Identification and molecular characterization of <i>Cathepsin L</i> gene and its expression analysis during early ontogenetic development of kuruma shrimp <i>Marsupenaeus japonicus</i>

Identification and molecular characterization of Cathepsin L gene and its expression analysis during early ontogenetic development of kuruma shrimp Marsupenaeus japonicus

PDF

Ying QIAO¹, Jun WANG¹, Yong MAO¹, Min LIU¹, Xiaohong SONG¹, Yongquan SU¹^,^*, Chunzhong WANG², Zhipeng ZHENG²

Acta Oceanologica Sinica | 2017, 36(6) : 52 - 60

Less

Acta Oceanologica Sinica | 2017, 36(6): 52-60

• •

Identification and molecular characterization of Cathepsin L gene and its expression analysis during early ontogenetic development of kuruma shrimp Marsupenaeus japonicus

Full

Ying QIAO¹, Jun WANG¹, Yong MAO¹, Min LIU¹, Xiaohong SONG¹, Yongquan SU¹^,^*, Chunzhong WANG², Zhipeng ZHENG²

Affiliations

¹ State Key Laboratory of Marine Environmental Sciences, Xiamen University, Xiamen 361102, China

² Putian Tian-ran-xing Agricultural Development Co. Ltd., Fujian 351100, China

Published: 2017-02-01 doi: 10.1007/s13131-017-0983-5

Outline

Abstract

Less

Cathepsin L gene is a member of the cysteine proteinase gene group. In this study Cathepsin L gene was isolated from Kuruma shrimp Marsupenaeus japonicus (Mj-Cathepsin L) and the full-length DNA sequence was 1 963 bp. Mj-Cathepsin L protein showed high homologies with other Cathepsin L proteins documented in vertebrates, mollusks and other crustaceans. Expression analysis of Mj-Cathepsin L gene in different tissues revealed that it was predominant in hepatopancreas. During early ontogenetic development stages Mj-Cathepsin L showed a development-regulated expression, and the Mj-Cathepsin L showed a molting stage-regulated expression during the five molting stages, inferring its role in the ontogenic development of M. japonicus. Two kinds of forms of Mj-Cathepsin L protein: pro-Cathepsin L and Cathepsin L were measured in hepatopancreas, stomach and intestine by Western Blotting.

Key words

Cathepsin L gene / larval development / molting cycle / tissue distribution / Marsupenaeus japonicus

Cite this Article

Ying QIAO, Jun WANG, Yong MAO, Min LIU, Xiaohong SONG, Yongquan SU, Chunzhong WANG, Zhipeng ZHENG. Identification and molecular characterization of Cathepsin L gene and its expression analysis during early ontogenetic development of kuruma shrimp Marsupenaeus japonicus[J]. Acta Oceanologica Sinica, 2017 , 36 (6) : 52 -60 . DOI: 10.1007/s13131-017-0983-5

Full Text

Less

1 Introduction

Less

Cathepsin L, a lysosomal cysteine proteinase belongs to the papain superfamily, is an abundant endopeptidase in eukaryotic organisms (Roth et al., 2000). Studies revealed that Cathepsin L plays important roles in crucial biological processes such as carcinogenesis process, antigen presentation, exopeptidase maturation and embryogenesis (Nakagawa et al., 1998; Dahl et al., 2001; Miyamoto et al., 2011; Zhang et al., 2014). In crustaceans, Cathepsin L was first purified from gastrointestinal juice of the American lobster Homarus americanus (Laycock, 1989). To date, Cathepsin L has been reported in shrimps, such as Litopenaeus (Penaeus) vannamei, Macrobrachium rosenbergii, Metapenaeus ensis, Nephrops norvegicus, Pandalus borealis and Penaeus monodon (Le Boulay et al., 1995, 1996, 1998; Aoki et al., 2004; Hu and Leung, 2004; Glenn et al., 2005; Arockiaraj et al., 2013).

Studies on Cathepsin L in shrimps focused on its functions during the food digestion and the molting process. In M. ensis, Cathepsin L was found in the B cells of the hepatopancreas, the most important organ responsible for the immune, enzyme secretion and digestion (Hu and Leung, 2004). During the digestion-related cell differentiation in M. ensis, however, Cathepsin L mRNA was found in the F-cell rather than in the mature B-cell, indicated that the Cathepsin L digestive model during the rapid digestion-related cell differentiation in hepatopancreas (Hu and Leung, 2007). In L. vannamei, Cathepsin L transcript levels were correlated with the molting stages (Le Boulay et al., 1996). Though the Cathepsin L was found nothing significantly associated with body weight in L. vannamei populations (Glenn et al., 2005), but Qian et al. (2013) suggest Cathepsin L may be one of candidates which significantly affect the growth of L. vannamei and the distribution of Cathepsin L mRNA may be related to its specific functions in different tissues and its positive roles in regulation of shrimp muscle growth.

Kuruma shrimp Marsupenaeus (Penaeus) japonicus is a commercial important fishery and mariculture species in China. The objectives of this study were to analyze the molecular characters of Cathepsin L and to evaluate the role of Cathepsin L throughout the early ontogenetic developmental stages of M. japonicus for the first time, giving its significant changes on morphology, physiology and behavior.

2 Materials and methods

Less

2.1 Sample collection of Marsupenaeus japonicus

To clone and analyze the tissue distribution of Mj-Cathepsin L gene, samples of M. japonicus (n=30, BW±SD, (21.0±2.54) g) were obtained from Dongshan (Fujian Province, China), and kept a concrete water tank with aeration for at least 7 d. Seven tissues (muscle, gill, heart, intestine, stomach, hepatopancreas and eye stalk) were collected, preserved immediately in Sample Protector for RNA (TaKaRa, Japan) and stored at –20°C for further RNA extraction.

