Proteomic Profiling of the Silkworm Skeletal Muscle Proteins during Larval-Pupal Metamorphosis Pingbo Zhang,† Yoichi Aso,‡ Hiroyuki Jikuya,# Takahiro Kusakabe,‡ Jae Man Lee,‡ Yutaka Kawaguchi,‡ Kohji Yamamoto,† Yutaka Banno,† and Hiroshi Fujii*,† Institute of Genetic Resources, Kyushu University, Fukuoka 812-8581, Japan, Faculty of Agriculture, Kyushu University, Fukuoka, Japan, and Bio-Architecture Center, Kyushu University, Fukuoka, Japan Received February 8, 2007
The silkworm is a typical holometabolous insect going through drastic morphological changes upon metamorphosis from larvae to pupae. Comprehensive studies focusing on the changes help elucidate understanding of a biogenic mechanism. Here, we report the initial profile of the intersegmental muscle (ISM) proteins of the silkworm during larval-pupal metamorphosis. In total, 258 protein spots were resolved by two-dimensional gel electrophoresis (2-DE). Fifty-seven larval proteins were identified, where 3 proteins were exclusively detected in larval samples. Fifty-four other proteins were common in pupal samples. Of these, 12 proteins belonging to the contractile apparatus, metabolism, regulation, and signal transduction were altered in their contents during the metamorphosis from larvae to pupae. Three pupa-defective proteins were identified as isoforms of troponin I, followed by an immunoblotting validation. This data will be helpful in understanding the biochemistry of an insect ISM. Keywords: Bombyx mori • Insect skeletal muscle • Metamorphosis • Two-dimensional gel electrophoresis • Matrixassisted laser desorption/ionization-time-of-flight mass spectrometry • Larva-specific protein
Introduction Proteomics is a rapidly advancing field of study relevant to protein expression including post-translational modification, protein-protein interaction, and intracellular compartmentalization.1-3 Recently, proteomic technology, in particular twodimensional gel electrophoresis (2-DE) in conjunction with mass spectrometry, has been used to catalog the proteins expressed in skeletal muscle tissue of humans,4 mice,5 rats,6 bovines,7 rabbits,8 chickens,9 and fish.10 However, most of the data is available on samples from vertebrate individuals, and an extensive variety of different muscle protein species are presented in detail. Although proteomic technology has also been successfully utilized in the characterization of the Drosophila indirect flight muscle proteome,11 this approach has not been previously applied to insect skeletal muscles (intersegmental muscles, ISM). The silkworm, Bombyx mori, is a typical holometabolous insect going through drastic morphological changes upon metamorphosis from larvae to pupae. ISM tissues span each of the abdominal segments, and their contractions allow the caterpillar to move forwardly. During larval-pupal metamorphosis, several larval tissues including muscles are destroyed and adult tissues are generated. For ISM tissues, the transition of larval-pupal metamorphosis starts from the early pupation, for example, day 1 of pupal stage. Although morphologic * To whom correspondence should be addressed. Institute of Genetic Resources, Kyushu University, Fukuoka 812-8581, Japan. Tel: (092) 621-4991. Fax: (092) 621-1011. E-mail:
[email protected]. † Institute of Genetic Resources, Kyushu University. ‡ Faculty of Agriculture, Kyushu University. # Bio-Architecture Center, Kyushu University. 10.1021/pr070071y CCC: $37.00
2007 American Chemical Society
changes of the early pupal ISM are not obvious compared with the larval ISM, important biochemical characteristics of ISM proteins are altered at this stage. In fact, the term “programmed cell death” was originally coined in 1965 to describe the degeneration of ISM during silkworm metamorphosis.12 Since that, the silkworm ISM has served as a model system in studying skeletal muscle atrophy and programmed cell death, becuase this system has unique aspects that are not observed in vertebrates. First, it does not suffer the limitation of multiple fiber types observed with vertebrate skeletal muscle tissue,13 and it is easy to obtain excellent specimens with a nearly homogeneous fiber type. Second, it allows a large amount of muscle samples to be taken as the life cycle of the silkworm is very short, about 50 days in one life cycle. In contrast, muscle disorders in vertebrates occur during a long period of atrophy from several months to years. Therefore, ISM is ideal in use for a study of various muscle disorders. Despite this importance, ISM proteins have been less well characterized during larval-pupal metamorphosis until recently. The aim of this study was to define ISM proteins present in larvae and pupae of the silkworm using a proteomic approach. ISM proteome analysis not only provides the basic information for an understanding of the broad properties and multifarious functions of skeletal muscle proteins, but also is an important step toward the development of an insect model system for human muscle disorders.
