Comparative Proteomic Analysis between the Domesticated Silkworm

Comparative Proteomic Analysis between the Domesticated Silkworm (Bombyx mori) Reared on Fresh Mulberry Leaves and on Artificial Diet Zhong-hua Zhou,† Hui-juan Yang,† Ming Chen,‡ Cheng-fu Lou,† Yao-zhou Zhang,§ Ke-ping Chen,| Yong Wang,| Mei-lan Yu,§ Fang Yu,† Jian-ying Li,† and Bo-xiong Zhong*,† College of Animal Sciences, Zhejiang University, Hangzhou 310029, P. R. China, College of Life Sciences, Zhejiang University, Hangzhou 310058, P. R. China, College of Life Sciences, Zhejiang University of Science and Technology, Hangzhou 310018, P. R. China, and Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, P. R. China Received May 26, 2008

To gain an insight into the effects of different diets on growth and development of the domesticated silkworm at protein level, we employed comparative proteomic approach to investigate the proteomic differences of midgut, hemolymph, fat body and posterior silk gland of the silkworms reared on fresh mulberry leaves and on artificial diet. Seventy-six differentially expressed proteins were identified by MALDI TOF/TOF MS, and among them, 41 proteins were up-regulated, and 35 proteins were downregulated. Database searches, combined with GO analysis and KEGG pathway analysis revealed that some hemolymph proteins such as Nuecin, Gloverin-like proteins, PGRP, P50 and β-N-acetylglucosamidase were related to innate immunity of the silkworm, and some proteins identified in silkworm midgut including Myosin 1 light chain, Tropomyosin 1, Profilin, Serpin-2 and GSH-Px were involved in digestion and nutrition absorption. Moreover, two up-regulated enzymes in fat body of larvae reared on artificial diet were identified as V-ATPase subunit B and Arginine kinase which participate in energy metabolism. Furthermore, 6 down-regulated proteins identified in posterior silk gland of silkworm larvae reared on artificial diet including Ribosomal protein SA, EF-2, EF-1γ, AspAT, ERp57 and PHB were related to silk synthesis. Our results suggested that the different diets could alter the expression of proteins related to immune system, digestion and absorption of nutrient, energy metabolism and silk synthesis poor nutrition and absorption of nutrition in silkworm. The results also confirmed that the poor nutrient absorption, weakened innate immunity, decreased energy metabolism and reduced silk synthesis are the main reasons for low cocoons yield, inferior filament quality, low survival rate of young larvae and insufficient resistance against specific pathogens in the silkworms fed on artificial diet. Keywords: Domesticated silkworm (Bombyx mori) • artificial diet • proteomic analysis • two-dimensional gel electrophoresis • MALDI-TOF/TOF MS

The silkworm Bombyx mori has been domesticated for silk production for about 5000 years. There are many farms raising silkworms in many countries including China, India, and Thailand. The domesticated silkworm is not only an agricultural important species with high economic value, but also a key model organism in Lepidoptera especially for genomics research.1 In addition, it has been used as an important bioreactor for the production of recombinant proteins.2,3 The silkworm is an oligophagous insect that mainly feeds on fresh mulberry leaves. Artificial diet for the silkworm has

been studied and applied extensively in Japan, China and some other countries as it contains essential nutrients for supporting the normal growth of the larval. Although artificial diet can obviate the serious drawbacks of mulberry leaves such as the seasonal limitation on supply of fresh leaves, possible harm from parasites or pesticides and high labor cost, the silkworms reared on artificial diet during all instars are not as good as those fed on fresh mulberry leaves, which have been reflected in many aspects such as the filament quality of cocoons, survival rate of young larvae, and resistance to bacterial and viral diseases.4-6

* To whom correspondence should be addressed. College of Animal Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, P. R. China. Tel/Fax: +86-571-8697-1302. E-mail: [email protected]. † College of Animal Sciences, Zhejiang University. ‡ College of Life Sciences, Zhejiang University. § College of Life Sciences, Zhejiang University of Science and Technology. | Institute of Life Sciences, Jiangsu University.

The fifth instar is a transition period for metamorphosis from larva to pupa, and for biosynthesizing and spinning, during which larvae take in almost the entire nutrition for the whole life process. Day 3 of the fifth instar has been found to be a boundary for larval development.7 Most biological processes may be similar before this time point, but after that, silkworms

Introduction

10.1021/pr800383r CCC: $40.75

 2008 American Chemical Society

Journal of Proteome Research 2008, 7, 5103–5111 5103 Published on Web 11/11/2008

research articles begin to synthesize silk proteins in mass. Thus, the study of this time point will be helpful to elucidate the regulatory mechanism of the mass synthesis of silk proteins and the growth of silkworm larva as well. Among all the body tissues of silkworm, midgut, hemolymph, fat body and posterior silk gland are all vital tissues during the growth and development of silkworm, which function in nutrient digestion and absorption, nutrient transportation and innate immunity, nutrient synthesis and storage, and fibroin synthesis, respectively. Proteomics is a large-scale study of gene expression at the protein level, which ultimately provides direct measurement of protein expression levels and insights into the activity of all relevant proteins.8 Two-dimensional gel electrophoresis (2-DE) is an efficient technique for quantitative analysis of complex protein mixtures, which provides a powerful tool for understanding complex organismal processes at the protein level,9,10 combined with mass spectrum (MS) and bioinformatics approaches. In this study, a comparative proteomic approach was employed to investigate the proteomic differences of midgut, hemolymph, fat body and posterior silk gland between silkworms reared on fresh mulberry leaves and on artificial diet. This is the first report on the utilization of a proteomic approach to study the effects of the two different kinds of diets on the growth and development of the silkworm.