Fertilized eggs of M. japonicus were collected from one broodstock and subsequently reared in a 5×5 m concrete tank following larval production procedures. The larvae were fed every four hours with prawn slices before post-larva 10 (P10) then switched to pellet feed. 0.2 g of fertilized egg, nauplius larva, zoeal larva (Z₁ to Z₃), mysis larva (M₁ to M₃) and post-larva (P₁–P₅, P₈, P₁₁, P₁₄, P₁₇, P₂₁, P₂₇) were collected and stored (as described above) for further analyses.

Determination of molt stage was based on the degree of setae development according to the method described by Oliveira Cesar (de Oliveira Cesar et al., 2006). Hepatopancreas were collected and preserved as mentioned above.

2.2 RNA isolation and cDNA synthesis

Total RNA was extracted from each 100 mg of hepatopancreas, intestine, muscle, heart, eye stalk, stomach and gill using the RNAiso Plus Reagent (TaKaRa, Code No. 9108, Japan) according to the manufacturer’s protocol. The concentration and quality of the total RNA were measured by NanoDrop 1000 Spectrophotometer V3.7 (Thermo Scientific) and 1% agarose-gel electrophoresis, respectively. Only RNAs with absorbance ratios (A260:A280) greater than 1.8 were used for further analyses.

Total RNA (1 μg) was reverse-transcribed in a 25 μL reverse transcription reaction using the TaKaRa RNA PCR Kit (AMV) Ver.3.0 (TaKaRa, Code No. RR019A, Japan) according to the manufacturer’s protocol for cDNA cloning.

2.3 Mj-Cathepsin L DNA full-length amplification and sequencing

The intermediate fragment of Mj-Cathepsin L was obtained via screening the hepatopancreas transcriptome of the Marsupenaeus japonicus which constructed by our laboratory. In order to confirm the intermediate fragment of Mj-Cathepsin L gene, a pair of primers Cathepsin L-F (5′-AGCAATGGCACAACTTCAAGGCTG-3′) and Cathepsin L-R (5′-CTACGGAAAAGATACA- GCAAAGGCA-3′) was designed using PrimerPrimer 5.0 software. PCR products were visualized on 1.5% agarose-gel using Gene Finder^TM (Zeeshan, China) staining. Amplicon was cloned directly into the pMD®19-T Vector Cloning Vector (TaKaRa, Code No. D102A, Japan) according to the manufacturer’s instructions and subsequently transformed to DH5α bacterial competent cells. The plasmid DNA was isolated using E.Z.N.A.® Plasmid Mini Kit I (OMEGA Bio-Tek, Cat. No. D6942-01, USA) then used for the PCR assay to screen the positive colony before sending for commercial sequencing (Sangon Biotech Shanghai Co., China) to confirm its identity.

For the full-length amplification of Mj-Cathepsin L gene, rapid amplification of cDNA ends assays (3′-RACE and 5′-RACE) were employed. PCR amplification of the 3′ end was conducted using the specific primer CF3 (5′-GACGCCTCTCAACCTAGCCTCCAGT-3′), and the 3′ adaptor primer offered by the 3′-Full RACE Core Set (TaKaRa, Code No. 6106). In the PCR reaction, 1 μL CF3 and 3′ adaptor primers, and 0.6 μL cDNA were incubated with the Taq polymerase for 5 min at 95°C, followed by 32 cycles of 30 s at 95°C, 45 s at 68°C, 1 min at 72°C, and the final extension for 7 min at 72°C. The 5′ RACE Kit (2nd Generation, Roche) was employed for the 5′ cDNA preparation while the CR5 primer (5′-CCCGTCGTAGAGAAAGCCCAGCAGG-3′) was designed to amplify the complete 5′ sequence following to the manufacturer’s guidelines. The screening and sequencing of PCR products followed the same methods above.

The genomic DNA was extracted from muscle using phenol-chloroform method. The specific primers genomeF (5′-GAAGTTCCTGTCAGTGTTGGCT-3′) and genomeR (5′-CACAGAACTCTAGACGAGCGGG-3′) were designed to amplify the Mj-Cathepsin L genomic DNA sequence based on the full-length Mj-Cathepsin L cDNA. The screening and sequencing of PCR products followed the same methods above.

2.4 Sequence analysis

Contig Express was employed to overlap all the confirmed sequences to get the full-length cDNA of Mj-Cathepsin L. The Open Reading Frame (ORF) was predicted using DNAMAN software (DNAMAN LynnonBiosoft, Santa Clara, CA, USA) and the signal peptide was predicted by the SignalP4.1 Server (http:// www.cbs.dtu.dk/services/SignalP-3.0/). The propeptide and mature enzyme, catalytic C-H-N triad and the six Cys-disulfide-bridges were predicted via sequence alignment with homology sequences. The potential serine, threonine and tyrosine phosphorylation sites of Mj-Cathepsin L protein were predicted by NetPhos 2.0 Server (http://www.cbs.dtu.dk/services/NetPhos/).

Multiple Cathepsin L sequences from M. japonicus, L. vannamei, M. ensis, P. camtschaticus, E. sinensis, H. americanus CYSP2_HOMAM, N. norvegicus and H. americanus CYSP3_HOMAM, were aligned, and neighbor-joining (NJ) phylogenetic tree were performed using MEGA 6.0 program. The phylogenetic tree was tested by Bootstrap analysis of 1 000 replicates according to the literature method. Identity and similarity sequences were determined using the BLASTP algorithm with the MEGA version 6.0 stained with red colors by the ESPript 3.0 (http://espript. ibcp.fr/ESPript/ ESPript/index.php).

