Báo cáo khoa học: Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes pot

Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes Mari Kawaguchi 1,2 , Shigeki Yasumasu 3 , Akio Shimizu 4 , Kaori Sano 5 , Ichiro Iuchi 3 and Mutsumi Nishida 1 1 Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba, Japan 2 Research Fellow of the Japan Society for the Promotion of Science (JSPS), Tokyo, Japan 3 Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan 4 National Research Institute of Fisheries Science, Fisheries Research Agency, Yokohama, Japan 5 Graduate Program of Biological Science, Graduate School of Science and Technology, Sophia University, Tokyo, Japan Introduction Molecular bases of formation, hardening (conversion) and breakdown of teleostean egg envelope have been comprehensively studied in medaka Oryzias latipes as a model animal [1–3]. The egg envelope (chorion) consists of a major thick inner layer and an extremely thin outer layer. The inner layer is constructed of fibrous macromolecules comprising two groups of sub- unit proteins: ZI-1,2 and ZI-3 [4]. ZI-1,2 are heteroge- neous glycoproteins derived from the precursor proteins, choriogenin H (ChgH) and choriogenin H Keywords chorion; egg envelope; euteleostean fish; Fundulus heteroclitus; hatching enzyme; ZP domain Correspondence M. Nishida, Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan Fax: +81 4 7136 6211 Tel: +81 4 7136 6210 E-mail: mnishida@aori.u-tokyo.ac.jp Database The nucleotide sequence data reported in this paper are available in the EMBL ⁄ GenBank ⁄ DDBJ databases under the accession numbers AB533328 to AB533330 (Received 6 July 2010, revised 1 October 2010, accepted 6 October 2010) doi:10.1111/j.1742-4658.2010.07907.x We purified two hatching enzymes, namely high choriolytic enzyme (HCE; EC 3.4.24.67) and low choriolytic enzyme (LCE; EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE-swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N-termi- nal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved differ- ent sites of the subunit proteins. FHCE efficiently cleaved the Pro-X-Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE- swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly con- served between Fundulus and medaka. Finally, we extended such a compar- ison to three other euteleosts (i.e. three-spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope diges- tion by HCE and LCE orthologs was established in the lineage of eu- teleosts, and the mechanism is suggested to be conserved. Abbreviations ChgH, choriogenin H; ChgHm, choriogenin H minor; ChgL, choriogenin L; FE, fertilized egg envelope; FHCE, Fundulus HCE; FhChgH, F. heteroclitus ChgH; FhChgHm, F. heteroclitus ChgHm; FhChgL, F. heteroclitus ChgL; FhZPB, F. heteroclitus ZPB; FhZPC, F. heteroclitus ZPC; FLCE, Fundulus LCE; HCE, high choriolytic enzyme; LCE, low choriolytic enzyme; TFA, trifluoroacetic acid; UFE, unfertilized egg envelope; ZP, zona pellucida. FEBS Journal 277 (2010) 4973–4987 ª 2010 The Authors Journal compilation ª 2010 FEBS 4973 minor (ChgHm), which are synthesized in the liver of spawning female, transported through the blood, pro- cessed at their C-terminal processing sites, and assem- bled around the egg [5,6]. ZI-3 is a homogeneous glycoprotein derived from another precursor, chorioge- nin L (ChgL) [7]. All of the subunit proteins contain a zona pellucida (ZP) domain that is the common struc- ture in all vertebrate egg envelope proteins [8]. ChgH and ChgHm of medaka (precursors of ZI-1,2) are clas- sified into ZPB, whereas ChgL (precursor of ZI-3) are classified into ZPC [9]. The unfertilized egg envelope is soft or fragile. After fertilization, the envelope becomes hard and protects the embryo from the mechanical and chemical stresses of the environment. Egg envelope hardening in medaka has been suggested to be a result of the forma- tion of e-(c-glutamyl) lysine cross-links between sub- unit proteins of the envelope, mainly between inner layer subunit proteins [10]. At the time of hatching of the embryo, the inner layer is digested by hatching enzyme [1]. The outer layer that remains undigested is ruptured by movement of the embryo. The breakdown of the inner layer by the enzyme is responsible for embryo hatching. Medaka hatching enzyme is composed of two asta- cin family metalloproteases: high choriolytic enzyme (HCE; choriolysin H; EC 3.4.24.67) and low choriolyt- ic enzyme (LCE; choriolysisn L; EC 3.4.24.66) [11,12]. At the time of hatching, the two enzymes act coopera- tively on envelope: HCE swells the inner layer of enve- lope and LCE completely digests or solubilizes the HCE-swollen part of the inner layer. A previous study has revealed that HCE and LCE cleave different sites on the egg envelope subunit proteins in addition to one common site [13]. Recently, cDNAs for Fundulus heteroclitus orthologs of HCE and LCE (Fundulus HCE, FHCE; Fundulus LCE, FLCE) were cloned, and their gene expression during