Báo cáo khoa học: Spermosin, a trypsin-like protease from ascidian sperm cDNA cloning, protein structures and functional analysis doc

Spermosin, a trypsin-like protease from ascidian sperm cDNA cloning, protein structures and functional analysis Eri Kodama’, Tadashi Baba’, Nobuhisa Kohno”, Sayaka Satoh”, Hideyoshi Yok

Spermosin, a trypsin-like protease from ascidian sperm

cDNA cloning, protein structures and functional analysis

Eri Kodama’, Tadashi Baba’, Nobuhisa Kohno”, Sayaka Satoh”, Hideyoshi Yokosawa’ and Hitoshi Sawada’ ' Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Japan;

*Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, Japan

We have previously reported that two trypsin-like enzymes,

acrosin and spermosin, play key roles in sperm penetration

through the vitelline coat of the ascidian (Urochordata)

Halocynthia roretzi|Sawada et al (1984), J Biol Chem 259,

2900-2904; Sawada et al (1984), Dev Biol 105, 246-249]

Here, we show the amino-acid sequence of the ascidian

preprospermosin, which is deduced from the nucleotide

sequence of the isolated cDNA clone The isolated ascidian

preprospermosin cDNA consisted of 1740 nucleotides, and

an open reading frame encoding 388 amino acids, which

corresponds to a molecular mass of 41 896 Da By sequence

alignment, it was suggested that His178, Asp230 and Ser324

make up a catalytic triad and that ascidian spermosin be

classified as a novel trypsin family member The mRNA of

preprospermosin is specifically expressed in ascidian gonads

but not in other tissues Purified spermosin consists of

33- and 40-kDa bands as determined by SDS/PAGE under

nonreducing conditions The 40-kDa spermosin consists of a heavy chain (residues 130-388) and a long light chain designated LI (residues 23-129), whereas the 33-kDa spermosin includes the same heavy chain and a shorter light chain designated L2 (residues 97-129) The LI chain contains a proline-rich region, designated L1(AL2) which is lacking in L2 Investigation with the glutathione-S-trans- ferase (GST)-spermosin-light-chain fusion proteins, includ- ing GST-L1, GST-L2, and GST-L1(AL2), revealed that the proline-rich region in the L1 chain binds to the vitelline coat

of ascidian eggs Thus, we propose that sperm spermosin is a novel trypsin-like protease that binds to the vitelline coat and also plays a part in penetration of sperm through the vitelline coat during ascidian fertilization

Keywords: acrosin; ascidian; fertilization; lysin; spermosin;

trypsin-like protease; vitelline coat

Fertilization is a pivotal event in the creation of new

individuals In order to accomplish species-specific sperm—

egg fusion, sperm binding to and penetration through the

extracellular coat of the eggs (the vitelline coat in marine

invertebrates and zona pellucida in mammals), must be

precisely controlled, as the vitelline coat-free eggs would

allow gamete fusion with sperm from different species

Upon primary binding of the sperm to the vitelline coat,

the sperm undergoes an acrosome reaction, which is an

exocytosis of the acrosomal vesicle located on the sperm

head [1] A lytic agent called a sperm lysin is exposed on the

surface of the sperm head and is partially released into the

surrounding seawater In mammals, a trypsin-like enzyme

called acrosin (EC 3.4.21.10) has long been believed to be a

Correspondence to H Sawada, Department of Biochemistry, Graduate

School of Pharmaceutical Sciences, Hokkaido University, Sapporo

060-0812, Japan Fax: + 81 11 706 4900, Tel.: + 81 11 706 3720,

E-mail: hswd@ pharm.hokudai.ac.jp

Abbreviations: Boc, t-butyloxycarbonyl, L1, long light chain (residues

23-129); L2, short light chain (residues 97-129); LI(AL2), L2-deleted

LI (residues 23-96); MCA, 4-methylcoumaryl-7-amide; GST,

glutathione S-transferase

Enzyme: ascidian sperm spermosin (EC 3.4.21.99)

