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Molecular cloning and characterization of hatching enzyme-like geneII (BmHELII) in the silkworm, Bombyx mori
Biochemical and Biophysical Research Communications 419 (2012) 194–199
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Molecular cloning and characterization of hatching enzyme-like geneII (BmHELII) in the silkworm, Bombyx mori Shunming Tang a,b,⇑, Jun Wu a, Xinhui Zhao a, Huanying Wang a, Zhiyong Qiu a,b, Xingjia Shen a,b, Xijie Guo a,b a
Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China The Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu 212018, China
b
a r t i c l e
i n f o
Article history: Received 19 January 2012 Available online 8 February 2012 Keywords: Bombyx mori Hatching enzyme-like gene II Bioinformatic analysis Expression pattern Spermatogenesis
a b s t r a c t Hatching enzyme (HE) is an enzyme that digests an egg envelop at the time of embryo hatching. Previously, we have reported a kind of Bombyx mori hatching enzyme-like gene (BmHEL). In this paper, the full length of another BmHEL cDNA sequence (BmHELII, GenBank ID: JN627443) was cloned from bluish-silkworm-eggs. The cDNA was 977 bp in length with an open reading frame of 885 bp which encodes a polypeptide of 294 amino acids including a putative signal peptide of 16 amino acid residues and a mature protein of 278 amino acids. The deduced BmHELII had a predicted molecular mass of 33.62 kDa, isoelectric point of 5.44 and two conserved signature sequences of astacin family. Bioinformatic analysis results showed that the deduced protease domain amino acid sequence of BmHELII had 29.5–87.0% identities to that of HE identified in the other species. The BmHELII gene structure was 6-exon–5-intron, and the promoter region harbored some basal promoter elements and some embryo development related transcription factor binding sites. Semi-quantitative RT-PCR analysis revealed that the relative level of BmHELII transcripts at different stages during egg incubation increased with the development of embryos and reached to a maximum just before hatching, hence declined gradually after hatching. The spatio-temporal expression pattern of BmHELII basically resembled that of hatching enzyme gene. Moreover, the BmHELII transcript was detected in testis of the silkworm, and semi-quantitative RT-PCR analysis showed that it kept at the high level in testis of silkworm from larvae to moth, which suggested that BmHELII might take part in the development of sperm. These results will be helpful to provide a molecular basis for understanding the mechanism underlying silkworm hatching as well as spermatogenesis. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Hatching enzyme (HE) is a general term for an enzyme (or enzymes) which participates in degradation of the egg envelope, releasing the embryo from its intracapsular life [1–4]. HE, secreted at the time of embryo hatching by embryonic cells, provides a good candidate for research on the cell differentiation, protein synthesis, and gene expression regulation during the special stage of early embryo at the molecular level. Therefore, it will be of great importance and interest to understand the hatching mechanism of animals intensively. Most of HEs are belonging to astacin family, a Zn-metalloproteinase well characterized by the consensus sequences HExxHxxGFxHExxRxDR (the zinc-binding motif) and SxMHY (the metionine turn) [5,6]. Some of the HEs have a modular structure ⇑ Corresponding author at: Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China. Fax: +86 511 85601051. E-mail address: [email protected] (S. Tang). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.144
with a N-terminal astacin-like protease module followed by one or more regulatory domains, such as CUB [complement subcomponent C1r/C1s/embryonic sea urchin protein Uegf/bone morphogenetic protein 1, (BMP-1)] [7], epidermal growth factor domains or membrane anchors. The biochemical properties, gene structure and evolutionary relationship of HE from various animal species, such as mammalian [8], avians [7], amphibian [9,10], teleostei [3,4,11,12], insect [13,14], echinoderm [15], human and mouse [16], had been investigated since 1980s. Among them, the hatching process of some animals, for instance the sea urchin (Paracentrotus lividus), is performed by a single enzyme [15]. Whereas medaka (Oryzias latipes) posses an enzyme system consisting of two similar but distinct proteases, HCE (high choriolytic enzyme) and LCE (low choriolytic enzyme) [3,4]. They cooperatively digest egg envelope, HCE partially digests the chorion by its limited proteolytic action, and LCE hardly digests the chorion directly but can digest the HCEswollen chorion efficiently. Furthermore, the exon–intron structure of HE genes is different from species to species, including
S. Tang et al. / Biochemical and Biophysical Research Communications 419 (2012) 194–199
9-exon–8-intron [17], 8-exon–7-intron [11], 5-exon–4-intron [6] and intron-less structure [18]. The HE gene of teleostei conserves the 9-exon–8-intron structure of an assumed ancestor [19], and only HCE has no introns, which is of 55% identity to LCE in amino acid sequence [11]. The introns loss events may result from adaptation to new environment during long evolutionary process, according to the molecular phylogenetic analysis [20]. Up to now, few HEs from insects have been reported. The cDNA of HE from the silkworm, Bombyx mori [14], Chinese Oak Silkworm, Antheraea pernyi [21], and Chinese wild silkworm, Bombyx mandarina [22] have been reported recently. In this paper, we reported the cloning and characterization of another hatching enzyme-like gene in the silkworm (BmHELII). More interesting, we found an phenomenon that the relative expression level of BmHELII always kept the high level from larvae to moth of the silkworm, suggested that BmHELII might be associated with spermatogenesis as a novel biological function. These results may provide some insights for further investigation about hatching mechanism in the silkworm and other insects. 2. Materials and methods 2.1. Materials and reagents Silkworm variety, p50, was preserved by the Sericultural Research Institute, Chinese Academy of Agricultural Sciences (SRICAAS). Escherichia coli (Top10) was maintained in our laboratory. DEPC was product of Invitrogen Company. RNAiso Plus, Primerscript Reverse Transcriptase, high fidelity Taq enzyme, restriction enzymes, pMD18-T vectors, T4 DNA ligase, 30 RACE Kit were purchased from Takara Company. All PCR primers and DNA sequencing were accomplished by Shanghai Sangong Biological Engineering Technology & Services Co., Ltd. And all other chemicals used were analytical grade reagents. 2.2. Total RNA isolation and reverse transcription According to the protocol of the manufacturer, total RNA was isolated from eggs or other examined tissues (about 100 lg) by using RNAiso Plus. The quality of the total RNA was determined with 260/280 absorbance ratio as well as electrophoresis method, and was stored at 80 °C until the subsequent experiments. Treated with DNase I, 1 lg of total RNA was used for synthesizing the first strand cDNA by Primerscript Reverse Transcriptase following the protocol of the manufacturer. 