A cell line is an important experimental platform for biological sciences as it can basically reflect the biology of its original organism. In this study, we firstly characterized the proteome of cultured BmN cells, derived from Bombyx mori. Total 1478 proteins were identified with two or more peptides by using 1D (one-dimensional) SDS/PAGE and LTQ-Orbitrap. According to the gene ontology annotation, these proteins presented diverse pI values and molecular masses, involved in various molecular functions, including catalytic activity, binding, molecular transducer activity, motor activity, transcription regulator activity, enzyme regulator activity and antioxidant activity. Some proteins related to virus infection were also identified. These results provided us with useful information to understand the molecular mechanism of B. mori as well as antiviral immunity.

INTRODUCTION

The silkworm, Bombyx mori, is an important economic insect for production of silk, and recently it is also being developed as a suitable model insect similar to the fruitfly for biological science due to its excellent biological characteristics such as ease of rearing, large body and abundant genomic information available [1,2]. However, the larva depends on its natural food, mulberry leaves, and thereby is limited by the seasons. For this reason, B. mori cell lines were established [3], and they provided the researchers great convenience to prepare experimental materials through the year without limitation of seasons. In other aspects, it is known that positive results could not be observed if the in vivo experiment is done first, so many experiments have to be done first in vitro using the cell line, and B. mori cell line is being applied extensively in various fields. Maeda et al. [4] first applied BmN cells to produce recombinant viruses and used it as vector to synthesize human α-IFN (α-interferon) in silkworm [4]. Previously, the silkworm cell line NISES-BoMo-Cam1 was used as an in vitro model of the immune system organs of B. mori to clarify the signalling pathway of antimicrobial peptide gene activation in lepidopteron insects [5]. Besides, the B. mori cell line was found to be highly susceptible to BmNPV (B. mori nucleopolyhedrovirus) [6], and thus the cultured cells could also be used as a good tool for studying pathology such as detecting the proteins involved in virus infection. Although the silkworm genome has already been completed [1,2], little is known about the proteome of its cultured cells.

In the present study, we first characterized the proteomics of Bm cell line in order to make clear the essential background information about this experimental platform. Developments in MS have dramatically increased the capability of analysing complex proteomes in depth. It is shown that LTQ-Orbitrap MS, which has been recently developed, is better than LTQ-FT MS [7] to identify protein spots. In the present paper, the proteins from BmN cells were analysed by using 1D SDS/PAGE and LTQ-Orbitrap, and the MS data were determined by using Bioworks (v3.2) search algorithm and TurboSequest™ (v3.2; ThermoFinnigan). A total of 1478 proteins were identified and annotated.

MATERIALS AND METHODS

Sample preparation

The B. mori cells (BmN), originally derived from B. mori ovary [3], were grown in TC-100 medium (AppliChem) containing 10% (v/v) FCS (fetal calf serum) and 0.26% bacto-tryptose (Gibco BRL, Gaithersburg, MD, U.S.A.) at 27°C. The cells were collected by centrifuging at 1000 g for 2 min and washed twice with PBS (137 mM NaCl, 10 mM phosphate and 2.7 mM KCl, pH 7.4). Further, the cells were disrupted by suspending in 1 ml of lysis buffer (8 M urea, 2 M thiourea and 4% CHAPS) for 1 h with vortex-mixing every 10 min. The sample was centrifuged (4°C, 40000 g and 1 h) and the supernatant was then precipitated by using the following method [8]: approx. 400 ml of the sample was added to 400 ml of methanol and 200 ml of chloroform. The sample was vortex-mixed and centrifuged for 2 min at 10000 g. The upper phase was then removed and 300 ml of methanol was added. The tube was inverted twice and centrifuged (15 min and 10000 g) and the supernatant was discarded. The pellet was allowed to air-dry for 5 min before the addition of SDS/PAGE loading buffer (0.06 M Tris/HCl, 2% SDS, 5% 2-mercaptoethanol and 0.01% Bromophenol Blue).