Note: nucleotide sequence reported in this paper has been submitted to

the DDBJ/GenBank/EBI Data Bank under accession number

ABO052776

(Received 28 August 2001, revised 16 November 2001, accepted 21

November 2001)

zona-lysin [2,3], as the purified acrosin is capable of dissolving the zona pellucida in i vitro experiments [2,3]

convincingly demonstrated that acrosin is not essential for

in vivo sperm penetration through the zona pellucida [4,5] It

is currently thought that acrosin is involved in the dispersal

of acrosomal contents during acrosome reaction [6] These results led us to propose that a sperm protease other than acrosin may play a key role in the penetration of sperm through the zona pellucida

Ascidians (Urochordata) occupy a phylogenetic position between invertebrates and segmented vertebrates [7] Whereas all the ascidians are hermaphrodites that release sperm and eggs simultaneously during the spawning season, self-fertilization is strictly prohibited in several species including Halocynthia roretzi [8] As the vitelline coat-free eggs of H roretzi are self-fertile [8], the interaction between sperm and the vitelline coat of the egg seems to be the process of self-nonself recognition in ascidian fertilization Therefore, the vitelline coat lysin system seems to be activated after the sperm recognizes the vitelline coat of the egg as nonself

To investigate the biological functions of sperm proteases, one of the largest solitary ascidians, H roretzi, was used in this study; fertilization experiments are more accessible than those in mammals, and large amounts of sperm and egg are obtainable from thousands of these animals which are cultivated in Onagawa Bay for human consumption

We have previously reported that H roretzi sperm con- tain a novel trypsin-like protease called ascidian spermosin

in addition to acrosin, an ascidian homologue of

mammalian acrosin Ascidian spermosin has unique prop-

erties, especially in substrate specificity: it hydrolyses only

t-butyloxycarbonyl (Boc)-Val-Pro-Arg-4-methylcoumaryl-

7-amide (MCA) among many fluorogenic substrates [9]

In addition, involvement of spermosin in ascidian fertiliza-

tion was revealed by examining the effects of leupeptin

analogues and anti-spermosin antibody on fertilization of

H roretzi [10-12] The presence of the spermosin-like

protease in the sperm of the other animals, including

mammals, has not yet been investigated Therefore, the

unique properties of ascidian spermosin led us to assume

that ascidian spermosin belongs to a novel trypsin-like

protease of sperm origin In order to clarify this issue, we

attempted to isolate a cDNA clone encoding ascidian

spermosin We found that ascidian spermosin consists of

two chains: a light chain encoded in the N-terminal portion

and a heavy chain encoded at the C-terminal portion We

also found that there are two forms of spermosin in sperm,

which share the same heavy chain but are distinct in the

length of light chains: one contains a long L1 chain and the

other contains a shorter L2 chain Furthermore, the proline-

rich region in the LI chain is capable of binding to the

vitelline coat, implying a role in sperm binding to the

vitelline coat

MATERIALS AND METHODS

Biologicals

The solitary ascidian (Urochordata) Halocynthia roretzi

type C was used in this study Sperm and eggs were

collected from dissected gonads as described previously

[13,14] Mature oocytes were homogenized with fivefold

diluted (20%) artificial seawater containing 0.1 mm diiso-

propylfluorophosphate The homogenate was filtered

through a nylon mesh (pore size, 150 1m), and the vitelline

coats on the blotting cloth were washed extensively with

20% artificial seawater Purity of the isolated vitelline coats

was examined under a light microscope

Purification and assay procedure of spermosin

The enzymatic activity of spermosin was determined using

Boc-Val-Pro-Arg-MCA as a substrate as described previ-

ously [9] Spermosin was highly purified from H roretzi

sperm by DEAE-cellulose chromatography, Sephadex

G-100 gel filtration, and soybean trypsin inhibitor-immobi-

lized Sepharose chromatography according to procedure

described previously [9]

Determination of N-terminal amino-acid sequences

SDS/PAGE was carried out on a slab gel containing 12.5%

polyacrylamide as described previously [15] Purified sperm-

osin was subjected to SDS/PAGE under reducing and

nonreducing conditions and was then electrophoretically

transferred to a PVDF 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 bands were cut from the

membrane The N-terminal sequence of the purified

spermosin was determined using a protein sequencer model

Procise 492 (Applied Biosystems) The solubilized vitelline coat component, which is able to bind to the glutathione S-transferase (GST)-L1 and GST-L1(AL2) fusion proteins, was subjected to SDS/PAGE and transferred to a PVDF membrane The 28-kDa band on a membrane was subjected

to amino-acid sequence analysis

Cloning of spermosin cDNA The primers (sense and antisense) used for PCR were designed from the N-terminal amino-acid sequence of