Materials and Methods Insects and Sample Preparation. Experiments were carried out on skeletal muscles of the final-instar larvae and pupae of the silkworm strain p22, an international standard strain of the Journal of Proteome Research 2007, 6, 2295-2303
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research articles silkworm.14 Larvae were reared on fresh mulberry leaves according to a standard technique.15 On day 5 of the fifth instar, the isolated 30 silkworms were grouped into three groups (10/ each). Larvae from each group were opened along the middorsal line, and the internal organs such as the silk gland, midgut, and fat body were removed. Tracheae were carefully snipped away to expose the laterally ventral ISM. In B. mori, the larval abdomen is constructed of 11 segments (A1-11). ISMs were carefully dissected from only A1 to A8 by means of a dissecting microscope because, in A9-11, the muscle is smaller. After pupation, the ISMs in the first two and last two abdominal segments die, whereas the muscles in the middle four segments keep completely intact and persist throughout metamorphosis. Therefore, beginning on the first day of the larval pupation, only fibers in A4 to A7 were sampled. The collected tissues were rinsed in ice-cold saline solution, and rapidly frozen in liquid nitrogen after blotting on a filter paper. Two-Dimensional Gel Electrophoresis. Frozen muscle specimens (fresh weight 0.45 g, each group of 10 animals) were ground in a buffer containing a protease inhibitor cocktail as previously described,16 and centrifuged at 15 000g for 15 min. The concentration of the resulting supernatant was measured according to the instruction of Bio-Rad protein assay kit (BioRad, Hercules, CA) with the Bio-Rad model 550 microplate reader. IPG strips (18-cm nonlinear pH 3-10, Amersham Pharmacia Biotech) containing 100 or 800 µg of proteins were rehydrated in a buffer consisting of 8 M urea, 2 M thiourea, 4% CHAPS, 30 mM 1,4-dithioerythreitol, and 2% pharmalytes 3-10, then subjected to IEF in an Ettan IPGphor isoelectric focusing unit (Amersham Pharmacia Biotech) at 50 V for 12 h, 500 V for 1 h, 1000 V for 1 h, 4000 V for 1 h, 6000 V for 1 h, and 8000 V for 10 h (a total of 90 000 Vh). For the second dimension, IPG strips were equilibrated in 6 M urea, 2% SDS, 30% glycerol, and 50 mM Tris-HCl, pH 8.8, reduced with 1% DTT, and alkylated with 4% iodoacetamide. Then, the strips were subjected to the second-dimensional gel electrophoresis under denaturing conditions (SDS-PAGE) by transferring onto 15% polyacrylamide gels (240 mm × 240 mm × 1 mm) with 0.27% SDS, overlaid with 5% stacking gels. Electrophoresis was performed in a vertical slab gel apparatus (Eido, Japan) at 20 mA per gel overnight at 4 °C.17 Protein Visualization and Image Analysis. Protein spot patterns were visualized by Coomassie blue G-250 staining8 or by silver staining.18 The resulting 2-D protein patterns were photographed at 1024 × 1024 pixel resolution using a Canon digital camera and assigned the apparent Mr and pI values using complex 2-D protein markers (pI 4-7, Daiichi Pure Chemicals, Tokyo, Japan, and pI 3-10, Serva Electrophoresis, Heidelberg, Germany). Digitized gel images were analyzed using the PDQuest software 7.40 (Bio-Rad Laboratories, Hercules, CA) for spot detection, quantification, and comparative and statistical analysis. The PDQuest software models protein spots mathematically as a 3-D Gaussian distribution and determines maximum absorption after raw image correction and background subtraction. To accurately compare spot quantities between gels, each 2D spot from Coomassie-stained gels was normalized by calculating the relative volume (%V) (V ) integration of OD over the spot area; %V ) Vsingle spot/ Vtotal spot). The Student’s t test was performed, comparing protein expression in the larval and pupal muscles (n ) 10, p e 0.05). Proteins with more than 2-fold change in larval ISM compared with pupal ISM were considered differentially expressed. 2296
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Protein Identification. Protein spots that were found to have interesting patterns of differential expression and a wide range of protein landmarks in the larval and pupal 2-D maps were subjected to identification of proteins using peptide mass fingerprinting as previously described.16 Protein spots were excised as circular plugs 2-3 mm in diameter, and transferred to 1.5 mL Eppendorf tubes. A piece of gel was separately cut out from a protein-free region, treated as above, and used for a control to identify autoproteolysis products. In-gel tryptic digestion was performed manually as previously described.17 Briefly, Coomassie blue-stained gel pieces were first destained by washing with 50 µL of 50% methanol and 10% acetic acid, followed by three washes with 100 µL of Milli-Q water. The gel pieces were then dehydrated with 100% acetonitrile for 5 min, and dried by centrifugation for 30 min under vacuum Speedvac Concentrator (Savant, Holbrook, NY), before addition of 10 µL of modified trypsin (5-10 ng/µL, Promega, Madison, WI) in ammonium bicarbonate (25 mM). After incubation at 37 °C for 17 h, the products were recovered by extraction (twice) with a solution consisting of ammonium hydrogen carbonate (25 mM), TFA (0.1%), and acetonitrile (60%). Extracts were lyophilized and resuspended in 5 µL of TFA (0.1%) and acetonitrile (10%), and 1 µL aliquots were removed for MALDI-TOF mass spectroscopy. MALDI mass spectra were recorded with a PerSeptive Biosystem MALDI-TOF Voyager-DE RP Biospectrometry workstation (Applied Biosystems, Framingham, MA). For acquisition of a mass spectrometric peptide map, aliquots (1 µL) from the in-gel tryptic digestion were premixed with 1 µL of 10 mg/mL CHCA (R-cyano-4-hydroxy-cinnamic acid in 35% acetonitrile and 0.1% TFA) matrix solution, dispensed onto the sample support, allowed to dry, and desalted by brief washing with a few microliters of 0.1% TFA. Measurements were externally calibrated with Sequazyme Peptide Mass Standards kit including angiotensin II ([M + H]+1046.54) and angiotensin I ([M + H]+1296.68) (PerSeptive Biosystms, Framingham, MA), and internally re-calibrated with peptide fragments arising from autoproteolysis of trypsin. The peptide masses were searched against the Swiss-Prot.2005.01.06 database and the NCBInr.2005.01.06 database using Aldente (http://au.expasy.org/tools/aldente/) search tool. The following parameters were used in all searches: the maximum number of missed cleavages allowed, 1; the mass tolerance, 1 Da; minimum peptides required to match, 4; and the monoisotopic masses of observation peaks were used to match the calculated monoisotopic fragment masses for protein identification. Possible covalent modifications considered in the search procedure were carbamidomethylation of cysteine, conversion of peptide N-terminal glutamine to pyroglutamate, oxidation of methionine, and acetylation of protein N-terminus. Immunoblotting Analysis. One-dimensional immunoblotting analysis was carried out with larval and pupal muscle proteins. Aliquots (50 µg protein) were resolved by SDS-PAGE and electroblotted onto a Hybond ECL (nitrocellulose) filter in 25 mM Tris-HCl buffer, pH 8.8, containing 0.19 M glycine, 0.1% SDS, and 20% methanol at 120 mA for 1 h. The membranes were soaked in TBST (20 mM Tris-HCl buffer, pH 7.6, 137 mM NaCl, and 1% Tween-20) containing 5% skimmed milk (Snow Brand Milk Products, Tokyo) for 1 h at room temperature, and probed with the rabbit anti-troponin I (Tn I) polyclonal antibody (1:2000 dilution) (catalog no. 4002, Cell Signaling Technology, Danvers, MA) in TBST for 1 h at room temperature. Primary antibodies were reacted with horseradish peroxidase-
Proteomic Profiling of the Silkworm Skeletal Muscle Proteins
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Figure 1. Representative two-dimensional electrophoretic image obtained from the silkworm larval and pupal skeletal muscles. 2-DE was performed using a pH range of 3-10 in the first dimension and SDS-PAGE (15%T) in the second. Protein loading was 800 µg, and the gel was stained using Coomassie blue G-250. Calibration of Mr and pI was performed using PDQuest 7.40 software. (A) Larval skeletal muscles; (B) pupal skeletal muscles. Fifteen protein spots are indicated by numbers, showing statistically significant differences on expression level between larvae and pupae (n ) 10, p e 0.05). Of these, 4 spots indicated by up-triangles are up-regulated in pupal ISM compared with larval ISM, while 8 spots indicated by down-triangles are down-regulated; 3 spots (spot nos. 7, 9, and 10) indicated by open circles are exclusively detected in larval samples.