Materials and Methods Experimental Animal. A hybrid strain of the silkworm (Qiufeng × Baiyu, Bymbox mori) was used for this experiment, which was maintained in our laboratory at Zhejiang University, Hangzhou, China. Larvae were reared in plastic containers placed in a chamber where the temperature was at 25 ( 1 °C and the relative humidity was 75 ( 2%. They were reared on either commercial artificial diet (purchased from Sericultural Research Institute of Shandong Province, China) or fresh mulberry leaves (picked from ‘Tongxiangqing’, a Chinese mulberry variety, cultivated in mulberry field at Zhejiang University, Hangzhou, China) from hatching to the fifth instar. On day 3 of the fifth instar, 10 larvae of the same size and age were collected from each group (artificial diet feeding group and fresh mulberry leaves feeding group). Silkworm hemolymph was collected by cutting caudal horn and stored at -80 °C for further use. Subsequently, the midgut, fat body and posterior silk gland were collected from the same larvae, washed in 0.75% ice-cold physiological salt solution, blotted on a filter paper, frozen in liquid nitrogen immediately, and stored at -80 °C for further use. Sample Preparation. Each of 20 mg tissues from the midgut, fat body and posterior silk gland (or 20 µL of the hemolymph) was homogenized in 200 µL of lysis buffer containing 8 M urea, 2 M thiourea, 4% (v/v) CHAPS, 2% (v/v) IPG buffer (pH 3-10) and 30 mM DTT using a sample grinding kit (Amersham Biosciences) on ice for 10 min. The homogenate was kept for 10 min at room temperature, sonicated in an ice bath for 2 min, and centrifuged twice at 15 000g for 15 min at 4 °C. The resulting supernatant was stored at -80 °C for later use, and its protein concentration was determined by the Bradford’s method.11 2-DE and Image Analysis. Isoelectric focusing (IEF) was performed using IPGphor isoelectric focusing system by following the manufacturer’s instructions (Amersham Biosciences). The sample proteins (60 µg each) were loaded onto 24 cm immobiline dry strips (pH 3-10, Linear) (Amersham Bio5104

Journal of Proteome Research • Vol. 7, No. 12, 2008

Zhou et al. sciences) with 450 µL of rehydration buffer containing 8.0 M Urea, 2% (w/v) CHAPS, 30 mM DTT, 0.5% (v/v) IPG buffer (pH 3-10) and 0.002% (w/v) bromphenol blue. IEF was conducted with a gradient procedure as: 30 V for 12 h, 200 V for 1 h, 500 V for 1 h, 2000 V for 1 h, 4000 V for 1 h, and 8000 V for 11 h (a total of 95 060 Vh). The isoelectric-focused strips were incubated for 15 min in an equilibration buffer containing 6 M urea, 30% glycerol, 2% SDS, 0.05 M Tris pH 8.8 and 1% DTT, and were incubated for another 15 min in the same buffer except that DTT was replaced with 2.5% iodoacetamide. The equilibrated IPG strips were transferred to 12.5% SDS polyacrylamide constant separation gel and sealed with 0.5% agarose. SDSPAGE was performed at 15 °C using an Ettan DALT six electrophoresis unit (Amersham Biosciences) at constant power of 5 W/gel for 45 min followed by 15 W/gel until the bromophenol blue frontier reached the bottom of the gels. The gels were visualized with silver staining as described by Amersham Biosciences and scanned at 300 dpi using highresolution image scanner. Spot detection, spot matching and quantitative intensity analysis were evaluated automatically with ImageMaster 2D software (version 6.0) supplied by the manufacturer. Triplicate replications were performed for each sample and a comparison of relative intensity abundance between artificial diet feeding group and fresh mulberry leaves feeding group was conducted. Expression intensity ratiosartificial diet/fresh mulberry leaves higher than 1.80 or lower than 0.55 were set as a threshold of prominent changes. The proteins with higher expression in artificial diet feeding group were regarded as up-regulated proteins, while those with higher expression in fresh mulberry leaves feeding group were regarded as down-regulated ones. In-Gel Digestion. In-gel digestion was performed as reported by Gharahdaghi et al.12 The protein spots with prominent ratio were excised from the silver-stained gels, washed twice in milli-Q water, destained with a 1:1 solution of 30 mM potassium ferrocyanide and 100 mM sodium thiosulfate, washed in milli-Q water 3 times followed by an equilibration in 200 mM ammonium bicarbonate for 20 min, washed twice in milli-Q water again, dehydrated by addition of acetonitrile, and dried in a SpeedVac (Thermo Savant) for 30 min. The dried gel particles were rehydrated at 4 °C for 40 min with 2.5 µL/well trypsin (sequencing grade; Promega, Madison, WI) in 50 mM ammonium bicarbonate (20 µg/mL), and then incubated at 37 °C overnight. The resulting peptides were extracted twice by adding 50 µL of the solution including 5% trifluoroacetic acid and 50% acetonitrile for 15 min, respectively. The peptides were dried in a SpeedVac, and then dissolved in 1.5 µL of 0.1% trifluoroacetic acid for later use. Protein Identification and Database Searching. The above prepared peptide mixtures (0.3 µL) were mixed with an equal volume of matrix solution (R-cyano-4-hydroxy-cinnamic acid (CHCA, Sigma, St. Louis, MO) in 0.1% TFA and 50% ACN) and spotted on the target plate. Samples were allowed to air-dry and analyzed by 4700 MALDI-TOF/TOF Proteomics Analyzer (Applied Biosystems, Foster City, CA). GPS Explorer software (Applied Biosystems, Foster City, CA; Version 3.0) was used to create and search files in the MASCOT search engine (version 2.0; Matrix Science, London, U.K.) for peptide and protein identification with the following parameters: NCBInr database (release date: July 20, 2007) or Swiss-Prot database (release date: June 7, 2007), taxonomy of all entries, trypsin digest with one missing cleavage, fixed modifications of carbamidomethyl,

