Molecular characterization and function of β-N-acetylglucosaminidase from ridgetail white prawn Exopalaemon carinicauda

Accepted Manuscript Molecular characterization and function of β-Nacetylglucosaminidase from ridgetail white prawn Exopalaemon carinicauda

Yuying Sun, Jiquan Zhang, Jianhai Xiang PII: DOI: Reference:

S0378-1119(18)30053-2 https://doi.org/10.1016/j.gene.2018.01.046 GENE 42496

To appear in:

Gene

Received date: Revised date: Accepted date:

29 August 2017 4 December 2017 11 January 2018

Please cite this article as: Yuying Sun, Jiquan Zhang, Jianhai Xiang , Molecular characterization and function of β-N-acetylglucosaminidase from ridgetail white prawn Exopalaemon carinicauda. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), https://doi.org/10.1016/ j.gene.2018.01.046

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ACCEPTED MANUSCRIPT Molecular characterization and function of β-N-acetylglucosaminidase from ridgetail white prawn Exopalaemon carinicauda

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Yuying Sun1, 3, Jiquan Zhang1, 2 *, Jianhai Xiang2, 4

College of Life Sciences, Hebei University, Baoding, Hebei, 071002, China

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Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences,

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Qingdao 266071, China

College of Marine Life and Fisheries, Huaihai Institute of Technology, 59 Cangwu Road, Lianyungang 222005,

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Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and

* Corresponding author:

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Tel: +86-0532-82898786

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Technology, Qingdao 266000, China

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Fax: +86-0532-82898578

E-mail: [email protected]

Running title: Characterization and function of prawn β-N-acetylglucosaminidase

ACCEPTED MANUSCRIPT Abstract Chitin degradation is catalyzed by a two-component chitinolytic enzyme system, chitinase and β-N-acetylglucosaminidase (NAGase). In this paper, the full-length cDNA sequence encoding NAGase (EcNAG) was obtained from Exopalaemon carinicauda. The deduced amino acid sequence of EcNAG open reading frame (ORF) contained one Glycohydro_20b2 domain and one Glyco_hydro_20 domain. Based on the cDNA sequence,

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the genomic structure of EcNAG was characterized and it was composed of six exons and five introns. EcNAG

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mRNA majorly expressed in the hepatopancreas and epidermis. During the molting stages, EcNAG mRNA

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expression was well-regulated and its expression reached the highest level at the molting stage E. In addition, EcNAG was recombinant expressed in Pichia pastoris and the partial enzymatic characterization of recombinant

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EcNAG was confirmed. After being challenged with Vibrio parahaemolyticus and Aeromonas hydrophila, the

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expression of EcNAG was up-regulated significantly at 6 h and reached the peak at 12 h. And then, the expression began to down-regulated and came to the normal level at 72 h. It is helpful to research the relationship between the

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molt-related hormones and chitinlytic enzymes.

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1. Introduction

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Keywords: β-N-acetylglucosaminidase; Exopalaemon carinicauda; Pichia pastoris; recombinant expression

Chitin, a water-insoluble homopolymer of β-(1, 4)-N-acetyl-D-glucosamine (GlcNAc), is one of the most important biopolymers in nature (Merzendorfer and Zimoch, 2003; Younes et al., 2014). It is mainly produced by fungi, arthropods and nematodes and it is also one of the most unique biochemical constituents found in the exoskeletons and gut linings of arthropods (Fernandes-Pedrosa et al., 2008). In Crustacean, molting is a very important physiological process because it not only allows for growth and development of these animals bearing a rigid, confining exoskeleton but also is tied to metamorphosis during the early stages of the life cycle and

ACCEPTED MANUSCRIPT reproduction during the adult stage (Zou and Bonvillain, 2004). Chitin degradation is catalyzed by a two-component chitinolytic enzyme system, chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (NAGase) (EC 3.2.1.52). Chitinase is the endo-splitting enzymes, and NAGase is the exo-splitting enzymes required for chitin degradation (Liu et al., 2012). Chitinase hydrolyzes chitin into oligosaccharides, whereas NAGase is involved in

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the hydrolysis of terminal N-acetyl-D-glucosamine residues in N-acetyl-β-D-glucosaminides.

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At present, many researches on the structure and function of chitinases in crustaceans have been reported. The

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chitinase-3 gene (FcChi-3) transcript in Fenneropenaeus chinensis was down-regulated significantly in response to the challenge of WSSV at early stage (Zhang et al., 2010). Seven chitinase family members were identified in

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Litopenaeus vannamei, whose domain architectures, evolutionary relationships and tissue expression patterns

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provide reasonable explanation for the existence of multiple genes in crustacean chitinase family (Huang et al., 2010). Five hepatopancreatic (Pj-Cht1, 3A, 3B, 3C, and 4) and one epidermal (Pj-Cht1) chitinases were isolated

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from Pandalopsis japonica and the Pj-Cht2 was confirmed to play a role in chitin metabolism during molt cycle

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(Salma et al., 2012). Similar results were also found in other crustaceans, including Penaeus monodon (Tan et al., 2000; Lehnert and Johnson, 2002; Proespraiwong et al., 2010; Zhou et al., 2017), Marsupenaeus japonicas