Figure 2. Statistically significant differences on expression level between larvae and pupae. The PDQuest software 7.40 (Bio-Rad Laboratories, Hercules, CA) was used for spot detection, quantification, comparison, and statistical analysis. The Student’s t test was performed, comparing protein expression in the larval and pupal muscles (n ) 10, p e 0.05). Fifteen muscle proteins exhibited a marked change (g2-fold) on their expression levels. Spot nos. 7, 9, and 10 were present in samples of larval muscles, but absent in those of pupal muscles.
labeled anti-rabbit IgG donkey serum (1:5000 dilution) for 1 h, and visualized using the super signal enhanced chemiluminescence kit from Amersham Biosciences, U.K.16
Analysis of Subcellular Localization. The subcellular localization of ISM proteins was predicted using established machine learning techniques for animal protein sequences at the Journal of Proteome Research • Vol. 6, No. 6, 2007 2297
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Figure 3. Peptide mass fingerprint for troponin I. MALDI-TOF MS spectrometry was done with in-gel-digested peptides of spot no. 7 from 2-DE gels of larval skeletal muscle proteins. Two of the major trypsin self-peptides are designated by the letter T and used for the internal calibration. A total of 17 monoisotopic peptide masses was submitted to the database search under the conditions described in Materials and Methods, and 14 peptides matched the troponin I, fast skeletal muscle, with 67% sequence coverage. Table 1. Results of NCBInr.2005.01.06 Database Search for the Identification of Spot 7 submitted database ∆mass mass mass (Da)
568.349 615.649 637.351 657.216 672.265 688.174 1024.003 1308.400 1333.346 1451.524 1467.511a 1587.623 1619.610 2044.930 a
568.261 615.419 637.268 657.222 672.404 688.374 1024.477 1308.610 1333.613 1451.760 1467.755 1587.809 1619.820 2045.062
0.088 0.230 0.083 -0.006 -0.139 -0.200 -0.474 -0.210 -0.267 -0.236 -0.244 -0.186 -0.210 -0.132
peptide
position
(E)EEKY(D) (E)IKVQK(S) (K)VCMDL(R) (R)MSADAM(L) (R)ANLKQV(K) (K)ERDLR(D) (L)EDMNQKLF(D) (K)SSKELEDMNQK(L) (S)LPGSMAEVQELC(K) (R)MSADAMLKALLGSK(H) (R)MSADAMLKALLGSK(H) (G)SKHKVCMDLRANL(K) (S)VMLQIAATELEKEE(G) (K)QVKKEDTEKERDLRDVG(D)
76-79 83-87 132-136 116-121 138-143 151-155 93-100 88-98 53-64 116-129 116-129 128-140 20-33 142-158
1 Met-ox (oxidation of methionine).
Proteome Analyst Specialized Subcellular Localization Server (http://www.cs.ualberta.ca/∼bioinfo/PA/Subcellular/).19
Results and Discussion Image Analysis of ISM Proteins. In this study, we focused on defining the proteins present in the silkworm ISM during larval-pupal metamorphosis. Muscle extracts from larvae and pupae were repeatedly subjected to 2-DE analysis. Figure 1 shows a representative 2-D electropherogram of ISM proteins obtained from larvae and pupae of the silkworm. A majority of the ISM proteins were present in the isoelectric point (pI) range of 5-9 and in the molecular mass (Mr) range of 14-100 kDa. In total, 258 ( 5 proteins were resolved per 2-D gel of ISM proteins. Densitometric image analysis of CBB-stained 2-D gel images from larval and pupal ISM proteins revealed differentially displayed or expressed proteins. Figure 2 shows a total of 15 proteins that were found to be altered in abundance during larval-pupal metamorphosis. Of these, 12 spots (spot nos. 2, 3, 4, 14, 15, 17, 21, 28, 33, 41, 48, and 56) 2298
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Figure 4. One-dimensional immunoblotting analysis of troponin I. Western blotting analysis of troponin I was performed on larval and pupal muscle homogenates. Frozen samples from the larval and pupal muscles were solubilized in lysis buffer containing 4% SDS, 125 mM Tris-HCl, pH 8.8, 40% glycerol, and 0.5 mM PMSF. After centrifugation, the samples (100 µg of protein) were separated on a 15% T SDS-PAGE gel and transferred onto nitrocellulose membranes. Blocked membranes were probed with anti-troponin I polyclonal antibodies (1:2000, Cell Signaling Technology, Danvers, MA), followed by horseradish peroxidaselabeled secondary antibody (1:5000, Amersham Biosciences, U.K.), and by detection with the Super Signal enhanced chemiluminescence kit (Amersham Biosciences, U.K.). Lane M, molecular weight markers; lanes A and B, larval and pupal muscle proteins stained by silver staining, respectively; lanes C and D, immunoblotting against anti-troponin I antibodies in larval and pupal muscle proteins, respectively.
were common with larval and pupal ISM samples yet regulated in expression. With respect to the quantitative variation, 8 spots (spot nos. 2, 3, 4, 14, 15, 17, 28, and 48) were found to have more than a 2-fold average down-regulation in pupal ISM compared with larval ISM, while 4 spots (spot nos. 21, 33, 41, and 56) were up-regulated. Regardless of the Coomassie blue
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Proteomic Profiling of the Silkworm Skeletal Muscle Proteins Table 2. Identification of Proteins from the Insect Skeletal Muscle of the Silkworma spot no.
