Identification of two isoforms of Pop in the domestic silkworm, Bombyx mori: Cloning, characterization and expression analysis

Gene 667 (2018) 101–111

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Gene journal homepage: www.elsevier.com/locate/gene

Research paper

Identification of two isoforms of Pop in the domestic silkworm, Bombyx mori: Cloning, characterization and expression analysis Ping Fua,b, Wei Suna, Juan Laia, Yi-Hong Shenc, Ze Zhanga,

T

⁎

a

School of Life Sciences, Chongqing University, Chongqing, China Postdoctoral Station of Biomedical Engineering, Chongqing University, Chongqing, China c State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Prolyl oligopeptidase Alternative splicing Enzyme activity Angiotensin Ecdysis

Two isoforms, Bmpop-a and Bmpop-b, were cloned and characterized, which were found to encode prolyl oligopeptidase (Pop) of the domestic silkworm Bombyx mori. The full lengths of Bmpop-a and Bmpop-b were 2497 and 2508 bp, deducing 707 and 740 amino acids, respectively. Both of them, possessing the typical characteristics of the Pop family of serine proteinase, were detected to be expressed among different tissues and development stages at the transcription and translation levels. Soluble recombinant BmPop-a (rBmPop-a) had oligopeptidase activity toward the substrates, Z-Gly-Pro-pNA, Z-Gly-Pro-AMC and angiotensin I. An inhibition assay showed that the activity of rBmPop-a was significantly inhibited by KYP-2047 and S17092 in vitro. BmPop-b was identified in the molting fluids at three different stages by Western blotting analysis, showing a predominant expression in the integument. Two isoforms of Bmpop gene and other three genes in the renin-angiotensin system (RAS) in the integument were down-regulated by starvation treatments but up-regulated by refeeding. These results suggested that BmPops may play an important role in balancing the molting fluid pressure to guarantee ecdysis normally. This study provides clues for further elucidating the function and regulation mechanisms of two isoforms of Bmpop gene.

1. Introduction

distributed, and is involved in various physiological events such as learning and memory (Brandt et al., 2008; Mantle et al., 1996; Savolainen et al., 2015), cell signaling (Duan et al., 2014; MorenoBaylach et al., 2011), sperm motility (Dotolo et al., 2015; Kimura et al., 2002), and cell proliferation and differentiation (Moreno-Baylach et al., 2008; Sakaguchi et al., 2011). Generally, this enzyme has been considered as a cytosolic enzyme, but a membrane-bound form has been characterized (O'Leary et al., 1996; Tenorio-Laranga et al., 2008). In fact, Pop can be released from the cells into the follicular fluid of porcine ovary (Takahashi et al., 1996), even though it lacks a secretion signal (Venäläinen et al., 2004). Previous studies suggested that the extracellular and intracellular Pops in the ovary of porcine were the different products of a single gene, which supported by that Pops isolated separately from the fluid and tissue fractions of porcine ovaries showed no significant difference in properties (Kimura et al., 1998;

Prolyl oligopeptidase (Pop; EC 3.4.21.26; also known as prolyl endopeptidase and post-proline cleaving enzyme) is a member of serine protease (family S9 of clan SC), which is conserved through the longterm evolution (Rawlings and Barrett, 1994; Venäläinen et al., 2004). This enzyme preferentially cleaves short proline residue-containing peptides (Myöhänen et al., 2009). Its structure includes a β-propeller domain, which excludes access of large peptides or proteins to the catalytic site (Fülöp et al., 1998; Fuxreiter et al., 2005). Previous study indicated that this enzyme was involved in the maturation and degradation of peptide hormones or neuropeptides (Mentlein, 1988). Besides oligopeptidase activity, this enzyme also possesses non-hydrolytic function, such as protein-protein interaction (Di Daniel et al., 2009; Savolainen et al., 2015). In mammals, Pop is ubiquitously

List of abbreviations: Aph, acylpeptide hydrolase; AA, alternative acceptor site; AD, alternative donor site; AS, alternative splicing; ANCE, angiotensin-converting enzyme; Ang I, angiotensin I; B. mori, Bombyx mori; BSA, bovin serum albumin; DTT, dithiothreitol; E. coli, Escherichia coli; EDTA, ethylenediaminetetraacetic acid; ES, exon skipping; HPLC, high performance liquid chromatography; IR, intron retention; pI, isoelectric point; IPTG, isopropyl β-D-thiogalactoside; LC-MS/MS, liquid chromatography tandem-mass spectrometry; LINE1, long interspersed element-1; NJ, neighbor-joining; ORF, open reading frame; PMSF, phenylmethylsulphonyl fluoride; PO, phenoloxidase; PBS, phosphate buffered saline; PVDF, polyvinylidene fluoride; Pop, prolyl oligopeptidase; RACE, rapid-amplification of cDNA ends; RAS, renin-angiotensin system; RT-PCR, reverse transcription-polymerase chain reaction; rpl3, ribosomal protein L3; NaN3, sodium azide; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; UTR, untranslated region ⁎ Corresponding author at: Laboratory of Evolutionary and Functional Genomics, School of Life Sciences, Chongqing University, No.55 Daxuecheng South Rd., Shapingba, Science Building, Chongqing University Huxi Campus, Chongqing 401331, China. E-mail address: [email protected] (Z. Zhang). https://doi.org/10.1016/j.gene.2018.05.021 Received 26 February 2018; Received in revised form 2 May 2018; Accepted 7 May 2018 Available online 09 May 2018 0378-1119/ © 2018 Elsevier B.V. All rights reserved.

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Takahashi et al., 1996). Alternative splicing (AS) is an important molecular process of generating multiple transcripts from a single gene. AS events are classified into four basic types depending on the regions affected: the intron retention (IR) events, the exon skipping (ES) events, the alternative donor site (AD) events, and the alternative acceptor site (AA) events (Breitbart et al., 1987). These events play a key regulatory role in modulating the transcriptome and proteome diversity (Blencowe, 2006; Graveley, 2001; Marquez et al., 2012; Nilsen and Graveley, 2010). Besides, AS affects different isoforms toward tissue-specific expression (Lazaridis et al., 2000; Mullen et al., 1999; Tabuchi and Südhof, 2002; Yeo et al., 2004), stage-specific expression (Mullen et al., 1999), and subcellular localization (Laity et al., 2000; Tong et al., 2003). Several studies have investigated the Pop in insects. Pop was shown to involve in differentiation of imaginal discs and DNA synthesis in Sarcophaga peregrina (flesh fly) (Ohtsuki et al., 1994; Ohtsuki et al., 1997; Ohtuski et al., 1997). Also in a proteomic research, prolyl oligopeptidase was found in the molting fluids of the domestic silkworm Bombyx mori (Zhang et al., 2014). Molting fluids fill the space between the old and new cuticles during periodical ecdysis in Ecdysozoa. Insects secrete molting fluid, which is a mixture containing various proteins for protection and regulation of ecdysis. Approximately 30% of these identified proteins are enzymes, and these enzymes are also related to the renin-angiotensin system (RAS) in molting fluids (Zhang et al., 2014). In mammals, the RAS is a major regulator of blood pressure, fluid, and electrolyte homeostasis (Pahlavani et al., 2017). In the RAS, active renin cleaves angiotensinogen into angiotensin I (Ang I), which is then cleaved into angiotensin II (Ang II) by the angiotensin-converting enzyme (ANCE); Pop is capable of hydrolyzing Ang I or Ang II into angiotensin-(1-7) (Ang-(1-7)) (Kehoe et al., 2016; Pereira et al., 2009). Ang II, a vasopressor hormone, has a critical role in the maintenance of the normal blood pressure (Murata et al., 2015). Insects may use the components of the RAS to balance the molting fluid pressure, which deserves further study. The aim of this study is to characterize the putative functions of Bmpop gene and to discuss the possible role of BmPops in the RAS of silkworm.