2.3. Cloning of BmHELII cDNA Using BmHEL cDNA sequence, we blasted against the 8.5-fold sequence coverage silkworm genome database released in 2008 [23]. The result showed that there existed another sequence (named BmHELII) with high homology over 90% in the silkworm genome. Based on the differences between the two sequences, specific primers TeHF1 (50 -CATCGAGGAGGCGATCGAAGAT-30 ) and TeHF2 (50 CCAGCATCGGATACTGGGAGC-30 ) were designed for BmHELII. To obtain the 30 -end sequence of BmHELII cDNA, 30 RACE PCR was performed from cDNAs synthesized from RNAs extracted from bluish-silkworm-eggs by using Takara 30 RACE Kit. Nested PCR was carried out, specific primers TeHF1 and 30 RACE outer primer, TeHF2 and 30 RACE inner primer were used as a set of primers in outer PCR and inner PCR, respectively. The PCR products was then inserted into pMD18-T vector and subjected to sequencing analysis. To obtain the 50 -end sequence of BmHELII cDNA, in silico cloning strategy was employed. According to the information from the silkworm genome database, we predicted the promoter region of
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BmHELII upstream, the sequence of the 50 UTR of BmHELII was identical with that of corresponding fragment on genome. So the 50 -end sequence of BmHELII was obtained. 2.4. Bioinformatic analysis The whole genomic sequence of BmHELII was obtained by blasting against the silkworm genome database (http://www.silkdb.org/ silkdb/). Promoter region was predicted by neutral network promoter program (NNPP) online (http://www.fruitfly.org/seq_tools/ promoter.html), and transcription factor binding sites in promoter region were predicted by TFSEARCH program (http:// www.cbrc.jp/research/db/TFSEARCH.html). The gene structure was analyzed by Spidey program online (http://www.ncbi.nlm.nih.gov/spidey/). Some properties of BmHELII were analyzed by DNAstar software, such as sequence splice, ORF search, nucleotide sequence translation as well as isoelectric point and molecular weight prediction. The prediction of signal peptide and protein domains was accomplished by SMART program online (http://smart.embl-heidelberg.de). All HE cDNA sequences of other species were obtained from Genbank, a multiple sequence alignment of amino acid sequences of mature protease domains was constructed by CLUSTAL W program [24]. Using astacin as an outgroup, the phylogenetic tree was established by MEGA4 program, and the reliability of the tree was evaluated by bootstrap values obtained with 1000 replicates according to the neighbor-joining method. 2.5. Semi-quantitative RT-PCR analysis of BmHELII transcript level at different development stages of silkworm To investigate the expression pattern of BmHELII, semi-quantitative RT-PCR was carried out. 1 lg of total RNA, separated from different embryo development stages and larvae, were converted into cDNAs according to the manufacturer’s instructions. Bmactin 3 gene (GenBank ID: NM_001126254), a house-keeping gene, was used as a reference to eliminate difference among samples. PCR reactions were carried out under the following conditions: 1 cycle of 94 °C for 3 min, 28 cycles of 94 °C for 30 s, 64 °C for 30 s and 72 °C for 1 min, with a final extension step of 10 min at 72 °C. The primers for BmHELII and Bmactin 3 were TeHF1 and TeHR (50 -TTAGTCCCASGCTCCTTTGCAGTTGTA-30 ), Bmactin3F (50 -GGATGTCCACGTCGC ACTT-30 ) and Bmactin3R (50 -GCGCGGCTACTCGTTCACT-30 ), respectively. About 5 ll of amplified DNA products were then separated on 1.0% agarose gels, and visualized with UV transilluminator. The band intensities of PCR products were analyzed by Labimage software. The ratios of the quantity of mRNA for BmHELII gene with Bmactin 3 were considered to reflect the relative transcript level of BmHELII at different development stages, before and after hatching. 2.6. Expression profile of BmHELII in different tissues during larval stage To investigate the expression profile of BmHELII in different tissues of the silkworm, RT-PCR was performed. Total RNA was from cuticle, malpighian tube, posterior silk gland, trachea, fatbody, midgut, testis and ovary, respectively. The PCR conditions were described above. 2.7. Semi-quantitative RT-PCR analysis of BmHELII transcript level in testis at different development stages of silkworm To analyze BmHELII mRNA content in testis, semi-quantitative RT-PCR was performed. The total RNA samples were from each
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day of three different development stages of silkworm, including 5th instar larvae, pupa and moth. The method was following the criteria mentioned as described previously.
Table 1 The size of exons and introns of the BmHELII gene, and neighbor-joining sequence of introns. Exon
3. Result 3.1. Full-length of BmHELII cDNA Using 30 RACE technique (Fig. 1A) combined with bioinformatic methods, the full-length of BmHELII cDNA sequence was successfully obtained. The cDNA was 977 bp in length, which harbored 50 UTR, 30 UTR and a complete ORF of 885 bp. The 30 UTR possessed the signal for polyadenylation, AATAAA. Furthermore, using FullTeHF (50 -GGATCCATGTTGCGTATTGCACTATTAGTTACTAT-30 ) (BamH I site underlined) and FullTeHR (50 -GTCGACTTAGTCCCAGCTCCTTT GCAGTTGTA-30 ) (Sal I site underlined) as specific primers, the ORF of BmHELII was achieved (Fig. 1B). The ORF encodes a polypeptide of 294 amino acids with its molecular weight 33.62 kDa and isoelectric point 5.44. The deduced BmHELII protein sequence has a signal peptide of 16 amino acids in N-terminal, which cut-site is located at Ala16/Thr17, and the mature protein is composed of 278 amino acids. The protease domain of BmHELII contains the basic features of the astacin family, such as the two signature sequences, the zinc-binding motif HEWMHILGFLHMHATYNR (in positions 187–204) and the metionine turn SCLHY (in positions 242–246), and four conserved cysteine residues that form disulfide bond, but there is no CUB domains in N-termination. 3.2. Genomic structure of BmHELII gene Blasting against the silkworm genome database with the fulllength of BmHELII cDNA sequence, we found that only one scaffold (nscaf2888) contained the intact cDNA with identity over 97%, which indicated that BmHELII gene was present in the whole silkworm genome as only a single copy. Analysis result with Spidey program showed that BmHELII gene contained 6 exons interrupted by 5 introns. Four introns splicing followed the classic GT-AG rules while the GT was replaced AG in the fourth intron (Table 1). A promoter region about 1.2 kb of BmHELII was predicted with NNPP program and TFSEARCH program. Six promoter regions with high scores over 0.85 were found, which harbored some basal promoter element, such as CAAT-box and TATA-box, and some embryo development related transcription factor binding sites, such as GATA-1 [25], HSF1 [26]. Furthermore, spermatogenesis related
Intron
Number
Size (bp)
Left-joining
Size (bp)
Right-joining
1 2 3 4 5 6
118 213 110 215 133 96
GTGAGATTTT GTAAGGCTGT GTGAGTATCA GTAAACAATA GTAAGAACTC
1093 7749 1560 787 342