protein name
5
24
Troponin C, slow skeletal and cardiac muscles (TN-C) Troponin I, fast skeletal muscle Troponin I, slow skeletal muscle Troponin I, slow skeletal muscle Myosin light chain 1, embryonic muscle/atrial isoform Myosin light chain 1, embryonic muscle/atrial isoform Myosin light chain 1, skeletal muscle isoform Myosin light chain 1, skeletal muscle isoform Myosin light chain 1, slow-twitch muscle B ventricular isoform Tropomyosin 2
25
Tropomyosin 2
29
Tropomyosin 1
27
CNN2
Calponin
CNN1
Calponin
34
Calponin-2 (Neutral calponin) (Calponin H2, smooth muscle) Calponin 1 (Basic calponin) (Calponin H1, smooth muscle) Actin, muscle A2
None
Actin
35
Actin, muscle A1
None
Actin
36
Actin, muscle A1
None
Actin
38
Tubulin beta chain (Beta tubulin) Tubulin alpha chain (Alpha tubulin) Myosin heavy chain, muscle Myosin heavy chain, muscle Myosin heavy chain, muscle
None
Tubulin
None
Tubulin
Mhc
Myosin head-like domain Myosin head-like domain Myosin head-like domain
7c 9c 10c 11 12 13 14d 15d
28d
59 52 53 54
30 32
33e
39 51 47
1 42 46
Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) Phosphoribosyl pyrophosphate synthetase-associated protein 2 1) Hydroxyacid oxidase 1 (HAOX1) (GOX) (EC 1.1.3.15) 2) Peptide-O-fucosyltransferase (EC 2.4.1.221) Pyruvate dehydrogenase kinase isoform 1 (EC 2.7.1.99) Glycogen phosphorylase Trehalase precursor (glycosyl hydrolase)
ATP synthase coupling factor 6 (EC 3.6.3.14) ATP synthase beta chain mitochondrial precursor ATP synthase alpha chain, mitochondrial precursor
17d 1) 60S ribosomal protein L9 2) Metalloproteinase inhibitor 3 18 1) 60S ribosomal protein L10a-2 2) Transcription cofactor HES-6 22 Transcription initiation factor TFIID subunit 9 (p42) 23 Transcription elongation factor A protein 2 40 Elongation factor 1-alpha (EF-1-alpha)
gene name
gene family
subcellular locationb
source
Contractile Proteins Human P63316/54042075
TNNC
Troponin C
Cytoplasm 100%
TNNI2
Troponin I Troponin I
Human
TNNI1
Troponin I
MLC1 (MYL4)
EF-hand calcium binding domains
Cytoplasm 98.8% Cytoplasm 98.8% Cytoplasm 98.8% Cytoplasm 100%
Rabbit
TNNI1
MLC1 (MYL4)
EF-hand calcium binding domain
MYL1
EF-hand calcium binding domain EF-hand calcium binding domain EF-hand calcium binding domain
MYL1 MYL3
Tm2 Tropomyosin (CG4843) Tm2 Tropomyosin (CG4843) Tm1 TmII Tropomyosin
Mhc Mhc
Gpdh (CG9042) PRPSAP2
3.10E+03 18403/4.04
18400/3.60
P02643/401209
14
21200/7.90
10
2.31 E+04 21693/9.60
22000/8.85
Human
P19237/1351298
10
2.31 E+04 21693/9.60
22000/8.97
Human
P12829/127138
8
67 (121/181AA’s) 37 (69/186AA’s) 37 (69/186AA’s) 39 (77/197AA’s)
8.50E+06 21083/8.86
P19237/1351298
2.35 E+03 21565/5.00
24600/4.38
Cytoplasm 100%
Human
P12829/127138
8
39 (77/197AA’s)
2.35 E+03 21565/5.00
22600/4.53
Cytoplasm 100% Cytoplasm 100% Cytoplasm 100%
Human
P05976/127128
14
21700/4.80
P05976/127128
18
1.18 E+04 21145/5.00
21200/5.00
Human
P08590/127149
8
38 (74/194AA’s) 54 (105/194AA’s) 38 (75/195AA’s)
2.48E+03 21145/5.00
Human
1.72 E+03 21932/5.00
21200/5.05
Cytoplasm 99.6% Cytoplasm 99.6% No prediction Cytoplasm 100%
Fruit fly
P09491/136072
11
32300/5.15
P09491/136072
7
6963
32981/4.80
32900/5.21
Fruit fly
P06754/76789674
21
9.79 E+05 32762/4.70
32800/5.10
Human
Q99439/6226844
10
42 (122/284AA’s) 32 (92/284AA’s) 56 (162/285AA’s) 31 (97/309AA’s)
1.19 E+05 32981/4.80
Fruit fly
1.01 E+03 33697/ 6.90
33700/7.35
Cytoplasm 100%
Human
P51911/829431
11
36 (108/297AA’s)
1.59 E+03 33171/9.10
33200/8.25
Cytoplasm 100% Cytoplasm 100% Cytoplasm 100% Cytoplasm 88% Cytoplasm 88% Cytoplasm 100% Cytoplasm 100% Cytoplasm 100%
Silkworm
P07837/113231
11
42000/5.07
P07836/113216
13
7.83 E+04 41876/5.30
41900/5.34
Silkworm
P07836/113216
12
1.56 E+04 41876/5.30
41900/5.69
Silkworm
P41385/174602
19
4.71 E+05 50335/4.70
50300/5.33
Silkworm
P52273/1729841
14
39 (150/376AA’s) 58 (220/376AA’s) 47 (177/376AA’s) 46 (207/450AA’s) 34 (156/450AA’s) 34 (682/1962AA’s) 44 (882/1962AA’s) 14 (277/1962AA’s)
1.22 E+04 41803/5.30
Silkworm
1.31 E+03 49906/5.00
50600/5.48
Fruit fly
P05661/0455497
20
Fruit fly
P05661/20455497
30
Fruit fly
P05661/0455497
13
Metabolism I. Carbohydrate and ATP Metabolism NAD-dependent Cytoplasm Fruit fly P13706/4286124 glycerol-3-phosphate 100% dehydrogenase Ribose-phosphate Cytoplasm Human O60256/4418492 pyrophosphokinase 88% Peroxisome 100%
C15C7.7
Glycosyltransferase 68 PDK/BCKDK protein kinase
Endoplasmic Nematode Q18014/0392863 reticulum 100% Mitochondrion Human Q15118/3183117 100%
Glycogen phosphorylase glycosyl hydrolase 37
Cytoplasm 99.8% Plasma membrane
ATPase subunit F6 ATPase alpha/beta chains
Metabolism I. Carbohydrate and ATP Metabolism Mitochondrion Fruit fly P21571/14690 100% Mitochondrion Fruit fly Q05825/7606749 100%
ATPase alpha/beta chains
