From the Department of Biochemistry, Graduate School
of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan and the § Institute of Applied Biochemistry,
University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan
Received for publication, December 18, 2000, and in revised form, April 16, 2001
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ABSTRACT |
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cDNA cloning and functional analysis of
proacrosin from the ascidian Halocynthia roretzi were
undertaken. The isolated cDNA of the ascidian preproacrosin
consists of 2367 nucleotides, and an open reading frame encodes 505 amino acids, which corresponds to the molecular mass of 55,003 Da. The
mRNA of proacrosin was found to be specifically expressed in the
gonad by Northern blotting and in the spermatocytes or spermatids by
in situ hybridization. The amino acid sequences
around His76, Asp132, and Ser227,
which make up a catalytic triad, showed high homology to those of the
trypsin family. Ascidian acrosin has paired basic residues (Lys56-His57) in the N-terminal region, which
is one of the most characteristic features of mammalian acrosin. This
region seems to play a key role in the binding of (pro)acrosin to the
vitelline coat, because the peptide containing the paired basic
residues, but not the peptide substituted with Ala, was capable of
binding to the vitelline coat. Unlike mammalian proacrosin,
ascidian proacrosin contains two CUB domains in the C-terminal region,
in which CUB domain 1 seems to be involved in its binding to the
vitelline coat. Four components of the vitelline coat that are capable
of binding to CUB domain 1 in proacrosin were identified. In response
to sperm activation, acrosin was released from sperm into the
surrounding seawater, suggesting that ascidian acrosin plays a key role
in sperm penetration through the coat. These results indicate that ascidian sperm contains a mammalian acrosin homologue, a
multi-functional protein working in fertilization.
In fertilization, sperm must bind to and penetrate through the
extracellular glycoprotein matrix surrounding the egg, which is called
the zona pellucida in mammals and the vitelline coat in marine
invertebrates. After this process, membrane fusion occurs between the
sperm and egg. Upon primary binding of the sperm to the vitelline coat,
it undergoes an acrosome reaction, which is an exocytosis of the
acrosomal vesicle located on the tip of the sperm head. A lytic agent
called lysin is exposed on the surface of the sperm head and partially
released into the surrounding seawater during the acrosome reaction. In
mammals, it has been believed that a trypsin-like enzyme called acrosin
(EC 3.4.21.10) is a zona lysin (1, 2). However, recent studies using
acrosin-gene knockout mice have revealed that acrosin is not essential
for mouse fertilization, although a significant delay (about 30 min) in
sperm penetration through the zona pellucida was observed (3, 4). From
these results it is currently thought that sperm proteases other than
acrosin may participate in sperm penetration through the mammalian egg
coat and that acrosin may be involved in the dispersal of the acrosomal
matrix (5).
Ascidians (Urochordata) occupy a phylogenetic position between
vertebrates and "true" invertebrates. Although all ascidians are
hermaphrodites, several ascidians including Halocynthia
roretzi are strictly self-sterile. The vitelline-coat lysin system
is thought to be activated after the sperm recognizes the vitelline coat of the egg as nonself. To elucidate the roles of sperm proteases in fertilization, we have been studying the sperm proteases of the
ascidian H. roretzi, one of the largest ascidians cultivated for food in Onagawa Bay, Japan. From this animal we can obtain a large
amount of sperm and eggs by controlling the seawater temperature and
light conditions. Fertilization experiments with this broadcast spawning animal are much easier than those with mammals. We have reported previously that the sperm trypsin-like and chymotrypsin-like proteases are indispensable for sperm penetration through the vitelline
coat in H. roretzi by examining the effects of various protease inhibitors on the fertilization of intact as well as naked
eggs (6, 7). We have purified two trypsin-like proteases (which we
designated as ascidian acrosin and spermosin) from H. roretzi sperm, and we showed that these two proteases play key roles in ascidian fertilization (8-10). The enzymatic properties, including the substrate specificity, of ascidian acrosin are very similar to those of mammalian acrosin. However, to determine whether ascidian acrosin is a mammalian acrosin homologue, information on its
amino acid sequence is required. In the present study, cDNA cloning
and functional analysis of ascidian proacrosin were carried out. It was
found that ascidian proacrosin has homology to mammalian proacrosin and
contains interesting sequences or domains, which were demonstrated to
be necessary for proacrosin to bind to the vitelline coat of the egg.
