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 1 , Tadashi Baba 2 , Nobuhisa Kohno 2 , Sayaka Satoh 2 , Hideyoshi Yokosawa 1 and Hitoshi Sawada 1 1 Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Japan; 2 Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City, Japan We have previously reported t hat two trypsin-like enzymes, acrosin a nd spermosin, play key roles in sperm penetration through the vitelline c oat of the ascidian (Urochordata) Halocynthia roretzi [Sawada et al. (1984), J. Biol. C hem. 25 9, 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. Th e isolated ascidian preprospermosin cDNA consisted of 1740 nucleotides, and an open reading frame encoding 388 amino acids, w hich corresponds to a molecular mass o f 41 896 Da. By sequence alignment, it was suggested that His178, A sp230 and Ser324 make up a catalytic triad and that ascidian spermosin be classi®ed as a novel trypsin family member. The mRNA of preprospermosin is speci®cally expressed in ascidian gonads but not in other tissues. P uri®ed spermosin c onsists of 33- and 40-kDa bands as determined by SDS/PAGE under nonreducing conditio ns. The 4 0-kDa s permosin consists of a heavy chain (residues 130±388) and a long light chain designated L1 (residues 23±129), whereas the 33-kDa spermosin includes the same heavy chain and a shorter light chain d esignated L 2 ( residues 97±129). T he L1 chain contains a proline-rich region, designated L1(DL2) which is lacking i n L 2. Investigation with the glutathione-S-trans- ferase (GST)±spermosin-light-chain fusion proteins, includ- ing GST±L1, GST±L2, and GST±L1(DL2), 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 tryp sin-like p rotease that binds to the vitelline coat and also plays a part in penetration of sperm through the vitelline coat during ascidian fe rtilization. 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-speci®c 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 p rimary binding of the sperm to the vitelline coat, the sperm undergoes an acrosome reaction, which is an exocytosis of the a crosomal vesicle l ocated on the s perm 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 zona-lysin [2,3], as the puri®ed acrosin is capable of dissolving the zona pellucida in in vitro experiments [2,3]. However, recent studies with acrosin-knockout mice 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 m ay play a k ey 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 o f the egg as nonself. To investigate the biological functions of sperm proteases, one of the largest solitary ascidians, H. roretzi,wasusedin this study; fertilization experiments are more a ccessible than those in mammals, and large amounts of s perm 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 nove l trypsin-like protease called ascidian spermosin 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); L 2, short light chain (residues 97±129); L1(DL2), L2-deleted L1 (residues 23±96); MCA, 4-methylcoumaryl-7-amide; GST, glutathione S-transferase. Enzyme: ascidian sperm spermosin (EC. 3.4.21.99). Note: nucleotide sequence reporte d in this paper has been submitted to the DDBJ/Ge nBank/E BI Data Bank under accession number AB052776. (Received 28 August 2001, revised 16 November 2001, accepted 21 November 2001) Eur. J. Biochem. 269, 657±663 (2002) Ó FEBS 2002 in addition to acrosin, an ascidian homologue of mammalian acrosin. Ascidian spermosin has unique prop- erties, especially in substrate speci®city: it hydrolyses only t-butyloxycarbonyl (Boc)-Val-Pro-Arg-4-methylcoumaryl- 7-amide (MCA) among many ¯uorogenic substrates [9]. In addition, involvement of spermosin in ascidian fertiliza- tion was r evealed by examining the effects o f 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 n ot yet been investigated. Therefore, t he 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 c DNA clone encoding ascidian spermos in. 