Mitochondrion Fruit fly 100%
ATPsyn-f6 (CG4412) ATPsynbeta CG11154 Blw CG3612
RpL9 (CG6141) Timp3 RpL10Ab (CG7283) HES6 TAF40 (CG6474)
Ribosomal protein L6P Protease inhibitor I35 tRNA-binding Helix-loop-helix domain DNA-binding
MOWSE theoretical estimated score Mr(Da)/pI Mr(Da)/pI
34 (55/161AA’s)
Hydroxy acid dehydrogenase
GlyP CG7254 None
matched peptide cover (%)
10
HAOX1 GOX1
PDK1
no. of peptide matched
accession no. Swiss-Prot/ NCBInr
Human
Q9UJM8/13124294
224483/5.90 224500/6.70
21 (78/363AA’s)
4.13E+03 39685/6.20
39700/6.21
10
33 (124/369AA’s)
2.64 E+03 40926/7.10
41000/7.10
8
17 (66/370AA’s)
91.2
40925/8.20
41000/7.47
7
14 (54/389AA’s) 24 (106/436AA’s)
1.80 E+03 41776/7.72
41000/7.47
3.11E+04 49245/8.90
50200/8.70
50 (425/844AA’s) 42 (246/579AA’s)
1.63 E+04 96997/6.10
97000/6.89
2.03 E+04 66542/4.90
66500/5.59
35 (38/106AA’s) 44 (226/505AA’s)
85.9
11936/9.00
11900/8.50
3.89 E+04 54108/5.10
54200/5.42
Fruit fly
Q9XTL9/4916636
27
Silkworm
P32358/18128
15
7 18
II. Protein Metabolism No Fruit fly P50882/4286156 prediction Extracellular Rat P48032/1351251 100% No Fruit fly Q9VTP4/51701866 prediction Nucleus 100% Human Q96HZ4-2/50400609
3842
9
13
P35381/5921205
2.21E+04 224483/5.90 224500/5.90 3.49E+04 224483/5.90 224500/6.33
18
42 (234/552AA’s)
1.01 E+04 59422/9.10
59000/8.14
9
18 (35/190AA’s) 20 (42/211AA’s) 25 (55/217AA’s) 14 (31/224AA’s) 20 (56/278AA’s)
6.00 E+04 21392/9.70
22000/9.35
7 10 6
Nucleus 100%
Fruit fly
Q27272/2498980
9
TCEA2
TFS-II
Nucleus 100%
Human
Q15560/28380177
13
None
GTP-binding elongation factor f
Cytoplasm 100%
Silkworm
P29520/232028
16
33 (100/299AA’s) 38 (176/463AA’s)
3371
21687/9.21
22000/9.35
682
24274/9.90
24300/9.43
2.60 E+04 24314/11.9
24300/9.43
145
29315/9.40
29300/9.05
107
33601/9.30
33600/9.06
5.47 E+04 50373/9.20
50400/9.20
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Table 2. (Continued) spot no. 44
protein name
gene name
gene family
Cytochrome P450 4p1 (CYPIVP1) Cytochrome P450 4p3 (EC 1.14.-.-)
Cyp4p1
Cytochrome P450
Cyp4p3 CG10843
Cytochrome P450
Myosin regulatory light chain isoform1 (Larval-non-IFM) Myosin regulatory light chain isoform2 Myosin regulatory light chain isoform2 Myosin regulatory light chain 2 (MLC-2) Synaptosomal-associat d protein 25 (SNAP-25)
Mlc1 (CG5596)
EF-hand calcium binding domain
Mlc1 (CG5596) Mlc1 (CG5596) Mlc2 (CG2184) SNAP25 (CG40452)
EF-hand calcium binding domain EF-hand calcium binding domain EF-hand calcium binding domain SNAP-25
20
Synaptosomal-associat d protein 29 (SNAP-29)
SNAP29
SNAP-25
26
Soluble NSF attachment protein (SNAP) Calpactin II (Annexin A1)(Annexin I) 1). G protein-coupled receptor GPR85 2). Polysialyltransferase-1 Synaptotagmin (p65)
Snap (CG6625) ANXA1 LPC1 GPR85;
SNAP
58 2d 3d 4d 6 8
31 41e
43 49 19 50
56e 21e 37 48d 55 57
MAP/microtubule affinity- regulating kinase 3 Hippocalcin-like protein 1 (Calcium-binding protein-1) Muscle-specific calcium-activated neutral protease 3 (Calpain 3) Calmodulin-dependent calcineurin A1 subunit Low molecular 30 kDa lipoprotein PBMHPC-23 precursor Wnt-2b protein precursor (Wnt-13) Maternal effect protein oskar T-complex protein 1, gamma subunit (TCP-1-gamma) T-complex protein 1, alpha subunit (TCP-1-alpha)
Annexin
subcellular locationb
source
accession no. Swiss-Prot/ NCBInr
III. Insect Hormone Metabolism Endoplasmic Fruit fly Q9V558/2643914 reticulum 100% Endoplasmic Fruit fly Q9V559/22096348 reticulum 100% Regulatory Proteins Cytoplasm 100% Fruit fly P06742-1/27923982
no. of peptide matched 14
matched peptide cover (%)
MOWSE theoretical estimated score Mr(Da)/pI Mr(Da)/pI
31 1.33 E+04 59433/6.80 (164/513AA’s) 45 2.15 E+04 59652/6.10 (234/515AA’s)
60000/6.69
11
47 (74/155AA’s)
1.60E+03
17524/4.30
17500/4.55
47 (74/155AA’s) 47 (74/155AA’s) 40 (88/221AA’s) 44 (95/212AA’s)
2.58E+04
17533/4.40
17400/4.80
3.37E+03
17533/4.40
17500/4.96
1.02E+03
23583/4.70
20600/5.14
71.2
23686/4.50
20600/3.69
28971/5.60
29000/6.00
16
Cytoplasm 100%
Fruit fly
P06742-2/27923982
13
Cytoplasm 100%
Fruit fly
P06742-2/27923982
12
Cytoplasm 100%
Fruit fly
P18432/27183
9
Plasma membrane 99.3% Plasma membrane 99.9% Endoplasmic reticulum 66.4% No prediction
Fruit fly
P36975/548941
10
Human
O95721/6685982
12
Fruit fly
Q23983/18202518
9
Human
P04083/29239
7
52 2.51E+03 (136/258AA’s) 19 (58/292AA’s) 15 (55/346AA’s) 24 (90/370AA’s) 19 (71/359 AA’s) 52 (251/474AA’s) 54 (422/776AA’s)
59700/6.69
310
33000/5.30
33000/5.78
85.7
38715/6.60
38700/6.68
189.0
41995/9.70
42000/9.70
91.2
41295/9.77
42000/9.70
1173
53265/5.70
53300/5.80
1366
86945/9.60
87000/8.66
G-protein coupled receptor 1 Glycosyltransferase 29 Synaptotagmin
Plasma Human membrane 100% Golgi 100% Human
P60893/6397442
12
Q92187/2494834
10
Cytoplasm 100%
Fruit fly