The genomic DNA sequence obtained was aligned with the cDNA to identify the exons and introns conforming to the splicing consensus GT-donor/AG-acceptor rule, and the genomic DNA sequences from other species were also consulted.

2.5 Quantitative real-time PCR (qRT-PCR)

Total RNA (1 μg) was reverse-transcribed using the PrimeScript^TM RT Master Mix (Perfect Real Time) (TaKaRa, Japan) and stored at –20°C for the subsequent analysis. Two Mj-Cathepsin L gene-specific primers F (5′-TCTCAACCTAGCCTCCAGTTCTACC-3′) and R (5′-ATGCCGCAGTTGTTCTTCTTGTTGC-3′) were designed to amplify a product of 209 bp. In shrimp glyceroldehyde-3 phosphate dehydrogenase (GAPDH) and Elongation factor 1-α (EF1-α) were demonstrated to be the more stable genes than β-actin and 18S rRNA (Dhar et al., 2009). Here the Elongation factor 1-α (EF1-α) was chosen to design a set of EF1-α primers Mj-EF1-αF (5′-GGAACTGGAGGCAGG ACC-3′) and Mj-EF1-αR (5′-AGCCACCGTTTG CTTCAT-3′) that approximately amplified a product of 158 bp, for the more similar amplification efficiency with the Mj-Cathepsin L gene.

The qRT-PCR amplifications were performed in triplicate using ABI 7500 Fast RT-PCR System (Applied Biosystems). Each reaction mixture had a total volume of 20 μL, which contained 10 μL of SYBR®PremixDimerEraserTM (2×), 0.4 μL ROX Reference Dye II (50×), 0.6 μL of each of the forward and reverse primers (10 mmol/L), 6.4 μL of PCR-grade water and 1 μL of the diluted cDNA (1:10). The 3-step PCR Standard Protocol was used which had an initial denaturation of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 30 s and 72°C for 30 s. Amplification efficiencies for Mj-Cathepsin L and EF1-α primers were determined via separate standard curves, and the specificities of the PCR amplification product were verified from the melting curves. The relative expression ratios of the target gene (Mj-Cathepsin L) versus the internal control gene in different tissues were calculated by the 2^-ΔΔCt method (Livak and Schmittgen, 2001).

2.6 Prokaryotic expression of Mj-Cathepsin L recombinant protein

The coding region of Mj-Cathepsin L was cloned into the prokaryotic expression ET-28a+ vector (Novegan, Germany) using the primers CTSLePF (5′-ATGGGTCGCGGATCCGAATTC ATGAAGTTCCTGTCAGTGTTGGCTT-3′) and CTSLePR (5′-CTCGAGTGCGGCCGCAAGCTTCACAGAACTCTAGACGAGCGGGTAC-3′) following the NovoRec PCR step directional cloning kit (Novoprotein, China). The recombinant Mj-Cathepsin L-pET-28a+ plasmid was transformed into Escherichia coli DH5α cells and screened the positive clones on Luria Broth (LB) culture medium supplied with 100 μg/mL of kanamycin. The positive clones were confirmed using sequencing (Sangon, China) and the confirmed plasmid was transformed into E. coli (BL21 DE3) pLysS cells for the expression of Mj-Cathepsin L recombinant protein. The log-phase bacterial cultures (5 mL) were added into the self-induced culture medium (500 mL) and incubated at 37°C with shaking at 150 r/min for 20 h. Before the induction, an aliquot of the cells was removed from the culture and cultured in the LB culture medium under the same condition for the none-induction control. The induction result was detected using 12% SDS-Polyacrylamide gel electrophoresis (SDS-PAGE).

2.7 Purification of Mj-Cathepsin L recombinant protein

The cultured cells were centrifuged at 5 000 r/min for 10 min at 4°C, and the supernatant was discarded. The precipitate was resuspended using the binding buffer with 0.2 mg/mL lysozyme, 20 μg/mL DNase, 1 mmol/L MgCl₂ and 1 mmol/L PMSF. The mixture was incubated at 22°C for 30 min and was ultrasonicated on ice in 5 s bursts, following 5 s rests between the bursts for approximately 90 cycles until the mixture was no longer viscous. After that, the mixture was centrifuged at 13 000 r/min for 20 min at 4°C and filtered using 0.45 μm micropore filter to discard the impurities for the subsequent operations.

The HisTrap^TM HP column (1 mL) (GE Healthcare Life Sciences, Sweden) that loaded with Ni Sepharose High Performance Linking to the AKTA purifier 100 workstation (GE Healthcare) as an immobilized metal affinity chromatography system was used for the recombinant protein purification. At least 5 mL binding buffer (20 mmol/L sodium phosphate, 0.5 mol/L sodium chloride, 40 mmol/L imidazole, 6 mol/L urea, pH 7.4) was loaded onto the HisTrap^TM HP column at the flow-rate of 1 mL/min for the equilibration of the column, then the sample was loaded at the flow-rate of 0.8 mL/min. The 5–10 mL binding buffer was used to wash the column at the flow-rate of 1 mL/min until the UV absorption baseline was reached. The recombinant protein was eluted using the elution buffer (20 mmol/L sodium phosphate, 0.5 mol/L sodium chloride, 500 mmol/L imidazole, 6 mol/L urea, pH7.4). The elution fractions were dialyzed in a linear gradient refolding buffer from 6 mol/L to 0 mol/L urea, finishing with the Milli-Q water. The purified Mj-Cathepsin L recombinant protein was frozen at –80°C for further studies.