H roretzi spermosin (heavy chain): sense primer, 5’-AT (T/C/A)GT(T/C/A/G)GG(T/C/A/G)GG(T/C/A/G)GC (T/C/A/G)GA(A/G)GC-3’; and antisense primer, 5’-AA (T/C/A/G)GG(T/C/A/G)GG(T/C)GT(T/C/A)TA(T/C) AG(T/C)A T-3’ The former and latter primers encoded the amino-acid sequences IVGGAEA and YDIXGGK, respec- tively The primers at concentrations of 10 [tm were mixed

in PCR to amplify the H roretzi gonad Agt11 cDNA library

as described previously [16] A DNA band migrating at 78 base pairs was isolated, cloned into a pCRII vector, and transformed into Escherichia coli DHS5a The spermosin cDNA clones were isolated from 3 x 10° clones of H rore- tzi gonad Agtl1 cDNA library by phage plaque hybridiza- tion using the above PCR-amplified DNA fragment encoding spermosin as a probe The probe was labelled with [o°P]dCTP (BcaBEST kit, Takara) by the random- priming procedure Briefly, plaque lifts were prehybridized

at 55 °C in 5 x NaCl/Cit (1 x NaCl/Cit, 15 mm sodium

citrate pH 7.0 and 0.15 m NaCl), 0.02% Ficoll 400, 0.02%

polyvinylpyrrolidone, 0.02% BSA, 0.1% SDS, and 0.1 mg-mL7’ salmon testis DNA Hybridization was carried out at 55 °C overnight in prehybridization buffer containing

*P-labelled probe The membranes were washed in 2x NaCl/Cit at room temperature for 10min, in

2 x NaCl/Cit containing 0.1% SDS at 60 °C for 10 min, and in 2 x NaCl/Cit at room temperature for 10 min before autoradiography at —80 °C The nucleotide sequence of spermosin cDNA clone was determined by a Big Dye Terminator Cycle Sequencing Ready Reaction using an ABI 377A DNA Sequencing Apparatus (Applied Biosys- tems)

Northern blot analysis Total RNA was extracted from the H roretzi gonad according to the standard method of acid/guanidinium thiocyanate/chloroform Poly(A) * RNA was isolated from total RNA by using oligotex-dT30 (Roche Diagnostics Co.) Two microgrmas of poly(A)" RNA were subjected to electrophoresis on 1.2% agarose gel containing 6% form- aldehyde, and RNA bands were transferred to a Hybond- N+ nylon membrane (Amersham) Stringency used for hybridization and washing, and the probe were the same as those, used in ‘cloning’ After washing, the blots were autoradiographed at —80 °C

Extraction of the vitelline coat

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

Almost all the protein components of the vitelline coat were

solubilized under these conditions

Expression of GST-spermosin light chain fusion proteins

DNA fragments corresponding to the L1, L2, and L1(AL2),

which is an LI lacking L2 region, were amplified by PCR

from the cDNAs using the following combinations of

forward and reverse primers designed to produce 5’ BamHI

and 3’ Xhol restriction sites to facilitate directional cloning

into the expression vector pGEX-6P-1 (Amersham Phar-

macia): two forward primers (a, 5-GGATCCTCT

GAATCTACAAATCC-3, b, 5-GGATCCTCTGAAG

GCCCGGTTC-3’) and two reverse primers (c, 5-CTC

GAGTCATTTTCCTTTCTTTAG-3; d, Š5-CTCGAGT

CAATTTTCAGATTCCG-3’) were designed and the com-

bination of primers for L1, L2, and L1{AL2) were (a + c)