Comparative Proteomic Analysis of the Domesticated Silkworm

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Figure 1. 2-DE pattern of the proteins in various silkworm tissues. The proteins of midgut (A and B), hemolymph (C and D), fat body (E and F) and posterior silk gland (G and H) of silkworms reared on fresh mulberry leaves (A, C, E, and G) and artificial diet (B, D, F, and H). Sixty micrograms of protein was applied to the IPG strip (24 cm, pH 3-10, L) and 12.5% SDS-PAGE was carried out for separation in the second dimension. The differentially expressed protein spots were shown in gels with a small letter and an Arabic numeral, the small letter ‘m’, ‘h’, ‘f’ and ‘s’ represent midgut, hemolymph, fat body and posterior silk gland, respectively. The circle or ellipse indicates the successfully identified protein spots.

variable modifications of oxidation, MS and MS/MS tolerance of 100 ppm. The searching results of the proteins and the individual MS/MS spectra with statistically significant (confidence level >95%) were accepted. Bioinformatics Analysis. According to the annotations of the identified proteins from UniProt knowledgebase (http://www. expasy.org/sprot/), corresponding Gene Ontology (GO) IDs of these proteins were obtained by InterproScan searching (http:// www.ebi.ac.uk/InterProScan/). Following the method described by Ye et al.13 and based on Gene Ontology Database (OBO v1.2 format: http://www.geneontology.org/GO.downloads.ontology. shtml), GO classification of these proteins were conducted with WEGO (http://wego.genomics.org.cn/). In addition, the related KEGG pathways of these proteins were sought by UniProtKB (http://pir.georgetown.edu/pirwww/search/blast.shtml) searching based on the annotations of the identified proteins from UniProt knowledgebase; then, these KEGG pathways were classified into several sorts in accordance with the KEGG database (http://www.genome.ad.jp/kegg/pathway.html).

Results 2D PAGE Patterns of Proteins from Various Tissues of the Silkworms Reared on Fresh Mulberry Leaves and on Artificial Diet. There were prominent differences between the 2D-PAGE patterns of proteins from midgut, hemolymph, fat body and posterior silk gland of silkworms reared on the two different diets (Figure 1), while three replications of each tissue were highly reproducible and acceptable (Table S1 in Supporting Information). In total, 1090 ( 7 spots, 669 ( 10 spots, 946 ( 17 spots and 798 ( 8 spots were detected in the midgut, hemolymph, fat body and posterior silk gland tissues from silkworms fed on fresh mulberry leaves, respectively. There were 1083 ( 6 spots, 676 ( 5 spots, 955 ( 6 spots and 814 ( 10 spots in the corresponding tissues from artificial diet rearing group. On the basis of average intensity ratios of protein spots between silkworms fed on different diets, 111 protein spots with a ratio higher than 1.8 or lower than 0.55 were identified as differentially expressed proteins, including 27 proteins of Journal of Proteome Research • Vol. 7, No. 12, 2008 5105