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(Watanabe and Kono, 1997; Watanabe et al., 1998), Carcinus maenas (Lunt and Kent, 1960), Uca pugilator (Zou

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and Bonvillain, 2004) and so on. In our previous research, two kinds of native chitinase protein (EcChi1 and EcChi2) had been isolated from the hepatopancreas of Exopalaemon carinicauda and the enzymatic characterization of EcChi1 were also been clarified (Wang et al., 2015). In addition, we succeeded in knocking out one chitinase gene (EcChi4) of E. carinicauda via CRISPR/Cas9 method and clarifying its function in vivo (Gui et al., 2016; Sun et al., 2017). Unlike the chitinases, however, there are few reports about structure and function of NAGase from crustaceans. In this research, we reported a NAGase gene (EcNAG) in E. carinicauda. The expression profiles of EcNAG gene

ACCEPTED MANUSCRIPT in different tissues and different molting stages were analyzed. Furthermore, the EcNAG was recombinant expressed in Pichia pastoris and the partial enzymatic characterization of recombinant EcNAG was analyzed.

2. Materials and methods

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2.1. Cultivation and immune challenge of the experimental animals

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Prawns for gene expression profile in different tissues: The ridgetail white prawns, E. carinicauda with body

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length of 5.5 ± 0.5 cm were bred in plastic tanks filled with aerated fresh seawater at 24-26 ºC, and fed twice per day in our laboratory. Ten healthy adults of E. carinicauda were dissected to separate eyestalk, intestine, muscle,

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cuticle, hepatopancreas, nerve cord, heart, stomach, and gill. Then, the samples were preserved in liquid nitrogen

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for RNA extraction.

The mutant ridgetail white prawns with 5 bp deletion in the fourth extron of EcChi4 were obtained by

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CRISPR/Cas9 tool in our laboratory reported in the previous research (Gui et al., 2016).

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Prawns for expression profiles in different molting stages: The wild-type and EcChi4-deleted mutant prawns with the same size (body length of 3.0 ± 0.5 cm) were bred in plastic tanks filled with aerated fresh seawater at

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24-26 ºC, and fed twice per day in our laboratory. Two hundred healthy wild-type prawns were divided into 7

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groups according to the different molting periods described by Cesar et al (Cesar et al., 2006). They were late postmolt (B), intermolt (C), onset of premolt (D0), early premolt (D1), intermediate premolt (D2), late premolt (D3) and molt (E) stages. Then the hepatopancreas and cuticle of prawns in each group were collected and preserved in liquid nitrogen for RNA extraction. For the EcChi4-deleted mutant prawns, the hepatopancreas at the stage E were collected and preserved in liquid nitrogen for RNA extraction. Prawns for expression profiles after immune challenge: The wild-type prawns with the same size were challenged with Vibrio parahaemolyticus and Aeromonas hydrophila according to the method (Yang et al., 2008;

ACCEPTED MANUSCRIPT Saejung et al., 2014). Experimental groups and the control group were set up for each sampling point and 200 prawns were sampled from each group. For the bacterial challenge experiment, the experimental group was injected individually with 10 μL phosphate buffer saline (PBS) containing V. parahaemolyticus or A. hydrophila (107 CFU mL-1). At the same time, the prawns which were injected with 10 μl sterile PBS were maintained as the control. The

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residual prawns were calculated at 0, 6, 12, 24, 48, 72, and 96 h. The hepatopancreas of five prawns from each

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group were collected at 0, 6, 12, 24, 48, 72, and 96 h. All the samples were preserved for semi-quantitative

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RT-PCR.

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2.2. RNA isolation and cDNA synthesis

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Total RNA was extracted from the collected tissues with Trizol® reagent. The extracted RNA was then treated with RQI RNase-Free DNase (Promega, USA) to remove contaminated DNA. Two micrograms of total RNA and

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0.2 μM random hexamer primers were used to synthesize cDNA from each sample by M-MLV reverse transcriptase

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(Promega, USA). Then SYBR Green-based qPCR was adopted to detect EcNAG expression levels at different

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tissues or different molting stages in the hepatopancreas and cuticle.

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2.3 Sequence analysis and phylogenetic tree Based on the transcriptomic and genomic data of E. carinicauda (Yuan et al., 2017), the full-length NAG cDNA sequence of E. carinicauda (EcNAG) was obtained and confirmed by the procedures of reverse transcription-polymerase chain reaction (RT-PCR). ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to analyze the complete open reading frame (ORF) regions of EcNAG and SMART (http://smart.embl-heidelberg.de/) was used to predict the functional domains on the deduced amino acid sequences. The multiple sequence alignments and phylogenetic analysis were

ACCEPTED MANUSCRIPT performed on amino acid sequences of known NAG molecules using CLUSTAL W and MEGA version 4.0 (Tamura et al., 2007). Based on the full-length cDNA sequence and the partial information from genomic data of E. carinicauda (Yuan et al., 2017), a pair of primers (EcNAG-InF and EcNAG-InR) was designed to amplify the gene

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fragments using the extracted genomic DNA.