GTTTCCGCAG TTTTTCAAAG ATGCGATCAG CTTCAGGTCT TCATTTTCAG
transcription factor binding sites were also appeared, such as Sox-5/SRY [27]. 3.3. Homologous alignment and phylogenetic analysis Fig. 2A showed that a alignment of amino acid sequences of mature enzymes in different species, including silkworm, wild silkworm, Chinese oak silkworm, fruit flies, mosquito, turtle, quail, fish, xenopus, and astacin. Homologous analysis results of the deduced protease domain showed that BmHELII has identity of 87.0%, 85.5%, 77.8%, 47.8%, 44.9%, 34.3%, 32.9%, 32.4%, 31.9%, 31.4%, 30.9%, 30.9%, and 29.5% to BmHEL, BmandHE, ApHEL, CuefHCE, DmHEHS, MLCE, MHCE23, ZHCE1, EHE12, XHE, TuHE, QHE and Astacin, respectively. Fig. 2B showed that the phylogenetic tree constructed using neighbor-joining method. According to the phylogenetic tree, HEs were clearly classified into two groups. The silkworm, wild silkworm, Chinese oak silkworm, fruit flies and mosquito were clustered together into a group, which were belong to the invertebrates. The vertebrates clustered in the other one. Furthermore, BmHELII branched off from BmHEL, the latter clustered with ApHEL together. The evolutionary relationship of BmHELII and BmHEL is similar to that of HCE and LCE in Teleostei. 3.4. Change of BmHELII transcript level in embryo and larvae at different development stages To investigate the relative of BmHELII transcript in embryo at different development stages, before and after hatching, semiquantitative PCR technique was employed. According to Fig. 3A, the transcript level of BmHELII was found to increase gradually until a maximum was reached just before hatching. During the following several days, a dramatical decline of BmHELII transcript was observed. The strict temporal pattern of BmHELII expression might be in accordance with a possible role of HE gene. 3.5. Expression profile of BmHELII in different tissues during larval stage Fig. 4 shows us the expression profile of BmHELII in different tissues of the silkworm at 4th day of 5th instar. 328 bp of expected DNA fragment was detected in testis and ovary, but was not amplified from other examined tissues, including cuticle, malpighian tube, posterior silk gland, trachea, fatbody and midgut. Moreover, the BmHELII expression level in testis was much higher than in ovary. The above results indicated that BmHELII might have other biological functions. 3.6. Change of BmHELII transcript level in testis at different development stages of silkworm (larvae, pupa and moth)
Fig. 1. The electrophoresis bands of PCR products. (A) 30 RACE product of about 530 bp; (B) the BmHELII ORF of 885 bp; (M) is DL2000 DNA Marker.
According to the result that BmHELII expressed abundantly in testis at 4th day of 5th instar, we further investigated the BmHELII
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Fig. 2. Comparison of BmHELII with other hatching enzymes. (A) A multiple alignment of amino acid sequences of mature protease. The zinc-binding motif and the metionine turn were underlined with solid line and dotted line, respectively, and four conservative cysteines were marked with circles. (B) A phylogenetic tree constructed from amino acid sequences of the mature enzyme by the neighbor-joining method. BmHEL Bombyx mori hatching enzyme-like (GenBank ID: FJ147197), BmandHE Bombyx mandarina hatching enzyme (JN620366), ApHEL Antheraea pernyi hatching enzyme-like (JN205047), CuefHCE house mosquito high choriolytic enzyme 1 (XP_001844559), DmHEHS Drosophila melanogaster hatching enzyme homology sequence (NM135911), MLCE madaka low choriolytic enzyme (NP_001098292), MHCE23 medaka high choriolytic enzyme (P31580), ZHCE1 zebrafish hatching enzyme (NP_998800), EHE12 eel hatching enzyme (BAB68517), XHE Xenopus hatching enzyme (BAA14003), TuHE turtle hatching enzyme (BAD95471), QHE quail hatching enzyme (BAD95472) and Astacin (CAB43519).
expression level at different development stages in testis. As Fig. 3B showed, BmHELII gene kept at the high level in testis of the whole of 5th instar larvae, pupa and moth, and it expressed higher in pupa and moth than that of 5th instar larvae. 4. Discussion Previously, we have reported a kind of B. mori hatching enzymelike gene [14], but the expressed BmHEL presented the weak degradation activity against the eggshells [22]. Whether there was other hatching enzyme in the silkworm, similar to that of ‘‘HCE– LCE’’ system in medaka? In medaka, HCE and LCE digested the envelope cooperatively [3,4]. In the present study, we focused on the identification of other kind of hatching enzyme gene in the silkworm. Using bioinformatic methods as well as RACE technique, we obtained the full length of BmHELII cDNA. Due to the ORF of BmHELII and BmHEL showed the identity as high as 93%, many primers could be shared by the two sequences. According to the information that the two sequences existed several discontinuous mutational sites in the 30 -terminal, specific primers TeHF1 and TeHR were designed for BmHELII. The amplified products were identical with the corresponding sequence of BmHELII, but not
BmHEL. Therefore, the specific primers, TeHF1 and TeHR, were reliable to detect the expression level of BmHELII gene. The expression level of BmHELII increased with the development of embryo before hatching, and reached to a maximum just prior to hatching, then declined sharply after hatching, and kept very low until 1st day of 2nd instar of larvae. The spatio-temporal expression pattern of BmHELII gene basically resembles that of hatching enzyme gene identified in other specie [28]. Bioinformatic analysis showed that BmHELII gene was a 6-exon–5-intron structure, and some embryo development related transcription factor binding sites were found in its promoter region, such as GATA-1 [25], HSF1 [26]. Moreover, the deduced BmHELII protein harbored the two signature sequences, HEWMHILGFLHMHATYNR and SCLHY, which were similar to that of conserved sequences in all astacin families [5,6]. The above researches indicate that BmHELII may play a role in hatching process of the silkworm. The gene structure and signature sequences of BmHELII were different from that of HEs identified in other species. The structure of HE gene differed from species to species, and it contained 9exon–8-intron [17], 8-exon–7-intron [11], 5-exon–4-intron [6], and no-intron structure [18]. The 9-exon–8-intron structure of Japanese eel was considered as an assumed ancestor [19]. But BmHELII
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Fig. 3. Change of BmHELII transcript level in embryo and testis at different development stages. (A) Change of BmHELII transcript level in embryo, before and after hatching; (1–9) the days before prior to hatching, at 9th day, hatching happened; (10) the new hatched larvae; (11 and 12) 1st day of 1st instar, 1st of 2nd instar larvae, respectively. (B) Change of BmHELII transcript level in testis at larva, pupa and moth stages of silkworm. (L17) Each day of 5th instar larvae stage; (P110) each day of pupa stage; (W1) the 1st day of moth. The eggs and testis were collected at 8 a.m. every day. Error bars represent standard deviation of three replicate samples.