were carried out with the outer primers provided in the kit, combined with the gene-specific primers AS1 and S1 for the 5′RACE and 3′RACE, respectively. The temperature program was used: 94 °C for 4 min followed by 35 cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 2 min with a final extension of 10 min at 72 °C. Additional steps were performed with an aliquot of the initial 5′RACE and 3′RACE PCR samples as templates and the inner primers provided in the kit, combined with the genespecific primers AS2 and S2 for the 5′ and 3′RACE, respectively. The temperature program was used: 94 °C for 4 min followed by 35 cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 2 min with a final extension of 10 min at 72 °C. All the amplified products were purified with Gel Extraction Kit (OMEGA, USA), and then cloned into the pEASY-T1 Simple Cloning Vector (Transgen Biotech, China). Orientation and presence of the inserted cDNA were confirmed by sequencing of the recombinant plasmid with an automated DNA Analyzer (Applied Biosystems 3730xl, Shanghai, China).

2. Materials and methods

The open reading frame (ORF) of Bmpop-a isoform was amplified using Phusion® High-Fidelity DNA Polymerases (New England Biolabs) from silkworm larvae cDNA using specific primers (Supplementary material: Table S1), and cloned into the pET-28(a) vector using NdeI and XhoI. The recombinant plasmids were verified by DNA sequencing and transformed into E. coli BL21 (Transetta, DE3) cells. The recombinant protein was induced by isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.2 mM in Luria-Bertani (LB) medium at 16 °C for 16 h. The cells were collected and disrupted by sonication. The supernatant was clarified by centrifugation and subjected to nickel affinity chromatography with ProteinIso® Ni-NTA Resin (TransGen Biotech, China). The bound recombinant BmPop-a (rBmPop-a) was eluted using a linear gradient of 0.01–0.25 M imidazole. The eluate containing rBmPop-a was buffer-exchanged into PBS, pH 7.6, and then concentrated with an Amicon Ultracel-15k ultrafiltration device (Millipore). Cell extracts of E. coli and the purified protein were analyzed by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and followed by staining with Coomassie Blue G250. The Coomassie stained band was excised from the gel and in-gel digestion was performed with trypsin. The peptide mixture was used for Maldi-TOF-TOF analysis. MASCOT (MatrixScience, UK) was used for database searching against NCBI nr database.

2.3. Phylogenetic analysis In order to construct the phylogenetic tree for pop genes, the putative protein sequence of the candidate gene was used as the query sequence to search for the homologous sequences of other species in the National Center for Biotechnology Information database (http://www. ncbi.nlm.nih.gov/). Previous study showed that Pop protein belongs to the prolyl oligopeptidase family of serine proteases. Thus, several proteins which had been reported to be Pop and other putative Pops were downloaded from NCBI. All the sequences were aligned by the MUSCLE 3.8.31 (Edgar, 2004). Neighbor-joining (NJ) tree was reconstructed by the program MEGA 6.0 (Tamura et al., 2013). Poisson model and pairwise deletion of gaps were selected. A total of 2000 bootstrap replications were performed. Bootstrap values (2000 replications) > 50% are indicated on the nodes of the NJ tree. Mammal acylpeptide hydrolase (Aph) sequences were used as the outgroup in this analysis, as this enzyme is another member of the Pop family. 2.4. Expression and purification of recombinant BmPop-a

2.1. Biological materials The silkworm strain DaZao (p50) was used in this study. The larvae were reared on fresh mulberry leaves at 25 °C with 75% ± 5% relative humidity and a photoperiod of 12 h light: 12 h dark. Heads, midgut, silk gland, fat body, integument, Malpighian tubule, gonads and hemocyte from the 3rd day of the 5th instar larvae were collected. More than four larvae, pupae, moths and eggs were collected at different development stages. These integuments from different treatment groups, which described in Section 2.8, were collected. All samples were immediately frozen using liquid nitrogen and stored at −80 °C. 2.2. cDNA cloning and sequencing of Bmpop gene Total RNA was extracted from silkworm larvae using TransZol Up, a commercial RNA extraction kit according to the manufacturer's instruction (TransGen Biotech, China). The purified RNA was quantified by NanoVue Plus spectrophotometer. First strand cDNA was synthesized using the FirstChoice RLM-RACE Kit according to the manufacturer's instruction (ThermoFisher Scientific, USA). Based on the bioinformatics analysis, Bmpop-specific primers were designed (Supplementary material: Table S1) and used to amplify the cDNA sequence of the putative Bmpop gene. The first PCR using the sense (S) and antisense (AS) gene-specific primers was performed as follows: 94 °C for 4 min followed by 32 cycles at 94 °C for 30 s, 58 °C for 30 s, 72 °C for 2 min with a final extension of 10 min at 72 °C. The 5′ rapid amplification of cDNA ends (RACE) and 3′RACE PCR procedures

2.5. Prolyl oligopeptidase activity assays Prolyl oligopeptidase activity assays were performed on rBmpop-a using an adapted method (Brandt et al., 2005). The reaction mixture (200 μl), containing the appropriate amount of enzyme in 100 mM potassium phosphate (pH 7.5), 1 mM DTT, 1 mM EDTA, 1 mM NaN3, 102

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instructions (TransGen Biotech, China). A CFX96™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and SYBR Green qRTPCR Mix (Bio-Rad) were used for qRT-PCR according to the manufacturer's instruction. The isoforms-specific primers of Bmpop gene were designed (Supplementary material: Table S1). B. mori ribosomal protein L3 (Bmrpl3) was used as an internal control for normalization of sample loading. qRT-PCR was performed under the following conditions: initial denaturation at 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s, 58 °C for 30 s. Relative expression levels of different isoforms of Bmpop gene were calculated based on the 2−ΔCt method. All experiments were carried out in triplicate, and all of the data were presented as mean ± standard deviation (SD) with n = 3. Student's t-test was used to evaluate statistical significance (*P < 0.05, **P < 0.01 and ***P < 0.001).

was pre-incubated under the assay condition for 30 min. Then, different concentrations of substrate, Z-Gly-Pro-pNA (Bachem, catalog number L1235), were added and the release of product was monitored at 405 nm with a Synergy™ HTX Multi-Mode Microplate Reader (Biotek). Data were fitted to the Michaelis-Menten equation by nonlinear regression (curve fit) with GraphPad Prism. Calculated activities were based on the initial linear phase of release. One unit of rBmPop-a activity (U) was defined as the amount of enzyme releasing 1 μM of substrate per minute under the assay conditions. A second assay involved incubating 0, 0.125, 0.25, 0.5, 1.0, 2.0 μg/ml of rBmpop-a with the volume made up to a total of 200 μl with 100 mM potassium phosphate (pH 7.5), 1 mM DTT, 1 mM EDTA, 1 mM NaN3. Bovine serum albumin (BSA) pre-incubation occurred for 30 min at 25 °C prior to the addition of 50 μM ZGly-Pro-AMC (Bachem, catalog number I-1145) and subsequent incubation at 25 °C for a further 30 min. The fluorescence intensity was measured at excitation and emission wave-lengths of 360 nm and 460 nm, respectively, with a Synergy™ HTX Multi-Mode Microplate Reader (Biotek). Stock solutions of substrates were prepared in DMSO and the final assay concentration of DMSO was 1.0%. All experiments were carried out in triplicate in 96-well microplates (Corning).