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2.4. SYBR Green-based quantitative real-time PCR (qPCR)

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SYBR Green-based qPCR (Yang et al., 2008) was adopted to detect expression levels of EcNAG mRNA at different tissues or in the hepatopancreas and cuticle of prawns during different molting stages. 18S rRNA from E.

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carinicauda was used as the internal control. Primers were shown in Table 1. The expected size of EcNAG and 18S

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rRNA was of 147 bp and 147 bp in length, respectively. The real-time PCR was carried out according to the program of 35 cycles of 95 °C for 15 s, 55 °C for 20 s and 72 °C for 20 s, following by an extension of 72 °C for 10

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min. The data were analyzed using the comparative CT method and then subjected to one-way analysis of variance

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(one-way ANOVA) using SPSS software 20.0. The p values less than 0.05 were considered statistically significant.

Sequences (5'-3')

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Primers

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Table 1 Primers mentioned in the paper Sequence information

EcNAGF

CGGAGGTGATGAGGTAAA

Real-time PCR

EcNAGR

GTCGTAAGTAGCTGGAGGG

Real-time PCR

18S-F

TATACGCTAGTGGAGCTGGAA

Real-time PCR

18S-R

GGGGAGGTAGTGACGAAAAAT

Real-time PCR

EcNAG-InF

CCCGTGAGAGGGTCTACACATGG

Confirm the genomic structure of EcNAG

EcNAG-InR

CCACAATTTTGTTTTATTCCTGAC

Confirm the genomic structure of EcNAG

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Construct the expression vector,

CAAGACTCTCCTTGGGGGTATAGA

restriction enzyme site for EcoR I and a 6×His-tag

GCGCGGCCGCTCAAGCATAGCAAAG

Construct the expression vector, introducing a

CCCTTCA

restriction enzyme site for Not I

5’AOX1

GACTGGTTCCAATTGACAAGC

Confirm the insert target gene

3’AOX1

GCAAATGGCATTCTGACATCC

Confirm the insert target gene

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9k-EcNAGR

introducing a

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GCGAATTCCATCATCACCATCACCAC

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9k-EcNAGF

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2.5. Recombinant expression and purification of EcNAG in Pichia pastoris

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Based on the information of the nucleotide sequence encoding the mature peptide of EcNAG and multiple cloning sites (MCS) in the pPIC9K, a pair of primers 9k-EcNAGF/9k-EcNAGR was designed to construct the

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recombinant plasmid according to our previous research (Zhang et al., 2015). The plasmid containing the

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full-length EcNAG sequence was used as the template. The PCR product was digested with EcoR I and Not I at the same reaction volume. The fragment of the digested PCR product was ligated into the linearized vector pPIC9k,

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which was precut with EcoR I and Not I and transformed into the DH5α. The resulting construct pPIC9k-EcNAG

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was verified by sequencing. The recombinant plasmid pPIC9k-EcNAG was extracted and linearized with Sal I followed by transformation with Pichia pastoris host strain KM71 using PEG1000 method. Transformants were selected for their ability to grow on histidine-deficient minimal dextrose agar plates. In addition, isolation of genomic DNA was performed following the Invitrogen’s protocol and PCR amplifications were then carried out to select positive clones according to Invitrogen’s recommendations with a pair of primers (5’AOX1/3’AOX1) (Table 1). For each positive clone, small-scale expression trials were initially performed to identify the most productive

ACCEPTED MANUSCRIPT transformants and secretion of EcNAG was determined by SDS-PAGE using 12% (w/v) separating gel and 5% (w/v) stacking gel at 96 h after induction with methanol. Once the most productive transformant was selected, a large-scale expression of rEcNAG was performed and the cells were pelleted out from the culture medium by centrifugation at 8, 000 r/min for 10 min at 4 ºC. The supernatant was used to purify the recombinant EcNAG by

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affinity chromatography using Co-NTA-agarose resin (Sun et al., 2015).

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2.6 Biological assay for recombinant EcNAG activities

Enzyme assay: The enzyme activity of purified rEcNAG was monitored using 4-Nitrophenyl

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N-acetyl-β-D-glucosaminide (Sigma, N9376) substrates and the hydrolyzed 4-Nitrophenol in the alkali region was

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quantified by measuring the absorbance at 405 nm. The enzyme activity was expressed in μmol of product formed per minute. The reaction mixture contained 100 mM pH5.0 citrate buffer and 1 mM 4-Nitrophenyl

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N-acetyl-β-D-glucosaminide. After incubation for an appropriate time, 800 μl of 0.1 M borate buffer pH 9.2 was

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added to 200 μl of the reaction mixture and the concentration of the liberated 4-Nitrophenyl was determined (Ryslava et al., 2014).

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The optimum pH of purified rEcNAG was determined by varying the pH of reaction mixture from 2.2 to 9.0.

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The buffer used to generate pH ranges were 50 mM glycine-HCl buffer (pH 2.2-3.0), citrate buffer (pH 3.0-6.0), sodium phosphate buffer (pH 6.0-8.0), and sodium carbonate buffer (pH8.0- 9.0). The optimum temperature was measured by putting reaction mixtures in different temperatures (ranging from 20 to 60 °C) at the optimal pH condition. Several metal ions (Ca2+, Mg2+, Cu2+, Hg2+, Mn2+, Fe3+, K+, Cd2+, Co2+ and Zn2+) were used to identify their effect on enzyme activity. Each metal ion was added into reaction mixture with a final concentration at 10 mM, and then the enzyme activity was determined immediately following the method described above.