(b + c), and (a + d), respectively All clones were

sequenced to confirm the reading frame and sequence

The GST fusion protein expression vectors were used for

expression in FE coli BL21 The expressed fusion proteins

were purified using glutathione-agarose beads (Amersham)

according to the manufacturer’s protocol

Binding of GST-spermosin light chain fusion proteins

to the vitelline coat

The purified GST-spermosin light chain fusion proteins

(3 ug) were mixed with the solubilized vitelline coat in

artificial seawater, and incubated at 4 °C for | h to form the

complex consisting of the fusion protein and the vitelline

coat The mixture was applied onto a glutathione-agarose

column and washed four times with artificial seawater The

GST-spermosin light chain fusion protein—vitelline coat

complex was eluted from the glutathione-agarose column

with 50 mmo Tris/HCl (pH 8.8) containing 20 mm glutathi-

one The eluted proteins were subjected to SDS/PAGE

followed by silver staining (Kanto Chemical Co Inc.,

Tokyo, Japan) or by blotting to a PVDF membrane The

bands detected were cut from the membrane and_ the

N-terminal amino-acid sequence was determined as

described above

RESULTS

CDNA cloning of ascidian spermosin

Spermosin was highly purified from H roretzi sperm as

described previously [9] Purified spermosin was subjected to

SDS/PAGE under reducing conditions, followed by blot-

ting toa PVDF membrane The sequence of 33 amino acid

residues from the N terminus of the 28-kDa spermosin

band, which corresponds to that of the heavy chain as

described below, was determined using a protein sequencer

(see Figs 1 and 3) The N-terminal sequence of the

spermosin (heavy chain) was used to design the degenerate

oligonucleotide primers for PCR of H roretzi gonad

cDNA The PCR product was used as a probe for screening

the gonad Agt11 cDNA library to isolate a spermosin clone

(Fig 1) A single open reading frame of the spermosin clone

encodes 388 amino acids The deduced amino-acid sequence

contains a region from residue 130 to residue 162 that

corresponds to the N-terminal amino-acid sequence deter-

-24/0 AGT ATA GTC AAG TGG TIT TGC AGC

ATG GCT GCA ATC AAC GTT ATA TTT ATC TCG GGA GCT ATA GCA TTA TTC GCT TIA ACG GGA

Met ala ala ile asn val ile phe ile ser gly ala ile ala leu phe ala leu thr gly

TCG TGT TCT GAA TCT ACA AAT CCT TTC ACT AAT AAA CCA TAT GCA ACC CAA AAT CCA TAC

AGC CCT CCA CAA ACC AAT CAG CCT ACA AAA CGC CCT TAC CAA CCT GGC CCC GCA CCC ACA

ser pro pro gln thr asn gln pro thr lys arg pro tyr gln pro gly pro ala pro thr

181/61 211/71

CCA GCT CCA TAC ATC CCA CAA AAA ACT AAT CCA CCT ACG AAA CGA CCG CTC AAC CCC ACT

241/81 271/91

CCT TCG CCT ACA GCG AAA CCT CCA TCT GAG AAT TCG GAA TCT GAA AAT TCT GAA GGC CCG

301/101 331/111

GTT CTA ATT GAG GAG GAC CAT TIT ACT GTC GAT GCC AAT TTC AAA TGT GGT ATT CCT CCA

GTC GAG CCG GAT CTA AAG AAA GGA AAA ATA GTG GGT GGA GCA GAG GCA GTG CCC AAT TCC

421/141 451/151 TGG CCG TAT GCT GCA GCA TIC GGT ACA TAC GAT ATT TCA GGC GGG AAA TTA GAA GTT TCC

trp pro tyr ala ala ala phe gly thr tyr asp ile ser gly gly lys leu glu val ser

481/161 511/171

CAG ATG TGC GGG TCT ACT ATT ATC ACT CCG AGG CAT GCC TTG ACG GCC GCC|CAT|TGT TIT

ATG ATG GAC CCG GAC ATA GAC CAG ACG TAC TAC ATA TTT ATG GGT CTT CAT GAC GAG ACT

met met asp pro asp ile asp gin thr tyr tyr ile phe met gly leu his asp glu thr

601/201 631/211