2.10. Molting fluid collection The molting fluids produced during larval-larval (fourth larval molting stage, L-L), larval-pupal (L-P) and pupal-adult (P-A) were collected as previously described (Qu et al., 2014; Zhang et al., 2014). The same volume of the molting fluids at three different stages was heated at 100 °C for 10 min with 2 × SDS buffer and the supernatant (centrifuged at 10,000g at room temperature for 5 min) was used for Western blotting analysis. Melanization was induced as previously described (Han et al., 2017), the supernatant and melanized debris were used for Western blotting analysis.

2.6. Temperature and pH influence on rBmPop-a activity Determination of temperature and pH optima of rBmPop-a were performed with Z-Gly-Pro-AMC as the substrate. Optimum temperature for the purified enzyme was determined under the assay conditions described above in the temperature range 20–50 °C. Effect of pH was determined between pH 5.5 and pH 10.0 under the standard assay conditions with optimum temperature. For pH between 5.5 and 9.0, different proportion of Na2HPO4 and NaH2PO4 buffer (50 mM) were selected. Na2HPO4 buffer with appropriate NaOH was used for pH 10.0.

2.11. Western blotting analysis Polyclonal antibody against BmPop-a were produced by immunizing female mice with the purified rBmPop-a protein as previously described (Fu et al., 2016). Samples were homogenized in RIPA Lysis Buffer (Beyotime Biotechnology, China) with a final concentration of 1 mM phenylmethylsulphonyl fluoride (PMSF). The supernatant was collected by centrifugation at 10,000g at 4 °C for 5 min. The protein concentration was determined by using Easy II Protein Quantitative Kit (BCA) following the manufacturer's instructions (TransGen Biotech, China). Total protein (50 μg) was separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare Life Sciences, Germany). The membranes were blocked with 5% skim milk at 37 °C for 1 h and then incubated with the polyclonal antibody against BmPop-a (diluted 1:8000) at 37 °C for 1 h. Membranes were washed three times and then incubated with horseradish peroxidase-labeled goat anti-mouse immunoglobulin G (IgG) antibody (diluted 1:10,000; Beyotime Biotechnology, China) at 37 °C for 1 h. Membranes were again washed three times, and then visualized using Western blotting detection kit (Advansta, USA).

2.7. Effect of inhibitors on rBmPop-a activity Two mammal's Pop inhibitors (KYP-2047 and S17092) were selected to determine the influence on rBmPop-a activity. The appropriate amount of enzyme was pre-incubated with inhibitors (from 0.5 to 300 nM) in the assay buffer at 25 °C for 30 min. Whereafter, Z-Gly-ProAMC was added to a final concentration of 50 μM. After 30 min, the incubation samples were directly assayed for residual activity (compared with that of the control). The concentration of compounds giving 50% inhibition (IC50) was calculated from plot of residual activity versus inhibitors concentrations. 2.8. Expression of two isoforms of Bmpop gene after starvation and refeeding To test the influence of starvation and refeeding on the expression of two isoforms of Bmpop gene, newly molted fifth instar larvae were divided into three groups. Feeding group: larvae were collected at 6, 12, 24, 48 and 72 h after being fed fresh mulberry leaves. Starvation group: larvae were collected at 6, 12, 24, 48 and 72 h post-starvation without mulberry leaves. Refeeding group: larvae that had been starved for 12, 24, 36 h were collected after being fed again for 12, 24, and 36 h, respectively. The larvae integument in each group was collected for quantitative RT-PCR (qRT-PCR) and Western blotting analysis. Meanwhile, the expression of the related genes of the RAS had been investigated after starvation and refeeding.

2.12. Determination of the cleavage site of angiotensin I The study of the hydrolysis of angiotensin I by rBmPop-a was based on the separation of the products of hydrolysis by high performance liquid chromatography (HPLC) on a HT-230A column. The appropriate amount of the purified rBmPop-a was pre-incubated in 50 mM of MES (pH 7.5) at 25 °C for 15 min. Then angiotensin I was added to a final concentration of 40 μM. After 2 h, the reactions were quenched by adding eluent A (0.065% trifluoroacetic acid in water) to the aliquot (1:1). The mixtures were immediately subjected to HPLC on a HT-230A column. The elution solvent system was composed of elution A and eluent B (0.05% trifluoroacetic acid in acetonitrile). The elution conditions were as follows: 75% elution A and 25% elution B; flow rate: 1 ml/min; detection wavelength: 220 nm; and column temperature: room temperature.

2.9. Real-time qPCR analysis Total RNA was extracted from silkworm DaZao strain at different developmental stages and different tissues of the 3rd day of the 5th instar larvae as described above, and 1 μg RNA was reverse-transcribed into complementary DNA (cDNA) using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix following the manufacturer's 103

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A 0.5 M

Nscaf 2529

+

1F

2F

1R

2R

Gap 1

_

Gap 2

BGIBMGA002592

BGIBMGA002593

Bmpop-a . Bmpop-b 5'

3' 0kb

Genome

5kb

Gap

10kb

CDS

UTR

15kb

20kb

25kb

30kb

35kb

Intron

B

(Bmpop-b)

(Bmpop-a) qPCR primer-F

qPCR primer-F

ORF primer-F

qPCR primer-R

ORF primer-F

qPCR primer-R

5’RACE nested primer

5’RACE nested primer

5’RACE

5’RACE

primer

primer

3’RACE primer

3’RACE primer

ORF primer-R

ORF primer-R

3’RACE nested primer

3’RACE nested primer

Fig. 1. Profile of the Bombyx mori prolyl oligopeptidase (Bmpop) gene. (A) Structures of the Bmpop gene alternative splicing isoforms. The visualization is carried out by gene structure display server 2.0 (Hu et al., 2015). (B) Nucleotide and deduced amino acid sequences of silkworm Pop two isoforms, BmPop-a and BmPop-b. The deduced amino acid sequence are shown below the nucleotide sequence, the amino acid resides are numbered starting from the first methionine residue, the nucleotides are numbered starting from the first letter of the Met codon, and the asterisk shows the termination codon. The putative polyadenylation signal is boxed. Black triangles represent the intron positions. The positions and directions of primers are indicated as arrows above the nucleotide sequence.