For the obtain of Antiserum, 100 μg purified Mj-Cathepsin L recombinant protein was mixed with Freund’s incomplete adjuvant (Sigma, St. Louis, MO) and injected three of eight weeks Kunming mice intra-peritoneally. After three additional boosters in the following three weeks (once a week) with 100 μg protein in Freund’s complete adjuvant by the same route, the anti-sera was collected and preserved in –20°C until use.

2.8 Western blot analysis

Crude extracts from stomach, hepatopancreas and intestine of M. japonicus were prepared by homogenizing the tissues in PBS buffer, then centrifugated at 13 000 r/min to remove the residual debris. Crude protein quantification was performed using an Easy Protein Quantitative Kit (TransGen Biotech). Different tissue lysates were boiled in 2× loading buffer, run on the mini-protein II-system and were transferred to Immobilon-P poly-vinylidene fluoride membranes (Millipore, Bedford, MA). The PVDF membrane was blocked in TBSTM (5% non-fat dried milk with 25 mmol/L Tris, pH 8.0, 125 mmol/L NaCl and 0.05% Tween 20 (v/v)) for 1 h at RT. Anti-Mj-Cathepsin L polyclonal mouse anti-sera at 1:1 000 and goat anti-mouse antibody conjugated to horseradish peroxidase (1:5 000) were used as antibody to incubate the PVDF membrane at 37°C for 1 h. Chemiluminescent detection was performed using the EasySee® Western Blot Kit (TransGen Biotech).

3 Results

Less

3.1 Mj-Cathepsin L full-length cDNA and DNA

The full-length cDNA sequence of Mj-Cathepsin L (No. KJ871613) was obtained, with 1191 bp in length which encompassed a 5′-untranslated region (5′-UTR) of 15 bp, an ORF of 984 bp and a 3′-UTR region of 192 bp with a consensus downstream poly-(A) tail. The 984 bp of ORF region started with the initiation codon ATG on 16–18 base, ended with the terminal codon TGA on 997–999 base, and encoded a polypeptide of 327 amino acids (aa) with an estimated molecular mass of 35.9 kDa and the theoretical isoelectric point of the polypeptide is 5.34 (Fig. 1).

1 963 bp of Mj-Cathepsin L genomic DNA was obtained and the coding region consists of six exons separated by five introns. The interspecies comparison of the Cathepsin L orthologs in Danio rerio, Homo sapiens, Mus musculus, Limpenaeus vannamei and Marsupenaeus japonicus revealed that the sizes of exons and introns between the vertebrates ortholog and that of the arthropods are different (Fig. 2). The intron/exon distribution, exon sizes are highly conserved among Penaeidae family, except Metapenaeus ensis.

The confirmed Mj-Cathepsin L preproprotein contained a typical signal peptide sequence of 16 aa (Met₁–Ala₁₆), a propeptide of 93 aa (Ser₁₇–Thr₁₀₉) and mature enzyme domain of 217 aa (Leu₁₁₀–Val₃₂₇). Cys₁₃₄, His₂₇₃ and Asn₂₉₄ formed the predicted highly conserved catalytic triad and the six Cysteine forming conserved disulfide-bridges were located at Cys₁₃₁, Cys₁₆₅, Cys₁₇₄, Cys₂₀₇, Cys₂₆₆ and Cys₃₁₆ (Fig. 1). ERFNIN (Position 40–59), GNFD (Position 60–78) and GCNGG (Position 173–177) motifs for typical Cathepsin L proteases were marked above the alignment sequences with bold black capital letters (Fig. 3). The phosphorylation sites of Cathepsin L protein were at Ser_{19, 37, 46, 81, 85, 142, 156, 158, 282}, Thr_{80, 123, 219 and Tyr43, 198, 289, 305} (Data not shown).

3.2 Phylogenetic tree of Cathepsin L genes

Multiple alignment of Mj-Cathepsin L with Cathepsin L amino acid sequences of other shrimps revealed the strong amino acid conservation in Cathepsin L proteins. Comparative analysis of the homology sequences revealed that the deduced amino acids sequence of Mj-Cathepsin L shared the highest identity with L. vannamei (90.5%), and 62.0%–79.8% identity to other shrimp Cathepsin L amino acids sequences (Table 1).

There were two large groups in the NJ tree of Cathepsin L proteins. One group consisted of two sub-groups, Crustacea and Arachnoidea, both belong to the Arthropoda (Fig. 4). Marsupenaeus japonicus was clustered with L.vannamei and formed the Crustacea sub-group with N. norvegicus, H. americanus, M. ensis, P. camtschaticus and E. sinensis. The two different parasite ticks Rhipicephalus haemaphysaloides haemaphysaloides and Hyalomma anatolicum anatolicum were clustered together forming the Arachnoidea sub-group. The other group is the Vertebrate sub-group, including Salmo salar, Lates calcarifer and Homo sapiens.

3.3 Tissue distribution expression of Mj-Cathepsin L

Mj-Cathepsin L mRNA was expressed significantly higher in hepatopancreas, followed by a moderate expression level in stomach, and relatively faint expression levels in intestine, muscle, heart, eye stalk and gill (P<0.05) (Fig. 5).

3.4 Mj-Cathepsin L mRNA expression levels during early developmental stages and the molt cycle

The relative expression levels of Mj-Cathepsin L mRNA were detected during the early developmental stages from fertilized egg to post-larva 27 (P₂₇) (Fig. 6). The expression levels of Mj-Cathepsin L divided into two parts; there were faintly expression levels before P₁ stage and maintained significantly high levels after P₁ stage (P<0.05). The expression levels increased steadily from P₁ to P₅ and sharply decreased at P_8–11; the expression level increased gradually to the highest value at P₂₁ and fell back to a low level at P₂₇ (P<0.05).