1D (one-dimensional) SDS/PAGE and in-gel digestion with trypsin

Regular 1D 12% Tricine SDS/PAGE was applied. Briefly, 100 μg of BmN cell proteins was loaded on to a single lane on a 7 cm long, 1 mm thick 12% Tricine SDS/PAGE gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 (GE Healthcare). Protein lanes were excised along the visible protein bands ranging from 14.4 to 97.0 kDa in molecular mass after staining; two lanes of the gel were combined and then sliced into 15 pieces; subsequently they were destained and in-gel digested with trypsin (Roche Applied Science, Indianapolis, IN, U.S.A.) following standard procedures. In brief, gel bands were soaked in 75 mM NH4HCO3 in 40% ethanol and vortex-mixed every 10 min. Fresh destaining buffer was added until Coomassie Brilliant Blue was completely removed. Gel bands were then washed with 25 mM NH4HCO3 followed by dehydration with acetonitrile. The process was repeated several times and the remaining acetonitrile was removed by vacuum centrifugation for 15 min. Dry gel bands were rehydrated with 50 mM NH4HCO3-containing trypsin. Digestion was carried out at 37°C overnight. After digestion, tryptic peptides were extracted with 50 mM NH4HCO3, acetonitrile/5% trifluoroacetic acid (1:1) and acetonitrile. The combined extracts were dried by vacuum centrifugation for 30–60 min and stored at −80°C until MS analysis.

MS

Separation of tryptic peptide mixtures was achieved by nanoscale reverse-phase HPLC, in combination with online LTQ-Orbitrap. For the HPLC separation, a nano-MDLC (multidimensional LC) system (Ettan MDLC; GE Healthcare) was used, employing a linear gradient of 5–45% buffer B (95% acetonitrile +5% water+0.1% formic acid) over 60 min. The column system consisted of a trap (0.5 mm×2 mm) and a separation column (Magic C18 AQ; 3 μm, 200 Å, 0.2×150 mm; 1 Å=0.1 nm), both purchased from Michrom Company. While column 1, trap 1 was running, column 2, trap 2 was equilibrated with buffer A (95% water+5% acetonitrile+0.1% formic acid) to allow continuous running of the sample through two columns.

LTQ-Orbitrap

The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap-MS and Orbitrap-MS/MS (tandem MS) acquired. Survey full scan MS spectra (from m/z 200 to 2000) were acquired in the Orbitrap with resolution R=60000 at m/z 400. The most intense ions (up to five, depending on signal intensity) were sequentially isolated for fragmentation; ions were recorded in the Orbitrap with resolution R=15000 at m/z 400.

For accurate mass measurements the lock mass option was enabled in both MS and MS/MS modes and the polydimethyl-cyclosiloxane ions generated in the electrospray process from ambient air {protonated [Si(CH3)2O]6; m/z 445.120025} were used for internal recalibration. For single SIM (selected ion monitoring) scan injection of the lock mass into the C-trap, the lock mass ‘ion gain’ was set as 10% of the target value of the full mass spectrum. When calibrating in MS/MS mode, the ion at 429.088735 [PCM (pneumatic control module) with neutral methane loss] was used instead for recalibration.

Target ions already selected for MS/MS were dynamically excluded for 180 s. General MS conditions were: electrospray voltage, 1.8 kV, no sheath and auxiliary gas flow; ion transfer tube temperature, 200°C; collision gas pressure, 1.3 mtorr (1 torr=0.133 kPa); normalized collision energy, 35% for MS, ion selection threshold was 500 counts for MS/MS. An activation q-value of 0.25 and activation time of 30 ms was applied for MS/MS acquisitions.

MS data interpretation

The derived MS data sets were converted into generic format (*.dta) files using the Bioworks Browser (3.2) and searched against the silkworm proteins database [downloaded from the website (ftp://ftp.genomics.org.cn/pub/SilkDB/Gene-Annotation/Proteins/SW-ge2k-BGF.pep)], containing all 21302 proteins in the data set using the Bioworks (v3.2) search algorithm, TurboSequest™ (v3.2; ThermoFinnigan). The species subset was set as B. mori. The number of allowed miscleavages was set to 2.0 and oxidation of methionine was selected. Intensity threshold was set to 500. The parent ion selection was set to 5 p.p.m., with fragment ion set to 1 p.p.m. The following filters were set for every peptide: (i) For a peptide charge of 1, the Xcorr value was a minimum of 1.5. For a peptide charge of 2, the Xcorr value was a minimum of 2.0, and for a peptide charge of 3, the minimum Xcorr value was set to 2.5; (ii) the difference between the first and second ranked assignments by simplified CorrX (ΔCN) value was greater than 0.1; and (iii) Rsp value was less than 5. Peak lists were generated using the TurboSequest™ (v3.2) algorithm. Each product assignment was made based on two unique spectra from the top hit.