ACG TAT AAA GGA GTA CGG CCT AAT AAG ATT GIC GGT GTT CGT TAT CAT CCT AAG ACC AAC

GTT TTC ACC GAT GAC CCC TGG CTA GTA TAT/GAC|TIT GCT ATA CTG ACT CTG AGG AAA AAA

721/241 751/251

GTA ATT GCA AAC TIT GCA TGG AAT TAT GCC TGT CTT CCA CAG CCA AAA AAG ATT CCT CCA

GAA GGA ACG ATC TGT TGG AGC GTT GGC TGG GGA GTT ACG CAG AAT ACA GGA GGA GAT AAT

GTI CTT AAA CAA GTA GCG ATC GAT CTC GTT TCA GAG AAG AGA TGC AAG GAG GAA TAC AGA

TCT ACT ATT ACA AGT AAA TCT ACT ATA TGC GGT GGA ACC ACA CCG GGA CAA GAT ACA TGC

CAG GGT GATJAGT|GGC GGC CCA CTA TTT TGT AAG GAA GAC GGC AAG TGG TAT CTT CAA GGC

ATC GIA AGC TAC GGT CCC TCG GTA TGC GGT TCG GGT CCA ATG GCA GCA TAT GCT GCG GIT ile val ser tyr gly pro ser val cys gly ser gly pro met ala ala tyr ala ala val

GCA TAC AAC TTG GAA TGG TTA TGC TGT TAC ATG CCG AAT TTA CCT TCT TGC GAA GAC ATT

GAA TGT GAC GAG AGC GGA GAA AAC TGA CAC GTG ATC AAA AGG CAT GCG TCG ATT TGA CTG glu cys asp glu ser gly glu asn OPA

ACA ACA GAA GAA AGC CGT ACA CAC GAA CAC ATG CAT AAA AGA AAG AAA TGC TTG AAA ATG GcG TGC CTI GIT TGA TGA GCT GAT TIT TTA TTG ATG TAT TGT GGT CGG AAA CCA ACT ACA AGA TTT GIT TTG GTT CAA AAT GAT TTT TTT TTA CAA TTG AGT TGT CTA TAT TAA TTC TTA CTG ACA GIT GCT AGT ATA ATA GAT CAA CTC ACC ATT AGT ATA AAC TGC TAT TAA CGA AGG AGC ATC AAT GGT TAT GTC ATT ATG ATA TAT TIT TAC TAA TGT TAT ACA AGA CAG TAA AGA AAA TAT GGC TAA AAA AAA AAA AAA AAA

Fig 1 Nucleotide and deduced amino-acid sequences of H roretzi spermosin The amino-acid sequence as determined by a protein sequencer is underlined The conserved catalytic triads in the serine protease are indicated by boxes The presumed cleavage sites in preprospermosin and prospermosin are indicated by an arrow and two arrowheads, respectively Note that the cleavage of prospermosin at the second arrowhead yields the L1 light chain and heavy chains, while the two cleavages at two arrowheads yields the L2 light chain and heavy chain (see Fig 3C)

mined by sequencing of the purified spermosin (heavy chain) protein The molecular mass of preprospermosin was estimated to be 41 896 Da The N-terminal sequence (22 residues) of the preprospermosin corresponds to a signal peptide for a nascent protein destined for initial transfer to the endoplasmic reticulum Thus, the pro-form of spermosin

Gn Hp In Bb

19kb >

Fig 2 Tissue-specific expression of spermosin mRNA in H roretzi

Gn, Gonad; Hp, hepatopancreas; In, intestine; Bb, branchial basket

Northern blots of poly(A)” RNA (2 ug each) from H roretzi tissues

were hybridized with radiolabelled cDNA probes A 1.9-kb mRNA

signal was detected only in the gonad

may start from Ser23 (see Figs 1 and 3) The active site

residues in serine proteases, histidine, aspartic acid, and

respectively, in preprospermosin This indicates that sperm-

osin is classified into a family S1 (trypsin family) of clan SA

in serine proteinases [17] The amino-acid sequence of

spermosin (heavy chain) showed 32% homology to that

of mouse plasma kallikrein and 27% homology to those of

mouse and ascidian acrosin (see Fig 5)

A dendrogram analysis showed that ascidian spermosin is

classified as a novel member of the SI trypsin family (data

not shown)

Expression of spermosin mRNA in ascidian gonads

Northern blotting was carried out with the same probe used

for cDNA cloning A single transcript of approximately

1.9 kb was detected in the gonad, but not in the hepato-

pancreas, intestine, or branchial basket, of H roretzi

(Fig 2)