development was observed by northern blotting as well as whole-mount in situ hybridization [14]. Their gene structures and expression patterns conserved those of medaka. In the previous study, we separately purified two isoforms of FHCE (FHCE1 and FHCE2). By contrast, FLCE was not fully purified. In vitro egg envelope digestion revealed that both the purified FHCE1 and FHCE2 swell the egg envelope, and the partially purified FLCE-fraction has the solubilizing activity of the FHCE1 ⁄ 2-swollen egg envelope. There- fore, it has been predicted that the mode of their proteolytic action toward the envelope is conserved between Fundulus and medaka. In the present study, we first purified FLCE as a major band by SDS ⁄PAGE. Next, we cloned Fundulus cDNA orthologs for egg envelope protein precursors, ChgH, ChgHm and ChgL. The cleavage sites of FHCE1 ⁄ 2 or FLCE on the egg envelope proteins were determined, and the amino acid sequences around the sites were compared with those of medaka. Finally, we extended the comparison to three other euteleosts: three-spined stickleback, spotted halibut and rainbow trout. Results Purification of FLCE from Fundulus hatching liquid The purity of the previously obtained FLCE-fraction was not sufficient to determine FLCE-cleavage sites on egg envelope protein. Therefore, we developed a new purification method. As shown in Fig. 1A, the Toyo- pearl HW-50S column chromatography of ammonium sulfate precipitate from hatching liquid revealed two proteolytically active peaks (fractions I and II). As shown in a previous study [14], fractions I and II con- tained FLCE and FHCE1 ⁄ 2, respectively. We employed fraction I for further purification procedures. Fraction I was applied to an S-Sepharose column, and adsorbed proteins were eluted once with 50 mm Tris-buffer containing 0.4 m NaCl. The eluate, named fraction IS, was applied to a Source 15S column. Most of the proteins were adsorbed and fractionated mainly into three peaks, named IS-a, IS-b and IS-c (Fig. 1B). When caseinolytic specific activity was examined, the highest activity was observed in IS-c (Fig. 1C). MALDI-TOF-MS analysis of IS-c showed a major peak of m ⁄ z at 23800.9 and the value was well concor- dant with the molecular weight calculated from FLCE cDNA (MW = 23805.65). SDS⁄ PAGE showed that the densities of IS-a, IS-b and IS-c bands at 23 kDa were comparable with the specific activities of the respective fractions (Fig. 1C). To confirm LCE activity for fraction IS-c, the envelopes swollen either by FHCE1 or by FHCE2 were incubated with IS-c and observed by microscopy. The swollen envelopes were efficiently solubilized by IS-c (data not shown). Thus, we concluded that the 23 kDa band in fraction IS-c is FLCE. Figure 1D shows the SDS ⁄ PAGE patterns of purified FHCE1, FHCE2 and FLCE. The electrophoretic mobil- ity of FLCE was slightly higher than those of FHCE1 and FHCE2, and clearly different from them. The caseinolytic specific activities of FHCE1, FHCE2 and FLCE were estimated as 16.0, 12.6 and 14.4 DA 280 min )1 Æmg protein )1 , respectively, which were similar to each other and approximately one half of Egg envelope digestion mechanism M. Kawaguchi et al. 4974 FEBS Journal 277 (2010) 4973–4987 ª 2010 The Authors Journal compilation ª 2010 FEBS those of medaka HCE (30.2 DA 280 min )1 Æmg protein )1 ) and medaka LCE (24.5 DA 280 min )1 Æmg protein )1 ). Fundulus orthologs of choriogenin H, H minor and L In medaka, it has been reported that choriogenins, which are precursors of egg envelope subunit proteins, are synthesized in the liver under the influence of estro- gen [15]. RNAs extracted from the spawning female liver of Fundulus were used as a template of RT-PCR and, finally, three kinds of full-length choriogenin cDNAs were cloned. According to the phylogenetic analysis, the three Fundulus cDNAs were separately located in the ChgH, ChgHm and ChgL clades (Fig. 2), and therefore named FhChgH (F. heteroclitus ChgH), FhChgHm (F. heteroclitus ChgHm) and FhChgL (F. heteroclitus ChgL) cDNAs, respectively. Amino acid sequences deduced from FhChgH, FhChgHm and FhChgL cDNAs are shown in Fig. 3, together with medaka orthologs OlChgH, OlChgHm and OlChgL. All of them possessed a hydrophobic signal peptide at their N-termini. The cleavage site of signal peptidase was deduced to be at Ala26 ⁄ Gln27 for FhChgH, Ala22 ⁄Gln23 for FhChgHm and Ala22 ⁄ Gln23 for FhChgL, according to signalp 3.0 software (http: ⁄⁄www.cbs.dtu.dk ⁄ services ⁄ SignalP ⁄ ). FhChgH, FhChgHm and FhChgL all had a ZP domain. The tre- foil domain was found at the N-terminal side of the ZP domain of FhChgH and FhChgHm. The consensus motif for the processing site, such as Arg-Lys-X-fl-Arg, was found near the C-termini of FhChgHm and FhChgL. In medaka, the C-terminal regions from those sites are excised before the assembly of water-soluble precursors into the water-insoluble egg envelope [16]. The site of FhChgH was Arg-Lys-Gly-Lys. Therefore, each processing