To the best of our knowledge, this is the first report showing the
existence of sperm acrosin or a mammalian acrosin homologue in invertebrates.
Purification of Ascidian Acrosin--
Sperm of the solitary
ascidian H. roretzi was collected as described previously
(6, 7). Ascidian acrosin was purified according to the procedure
described previously (8). The enzymatic activity was determined using
t-butoxycarbonyl-Val-Pro-Arg-4-methylcoumaryl-7-amide as a
substrate (7, 8).
Analysis of the N-terminal Amino Acid Sequence of
Acrosin--
SDS-PAGE1 was
carried out in a slab gel (12.5%) according to the method of Laemmli
(11). Purified acrosin was subjected to SDS-PAGE and
electrophoretically transferred to a polyvinylidene difluoride membrane
(Millipore). The blotted membrane was stained with 0.1% Coomassie
Brilliant Blue R-250 containing 1% acetic acid and 40% methanol.
After washing with 50% methanol, the band was cut off from the
membrane. The N-terminal sequence of the purified acrosin was
determined using a Procise 492 Protein Sequencer (PerkinElmer Life Sciences).
Construction of an Ascidian Gonad cDNA Library--
Total
RNA was extracted from one gonad of H. roretzi type C by the
acid guanidinium-phenol-chloroform procedure. Poly(A)+ RNA
was prepared from the total RNA with oligotex-dT30 (Roche). cDNAs
were synthesized by SuperScript II reverse transcriptase using 4 µg
of poly(A)+ RNA (a template), 1 µg of oligo(dT), and 0.1 µg of random hexamers (primers) according to the protocol of the
manufacturer of the SuperScript Choice System for cDNA synthesis
(Life Technologies, Inc.). The cDNAs thus obtained were subjected
to cDNA size-fractionation column chromatography followed by
ligation to an EcoRI-digested and dephosphorylated Cloning and Sequencing of Ascidian Acrosin cDNA--
The
sense primer used for PCR was designed from the N-terminal sequence of
ascidian acrosin
(5'-GG(T/C/A/G)GA(A/G)TT(T/C)CC(T/C/A/G)TGGCA(A/G)GC-3'). This primer
encodes the amino acid sequence of GEFPWQ. The antisense primer was
designed from the consensus sequence in the vicinity of the serine
residue in the catalytic triad of the trypsin family (5'-GGICCICCI(C/G)(A/T)(A/G)TCIC(C/T)CTG(A/G)CA-3'). The antisense primer encodes the amino acid sequence of CQGDSGGP. The primers, each
at a concentration of 10 µM, were mixed in PCR to amplify the ascidian gonad cDNA library. PCR was done in 10 mM
Tris-HCl (pH 9.5) containing 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 4 mM
deoxynucleotides, and 5 units/ml AmpliTaq Gold DNA polymerase (PerkinElmer Life Sciences). After denaturation at 94 °C for 3 min,
30 cycles were run with annealing at 45 °C for 2 min, elongation at
72 °C for 3 min, and denaturation at 94 °C for 1 min. A DNA band
migrating at about 600 base pairs was isolated, cloned into a pCRII
vector (Invitrogen), and transformed into Escherichia coli
DH5 Northern Blot Analysis--
Total RNA was extracted from each
tissue according to the standard acid guanidinium-phenol-chloroform
procedure. Poly(A)+ RNA was isolated from the total RNA by
using oligotex-dT30. Two µg each of poly(A)+ RNA of the
ascidian gonad, hepatopancreas, intestine, and gill were subjected to
electrophoresis on 1.2% agarose gel containing 6% formaldehyde, and
RNA bands were transferred to a Hybond N+ nylon membrane
(Amersham Pharmacia Biotech). The stringency used for hybridization and
washing and the probe was the same as that used in cloning.