3. Results and discussion

predicted the Bmpop gene in the scaffold 2529 on the 5th chromosome of silkworm using softberry (www.softberry.com) by cloning and sequencing the gap2 and found that these two ORFs should be merged as one gene, that is, the putative Bmpop gene. The full-length cDNA cloning also confirmed this prediction based on the specific primers (Supplementary material: Table S1). Furthermore, the 5′RACE indicated that this gene has two isoforms, named Bmpop-a and Bmpop-b, with different initiation sites both for transcription and translation (Fig. 1A). In previous version of assembly of the domestic silkworm genome, there is another gap (Gap1) between exon1 and exon2 of Bmpop-a

3.1. Cloning and sequence analysis The protein sequence of Pop in Sarcophaga peregrina (SpPop) (Ohtsuki et al., 1997) was used as a query to do Blastp against the silkworm genome database (Duan et al., 2010). Two significant hits (BGIBMGA002592 and BGIBMGA002593) were obtained. These two predicted ORFs were distributed adjacently to each other in the same scaffold (scaffold 2529) on the 5th chromosome of silkworm (Fig. 1A) and there is a gap (Gap2, in Fig. 1A) between these two ORFs. We re104

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BmPop-a BmPop-b SpPop MmPop HsPop SsPop

: : : : : :

---------------------------------MLYDYPAVRRDETVVDDYHGTKIKDPYRWLEDPDSNETKEFVEAQNKITRPYLDACPVQKSINERLTELWNYPKYSCPFRKGSRYFF MGLVFAMRACIICVLIACAFLQCTGTTILSSRNMLYDYPAVRRDETVVDDYHGTKIKDPYRWLEDPDSNETKEFVEAQNKITRPYLDACPVQKSINERLTELWNYPKYSCPFRKGSRYFF --------------------------MTEETKNVQINYPTPRKDESYVDNFHGVEIKDVYRWLEDPDSEETKKYVDLQNEISQPFLENCEAWKKINEKLSKLWNYEKYGCPVRHGNFYYF --------------------------------MLSFQYPDVYRDETSVQEYHGHKICDPYSWLEDPDSEQTKAFVEAQNKITVPFLEQCPIRGLYKERMTELYDYPKYSCHFKKGKRYFY --------------------------------MLSLQYPDVYRDETAVQDYHGHKICDPYAWLEDPDSEQTKAFVEAQNKITVPFLEQCPIRGLYKERMTELYDYPKYSCHFKKGKRYFY --------------------------------MLSFQYPDVYRDETAIQDYHGHKVCDPYAWLEDPDSEQTKAFVEAQNKITVPFLEQCPIRGLYKERMTELYDYPKYSCHFKKGKRYFY

: 87 : 120 : 94 : 88 : 88 : 88

BmPop-a BmPop-b SpPop MmPop HsPop SsPop

: : : : : :

FKNTGLQNQNALYVQDGLDGEPRVFLDPNTLSEDGTVALSGTKFTEDGSTLAYGLSASGSDWIAIHFKDVATGEDYPEVLEKVKFASMSWTKDNKGLFYSMYPKQTGKTDGSETEVNHDQ FKNTGLQNQNALYVQDGLDGEPRVFLDPNTLSEDGTVALSGTKFTEDGSTLAYGLSASGSDWIAIHFKDVATGEDYPEVLEKVKFASMSWTKDNKGLFYSMYPKQTGKTDGSETEVNHDQ YKNTGLQNQSVLYQQDTLEWRARLFFDPNALSTDGTIALAQKSFSDDGKYMAYGLSESGSDWVKILVRDVDSGKDLDEVLEKVKFSEISWTKDNKGFFYVRYPAQEGKTDGSETKTNEFQ FYNTGLQNQRVLYVQDSLEGEARVFLDPNTLSDDGTVALRGYAFSEDGEYFAYGLSASGSDWVTIKFMKVDGAKELPDVLERVKFTCMAWTHDGKGMFYNSYPQQDGKSDGTETSTNLHQ FYNTGLQNQRVLYVQDSLEGEARVFLDPNILSDDGTVALRGYAFSEDGEYFAYGLSASGSDWVTIKFMKVDGAKELPDVLERVKFSCMAWTHDGKGMFYNSYPQQDGKSDGTETSTNLHQ FYNTGLQNQRVLYVQDSLEGEARVFLDPNILSDDGTVALRGYAFSEDGEYFAYGLSASGSDWVTIKFMKVDGAKELPDVLERVKFSCMAWTHDGKGMFYNAYPQQDGKSDGTETSTNLHQ

: : : : : :

207 240 214 208 208 208

BmPop-a BmPop-b SpPop MmPop HsPop SsPop

: : : : : :

KLCYHRINTSQSEDVVVVEFPEEPLWRIVADVSDCGRYLLVYPVRDSRD-NLLFFADLSKHPE-INGKLPLMPVVEEFEADYEYVTNEDSVCIIRTNKNAPNYRLIMIDLQNPAKENWRT KLCYHRINTSQSEDVVVVEFPEEPLWRIVADVSDCGRYLLVYPVRDSRD-NLLFFADLSKHPE-INGKLPLMPVVEEFEADYEYVTNEDSVCIIRTNKNAPNYRLIMIDLQNPAKENWRT KLYYHYVGQPQEKDMLMVEFPEEPTWRIESVVSDCGKYLIMAIVKDCRD-NIVYYANLEEAGE-ITGKLKVHKIVEKFESDYQYITNIGSRVFFRTNKNAPNYRIIGIDFENHAEENWET KLCYHVLGTDQSEDILCAEFPDEPKWMGGAELSDDGRYVLLSIWEGCDPVNRLWYCDLQQEPNGITGILKWVKLIDNFEGEYDYVTNEGTVFTFKTNRNSPNYRLINIDFTDPDESKWKV KLYYHVLGTDQSEDILCAEFPDEPKWMGGAELSDDGRYVLLSIREGCDPVNRLWYCDLQQESSGIAGILKWVKLIDNFEGEYDYVTNEGTVFTFKTNRQSPNYRVINIDFRDPEESKWKV KLYYHVLGTDQSEDILCAEFPDEPKWMGGAELSDDGRYVLLSIREGCDPVNRLWYCDLQQESNGITGILKWVKLIDNFEGEYDYVTNEGTVFTFKTNRHSPNYRLINIDFTDPEESKWKV

: : : : : :

325 358 332 328 328 328

BmPop-a BmPop-b SpPop MmPop HsPop SsPop

: : : : : :

LVPEHPTDVLDWAAAVDQDKLVIHYIRDVKSVLQLHDLKTGEFIQTFPLDVGSVVGFTGKKNQTEIFYHFMSLLTPGVIYHVDFKQKPYTPKVFREV--TVKGFDASQYEAKQIFYSSKD LVPEHPTDVLDWAAAVDQDKLVIHYIRDVKSVLQLHDLKTGEFIQTFPLDVGSVVGFTGKKNQTEIFYHFMSFLTPGVIYHVDFKQKPYTPKVFREV--TVKGFDASQYEAKQIFYSSKD LVAEHETDVLDWANCVDQDKLVLCYIQDVKSALQVNSLKDGKFISKFDLDIGTVVSFSGEKKYSEIFYNFSSFLNPGTIYHYDFKWNDFKPKVLREIKLNLENFSAAKYKVEQKFYNSKD LVPEHEKDVLEWVACVRSNFLVLCYLHDVKNILQLHDLTTGALLKTFPLDVGSVVGYSGRKKDSEIFYQFTSFLSPGVIYHCDLTKEELEPMVFREV--TVKGIDAADYQTIQIFYPSKD LVPEHEKDVLEWIACVRSNFLVLCYLHDVKNILQLHDLTTGALLKTFPLDVGSIVGYSGQKKDTEIFYQFTSFLSPGIIYHCDLTKEELEPRVFREV--TVKGIDASDYQTVQIFYPSKD LVPEHEKDVLEWVACVRSNFLVLCYLHDVKNTLQLHDLATGALLKIFPLEVGSVVGYSGQKKDTEIFYQFTSFLSPGIIYHCDLTKEELEPRVFREV--TVKGIDASDYQTVQIFYPSKD