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3. Results 3.1. Amplification and characterization of EcNAG Based on the transcriptomic and genomic data of E. carinicauda (Yuan et al., 2017), the full-length cDNA sequence of EcNAG was obtained with 2293 bp (GenBank accession no. MF319599). As shown in Fig.1A, the

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complete nucleotide sequence of EcNAG contained an 1851 bp open reading frame (ORF) encoding EcNAG

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precursor of 616 amino acids with a predicted molecular weight (MW) about 69778.26 Da and theoretical

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isoelectric point (pI) of 4.82. The analysis with the SignalP 4.0 software revealed the presence of a signal peptide with 25 amino acids at the N-terminal of EcNAG precursor. The predicted MW and PI of the mature peptide was

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about 66952.87 Da and 4.81, respectively.

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The domain architecture prediction of EcNAG protein by SMART software online showed that there were two functional domains, one Glycohydro_20b2 domain (at the position of 68-195) and one Glyco_hydro_20 domain (at

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the position of 219-574) (Fig. 1B). To determine the presence of introns in EcNAG gene, the genomic DNA

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fragment of EcNAG with the corresponding cDNA sequence was obtained by PCR and the result showed that it was composed of five exons and four introns (Fig. 1C). All intron-exon boundaries were consistent with the consensus

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splicing junctions at both the 5’ splice donor site (GT) and the 3’ splice acceptor sites (AG) of each intron. The

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genomic organization of EcNAG is similar with that of HaztNAG from Hyalella azteca (Fig. 1C).

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(A)

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(C)

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(B)

Fig.1 Nucleotide and deduced amino acid sequences of EcNAG gene (A), the schematic representation of

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functional domain of EcNAG (B) and genomic structure of genomic structure of EcNAG from E. carinicauda and HaztNAG from H. azteca (C).

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Nucleotides are numbered on the both sides of the sequence. The letters in box represented the peptide sequence of deduced EcNAG; the letters marked with wavy underline and single underline represented the Glycohydro_20b and

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Glyco_hydro_20 domains respectively.

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A multiple sequence alignment of EcNAG with other shrimp EcNAG sequences is shown in Fig. 2. EcNAG is closely related to other shrimp NAG proteins and it has the highest homology to that of Macrobrachium nipponense (65%) at the amino acid level. The phylogenetic tree analysis showed that Arthropoda NAG could be divided into two groups, Malacostraca NAG and Insecta NAG. Decapoda NAG could furtherly form a subgroup and EcNAG was divided into this branch (Fig. 3).

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Fig.2 Alignment of the amino acid sequences of EcNAG with known Decapoda NAGs.

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The identical residues are shown in solid boxes. Sequences start at the first methionine residue. Macrobrachium nipponense (MnNAG, ANV82809.1) Litopenaeus vannamei (LvNAG, ACR23316.1); Fenneropenaeus chinensis

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(FcNAG, ABB86961.1); Exopalaemon carinicauda (EcNAG, MF319599, in this research).

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Fig.3 Phylogenetic tree of NAGs.

Agrotis ipsilon (AiNAG, GenBank accession no. ADF56765.1); Athalia rosae (ArNAG, XP_012269195.1);

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Camponotus floridanus (CfNAG, XP_011255300.1); Cerapachys biroi (CbNAG, XP_011347841.2); Cherax quadricarinatus (CqNAG, ALC79577.1); Dinoponera quadriceps (DqNAG, XP_014478370.1); Fenneropenaeus chinensis (FcNAG, ABB86961.1); Habropoda laboriosa (HlNAG, KOC64317.1); Harpegnathos saltator (HsNAG, XP_011141421.1); Hyalella azteca (HaNAG, XP_018025933.1); Lasius niger (LnNAG, KMQ97117.1); Linepithema humile (LhNAG, XP_012220111.1); Litopenaeus vannamei (LvNAG, ACR23316.1); Locusta migratoria (LmNAG, AFZ76982.1); Macrobrachium nipponense (MnNAG, ANV82809.1); Mamestra brassicae (MbNAG, AKR06190.1); Pogonomyrmex barbatus (PbNAG, XP_011635262.1); Polistes canadensis (PcNAG,

ACCEPTED MANUSCRIPT XP_014613786.1); AHJ81101.1);

Polistes

dominula

Pseudomyrmex

(PdNAG,

gracilis

(PgNAG,

XP_015189390.1);

Portunus

XP_020286223.1);

trituberculatus

Vollenhovia

emeryi

(PtNAG, (VeNAG,

XP_011862497.1); Exopalaemon carinicauda (EcNAG, MF319599, in this research). Values on the line are

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bootstrap values showing percentage confidence of relatedness.