After washing, the blotted membranes were autoradiographed at
In Situ Hybridization--
Ascidian acrosin cDNA was
amplified with two primers. The sense primer was
5'-GGTGAAATGGCAAAATTGGGC-3', which corresponds to GEMAKLG in the
N-terminal sequence, and the antisense primer was
5'-TAAATCAGCTGCGTCTGCGCT-3', which corresponds to SADAADL in the
C-terminal sequence. A DNA band migrating at 1010 base pairs was
isolated, cloned into a pCRII vector (Invitrogen), and transformed into
E. coli DH5 Preparation of Antibody--
A 14-residue oligopeptide, A2
(RVADLDKTDDTDEG), corresponding to residues 96-109 in the ascidian
acrosin sequence was synthesized and coupled to keyhole limpet
hemocyanin according to the method described previously (13). An
antibody raised against ascidian acrosin peptide A2 was purified by
ammonium sulfate fractionation followed by affinity chromatography with
peptide A2-immobilized agarose beads (Affi-Gel 10, Bio-Rad).
Sperm Reaction--
The activation of ascidian sperm was
elicited by gentle stirring at 13 °C for 20 min in alkaline
artificial seawater (pH 9.0). After stirring, the sperm suspension was
centrifuged at 5000 × g for 20 min to obtain the
supernatant, which is called the sperm exudate. Sperm suspension in
artificial seawater (pH 8.0) was used as a control.
Extraction of the Vitelline Coat--
The ascidian eggs were
homogenized with 5-fold diluted (20%) artificial seawater containing
0.1 mM diisopropylfluorophosphate. The homogenate was
filtered through nylon mesh (pore size, 150 µm), and the vitelline
coat on the blotting cloth was washed extensively with 20% artificial
seawater. The purity of the isolated vitelline coats was examined under
a light microscope. The vitelline coats were suspended in artificial
seawater containing 0.5% Triton X-100. After stirring for 30 min at
4 °C, the suspension was centrifuged at 10,000 × g
for 30 min to obtain the supernatant as the solubilized vitelline coat.
Vitelline Coat Binding Assay--
Five hundred µg of
sulfosuccinimidyl-6-(biotinamido)hexanoate biotinylating agent
(Pierce) was added to 1 ml of the solubilized vitelline coats in
artificial seawater (~20% suspension). After incubation for 30 min
at 4 °C, the solubilized vitelline coat was dialyzed against
artificial seawater to remove nonreacted sulfosuccinimidyl-6(biotinamido)-hexanoate biotinylating agent. The
following two 12-residue oligopeptides corresponding to residues 51-62
in the ascidian proacrosin were synthesized and purified by reverse
phase high pressure liquid chromatography: A1, AAFLYKHVQVCG (residues
51-62) and A1 (KH/AA), AAFLYAAVQVCG. One hundred µl of 1 mM oligopeptide was added to each well of a 96-well plate, and the plate was allowed to stand overnight to adsorb the peptide. After adsorption, each well was blocked with 5% bovine serum albumin and then treated with the biotinylated solubilized vitelline coat. After incubation for 2 h at 4 °C, the plate was washed four
times with artificial seawater and incubated with avidin-biotinylated peroxidase complex (Vectastain) in artificial seawater for 30 min
according to the manufacturer's protocol. The plate was washed four
times with artificial seawater followed by treatment with 0.05%
3,3'-diaminobenzidine and 0.02% H2O2 to
develop the color. Finally, the developed color was measured at 490 nm
using a Bio-Rad Model 450 microplate reader.
Effect of the CUB Domain on Fertilization--
Two 15-residue
oligopeptides at residues 348-362 in the CUB domain 1 and 443-456 in
the CUB domain 2, respectively, of ascidian proacrosin were
synthesized: CUB1 (TEFGVEYHTFCWYDD) and CUB2 (CGEFSSKHYPNYYDA). The
eggs were incubated previously with each oligopeptide, CUB1 or CUB2, in
seawater for 30 min at 13 °C and were then inseminated. The
fertilization ratio was determined as described previously (6, 7).
Isolation of CUB Domain-binding Components of the Vitelline
Coat--
CUB1 or CUB2 peptide-immobilized agarose was prepared using
Affi-Gel 10 (Bio-Rad) according to the manufacturer's protocol. The
biotinylated solubilized vitelline coat was incubated with CUB1 or CUB2
peptide-immobilized agarose for 1 h at 4 °C. After washing with
artificial seawater, the components of the vitelline coat were eluted
with SDS-PAGE sample buffer and subjected to SDS-PAGE. The protein
bands were transferred to the polyvinylidene difluoride membrane and
subjected to determination of the N-terminal amino acid sequences as
described previously.