The transcription analysis revealed that Mj-Cathepsin L predominantly expressed during preecdysis (premolt) stages of the molt cycle, and maintained a relatively low level in other four stages (Fig. 7).

3.5 Mj-Cathepsin L recombinant protein

SDS-PAGE analysis showed that almost all the recombinant Mj-Cathepsin L was expressed as inclusion bodies contained in the cell lysate. The purified recombinant Mj-Cathepsin L product was detected using SDS-PAGE and showed a clear band with the predicted molecular mass of about 40.0 kDa (Fig. 8). The recombinant Mj-Cathepsin L was used to raise antibodies in mice for western blotting.

3.6 Western blotting analysis of Mj-Cathepsin L in stomach, hepatopancreas and intestine of M. japonicus

Crude extracts of stomach, hepatopancreas and intestine were analyzed by western blot. The protein level of Mj-Cathepsin L in stomach, hepatopancreas and intestine of M. japonicus were detected using antibodies against recombinant Mj-Cathepsin L. The results showed that the anti-Mj-Cathepsin L serum positively identified two bands in ST and HP: the upper Mj-Cathepsin L protein (CathL) bands about 40 kDa and the lower mature-Mj-Cathepsin L protein (mat-CathL) bands about 22 kDa (Fig. 9).

4 Discussion

Less

In this study, we for the first time identified Cathepsin L gene from M. japonicus and put insight into the Mj-Cathepsin L mRNA changes during ontogenetic development, which is the most crucial physiological and morphological processes in the life history of shrimps. The cDNA encoding Mj-Cathepsin L gene was 1 191 bp in full-length, and contained a typical signal peptide sequence of 16 aa (Met₁-Ala₁₆). The signal peptide is participated in the protein secretary process in many eukaryotic organisms including crustaceans (Le Boulay et al., 1998), indicating that the secretary mechanism of Mj-Cathepsin L. Interestingly, in some organisms the signal peptide is either absent or shorter in length (McIntyre et al., 1994; Chauhan et al., 1998), suggested that the secretary mechanism of Cathepsin L may be absent.

To our knowledges, Cathepsin L genes have diverse gene structures with the number of introns, ranging from zero to nine. The Mj-Cathepsin L with five introns was found to possess a relatively classic exon/intron structure among most of the shrimp. But interestingly some Cathepsin L genes have asymmetric distribution of introns and paralog of Mj-Cathepsin L in Metapenaeus ensis in the same family Penaeidae is intronless (Hu and Leung, 2006). The mechanism of the intron loss in Cathepsin L gene and the implicit information about deep evolutionary history need further research.

High similarity and identity of Cathepsin L genes in organisms including M. japonicus were revealed with the propeptide and mature enzyme, catalytic C-H-N triad and the six Cys-disulfide-bridges were predicted (Ma et al., 2010). ERF/WNIN-like motif is believed as the principal characteristic feature of Cathepsin L (Karrer et al., 1993). The deduced Mj-Cathepsin L protein presented in ERFNIN (Position 40–59), GNFD (Position 60–78) and GCNGG (Position 173–177) motifs which could be used to identify it as a member of the typical Cathepsin L family. The phylogenetic analysis of the Cathepsin L genes showed that Mj-Cathepsin L clustered with crustaceans, suggesting the functional conservation of Cathepsin L in the same species group.

Cathepsin L was presumed to play an extracellular digestive role in guts of invertebrates including shrimps (Johnson and Rabosky, 2000) (Lima et al., 2001). Mj-Cathepsin L mRNA was predominantly expressed in hepatopancreas; same pattern was documented in P. borealis and M. ensis (Aoki et al., 2004; Hu and Leung, 2004). Hepatopancreas is considered as the most crucial organ of the crustaceans that participates in the secretion and excretion, storage of nutrients and even in the immune responses.

Morphological and physiological processes during ontogenetic development in shrimps consumed a quantity of energy for swim and predation, and young larvae took advantage of carbohydrates and lipids preferentially, conserved the proteins for the succeeding metamorphosis and development (Johnston, 2003; Darias et al., 2006). In this study, larvae were fed with the same prawn slices before P10 to reduce the diet effect to the expression of the Mj-Cathepsin L based on the assumption that the expression of Cathepsin L was regulated by the internal mechanism. Mj-Cathepsin L mRNA expression was demonstrated to be ontogenetic developmental-regulated in different larval stages.

First, the Nauplius stage, known as the none-feeding stage and started its preliminary differentiation of hepatopancreas, had a low expression of Mj-Cathepsin L. The nauplius larvae can secrete Cathepsin L to utilize the vitellogenin, promote the development and to prepare for the raptorial-feeding life after metamorphosis (Pan et al., 2006). Second, in Zoea and Mysis stages, the expressions of Mj-Cathepsin L presented an “M” shape trend with sharp peaks in Zoea 2 and Mysis 2 stages (Fig. 4). The digestive enzyme activities approached a high level from Z₃ to M₁ development in M. japonicus (Rodriguez et al., 1994). In this study the expressions of Mj-Cathepsin L mRNA in Z₃ to M₁ were relatively low, inferring that the Mj-Cathepsin L contribute to other functions than digestion during early development. Third, in the post-larval stages the carapace length showed a positive relationship with body length, responding with the increase of food intake (data not shown). The expression of Mj-Cathepsin L approached a relatively high level in the post-larval stages, with an obvious decrease from P₆ to P₁₁ (Fig. 6), suggested that it may be associated with the behavioral change of M. japonicas from pelagic to benthic life around P₈ stage, and with the physiological changes of feeding habit, digestive system change and energy distribution (Lemos and Rodríguez, 1998). Interestingly, under the same food intake conditions, we noticed that mRNA expressions of Mj-Cathepsin L in different larvae stages are opposite to that of α-amylase in our previous studies, which is known as a major glucosidase involved in the digestive function of shrimps (unpublished data). This demonstrates the different nutrient intake in different larvae stages and suggests the development-regulated expression of the Mj-Cathepsin L.