Bioinformatics

The output including the protein IDs from silkworm database were obtained through TurboSequest. Microarray database (http://silkworm.swu.edu.cn/microarray) was used to find mRNA expression profiles corresponding to the protein IDs. In order to characterize the complete protein data set, Wego software (http://wego.genomics.org.cn/cgi-bin/wego/index.pl) was used to investigate and categorize the GO (gene ontology) annotations (cellular components, molecular functions and biological processes). Finally, the newly identified proteins were annotated by pBLASTp and the pI and molecular mass of proteins were analysed by using ProtParam software (http://www.expasy.ch/tool/protparam.html).

RESULTS

Nanospray MS is a very sensitive and versatile method for protein identification. 1D SDS/PAGE was used to prefractionate the BmN cell protein sample for detecting more low-abundance proteins. By dissecting the separation gel into slices of approx. 1 mm, the entire spectrum of proteins in the sample could be recovered from the gel. A total of 15 fractions were individually resolved by reverse-phase chromatography and proteolytically digested before directly spraying into the mass spectrometer. In order to generate a final list of protein identifications from the information obtained by MS, we run the data against the silkworm protein database, which was downloaded from the website (ftp://ftp.genomics.org.cn/pub/SilkDB/Gene-Annotation/Proteins/SW-ge2k-BGF.pep) and predicted by silkworm genome. This is a complete and detailed protein database with minimal redundancy. We set specific filter criteria to ensure that questionable peptide identification was not considered in our results list. The criteria were largely based on Kapp et al. [9], who compared several criteria for different search engines and the associated false-positive outcomes. The criteria have also been applied to analyse the sperm proteomics of human [8] and rat [10]. The peptide information, together with the associated TurboSequest™ information, is given in Supplementary Table S1 (http://www.bioscirep.org/bsr/030/bsr0300209add.htm).

In the newly constructed BmN cell proteomic database, each protein contains at least two peptides. The number of unique peptides, for each of the reported proteins, can be found in Supplementary Table S2 (http://www.bioscirep.org/bsr/030/bsr0300209add.htm). Figure 1 indicates the relation between the newly identified proteins and the number of peptides used for protein identification. From Figure 1, we can learn that as the number of peptides increases, the number of proteins is close to exponential distribution. There are only six proteins, Bmb004930, Bmb008789, Bmb007857, Bmb012614, Bmb016644 and Bmb009360, which contain more than 20 peptides.

Distribution of identified proteins of BmN cells based on the number of peptides used for protein identification

Figure 1
Distribution of identified proteins of BmN cells based on the number of peptides used for protein identification
Figure 1
Distribution of identified proteins of BmN cells based on the number of peptides used for protein identification