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 i n sperm, which share the same h eavy chain but are distinct i n the length of light chains: one contains a long L1 chain and the other contains a shorter L 2 chain. Furthermore, the proline- rich region in the L 1 chain is capable of binding to the vitelline coat, implying a r ole 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 previou sly [13,14]. Mature oocytes wer e homogenized with ®vefold diluted (20%) arti®cial seawater containing 0.1 m M diiso- propyl¯uorophosphate. The homogenate was ®ltered through a nylon mesh (pore size, 150 lm), and the vitelline coats o n t he blotting cloth w ere w ashed e xtensively with 20% arti®cial seawater. Purity of the isolated vitelline coats was examined under a light microscope. Puri®cation 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 puri®ed from H. roretzi sperm b y DEAE-cellulose chromatography, S ephadex G-100 gel ®ltration, and soybean trypsin inhibitor-immobi- lized Sepharose chromatography according to procedure described previously [9]. Determination of N-terminal amino-acid sequences SDS/PAGE was c arried out on a slab gel containing 12.5% polyacrylamide as described previously [15]. Puri®ed sperm- osin was subjected to SDS/PAGE under reducing and nonreducing conditions and was then electrophoretically transferred to a PVDF me mbrane (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 t he puri®ed 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(DL2) 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 p rimers encoded t he amino-acid sequences IVGGAEA and YDIXGGK, respec- tively. The primers at concentrations of 10 l M were mixed in PCR to amplify the H. roretzi gonad kgt11 cDNA library as described p reviously [16]. A DNA band migrating at 78 base pairs was isolated, cloned into a pCRII v ector, and transformed into Escherichia c oli DH5a. The spermosin cDNA clones were isolated from 3 ´ 10 5 clones of H. rore- tzi gonad kgt11 cDNA library by phage plaque hybridiza- tion using the above PCR-ampli®ed DNA fragment encoding spermosin as a probe. The probe was labelled with [a 32 P]dCTP (BcaBEST kit, Takara) by the random- priming procedure. Brie¯y, plaque lifts were prehybridized at 55 °Cin5´ NaCl/Cit (1 ´ NaCl/Cit, 15 m M sodium citrate p H 7.0 and 0.15 M NaCl), 0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, 0.02% BSA, 0.1% SDS, and 0.1 mgámL )1 salmon testis DNA. Hybridization was carried out at 55 °C overnight in prehybridization buffer containing 32 P-labelled probe. The membranes were w ashed i n 2 ´ NaCl/Cit at room temperature for 10 min, in 2 ´ NaCl/Cit containing 0.1% SDS at 6 0 °C for 10 min, and in 2 ´ NaCl/Cit at room temperature for 10 min before autoradiography at )80 °C. The nucleotide s equence of spermosin cDNA clone was determined by a Big Dye Terminator Cycle Sequencing R eady Reaction using an ABI 377A DNA Sequencing A pparatus (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 w ere 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 ÔcloningÕ. After washing, the blots were autoradiographed at )80 °C. Extraction of the vitelline coat The v itelline coats were suspended i n arti®cial seawater containing 0.5% Triton X-100. After stirring for 30 min at 4 °C, the suspension was centrifuged at 1 0 000 g for 30 min to obtain the supernatant as the solubilized vitelline coat. 658 E. Kodama et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Almost all the protein c omponents o f 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(DL2 ), which is an L1 lacking L2 region, were ampli®ed by PCR from the cDNAs using the following combinations of forward and reverse p rimers designed to produce 5¢ BamHI and 3¢ XhoI restriction sites to facilitate directional cloning into the expression v ector pGEX-6P-1 (Amersham Phar- macia): two forward primers (a, 5¢-GGATCCTCT GAATCTACAAATCC-3¢;b,5¢-GGATCCTCTGAAG GCCCGGTTC-3¢) a nd two reverse primers (c, 5¢-CTC GAGTCATTTTCCTTTCTTTAG-3¢;d,5¢-CTCGAGT CAATTTTCAGATTCCG-3¢) w ere designed and th e com- bination of primers for L1, L2, and L1(DL2) were (a + c) (b + c), and (a + d), respectively. All clones were sequenced to con®rm the reading frame and sequence. The GST fusion protein expression vectors were used for expression in E. coli BL21. The e xpressed fusion proteins were puri®ed using glutathione±agarose beads (Amersham) according to the manufacturer's protocol. Binding of GST±spermosin light chain fusion proteins to the vitelline coat The puri®ed GST±spermosin light chain fusion proteins (3 lg) were mixed with the solubilized vitelline c oat i n arti®cial seawater, and incubated at 4 °C for 1 h to form the complex consisting of the fusion p rotein and the vite lline coat. The mixture was applied onto