: : : : : :

443 476 452 446 446 446

BmPop-a BmPop-b SpPop MmPop HsPop SsPop

: : : : : :

GTKVPMFIISKKDLPRDGSNPVLLYGYGGFNINVQPGFSVTRLVFMQHMNGIVAIPNIRGGGEYGERWHNAGRLLNKQNVFDDFQAAAEYMVSERYTRPALLTIQGGSNGGLLVAACINQ GTKVPMFIISKKDLPRDGSNPVLLYGYGGFNINVQPGFSVTRLVFMQHMNGIVAIPNIRGGGEYGERWHNAGRLLNKQNVFDDFQAAAEYMVSERYTRPALLTIQGGSNGGLLVAACINQ GTKVPMFIIYKNTDAIK-PRPCLLYGYGGFNISVQPAFSISGLLFIDSFDGILAYPNLRGGGEYGEKWHNAGRLLNKQNVFDDFQAAAEYLVTNNYTTKDRLVIQGGSNGGLLVGACINQ GTKIPMFIVHKKGIKLDGSHPAFLYGYGGFNISITPNYSVSRLIFVRHMGGVLAVANIRGGGEYGETWHKGGILANKQNCFDDFQCAAEYLIKEGYTSPKRLTINGGSNGGLLVAACANQ GTKIPMFIVHKKGIKLDGSHPAFLYGYGGFNISITPNYSVSRLIFVRHMGGILAVANIRGGGEYGETWHKGGILANKQNCFDDFQCAAEYLIKEGYTSPKRLTINGGSNGGLLVAACANQ GTKIPMFIVHKKGIKLDGSHPAFLYGYGGFNISITPNYSVSRLIFVRHMGGVLAVANIRGGGEYGETWHKGGILANKQNCFDDFQCAAEYLIKEGYTSPKRLTINGGSNGGLLVATCANQ

: : : : : :

563 596 571 566 566 566

BmPop-a BmPop-b SpPop MmPop HsPop SsPop

: : : : : :

RPDLYGAAVVQVGVLDMLRFQKFTIGHAWVSDYGSSDNKTQFEYLLKYSPLHNIQPPSENRPEYPATLILSADHDDRVVPLHSLKFVAELQHVAGRSPAQRAPLLARFDTKAGHGGGKPT RPDLYGAAVVQVGVLDMLRFQKFTIGHAWVSDYGSSDNKTQFEYLLKYSPLHNIQPPSENRPEYPATLILSADHDDRVVPLHSLKFVAELQHVAGRSPAQRAPLLARFDTKAGHGGGKPT RPDLFGASFSQVGVIGMLRFHKFTIGHAWCSDYGNPSEKDHFDNLIKYSPLHNVHTPQTEMEEYPSTLILTADHDDRVSPLHSLKFTAALQEAVRESKHQKNPILLRVYSKAGHGAGKPT RPDLFGCVIAQVGVMDMLKFHKFTIGHAWTTDYGCSDTKQHFEWLLKYSPLHNVKLPEADDIQYPSMLLLTADHDDRVVPLHSLKFIATLQYIVGRSRKQSNPLLIHVDTKAGHGAGKPT RPDLFGCVIAQVGVMDMLKFHKYTIGHAWTTDYGCSDSKQHFEWLVKYSPLHNVKLPEADDIQYPSMLLLTADHDDRVVPLHSLKFIATLQYIVGRSRKQSNPLLIHVDTKAGHGAGKPT RPDLFGCVIAQVGVMDMLKFHKYTIGHAWTTDYGCSDSKQHFEWLIKYSPLHNVKLPEADDIQYPSMLLLTADHDDRVVPLHSLKFIATLQYIVGRSRKQNNPLLIHVDTKAGHGAGKPT

: : : : : :

683 716 691 686 686 686

BmPop-a BmPop-b SpPop MmPop HsPop SsPop

: : : : : :

TKIIDEHTDILCFMTQALGLKFVK TKIIDEHTDILCFMTQALGLKFVK SKRIEEATDVLTFMLKSLECGKCAKVIEEVSDMFAFIARCLNIEWIQ AKVIEEVSDMFAFIARCLNVDWIP AKVIEEVSDMFAFIARCLNIDWIP

: : : : : :

707 740 714 710 710 710

Identity(%) 100 95 59 59 59 58

Fig. 2. Alignment of B. mori Pops with other known Pops. Deduced amino acid sequences from Bombyx mori Pop isoforms BmPop-a, BmPop-b, Sarcophaga peregrina Pop (SpPop), Homo sapiens Pop (HsPop), Mus musculus Pop (MmPop) and Sus scrofa Pop (SsPop) were aligned by the MUSCLE (Edgar, 2004). Gaps were inserted for optimal matching, conserved and similar residues have been highlighted in black and gray backgrounds, respectively. The putative signal peptide sequence in the Nterminal region is boxed and the residues essential for enzyme activity are indicated by black triangles. The consensus sequence containing catalytic residue (Ser) is underlined by gray hexagon. The Peptidase_S9_N domain (PF02897) and Peptidase_S9 domain (PF00326) are underlined by dotted line and solid line, respectively.

isoform (Fig. 1A). We filled this gap (Gap1 in Fig. 1A) by Sanger sequencing and identified a long interspersed element-1 (LINE-1) in this region. LINE-1 inserted into intron 14 of the RB1 gene that caused a highly heterogeneous splicing pattern of RB1 mRNA (Rodríguez-Martín et al., 2016). In B. mori, whether LINE-1 insertion causes alternative splicing of Bmpop gene needs further investigation. The 3′RACE produced a 189 bp downstream-untranslated region (UTR) with a putative polyadenylation signal (AATAA) followed by a short poly(A) tail (Fig. 1B). The sequences of two isoforms of Bmpop gene were deposited in the GenBank database under the accession number MF538581 and MF538582, respectively. The full-length cDNA sequence of Bmpop-a was 2497 bp long with a 5′UTR of 184 bp and interrupted by 15 introns (black triangles, Fig. 1B). Its ORF was 2124 bp that encodes a polypeptide of 707 amino acids, having a predicted molecular weight of 80.21 kDa and a theoretical pI of 5.74. The full-length cDNA sequence of Bmpop-b was 2508 bp with a 5′UTR of 96 bp and interrupted by 14 introns (black triangles, Fig. 1B). Its ORF was 2223 bp containing the intact ORF of Bmpop-a, encoding a polypeptide of 740 amino acids. The initial 25 amino acids of BmPop-b were predicted as a signal peptide, but no signal peptide was predicted from BmPop-a (Fig. 2). The