The N-terminal 44-amino acid sequence of the purified acrosin was
determined using a protein sequencer (Fig.
1). The N-terminal sequence of acrosin
was used to design the degenerate oligonucleotide sense primers for PCR
of the H. roretzi gonad cDNA library. The antisense
primer was designed from the consensus sequence around the active site
serine residue of the trypsin family. The deduced amino acid sequence
of the PCR product contained the N-terminal peptide sequence of acrosin
determined by N-terminal amino acid sequencing. The PCR product was
used as a probe for screening the ascidian gonad
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gt11
vector (Stratagene). Then, in vitro packaging of the DNA was
carried out using a Gigapack III Gold kit (Stratagene).
. Ascidian acrosin cDNA clones were picked from 2 × 106 clones of ascidian gonad
gt11 cDNA liberally by
phage plaque hybridization using the PCR-amplified DNA fragment
encoding acrosin as a probe (12). The probe was labeled with
[
32P]dCTP (BcaBEST kit, Takara) by the random-priming
procedure. Briefly, the plaque lifts were prehybridized at 42 °C in
5× SSC (1× SSC: 15 mM sodium citrate-HCl (pH 7.0) and
0.15 M NaCl) containing 0.02% Ficoll 400, 0.02%
polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.02% SDS, 50%
formamide, and 0.1 mg/ml yeast tRNA. Hybridization was carried out at
42 °C overnight in a prehybridization buffer containing a
32P-labeled probe. The membranes were washed with 2× SSC
containing 0.1% SDS at room temperature for 10 min and twice at
60 °C for 15 min before autoradiography at
80 °C. Three
independent positive clones were obtained. The nucleotide sequence of
the acrosin cDNA clone was determined by a Big Dye Terminator Cycle
Sequencing Ready Reaction kit (Applied Biosystems) using an ABI 377A
DNA sequencing apparatus (Applied Biosystems).
80 °C (Fuji x-ray film).
. An acrosin clone in the pCRII vector was
reacted with EcoRI. An ~1020-base pair fragment was
gel-purified from an agarose gel and ligated into a pBluescript SK(+)
plasmid vector. The recombinant plasmid was transformed into E. coli DH5
. The sense and antisense cRNAs were synthesized by
using T3 and T7 RNA polymerases, respectively. During the synthesis of
the cRNAs, digoxigenin-labeled uridine triphosphate was incorporated according to the protocol of Roche Molecular Biochemicals. A gonad of
H. roretzi was fixed with 4% paraformaldehyde in artificial seawater and embedded in paraffin. Sections of 5 µm in width were prepared. A subsequent enzyme-catalyzed color reaction was conducted by
the addition of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt, and the mixture was incubated overnight at room temperature in the dark.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gt11 cDNA
library to isolate a cDNA clone encoding ascidian acrosin (Fig. 1).
The cDNA clone consisted of 2367 nucleotides. A single open reading
frame encoded 505 amino acids. The deduced protein sequence contained a
sequence from residues 36 to 79, which corresponds to the N-terminal
amino acid sequence determined from the isolated acrosin protein. The
molecular mass of preproacrosin was estimated to be 55,003 Da. The
sequence of the N-terminal 19 residues of preproacrosin contained a
highly hydrophobic region, probably corresponding to a signal peptide for a nascent protein destined for initial transfer to the endoplasmic reticulum. Thus, the N terminus of proacrosin may start at Asp-20. His,
Asp, and Ser, which form a catalytic triad in serine proteases, are
located at residues 76, 132, and 227, respectively, in ascidian proacrosin. As compared with mammalian proacrosin, ascidian proacrosin has three consensus sequences for N-linked carbohydrate
chain modification in the C-terminal region but not in the region
commonly observed in mammalian proacrosin (e.g. mouse
acrosin, Cys-Asn211-Ser-Thr; ascidian acrosin,
Cys-Leu200-Ala-Thr). Ascidian acrosin also has paired basic
residues in the N-terminal region, which are thought to be critical for
the binding of acrosin to the zona pellucida (14). In addition, ascidian proacrosin, but not mammalian proacrosin, has two CUB domains
in the C-terminal region. It is known that CUB domains are involved in
sperm binding to the zona pellucida in the mammalian spermadhesin
molecule (15-18).