research articles midgut, 39 of hemolymph, 31 of fat body and 14 of posterior silk gland (Table S1 in Supporting Information). Mass-Spectrum Identification of Differentially Expressed Proteins. The above 111 protein spots were excised from 2-DE gels and subjected to in-gel trypsin digestion and subsequent MALDI-TOF/TOF identification. Finally, 76 proteins were successfully identified as shown in Table 1 and Figure 1 (the MS and MS/MS spectra were listed in the supplemental figures in Supporting Information), among which 18 spots were of midgut, 28 of hemolymph, 19 of fat body and 11 of posterior silk gland. According to the average expression intensity abundance ratio, 41 proteins were up-regulated proteins (ratiosartificial diet/ fresh mulberry leaves g 1.8) while the rest 35 proteins were down-regulated ones (ratiosartificial diet/ fresh mulberry leaves e 0.55). Remarkably, a number of different spots in the same gel were identified as the same protein or similar one, including Nuecin (Table 1 and Figure 1, h25, h27, h28), Gloverin-like protein (Table 1 and Figure 1, h4, h18, h22, h23), β-Nacetylglucosaminidase (Table 1 and Figure 1, h7, h8), P50 protein (Table 1 and Figure 1, h13, h16) and Elongation factor 1 gamma subunit (Table 1 and Figure 1, s4, s5), which reflected the shifts of protein pI or molecular weight and were probably caused by post-translation modification or protein degradation. Bioinformatics Analysis of Differentially Expressed Proteins. The GO analysis indicated that 51 proteins of all the 76 identified proteins were found with at least one matched GO. The matched GO related to the differentially expressed proteins of midgut, hemolymph, fat body and posterior silk gland were 76, 67, 61 and 46, respectively (Figure 2). Most of the differentially expressed proteins were involved in cell, cell part, macromolecular complex, organelle, binding activity, catalytic activity, biological regulation, cellular process, developmental process, metabolic process, while no obvious differences were observed among the percentages of those categories in various tissues. However, a few of them showed tissue specificity. For instances, a few midgut proteins were involved in the antioxidant activity as well as molecular transducer activity uniquely, some hemolymph proteins were classified into extracellular region, immune system process and multicellular organismal process. A few fat body proteins were involved in the reproduction, reproductive process and viral reproduction as well. Among the 76 identified proteins, 30 proteins were involved in certain kinds of KEGG pathways (Figure 3). Some upregulated proteins of midgut fell into pathways related to metabolism and cellular process, while the down-regulated proteins were involved in the pathways dealing with environment information process, cellular process and human diseases. In addition, almost all hemolymph proteins involved in KEGG pathways were up-regulated and were mainly implicated in special metabolic processes such as glycan biosynthesis and metabolism, cellular process and human diseases. However, nearly all fat body proteins included in KEGG pathways were down-regulated and fell into pathways related to lipid mechanism, signal transduction, various cellular process and human diseases. In the same way, all posterior silk gland proteins involved in KEGG pathways were down-regulated and were implicated in various pathways especially in amino acid metabolism. Interestingly, we found that a few up-regulated proteins together with other down-regulated proteins in the same tissue fell into the same KEGG pathways including 5106


Zhou et al. nucleotide metabolism, cell motility, endocrine system and immune system (Figure 3 and Table S2 in Supporting Information).

Discussions Differences in Digestion and Absorption of Nutrient between the Silkworms Fed on Different Diets. In this study, 5 proteins were identified in silkworm midgut that may function in digestion and absorption of nutrient including Myosin 1 light chain (m1), Tropomyosin 1 (m7), Profilin (m11), Serpin-2 (m3) and Glutathione peroxidase (m15) (Figure 1A,B and Table 1). Among these proteins, Myosin 1 light chain, Tropomyosin 1 and Profilin are related to actin filament through driving the myosin molecules to move along the actin filaments,14 stabilizing actin filaments,15 and inhibiting actin filament formation,16 respectively, whereas Serpin-2 and Glutathione peroxidase (GSH-Px) are two key enzymes and are involved in the inactivation of proteases17 and elimination of oxidation byproducts, respectively.18 We also found that the expression of Profilin, Serpin-2 and Glutathione peroxidase were up-regulated in midgut of silkworm fed on artificial diet, while Myosin 1 light chain and Tropomyosin were downregulated. The proteomic data together with previous studies demonstrated that the decreased expression of Myosin 1 light chain means the destabilization of the apical brush border membrane, which will result in the enhanced sensitivity to oral infection of bacterial pathogen.19 In addition, the overexpression of Profilin and the decreased expression of Tropomyosin 1 might inhibit the formation of actin filament therefore weakening contraction ability of the smooth muscle in the silkworm midgut.16 Moreover, the increased expression of Serpin-2 might also weaken the activity of enzymes related to the digestion and absorption of nutrients. However, the increased expression of Glutathione peroxidase probably reflected a rise of oxidative radical environment in midgut of the silkworms fed on artificial diet.20 The above results which indicated low ability in digestion and absorption of nutrients in the silkworms reared on artificial diet compared with that fed on fresh mulberry leaves may probably be due to the weakened peristalsis of midgut, decreased activity of relative enzymes and high concentration of oxidative radicals. Factually, bioinformation analysis also suggested that some vital functional pathways in silkworm midgut such as calcium signaling pathway, phosphatidylinositol signaling system, regulation of actin cytoskeleton, arachidonic acid metabolism and glutathione metabolism had been affected by artificial diet (Figure 3 and Table S2 in Supporting Information), which also confirmed our conclusion. In addition, we identified a number of other interesting proteins, such as NADP-dependent isocitrate dehydrogenase (m6), N-acetylglucosamine kinase (m14), Ezrin (m5) and polyribonucleotide nucleotidyltransferase (m9). We speculated that all these proteins may play a potential role in nutrient absorption of silkworm, though it is unclear why and how the two kinds of diet can alter expression of those proteins markedly. Effects of Nutrient on the Immune System. Like other kinds of insect, silkworm has an efficient innate immune system which can recognize an invading organism as foreign objects followed by the activation of its antimicrobial peptides.21 In addition, a red fluorescent protein complex plays vital roles in immune system of silkworm due to its antiviral property.22 This protein complex contains a special protein secreted by silk-

research articles

Comparative Proteomic Analysis of the Domesticated Silkworm Table 1. List of the Differentially Expressed Protein Spots Identified by MALDI-TOF/TOF spot no.a

m5 m9 m1 m2 m3 m4 m6 m7 m8 m10 m11 m12 m13 m14 m15 m16 m17

m18 h5 h4 h14 h18 h21 h22 h23 h25 h26 h27 h28 h1 h2 h3 h6 h7 h8 h9 h10 h11 h12 h13 h15 h16 h17 h19 h20 h24 f2 f8 f14 f16 f10 f1

