less
than that of MHCE because a considerable amount of
undigested bands remained in the digest after a 10 min
incubation, and the 36.5 kDa band was not easily
detected even after a 120 min incubation. The sequence
analyses revealed that the N-terminal sequence of
43 kDa product digested by MLCE was identical to
that of site E cleaved by ZHE1 (Fig. 6). However, the
site in the 39 kDa product was not identical to the
ZHE1-cleaving site of the 39 kDa product (site A),
and the cleaving site was shifted one amino acid resi-
due to the N-terminal side from site A. Therefore, the
specificity of MLCE toward zebrafish egg envelope is
similar to, but somewhat different from, that of
ZHE1.
By contrast, the medaka unfertilized egg envelope
was digested by ZHE1. The SDS ⁄ PAGE pattern was
similar to that by MHCE (Fig. 8B, lanes 10 and 11).
The N-terminal sequences of the 47 and 35 kDa prod-
ucts in ZHE1 digest matched with those of MHCE
digests, suggesting that ZHE1-cleaving specificity
toward medaka unfertilized egg envelope is similar to
that of MHCE, and not MLCE (Fig. 8B, lane 12).
Comparison of specific activities of ZHE1,

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

Fig. 8. Cross-species digestion using hatching enzyme and the egg envelope of zebrafish and medaka. (A) Zebrafish unfertilized egg enve-
lopes (lane 1) were incubated with either ZHE1 (lane 2), MHCE (lanes 3–5) or MLCE (lanes 6–8). After incubation for 10 min (lanes 2, 3 and
6), 40 min (lanes 4 and 7) and 120 min (lanes 5 and 8), each digest was separated by SDS ⁄ PAGE. (B) Medaka unfertilized egg envelopes
(lane 9) were incubated with either ZHE1 (lane 10), MHCE (lane 11) or MLCE (lane 12) for 15 min. Each digest was separated by
SDS ⁄ PAGE. Numbers on the left are the sizes (kDa) of molecular markers, and those on the right are the molecular masses for major
bands.
K. Sano et al. Hatching enzyme of zebrafish
FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS 5941
that ZI1,2 possesses Pro-X-Y repeat sequences in its
N-terminal region, which are not found in that of
zebrafish ZP2. MHCE and MLCE are known to have
different cleaving specificity toward the medaka egg
envelope. MHCE mainly cleaves Pro-X-Y repeat
sequences present in the N-terminal region of ZI-1,2
[7]. By contrast, the most efficient cleaving site of
MLCE is in the center of the ZP domain of ZI-1,2.
Therefore, two peptide substrates were designed from
the amino acid sequences of Pro-X-Y repeat region,
named Pro-X-Y-1 and Pro-X-Y-2, for MHCE, and
another peptide substrate was designed from the amino
acid sequence of ZP domain, named ZP domain
(ZPD)-center for MLCE. As we expected, MHCE effi-
ciently cleaved Pro-X-Y-1 and Pro-X-Y-2, whereas
MLCE efficiently cleaved ZPD center (Fig. 9). In addi-
tion, ZHE1 well cleaved Pro-X-Y-1 and Pro-X-Y-2,
the sites for MHCE, and their specific activity values
were approximately 80% of those of MHCE, whereas
ZHE1 did not easily cleave the ZPD-center, the site
for MLCE. The results suggest that ZHE1 has the
MHCE-like activity toward medaka egg envelope; this

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-

tionary process, namely a mutation(s) in the amino
acid sequence of ZHE2 changed its substrate specificity
as a proteolytic enzyme and, eventually, ZHE2 become
uninvolved in the egg envelope digestion. Subse-
quently, the amount of its expression would be
decreased by accumulation of a mutation(s) in a regu-
latory region(s) responsible for gene expression.
100
50
0
100
50
0
0
25
50
0
25
50
0
25
50
10
5
0
Site-A
Site-B
n mole
–1
·30 min