The presence of two forms of spermosin in ascidian

sperm

SDS/PAGE of the purified spermosin gave a single band of

28 kDa under reducing conditions, whereas it showed two

bands of 33 and 40 kDa under nonreducing conditions

(Fig 3A) The N-terminal sequence determination of these

2-ME +

(kDa) (kDa) 2skDa:IVGG 45¬ 340

33 kDa: IVGG

my) <33 SEGP

SEST

C

Signal

Preprospermosin H D S |

Cleavage site

Spermosin type 1 | LI | 28 kDa |

SEGP IVGG

Spermosin type 2 | L2 | 28 kDa |

S—————s Fig 3 The presence of two forms of spermosin in H roretzi sperm (A) SDS/PAGE of the purified spermosin SDS/PAGE gave a band of

28 kDa under reducing conditions and two bands of 33 and 40 kDa under nonreducing conditions 2-ME, 2-mercaptoethanol (B) The N-terminal amino-acid sequences of three bands The 28-kDa protein had a single amino-acid sequence and was determined as a heavy chain

of spermosin, whereas the 40-kDa protein consisted of a heavy chain (residues 130-388) and a light chain designated as L1 (residues 23-129, designated L1), and the 33-kDa protein consisted of the heavy chain (residues 130-388) and a light chain designated as L2 (residues 97-129) There were no distinct bands of L1 and L2 chains on SDS/ PAGE (12.5% gel) under the reducing conditions (see also Fig 2 in [9]), probably because of their high electrophoretic mobility that is indistinguishable from the migration front Alternatively, the low molecular mass proteins (11-kDa L1 chain and 3-kDa L2 chain) might not be sufficiently fixed within the gel under our experimental condi- tions (C) Protein structures of ascidian preprospermosin and sperm- osin type | and 2 The N-terminal amino-acid sequences of L1, L2, and heavy chains are shown above the respective models The putative disulfide bond is indicated by analogy to the other trypsin family [17] bands (Fig 3B) revealed that the 28-kDa protein is the heavy chain of spermosin, while the 33-kDa spermosin is made up

of the heavy chain (residues 130-388) and the light chain designated as L2 (residues 97-129), and the 40-kDa sperm- osin consists of the heavy chain (residues 130-388) and the light chain designated as L1 (residues 23-129) (Fig 3C)

From these results, it was concluded that there are two forms

of spermosin in ascidian sperm and that the amount of the 33-kDa form is higher than that of the 40-kDa form Models for protein structures of preprospermosin and spermosin type 1 and type 2 are depicted in Fig 3C

Binding of spermosin light chains to the vitelline coat Comparison of the sequences between L1 and L2 light chains revealed that the LI but not the L2 had a region