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3.2. Expression profile of EcNAG

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Among all the tested tissues, EcNAG mainly expressed in hepatopancreas and epidermis, with a lower level in intestine and stomach, but poorly in other tissues (Fig. 4). EcNAG in intestine and stomach during different molting

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stages appeared to be expressed continuously throughout the molting cycle; mRNA expression reached the highest

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level at the molting stage (E) (Fig. 5). After molting (late postmolt, stage B), the level of EcNAG was remarkably decreased from the highest level observed at the molting stage (E). Then, a slightly decline of EcNAG expression

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was detected during the intermolt stage (C). The expression pattern of EcNAG throughout the C, D0, D1 and D2

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stages in epdimers was different with that in hepatopancreas. The expression of EcNAG in epidermis had no significant change throughout the C, D0, D1 and D2 stages (p > 0.05). However, the expression of EcNAG in

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hepatopancreas reached the low point at stage D0 and then began to increase significantly from stage D1 to stage E

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(p<0.05). At the stage E, the expression of EcNAG in hepatopancreas had no significant difference between the wild-type and EcChi4-deleted mutant prawns (p > 0.05) (Data not shown).

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Fig.4 Detection of EcNAG transcripts in the different tissues from healthy E. carinicauda Complementary DNAs from various tissues were amplified by real-time PCR. Tissues were shown in the abscissa.

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Each bar represented the mean + S.D. (n=3).

ACCEPTED MANUSCRIPT Fig. 5 Expression profiles of EcNAG at different molting stages. B, C, D0, D1, D2, D3, and E represent molting stages. Late postmolt (B), intermolt (C), onset of premolt (D0), early premolt (D1), intermediate premolt (D2), late premolt (D3) and molt (E) stages. 18S rRNA served as internal control. Statistical analyses were performed using one-way ANOVA. Different letters shown above the error bars

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indicate significant difference.

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3.3. Recombinant expression of EcNAG in P. pastoris

Expression cassette-positive clones were cultured first in BMGY medium and then induced with methanol to a

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final concentration of 0.5% in BMMY for a total induction time of 96 h. Among the proteins secreted into the

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medium and visualized by SDS-PAGE, there was a protein band specifically induced by methanol migrating

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approximately at the position on the gel expected for the predicted size of rEcNAG of 68314.32 Da (Fig.6).

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Fig.6 SDS-PAGE analysis of EcNAG recombinant expression in Pichia pastoris

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M: protein marker; T0: without inducer; T1: expression with inducer for 96 h. 3.4 Enzymatic characterization of rEcNAG

In the native conditions, HisTrap™ FF Crude (5 mL) column was used to purify the rEcNAG in the broth

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supernant and the purified rEcNAG was selected to study its characteristic, including the optimal pH and temperature, the effects of metal ions.

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The analysis results showed that the optimum pH and temperature of rEcNAG was pH5.0 and 40°C (Fig.7, 8). Ten kinds of metal ions were used to analyze their effect on enzyme activity of rEcNAG (Fig.9). Co2+ and Mn2+ had an obvious promoting effect upon enzyme activity of rEcNAG. Hg2+ had an inhibiting effect on enzyme activity of rEcNAG and residual enzyme activity decreased 50%.

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Fig.7 The optimum pH of the purified rEcNAG

Fig.8 The optimum temperature of the purified rEcNAG

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Fig.9 Effect of 10 mM metal ions on enzyme activity of the purified rEcNAG

3.5 Expression profile of EcNAG after challenged with bacteria

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The expression of EcNAG in hepatopancreas of prawns responding to bacteria challenge was measured through a semi-quantitative RT-PCR method with 18S rRNA gene as the internal control. The expression profiles

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of EcNAG in the Vibrio-challenged or Aeromonas-challenged samples are shown in Fig.10A, B. The expression of EcNAG had no significant change in the control samples (p > 0.05). After the prawns being challenged with Vibrio, the expression of EcNAG was up-regulated significantly at 6 h (p<0.05) and reached the peak at 12 h. And then its expression followed with a comeback step by step at 72 h (p > 0.05) (Fig.10A). The expression profile in Aeromonas-challenged group was the same as that in Vibrio-challenge group (Fig. 10B).

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(A)

(B) Fig.10 Expression profiles of EcNAG in the hepatopancreas after the prawns were challenged with Vibrio, Aeromonas or equal volume of PBS at 0, 6, 12, 24, 48, 72, and 96 h

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4. Discussion In this research, we cloned the full-length cDNA sequene of β-N-acetylglucosaminidase gene (EcNAG) from E. carinicauda. Just as other Decapoda NAGs, the deduced amino acid sequence of EcNAG had the typical characteristics of β-N-acetylglucosaminidase protein with a peptide sequence, a Glycohydro_20b domain, and a

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Glyco_hydro_20 domain. Among the different Decapoda NAGs, the Glycohydro_20b and Glyco_hydro_20

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domains are highly conserved. Glycohydro_20b2 represents the N-terminal domain of the eukaryotic

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β-N-acetylglucosaminidase and Glyco_hydro_20 represents the glycoside hydrolase family 20 catalytic domain. At present, an annotated reference transcriptome and genome for the amphipod Hyalella Azteca had been submitted in NCBI database by Baylor College of Medicine. The

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the

full-length cDNA sequence

encoding

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beta-N-acetylglucosaminidase of H. azteca (HaztNAG, GenBank accession no. XM_018170444.1) and one scaffold822 with 168, 387 bp in length (GenBank accession no. NW_017251162.1) was found in the NCBI

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database. Our result showed that the genomic organization of EcNAG was similar with that of HaztNAGa. The gene

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contains 5 exons and 4 introns.