protein name

Ezrin Polyribonucleotide nucleotidyltransferase myosin 1 light chain phosphohistidine phosphatase 1 serpin-2 autoantigen La NADP-dependent isocitrate dehydrogenase tropomyosin 1 phosphatidylinositol transfer protein cellular retinoic acid binding protein profilin eukaryotic translation elongation factor 1 protein phosphatase I alpha subunit N-Acetylglucosamine kinase glutathione peroxidase radixin propionyl-Coenzyme A carboxylase, alpha polypeptide precursor Moesin isoform 3 NM23A protein gloverin-like protein 1 pericentrin 2 gloverin-like protein 3 AGAP010319-PA gloverin-like protein 4 gloverin-like protein 2 nuecin acidic ribosomal phosphoprotein PO nuecin nuecin chemosensory protein 11 antitrypsin precursor low molecular mass 30 kDa lipoprotein 21G1 precursor dihydropyrimidinase-like 2 beta-N-acetylglucosaminidase isoform A β-N-acetylglucosaminidase B BmLSP-T(Bombyx mori larval serum protein) mature 30K lipoprotein 30K protein precursor heat shock protein 27 p50 protein heterogeneous nuclear ribonucleoprotein K p50 protein diapause bioclock protein imaginal disk growth factor peptidoglycan recognition protein cyclophilin-like protein myosin 1 light chain vacuolar ATP synthase subunit B peptidylprolyl isomerase A LOC445855 protein β-actin heat shock protein 27

calculated confidence peptides sequence experimental protein interval matched covered (2-D) accession no. database (MS) (pI/Mr)(kDa) (pI/Mr)(kDa) score (%) (n) (%) ratiob

gi|46249758 gi|83746292

NCBInr NCBInr

5.9/69.2 5.6/77.5

3.7/24 4.2/29

190 83

100 97.1

21 9

28.0 12.0

Pm Pa

gi|56462256 gi|24475861 gi|112983210 gi|10835067 gi|3641398

NCBInr NCBInr NCBInr NCBInr NCBInr

4.6/16.7 5.7/14 4.9/41.7 6.7/47 6.3/46.7

3.4/20 35/25 3.6/39 3.7/25 3.9/18

192 84 190 101 117

100 97.9 100 100 100

5 10 12 10 11

33.3 87.0 28.5 24.0 29.0

0.43 2.82 2.78 0.28 8.72

gi|114052020 NCBInr gi|6912594 NCBInr

4.8/32.5 6.4/31.5

3.9/36 4.1/16

243 115

100 100

16 10

52.1 41.0

0.42 2.08

gi|112983600 NCBInr

5.7/14.8

4.9/18

315

100

13

78.8

2.79


5.9/13.7 4.9/31.2

5.6/16 5.4/31

211 119

100 100

5 9

67.5 37.0

2.20 0.54

gi|190281

NCBInr

6.4/35.8

5.4/51

91

7

25.0

0.45

gi|49574508 gi|112983348 gi|4388775 gi|109121193

NCBInr NCBInr NCBInr NCBInr

5.8/37.4 8.7/22.4 5.8/68.5 7.0/79.9

5.8/18 6.9/24 7.4/32 8.0/28

141 205 84 112

100 100 98 100

11 9 11 15

38.0 63.3 17.0 23.0

5.75 2.66 1.91 3.11

gi|74007502 gi|38045913 gi|112982990 gi|74001604 gi|112983026 gi|158298104 gi|112982699 gi|112982659 gi|112983296 gi|28189747

NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr

9.1/32.6 5.4/19.9 7.0/19.2 5.6/353.3 6.9/19.0 8.6/34.0 6.8/18.8 6.4/18.9 9.9/22.5 9.7/18.5

8.1/26 5.3/53 5.2/18 6.5/34 6.9/18 7.4/32 8.0/18 8.1/18 9.8/17 9.9/17

109 101 270 85 346 81 365 226 126 159

100 100 100 98.1 100 95.1 100 100 100 100

9 6 13 38 11 5 13 11 6 13

29.0 36.0 62.4 9.0 48.6 17.0 63.7 45.1 41.1 55.0

3.87 Pm Pa Pa Pa Pa Pa Pa Pa Pa

gi|112983296 gi|112983296 gi|112983052 gi|112983770 gi|266439


9.9/22.5 9.9/22.5 5.0/13.5 5.4/43.4 6.3/30.2

9.8/23 9.9/23 4.2/16 4.8/44 5.2/30

281 162 293 284 92

100 100 100 100 99.6

13 10 8 14 12

53.7 42.1 48.4 47.4 54.4

Pa Pa 2.85 1.84 1.88


6.0/62.3 5.3/61.5

5.3/55 5.3/65

168 134

100 100

12 8

26.0 18.1

1.82 2.09


5.5/61.7 5.6/30.9

5.4/65 5.6/33

173 227

100 100

13 13

23.9 42.4

3.73 1.96

gi|1335608 gi|112984502 gi|662841 gi|112984204 gi|71897277


6.1/29.7 6.4/28.4 7.8/22.3 6.2/52.2 6.0/47.2

5.8/32 5.8/31 5.8/55 6.2/58 6.5/27

142 145 94 138 110

100 100 99.8 100 100

12 10 7 5 10

41.9 44.1 33.0 15.0 31.0

1.91 1.87 0.45 4.52 1.83

gi|112984204 gi|116175238 gi|152061158 gi|112983994 gi|60592747 gi|56462256 gi|148298717 gi|10863927 gi|124481909 gi|119943232 gi|662841

NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr

6.2/52.2 6.1/18.2 7.6/48.1 6.7/21.6 7.7/17.9 4.6/16.7 5.3/54.4 7.7/18.2 5.9/43.4 5.3/42 7.8/22.4

6.6/58 6.6/20 6.7/55 7.2/19 7.8/18 3.7/16 5.0/64 5.7/31 6.9/25 5.6/43 3.4/30

88 261 231 228 96 236 114 155 90 117 106

99 100 100 100 99.9 100 100 100 99.4 100 100

4 6 16 12 4 5 7 16 9 10 10

12.4 58.7 35.0 43.9 39.4 37.3 15.9 73.0 24.0 37.0 49.0

4.17 2.53 1.93 1.90 0.29 Pm Pm Pm Pm Pa 0.45

99.5

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Table 1. Continued spot no.a

f3 f4 f5

f6 f7 f9 f11 f12 f13 f15 f17 f18 f19 s2 s1 s3 s4 s5 s6 s7 s8 s9 s10 s11

protein name

β-tubulin myosin light chain 2 phosphatidylserine/ phosphatidylglycerophosphate/ cardiolipin synthases and related enzyme tropomyosin 1 lysophospholipase I ribosomal protein P0 glucose-methanol-choline oxidoreductase cytokine induced apoptosis inhibitor 1, isoform CRA_b arginine kinase PREDICTED: similar to CG1341-PA beta Actin peptidylprolyl isomerase B vacuolar ATP synthase subunit B ribosomal protein SA protein disulfide-isomerase like protein ERp57 cofilin 1 (nonmuscle) elongation factor 1 gamma elongation factor 1 gamma-subunit prohibitin protein WPH tropomyosin 4 isoform 2 glial fibrillary acidic protein elongation factor 2 aspartate aminotransferase mago-nashi homologue

accession no.

database

calculated confidence peptides sequence experimental protein interval matched covered (2-D) (MS) (pI/Mr)(kDa) (pI/Mr)(kDa) score (%) (n) (%) ratiob

gi|3399724 NCBInr gi|148298826 NCBInr gi|48870799 NCBInr

4.8/50.1 4.7/22.0 9.3/56.5

3.7/31 3.6/28 3.7/28

93 226 84

99.7 100 99.1

4 9 9

11.9 45.3 20.1

0.47 0.49 0.35

gi|114052020 gi|5453722 gi|4506667 gi|162283347

NCBInr NCBInr NCBInr NCBInr

4.8/32.5 6.3/25.0 5.7/34.3 8.0/58.1

3.8/44 4.1/34 5.2/47 5.4/34

313 97 109 110

100 99.9 100 100

18 7 8 9

54.9 44.0 36.0 25.0

0.54 0.44 2.05 0.53

gi|119603329 NCBInr

5.5/32.8

5.6/36

152

100

10

42.0

0.39

gi|46401508 gi|91088885 gi|29603621 gi|114052472 gi|95102856 gi|54609281 gi|62241290

NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr NCBInr

6.1/30.0 5.8/48.5 5.3/34 7.9/22.4 9.6/21.4 4.9/33.4 5.3/55.1

5.6/48 6.1/60 7.0/34 7.9/23 10.0/25 5.1/36 4.9/60

99 93 99 176 117 87 83

100 99.7 99.9 100 100 99.5 95.2

10 15 7 14 12 6 11

37.3 30.9 36.0 64.4 55.0 24.5 25.7

0.37 0.52 0.32 0.55 0.46 Pm 0.51

gi|5031635 gi|112983898 gi|25528911 gi|114053221 gi|47085929 Q28115 P15112 gi|114053127 gi|109472730

NCBInr NCBInr NCBInr NCBInr NCBInr Swiss-Prot Swiss-Prot NCBInr NCBInr

8.2/18.7 5.8/48.4 5.8/48.4 6.5/30.1 4.6/23.4 5.3/49.4 6.2/91.6 8.8/47.8 8.4/23.4

5.4/57 5.5/50 5.7/50 6.2/30 7.7/52 8.1/32 8.8/62 9.0/48 9.7/37

118 84 129 126 96 90 70 120 98

100 97.8 100 100 99.9 99.9 98.3 100 99.9

8 8 14 9 14 8 15 9 16

62.0 21.0 33.1 39.0 42.7 19.6 14.6 26.0 50.0

0.54 0.50 0.53 0.52 0.53 0.50 0.53 0.40 0.55

a The small letter ‘m’, ‘h’, ‘f’ and ‘s’ represent midgut, hemolymph, fat body and posterior silk gland, respectively. b The expression intensity ratio of the silkworms fed on artificial diet to that fed on fresh mulberry leaves; Pm and Pa mean that the protein spot was the unique spot in gel of silkworm fed on fresh mulberry and on artificial diet, respectively.