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Fig. 1.
Nucleotide and deduced amino acid sequences
of H. roretzi preproacrosin. The presumed
cleavage sites in preproacrosin and proacrosin are indicated by an
arrow and arrowhead, respectively. The
deduced amino acid sequence matching the N-terminal sequence of purified acrosin is underlined by a solid line. Paired
basic amino acid residues are indicated by an open square
box. The conserved active site residues (His, Asp, and Ser) in the
trypsin family are indicated by shaded square boxes. The
putative N-linked glycosylation sites are indicated by
asterisks. The CUB domains 1 and 2 are underlined by a
broken line and a dotted line,
respectively.
Northern blot analysis was carried out using the same probe as that
used for the screening of cDNA. As shown in Fig.
2, a single transcript of ~4.7
kilobases was detected only in the gonads of H. roretzi. The
expression of proacrosin mRNA was also investigated by in
situ hybridization. A specific signal was observed in
spermatids/spermatocytes (spermatids and spermatocytes are
indistinguishable in ascidians by light microscopy) but not in mature
sperm with the antisense riboprobe. A specific signal was not observed
with the sense riboprobe. These results indicate that ascidian
proacrosin mRNA is definitely expressed in both or either of
spermatids and spermatocytes (data not shown).
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Western blot analysis of the sperm extract demonstrated the presence of
35-, 40-, and 50-kDa proteins that immunoreacted with the anti-acrosin
antibody under nonreducing conditions (Fig.
3). Because proacrosin has a molecular
mass of 53,052 Da, the 50-kDa protein seems to be proacrosin. It is
inferred that proacrosin with a molecular mass of 53 kDa is processed
into the 35-kDa acrosin through the 40-kDa protein as an
intermediate.
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Ascidian sperm undergoes sperm reaction, accompanied by vigorous sperm
movement and mitochondrial shedding, upon sperm binding to the
vitelline coat during fertilization (19). This sperm reaction is
mimicked by increasing the extracellular pH to 9.0. When sperm of
H. roretzi were treated with alkaline artificial seawater
(pH 9.0), a similar morphological change was induced. As shown in Fig.
4, ascidian acrosin was found to be
released from sperm treated with the artificial seawater (pH 9.0). This result suggests that ascidian acrosin is released from sperm during fertilization. In contrast, proacrosin was not detected in the sperm
exudate, suggesting that only the active form of acrosin is released
from the reacted sperm.
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Ascidian proacrosin contains two interesting sequences: one is the paired basic residues-containing sequence, and the other is the CUB domain. First, the role of the former sequence was investigated. Peptide A1 containing the paired basic residues of ascidian acrosin and the peptide A1(KH/AA), the paired basic residues of which had been substituted with Ala-Ala, were synthesized and tested for their abilities to bind to the isolated vitelline coats. The binding ability of peptide A1 containing the paired basic residues (Lys-His) to the vitelline coat was found to be 2.5-fold higher than that of peptide A1(KH/AA).
Next, the role of the CUB domain was investigated. Ascidian
proacrosin contains two CUB domains in the C-terminal region, one being
complete and the other being incomplete (Fig. 1). Two peptides called
CUB1 and CUB2, which are derived from CUB domains 1 and 2, respectively, were synthesized, and their ability to inhibit
fertilization of H. roretzi was tested. As shown in Fig. 5A, the CUB1 peptide inhibited
the fertilization more strongly than did the CUB2 peptide. The fact
that fertilization is strongly inhibited by the CUB1 peptide suggests
that the CUB domain 1 can interact with the vitelline coat.
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To identify the CUB domain 1-binding components of the vitelline coat,
a pull-down assay using CUB1 peptide-immobilized agarose beads was
carried out. Five components (90-, 85-, 30-, 28-, and 25-kDa proteins)
of the vitelline coat were found to be capable of binding to CUB1
peptide-immobilized agarose beads but not to CUB2 peptide-immobilized
agarose beads (Fig. 5B). The N-terminal sequences of the
90-, 85-, 30-, and 25-kDa proteins were determined (Fig.
5C). However, we could not find homologous or similar
proteins to the above proteins, which are involved in cell-cell
interaction as determined by FASTA and BLAST search analyses.