Molting, a phenomenon of shedding the old cuticle and re-generating the new one, was considered as the most important process during larvae stages or even the whole life cycle. Le Boulay et al. (1996) demonstrated that variations in cysteine protease activity were correlated with the variations in the levels of specific mRNA during intermolt cycles of Penaeus vannamei. Here we found Mj-Cathepsin L predominantly expressed during preecdysis/premolt (D₀–D₃) stages of the molt cycle, and it maintained a relatively low level in other four stages. There was a gradually rising trend from Stages A to C, following the well-known Stage E (ecdysis). The expression of Mj-Cathepsin L during molt cycle suggested that Mj-Cathepsin L might be required for the molting process, especially in preecdysis/premolt (D) stages.

Western blotting confirmed the results of tissue distribution, we also demonstrated the nature Mj-Cathepsin L protein presents two forms in the shrimp tissues, the pro-Mj-Cathepsin L about 40 kDa and the mature-Mj-Cathepsin L (mat-CathL) about 22 kDa. The hepatopancreas showed two very strong bands as compared with stomach and intestine, and the upper pro-Mj-Cathepsin L was relatively stronger than lower mature-Mj-Cathepsin L. The results revealed the Mj-Cathepsin L protein mainly existed in the form of pro-Mj-Cathepsin L, but not the activity mature-Mj-Cathepsin L.

In summary, Mj-Cathepsin L gene was isolated from Kuruma shrimp Marsupenaeus japonicus and was demonstrated expressing in the hepatopancreas and stomach. The mRNA levels of Mj-Cathepsin L were studied during early ontogenetic development stages from eggs to post-larvae 28 and during the five molt cycles. The results suggest the Mj-Cathepsin L seems to be regulated by digest and molting processes during the early ontogenetic development stages of Marsupenaeus japonicus. Western blot also revealed two forms of Mj-Cathepsin L protein existing in three tissues. Further studies will focus on the localization of the Mj-Cathepsin L.

Acknowledgements

Less

The authors thank Dong-shan Maoxin Aquaculture Company (Dongshan County, Fujian Province) for providing the experimental shrimp and equipment.

Funding

Less

The National High-tech R&D Program of China (863 Program) under contract No. 2012AA10A409-03; the Project of China Agriculture Research System under contract No. CARS-47; the Project of Xiamen Southern Ocean Research Center under contract No. 14CZY033HJ07; China Spark Program under contract No. 2015GA720002.

References

Less

Aoki H, Ahsan M N, Watabe S. 2004. Molecular and enzymatic properties of a cathepsin L-like proteinase with distinct substrate specificity from northern shrimp (Pandalus borealis). J Comp Physiol B, 174(1): 59–69

Arockiaraj J, Gnanam A J, Muthukrishnan D, et al. 2013. Macrobrachium rosenbergii cathepsin L: molecular characterization and gene expression in response to viral and bacterial infections. Microbiol Res, 168(9): 569–579

Chauhan S S, Ray D, Kane S E, et al. 1998. Involvement of carboxy-terminal amino acids in secretion of human lysosomal protease cathepsin L. Biochemistry, 37(23): 8584–8594

Dahl S W, Halkier T, Lauritzen C, et al. 2001. Human recombinant pro-dipeptidyl peptidase I (cathepsin C) can be activated by cathepsins L and S but not by autocatalytic processing. Biochemistry, 40(6): 1671–1678

Darias M J, Murray H M, Gallant J W, et al. 2006. Characterization of a partial α-amylase clone from red porgy (Pagrus pagrus): expression during larval development. Comp Biochem Physiol B Biochem Mol Bio, 143(2): 209–218

de Oliveira Cesar J R, Zhao Baoping, Malecha S, et al. 2006. Morphological and biochemical changes in the muscle of the marine shrimp Litopenaeus vannamei during the molt cycle. Aquaculture, 261(2): 688–694

Dhar A K, Bowers R M, Licon K S, et al. 2009. Validation of reference genes for quantitative measurement of immune gene expression in shrimp. Mol Immunol, 46(8–9): 1688–1695

Glenn K L, Grapes L, Suwanasopee T, et al. 2005. SNP analysis of AMY2 and CTSL genes in Litopenaeus vannamei and Penaeus monodon shrimp. Anim Genet, 36(3): 235–236

Hu Kejin, Leung P C. 2004. Shrimp cathepsin L encoded by an intronless gene has predominant expression in hepatopancreas, and occurs in the nucleus of oocyte. Comp Biochem Physiol B Biochem Mol Biol, 137(1): 21–33

Hu Kejin, Leung P C. 2007. Food digestion by cathepsin L and digestion-related rapid cell differentiation in shrimp hepatopancreas. Comp Biochem Physiol B Biochem Mol Biol, 146(1): 69–80

Hu K J, Leung P C. 2006. Complete, precise, and innocuous loss of multiple introns in the currently intronless, active cathepsin L-like genes, and inference from this event. Mol Phylogenet Evol, 38(3): 685–696

Johnson K S, Rabosky D. 2000. Phylogenetic distribution of cysteine proteinases in beetles: evidence for an evolutionary shift to an alkaline digestive strategy in Cerambycidae. Comp Biochem Physiol B Biochem Mol Biol, 126(4): 609–619

Johnston D J. 2003. Ontogenetic changes in digestive enzyme activity of the spiny lobster, Jasus edwardsii (Decapoda; Palinuridae). Marine Biology, 143(6): 1071–1082