Fig 4 Expression of recombinant H roretzi GST-L1

spermosin light chain-GST fusion proteins

(A) GST fusion proteins including L1, L2,

and LI(AL2) (B) The fusion proteins were

expressed in clones generated by PCR from

GST-L2

the spermosin cDNA as described in Mate-

rials and methods GST-spermosin light

chain fusion proteins (3 pg each), which had

been previously purified by glutathione—

agarose chromatography, were mixed with

the solubilized vitelline coat in artificial sea-

water After incubation at 4 °C for 1 h, the

fusion protein was applied to a column of

glutathione—agarose beads The proteins

eluted with 20 mm glutathione were sub-

jected to SDS/PAGE and visualized by silver

staining The N-terminal amino-acid

sequence of the 28-kDa protein, which was

detected in both cases of GST-L1 and GST-

L1(AL2) fusion proteins, was determined as

described in Materials and methods

containing high amounts of proline residues (residues

28-88, see Fig 1) It has been suggested that the proline-

rich regions located in the C terminals of human and

porcine proacrosins play a key role in interaction between

proacrosin and the zona pellucida [18] To investigate the

binding ability of the proline-rich region in ascidian

spermosin to the vitelline coat, three GST fusion proteins

(Fig 4A), including L1, L2, and LI(AL2), an L1 lacking

the L2 region, were expressed and purified by glutathione—

agarose chromatography The purified GST fusion

proteins were incubated with the solubilized vitelline coat

and the complex formed was adsorbed to the glutathione—

agarose beads After washing, the vitelline coat protein

components, which can interact with the fusion proteins,

were eluted with 20 mm glutathione and analysed by SDS/

PAGE By comparison of protein patterns in SDS/PAGE,

it was found that the 28-kDa band was detected only with

the GST-LI or GST-LI(AL2) Fusion proteins, but not with

the GST-L2 fusion protein (Fig 4B), indicating that the

28-kDa protein of the vitelline coat has an ability to bind

to the proline-rich region present in the L1(AL2) domain of

the spermosin light chain The predicted N-terminal

amino-acid sequence (SAXARNQNFG) showed no

appreciable identity with any proteins by FAsTA and BLAST

database search analyses

DISCUSSION

The present study demonstrated the amino-acid sequence of

spermosin from sperm of the ascidian H roretzi for the first

time Here we show that there are two molecular forms of

the molecule made up of a common heavy chain and either

a short or a long light chain depending on the processing

sites We previously reported that ascidian spermosin is a

ctu.) ay

GST alone

' 97-129

B

28 kDa > | — _ Ý

novel sperm trypsin-like protease, distinct from acrosin, a well-known sperm trypsin-like protease that is widely distributed in mammalian sperm, in terms of substrate specificity and inhibitor susceptibility [9,12] The present study clearly showed that ascidian spermosin is a novel protease and is distinct from ascidian acrosin on the basis of amino-acid sequence [16] Whereas the acrosin family has a C-terminal extension as a pro-piece of proacrosin, sperm- osin has no such C-terminal region In contrast, the N-terminal region of the light chain of ascidian spermosin contains a proline-rich region, which is observed in the C-terminal region of proacrosin of nonrodent mammals

[19] We identified the vitelline coat component, to which the

proline-rich region of the L1 chain of spermosin binds, for the first time

The sequence alignments of trypsin family proteases (Fig 5), suggested that the active site residues, histidine, aspartic acid, and serine, are located at residues 178, 230, and 324, respectively, in preprospermosin (see Figs | and 5) Although spermosin heavy chain showed the highest homology to mouse plasma kallikrein (32% identity) among the trypsin family, spermosin is unlikely to be a functional homologue of kallikrein: spermosin is not able to hydrolyse Pro-Phe-Arg-MCA [9], a preferred substrate for plasma kallikrein, but is able to efficiently hydrolyse Boc-

Val-Pro-Arg-MCA [9], a preferred substrate for thrombin

Ascidian spermosin is initially synthesized as a 388-residue preproprotein with a 22-residue signal peptide in the terminus (Figs | and 3) The N-terminal region from residue N-23 to residue 129 (i.e the LI light chain) in the pro-form of ascidian spermosin, which precedes the IVGG sequence, is thought to be cleaved off at the bond between Lys129 and Ile130 by the action of a putative trypsin-like protease As the sequence around the scissile bond is

Vv

IVGGAEAVPNSWP YAAAFGTYDISGGKLEVSQMCGSTIITPRHALTAAHCFMMDPDIDOQT

Fig 5 Sequence alignment of ascidian

MmAcrosin 96 DWRLVFGAQEIEYGRNKPVKEPQQERYVOKIVIHEKYNVVTEG NDIALLKITPPVT active site residues in the $1 subfamily [17] of

HrA raeressn 88 IKKEDALIRVADLDKTDDTDEGEMTFEVED & VAD I I ET TREOYNR® [HEQYNRQTFD NDIMLIEILGSIT DEMETETEGS trypsin-like protease are indicated by closed .r _

triangles The locations of paired basic resi-

HrAcrosin 144 YGPTVQPACIP-GANDAVADGTKCLISGWGDTQDHVHNRWPDKLOKAQVEVFARAQC - pace mm PORAQVEN % circles Asterisks indicate the positions of ¬ di x

MmAcrosin 211 QWYNGRVTSTNVCAGYPEGKIDTCQGDSGGPLMCRDNVDSP-—-FVVVGITSWG-VGCAR of Gonnet Pam250, > 0.5) and “weak”