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DISCUSSION |
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Here we cloned a cDNA of mammalian acrosin
homologue from the solitary ascidian H. roretzi
(Urochordata), and also we studied a functional analysis of the
ascidian acrosin. This is a first report showing the occurrence of
acrosin homologue in nonmammalian species. Ascidian acrosin seems to be
synthesized as a preproprotein with 505 amino acid residues and with a
signal sequence consisting of 19 putative residues in the N-terminal
region. By motif search analysis, it was found that ascidian proacrosin
has three consensus sequences of N-linked sugar attachment,
paired basic residues in the N-terminal region that are thought to be
critical for the binding of acrosin to the zona pellucida (14), a
protease domain in the middle region, and two CUB domains in the
C-terminal region. The amino acid sequences around the active site
residues in ascidian acrosin, His76, Asp132,
and Ser227, showed high homology to those of the trypsin
family including mammalian acrosin (Fig.
6). Identity in the total amino acid
sequence between mammalian proacrosins and ascidian proacrosin is
33-35%. A dendrogram analysis revealed that H. roretzi
acrosin is most similar to mammalian acrosin family members (see Fig.
7). The result that the branching point
of ascidian acrosin in the acrosin clade is far from the cluster of
mammalian acrosin may reflect the phylogenetic relationship between
these two classes (Ascidiacea and Mammalia). In addition to the
homology in the amino acid sequences, the ascidian proacrosin is
thought to be a homologue of mammalian proacrosin for the following
three reasons. First, ascidian proacrosin was expressed in the gonad
but not in the other tissues tested. Second, this protease contains
vitelline coat-interactable paired basic residues in the N-terminal
region, the position of which coincides well with that of mammalian
acrosin. Third, the existence of the C-terminal extension (two CUB
domains in the case of ascidian proacrosin) of zymogen is a
characteristic feature observed in mammalian proacrosin among members
of the trypsin family.
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The purified acrosin gave bands of 34 and 35 kDa under reducing and nonreducing conditions, respectively (data not shown). By analogy to the mammalian acrosin, it is thought that the processed ascidian acrosin probably consists of a short (16 residues) light chain and a heavy chain, both of which are disulfide linked. Taking into account the conserved positions of the cysteine residues in mammalian acrosins, it can be inferred that Cys21 in the light chain is disulfide-bonded to Cys152 in the heavy chain in ascidian acrosin.
The results of the amino acid composition analysis (data not shown) of the purified ascidian acrosin suggested that the C-terminal amino acid is situated in the vicinity of residue 290. If ascidian proacrosin is cleaved autocatalytically, as are mammalian proacrosins, the C-terminal amino acid residue of activated acrosin would be Arg286, because the Arg residues are located at 286, 269, and 254 in the presumed C-terminal region of active acrosin. If it is the case, the C-terminal CUB domains would be completely removed by autocatalytic activation.
In mammalian proacrosin, the participation of the N-terminal region in the binding to the zona pellucida was confirmed by using synthetic peptides and recombinant proteins, and recent studies on site-directed mutagenesis have revealed that the paired basic residues in the N-terminal region of acrosin are crucial for maintenance of the binding of proacrosin to the zona pellucida (14). It was found that ascidian acrosin also has paired basic residues, Lys56-His57, in the N-terminal region that are involved in the binding to the vitelline coat; the binding ability of the peptide containing paired basic residues (Lys-His) to the vitelline coat was found to be higher than that of the peptide containing Ala-Ala. The present findings agree with the above-mentioned finding on the role of paired basic residues in mammalian proacrosin. (14).