worm midgut and chlorophyllide a, the prosthetic group of chlorophyll a (Ch-a), which is produced by chlorophyllase, an enzyme released from mulberry leaves.23,24 Usually, fresh mulberry leaves contain activated chlorophyllase, while almost all kinds of artificial diet only contain inactivated chlorophyllase, due to long-term storage and cooking process of artificial diet before feeding. In this experiment, 8 proteins including Nuecin (h25, h27, h28), P50 (h13, h16), PGRP (h20), β-Nacetylglucosamidase (h7, h8) and 4 isoforms of Gloverin-like proteins (h4, h18, h22, h23) related to silkworm innate immunity were identified (Figure 1C,D and Table 1). Nuecin and Gloverin-like proteins belong to of antibacterial peptides of silkworm.25,26 P50 is a kind of Gram-negative-binding protein.27 PGRP is a recognition molecule that can recognize and bind Gram-positive bacteria or fungi. β-N-acetylglucosamidase (GlcNAcase) is a widely distributed enzyme in insect that has an important role in glycoprotein biosynthesis.28 Wherein, two antibacterial peptides were only identified in silkworm fed on artificial diet, while the other 3 proteins were up-regulated. GO analysis and KEGG pathway analysis also confirmed that some hemolymph proteins were involved in silkworm immune process (Figures 2and 3 and Table S2 in Supporting Information). On the basis of our data together with some models for mechanism of microbial infection and subsequent activation in other insect,29-31 we concluded that when silkworms were fed with fresh mulberry leaves, the proper and enough nutrient diet, they could protect themselves with a layer of antimicrobial 5108


secretions on their exterior as well as with abundant red fluorescent protein complex in gut, which is effective to control infection from all kinds of pathogens. In contrast, silkworms could not form the red fluorescent protein complex in midgut when reared with artificial diet, due to the inactivation of chlorophyllase in artificial diet; thus, this weakened their resistance to various invading microbes, which made the larvae more sensitive to infections from Gram-positive bacteria, Gramnegative bacteria or fungi. Upon this infection, the invaded microbes were recognized and attached by PGRP or P50, which induced the activation of the innate immune system and subsequently generated the antimicrobial peptides to restrain bacterial proliferation in silkworm. Effects of Nutrient on the Metabolism of Energy. Two energy metabolism-associated proteins in our experiment were identified as vacuolar ATP synthase subunit B (V-ATPase subunit B) (Figure 1E,F and Table 1, f19) and Arginine kinase (Figure 1E,F and Table 1, f13). Both proteins were downregulated in fat body of silkworm larvae reared on artificial diet. V-ATPase subunit B is the subunit of ATP synthesis V1 sector as its name implies,32 which possesses the activity of ATP hydrolysis and actin binding.33 Arginine kinase (Arg N-phosphotransferase), the analog of creatine kinase in vertebrates, can catalyze arginine to form phosphoarginine,34 a vital energy reserve, and plays a key role in the interconnection of energy production and utilization in invertebrates.35 The proteomic data demonstrated that the metabolism of energy in the silkworms fed on artificial diet was less active than that in

Comparative Proteomic Analysis of the Domesticated Silkworm

research articles

Figure 2. Classification of the differentially expressed proteins in various tissues. These proteins were classified into 3 main categories or 26 subcategories. Total number of matched GOs responding to differentially expressed proteins of midgut, hemolymph, fat body and posterior silk gland reached 76, 67, 61 and 46, respectively.

Figure 3. Classification of KEGG pathways related to the differentially expressed proteins in various tissues. The related pathways were classified into 5 main categories, 21 subcategories or 58 basic categories according to KEGG. The positive number represented the total number of all up-regulated proteins involved in the same pathway, while the negative number represented that of downregulated proteins.

silkworms reared on fresh mulberry leaves. Therefore, less energy could be saved and be converted for other metabolic

processes like normal growth and development as well as silk synthesis. In addition, GO analysis and KEGG pathway analysis Journal of Proteome Research • Vol. 7, No. 12, 2008 5109

research articles also showed that some functional pathways such as signal transduction, cell communication and endocrine system were down-regulated (Figures 2 and 3 and Table S2 in Supporting Information), which may be responsible for reduced expression of vacuolar ATP synthase subunit B and Arginine kinase. Effects of Nutrient on Silk Synthesis. Ribosomal protein SA is a vital component of eukaryotic ribosome 40S subunit and plays an important role in modulating activity of ribosome.36 EF-2 is involved in translocation step of the eukaryotic polypeptide chain elongation and selectively binds to the pretranslocational ribosome.37 EF-1γ belongs to a subunit of silk gland EF-1 L (the lighter form) and can facilitate the exchange of EF1R bound GDP for GTP together with EF-1β and combine glutathione.38 AspAT, a vital enzyme that catalyzes the reversible reaction between a-ketoglutarate and oxaloacetate, has been proposed to serve as a strategic link between the carbohydrate and protein metabolism along with other transaminases.39 ERp57 is a protein disulfide isomerase-related polypeptide that is specifically involved in the modulation of glycoprotein folding.40 As a highly conserved protein, PHB has various roles in maintaining function of mitochondria, protection against senescence, tumor suppressor, regulation of cell cycle progression and apoptosis.41 This study identified 6 silk synthesis-related proteins in posterior silk gland including Ribosomal protein SA (s2), Elongation factor 2 (EF-2) (s9), Elongation factor 1 gamma subunit (EF-1γ) (s4, s5), Aspartate aminotransferase (AspAT) (s10), ERp57 (s1) and Prohibitin protein (PHB) (s6) (Figure 1G,H and Table 1). All these 6 proteins were down-regulated in silkworms reared on artificial diet. These results suggested that, in the posterior silk gland of silkworms fed on artificial diet, the decreased expression of chaperone proteins PHB may inhibit the normal function of silk gland cells. In addition, the low expression of ERp57 prevented various glycoproteins from being folded and forming all kinds of functional molecules. Furthermore, down-regulated expression of ribosomal protein SA, EF-2 and EF-1γ could reduce the protein synthesis ability. Moreover, the decreased AspAT may reduce the transformation from carbohydrate to all kinds of amino acids. We also found that many pathways such as biotin metabolism, lysine degradation, pyrimidine metabolism, alanine and aspartate metabolism, alkaloid biosynthesis, arginine and praline metabolism, cysteine metabolism, glutamate metabolism and tyrosine metabolism were down-regulated in the silkworms fed on artificial diet according to the GO analysis and KEGG pathway analysis (Figures 2 and 3 and Table S2 in Supporting Information), most of which were related to silk synthesis. Finally, we concluded that silk synthesis in the silkworm was affected by nutrient. Gene-Nutrient Interactions. Genes can interact not only with other genes, but also with environmental factors. Many reports have shown that the interactions between environmental factors including nutrient and genes have close relations with economic traits of crops, economic traits of animals, and human diseases.41-44 Nutrient is regarded as a vital environmental factor and studies on gene-nutrient interactions have become increasingly documented in the literature recently.46,47 In this study, we also found that diet, as a kind of nutrient form, could alter the expression of proteins related to immune system, digestion and absorption of nutrient, energy metabolism and silk synthesis, which may be caused by gene-nutrient 5110