a glutathione±agarose column and washed four times with arti®cial seawater. The GST±spermosin light chain f usion p rotein±vitelline coat complex was eluted from the glutathione±agarose column with 50 m M Tris/HCl (pH 8.8) containing 20 m M glutathi- one. T he eluted proteins were subjected to SDS/PAGE followed by silve r 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 puri®ed from H. roretzi sperm as described previously [9]. Puri®ed spermosin was subjected to SDS/PAGE under reducing conditions, f ollowed by blot- ting to a PVDF membrane. The s equence of 33 amino acid residues from the N terminus of th e 28-kDa spermosin band, which corresponds to that of the heavy chain as described b elow, was determined using a protein sequencer (see Figs 1 and 3). The N-terminal sequence o f the spermosin (heavy chain) was used to design the degenerate oligonucleotide primers for P CR of H. roretzi gonad cDNA. The PCR product was used as a probe for screening the gonad kgt11 cDNA library to isolate a spermosin clone (Fig. 1). A single open reading frame o f the spermosin clone encodes 388 amino acid s. The deduced amino-acid sequence contains a region f rom residue 130 to residue 162 t hat corresponds to the N-terminal amino-acid sequence deter- mined by sequencing of the puri®ed spermosin (heavy chain) protein. The molecular mass of preprospermosin was estimated to be 41 896 Da. T he 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 Fig. 1. Nucleotide and d educed amino-acid se quences of H. roretzi spermosin. The a mino-acid sequence as d etermined by a p rotein 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). Ó FEBS 2002 Cloning and characterization of ascidian spermosin (Eur. J. Biochem. 269) 659 may start from Ser23 (see F igs 1 and 3 ). The active site residues in serine proteases, histidine, aspartic acid, a nd serine, were located at residues 178, 230, and 324, respectively, in preprospermosin. This indicates that sperm- osin is classi®ed i nto 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 classi®ed as a n ovel member of the S1 trypsin family (data not shown). Expression of spermosin mRNA in ascidian gonads Northern blotting was c arried ou t 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 puri®ed spermosin gave a s ingle band of 28 kDa under reducing conditions, whereas it showed two bands of 33 and 4 0 kDa under nonreducing c onditions (Fig. 3A). The N-terminal sequence determination of these 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 2 3±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 s tructures o f preprospermosin a nd spermosin type 1 and type 2 are depicted in Fig. 3C. Binding of spermosin light chains to the vitelline coat Comparison of th e sequences between L1 a nd L2 light chains revealed that the L1 but not the L2 had a region 1.9 kb Gn Hp In Bb Fig. 2. Tissue-speci®c e xpression of spermosin mRNA i n H. roretzi. Gn, Gonad; Hp, hepatopancreas; In, intestine; Bb, branchial basket. Northern blots of poly(A) + RNA (2 lg each) from H. roretzi tissues were hybridized with radiolabelled cDNA probes. A 1.9-kb mRNA signal was detected only in the gonad. SEST 40 kDa: IVGG 33 kDa: SEGP IVGG 28 kDa: IVGG BA 29 45 2-ME + - (kDa) 28 33 40 (kDa) C Preprospermosin Spermosin type 1 Spermosin type 2 Signal peptide Light chain Heavy chain S S SEST IVGG S S SEGP IVGG 1 23 97 130 388 Cleavage site H D S 178 230 324 28 kDa (33 kDa) L1 28 kDaL2 (40 kDa) Fig. 3. The presence of two forms of spermosin in H. roretzi sperm. (A) SDS/PAGE of the puri®ed spermosin. SDS/PAGE gave a band of 28 kDa under red ucing conditions and two b ands of 33 and 40 kDa under nonreducing co nd itions. 2-ME, 2-mercaptoeth anol. (B) The N-terminal amino-acid seq uences of three bands. T he 28-kDa protein had a single amino-acid sequence and was determined as a heavy chain of spermosin, whereas the 40-kD a protein con siste d of a he avy 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 distinc t bands of L1 and L 2 chains on SDS/ PAGE (12.5% gel) under the reducing conditions (see also Fig. 2 in [9]), probably because of their high electrophore tic mobility that is indistinguishable from the migration front. Alternatively, the low molecular mass p roteins (11-kDa L1 chain and 3-kDa L2 chain) m ight not be suciently ®xed within the g el under our experimental condi- tions. (C) Protein structure s of ascidian preprospermosin and sperm- osin type 1 and 2. The N-terminal amino-acid sequences of L1, L2, and heavy chains are shown above the respective models. The putative disul®de bond is indicated by analogy to the oth er trypsin family [17]. 