site is predicted to be at Gly564 ⁄ Lys565 for FhChgH, Lys423 ⁄ Arg424 for FhChgHm and Val400 ⁄Arg401 for FhChgL. One of the characteristics of OlChgH and OlChgHm is the presence of Pro-X-Y repeat sequences in their N-terminal regions [5,6]. Such Fig. 1. Purification of Fundulus hatching enzyme. (A) Toyopearl HW-50S column chromatogram of hatching liquid. Solid line, A 280 ; dashed line, caseinolytic activity indicated by A 280 . (B) Elution pattern of fraction IS, which was obtained from fraction I via an S-Sepharose column, by cation exchange HPLC with a linear gradient of 0–400 m M NaCl. (C) Caseinolytic specific activity (DA 280 min )1 Æmg protein )1 ) of fractions IS-a, IS-b and IS-c, as well as their SDS ⁄ PAGE patterns detected by silver staining. (D) SDS ⁄ PAGE patterns of purified FHCE1, FHCE2 and FLCE (fraction IS-c), detected by silver staining. Numbers on the left refer to the size (kDa) of the molecular markers. Fig. 2. A phylogenetic tree of the ZP domain of choriogenins. The tree was constructed by the maximum likelihood method using the nucleotide sequences. Numbers at the nodes represent bootstrap values (shown as percentages). Accession numbers: F. heteroclitus (FhChgH, AB533328; FhChgHm, AB533329; FhChgL, AB533330); O. latipes (OlChgH, D89609; OlChgHm, AB025967; OlChgL, D38630); Oryzias javanicus (OjChgH, AY913759; OjChgL, AY913760); Oryzias dancena (OdChgH, EF392363; OdChgL, EF392364); Oryzias sinensis (OsChgL, AY758411); Cyprinodon variegatus (zona radiata-2 CvZR2, AY598615; zona radiata-3 CvZR3, AY598616). M. Kawaguchi et al. Egg envelope digestion mechanism FEBS Journal 277 (2010) 4973–4987 ª 2010 The Authors Journal compilation ª 2010 FEBS 4975 Egg envelope digestion mechanism M. Kawaguchi et al. 4976 FEBS Journal 277 (2010) 4973–4987 ª 2010 The Authors Journal compilation ª 2010 FEBS regions were also found in FhChgH and FhChgHm, and their repetitive units were YPQQPQ(T ⁄ K ⁄ Q)PS and YP(K ⁄ N)PQTPPSKPQ for FhChgH and YPSKP- QQPQQPQ and YPQQPQQPQ for FhChgHm (Fig. 3). Expression of choriogenin genes Choriogenin gene expression was observed by northern blotting (Fig. 4). Each FhChgH, FhChgHm and FhChgL probe was hybridized with two transcripts, and the sizes were approximately 2 and 5 kb (FhChgH), 1.6 and 5 kb (FhChgHm) and 1.4 and 5 kb (FhChgL). The sizes of the smaller bands hybridized with all of the probe (Fig. 4, asterisks) were similar to those of cloned cDNAs: 2037 bp for FhChgH, 1518 bp for FhChgHm and 1428 bp for FhChgL. Therefore, the smaller bands represented choriogenin genes. Each of the larger bands, obtained from the three probes, was assumed to be the choriogenin gene-related RNAs, such as pre-mRNA for choriogenin genes. When com- paring the choriogenin gene signals, strongest expres- sion was found in the FhChgL gene, followed by the FhChgH gene. This relationship was similar to that of medaka (i.e. strongest in the OlChgL gene, followed by the OlChgH gene) (Fig. 4). Thus, the relative expression level of choriogenin genes was conserved between Fundulus and medaka. Cleavage sites of hatching enzyme on unfertilized egg envelope One of the goals of the present study was to deter- mine the cleavage sites of hatching enzyme on egg envelope proteins. The natural substrate of hatching enzyme is fertilized egg envelope (FE), as described in the Introduction. FE was digested or solubilized only by the combined action of two enzymes, and not by any one of the two enzymes, nor by SDS. Alternatively, unfertilized egg envelope (UFE) was digested by one of the enzymes FHCE1 ⁄ 2 or FLCE, and was solubilized by SDS. Therefore, UFE was first used as a substrate to determine the cleavage sites. When UFE isolated from Fundulus was solubilized by SDS and applied onto SDS ⁄ PAGE, two major bands were found at molecular masses of 60 and 48 kDa (Fig. 5B). This pattern was similar to that of medaka UFE [4,13]. The 60 and 48 kDa bands are regarded as Fundulus homologs of ZI-1,2 (ZPB groups of medaka) and ZI-3 (a ZPC group of medaka), respectively, and were designated as FhZPB (F. hetero- clitus ZPB) and FhZPC (F. heteroclitus ZPC), respec- tively. FhZPB is considered to be a group of egg envelope subunit proteins derived from their precur- sors FhChgH and FhChgHm, and FhZPC is an egg envelope subunit protein derived from its precursor FhChgL. Digests of Fundulus UFE by FHCE1 and those by FHCE2 showed the same SDS ⁄ PAGE pattern (data not shown), and were observed at 46, 36 and 32 kDa (Fig. 5B). An N-terminal amino acid sequence obtained from the 46 kDa digest was NQQQLQTFK and was found from Asn41 of FhChgL (Table 1). The sequence Fig. 3. Alignment of amino acid sequences of choriogenin H (A), H minor (B) and L (C) of Fundulus (FhChgH, FhChgHm and FhChgL) and medaka (OlChgH, OlChgHm and OlChgL). Identical residues are indicated by asterisks below the sequences, and dashes represent gaps. The trefoil domain and ZP domain are shown within dark and light gray boxes, respectively. Conserved