The ridgetail white prawn, E. carincauda was used as an experimental animal which can be maintained with

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reproductive capacity all the year round in laboratory environment with an about 60-day reproduction cycle (Zhang

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et al., 2014; Zhang et al., 2015). In our previous research, we succeeded in realizing the genome editing in E. carinicauda via CRISPR/Cas9 system and knocking out a specific gene (EcChi4) in shrimp for the first time (Gui et al., 2016). As we known, EcChi4 was a chitinase gene. Generally, chitin degradation is catalyzed by a two-component chitinolytic enzyme system, chitinase and β-N-acetylglucosaminidase. In addition, it is reported that NAGase is mainly a molting enzyme that degrades chitin or chitinoligosaccharides in insects and cuticular chitin degradation is extremely important for insect growth and development (Hogenkamp et al., 2008; Xi et al., 2015). In our results, we found that EcNAG was highly expressed in the cuticle and its expression level was

ACCEPTED MANUSCRIPT significantly different during the molting process. In addition, NAGase may be involved in carbohydrate metabolism. Our results showed that the expression of EcNAG mRNA was highest in the hepatopancreas of the adult prawns. There are a lot of chitinlytic enzymes in the hepatopancreas and we had isolated two kinds of native chitinase (EcChi1 and EcChi2) from the hepatopancreas of E. carinicauda (Wang et al., 2015). By analyzing the

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expression difference of EcNAG at the molting stage E, there was no significant difference between the wild-type

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clue that there may be no relationship between EcChi4 and EcNAG.

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and EcChi4-deleted mutant prawns. In addition, the EcChi4 was a hepatopancreas specific gene which provided a

It is a common method to obtain the recombinant protein to clarify the function of the target gene in organism

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at the protein levels. In this research, EcNAG was recombinantly expressed in Pichia pastoris and the recombinant

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EcNAG was purified on the HisTrap™ FF Crude column at the native condition. The optimum temperature and pH were two important enzymatic characterization and they sometimes were used to compare the difference of the

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enzymes from different organisms. For the enzymatic characterizations of chitinlytic enzymes, it was reported that

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the native EcChi1 from the hepatopancreas of E. carinicauda had the optimum temperature and pH at 37 ºC and pH 4.0 (Wang et al., 2015). Similarly, the recombinant EcNAG had the optimum temperature and pH at 40 ºC and pH

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5.0, which was consistence with most chitinlytic enzymes from crustaceans. At present, a lot of NAGase from

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different organisms including bacteria, fungi, plants, insects and other animals, had been purified or recombinantly expressed to study their enzymatic characterizations (Calvo et al., 1978; Koga et al., 1991; Lovatt and Roberts, 1994; Sakai et al., 1994; Chang et al., 1998; Pierce et al., 2001; Jin et al., 2002; Taylor et al., 2002; Gomes Junior et al., 2010; Ryslava et al., 2014). It was reported that NAGase expression was regulated by the molting hormone in crustaceans and 20-hydroxyecdysone significantly increased the activity of NAGase in the epidermis of Uca pugilator (Zou and Bonvillain, 2004). Molt-inhibiting hormone (MIH) was also an important hormone to regulate the crustacean

ACCEPTED MANUSCRIPT molting (Chan et al., 2003). How about the relationship between the chitinlytic enzymes and MIH? At present, we are trying to knock out the MIH gene of E. carinicauda. Once the MIH-deleted mutant prawns being obtained, EcNAG as an important chitinlytic enzyme, will be selected to clarify the relationship between MIH and chitinlytic

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enzymes.

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Conflict of interest

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There is no conflict of interest.

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Acknowledgments

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The project was supported by The National Natural Science Foundation of China (Nos. 31172449, 41376165), The National High Technology Research and Development Program of China (No.

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2012AA10A401).

References

Calvo, P., Reglero, A. and Cabezas, J.A., 1978. Purification and properties of beta-N-acetylhexosaminidase from the

CE

mollusc Helicella ericetorum Muller. Biochem J 175, 743-750. Cesar, J.R.D., Zhao, B.Q., Malecha, S., Ako, H. and Yang, J.Z., 2006. Morphological and biochemical changes in the muscle of the marine shrimp Litopenaeus vannamei during the molt cycle. Aquaculture 261, 688-694.

AC

Chan, S.M., Gu, P.L., Chu, K.H. and Tobe, S.S., 2003. Crustacean neuropeptide genes of the CHH/MIH/GIH family: implications from molecular studies. Gen Comp Endocrinol 134, 214-219. Chang,

C.T.,

Young,

F.P.,

Chang,

M.H.

and

Sung,

H.Y.,

1998.