Fig. 4. Expression profile of BmHELII in various tissues at 4th day of 5th instar of larval stage. 1% agarose gel analyses of BmHELII amplifications in comparison with those of controls (Bmactin 3). (1) cuticle; (2) malpighian tube; (3) posterior silk gland; (4) trachea; (5) fatbody; (6) midgut; (7) testis (8) ovary.
gene was composed of 6 exons and 5 introns. BmHELII might have diverged and lose some of its introns during the evolutionary process to the silkworm. Besides, there were some subtle differences of signature sequences between BmHELII and HEs in astacin family. In astacin family, they were HExxHxxGFxHExxRxDR and SxMHY [5,6]. But in the silkworm, the corresponding sequences were HEWMHILGFLHMHATYNR and SCLHY, respectively. Moreover, in insects, the ‘‘L’’ replaced the ‘‘M’’ in the ‘‘Met-turn’’ SxMHY. The differences maybe resulted from the gene mutation and diversification
events to adapt to the different environment during the long evolutionary process [19]. To date, the two hatching enzyme-like genes, BmHEL and BmHELII, have been identified in the silkworm, and they show an identify of 88.8% in amino acid sequence. They have the same exon–intron structure. In medaka, LCE has introns, while HCE has no introns [18]. These differences indicated that the digestion manners of BmHELs in the silkworm might differ from ‘‘HCE-LCE’’ system in medaka. In medaka, HCE digested the chorion partially at the first step, then LCE continued to digest the HCE-swollen chorion efficiently [3,4]. Recently, the expressed BmHEL presented very low degradation activity against the egg envelope. While the same amount of BmHELII and the mixture of the two proteases with ratio 1:1 showed that the activity is 3.67 times and 5.67 times as high as BmHEL, respectively (date was not showed). These results suggested that BmHEL and BmHELII play a role in the silkworm hatching process. More interesting, the research results suggested that BmHELII might be associated with a novel function as spermatogenesis of the silkworm. Our presumption could be supported by the following reasons. Firstly, the relative expression level of BmHELII in testis kept high through the whole different development stages of
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silkworm, and it maintained a little higher level at pupa and moth stages than that of 5th instar larvae. While at 1st day of 2nd instar of larvae, the BmHELII transcripts were almost not detected. The change of BmHELII transcript level in testis indicates that BmHELII might be associated with spermatogenesis [29]. Secondly, the BmHELII promoter region harbors high-frequently spermatogenesis related transcription factor binding sites, Sox-5/SRY [27]. Lastly, astacin family had various physiological functions, and one of them was morphogenesis [30]. Therefore, there was a possibility that BmHELII belonging to astacin family might have similar function as spermatogenesis during a special development of the silkworm. Besides, it was reported that fertilin a and fertilin b, which were belong to the metalloprotease–disintegrin protein family (ADAMs), played a critical role of proteolysis in sperm maturation and activation [31,32]. Similarity, as a Zn-metalloproteinase, BmHELII might exert the physiological function as spermatogenesis. To further elucidate this function of the BmHELII protein, RNAi or microRNAs strategy could be employed in the future. Acknowledgments This work was supported partly by the National Natural Science Foundation of China (Grant No. 31172266) & the National Basic Research Program of China (Grant No. 2012CB114605). References [1] E.H. Slifer, A cytological study of the pleuropodia of Melanoplus differentialis (Orthoptera, Acrididae) which furnishes new evidence that they produce the hatching enzyme, J. Morphol. 63 (1938) 181–205. [2] D. Barrett, B.F. Edwards, Hatching enzyme of the sea urchin Strongylocentrotus purpuratus, Methods Enzymol. 45 (1976) 354–373. [3] S. Yasumasu, I. Iuchi, K. Yamagami, Purification and partial characterization of high choriolytic enzyme (HCE), a component of the hatching enzyme of the teleost, Oryzias latipes, J. Biochem. 105 (1989) 204–211. [4] S. Yasumasu, I. Iuchi, K. Yamagami, Isolation and some properties of low choriolytic enzyme (LCE), a component of the hatching enzyme of the teleost, Oryzias latipes, J. Biochem. 105 (1989) 212–218. [5] J.S. Bond, R.J. Beynon, The astacin family of metalloendopeptidases, Protein Sci. 4 (1995) 1247–1261. [6] K. Inohaya, S. Yasumasu, K. Araki, K. Naruse, K. Yamazaki, I. Yasumasu, I. Iuchi, K. Yamagami, Species-dependent migration of fish hatching gland cells that express astacin-like proteases in common [corrected], Dev. Growth Differ. 39 (1997) 191–197. [7] S. Yasumasu, K.M. Mao, F. Sultana, H. Sakaguchi, N. Yoshizaki, Cloning of a quail homologue of hatching enzyme: its conserved function and additional function in egg envelope digestion, Dev. Genes Evol. 215 (2005) 489–498. [8] H. Sawada, K. Yamazaki, M. Hoshi, Trypsin-like hatching protease from mouse embryos: evidence for the presence in culture medium and its enzymatic properties, J. Exp. Zool. 254 (1990) 83–87. [9] Y. Kitamura, C. Katagiri, Characterization of the hatching enzyme from embryos of an anuran amphibian, Rana pirica, Biochim. Biophys. Acta 1387 (1998) 153–164. [10] T.J. Fan, C. Katagiri, Properties of the hatching enzyme from Xenopus laevis, Eur. J. Biochem. 268 (2001) 4892–4898. [11] S. Yasumasu, I. Luchi, K. Yamagami, cDNAs and the genes of HCE and LCE, two constituents of the medaka hatching enzyme, Dev. Growth Differ. 36 (1994) 241–252. [12] M. Kawaguchi, S. Yasumasu, J. Hiroi, K. Naruse, M. Inoue, I. Iuchi, Evolution of teleostean hatching enzyme genes and their paralogous genes, Dev. Genes Evol. 216 (2006) 769–784.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Molecular cloning and characterization of hatching enzyme-like geneII (BmHELII) in the silkworm, Bombyx mori Shunming Tang a,b,⇑, Jun Wu a, Xinhui Zhao a, Huanying Wang a, Zhiyong Qiu a,b, Xingjia Shen a,b, Xijie Guo a,b a
Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China The Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu 212018, China
b
a r t i c l e
i n f o
Article history: Received 19 January 2012 Available online 8 February 2012 Keywords: Bombyx mori Hatching enzyme-like gene II Bioinformatic analysis Expression pattern Spermatogenesis