cysteine residues are highlighted in white with a black background. Conserved cysteine residues 1–8 of ZP proteins and additional conserved cysteine residues a and b of ZPB proteins are labeled. The black arrowheads and black diamonds are HCE and LCE cleavage sites determined using unfertilized egg enve- lopes, respectively. The white diamonds represent the cleavage sites determined using hatching liquid. The names of the cleavage sites are shown to the left of the marks. Four types of dashed ⁄ dotted lines above the sequences indicate the four types of repeating units found in the Pro-X-Y repeat region of FhChgH and FhChgHm. White and black triangles indicate putative signal sequence cleavage sites and process- ing sites, respectively. Italicized, underlined letters indicate the consensus C-terminal processing site, Arg-Lys-X-fl-Arg. Fig. 4. Northern blot analysis of Fundulus or medaka choriogenin gene expression. Gene names are shown at the top. Bands show- ing choriogenin genes are indicated by asterisks. Numbers on the left refer to the size (base) of the molecular markers. Gel images of 28S and 18S rRNA are shown at the bottom. M. Kawaguchi et al. Egg envelope digestion mechanism FEBS Journal 277 (2010) 4973–4987 ª 2010 The Authors Journal compilation ª 2010 FEBS 4977 of the 36 kDa digest, APGVPT, was found from Ala230 residing near the N-terminal side of the trefoil domain of FhChgH (Table 1). Two bands were observed at 32 kDa. However, only a single sequence was obtained from such two bands, and the sequence, TPTET, was found from Thr77 residing at the N-terminal of the trefoil domain of FhChgHm (Table 1). The FHCE1 ⁄ 2 cleavage sites on FhChgH and FhChgL thus determined were located at positions similar to those of the cleavage sites of medaka HCE (Fig. 3) [13]. The digests by the mixture of FHCE1 ⁄ 2 and FLCE were observed at 60, 46, 38, 32 and 17 kDa (Fig. 5B). The 60 kDa band was that of undigested FhZPB, and the 46 and 32 kDa bands were those of FHCE1 ⁄2- digests. We could determine two FLCE sites that were not found in FHCE1 ⁄ 2 sites. An amino acid sequence obtained from the 38 kDa digest was YPVPAATVA and matched the sequence from Tyr74 of FhChgL (Table 1). The N-terminal sequence of the 17 kDa pro- duct was a mixture of two products. By comparison Fig. 5. The egg envelope digestion pattern of hatching enzyme. (A) Schematic presentation of the egg envelope digestion processes by FHCE1 ⁄ 2 and FLCE, together with the respective morphological changes of the fertilized egg envelope of Fundulus. FhZPB is the Fundulus ZPB ortholog derived from FhChgH and FhChgHm, and FhZPC is the Fundulus ZPC ortholog derived from FhChgL. Black, dark gray and light gray boxes indicate the Pro-X-Y repeat region, trefoil domain and ZP domain, respectively. Arrowheads indicate the cleavage sites of FHCE1 ⁄ 2 or FLCE. The length between the two arrows in the images indicates the thickness of the egg envelope. Scale bar = 0.1 mm. (B) SDS ⁄ PAGE pattern of envelopes isolated from Fundulus unfertilized egg (as a control), digests of the envelope by FHCE2, the digests by the mixture of the FHCE1 ⁄ 2 and FLCE, and the digests in Fundulus hatching liquid. Numbers on the left refer to the size of the molecular markers (kDa). Table 1. Cleavage sites of hatching enzymes on egg envelope determined after the incubation of unfertilized egg envelope with FHCE1, FHCE2 or the mixture of FHCE1 ⁄ 2 and FLCE. The sites found at natural hatching were determined using hatching liquid. Enzyme Size (kDa) N-terminal sequence Choriogenin Site Site name FHCE1 ⁄ FHCE2 46 NQQQLQTFK FhChgL Q40 ⁄ N41 FhZPC1 36 APGVPT FhChgH E229 ⁄ A230 FhZPB1 32 TPTET FhChgHm Q76 ⁄ T77 FhZPB3 FHCE ⁄ FLCE 38 YPVPAATVA FhChgL R73 ⁄ Y74 FhZPC2 32 TPTETFHTxDVPAPF FhChgHm Q76 ⁄ T77 FhZPB3 17 NPPPAVAELGPIRVA FhChgH D394 ⁄ N395 FhZPB2 APGVPTPKSxDVEVA FhChgH E229 ⁄ A230 FhZPB1 Hatching liquid 35 YPVPAATVAV FhChgL R73 ⁄ Y74 FhZPC2 PVPAATVAVE FhChgL Y74 ⁄ P75 32 TPTETFHTxD FhChgHm Q76 ⁄ T77 FhZPB3 25 TSQAAVIVE FhChgL R167 ⁄ T168 FhZPC3 18 NPPPAVAELG FhChgH D394 ⁄ N395 FhZPB2 16 VPTPKSxDVE FhChgH G232 ⁄ V233 Egg envelope digestion mechanism M. Kawaguchi et al. 4978 FEBS Journal 277 (2010) 4973–4987 ª 2010 The Authors Journal compilation ª 2010 FEBS with the amino acid sequence deduced from cDNA, they were predicted to be the sequences from Asn395 residing inside of the ZP domain of FhChgH (NPPPAVAELG- PIRVA) and from Ala230 located near the N-terminal side of the trefoil domain of FhChgH (AP- GVPTPKSxDVEVA) (Table 1). The latter sequence corresponded to that of the 36 kDa digest in FHCE1 ⁄ 2- digests. Therefore, the 36 kDa digest could be further cleaved by FLCE and divided into two 17 kDa digests. The positions of two FLCE cleavage sites thus deter- mined were well concordant with LCE sites of medaka (Fig. 3) [13]. To clarify hatching enzyme cleavage sites in natural hatching, we finally determined the N-terminal sequence of the digests of FE