–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

·30 min
–1
·µg
–1
enzyme
Site-E Pro
XY-1
Pro
XY-2
ZPD
-Center
Pro
XY-1
Pro
XY-2
ZPD
-Center
Pro
XY-1
Pro
XY-2
ZPD
-Center
Site-A
Site-B
Site-E
Site-A
Site-B
Site-E
ZHE

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

that ZHE1 and MHCE cleaved the same sites on both
zebrafish and medaka egg envelopes with a similar effi-
ciency. ZHE1 swelled the medaka egg envelope but did
not solublize its swollen envelope (data not shown).
Such an agreement is surprising when it is considered
that zebrafish and medaka diverged 140 million years
ago [27]. From an evolutionary aspect on egg envelope
digestion, ZHE1 and MHCE are presumed to maintain
the substrate specificity of a common ancestral hatching
enzyme. By contrast, the cleaving specificity of MLCE
toward the zebrafish egg envelope was similar to those
of ZHE1 and MHCE, but its cleaving efficiency was
approximately ten-fold lower. Furthermore, MLCE had
another efficient cleaving site, the center of ZP domain,
where ZHE1 and MHCE hardly cleave. These results
imply that MLCE changed its substrate specificity to
one different from that of an ancestral enzyme, although
its substrate specificity still remains to a certain extent.
Therefore, we consider that the single enzyme-depen-
dent swelling of the egg envelope in zebrafish is closely
related to an original or ancestral form of egg envelope
digestion, and the HCE-LCE system comprises a more
developed form. HCE and ZHE1 would inherit the
character of the ancestral enzyme with respect to the
swelling of egg envelope. After gene duplication and
diversification, LCE would be produced by changing its
substrate specificity and would acquire a new function,
the digestion of the swollen egg envelope.
On comparing amino acid sequences between zebra-
fish and medaka egg envelope subunits, we see that the

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

co-evolve hatching enzyme and egg envelope subunit
protein. To understand such a rule, it is necessary to
obtain more information from other fish species, such
as the Japanese eel belonging to Elopomorpha that is
sister to the common ancestor of zebrafish and
medaka. The present study is the first approach aiming
to fully understand the molecular mechanism of
co-evolution of hatching enzyme and egg envelope.
A further study is now in progress.
Experimental procedures
Fish
Wild-type embryos of the Ab strain of zebrafish were used.
Embryos were obtained from natural mating and cultured
in tap water at 30 °C. After 42 h of culture, the embryos
were transferred into a beaker with a small amount of med-
ium containing 10 mm NaCl and 2 mm NaHCO
3
and
allowed to hatch. When 80–90% of the embryo hatched
out, the culture medium, now termed hatching liquid, was
filtered, frozen and stored at )20 °C.
Northern blot analysis
Ten microgram of total RNA extracted from embryos at
11.5 or 24 h, or after hatching, were electrophoresed on a
K. Sano et al. Hatching enzyme of zebrafish
FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS 5943
1% formaldehyde-agarose gel and transferred to nylon
membrane (Hybond-N+; GE Healthcare, Amersham,
UK). A digoxigenin-labeled DNA probe was synthesized
with PCR Probe Synthesis Kit (Roche, Indianapolis, IN,

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

CTGAGACTAAATGG-3¢, reverse: 5¢-GGAAATAAGAG
CACGTACTGTGG-3¢.
The cycle number of PCR was adjusted for each gene so
that it to barely showed visible bands on agarose gels.
Aliquots of PCR were loaded on 1% agarose gels and
stained with ethidium bromide.
Whole mount in situ hybridization
Whole mount in situ hybridization was performed
according to the procedures described previously [9]. The
DIG-labeled RNA probes were hybridized to 24 h
embryos.
Purification of hatching enzyme
Approximately 50 mL of hatching liquid derived from
approximately 1000 embryos was concentrated by Amicon
Ultra 15 Ultracel-10K (Millipore Co., Billerica, MA, USA).
Approximately 500 lL of the concentrated hatching liquid
was applied to a Superdex 75 10 ⁄ 300 GL column (GE
Healthcare) in the HPLC system (Gilson, Middleton, WI,
USA) equilibrated with a 50 mm bicarbonate buffer (pH
10.0). The fractions having proteolytic activity were
collected and dialyzed against a 25 mm Tris–HCl buffer
(pH 7.5). The sample was applied to a Source 15S
column (GE Healthcare) in the HPLC system and eluted
with a linear gradient of 0–1 m NaCl in a 25 mm Tris–HCl
buffer (pH 7.5). The fraction having proteolytic
activity was dialyzed against a 25 mm Tris–HCl buffer
(pH 7.5).
Medaka hatching enzymes, MHCE and MLCE, were
purified from hatching liquid as previously described [4,5].
Recombinant ZHE1