a b s t r a c t Hatching enzyme (HE) is an enzyme that digests an egg envelop at the time of embryo hatching. Previously, we have reported a kind of Bombyx mori hatching enzyme-like gene (BmHEL). In this paper, the full length of another BmHEL cDNA sequence (BmHELII, GenBank ID: JN627443) was cloned from bluish-silkworm-eggs. The cDNA was 977 bp in length with an open reading frame of 885 bp which encodes a polypeptide of 294 amino acids including a putative signal peptide of 16 amino acid residues and a mature protein of 278 amino acids. The deduced BmHELII had a predicted molecular mass of 33.62 kDa, isoelectric point of 5.44 and two conserved signature sequences of astacin family. Bioinformatic analysis results showed that the deduced protease domain amino acid sequence of BmHELII had 29.5–87.0% identities to that of HE identified in the other species. The BmHELII gene structure was 6-exon–5-intron, and the promoter region harbored some basal promoter elements and some embryo development related transcription factor binding sites. Semi-quantitative RT-PCR analysis revealed that the relative level of BmHELII transcripts at different stages during egg incubation increased with the development of embryos and reached to a maximum just before hatching, hence declined gradually after hatching. The spatio-temporal expression pattern of BmHELII basically resembled that of hatching enzyme gene. Moreover, the BmHELII transcript was detected in testis of the silkworm, and semi-quantitative RT-PCR analysis showed that it kept at the high level in testis of silkworm from larvae to moth, which suggested that BmHELII might take part in the development of sperm. These results will be helpful to provide a molecular basis for understanding the mechanism underlying silkworm hatching as well as spermatogenesis. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Hatching enzyme (HE) is a general term for an enzyme (or enzymes) which participates in degradation of the egg envelope, releasing the embryo from its intracapsular life [1–4]. HE, secreted at the time of embryo hatching by embryonic cells, provides a good candidate for research on the cell differentiation, protein synthesis, and gene expression regulation during the special stage of early embryo at the molecular level. Therefore, it will be of great importance and interest to understand the hatching mechanism of animals intensively. Most of HEs are belonging to astacin family, a Zn-metalloproteinase well characterized by the consensus sequences HExxHxxGFxHExxRxDR (the zinc-binding motif) and SxMHY (the metionine turn) [5,6]. Some of the HEs have a modular structure ⇑ Corresponding author at: Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China. Fax: +86 511 85601051. E-mail address: [email protected] (S. Tang). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.144
with a N-terminal astacin-like protease module followed by one or more regulatory domains, such as CUB [complement subcomponent C1r/C1s/embryonic sea urchin protein Uegf/bone morphogenetic protein 1, (BMP-1)] [7], epidermal growth factor domains or membrane anchors. The biochemical properties, gene structure and evolutionary relationship of HE from various animal species, such as mammalian [8], avians [7], amphibian [9,10], teleostei [3,4,11,12], insect [13,14], echinoderm [15], human and mouse [16], had been investigated since 1980s. Among them, the hatching process of some animals, for instance the sea urchin (Paracentrotus lividus), is performed by a single enzyme [15]. Whereas medaka (Oryzias latipes) posses an enzyme system consisting of two similar but distinct proteases, HCE (high choriolytic enzyme) and LCE (low choriolytic enzyme) [3,4]. They cooperatively digest egg envelope, HCE partially digests the chorion by its limited proteolytic action, and LCE hardly digests the chorion directly but can digest the HCEswollen chorion efficiently. Furthermore, the exon–intron structure of HE genes is different from species to species, including
S. Tang et al. / Biochemical and Biophysical Research Communications 419 (2012) 194–199
9-exon–8-intron [17], 8-exon–7-intron [11], 5-exon–4-intron [6] and intron-less structure [18]. The HE gene of teleostei conserves the 9-exon–8-intron structure of an assumed ancestor [19], and only HCE has no introns, which is of 55% identity to LCE in amino acid sequence [11]. The introns loss events may result from adaptation to new environment during long evolutionary process, according to the molecular phylogenetic analysis [20]. Up to now, few HEs from insects have been reported. The cDNA of HE from the silkworm, Bombyx mori [14], Chinese Oak Silkworm, Antheraea pernyi [21], and Chinese wild silkworm, Bombyx mandarina [22] have been reported recently. In this paper, we reported the cloning and characterization of another hatching enzyme-like gene in the silkworm (BmHELII). More interesting, we found an phenomenon that the relative expression level of BmHELII always kept the high level from larvae to moth of the silkworm, suggested that BmHELII might be associated with spermatogenesis as a novel biological function. These results may provide some insights for further investigation about hatching mechanism in the silkworm and other insects. 2. Materials and methods 2.1. Materials and reagents Silkworm variety, p50, was preserved by the Sericultural Research Institute, Chinese Academy of Agricultural Sciences (SRICAAS). Escherichia coli (Top10) was maintained in our laboratory. DEPC was product of Invitrogen Company. RNAiso Plus, Primerscript Reverse Transcriptase, high fidelity Taq enzyme, restriction enzymes, pMD18-T vectors, T4 DNA ligase, 30 RACE Kit were purchased from Takara Company. All PCR primers and DNA sequencing were accomplished by Shanghai Sangong Biological Engineering Technology & Services Co., Ltd. And all other chemicals used were analytical grade reagents. 2.2. Total RNA isolation and reverse transcription According to the protocol of the manufacturer, total RNA was isolated from eggs or other examined tissues (about 100 lg) by using RNAiso Plus. The quality of the total RNA was determined with 260/280 absorbance ratio as well as electrophoresis method, and was stored at 80 °C until the subsequent experiments. Treated with DNase I, 1 lg of total RNA was used for synthesizing the first strand cDNA by Primerscript Reverse Transcriptase following the protocol of the manufacturer. 2.3. Cloning of BmHELII cDNA Using BmHEL cDNA sequence, we blasted against the 8.5-fold sequence coverage silkworm genome database released in 2008 [23]. The result showed that there existed another sequence (named BmHELII) with high homology over 90% in the silkworm genome. Based on the differences between the two sequences, specific primers TeHF1 (50 -CATCGAGGAGGCGATCGAAGAT-30 ) and TeHF2 (50 CCAGCATCGGATACTGGGAGC-30 ) were designed for BmHELII. To obtain the 30 -end sequence of BmHELII cDNA, 30 RACE PCR was performed from cDNAs synthesized from RNAs extracted from bluish-silkworm-eggs by using Takara 30 RACE Kit. Nested PCR was carried out, specific primers TeHF1 and 30 RACE outer primer, TeHF2 and 30 RACE inner primer were used as a set of primers in outer PCR and inner PCR, respectively. The PCR products was then inserted into pMD18-T vector and subjected to sequencing analysis. To obtain the 50 -end sequence of BmHELII cDNA, in silico cloning strategy was employed. According to the information from the silkworm genome database, we predicted the promoter region of