contained in hatching liquid. As shown in Fig. 5B, the SDS ⁄ PAGE pattern of Fundulus hatching liquid was somewhat different from that of FHCE ⁄FLCE-digests of UFE (i.e. the digests of UFE by the mixture of FHCE1 ⁄2 and FLCE). However, the cleavage sites determined using major digests in hatching liquid were essentially con- cordant with those of UFE as summarized in Table 1. (a) The sequence of the 35 kDa digest was a mixture of YPVPAATVAV and PVPAATVAVE. The former sequence corresponded to that of the digest cleaved at the site FhZPC2 of UFE, and the latter was that cleaved at one amino acid residue from C-terminal side of the FhZPC2 site (Table 1). (b) The sequence of the 32 kDa digest was TPTETFHTxD, which corresponds to that of the digest cleaved at the site FhZPB3 of UFE (Table 1). (c) Two bands were found at 18 and 16 kDa. The sequence of the 18 kDa digest was NPPPAVAELG found from Asn395 in FhChgH, and the cleavage site Asp394 ⁄ Asn395 matched with the FLCE cleavage site, FhZPB2, determined with UFE (Table 1). The sequence of the 16 kDa digest was VPTPKSxDVE found from Val233 in FhChgH. This site was located at three amino acid residues from C-terminal side of the FHCE1 ⁄ 2 cleavage site, FhZPB1, determined with UFE. Discrepancy between digests of UFE and those of FE in hatching liquid, such as minor differences with respect to electropho- retic mobility and cleavage sites, might result from the structural difference between UFE and FE, probably as a result of the existence of e-(c-glutamyl)-lysine cross-links in FE. (d) The 25 kDa band was observed in the hatching liquid but not in the FHCE ⁄FLCE- digests of UFE. The N-terminal sequence of the digest was TSQAAVIVE and was located inside of ZP-domain of FhChgL. In natural hatching, a part of the 35 kDa digest was further digested and degraded into the 25 kDa digest. The SDS ⁄ PAGE patterns of the digests of isolated FE by purified FHCE1 ⁄ 2 and FLCE were the same as that of the hatching liquid (data not shown). Thus, the results obtained show that the hatching enzyme cleavage sites determined with FE reflect well those determined with UFE. Next, FHCE1 ⁄ 2 cleavage sites that are present in the Pro-X-Y repeat region were determined. The HCE- inducing swelling of FE in medaka releases water- soluble peptides that are excised from the Pro-X-Y repeat region [10]. The previous study showed that this region was broken into small peptides that can not be detected by SDS ⁄ PAGE. Therefore, after UFE was digested with FHCE1 alone, the supernatant was applied to the reverse phase HPLC system. Seven major peaks were obtained and subjected to N-terminal sequencing and MALDI-TOF-MS. We obtained sequences such as YPQQPQ, YPSKPQ, YPNPQ, YP- KPQ and YPRPQ, suggesting that FHCE1 cleaved the sites locating the tyrosine residue at the P1¢ site and the proline residue at the P2¢ site [17,18]. To further study the FHCE1 cleavage sites, all the peaks eluted with chromatography were subjected to MALDI-TOF-MS. As shown in Fig. 6A,B, all of the monoisotopic molecu- lar weights thus determined matched the molecular weights calculated from either FhChgH and FhChgHm cDNA. In addition, the results obtained were confirmed using a recombinant protein of the Pro-X-Y repeat region of FhChgH, called rec.FhChgH_ProXY. After rec.FhChgH_ProXY was digested by FHCE1 ⁄ 2, the digests were fractionated by reverse phase column chro- matography, and analyzed by MALDI-TOF-MS. The result obtained was consistent with the FHCE1-cleavage pattern of the Pro-X-Y repeat region of FhChgH obtained from UFE (Fig. 6C). This clearly indicates that the Pro-X-Y repeat region was broken into small pieces, the size of which was three, four, five, six, nine or 12 amino acids in length. FHCE1 ⁄ 2 cleaved a bond between Gln and Tyr of the Pro-X-Y region or between Ser and Tyr, and occasionally also cleaved a bond between Ser and Lys or between Gln and Thr. Estimation of the egg envelope digestion efficiency of FHCE1 ⁄ 2 and FLCE The substrate preferences of FHCE1 ⁄ 2 and FLCE were quantitatively estimated using synthetic peptides. The peptide sequences were designed from three FHCE1 ⁄2 cleavage sites (FhZPB1, FhZPB3 and FhZPC1); two FLCE sites (FhZPB2 and FhZPC2); and one site (FhZPC3) determined using hatching liquid. The names of the synthetic peptides correspond to those of the sites (Table 2). In addition, six peptides were designed from the Pro-X-Y repeat region. Four of them (PSYP, PQYP, PQTP and PSKP) were M. Kawaguchi et al. Egg envelope digestion mechanism FEBS Journal 277 (2010) 4973–4987 ª 2010 The Authors Journal compilation ª 2010 FEBS 4979 designed from FHCE1 ⁄ 2 sites. As a control, two (PQQP and PQKP) were designed from the sites that were not cleaved by any type of hatching enzymes. As shown in Table 2, FLCE showed high activity toward the peptides for the FLCE sites (FhZPB2 and FhZPC2) but no activity toward all of the peptides designed from the Pro-X-Y repeat region, or low activ- ity