The identified proteins were annotated based on various aspects. Supplementary Table S3 (http://www.bioscirep.org/bsr/030/bsr0300209add.htm) shows the results for the values of pI and molecular mass predicted by ProtParam software. From Supplementary Table S3, it can be observed that the proteins of BmN cells are distributed in the pI ranges 5–7 and 8–10 and in the molecular mass range 8–80 kDa. The tissue distribution of the proteins was plotted based on a DNA microarray. By using the microarray data to annotate the identified proteins (Supplementary Table S4 at http://www.bioscirep.org/bsr/030/bsr0300209add.htm), it is found that out of 1478 proteins, 509 proteins cannot be found in ten tissues. A total of 790 proteins can be found in testis and this is the largest number of proteins identified in all tissues. The number of identified proteins in haemolymph is 391, which is the least number for all tissues (Figure 2). Using Wego software, we were able to annotate 778 proteins out of 1478 proteins. An analysis of the subcellular distribution of BmN proteins based on GO categories (Supplementary Table S5 at http://www.bioscirep.org/bsr/030/bsr0300209add.htm); Figure 3) revealed 294 proteins belonging to cell parts in the cellular component, with 127 proteins belonging to organelles. A total of 500 proteins could be annotated based on their molecular function (Figure 3). From this analysis, 76% of this population possessed a catalytic domain. Proteins containing a binding domain represented 70% of the annotatable population, corresponding to the second largest category. This is important for two reasons: first, by identifying which catalytic reactions are occurring in the cell, it should be possible to determine the mechanisms responsible for the relationship between BmNPV and its host BmN cells and, secondly, the ability to identify novel proteins related to antiviral activity or other immune activity relies heavily on whether proteins possess cell-specific catalytic domains and binding domains. An analysis of the biological process (636 proteins) demonstrated that, behind the broader GO definitions of the ‘cellular process’, 384 ranked the highest (Figure 3). Supplementary Table S5 reveals the identity of more than 360 proteins involved in the metabolism of BmN cells. In addition, 1274 proteins of the BmN cells were annotated by protein BLAST searching against non-redundant protein sequences (nr) from all organisms (Supplementary Table S6 at http://www.bioscirep.org/bsr/030/bsr0300209add.htm).

Tissue distribution of the protein hits reported

Figure 2
Tissue distribution of the protein hits reported
Figure 2
Tissue distribution of the protein hits reported

GO categories of the identified protein of the BmN cells by using Wego software

Figure 3
GO categories of the identified protein of the BmN cells by using Wego software
Figure 3
GO categories of the identified protein of the BmN cells by using Wego software

DISCUSSION

GO functional analysis and protein BLAST search of the annotation results of the identified proteins revealed that many of the proteins related to BmNPV infection are found in our BmN cell proteome.

Selected examples of these proteins are discussed in detail below.

Clathrin is a protein that is the major constituent of the ‘coat’ of the clathrin-coated pits and coated vesicles formed during endocytosis of materials at the surface of cells [11,12]. Different inhibitors proved that functional entry of the budded virus form of baculovirus into insect and mammalian cells is dependent on clathrin-mediated endocytosis, which is generally thought to occur through a low-pH-dependent endocytosis pathway, clathrin-coated pits [13]. Clathrin is made up of ‘three legs’, each leg comprising a heavy chain and a light chain [14]. A total of 21 peptides were identified (Supplementary Table S2). Its number in the protein database is Bmb007857, which was found to be similar to CLH-17 (clathrin heavy-17) isoform 6 of dog by using protein BLAST software. The score of the results of protein BLAST software is 0 (Supplementary Table S6).

Microfilament, microtubule and intermediate filament are the main components of the cytoskeleton and nuclear matrix of animal cells. They are also the factors required for baculovirus entering the insect cell nucleus. The ultra-microstructure of midgut, fat and haemolymph cells indicates that nucleocapsids of PDV (polyhedra-derived virus) or CRV (cell-released virus) are related to the tracks of the microtubule [15,16]. Formation of actin filaments facilitates the nuclear import of viral nucleocapsid [17]. In the present study, we identified a large number of proteins related to actin filaments and microtubules shown in Table 1.

Table 1
List of proteins identified related to actin filaments and microtubules

actin-RPV, vertebrate actin-related protein; ARP1, actin-related protein 1; RalBP1, RalA-binding protein 1; TSNAXIP1, translin-associated factor X-interacting protein 1.