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BmHELII upstream, the sequence of the 50 UTR of BmHELII was identical with that of corresponding fragment on genome. So the 50 -end sequence of BmHELII was obtained. 2.4. Bioinformatic analysis The whole genomic sequence of BmHELII was obtained by blasting against the silkworm genome database (http://www.silkdb.org/ silkdb/). Promoter region was predicted by neutral network promoter program (NNPP) online (http://www.fruitfly.org/seq_tools/ promoter.html), and transcription factor binding sites in promoter region were predicted by TFSEARCH program (http:// www.cbrc.jp/research/db/TFSEARCH.html). The gene structure was analyzed by Spidey program online (http://www.ncbi.nlm.nih.gov/spidey/). Some properties of BmHELII were analyzed by DNAstar software, such as sequence splice, ORF search, nucleotide sequence translation as well as isoelectric point and molecular weight prediction. The prediction of signal peptide and protein domains was accomplished by SMART program online (http://smart.embl-heidelberg.de). All HE cDNA sequences of other species were obtained from Genbank, a multiple sequence alignment of amino acid sequences of mature protease domains was constructed by CLUSTAL W program [24]. Using astacin as an outgroup, the phylogenetic tree was established by MEGA4 program, and the reliability of the tree was evaluated by bootstrap values obtained with 1000 replicates according to the neighbor-joining method. 2.5. Semi-quantitative RT-PCR analysis of BmHELII transcript level at different development stages of silkworm To investigate the expression pattern of BmHELII, semi-quantitative RT-PCR was carried out. 1 lg of total RNA, separated from different embryo development stages and larvae, were converted into cDNAs according to the manufacturer’s instructions. Bmactin 3 gene (GenBank ID: NM_001126254), a house-keeping gene, was used as a reference to eliminate difference among samples. PCR reactions were carried out under the following conditions: 1 cycle of 94 °C for 3 min, 28 cycles of 94 °C for 30 s, 64 °C for 30 s and 72 °C for 1 min, with a final extension step of 10 min at 72 °C. The primers for BmHELII and Bmactin 3 were TeHF1 and TeHR (50 -TTAGTCCCASGCTCCTTTGCAGTTGTA-30 ), Bmactin3F (50 -GGATGTCCACGTCGC ACTT-30 ) and Bmactin3R (50 -GCGCGGCTACTCGTTCACT-30 ), respectively. About 5 ll of amplified DNA products were then separated on 1.0% agarose gels, and visualized with UV transilluminator. The band intensities of PCR products were analyzed by Labimage software. The ratios of the quantity of mRNA for BmHELII gene with Bmactin 3 were considered to reflect the relative transcript level of BmHELII at different development stages, before and after hatching. 2.6. Expression profile of BmHELII in different tissues during larval stage To investigate the expression profile of BmHELII in different tissues of the silkworm, RT-PCR was performed. Total RNA was from cuticle, malpighian tube, posterior silk gland, trachea, fatbody, midgut, testis and ovary, respectively. The PCR conditions were described above. 2.7. Semi-quantitative RT-PCR analysis of BmHELII transcript level in testis at different development stages of silkworm To analyze BmHELII mRNA content in testis, semi-quantitative RT-PCR was performed. The total RNA samples were from each
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day of three different development stages of silkworm, including 5th instar larvae, pupa and moth. The method was following the criteria mentioned as described previously.
Table 1 The size of exons and introns of the BmHELII gene, and neighbor-joining sequence of introns. Exon
3. Result 3.1. Full-length of BmHELII cDNA Using 30 RACE technique (Fig. 1A) combined with bioinformatic methods, the full-length of BmHELII cDNA sequence was successfully obtained. The cDNA was 977 bp in length, which harbored 50 UTR, 30 UTR and a complete ORF of 885 bp. The 30 UTR possessed the signal for polyadenylation, AATAAA. Furthermore, using FullTeHF (50 -GGATCCATGTTGCGTATTGCACTATTAGTTACTAT-30 ) (BamH I site underlined) and FullTeHR (50 -GTCGACTTAGTCCCAGCTCCTTT GCAGTTGTA-30 ) (Sal I site underlined) as specific primers, the ORF of BmHELII was achieved (Fig. 1B). The ORF encodes a polypeptide of 294 amino acids with its molecular weight 33.62 kDa and isoelectric point 5.44. The deduced BmHELII protein sequence has a signal peptide of 16 amino acids in N-terminal, which cut-site is located at Ala16/Thr17, and the mature protein is composed of 278 amino acids. The protease domain of BmHELII contains the basic features of the astacin family, such as the two signature sequences, the zinc-binding motif HEWMHILGFLHMHATYNR (in positions 187–204) and the metionine turn SCLHY (in positions 242–246), and four conserved cysteine residues that form disulfide bond, but there is no CUB domains in N-termination. 3.2. Genomic structure of BmHELII gene Blasting against the silkworm genome database with the fulllength of BmHELII cDNA sequence, we found that only one scaffold (nscaf2888) contained the intact cDNA with identity over 97%, which indicated that BmHELII gene was present in the whole silkworm genome as only a single copy. Analysis result with Spidey program showed that BmHELII gene contained 6 exons interrupted by 5 introns. Four introns splicing followed the classic GT-AG rules while the GT was replaced AG in the fourth intron (Table 1). A promoter region about 1.2 kb of BmHELII was predicted with NNPP program and TFSEARCH program. Six promoter regions with high scores over 0.85 were found, which harbored some basal promoter element, such as CAAT-box and TATA-box, and some embryo development related transcription factor binding sites, such as GATA-1 [25], HSF1 [26]. Furthermore, spermatogenesis related
Intron
Number
Size (bp)
Left-joining
Size (bp)
Right-joining
1 2 3 4 5 6
118 213 110 215 133 96
GTGAGATTTT GTAAGGCTGT GTGAGTATCA GTAAACAATA GTAAGAACTC
1093 7749 1560 787 342
GTTTCCGCAG TTTTTCAAAG ATGCGATCAG CTTCAGGTCT TCATTTTCAG