presence of putative signal peptide suggested BmPop-b might be a secretory protein. We examined whether the signal peptide sequence is present also in other lepidopteran insects, using the lepidopteran genome database (http://lepbase.org/), and found that the Pop of Papilio xuthus, Spodoptera frugiperda and Heliconius erato demophoon have this sequence. Especially, the alternative spicing was found to be occurring in the former two species (data not shown). Moreover, when aligned with other known Pops, the deduced amino acid sequences of two isoforms of Bmpop gene contained the Peptidase_S9_N domain (Pfam accession number: PF02897, dotted line) and Peptidase_S9 domain (Pfam accession number: PF00326, solid line) of the Pop family of serine proteinases (Fig. 2). Additionally, two BmPops also possessed the typical structural features of the Pop family of serine proteinases, such as the highly conserved catalytic triad Ser551, Asp638 and His677 in BmPop-a (Ser584, Asp671, and His710 in BmPop-b) and the consensus sequence G-G-S-N-G-G-L-L, including the active site serine residue (Fig. 2) (Ishino et al., 1998; Ohtsuki et al., 1997; Rennex et al., 1991; Shirasawa et al., 1994). These results suggested that the predicted BmPops should belong to the prolyl oligopeptidase family. To perform the phylogenetic analysis of BmPop, the amino acid sequence 105

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kDa 1

2

3

120 100 80

4

5

6

7

Table 1 Peptides of recombinant BmPop-a identified by Maldi-TOF-TOF.

82 kDa

60 50 40 30 20 Fig. 3. Expression and purification of rBmPop-a. BL21 bacteria either containing pET-28a/BmPop-a plasmid (lane 5) or empty vector (lane 3) were induced by 0.2 mM IPTG for 16 h at 16 °C. BL21 bacteria either containing pET28a/BmPop-a plasmid (lane 4) or empty vector (lane 2) were induced without IPTG for 16 h at 16 °C. The rBmPop-a protein was purified by affinity chromatography on a nickel-agarose resin (lane 6) and analyzed by SDS-PAGE (10%) under reducing conditioned and Coomassie Blue staining. Western blotting was performed as described in the Materials and methods section using anti-BmPop-a antibody (lane 7, the purified rBmPop-a). Lane 1, molecular-mass standards.

Number

Peptides

Length (amino acids)

Position (start-end)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total

RDETVVDDYHGTK WLEDPDSNETKEFVEAQNK LTELWNYPK NTGLQNQNALYVQDGLDGEPR VFLDPNTLSEDGTVALSGTK FTEDGSTLAYGLSASGSDWIAIHFK DVATGEDYPEVLEK LIMIDLQNPAK TLVPEHPTDVLDWAAAVDQDK SVLQLHDLK TGEFIQTFPLDVGSVVGFTGK GFDASQYEAK DGSNPVLLYGYGGFNINVQPGFSVTR LVFMQHMNGIVAIPNIR QNVFDDFQAAAEYMVSER FTIGHAWVSDYGSSDNK TQFEYLLK YSPLHNIQPPSENRPEYPATLILSADHDDR FVAELQHVAGR AGHGGGKPTTK

13 19 9 21 20 25 14 11 21 9 21 10 26 17 18 17 8 30 11 11 331

10–22 29–47 66–74 90–110 111–130 131–155 156–169 310–320 325–345 356–364 365–385 426–435 460–485 486–502 521–538 586–602 603–610 611–640 649–659 675–685

specific inhibitors (KYP-2047 and S17092) inhibited significantly mammal's Pop enzyme activity (Barelli et al., 1999; Myöhänen et al., 2012). Therefore, these two inhibitors were selected to investigate the influence on the activity of rBmPop-a in vitro. The results from inhibition experiments of rBmPop-a were shown in Fig. 4B. The inhibitory patterns of the two inhibitors on rBmPop-a were similar, which the residual activities gradually decreased with increasing amounts of each inhibitor. The IC50 values of KYP-2047 and S17092 were 43.5 ± 0.9 nM and 79.6 ± 0.7 nM, respectively. Relatively, KYP-2047 was a more effective inhibitor on rBmPop-a. In evaluating the pH-activity profile of the rBmPop-a, the results showed that the substrate was cleaved between pH 5.5 and 10.0 with optimal activity around pH 7.5 (Fig. 4C). No Pop enzyme activity was detected below pH 5.5. However, under the case that pH value was lower than 7.0 or higher than 8.0, rBmPop-a activity drastically decreased under the assay conditions. The temperature-activity profile of the rBmPop-a showed a limited range (from 20 °C to 30 °C) with optimal activity around 25 °C (Fig. 4D). In conclusion, these results confirm that BmPop-a is a specific and functionally-active prolyl oligopeptidase.

of the putative BmPop-a was aligned with Pops of other species (Supplementary material: Table S2). The phylogenetic tree formed four clades, namely insect, higher organism, bacteria and archaea, and the nodes were supported by bootstrapping values of 99, 79, 97 and 99, respectively. These results showed that the putative BmPop-a clustered together with other lepidopteran insect Pops, and then grouped with other insects (Fig. S1), suggesting that this enzyme is conserved among different species. Taken together, the putative BmPops may have Pop activity. 3.2. Heterologous expression and purification of BmPop-a isoform Excepting the signal peptide sequence and that BmPop-b has only more eight amino acids (TTILSSRN) in N-terminal than BmPop-a, the amino acid sequences of the two isoforms are the same (Fig. 2). So BmPop-a isoform was heterologously expressed as a His-tagged fusion protein in E. coli. As compared to the control, additional band around 80 kDa was detected in the recombinant pET-28(a) plasmids infected cells by SDS-PAGE analysis (Fig. 3, Lane 5). The soluble rBmPop-a protein was purified by nickel affinity chromatography and dialysed, and homogenized to a single band in the SDS-PAGE analysis (Fig. 3, Lane 6). The purified protein was identified by the mass spectrometry Maldi-TOF-TOF. The protein sequence coverage was about 46.8% and matched peptides were listed in Table 1. This verified that BmPop-a isoform was expressed in E. coli. Then the purified protein was used to analyze the specificity of the BmPop-a antibody. One specific band approximately 80 kDa (the putative BmPop-a) was detected by Western blotting analysis (Fig. 3, Lane 7). Therefore, the BmPop-a antibody was suitable to do further research.