660 E. Kodama et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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 t erminals 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), i ncluding L1, L2, and L1(DL2), an L1 lacking the L2 region, were expressed and puri®ed by glutathione± agarose chromatography. The puri®ed 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 c oat protein components, which can interact with the f usion proteins, were eluted with 20 m M glutathione and analysed by SDS/ PAGE. By comparison of protein patterns in SDS/PAGE, it was found that th e 28-kDa band was detected only with the GST-L1 or GST-L1(DL2) 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(DL2) domain of the spermosin light chain. The predicted N-terminal amino-acid sequence (SAXARNQNFG) showed no appreciable identity with an y 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 ®rst time. Here we show that there are two molecular forms of the molecule made up of a common h eavy chain and either a short or a long light chain depending on the processing sites. We previously reported that ascidian spermosin is a novel sperm trypsin-like protease, distinct from acrosin, a well-known sperm trypsin-like protease that is widely distributed in mammalian s perm, in terms of substrate speci®city 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 n o such C-terminal region. In contrast, the N-terminal region of the light chain of ascidian spermosin contains a p roline-rich region, which is observed in the C-terminal region of proacrosin of nonrodent mammals [19]. We identi®ed the vitelline coat component, to which the proline-rich region of the L1 chain of spermosin binds, for the ®rst 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 1 and 5 ). Although spermosin heavy chain showed the highest homology to mouse plasma kallikrein (32% ide ntity) 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 ef®ciently 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 1 and 3). The N-terminal region from residue N-23 to residue 129 (i.e. the L1 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 A B GST-L1 GST-L1 (∆L2) GST-L2 23 129 9623 97 129 GST GST GST GST alone GST-L1( GST-L1 GST-L1(∆L2)GST-L2 28 kDa Fig. 4. Expression of recombinant H. roretzi spermosin light chain-GST fusion proteins. (A) GST fusion prote ins inc luding L1, L2, and L1(DL2). (B) The fusion proteins were expressed in clones generated by PCR from the spermosin c DNA as described in Mate- rials and methods. GST±spermosin light chain fusion proteins (3 lg each), which had been previously puri®ed by glutathione± agarose chromatography, were mixed with the solubilized vitelline coat in arti®cial sea- water. After incub ation at 4 °C for 1 h, the fusion protein was applied to a column of glutathione±agarose beads. The proteins eluted with 20 m M glutathione w ere 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 b oth cases of G ST-L1 and GST- L1(DL2) fusion p roteins, was determined as describedinMaterialsandmethods. Ó FEBS 2002 Cloning and characterization of ascidian spermosin (Eur. J. Biochem. 269) 661 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±Ile130 bond of prospermosin. Here we showed the existence of two f orms of spermosin in ascidian sperm: the type 1 form containing the L1 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 endop eptidase that cleaves the C-terminal side of the asparagine residue may be responsible for this processing. It seems unlikely that spermosin type 1 is an inactive precursor of spermosin type 2, as spermosin type 1 and 2 are capable of binding to a soybean trypsin inhibitor-immobilized Sepharose column. With respect to the disul®de bond between the light chain and the heavy chain of ascidian spermosin, it is plausible that the C ys116 residue in the light chain is disul®de-bonded to the C ys251 residue in the heavy chain by analogy to other trypsin family proteins: the nearest cysteine residue to the active s ite aspartic acid residue is known to b e d isul®de- 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 h eavy chains of mammalian acrosin are bonded by two disul®de bridges, a single S±S bridge between light and heavy chains is rather common in serine proteases including mouse testis-speci®c proteases (TESP-I and -II) [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 o f an N-linked sugar attachment and paired basic residues in the N-terminal region, t he 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 L1 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 p roacrosins. 