transcription factor binding sites were also appeared, such as Sox-5/SRY [27]. 3.3. Homologous alignment and phylogenetic analysis Fig. 2A showed that a alignment of amino acid sequences of mature enzymes in different species, including silkworm, wild silkworm, Chinese oak silkworm, fruit flies, mosquito, turtle, quail, fish, xenopus, and astacin. Homologous analysis results of the deduced protease domain showed that BmHELII has identity of 87.0%, 85.5%, 77.8%, 47.8%, 44.9%, 34.3%, 32.9%, 32.4%, 31.9%, 31.4%, 30.9%, 30.9%, and 29.5% to BmHEL, BmandHE, ApHEL, CuefHCE, DmHEHS, MLCE, MHCE23, ZHCE1, EHE12, XHE, TuHE, QHE and Astacin, respectively. Fig. 2B showed that the phylogenetic tree constructed using neighbor-joining method. According to the phylogenetic tree, HEs were clearly classified into two groups. The silkworm, wild silkworm, Chinese oak silkworm, fruit flies and mosquito were clustered together into a group, which were belong to the invertebrates. The vertebrates clustered in the other one. Furthermore, BmHELII branched off from BmHEL, the latter clustered with ApHEL together. The evolutionary relationship of BmHELII and BmHEL is similar to that of HCE and LCE in Teleostei. 3.4. Change of BmHELII transcript level in embryo and larvae at different development stages To investigate the relative of BmHELII transcript in embryo at different development stages, before and after hatching, semiquantitative PCR technique was employed. According to Fig. 3A, the transcript level of BmHELII was found to increase gradually until a maximum was reached just before hatching. During the following several days, a dramatical decline of BmHELII transcript was observed. The strict temporal pattern of BmHELII expression might be in accordance with a possible role of HE gene. 3.5. Expression profile of BmHELII in different tissues during larval stage Fig. 4 shows us the expression profile of BmHELII in different tissues of the silkworm at 4th day of 5th instar. 328 bp of expected DNA fragment was detected in testis and ovary, but was not amplified from other examined tissues, including cuticle, malpighian tube, posterior silk gland, trachea, fatbody and midgut. Moreover, the BmHELII expression level in testis was much higher than in ovary. The above results indicated that BmHELII might have other biological functions. 3.6. Change of BmHELII transcript level in testis at different development stages of silkworm (larvae, pupa and moth)
Fig. 1. The electrophoresis bands of PCR products. (A) 30 RACE product of about 530 bp; (B) the BmHELII ORF of 885 bp; (M) is DL2000 DNA Marker.
According to the result that BmHELII expressed abundantly in testis at 4th day of 5th instar, we further investigated the BmHELII
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Fig. 2. Comparison of BmHELII with other hatching enzymes. (A) A multiple alignment of amino acid sequences of mature protease. The zinc-binding motif and the metionine turn were underlined with solid line and dotted line, respectively, and four conservative cysteines were marked with circles. (B) A phylogenetic tree constructed from amino acid sequences of the mature enzyme by the neighbor-joining method. BmHEL Bombyx mori hatching enzyme-like (GenBank ID: FJ147197), BmandHE Bombyx mandarina hatching enzyme (JN620366), ApHEL Antheraea pernyi hatching enzyme-like (JN205047), CuefHCE house mosquito high choriolytic enzyme 1 (XP_001844559), DmHEHS Drosophila melanogaster hatching enzyme homology sequence (NM135911), MLCE madaka low choriolytic enzyme (NP_001098292), MHCE23 medaka high choriolytic enzyme (P31580), ZHCE1 zebrafish hatching enzyme (NP_998800), EHE12 eel hatching enzyme (BAB68517), XHE Xenopus hatching enzyme (BAA14003), TuHE turtle hatching enzyme (BAD95471), QHE quail hatching enzyme (BAD95472) and Astacin (CAB43519).
expression level at different development stages in testis. As Fig. 3B showed, BmHELII gene kept at the high level in testis of the whole of 5th instar larvae, pupa and moth, and it expressed higher in pupa and moth than that of 5th instar larvae. 4. Discussion Previously, we have reported a kind of B. mori hatching enzymelike gene [14], but the expressed BmHEL presented the weak degradation activity against the eggshells [22]. Whether there was other hatching enzyme in the silkworm, similar to that of ‘‘HCE– LCE’’ system in medaka? In medaka, HCE and LCE digested the envelope cooperatively [3,4]. In the present study, we focused on the identification of other kind of hatching enzyme gene in the silkworm. Using bioinformatic methods as well as RACE technique, we obtained the full length of BmHELII cDNA. Due to the ORF of BmHELII and BmHEL showed the identity as high as 93%, many primers could be shared by the two sequences. According to the information that the two sequences existed several discontinuous mutational sites in the 30 -terminal, specific primers TeHF1 and TeHR were designed for BmHELII. The amplified products were identical with the corresponding sequence of BmHELII, but not
BmHEL. Therefore, the specific primers, TeHF1 and TeHR, were reliable to detect the expression level of BmHELII gene. The expression level of BmHELII increased with the development of embryo before hatching, and reached to a maximum just prior to hatching, then declined sharply after hatching, and kept very low until 1st day of 2nd instar of larvae. The spatio-temporal expression pattern of BmHELII gene basically resembles that of hatching enzyme gene identified in other specie [28]. Bioinformatic analysis showed that BmHELII gene was a 6-exon–5-intron structure, and some embryo development related transcription factor binding sites were found in its promoter region, such as GATA-1 [25], HSF1 [26]. Moreover, the deduced BmHELII protein harbored the two signature sequences, HEWMHILGFLHMHATYNR and SCLHY, which were similar to that of conserved sequences in all astacin families [5,6]. The above researches indicate that BmHELII may play a role in hatching process of the silkworm. The gene structure and signature sequences of BmHELII were different from that of HEs identified in other species. The structure of HE gene differed from species to species, and it contained 9exon–8-intron [17], 8-exon–7-intron [11], 5-exon–4-intron [6], and no-intron structure [18]. The 9-exon–8-intron structure of Japanese eel was considered as an assumed ancestor [19]. But BmHELII
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Fig. 3. Change of BmHELII transcript level in embryo and testis at different development stages. (A) Change of BmHELII transcript level in embryo, before and after hatching; (1–9) the days before prior to hatching, at 9th day, hatching happened; (10) the new hatched larvae; (11 and 12) 1st day of 1st instar, 1st of 2nd instar larvae, respectively. (B) Change of BmHELII transcript level in testis at larva, pupa and moth stages of silkworm. (L17) Each day of 5th instar larvae stage; (P110) each day of pupa stage; (W1) the 1st day of moth. The eggs and testis were collected at 8 a.m. every day. Error bars represent standard deviation of three replicate samples.