Protein IDProtein description
Bmb001619 Similar to exosome complex exonuclease RRP43 (ribosomal RNA processing protein 43) 
 (exosome component 8) (p9) (Opa-interacting protein 2) [Apis mellifera
Bmb001856 Similar to huntingtin interacting protein E; huntingtin interactor protein E [Pan troglodytes
Bmb002021 Similar to MAPK8 (mitogen-activated protein kinase 8) interacting protein; MAPK8 interacting protein 1 [Gallus gallus
Bmb002802 Similar to α-centractin (centractin) (centrosome-associated actin homologue) (actin-RPV) (ARP1) isoform 9 [Bos taurus
Bmb004087 α-Actinin – fruitfly (Drosophila melanogaster
Bmb004470 Translin-associated factor X interacting protein 1 [Homo sapiens] unknown [H. sapiens] TSNAXIP1 protein [H. sapiens
Bmb004752 Similar to phosphatase and actin regulator 1 isoform 1 [Canis familiaris
Bmb006176 RalBP1-associated Eps domain-containing protein 1 (RalBP1-interacting protein 1) 
 RalBP1-associated Eps domain-containing protein [Mus musculus] RalBP1-associated EH domain protein Reps1 [M. musculus
Bmb006203 Hypothetical protein Aple02000468 [Actinobacillus pleuropneumoniae serovar 1 str. 4074] 
Bmb006486 Similar to F-actin capping protein α-subunit [Ap. mellifera
Bmb006636 Glutamate receptor interacting protein 1 isoform 2 [M. musculus] glutamate receptor interacting protein 1a-s [M. musculus
Bmb007009 Similar to regulating synaptic membrane exocytosis protein 2 [RIM2 (Rab3-interacting molecule 2)] [Ap. mellifera
Bmb007404 Membrane interacting protein of RGS16 [H. sapiens] membrane interacting protein of RGS16 [H. sapiens
Bmb007769 Similar to Numb-interacting protein 2 isoform 1 [B. taurus
Bmb008129 PREDICTED: similar to SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (sucrose non-fermenting protein (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin A5) (sucrose non-fermenting protein 
Bmb008813 Similar to TBP (TATA-box-binding protein)-interacting protein 120A, partial [Strongylocentrotus purpuratus
Bmb009416 Similar to TNF (tumour necrosis factor) receptor-associated factor 3 interacting protein 1 (predicted), partial [S. purpuratus
Bmb010478 Similar to myosin phosphatase-Rho interacting protein isoform 2 [C. familiaris
Bmb011097 Similar to actin related protein 2/3 complex subunit 2 [C. familiaris
Bmb011236 Smad- and Olf-interacting zinc finger protein [H. sapiens] KIAA0760 protein [H. sapiens
Bmb012212 Actin 6 [Aedes aegypti
Bmb014240 Similar to MAPK kinase 7 interacting protein 1 [Rattus norvegicus
Bmb014548 Sec23-interacting protein p125 [H. sapiens] Sec23-interacting protein p125 [H. sapiens
 SEC23-interacting protein (p125) phospholipase [H. sapiens
Bmb015457 Similar to schwannomin interacting protein 1 [Ap. mellifera
Bmb017622 RAB5-interacting protein [R. norvegicus
Protein IDProtein description
Bmb001619 Similar to exosome complex exonuclease RRP43 (ribosomal RNA processing protein 43) 
 (exosome component 8) (p9) (Opa-interacting protein 2) [Apis mellifera
Bmb001856 Similar to huntingtin interacting protein E; huntingtin interactor protein E [Pan troglodytes
Bmb002021 Similar to MAPK8 (mitogen-activated protein kinase 8) interacting protein; MAPK8 interacting protein 1 [Gallus gallus
Bmb002802 Similar to α-centractin (centractin) (centrosome-associated actin homologue) (actin-RPV) (ARP1) isoform 9 [Bos taurus
Bmb004087 α-Actinin – fruitfly (Drosophila melanogaster
Bmb004470 Translin-associated factor X interacting protein 1 [Homo sapiens] unknown [H. sapiens] TSNAXIP1 protein [H. sapiens
Bmb004752 Similar to phosphatase and actin regulator 1 isoform 1 [Canis familiaris
Bmb006176 RalBP1-associated Eps domain-containing protein 1 (RalBP1-interacting protein 1) 
 RalBP1-associated Eps domain-containing protein [Mus musculus] RalBP1-associated EH domain protein Reps1 [M. musculus
Bmb006203 Hypothetical protein Aple02000468 [Actinobacillus pleuropneumoniae serovar 1 str. 4074] 
Bmb006486 Similar to F-actin capping protein α-subunit [Ap. mellifera
Bmb006636 Glutamate receptor interacting protein 1 isoform 2 [M. musculus] glutamate receptor interacting protein 1a-s [M. musculus
Bmb007009 Similar to regulating synaptic membrane exocytosis protein 2 [RIM2 (Rab3-interacting molecule 2)] [Ap. mellifera
Bmb007404 Membrane interacting protein of RGS16 [H. sapiens] membrane interacting protein of RGS16 [H. sapiens
Bmb007769 Similar to Numb-interacting protein 2 isoform 1 [B. taurus
Bmb008129 PREDICTED: similar to SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (sucrose non-fermenting protein (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin A5) (sucrose non-fermenting protein 
Bmb008813 Similar to TBP (TATA-box-binding protein)-interacting protein 120A, partial [Strongylocentrotus purpuratus
Bmb009416 Similar to TNF (tumour necrosis factor) receptor-associated factor 3 interacting protein 1 (predicted), partial [S. purpuratus
Bmb010478 Similar to myosin phosphatase-Rho interacting protein isoform 2 [C. familiaris
Bmb011097 Similar to actin related protein 2/3 complex subunit 2 [C. familiaris
Bmb011236 Smad- and Olf-interacting zinc finger protein [H. sapiens] KIAA0760 protein [H. sapiens
Bmb012212 Actin 6 [Aedes aegypti
Bmb014240 Similar to MAPK kinase 7 interacting protein 1 [Rattus norvegicus
Bmb014548 Sec23-interacting protein p125 [H. sapiens] Sec23-interacting protein p125 [H. sapiens
 SEC23-interacting protein (p125) phospholipase [H. sapiens
Bmb015457 Similar to schwannomin interacting protein 1 [Ap. mellifera
Bmb017622 RAB5-interacting protein [R. norvegicus