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 L1 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 th e sperm binding to the vitelline coat. Whether s permosin or its homologue is present in mammalian sperm is an intriguing issue, as a sperm protease(s) other than acrosin i s 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 p rotein in mouse epididymis extract i s speci®cally recognized by anti-ascidian spermosin antibody on the basis of Western blot analysis (E. Kodama, H. Yokosawa & H. S awada, unpublished data). Further studies are needed to search for a spermosin homologue in mammalian sperm and to elucidate its role in mammalian fertilization. HrSpermosin 130 IVGGAEAVPNSWPYAAAFGTYDISGGKLEVSQMCGSTIITPRHALTAAHCFMMDPDIDQT MmKallikrein 391 IVGGTNASLGEWPWQVSLQV KLVSQTHLCGGSIIGRQWVLTAAHCFDGIPY PD MmAcrosin 40 IVSGQSAQLGAWPWMVSLQI FTSHNSRRYHACGGSLLNSHWVLTAAHCFDNKKK VY HrAcrosin 36 IVGGEMAKLGEFPWQAAFLY KHVQVCGGTIIDTTWILSAAHCFDPHMYNLQS **.* * . :*: .:: **.::: *:***** HrSpermosin 190 YYIFMGLHDETT YKGVRP-NKIVGVRYHPKTNVFTDDPWLVYDFAILTLRKKVI MmKallikrein 444 VWRIYGGILSLS EITKETPSSRIKELIIHQEYKVSEGN YDIALIKLQTPLN MmAcrosin 96 DWRLVFGAQEIEYGRNKPVKEPQQERYVQKIVIHEKYNVVTEG NDIALLKITPPVT HrAcrosin 88 IKKEDALIRVADLDKTDDTDEGEMTFEVKDIIIHEQYNRQTFD NDIMLIEILGSIT : : * : : . *: :: : : HrSpermosin 243 ANFAWNYACLP-QPKKIPPEGTICWSVGWGVTQNTGGDNV LKQVAIDLVSEKRCK-E MmKallikrein 495 YTEFQKPICLP-SKADTNTIYTNCWVTGWGYTKEQGETQN ILQKATIPLVPNEEC-QK MmAcrosin 152 CGNFIGPCCLPHFKAGPPQIPHTCYVTGWGYIKEKAPRPSP-VLMEARVDLIDLDLCNST HrAcrosin 144 YGPTVQPACIP-GANDAVADGTKCLISGWGDTQDHVHNRWPDKLQKAQVEVFARAQC *:* * *** :: * :. : :. * HrSpermosin 298 EYRSTITSKSTICGGTTPG-QDTCQGDSGGPLFCKEDGK WYLQGIVSYGPSVCG- MmKallikrein 551 KYRDYVINKQMICAGYKEGGTDACKGDSGGPLVCKHSGR WQLVGITSWG-EGCGR MmAcrosin 211 QWYNGRVTSTNVCAGYPEGKIDTCQGDSGGPLMCRDNVDSP FVVVGITSWG-VGCAR HrAcrosin 200 LATYPESTENMICAGLRTGGIDSCQGDSGGPLACPFTENTAQPTFFLQGIVSWG-RGCAL :*.* * *:*:******* * : : **.*:* *. HrSpermosin 351 SGPMAAYAAVAYNLEWLCCYMP NLPSCEDIECDESGEN MmKallikrein 605 KDQPGVYTKVSEYMDWILEKTQ SSDVRALETSSA MmAcrosin 267 AKRPGVYTATWDYLDWIASKIG PNALHLIQPATPHPPTTRHPMVSFHPPSLRPPW HrAcrosin 259 DGFPGVYTEVRKYSSWIANYTQHLLQDRNADVATFTITGDPCSSNGSIISGSEGDFSSPG *: . .*: MmAcrosin 322 YFQHLPSRPLYLRPLRPLLHRPSSTQTSSSLMPLLSPPTPAQPASFTIATQHMRHRTTLS HrAcrosin 319 FYSGSYTDNLDCKWIIQIPDIGSRIQLSFTEFGVEYHTFCWYDDVKVYSGAVGNIASADA MmAcrosin 382 FARRLQRLIEALKMRTYPMKHPSQYSGPRNYHYRFSTFEPLSNKPSEPFLHS HrAcrosin 379 ADLLGSHCGMNIPSDLLSDGSSMTVIFHSDYMTHTLGFRAVFHAVSADVSQSGCGGIREL HrAcrosin 439 LTDHGEFSSKHYPNYYDADSNCEWLITAPTGKTIELNFLSFRLAGSDCADNVAIYDGLNS HrAcrosin 499 SQLPKNN Fig. 5. Sequence alignment of ascidian spermosin heavy chain with those of trypsin- family proteases including ascidian acrosin by the CLUSTALW program. Identical residues in the sequenc es amon g se rine protease are indicated by asterisks. Three co nserved active site residues in the S1 subfamily [17] of trypsin-like pr otease are indicated by closed triangles. The loc ations of paired basic resi- dues in the N-terminal regions of mouse and ascidian acrosin are indicated by c los ed circles. Asterisks indicate the positions o f fully conserved amino-acid residue s. Double and single dots represent the ``strong'' (score of Gonnet Pam250, > 0.5) and ``weak'' (£ 0.5) consensus positions, respectively. For details of CLUSTAL W and Gonnet Pam250, access the URLs o f http:// hypernig.nig.ac.jp/homology/clustalw.shtml and http://bioinformer.ebi.ac.uk/n ewsletter/ archives/2/clustalw17.html, respectively. HrSpermosin, H. roretzi spermosin (DDBJ/ GenBank/EBI accession number, AB052776); MmKallikrein, Mus musculus plasma kallikrein (M58588); MmAcrosin, Mus musculus acrosin (D00574); HrAcrosin, H. roretzi acrosin (AB052635). 662 E. Kodama et al. (Eur. J. 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