toward FHCE1 ⁄2 sites. Therefore, FLCE is con- firmed to specifically cleave the N-terminal side of the ZP domain of FhZPC and the center of ZP domain of FhZPB. In addition, FLCE showed high specific activ- ity toward the peptide deduced from hatching liquid (FhZPC3) but FHCE1 ⁄ 2 had no activity toward the peptide, suggesting that FLCE specifically cleaves the FhZPC3 site on FE, but that FHCE1 ⁄ 2 do not. FHCE1 ⁄ 2 showed high specific activity toward two peptides designed from the Pro-X-Y repeat region, PQYP and PSYP, confirming that the tyrosine residue at the P1¢ site is preferred by FHCE1 ⁄ 2. Therefore, FHCE1 ⁄ 2 have a high specific activity for digesting the center of Pro-Gln ⁄ Ser-Tyr-Pro sequence in the Pro-X-Y repeat. However, no activity of FHCE1 ⁄ 2 toward PSKP and PQTP peptides was observed, suggesting that a bond between Ser and Lys or between Gln and Thr in UFE and in rec.FhChgH_ProXY is not so efficiently cleaved by FHCE1 ⁄ 2. In addition, FHCE1 ⁄ 2 showed no activity or only low activity toward the peptides designed from FHCE1 ⁄ 2 cleavage sites (FhZPB1, FhZPB3 and FhZPC1). This difference in substrate Fig. 6. FHCE1 ⁄ 2 cleavage sites found in the Pro-X-Y repeat region. The cleavage sites in FhChgH (A) and FhChgHm (B) were deter- mined using the unfertilized egg envelope and recombinant protein, rec.FhChgH_ProXY (C). Black arrowheads indicate FHCE1 ⁄ 2 cleav- age sites. FhZPB1 and FhZPB3 shown next to the white arrow- heads indicate FHCE1 ⁄ 2 cleavage sites, as described in Fig. 3. Values under the lines indicate observed monoisotopic masses together with their calculated monoisotopic masses (given in paren- theses). rec.FhChgH_ProXY possesses additional methionine and histidine residues at the N- and C-terminus, respectively. Table 2. Specific activity of FHCE1, FHCE2 and FLCE estimated using synthetic peptide substrates. The cleavage site on each pep- tide is indicated by an arrow. ND, not detected. Substrate Sequence Specific activity (nmolÆ30 min )1 Ælg protein )1 ) FHCE1 FHCE2 FLCE FHCE1 ⁄ 2 cleavage sites FhZPB1 PSKRPEflAPGVP 1.40 0.95 0.56 FhZPB3 YPSKPQflTPTET 0.66 0.35 2.71 FhZPC1 QSPPTQflNQQQL ND ND ND FLCE cleavage sites FhZPB2 EVLPLDflNPPPA 0.98 1.07 8.66 FhZPC2 VPFELRflYPVPA 0.05 0.05 10.3 Cleavage site deduced from hatching liquid FhZPC3 SVPVVRflTSQAA ND ND 19.5 Pro-X-Y region PSYP QTPSflYPQQ 13.7 11.6 ND PQYP SKPQflYPNP 23.2 16.0 ND PQTP PNPQTPPS ND ND ND PSKP TPPSKPQY ND ND ND PQQP SYPQQPQT ND ND ND PQKP QQPQKPSY ND ND ND Egg envelope digestion mechanism M. Kawaguchi et al. 4980 FEBS Journal 277 (2010) 4973–4987 ª 2010 The Authors Journal compilation ª 2010 FEBS preference may be a result of structural differences of the substrate, such as the macromolecular egg envelope and small peptides, and FHCE1 ⁄ 2 are able to cleave these sites only when egg envelope was used as substrate. Conservation of the egg envelope digestion mechanism of hatching enzymes in euteleosts LCE cleavage sites The present study suggests that the egg envelope diges- tion mechanism of hatching enzymes is conserved between two euteleosts, Fundulus and medaka. To extend the comparison from lower to higher euteleosts, we collected the hatching liquid of several fishes and determined the N-terminal amino acid sequences of their digests. Figure 7A shows the tricine-SDS ⁄ PAGE patterns of hatching liquid obtained from higher euteleosts, such as Fundulus, medaka, three-spined stickleback and spotted halibut, as well as the lower euteleost, rainbow trout. First, we focused on the three digests in Fundulus hatching liquid, indicated by *1, *2a and *3a in Fig. 7A. When the N-terminal sequences of the bands in hatching liquid of four fishes were compared with those of Fundulus, the respective bands were revealed to correspond well with those of the digests of Fundu- lus. (a) The 35 kDa digest of Fundulus was generated by cleavage at FhZPC2. The site was found in the 35 kDa product of medaka and spotted halibut or in the 27 kDa of rainbow trout (Fig. 7A, *1). (b) A part of the 35 kDa digest of Fundulus was cleaved at the site FhZPC3 to generate the C-terminal 27 kDa and N-terminal 9 kDa digests (Fig. 7A, *2a and *2b). The bands corresponding to them were observed at the 27 and 10 kDa digests of medaka and three-spined stickle- back or at the 27 and 9 kDa digests of spotted halibut, except rainbow trout. In three-spined stickleback, no bands around 35 kDa were observed, suggesting that this 35 kDa product is completely digested into the 27 and 10 kDa bands. These results suggest that cleavage efficiency at the site corresponding to FhZPC3 site is different from species to species. (c) The 18 kDa digest, as well as the 16 kDa digest, of Fundulus was generated by cleavage at the site FhZPB2. The site was found in the 17 kDa products in medaka, in the 14 kDa prod- ucts in three-spined stickleback and in the 18 kDa products in spotted halibut and rainbow trout (Fig. 7A, *3a). Alignment of the sequences