Ck kepada kk kek, (< 0.5) consensus positions, respectively For

details of CLUSTAL W and Gonnet

MmAcrosin 267 AKRPGVYTATWDYLDWTASKTG ~ PNALHLIQPATPHPPTTRHPMVSFHPPSLRPPW hypernig.nig.ac.jp/homology/clustalw.shtml

ke " and http://bioinformer.ebi.ac.uk/newsletter/

archives/2/clustalw17.html, respectively

GenBank/EBI accession number,

plasma kallikrein (M58588); MmAcrosin,

HrAcros¿n 499 5QLPKNN H roretzi acrosin (AB052635)

Lys-Lys-Gly-Lys-Ile-Val-Gly-Gly(126-133), together with

the fact that spermosin hydrolyses only Boc-Val-Pro-Arg-

MCA among peptidyl-MCA substrates, autocatalytic acti-

vation of prospermosin seems unlikely Acrosin is a

candidate protease that cleaves the Lys129-Ie130 bond of

prospermosin

Here we showed the existence of two forms of spermosin

in ascidian sperm: the type 1 form containing the LI light

chain and the type 2 form containing the L2 light chain in

addition to the heavy chain (Fig 3C) The Asn96—Ser97

bond should be cleaved to produce the lower molecular

weight form, and therefore, a putative sperm endopeptidase

that cleaves the C-terminal side of the asparagine residue

may be responsible for this processing It seems unlikely that

spermosin type | is an inactive precursor of spermosin type

2, aS spermosin type | and 2 are capable of binding to a

soybean trypsin inhibitor-immobilized Sepharose column

With respect to the disulfide bond between the light chain

and the heavy chain of ascidian spermosin, it is plausible

that the Cys116 residue in the light chain is disulfide-bonded

to the Cys251 residue in the heavy chain by analogy to other

trypsin family proteins: the nearest cysteine residue to the

active site aspartic acid residue is known to be disulfide-

bonded to the light chain in many serine proteases including

acrosin, thrombin, kallikrein, factor X and plasmin [17] (see

Figs 1, 3C and 5) Although light and heavy chains of

mammalian acrosin are bonded by two disulfide bridges, a

single S—S bridge between light and heavy chains is rather

common in serine proteases including mouse testis-specific

proteases (TESP-I and -I]) [20], ascidian acrosin [16], and

mammalian proteases such as thrombin, kallikrein, factor X

and plasmin [17]

Homology between ascidian spermosin and human

acrosin, and that between ascidian spermosin and ascidian

acrosin were 27% in both cases In contrast with acrosin,

spermosin did not have a consensus sequence of an N-linked sugar attachment and paired basic residues in the N-terminal region, the latter of which is proposed to be responsible for the binding of (pro)acrosin to the zona pellucida [21] In place of the paired basic residues, spermosin contained a proline-rich region in the L1 light chain We demonstrated that the proline-rich region in the

LI chain binds to the vitelline coat, as is the case for the

paired basic residues in the N termini and the proline-rich regions in the C termini of mammalian proacrosins In addition, we found that the 28-kDa vitelline coat protein is capable of binding to the above proline-rich region

We have reported previously that spermosin inhibitors, Z-Val-Pro-Arg-H [10] and Dns-Val-Pro-Arg-H [12], and anti-spermosin antibody [11] are capable of inhibiting fertilization in a concentration-dependent manner indicat- ing that spermosin plays an important extracellular role in ascidian fertilization and that the proteolytic activity of spermosin is required for ascidian fertilization As a proline-rich region (residues 28-88) of spermosin LI light chain is able to associate with the vitelline coat of the egg, it

is inferred that spermosin is involved not only in the sperm penetration of the vitelline coat but also in the sperm binding to the vitelline coat Whether spermosin or its homologue is present in mammalian sperm is an intriguing issue, aS a sperm protease(s) other than acrosin is considered to play an essential role in the sperm penetra- tion of the zona pellucida in mammals In connection with this, it should be noted that a 27-kDa protein in mouse epididymis extract is specifically recognized by anti-ascidian spermosin antibody on the basis of Western blot analysis (E Kodama, H Yokosawa & H Sawada, unpublished data) Further studies are needed to search for a spermosin homologue in mammalian sperm and to elucidate its role

in mammalian fertilization

ACKNOWLEDGEMENTS

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 We are grateful to C C

Lambert (California State University Fullerton) for his critical reading

of this manuscript and valuable advice

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