Karrer K M, Peiffer S L, Ditomas M E. 1993. Two distinct gene subfamilies within the family of cysteine protease genes. Proc Natl Acad Sci U S A, 90(7): 3063–3067

Laycock M V, Hirama T, Hasnain S, et al. 1989. Purification and characterization of a digestive cysteine proteinase from the American lobster (Homarus americanus). Biochem J, 263(2): 439–444

Le Boulay C, Van Wormhoudt A, Sellos D. 1995. Molecular cloning and sequencing of two cDNAs encoding cathepsin L-related cysteine proteinases in the nervous system and in the stomach of the Norway lobster (Nephrops norvegicus). Comp Biochem Physiol B Biochem Mol Bio, 111(3): 353–359

Le Boulay C, Van Wormhoudt A, Sellos D. 1996. Cloning and expression of cathepsin L-like proteinases in the hepatopancreas of the shrimp Penaeus vannamei during the intermolt cycle. J Comp Physiol B, 166(5): 310–318

Le Boulay C, Sellos D, Van Wormhoudt A. 1998. Cathepsin L gene organization in crustaceans. Gene, 218(1–2): 77–84

Lemos D, Rodríguez A. 1998. Nutritional effects on body composition, energy content and trypsin activity of Penaeus japonicus during early postlarval development. Aquaculture, 160(1–2): 103–116

Lima A P C A, dos Reis F C G, Serveau C, et al. 2001. Cysteine protease isoforms from Trypanosoma cruzi, cruzipain 2 and cruzain, present different substrate preference and susceptibility to inhibitors. Mol Biochem Parasitol, 114(1): 41–52

Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25: 402–408

Ma Jianjun, Zhang Dianchang, Jiang Jingjing, et al. 2010. Molecular characterization and expression analysis of cathepsin L1 cysteine protease from pearl oyster Pinctada fucata. Fish Shellfish Immunol, 29(3): 501–507

McIntyre G F, Godbold G D, Erickson A H. 1994. The pH-dependent membrane association of procathepsin L is mediated by a 9-residue sequence within the propeptide. J Biol Chem, 269(1): 567–572

Miyamoto K, Iwadate M, Yanagisawa Y, et al. 2011. Cathepsin L is highly expressed in gastrointestinal stromal tumors. Int J Oncol, 39(5): 1109–1115

Nakagawa T, Roth W, Wong P, et al. 1998. Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus. Science, 280(5362): 450–453

Pan Luqing, Liu Hong yu, Xiao Guoqiang. 2006. A review on digestive enzyme of crustacean larvae. J Fishery Sci China, 13(3): 492–501

Qian Zhaoying, Li Xilian, Xin Jingjing, et al. 2013. PCR-SSCP Polymorphism of CTSL gene and its correlation with growth traits of Litopenaeus vannamei and the different mRNA expressions of CTSL. Haiyang Xuebao, 35(6): 121–127

Rodriguez A, Le Vay L, Mourente G, et al. 1994. Biochemical composition and digestive enzyme activity in larvae and postlarvae of Penaeus japonicus during herbivorous and carnivorous feeding. Marine Biology, 118(1): 45–51

Roth W, Deussing J, Botchkarev V A, et al. 2000. Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and pertubation of hair follicle cycling. FASEB J, 14(13): 2075–2086

Zhang W, Wang S, Wang Q, et al. 2014. Overexpression of cysteine cathepsin L is a marker of invasion and metastasis in ovarian cancer. Oncol Rep, 31(3): 1334–1342

Appendix

Less

Year 2017 volume 36 Issue 6

PDF

Cite this Article

BibTeX

Article Info

doi: 10.1007/s13131-017-0983-5

Receive Date：2016-02-16
Online Date：2026-04-14
Published：2017-02-01

Article Data

Affiliations

History

Received：2016-02-16
Accepted：2016-03-29

Funding

The National High-tech R&D Program of China (863 Program) under contract No. 2012AA10A409-03; the Project of China Agriculture Research System under contract No. CARS-47; the Project of Xiamen Southern Ocean Research Center under contract No. 14CZY033HJ07; China Spark Program under contract No. 2015GA720002.

Affiliations

¹ State Key Laboratory of Marine Environmental Sciences, Xiamen University, Xiamen 361102, China

² Putian Tian-ran-xing Agricultural Development Co. Ltd., Fujian 351100, China

Corresponding:

*E-mail: yqsu@xmu.edu.cn

References

Share

https://castjournals.cast.org.cn/joweb/aos/EN/10.1007/s13131-017-0983-5

Share to

Scan QR to access full text

Cite this article

BibTeX

Citations

表12种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

关闭全屏

BibTeX
EndNote
RefWorks
TxT

	M. japonicus	L. vannamei	M. ensis	P. camtschaticus	E. sinensis	H. americanus CYSP2	N. norvegicus	H. americanus CYSP3
M. japonicus	–	0.174 0	0.286 8	0.638 0	0.737 4	0.835 1	0.787 9	0.833 5
L. vannamei	0.905	–	0.298 7	0.654 8	0.753 8	0.876 1	0.766 9	0.832 9
M. ensis	0.798	0.789	–	0.596 6	0.705 3	0.794 2	0.719 2	0.751 3
P. camtschaticus	0.685	0.685	0.662	–	0.681 6	0.902 7	0.784 4	0.852 0
E. sinensis	0.664	0.661	0.638	0.686	–	0.934 9	0.903 1	0.973 9
H. americanus CYSP2	0.623	0.606	0.609	0.597	0.588	–	0.680 9	0.691 9
N. norvegicus	0.617	0.621	0.643	0.622	0.581	0.669	–	0.150 9
H. americanus CYSP3	0.620	0.615	0.618	0.614	0.573	0.682	0.897	–