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

times with 5% Triton X-100 in a 50 mm Tris–HCl buffer
(pH 8.0) with 1 mm EDTA, followed by sonication, incuba-
tion (37 °C for 30 min) and centrifugation (5800 g for
10 min). One-fourth of the resulting pellet, predominantly
consisting of the inclusion body, was dissolved in 200 lLof
8 m urea and 0.1 m 2-mercaptoethanol in a 50 mm
Tris–HCl buffer (pH 8.0) and incubated at 37 °C for 1 h.
After centrifugation (5800 g for 10 min), the supernatant
was added to 150 mL of a 50 mm Tris–HCl buffer (pH 8.0)
containing 1 mm glutathione, 0.1 mm oxidized glutathione,
0.8 ml-arginine and 5 lm ZnSO
4
, stored at 4 °C for more
than 24 h and dialyzed against a 50 mm Tris–HCl buffer
(pH 8.0). After filtration, the folding mixture was loaded
onto Ni-NTA Superflow (Qiagen), and the elution was
achieved by a 50 mm Tris–HCl buffer (pH 8.0) containing
0.15 m NaCl and 0.4 m imidazole.
Hatching enzyme of zebrafish K. Sano et al.
5944 FEBS Journal 275 (2008) 5934–5946 ª 2008 The Authors Journal compilation ª 2008 FEBS
Digestion of egg envelope by ZHE1
Unfertilized eggs were isolated from spawning female fish
and homogenized in a saline mixture containing 5 mm
EDTA and 5 mm iodoacetic acid. After centrifugation
(2000 g for 30 s), the supernatant was decanted. This proce-
dure was repeated several times to completely remove cell
debris. Egg envelopes thus isolated were used for digestion
experiments. Six unfertilized egg envelopes were incubated
in 10 lLof50mm Tris–HCl (pH 7.5) containing 0.3 lgof
rec. ZHE1 or purified ZHE at 30 °C, and the resulting

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,

Foster City, CA, USA). The purified ZHE1 was also dotted
to the poly(vinylidene difluoride) membrane and applied to
the sequencer.
Caseinolytic activity
The caseinolytic activity of enzyme was measured using a
750 lL reaction mixture comprising 3.3 mgÆmL
)1
of casein
and enzyme in a 50 mm Tris–HCl (pH 7.5). The mixture
was incubated for 1 h at 30 °C. The reaction was stopped
by adding 250 lL of 20% perchloric acid, and the material
was allowed to stand in ice for 10 min. The mixture was
centrifuged at 18 500 g for 5 min at 4 °C. A
280
of the super-
natant was measured.
MCA cleaving activity
A 250 lL reaction mixture containing 100 lm MCA
peptide, peptidyl-4-methylcoumaryl-7-amides (Peptide Insti-
tute, Inc., Osaka, Japan) and enzyme in a 50 mm Tris–HCl
buffer (pH 7.5) was incubated at 30 °C for 30 min. After
the reaction was stopped by adding 500 l L of 20% acetic
acid, fluorescence was measured with a fluorescence
spectrophotometer at 380 nm (excitation) and 460 nm
(emission).
SDS ⁄ PAGE
SDS ⁄ PAGE was performed by the method of Laemmli
using a 12.5% gel [30]. The gel was stained with Coomassie
Brilliant Blue G or using a Silver Stain Kit (Wako, Osaka,
Japan).

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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