Zhou et al. interaction, therefore, affecting the growth and development, quality and yield of cocoons, and resistance to pathogens in silkworm.

Conclusion In this work, we employed the comparative proteomic approach to investigate the proteome of midgut, hemolymph, fat body and posterior silk gland of the silkworms reared on fresh mulberry leaves and on artificial diet, respectively. The results confirmed gene-nutrient interaction at protein level and suggested that the poor nutrient absorption, weakened innate immunity, decreased energy metabolism and reduced silk synthesis are the main reasons for poor filament quality and low yield of cocoons, low survival rate of young larvae and weakened resistance to various invading microbes in the silkworms fed on artificial diet. The results also provided some useful information for the relative studies. However, further studies such as genomic investigation, systematic biology-based researches and other extensive proteomic studies are necessary to elucidate more aspects in this area.

Acknowledgment. This works was supported by the National Basic Research Program of China (Grant No. 2005CB121000), National Hi-Tech Research and Development Program of China (Grant No. 2006AA10A118), National Postdoctoral Fund of China (Grant No. 20070411196), and Doctoral Fund of Ministry of Education of China (Grant No. 20070335148). We thank Mr. Li Jun (Jiangsu University, China) for his help on protein analyses with MALDI-TOF/TOF MS. We also thank Professor Jian-ke Li (Institute of Apicultural Research, CAAS, China) and Mr. Hossein Hosseini (Zhejiang University, China) for their revision of our manuscript. Supporting Information Available: Tables of total proteins and differentially expressed proteins on 2-DE, and classification of KEGG pathways related to the differentially expressed proteins in various tissues; figures of MALDI-TOF and confirming MALDI-TOF/TOF spectra of 76 differentially expressed protein spots. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Nagaraju, J.; Goldsmith, M. R. Silkworm genomics-progress and prospects. Curr. Sci. 2002, 83 (4), 415–425. (2) Tamura, T.; Thibert, C.; Royer, C.; Kanda, T.; Abraham, E.; Kamba, M.; Komoto, N.; Thomas, J. L.; Mauchamp, B.; Chavancy, G.; Shirk, P.; Fraser, M.; Prudhomme, J. C.; Couble, P. Germline transformation of the silkworm Bombyx mori L. using a piggyback transposonderived vector. Nat. Biotechnol. 2000, 18 (1), 81–84. (3) Tomita, M.; Munetsuna, H.; Sato, T.; Adachi, T.; Hino, R.; Hayashi, M.; Shimizu, K.; Nakamura, N.; Tamura, T.; Yoshizato, K. Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nat. Biotechnol. 2003, 21 (1), 52–56. (4) Hamamura, Y.; Hayashi, K.; Naito, K. Food selection by silkworm larvae, Bombyx mori, β-sitosterol as one of the binding factors. Nature 1961, 190 (4779), 880–881. (5) Ito, T. Application of artificial diets in sericulture. Jpn. Agric. Res. Q. 1980, 14, 119–136. (6) Kataoka, K.; Imai, T. Cocoon quality and physiological properties of the cocoon filament produced by silkworms reared on mulberry leaves and on an artificial diet. J. Seric. Sci. Jpn. 1986, 55 (2), 112. (7) Grzelak, K. Control of expression of silk protein genes. Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 1995, 110 (4), 671– 681. (8) Nabby-Hansen, S.; Waterfield, M. D.; Cramer, R. Proteomics-postgenomic cartography to understand gene function. Trends Pharmacol. Sci. 2001, 22 (7), 376–384. (9) Gorg, A.; Weiss, W.; Dunn, M. J. Current two-dimensional electrophoresis technology for proteomics. Proteomics 2004, 4 (12), 3665–3685.

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Comparative Proteomic Analysis between the Domesticated Silkworm

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