Purification

and

properties

of

beta-N-Acetylhexosaminidase from cabbage. Biochem Mol Biol Int 45, 371-380. Fernandes-Pedrosa, M.D., Junqueira-de-Azevedo, I.D.M., Goncalves-de-Andrade, R.M., Kobashi, L.S., Almeida, D.D., Ho, P.L. and Tambourgi, D.V., 2008. Transcriptome analysis of Loxosceles laeta (Araneae, Sicariidae) spider venomous gland using expressed sequence tags. BMC Genomics 9, 279-279. Gomes Junior, J.E., Souza, D.S., Nascimento, R.M., Lima, A.L., Melo, J.A., Rocha, T.L., Miller, R.N., Franco, O.L., Grossi-de-Sa, M.F. and Abreu, L.R., 2010. Purification and characterization of a liver-derived beta-N-Acetylhexosaminidase from marine mammal Sotalia fluviatilis. Protein J 29, 188-194. Gui, T., Zhang, J., Song, F., Sun, Y., Xie, S., Yu, K. and Xiang, J., 2016. CRISPR/Cas9-mediated genome editing and mutagenesis of EcChi4 in Exopalaemon carinicauda. G3-Genes Genom Genet 6, 3757-3764.

ACCEPTED MANUSCRIPT Hogenkamp, D.G., Arakane, Y., Kramer, K.J., Muthukrishnan, S. and Beeman, R.W., 2008. Characterization and expression of the beta-N-acetylhexosaminidase gene family of Tribolium castaneum. Insect Biochem Mol Biol 38, 478-489. Huang, Q., Yan, J., Tang, J., Tao, Y., Xie, X., Wang, Y., Wei, X., Yan, Q. and Chen, Q., 2010. Cloning and tissue expressions of seven chitinase family genes in Litopenaeus vannamei. Fish Shellfish Immun 29, 75-81. Jin, Y.L., Jo, Y.Y., Kim, K.Y., Shim, J.H., Kim, Y.W. and Park, R.D., 2002. Purification and characterization of beta-N-acetylhexosaminidase from rice seeds. J Biochem Mol Biol 35, 313-319. Koga, K., Iwamoto, Y., Sakamoto, H., Hatano, K., Sano, M. and Kato, I., 1991. Purification and characterization of beta-N-acetylhexosaminidase from Trichoderma harzianum. Agric Biol Chem 55, 2817-2823.

PT

Lehnert, S.A. and Johnson, S.E., 2002. Expression of hemocyanin and digestive enzyme messenger RNAs in the hepatopancreas of the black tiger shrimp Penaeus monodon. Comp Biochem Phys B 133, 163-171.

RI

Liu, T., Zhou, Y., Chen, L., Chen, W., Liu, L., Shen, X., Zhang, W., Zhang, J. and Yang, Q., 2012. Structural insights into cellulolytic and chitinolytic enzymes revealing crucial residues of insect beta-N-acetyl-D-hexosaminidase. Plos

SC

One 7, e52225.

Lovatt, A. and Roberts, I.S., 1994. Cloning and expression in Escherichia coli of the nahA gene from Porphyromonas gingivalis

indicates

that

beta-N-acetylhexosaminidase

an

outer-membrane-associated

lipoprotein.

NU

Microbiology 140, 3399-3406.

is

Lunt, M.R. and Kent, P.W., 1960. A chitinase system from Carcinus maenas. Biochim Biophys Acta 44, 371-373. Merzendorfer, H. and Zimoch, L., 2003. Chitin metabolism in insects: structure, function and regulation of chitin

MA

synthases and chitinases. J Exp Biol 206, 4393-4412.

Pierce, D.C., Butler, K.D. and Roer, R.D., 2001. Effects of exogenous N-acetylhexosaminidase on the structure and mineralization of the post-ecdysial exoskeleton of the blue crab, Callinectes sapidus. Comp Biochem Physiol B 128, 691-700.

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Proespraiwong, P., Tassanakajon, A. and Rimphanitchayakit, V., 2010. Chitinases from the black tiger shrimp Penaeus monodon: Phylogenetics, expression and activities. Comp Biochem Phys B 156, 86-96.

PT E

Ryslava, H., Valenta, R., Hyskova, V., Krizek, T., Liberda, J. and Coufal, P., 2014. Purification and enzymatic characterization of tobacco leaf beta-N-acetylhexosaminidase. Biochimie 107, 263-269. Saejung, C., Hatai, K. and Sanoamuang, L., 2014. The in-vitro antibacterial effects of organic salts, chemical disinfectants and antibiotics against pathogens of black disease in fairy shrimp of Thailand. J Fish Dis 37, 33-41.

CE

Sakai, K., Narihara, M., Kasama, Y., Wakayama, M. and Moriguchi, M., 1994. Purification and characterization of thermostable beta-N-acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost. Appl Environ Microbiol 60, 2911-2915.