Fig. 4. Expression profile of BmHELII in various tissues at 4th day of 5th instar of larval stage. 1% agarose gel analyses of BmHELII amplifications in comparison with those of controls (Bmactin 3). (1) cuticle; (2) malpighian tube; (3) posterior silk gland; (4) trachea; (5) fatbody; (6) midgut; (7) testis (8) ovary.
gene was composed of 6 exons and 5 introns. BmHELII might have diverged and lose some of its introns during the evolutionary process to the silkworm. Besides, there were some subtle differences of signature sequences between BmHELII and HEs in astacin family. In astacin family, they were HExxHxxGFxHExxRxDR and SxMHY [5,6]. But in the silkworm, the corresponding sequences were HEWMHILGFLHMHATYNR and SCLHY, respectively. Moreover, in insects, the ‘‘L’’ replaced the ‘‘M’’ in the ‘‘Met-turn’’ SxMHY. The differences maybe resulted from the gene mutation and diversification
events to adapt to the different environment during the long evolutionary process [19]. To date, the two hatching enzyme-like genes, BmHEL and BmHELII, have been identified in the silkworm, and they show an identify of 88.8% in amino acid sequence. They have the same exon–intron structure. In medaka, LCE has introns, while HCE has no introns [18]. These differences indicated that the digestion manners of BmHELs in the silkworm might differ from ‘‘HCE-LCE’’ system in medaka. In medaka, HCE digested the chorion partially at the first step, then LCE continued to digest the HCE-swollen chorion efficiently [3,4]. Recently, the expressed BmHEL presented very low degradation activity against the egg envelope. While the same amount of BmHELII and the mixture of the two proteases with ratio 1:1 showed that the activity is 3.67 times and 5.67 times as high as BmHEL, respectively (date was not showed). These results suggested that BmHEL and BmHELII play a role in the silkworm hatching process. More interesting, the research results suggested that BmHELII might be associated with a novel function as spermatogenesis of the silkworm. Our presumption could be supported by the following reasons. Firstly, the relative expression level of BmHELII in testis kept high through the whole different development stages of
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silkworm, and it maintained a little higher level at pupa and moth stages than that of 5th instar larvae. While at 1st day of 2nd instar of larvae, the BmHELII transcripts were almost not detected. The change of BmHELII transcript level in testis indicates that BmHELII might be associated with spermatogenesis [29]. Secondly, the BmHELII promoter region harbors high-frequently spermatogenesis related transcription factor binding sites, Sox-5/SRY [27]. Lastly, astacin family had various physiological functions, and one of them was morphogenesis [30]. Therefore, there was a possibility that BmHELII belonging to astacin family might have similar function as spermatogenesis during a special development of the silkworm. Besides, it was reported that fertilin a and fertilin b, which were belong to the metalloprotease–disintegrin protein family (ADAMs), played a critical role of proteolysis in sperm maturation and activation [31,32]. Similarity, as a Zn-metalloproteinase, BmHELII might exert the physiological function as spermatogenesis. To further elucidate this function of the BmHELII protein, RNAi or microRNAs strategy could be employed in the future. Acknowledgments This work was supported partly by the National Natural Science Foundation of China (Grant No. 31172266) & the National Basic Research Program of China (Grant No. 2012CB114605). References [1] E.H. Slifer, A cytological study of the pleuropodia of Melanoplus differentialis (Orthoptera, Acrididae) which furnishes new evidence that they produce the hatching enzyme, J. Morphol. 63 (1938) 181–205. [2] D. Barrett, B.F. Edwards, Hatching enzyme of the sea urchin Strongylocentrotus purpuratus, Methods Enzymol. 45 (1976) 354–373. [3] S. Yasumasu, I. Iuchi, K. Yamagami, Purification and partial characterization of high choriolytic enzyme (HCE), a component of the hatching enzyme of the teleost, Oryzias latipes, J. Biochem. 105 (1989) 204–211. [4] S. Yasumasu, I. Iuchi, K. Yamagami, Isolation and some properties of low choriolytic enzyme (LCE), a component of the hatching enzyme of the teleost, Oryzias latipes, J. Biochem. 105 (1989) 212–218. [5] J.S. Bond, R.J. Beynon, The astacin family of metalloendopeptidases, Protein Sci. 4 (1995) 1247–1261. [6] K. Inohaya, S. Yasumasu, K. Araki, K. Naruse, K. Yamazaki, I. Yasumasu, I. Iuchi, K. Yamagami, Species-dependent migration of fish hatching gland cells that express astacin-like proteases in common [corrected], Dev. Growth Differ. 39 (1997) 191–197. [7] S. Yasumasu, K.M. Mao, F. Sultana, H. Sakaguchi, N. Yoshizaki, Cloning of a quail homologue of hatching enzyme: its conserved function and additional function in egg envelope digestion, Dev. Genes Evol. 215 (2005) 489–498. [8] H. Sawada, K. Yamazaki, M. Hoshi, Trypsin-like hatching protease from mouse embryos: evidence for the presence in culture medium and its enzymatic properties, J. Exp. Zool. 254 (1990) 83–87. [9] Y. Kitamura, C. Katagiri, Characterization of the hatching enzyme from embryos of an anuran amphibian, Rana pirica, Biochim. Biophys. Acta 1387 (1998) 153–164. [10] T.J. Fan, C. Katagiri, Properties of the hatching enzyme from Xenopus laevis, Eur. J. Biochem. 268 (2001) 4892–4898. [11] S. Yasumasu, I. Luchi, K. Yamagami, cDNAs and the genes of HCE and LCE, two constituents of the medaka hatching enzyme, Dev. Growth Differ. 36 (1994) 241–252. [12] M. Kawaguchi, S. Yasumasu, J. Hiroi, K. Naruse, M. Inoue, I. Iuchi, Evolution of teleostean hatching enzyme genes and their paralogous genes, Dev. Genes Evol. 216 (2006) 769–784.
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