Several mammalian proacrosins, including human and porcine
proacrosins but neither mouse nor rat proacrosin, contain a Pro-rich region in the C terminus that is cleaved off during proacrosin activation (20). Because proacrosin and -acrosin, unlike the fully
activated form
-acrosin, are able to strongly bind to the zona
pellucida (21), it is thought that the C-terminal region may also be
involved in the binding to the zona pellucida (21). Ascidian
proacrosin, but not mammalian proacrosins, has two CUB domains in the
C-terminal region. The CUB domain is defined as a 100-110
residue-spanning extracellular module, the name of which is derived
from the following three proteins: complement subcomponents (C1r/C1s) (22, 23), an embryonic sea urchin protein
(Uegf) (24), and bone morphogenetic protein I
(BMP-I) (25, 26). The most characteristic features of the
CUB domain are the existence of three or four conserved Cys residues
that form two disulfide bonds and the existence of various hydrophobic
and aromatic amino acid residues that participate in the formation of
the antiparallel
-barrel topography of the molecule. CUB
domains in mammalian spermadhesin are known to be involved in sperm
binding to the zona pellucida (15-18). Our present results showing
that the C-terminal CUB domain 1 of ascidian proacrosin is involved in
the binding to the vitelline coat coincide well with the
above-mentioned fact regarding the CUB domain in mammalian
spermadhesin. Although ascidian proacrosin does not possess a Pro-rich
region in the C-terminal region, the CUB domains seem to work in the
binding of proacrosin to the vitelline coat as a functional homologue
of the Pro-rich region in mammalian proacrosin.
Of two peptides derived from CUB domains 1 and 2, the peptide of CUB domain 1 strongly inhibited fertilization. Five components (90-, 85-, 30-, 28-, and 25-kDa proteins) in the vitelline coat were found to be able to bind to CUB1 peptide-immobilized agarose beads. It is likely that the CUB1 peptide binds to the vitelline coat, resulting in the inhibition of fertilization. Although we failed to identify the homologous proteins to the above-mentioned five components, it is intriguing to note that the 30-kDa component contains an HAV motif, which is a key element in homophilic cadherin interactions (27). This motif may play an important role in the interaction between the 30-kDa component of the vitelline coat and the CUB domain 1 of ascidian proacrosin. In connection with this, it is also interesting that the CUB domain 2 also contains a His421-Ala422-Val423 sequence. Therefore, we cannot rule out the possibility that CUB domain 2 is also involved in the binding to the vitelline coat.
Concerning the reason why mammalian sperm exposes acrosin and
proacrosin after the acrosome reaction, an interesting hypothesis called "binding-releasing mechanism" in mammals has been proposed (28). After the acrosome reaction, exposed proacrosin in sperm binds to
the zona pellucida. Once this binding is established, autodigestion of
proacrosin takes place, leaving the sperm free to bind again. This
sequential mechanism would facilitate sperm penetration through the
zona pellucida. In H. roretzi, we could not detect a
substantial amount of proacrosin species in the sperm exudate by
Western blotting, implying that acrosin released from sperm upon sperm
activation may bind to the vitelline coat through the N-terminal
paired basic residues and digest the vitelline coat. Alternatively, if
the above mechanism in mammals works in ascidians, it can be inferred
that the residual membrane-associated proacrosin may bind to the
vitelline coat via the C-terminal CUB domain followed by autodigestion,
which enables sperm to freely bind to the vitelline coat again. Further
detailed study is necessary to clarify this point.
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ACKNOWLEDGEMENTS |
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We thank Yukichi Abe (Center for Instrumental Analysis, Hokkaido University) for cooperation in amino acid composition analysis and protein sequence determination. We are also indebted to Drs. Masato Sumi (Hokkaido University) and Charles C. Lambert (California State University at Fullerton) for valuable advice.
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FOOTNOTES |
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* This work was supported in part by Grant-in-aids for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and the Akiyama Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB052635.
¶ To whom correspondence should be addressed. Tel.: 81-11-706-3720; Fax: 81-11-706-4900; E-mail: hswd@pharm.hokudai.ac.jp.
Published, JBC Papers in Press, April 19, 2001, DOI 10.1074/jbc.M011370200
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ABBREVIATIONS |
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The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
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REFERENCES |
---|
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---|
1. | Müller-Esterl, W., and Fritz, H. (1981) Methods Enzymol. 80, 621-632[Medline] [Order article via Infotrieve] |
2. | Urch, U. A., Wardrip, N. J., and Hedrick, J. L. (1985) J. Exp. Zool. 233, 479-483[Medline] [Order article via Infotrieve] |
3. |
Baba, T.,
Azuma, S.,
Kashiwabara, S.,
and Toyoda, Y.
(1994)
J. Biol. Chem.
269,
31845-31849 |
4. | Adham, L. M., Nayernia, K., and Engel, W. (1997) Mol. Reprod. Dev. 46, 370-376[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Yamagata, K.,
Murayama, M.,
Okabe, M.,
Toshimori, K.,
Nakanishi, T.,
Kashiwabara, S.,
and Baba, T.