AC

Salma, U., Uddowla, M.H., Kim, M., Kim, J.M., Kim, B.K., Baek, H.-J., Park, H., Mykles, D.L. and Kim, H.-W., 2012. Five hepatopancreatic and one epidermal chitinases from a pandalid shrimp (Pandalopsis japonica): Cloning and effects of eyestalk ablation on gene expression. Comp Biochem Physiol B 161, 197-207. Sun, Y., Zhang, J. and Wang, S., 2015. Heterologous expression and efficient secretion of chitosanase from Microbacterium sp in Escherichia coli. Indian J Microbiol 55, 194-199. Sun, Y., Zhang, J. and Xiang, J., 2017. A CRISPR/Cas9-mediated mutation in chitinase changes immune response to bacteria in Exopalaemon carinicauda. Fish Shellfish Immunol 71, 43-49. Tamura, K., Dudley, J., Nei, M. and Kumar, S., 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596-9. Tan, S.H., Degnan, B.M. and Lehnert, S.A., 2000. The Penaeus monodon chitinase 1 gene is differentially expressed in the hepatopancreas during the molt cycle. Mar Biotechnol 2, 126-135. Taylor, G., Jabaji-Hare, S., Charest, P.M. and Khan, W., 2002. Purification and characterization of an extracellular

ACCEPTED MANUSCRIPT exochitinase, beta-N-acetylhexosaminidase, from the fungal mycoparasite Stachybotrys elegans. Can J Microbiol 48, 311-9. Wang, J., Zhang, J., Song, F., Gui, T. and Xiang, J., 2015. Purification and characterization of chitinases from ridgetail white prawn Exopalaemon carinicauda. Molecules 20, 1955-1967. Watanabe, T. and Kono, M., 1997. Isolation of a cDNA encoding a chitinase family protein from cuticular tissues of the Kuruma prawn Penaeus japonicus. Zool Sci 14, 65-68. Watanabe, T., Kono, M., Aida, K. and Nagasawa, H., 1998. Purification and molecular cloning of a chitinase expressed in the hepatopancreas of the penaeid prawn Penaeus japonicus. BBA-Protein Struct M 1382, 181-185. Xi, Y., Pan, P. and Zhang, C., 2015. The beta-N-acetylhexosaminidase gene family in the brown planthopper, Nilaparvata

PT

lugens. Insect Mol Biol 24, 601-10.

Yang, C., Zhang, J., Li, F., Ma, H., Zhang, Q., Jose Priya, T.A., Zhang, X. and Xiang, J., 2008. A Toll receptor from

RI

Chinese shrimp Fenneropenaeus chinensis is responsive to Vibrio anguillarum infection. Fish Shellfish Immunol 24, 564-74.

SC

Younes, I., Hajji, S., Frachet, V., Rinaudo, M., Jellouli, K. and Nasri, M., 2014. Chitin extraction from shrimp shell using enzymatic treatment. Antitumor, antioxidant and antimicrobial activities of chitosan. Int J Biol Macromol 69, 489-98.

NU

Yuan, J., Gao, Y., Zhang, X., Wei, J., Liu, C., Li, F. and Xiang, J., 2017. Genome sequences of marine shrimp Exopalaemon carinicauda Holthuis provide insights into genome size evolution of Caridea. Mar Drugs 15. Zhang, J., Gui, T., Wang, J. and Xiang, J., 2015. The ferritin gene in ridgetail white prawn Exopalaemon carinicauda:

MA

Cloning, expression and function. Int J Biol Macromol 72, 320-325. Zhang, J., Sun, Y., Li, F., Huang, B. and Xiang, J., 2010. Molecular characterization and expression analysis of chitinase (Fcchi-3) from Chinese shrimp, Fenneropenaeus chinensis. Mol Biol Rep 37, 1913-21. Zhang, J., Wang, J., Gui, T., Sun, Z. and Xiang, J., 2014. A copper-induced metallothionein gene from Exopalaemon

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carinicauda and its response to heavy metal ions. Int J Biol Macromol 70, 246-50. Zhou, K., Zhou, F., Huang, J., Yang, Q., Jiang, S., Qiu, L., Yang, L., Zhu, C. and Jiang, S., 2017. Characterization and

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expression analysis of a chitinase gene (PmChi-4) from black tiger shrimp (Penaeus monodon) under pathogen infection and ambient ammonia nitrogen stress. Fish Shellfish Immunol 62, 31-40. Zou, E.M. and Bonvillain, R., 2004. Chitinase activity in the epidermis of the fiddler crab, Uca pugilator, as an in vivo

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CE

screen for molt-interfering xenobiotics. Comp Biochem Phys C 139, 225-230.

ACCEPTED MANUSCRIPT Highlights

 It is the first time to clarify the function of β-N-acetylglucosaminidase (NAGase) in ridgetail white prawn Exopalaemon carinicauda.

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 qRT-PCR was performed to study the EcNAG expression pattern in different tissues and different

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molting stages.

parahaemolyticus and Aeromonas hydrophila,

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 EcNAG expression pattern was clarified after the prawns were challenged with Vibrio

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recombinant EcNAG was characterized.

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 The EcNAG was recombinant expressed in Pichia pastoris and the enzymatic characterization of

ACCEPTED MANUSCRIPT Abbreviation list

E. carinicauda: Exopalaemon carinicauda NAG: β-N-acetylglucosaminidase

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EcNAG: Exopalaemon carinicauda β-N-acetylglucosaminidase gene ORF: open reading frame

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V. parahaemolyticus: Vibrio parahaemolyticus

EcChi4: Exopalaemon carinicauda chitinase 4 gene

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A. hydrophila: Aeromonas hydrophila

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HaztNAG: Hyalella azteca β-N-acetylglucosaminidase gene