Fig. 1. Compiled genomic organization of the Marsupenaeus japonicus Cathepsin L gene. The open reading frame of Mj-Cathepsin L is marked with capital letters, the 15 bp 3′-UTR and 192 bp of the 5′-UTR are marked with lowercase letters. The putative signal peptide is underlined. Black down arrows indicate the cleavage site between signal peptide (SP) and propeptide, and between propeptide and mature enzyme. Black bold letters in the [ ] denote the catalytic C-H-N triad. The six Cys forming possible disulfide-bridges are marked with bold black and underlined with emphasis character. Stop condon is marked with *. Poly (A) sequence is marked with strikeout. The introns are in lowercase and intron splice sites GT-donor/AG-acceptor are boxed. Numbering of the nucleotide and the amino acid are given on the right-hand.

Fig. 2. Comparison of the gene structures of Mj-Cathepsin L genes with other species. The black boxes depict the coding regions, and the horizontal line represents the introns. The 5′ and 3′ UTR are shown as white boxes. The lengths of the elements are shown in nucleotides (bp). The genomic DNA sequences were obtained from the NCBI database (http://www.ncbi.nlm.nih.gov/) and ENSEMBL genomic database (http://www.ensembl.org).

Fig. 3. Multiple alignment of the predicted amino acid sequence of Mj-Cathepsin L with other shrimp Cathepsin L amino acid sequences. Shading indicates residues that are identical (red paper-white lettered) and blue rim-red lettered indicates conserved residues. The homology sequences used were as the following order: Litopenaeus vannamei (CAA68066.1), Metapenaeus ensis (AAM96001.1), Paralithodes camtschaticus (ADQ73946.1), Eriocheir sinensis (ADO65978.1), Homarus americanus CYSP2_HOMAM (P25782.1), Nephrops norvegicus (CAA56915.1), and Homarus americanus CYSP3_HOMAM (P25784.1).

Fig. 4. Neighbor-joining phylogenetic tree of Mj-Cathepsin L and similar Cathepsin L proteins. Amino acid distances were calculated using the Poisson correction model in the MEGA program Version 6.0., and the phylogram topology was representative for 1000 bootstrap replicates. Sequences used in the alignment were: Marsupenaeus japonicus (No. KJ871613), Litopenaeus vannamei (No. CAA68066.1), Metapenaeus ensis (No. AAM96001.1), Paralithodes camtschaticus (No. ADQ73946.1), Eriocheir sinensis (No. ADO65978.1), Homarus americanus CYSP2_HOMAM (No. P25782.1), Nephrops norvegicus (No. CAA56915.1), Rhipicephalus haemaphysaloides haemaphysaloides (No. AAQ16117.1), Hyalomma anatolicum anatolicum (No. AFQ98384.1), Salmo salar (No. NP_001140018.1), Lates calcarifer (No. ABV59078.1), and Homo sapiens (No. NP_001903.1).

Fig. 5. Relative expression levels (n=3, mean±SD) of Mj-Cathepsin L gene in various adult tissues of Marsupenaeus japonicus. Relative expression levels of the target gene were normalized by EF1-α gene and calculated based on the 2^-ΔΔCt method using hepatopancreas as the calibrator. * above the bar indicates the significant difference at the level of 0.05. HP represents hepatopancreas, IT intestine, MU muscle, HE heart, ES eye stalk, ST stomach, and GL gill.

Fig. 6. Quantitative expressions (n=3, mean±SD) of Mj-Cathepsin L gene at early developmental stages of Marsupenaeus japonicus. Relative expression levels of the target gene were normalized against EF1-α gene and the fertilized eggs were chosen as the calibrator. Different letters above the bars indicate the significant differences at the level of 0.05. E represents fertilized eggs, N nauplius, Z₁–Z₃ zoea 1–3, M₁–M₃ mysis 1–3, and P₁–P₂₇ post-larvae 1–27.

Fig. 7. Quantitative expressions of Mj-Cathepsin L gene during molt cycle. Stages A and B represent postecdysis/postmolt stages, Stage C intermolting stage, Stages D₀–D₄ preecdysis/ premolt stages, and Stage E ecdysis stage.

Fig. 8. Expression and purification analyses of recombinant Mj-Cathepsin L. SDS-PAGE analysis of expression and purification of recombinant Mj-Cathepsin L. M represents protein marker, 1 PET28a-Mj-Cathepsin L (BL21) (uninduced), 2 PET28a-Mj-Cathepsin L (BL21) (induced), 3 precipitation (the recombinant protein existed in the precipitations), 4 supernatant, and 5 the purified recombinant protein Mj-Cathepsin L. Left panel shows the position of the molecular mass markers (kDa).

Fig. 9. The expression of Mj-Cathepsin L in different tissues of Marsupenaeus japonicus detected with the antibody against the recombinant Mj-Cathepsin L protein. ST represents stomach, HP hepatopancreas, and IT intestine. The upper lanes were probed with anti-tubulin as the protein-loading control, and the lower lanes represent the Mj-Cathepsin L protein (CathL) and mature-Mj-Cathepsin L protein (mat-CathL) levels.

Articles: Latest Articles; Most Read; Collections

Updates: Events; News; Multimedia

About: About Us

Contact

No. 86 Xueyuan South Road, Haidian District, Beijing

100081

010-62199257

qkjq@cast.org.cn

Copyright © 2025 China Association for Science and Technology. All rights reserved. For all open access content, the relevant licensing terms apply.
Sponsored by the Office of the Leading Group for Cybersecurity and Informatization of CAST, and supported by Science and Technology Review Publishing House