3.4. Expression analysis of two isoforms of Bmpop gene The expression profiles of alternative splice isoforms of Bmpop gene at the transcription and translation levels were examined (Fig. 5). The temporal expression profiles of the two isoforms were examined from the 5th instar to the egg stage by the qRT-PCR (Fig. 5A). Although Bmpop-a isoform was continuously expressed in all tested developmental stages, it was highly expressed at the late pupal stage (P-9 d) as well as the early adult stage (A-0 h). However, Bmpop-b isoform displayed a temporal expression pattern that was different from Bmpop-a. Bmpop-b gradually increased from newly molted of 5th instar (V-0 h) to the late 5th instar (V-6 d). Then it showed a low expression at the early wandering stage (W-0 h) but highly expressed at the late wandering stage (W-48 h). Bmpop-b decreased at the 2nd day after pupating (P2 d), with a sharp increase at the late pupal stage (P-9 d). Finally, the transcription level of Bmpop-b isoform decreased at the adult stage, and almost reached to zero at the egg stage. Then, eight main tissues from the 3rd day of the 5th instar larvae,

3.3. Enzyme activity assay To test the kinetic parameters of rBmPop-a, an enzyme assay was performed with Z-Gly-Pro-pNA as the substrate. rBmPop-a showed affinity for Z-Gly-Pro-pNA (Km = 7.4 ± 0.6 μM). Meanwhile, rBmPop-a was found to be active in a prolyl oligopeptidase activity assay against another synthetic peptide substrate, Z-Gly-Pro-AMC (Fig. 4A). Two Pop

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100

100

80

80

Relative activity (%)

Relative activity (%)

S17092

rBmPop-a

KYP-2047

B

A

60 40 20 0 0.0

0.5

1.0

1.5

60 40 20 0 -3.5

2.0

-3.0

Enzyme concentration (µg/ml)

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

Log [inhibitor] µM

C

D 100

100

80

80

Relative activity (%)

Relative activity (%)

BSA

60 40

60 40 20

20

0

0 5.0

6.0

7.0

8.0

9.0

15

10.0

pH

20

25 30 35 40 Temperature ( )

45

50

Fig. 4. Enzyme activity assays of rBmPop-a using Z-Gly-Pro-AMC as the substrate. (A) An increase in activity was shown as the concentration of rBmPop-a also increases from 0.125 to 2.0 μg/ml. (B) The progressive inhibition of rBmPop-a (0.25 μg/ml) oligopeptidase activity in the presence of increasing concentrations (0.5–150 nM) of the specific inhibitors, KYP-2047 and S17092 were shown. (C) pH-activity profile of rBmPop-a. (D) Temperature-activity of rBmPop-a. Three independent experiments were performed. Relative activity values expressed as percentages relative to the maximum of the enzyme activity are means of three observations; error bars are standard deviations.

3.5. Induced expression of two isoforms of Bmpop gene by starvation and refeeding

when males can be distinct from females, were used to examine the expression profiles of the two isoforms of Bmpop gene. Bmpop-a was extensively expressed in all tested tissues, with the lowest expression level in the silk gland. Furthermore, there was a significant difference of Bmpop-a in expression between the tissues of male and female, excepting head and midgut (Fig. 5B). Bmpop-b was mainly expressed in the head, integument and testis among the tested tissues, furthermore, it had the highest expression level in the integument. However, the expression signal of Bmpop-b isoform was not detected in other tissues including midgut, silk gland, fat body, Malpighian tubule and ovary (Fig. 5B). These results were also confirmed by the Western blotting analysis (Fig. 5C). In Sarcophaga peregrina, Pop is involved in differentiation of imaginal discs and DNA synthesis (Ohtsuki et al., 1994; Ohtsuki et al., 1997; Ohtuski et al., 1997). Although the role of prolyl oligopeptidase in B. mori is still unclear now, the wide expression pattern of Bmpop-a isoform implies an important role among different tissues and developmental stages for the silkworm. Bmpop-b isoform was highly expressed before ecdysis and down-regulated later, implying that BmPop-b could involve in molting and metamorphosis development of the silkworm.

In this study, the temporal and spatial expression profiles of two isoforms of Bmpop gene showed that Bmpop-b isoform was highly expressed in the integument during the feeding stage. We hypothesized that BmPops might be involved in material metabolism in the integument. To determine the regulation of BmPops expression by feeding or starvation treatments and the function of BmPops during material metabolism, we examined the influence of starvation and refeeding on the expression of BmPops mRNA and protein in the integument. Analysis of qRT-PCR and Western blotting revealed that BmPops mRNA and protein were markedly down-regulated by starvation in all of the treatments and up-regulated again by refeeding (Fig. 6A, C). These results indicated that BmPops might be involved in the metabolism of secondary metabolites from dietary. Pop plays a key role in the RAS and can convert Ang I or Ang II to Ang-(1-7) (Kehoe et al., 2016; Pereira et al., 2009). The RAS regulates blood pressure and fluid balance in mammal (Nguyen et al., 2002; Pahlavani et al., 2017). The angiotensin-converting enzyme (ANCE)related gene, prolyl oligopeptidase and renin receptor were identified in

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A

Relative expression level

0.15 0.12 0.09 0.06 0.03 0.00

h V-0

B 0.05 Relative expression level

Fig. 5. Expression profiles of two isoforms of Bmpop gene in different development stages and tissues of B. mori. (A) Transcription levels of two isoforms of Bmpop gene in different development stages. V-0 h, newly molted of 5th instar; V-3 d, 3rd day of 5th instar; V-6 d, 6th day of 5th instar; W-0 h, 0 h after mounting; W-24 h, 24 h after mounting; W-48 h, 48 h after mounting; P-2 d, 2nd day after pupating; P-9 d, 9th day after pupating; A-0 h, 0 h after eclosion; E-1 d, 1st day after oviposition. Transcript levels (B) and translation levels (C) of two isoforms of Bmpop gene in different tissues of the 3rd day of the 5th instar larvae of B. mori. He, head; Mg, midgut; Sg, silk gland; Fb, fat body; In, integument; Mt., Malpighian tubule; Go, testis or ovary; Hc, hemocyte. Total RNA of different development stages and tissues were used in the qRT-PCR analysis. Silkworm ribosomal protein L3 was used as the control. Western blotting analysis were performed to detect the expression of the protein. Approximately 50 μg total protein of different tissues from the 3rd day of the 5th instar larvae were used in this analysis.

Bmpop-a Bmpop-b

d d 4h 8h 0h V-3 V-6 W- W-2 W-4

P-2

d

0h

d

P-9

A-

d

E-1

and 12 h (Fig. 6B), they might participate in multiple steps of the RAS pathway (Pereira et al., 2009). These results indicated that BmPops might be involved in the metabolism of Ang I or Ang II in the integument, thus regulate ecdysis.

Bmpop-a

0.04

**

0.03

**

3.6. BmPop-b in molting fluids cannot form a complex with other proteins after phenoloxidase-induced melanization

**

**

0.02

Prolyl oligopeptidase (BGIBMGA002593) was identified in molting fluids by Liquid chromatography tandem-mass spectrometry (LC-MS/ MS) (Zhang et al., 2014). In this study, the putative BmPop-b had a signal peptide sequence in the N-terminal, and might be secreted into molting fluids from epidermal cells. The molting fluids (L-L, L-P and PA) at three different stages were collected and analyzed by Western blotting. The results showed that there was BmPop protein in the molting fluids as previously identified by LC-MS/MS (Fig. 7A). However, a previous study did not identify this protein in the molting fluid during metamorphosis from pupa to adult (Zhang et al., 2014). Was this protein either BmPop-a or BmPop-b? Western blotting analysis confirmed BmPop-b in the molting fluids (Fig. S2, Line 4). In general, the matured BmPop-b would not have a signal peptide, in this case the length of BmPop-b is speculated to be longer than that of BmPop-a just by eight amino acids. However, our Western blotting analysis showed that there was a clear difference of the band size between BmPop-a and BmPop-b (Fig. S2, Line 1, 3). This can be explained if we assume that BmPop-b still retains the signal peptide after secretion. The molecular weight of BmPop-b is consistent with the predicted molecular weight of the putative BmPop-b (83.72 kDa). Insect molting fluids had functions of immunity protection and ecdysis regulation (Zhang et al., 2014). Phenoloxidase (PO) induced melanization was an important type of immune response against pathogens (Lu et al., 2014; Zhao et al., 2007). During melanization, activated PO can form a large complex after coupling with other proteins (Lu et al., 2014). In this study, there was BmPop-b protein in the molting fluids (L-L) both before and after melanization (supernatant after centrifugation; Fig. 7B, Lane 1 and Lane 2). The melanized debris was washed several times with PBS and suspended in 1 × SDS buffer. No obvious protein band was detected by the antibody directed against BmPop-a (Fig. 7B, Lane 3). As the level of BmPop-b in the supernatant of the molting fluids did not change significantly before and after melanization (Fig. 7B), this protein didn't not appear to be coupled with PO or other proteins during the process of melanization. These results indicated BmPop-b might not be involved in immune response, but ecdysis regulation.