P21521/55584156
16
Ser/Thr protein kinase
Cytoplasm 100%
Human
P27448/29337165
32
HPCAL1 (BDR1)
Recoverin
Cytoplasm 99.4% Human
P37235/20455519
10
17 (33/193AA’s)
157.0
22313/5.20
24600/5.84
Capn3 NCL1
Peptidase C2
Cytoplasm 100%
Human
P20807/1345664
16
28 4409 (231/821AA’s)
94254/5.80
94300/5.80
CanA1
PPP phosphatase
Cytoplasm 100%
Fruit fly
P48456/73920244
10
31 752.0 (182/577AA’s)
64588/6.10
64600/6.66
LP
30 kDa lipoprotein
Developmental and Signaling Proteins Extracellular Silkworm P09338/126419 100%
13
22 (59/264AA’s)
2.65 E+03 30344/8.50
29700/7.79
41 560 43770/9.30 (164/391AA’s) 58 7794 69285/9.30 (356/606AA’s) 27 1.48 E+03 59395/6.40 (150/544AA’s)
PST syt (CG3139) MARK3 CTAK1
WNT2B
Wnt
Osk CG10901 Cctamma CG8977 T-cp1 CG5374
Osk TCP-1 chaperonin TCP-1 chaperonin
Extracellular 100% No prediction Cytoplasm 100% Cytoplasm 100%
Human
Q93097/4424481
10
Fruit fly
P25158/129239
25
Fruit fly
P48605/1729872
10
Fruit fly
P12613/13959710
10
35 874 (198/557AA’s)
59557/6.00
43800/8.80 69300/8.94 59700/6.86 65000/6.89
a Our spot number, protein name, gene name, gene family, subcellular location, source, access numbers in Swiss-Prot.2005.01.06 and NCBInr.2005.01.06 database, matching peptide number, sequence coverage (%), theoretical, and estimated molecular weight (Mr)/isoelectric point (pI) are indicated. b Subcellular location is deduced according to the established machine learning techniques for animal protein sequences at the Proteome Analyst Specialized Subcellular Localization Server (PA-SUB) (http://www.cs.ualberta.ca/∼bioinfo/PA/Subcellular/), c Larva-specific proteins. d Down-regulated proteins in pupal ISM compared with larval ISM, e Up-regulated proteins in pupal ISM compared with larval ISM.
staining or silver staining (data not shown), proteins corresponding to three spots (spot nos. 7, 9, and 10) were present only in the larval ISM yet absent in the ISM of pupal samples, thus, suggesting that they are larva-specific proteins. Identification of ISM Proteins. A representative mass spectrum of troponin I (spot no. 7) is presented in Figure 3, and the result of a database search for this protein is listed in Table 1. A total of 17 monoisotopic peptide masses was submitted to the database search under the conditions described in Materials and Methods, and 14 peptides matched the troponin I, fast skeletal muscle, with 67% sequence coverage. To further verify this developmental biomarker, we also completed the Western blotting analysis of troponin I on larval and pupal ISM homogenates. Figure 4 shows a representative immunoblot for troponin I after SDS-PAGE and electrotransfer from larval and pupal samples. The signal of troponin I is completely absent from the pupal ISM; however, it is easily detected in the larval sample. The gene product of troponin I is part of the thin filament troponin complex, which regulates muscle contraction and muscle development such as myofibrillogenesis and sar2300
Journal of Proteome Research • Vol. 6, No. 6, 2007
comere formation in the Drosophila flight muscle.20 Some mutants of troponin I are known to cause hypercontraction and collapse of the indirect flight muscles in the pupal or early adult stages.21 In humans, troponin I is also a relevant indicator of heart failure.22 As is well-known, silkworms at the larval stage are active in behaviors such as creeping, feeding, and cocooning, whereas pupae keep themselves relatively inactive. Hence, we conjectured that the deletion of these key proteins in pupal individual ISM is consistent with the developmental transition from the larva to pupa. Apart from this result drawn from the standard race of p22, we found in fact that these larva-specific proteins are not dependent on the race of the silkworm, but all of them disappeared from pupal ISM (data not shown). This indicates that troponin I isoforms play an important role in the transition of larval-pupal metamorphosis. Conversely, it is not known whether their disappearances at pupal stages are due to the histolysis or programmed cell death. Nevertheless, comparative results of proteomics conducted by this study will guide us to systemically analyze these larva-specific proteins, using tech-
Proteomic Profiling of the Silkworm Skeletal Muscle Proteins
research articles
Figure 5. Representative two-dimensional electrophoretic image obtained from the silkworm larval skeletal muscles. 2-DE was performed using a pH range of 3-10 in the first dimension and SDS-PAGE (15%T) in the second. Protein loading was 800 µg, and the gel was stained using Coomassie blue G-250. Calibration of Mr and pI was performed using PDQuest 7.40 software. Proteins identified are marked, and spot numbers refer to Table 2.