(1998)
J. Biol. Chem.
273,
10470-10474 |
6. | Hoshi, M., Numakunai, T., and Sawada, H. (1981) Dev. Biol. 86, 117-121[Medline] [Order article via Infotrieve] |
7. | Sawada, H., Yokosawa, H., Hoshi, M., and Ishii, S. (1982) Gamete Res. 5, 291-301 |
8. |
Sawada, H.,
Yokosawa, H.,
and Ishii, S.
(1984)
J. Biol. Chem.
259,
2900-2904 |
9. | Sawada, H., Yokosawa, H., Someno, T., Saino, T., and Ishii, S. (1984) Dev. Biol. 105, 246-249[Medline] [Order article via Infotrieve] |
10. | Sawada, H., Iwasaki, K., Kihara-Negishi, F., Ariga, H., and Yokosawa, H. (1996) Biochem. Biophys. Res. Commun. 222, 499-504[CrossRef][Medline] [Order article via Infotrieve] |
11. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
12. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Labolatory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
13. | Liu, F.-T., Zinnecker, M., Hamaoka, T., and Katz, D. H. (1979) Biochemistry 18, 690-697[Medline] [Order article via Infotrieve] |
14. |
Richardson, R. T.,
and O'land, M. G.
(1996)
J. Biol. Chem.
271,
24069-24074 |
15. | Calvete, J. J., Sanz, L., Dostalova, Z., and Töpfer-Petersen, E. (1993) FEBS Lett. 334, 37-40[CrossRef][Medline] [Order article via Infotrieve] |
16. | Clavete, J. J., Mann, K., Schafer, W., Raida, M., Sanz, L., and Töpfer-Petersen, E. (1995) FEBS Lett. 365, 179-182[CrossRef][Medline] [Order article via Infotrieve] |
17. | Dostalova, Z., Clavete, J. J., Sanz, L., Hettel, C., Riedel, D., Schoneck, C., Einspanier, R., and Töpfer-Peteresen, E. (1994) Biol. Chem. Hoppe-Seyler 375, 457-461[Medline] [Order article via Infotrieve] |
18. | Töpfer-Petersen, E., and Calvete, J. J. (1996) J. Reprod. Fertil. Suppl. 50, 55-61[Medline] [Order article via Infotrieve] |
19. | Lambert, C. C., and Epel, D. (1979) Dev. Biol. 69, 296-304[Medline] [Order article via Infotrieve] |
20. |
Baba, T.,
Kashiwabara, S.,
Watanabe, K.,
Itoh, H.,
Michikawa, Y.,
Kimura, K.,
Takada, M.,
Fukamizu, A.,
and Arai, Y.
(1989)
J. Biol. Chem.
264,
11920-11927 |
21. | Urch, U. A., and Patel, H. (1991) Development 111, 1165-1172[Abstract] |
22. | Leytus, S. P., Kurachi, K., Sakariassen, K. S., and Davie, E. W. (1986) Biochemistry 25, 4855-4863[Medline] [Order article via Infotrieve] |
23. | Tosi, M., Duponchel, C., Meo, T., and Julier, C. (1987) Biochemistry 26, 8516-8524[Medline] [Order article via Infotrieve] |
24. | Delgadillo-Reynoso, M. G., Rollo, D. R., Hursh, D. A., and Raff, R. A. (1989) J. Mol. Evol. 29, 314-327[Medline] [Order article via Infotrieve] |
25. | Bork, P., and Beckmann, G. (1993) J. Mol. Biol. 231, 539-545[CrossRef][Medline] [Order article via Infotrieve] |
26. | Romero, A., Romao, M. J., Varela, P. F., Kolln, I., Dias, J. M., Carvalho, A., Sanz, L., Topfer-Petersen, E., and Calvete, J. J. (1997) Nat. Struct. Biol. 4, 783-788[Medline] [Order article via Infotrieve] |
27. | Blaschuk, O. W., Sullivan, R., David, S., and Pouliot, Y. (1990) Dev. Biol. 139, 227-229[Medline] [Order article via Infotrieve] |
28. | O'land, R., Welch, J. E., and Fisher, S. J. (1986) Adv. Exp. Med. Biol. 205, 131-144[Medline] [Order article via Infotrieve] |