*

0.01

**

0.00

Relative expression level

0.25

Bmpop-b **

0.20 0.15 0.10

**

0.05 0.00

C

kDa 150 120 90 60

150 120 90 60

He

M

Mg

He

Mg

Sg

Sg

Fb

Fb

In

In

Mt

Mt

Go

Go

Hc

Hc BmPop-b BmPop-a

BmPop-b BmPop-a

molting fluids of silkworm (Zhang et al., 2014). So we speculated that the RAS existed in the integument of silkworm and balanced the molting fluids pressure. Meanwhile, we examined the influence of starvation and refeeding on the expression of the related genes of the RAS in the integument. qRT-PCR revealed that renin receptor (BGIBMGA006497) was down-regulated by starvation and up-regulated again by refeeding with significant difference at 24 h, 48 h and 72 h (Fig. 6B), its expression profile was similar to that of Bmpops. But the expression profiles of ANCE-related genes (BGIBMGA002526 and BGIBMGA002527) were different from that of Bmpops at starvation 6 h

3.7. Angiotensin I cleaved by BmPops Pop can convert Ang I or Ang II to Ang-(1-7) (Mäkinen et al., 1994; Pereira et al., 2009). BmPop-a and BmPop-b contained identical peptidase domains (Peptidase_S9_N domain and Peptidase_S9 domain), and 108

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S

F

Relative expression level

0.018

B

Bmpop-a *** ***

0.015

0.036

**

***

0.012

**

*** Relative expression level

A

*

0.009

***

0.006 0.003 0.000 Bmpop-b

***

***

***

0.018 0.012 0.006

0.024

*** 0.28

***

***

0.21 ***

0.14

*** **

**

6h

12h

0.00

BGIBMGA002526 ***

0.020 ** 0.016

**

** ***

0.012

**

0.008 0.004 0.000

24h

48h

72h

6h

12h

24h

48h

Relative expression level

0.0018

C

***

0.024

***

0.35

0.07

0.030

**

**

0.000

Relative expression level

Relative expression level

0.42

BGIBMGA006497

RF

72h BmPop-b

F

BmPop-a BmPop-b

S

BmPop-a BmPop-b

RF

BGIBMGA002527

0.0015 ** 0.0012

** **

0.0009

***

0.0006

***

** ***

0.0003 0.0000

BmPop-a

6h

12h

24h

48h

72h

Fig. 6. Effect of starvation and refeeding on the expression of the related genes of the renin-angiotensin system in the integument of B. mori. (A) qRT-PCR analysis of two isoforms of Bmpop gene expression induced by starvation and refeeding. (B) qRT-PCR analysis of renin receptor and two ANCE-related genes expression induced by starvation and refeeding. F, feeding; S, starvation; RF, refeeding. Student's t-test was used to evaluate statistical significance differences (*P < 0.05, **P < 0.01 and ***P < 0.001). Error bars indicated the standard deviation (SD) of the mean (n = 3). (C) Western blotting analysis of two isoforms of BmPop expression induced by starvation and refeeding. Approximately 50 μg total protein in each sample was used in this analysis.

A

Moulting fluids L-L L-P P-A

B

kDa 150

kDa 150

120

120

90

90

60

60

there was only more 33 amino acids in the N-terminal of BmPop-b than BmPop-a (Fig. 2). AS could affect subcellular localization of different isoforms (Laity et al., 2000; Tong et al., 2003). Our Western blotting analysis showed that BmPop-b was secreted into the molting fluids (Fig. 7A). AS should only affect the subcellular localization of BmPop-b, while BmPop-b's activity would not be affected. So we determined the cleavage site of angiotensin I by using rBmPop-a. The result was shown in Fig. 8D. Indeed, the apparent product (Ang-(1-7)) peak with adding the rBmPop-a was detected in the reaction mixture. Meanwhile, the amount of substrate (Ang I) significantly decreased compared with the controls (Fig. 8D). These results verified that BmPops were involved in the metabolism of Ang I in the integument, thus regulating silkworm ecdysis.

L-L (MF) 1 3 2

4. Conclusion In this study, two isoforms of Bmpop gene, Bmpop-a and Bmpop-b, were successfully cloned and characterized. A signal peptide was found in the sequence of Pop for the first time. The recombinant BmPop-a, heterologously expressed in E. coli, showed the activity of prolyl oligopeptidase toward oligopeptides containing proline residue. Its activity was inhibited significantly by Pop specific inhibitors, and sensitive to pH and temperature in vitro. BmPop-b isoform was highly expressed in the integument, and could be secreted into molting fluids. In conclusion, our results suggested that BmPops may be involved in

Fig. 7. Western blotting analysis of BmPop-b in the molting fluids. (A) BmPop-b in the molting fluids. Molting fluids were collected from L-L, L-P and P-A ecdysis stages as described in main text. Approximately 5 μl molting fluids from the different types were loaded respectively. (B) BmPop-b didn't couple with other proteins during the process of melanization. Melanization was induced within the larvae molting fluid (MF). The original molting fluids (Lane 1) and the same volumes of supernatant (Lane 2) were loaded. The melanized debris was washed and heated in 1 × SDS buffer (Lane 3). BmPop-b didn't couple with other proteins during the process of melanization. 109

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Ang-(1-7)

A

Ang I

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2018.05.021.

600

Acknowledgements

520

Intensity (mV)

440

This work was supported by the National Natural Science Foundation of China (No. 31772524 to ZZ).

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References

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Fig. 8. Enzymic activity assay of rBmPop-a using angiotensin I as the substrate by HPLC. (A) The peak of substrate, angiotensin I (Ang I). (B) The peak of product, angiotensin-(1-7) (Ang-(1-7)). (C) The peak of a mixture of the same concentration of Ang I and Ang-(1-7). (D) The enzyme assay of rBmPop-a with the Ang I. Control, without any protein. BSA, with BSA. rBmPop-a, with recombinant BmPop-a.

biologically active peptides containing proline residue metabolism, BmPop-b may play an important role in the balance of molting fluid pressure for guaranteeing successful ecdysis. Although the precise physiological role of BmPops remains to be elucidated, our findings could provide a basis for future studies of Pop in silkworm ecdysis. 110

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