nologies such as gene cloning and microarray analysis in the future. In addition to 3 larva-specific troponin I proteins, identification of the proteins revealed that the differentially expressed genes that play an essential role in the metamorphosis of larval-pupal ISM tissues (Table 2) were involved in contractile apparatus (spot nos. 14, 15, and 28), metabolism (spot nos. 17 and 33), regulation (spot nos. 2-4, 41, and 56), and signal transduction (spot nos. 21 and 48). To obtain an overview of proteins expressed by ISM, we randomly harvested 57 spots for MS analysis by MALDI-TOF MS, including the differential proteins during larval-pupal metamorphosis (Figure 5). Table 2 summarizes the peptide mass fingerprinting and database searching results. All the proteins listed in Table 2 were the top candidates retrieved by the bioinformatics searches. Each of the identified proteins was classified and listed with categories according to specific homological domains that are shared with proteins in the fruit fly and other vertebrate animals. Proteins were annotated with the gene name, gene family, subcellular localization, source, Swiss-Prot and NCBI accession number, matched peptide number, sequence coverage, MOWSE score, theoretical, and estimated Mr/pI value. All predicted molecular weights and isoelectric points were obtained by using the Compute Mr/pI tool on the ExPASy server, based on amino acid sequences deposited in the Swiss-Prot amino acid sequence database. In the ISM context, we successfully identified 57 spots corresponding to 46 distinct gene products. Most of the ISM proteins identified in the present study are novel to the silkworm, and their homologues are mainly distributed in the fruit fly, human, and other animals. Eight proteins from 4 spots (spot nos. 17, 18, 33, and 41) represented a spot heterogeneity (multiple proteins within a single spot). The identified ISM proteins are of 39 acid proteins (68.4%, pI < 7.0), three times that of 13 basic proteins (22.8%, pI > 9.0). The majority of estimated gel and theoretical Mr/pI values matched quite well. More than 45 proteins (75%) showed the difference of the theoretical and estimated values within ∆Mr < 10% and ∆pI < 0.4. The presence of several isoforms with different Mr and pI
mostly results from post-translational modifications such as phosphorylation, glycosylation, proteolysis, and/or mRNA alternative splicing. ISM Is Enriched in Isoforms. Similar to vertebrate species, the ISM proteome is characteristic of enriched isoforms. A total of 32 ISM proteins (53%) was found to be present as multiple isoforms. The distributions of the proteins presented on 2-D gels are consistent with those already published in vertebrates such as humans,4 rats,6 bovines,7 chickens,9 fish,10 and fruit flies.11,23 We conjectured that a large number of similar regions of these proteins are present in both vertebrates and insects and generate a similar distribution on 2-D gels. With this in mind, we selected some identified ISM isoform sequences and compared them with the homologues of several vertebrate skeletal muscles in humans, rabbits, mice, rats, and the Drosophila indirect flight muscles. The comparative result of homologous sequences is listed in Table 3. Most ISM isoforms share a high degree of homologues of vertebrate skeletal muscles, such as those of the fruit fly’s indirect flight muscles. In general, this is consistent with the characteristics of skeletal muscles. Skeletal muscle, unlike indirect flight muscle, is largely super-contracting muscle. It has a greater ratio of thin-to-thick filament, and the filaments can penetrate the Z-disk during contraction.24 As a result, the expression of different ISM isoforms correlates with differences in muscle fiber type. Compared with other animal skeletal muscles, some silkworm ISM isoforms appear more similar to human homologues. For example, ISM isoforms belong to actins A1 and A2 (spot nos. 34 and 36), calponins 2 and 3 (spot nos. 27 and 50), myosin light chains (spot nos. 12-15), myosin heavy chains I and III (spot nos. 52 and 54), myosin regulatory light chains (spot nos. 2, 3, 4, and 6), SNAP proteins (spot nos. 8, 20, and 26), troponins (spot nos. 5 and 9), and tubulin beta chain (spot no. 38). Overall, the data presented here show that the insect ISM proteins are very similar to those found in mammalian muscles, despite their relatively great divergence in evolution. Accordingly, the insect ISM may provide a unique Journal of Proteome Research • Vol. 6, No. 6, 2007 2301
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Zhang et al.
Table 3. Homology Comparison of Some ISM Proteins among Some Organismsa ISM protein isoforms
spot no. human
accession no./coverage (%) rabbit
mouse
Actin P68032 P62740 P60710 Actin, muscle A2 34 37.0 34.0 34.0 Actin, muscle A1 35 42.0 34.0 46.0 Actin, muscle A1 36 54.0 46.0 48.0 Calponin Calponin 1 28 P51911 Q08091 36.0 37.0 Calponin 2 27 Q99439 Q08093 31.0 28.0 Calpain-3 50 P20807 Q64691 28.0 16.0 Myosin Light Chain, Skeletal Muscle P12829 P02602 P09541 Myosin light chain 4 12 39.0 30.0 34.0 Myosin light chain 1 13 38.0 20.0 24.0 Myosin light chain 2 14 54.0 27.0 30.0 Myosin light chain 3 15 38.0 32.0 Myosin Heavy Chain, Skeletal Muscle Q9UKX2 Q28641 Q02566 Isoform I 52 39.0 39.0 37.0 Isoform II 53 36.0 39.0 38.0 Isoform III 54 24.0 14.0 19.0 Myosin Regulatory Light Chain Q01449 P02608 P97457 Isoform I 2 56.0 41.0 44.0 Isoform II 3 56.0 41.0 44.0 Isoform III 4 56.0 41.0 44.0 Isoform IV 6 47.0 38.0 43.0 SNAP SNAP-25 8 P60880 49.0 SNAP-29 20 O95721 52.0 SNAP 26 P54920 Q9DB05 25.0 21.0 Tropomyosin P07951 P58776 P58771 Tropomyosin 2 24 29.0 29.0 25.0 Tropomyosin 2 25 Tropomyosin 1 29 P06753 P58776 P21107 34.0 38.0 36.0 Troponin Troponin C 5 P63316 P02591 P19123 34.0 34.0 34.0 Troponin I 9 P19237 P02645 Q9WUZ5 37.0 26.0 36.0 Tubulin Tubulin beta chain 38 Q9BQE3 P05214 26.0 22.0 Tubulin alpha chain 59 Q13748 P68368 22.0 33.0 a
rat
fruit fly
P68035 34.0 46.0 48.0
P02574 34.0 42.0 53.0
Q08290 41.0 P16259 18.0
-
P17209 34.0 24.0 30.0 35.0
-
P12847 37.0 40.0 19.0
P05661 34.0 44.0 18.0
Q9QVP4 53.0 53.0 53.0 43.0
P06742 47.0 47.0 47.0 40.0
Q9Z2P6 49.0 P54921 21.0
P36957 44.0 Q23983 19.0
P04692 31.0 P58775 36.0
P09491 42.0 32.0 P06754 56.0
-
P47949 16.0 -
Q68FR8 22.0 Q6AYZ1 21.0
P06604 26.0 P06603 31.0
-: No data.