around the three cleavage sites (Fig. 7B,D) suggests that the position of each cleavage site is well conserved in higher euteleosts, and two of three sites also coincide with each other in lower euteleosts. In addition to the digests described above, the 35 kDa digest was observed in rainbow trout hatching liquid (Fig. 7A, *4). The sequence analysis revealed Fig. 7. The conservation of the egg envelope digestion mechanism of hatching enzyme in euteleosts. (A) Tricine-SDS ⁄ PAGE pattern of hatching liquid for Fundulus, medaka, three-spined stickleback, spotted halibut and rainbow trout. The bands comparable to each other are indicated by *1 to *4. (C) The regions of products *1 to *3 are indicated in the schematic presentation of ZPB and ZPC, together with the cleavage sites of HCE and LCE. Arrowheads in gray and black indicate cleavage sites of HCE and LCE, respectively. Partial amino acid sequence alignment around the LCE cleavage sites on ZPB and ZPC is shown in (B) and (D), respectively. The names of the cleavage sites of Fundulus LCE are shown next to the arrowheads. M. Kawaguchi et al. Egg envelope digestion mechanism FEBS Journal 277 (2010) 4973–4987 ª 2010 The Authors Journal compilation ª 2010 FEBS 4981 that the 35 kDa product was derived from VEPb, one of two precursors of rainbow trout ZPB. The cleavage site deduced from the 35 kDa digest of rainbow trout corresponded well with that of the 32 kDa digest of Fundulus, the digest of FhZPB derived from FhChgHm. At present, the digests derived from two kinds of ZPBs were only detected in rainbow trout and Fundulus hatching liquids, whereas the digests derived from only ChgH orthologs were detected in the other species. This is probably a result of differences with respect to the content of ZPBs in egg envelope among species. Therefore, major cleavage patterns and their cleavage sites are conserved among the euteleosts examined in the present study. HCE cleavage sites The proline-rich Pro-X-Y repeat region has been also found in the N-terminal region of precursors of ZPB of many euteleosts [9]. To determine whether the frag- mentation of the region is a universal feature in eu- teleosts, we further determined cleavage sites on the Pro-X-Y repeat region of rainbow trout egg envelope protein. The digests in rainbow trout hatching liquid were fractionated by reverse phase column chromatog- raphy, and the small peptides that eluted with a low acetonitril concentration (30–40%) were collected. Six major peaks were obtained and subjected to N-termi- nal sequencing. The obtained sequences were WP(A ⁄V), WPPI, WPVQPG, QPPQRPA and (Q ⁄E)P(L ⁄F)P(Q ⁄P)RPA. These sequences were found in the Pro-X-Y repeat regions of VEPa and VEPb (Fig. 8), which are ZPB precursors of rainbow trout. The result clearly indicates that the Pro-X-Y repeat region of rainbow trout egg envelope proteins is bro- ken into small pieces, with notable cleavage of the sites locating the tryptophan, glutamine and glutamic acid residues at the P1¢ site and the proline residue at the P2¢ site. Considering that the digestion pattern in the Pro-X-Y repeat region was similar among two of the higher euteleosts (Fundulus and medaka) and one of lower euteleosts (rainbow trout), the cleavage pat- tern of HCE is also suggested to be conserved among euteleosts. Discussion The present study investigated the egg envelope diges- tion mechanism of Fundulus hatching enzyme. Three cDNA orthologs of egg envelope precursor proteins, ChgH, ChgHm and ChgL cDNAs, were cloned. By comparing the N-terminal amino acid sequences of HCE- and LCE-digests with the sequences deduced from cDNAs, the cleavage sites of HCE and LCE on egg envelope subunit proteins were determined. The results obtained showed that not only genes of hatch- ing enzymes and egg envelope proteins, but also cleav- age sites of hatching enzymes are well conserved between Fundulus and medaka. Below, we discuss the mechanism of egg envelope digestion by hatching enzyme, mainly based on the structural characteristics of egg envelope protein. In medaka, HCE swells the hardened fertilized egg envelope to convert its compact structure into a loose structure. This conversion results from medaka HCE cleaving the Pro-X-Y region of ZI-1,2 in the envelope, leading to the release of small peptide fragments, with notable cleavage of the sites locating tyrosine and asparagine residues at P1¢ site within the repeats [10,13]. The released fragments contain e -(c-glutamyl) lysine isopeptide cross-links that are responsible for egg envelope hardening after fertilization [10]. Although we did not determine the content of e-(c- glutamyl) lysine isopeptides in the Pro-X-Y region of FhZPB, the content of glutamine (25%) and lysine (7%) in the region resembled that of medaka (gluta- mine, 21%; lysine, 6%). The present study showed that FHCE1 ⁄ 2 also cleaved the Pro-X-Y region into small fragments (three to twelve amino acids in Fig. 8. Cleavage sites found in