ICE5 (interleukin-1β-converting enzyme 5), a member of the caspase protein family, is also named Caspase-1 (cysteinylaspartate-specific proteinase-1) [18] and plays an important role in the process of cell apoptosis [19]. In the process of viral infection, caspase in the form of zymogen can self-cut or can be cut by other members of the caspase family [20] and results in the production of long chains and short chains and forms a heterodimer containing the amino-acid sequence QACRG [21], which brings apoptosis of cells and prevents the production of new virus. Bmb001418 is the ICE5 protein identified in BmN cells. DNA microarray data indicated that it can be expressed not only in the midgut of the silkworm, but also in the head and haemolymph (Supplementary Table S4). In addition, GO annotation of all proteins indicated that Bmb004188 [Apaf-1 (apoptotic protease-activating factor 1)], Bmb014676 and Bmb007835 newly identified also exert much influence on BmN cell apoptosis.

From Supplementary Table S6, it can be learned that Bmb002206 is a kind of GBP-1 (IFN-induced guanylate-binding protein-1), which is the factor required for the replication of some virus [VSV (vesicular-stomatitis virus) and EMCV (encephalomyocarditis virus)] [22]. GBP-1 mediates an antiviral effect against VSV and EMCV and plays a role in the IFN-mediated antiviral response against these viruses [23]. This is the first time that GBP-1 of silkworm is found in BmN cells. We predicted that it may have antiviral function.

Abbreviations

     
  • 1D

    one-dimensional

  •  
  • EMCV

    encephalomyocarditis virus

  •  
  • IFN

    interferon

  •  
  • GBP-1

    IFN-induced guanylate binding protein-1

  •  
  • ICE5

    interleukin-1β-converting enzyme 5

  •  
  • MAPK8

    mitogen-activated protein kinase 8

  •  
  • MS/MS

    tandem MS

  •  
  • BmNPV

    Bombyx mori nucleopolyhedrovirus

  •  
  • GO

    gene ontology

  •  
  • VSV

    vesicular-stomatitis virus

FUNDING

This work was supported by National 863 [grant number 2007AA10Z159]; the Basic Research Program [grant number 2005CB121003]; and the New-Century Training Programme Foundation for the Talents, Ministry of Education, China [grant number NCET-06-0524]. The authors declare no conflict of interest.

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