model system for the study of developmentally regulated skeletal muscle disorders. For ISM isoforms, we have not yet learned the mechanisms of production in detail. On the basis of a bioinformatics search, it is highly possible that isoforms of myosin regulatory light chain (spot nos. 2-4, and 6), myosin light chain 1 (spot nos. 11-15), tropomyosin (spot nos. 24, 25, and 29), myosin heavychain (spot nos. 52-54), and troponins (spot nos. 7, 9, and 10) are due to post-translational modifications. On the whole, the diversity of muscle isoforms may allow for the generation of muscle fibers with different physiological properties during larval-pupal metamorphosis, for example, the speed and force of muscle contraction, the consumption of ATP, calcium sensitivity, and even the structure of the myofibril. 2302
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Concluding Remarks This paper demonstrates the feasibility of adopting a proteomic approach to analyzing insect skeletal muscle proteins during larval-pupal metamorphosis. The larva-specific protein troponin I was identified by peptide mass fingerprinting, followed by immunoblotting certification as a developmental biomarker that is involved in the muscle re-modeling during larval-pupal metamorphosis. Protein profiling and image analysis of 2-D gel images from larval and pupal ISM tissues revealed 12 differentially displayed or expressed proteins belonging to the contractile apparatus, metabolism, regulation, and signal transduction, suggesting that these different proteins may play an essential role in the metamorphosis of larval-pupal ISM tissues. These data will provide a valuable resource for investigations into the biochemical basis of insect skeletal muscle pathologies in general. Abbreviations: ISM, intersegmental muscles; SNAP, synaptosomal-associated proteins; TCP, T-complex protein; Tn I, troponin I; TSR, twinster protein.
Acknowledgment. This work was supported by the National Bioresource Project (Silkworm RR2002) from the Ministry of Education, Science, Sports and Culture of Japan. References (1) Yates, J. R.; Gilchrist, A.; Howell, K. E.; Bergeron, J. J. Nat. Rev. Mol. Cell. Biol. 2005, 6, 702-714. (2) Doran, P.; Dowling, P.; Donoghue, P.; Buffini, M.; Ohlendieck, K. Biochim. Biophys. Acta 2006, 1764, 773-785. (3) Faber, M. J.; Agnetti, G.; Bezstarosti, K.; Lankhuizen, I. M.; Dalinghaus, M.; Guarnieri, C.; Caldarera, C. M.; Helbing, W. A.; Lamers, J. M. Cell Biochem. Biophys. 2006, 44, 11-29. (4) Gelfi, C.; Vigano, A.; Ripamonti, M.; Pontoglio, A.; Begum, S.; Pellegrino, M. A.; Grassi, B.; Bottinelli, R.; Wait, R.; Cerretelli, P. J. Proteome Res. 2006, 5, 1344-1353. (5) Sanchez, J. C.; Chiappe, D.; Converset, V.; Hoogland, C.; Binz, P. A.; Paesano, S.; Appel, R. D.; Wang, S.; Sennitt, M.; Nolan, A.; Cawthorne, M. A.; Hochstrasser, D. F. Proteomics 2001, 1, 136163. (6) Yan, J. X.; Harry, R. A.; Wait, R.; Welson, S. Y.; Emery, P. W.; Preedy, V. R.; Dunn, M. J. Proteomics 2001, 1, 424-434. (7) Bouley, J.; Chambon, C.; Picard, B. Proteomics 2004, 4, 18111824. (8) Donoghue, P.; Doran, P.; Dowling, P.; Ohlendieck, K. Biochim. Biophys. Acta 2005, 1752, 166-176. (9) Doherty, M. K.; McLean, L.; Hayter, J. R.; Pratt, J. M.; Robertson, D. H.; El-Shafei, A.; Gaskell, S. J.; Beynon, R. J. Proteomics 2004, 4, 2082-2093. (10) Kjaersgard, I. V.; Norrelykke, M. R.; Jessen, F. Proteomics 2006, 6, 1606-1618. (11) Ashman, K.; Houthaeve, T.; Clayton, J.; Wilm, M.; Podtelejnikov, A.; Jensen, O. N.; Mann, M. Lett. Pept. Sci. 1997, 4, 57-65. (12) Lockshrn, R. A.; Williams, C. M. J. Insect Physiol. 1964, 10, 643 -649. (13) Schwartz, L. M. J. Neurobiol. 1992, 23, 1312-1326. (14) Banno, Y.; Fujii, H.; Kawaguchi, Y.; Yamamoto, K.; Nishikawa, K.; Nishisaka, A.; Tamura, K.; Eguchi, S. A Guide to the Silkworm Mutants; Institute of Genetic Resources, Kyushu University: Fukuoka, Japan, 2005. (15) Ito, T.; Kobayashi, M. Rearing of the silkworm. In The Silkworm: An Important Laboratory Tool; Tazima, Y., Ed.; KODANSHA Press: Tokyo, Japan, 1978; pp 83-88. (16) Zhang, P.; Aso, Y.; Yamamoto, K.; Banno, Y.; Wang, Y.; Tsuchida, K.; Kawaguchi, Y.; Fujii, H. Proteomics 2006, 6, 25862599. (17) Zhang, P.; Yamamoto, K.; Aso, Y.; Banno, Y.; Sakano, D.; Wang, Y.; Fujii, H. Biosci. Biotechnol. Biochem. 2005, 69, 20862093. (18) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850-858.
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