Pro-X-Y repeat regions in rainbow trout egg envelope protein. Amino acid sequences of the Pro-X-Y repeat regions of rainbow trout ZPB, VEPa (A) and VEPb (B), are shown. Broken lines above the sequences indicate repeating units. The sequences determined from low molecular weight products in the hatching liquid are underlined. The cleavage sites are predicted to be AflQ, QflW and AflE, and are indicated by arrowheads. Egg envelope digestion mechanism M. Kawaguchi et al. 4982 FEBS Journal 277 (2010) 4973–4987 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... basis of formation, hardening, and breakdown of the egg envelope in fish Int Rev Cytol 136, 51–92 2 Yamagami K (1992) Studies on the hatching enzyme and its substrate, egg envelope of Oryzias latipes Zool Sci 9, 1131 3 Yamagami K (1996) Studies on the hatching enzyme (choriolysin) and its substrate, egg envelope, constructed of the precursors (choriogenins) in Oryzias latipes: a sequel to the information... envelope, and swelling or loosening of the compact structure of the envelope This contribution of HCE would make the envelope accessible to LCE The HCE-swollen envelope, as a result of a lack of Pro-X-Y repeat regions, as described above, is considered to comprise mainly the ZP domain, namely the ZP domain of the ZPC and trefoil ⁄ ZP domains of ZPB (Fig 5A) Such ZP domains are assembled together to form... through their noncovalent interaction [19,20] In medaka, it has been proposed that the swollen envelope is formed as filaments by the assembly of ZP domains, and cleavage of the LCE site at the center of the ZP domain contributes to the complete solubilization of the HCEswollen envelope [13] Another study [21] has shown that the ZP domain consists of two sub-domains, ZPN and ZP-C sub-domains, and the two... sequence in the Pro-X-Y repeat region, similar to Fundulus and medaka HCEs Therefore, the egg envelope digestion mechanism of the HCE-LCE system was considered to be maintained during the evolution of euteleosts To provide evidence for this hypothesis, further studies using other species located between the higher and lower euteleosts will be conducted in the future The present study also suggests that the. .. cleave the site with a tyrosine residue at the P1¢ site of the Pro-X-Y regions These results suggest that not only the digestion manner of Fundulus HCE, but also its substrate specificity is similar to that of medaka HCE Thus, the contribution of HCE to egg envelope digestion acts to fragment the Pro-X-Y regions of the hardened egg envelope, leading to the release of small fragments from the envelope, ... sub-domains are connected by an intervening sequence This intervening sequence is proposed to be protease-sensitive [21] Applying this information to the results obtained in the present study, it is reasonable to assume that the LCE site found at the center of ZP domain is located within such a protease-sensitive intervening sequence [13] Cleavage of this site results in the complete solubilization of the. .. performed, the gel was stained with Coomassie Brilliant Blue G or using a Silver Stain II Kit (Wako, Osaka, Japan) Estimation of caseinolytic activity The caseinolytic activity of hatching enzyme was measured using a 750 lL reaction mixture consisting of 83 mm TrisHCl (pH 8.0) and 3.3 mgÆmL)1 of casein The mixture was incubated for 30 min at 30 °C After the reaction was stopped by adding 250 lL of 20%... acetonitrile in 0.1% TFA The elution was monitored by measuring A215 The activity was calculated from the ratio of peak area of digested peptides relative to that of digested and undigested peptides The cleavage sites were confirmed either with amino acid sequencing or by MALDI-TOF-MS analysis Determination of cleavage sites in the Pro-X-Y repeat region from rainbow trout hatching liquid Rainbow trout hatching. .. the gene occurred, and two types of hatching enzyme genes were established Consequently, all the euteleosts possess two hatching enzymes: HCE and LCE However, it remained to be clarified whether their molecular mechanism of egg envelope digestion is conserved among euteleosts In the present study, we compared the sites cleaved by hatching enzymes from lower euteleost (rainbow trout) to higher euteleosts... swollen envelope, and therefore this site comprises the ‘key site’ for egg envelope solubilization We have been studying the molecular evolution of teleostean hatching enzyme genes [22–24] The phylogenetic tree of hatching enzyme genes suggests that elopomorphs (basal teleosts) possess a single type of gene After elopomorphs branched off from the ancestor, duplication and diversification of the gene . of hatching enzyme on unfertilized egg envelope One of the goals of the present study was to deter- mine the cleavage sites of hatching enzyme on egg envelope. of the envelope, mainly between inner layer subunit proteins [10]. At the time of hatching of the embryo, the inner layer is digested by hatching enzyme