Proteomic Analysis of the Silkworm (Bombyx mori L.) Hemolymph during Developmental Stage Xing-hua Li, Xiao-feng Wu, Wan-fu Yue, Jian-mei Liu, Guang-li Li, and Yun-gen Miao* College of Animal Sciences, Zhejiang University, Hangzhou 310029, P. R. China Received June 25, 2006
We utilized the proteomic approach to investigate the proteome of the fifth instar hemolymph during growth and development, and to improve the understanding of this important bioprocess and gene expression situation. A total of 25 µL of hemolymph was used for 2D analysis, and the separated proteins were visualized by silver staining and analyzed using the ImageMaster 2D software. The report showed as many as 241 of protein spots were expressed in the beginning of the fifth instar. Among them, most were concentrated in pI 3.5-6.5, which reached 76% of the total protein spots. As for the protein molecular sizes, 182 protein spots concentrated between 35 and 90 kDa, which comes to 75% of the total spots. When the larvae grow to the seventh day (total fifth instar duration was 9 days), 298 protein spots were visualized through 2D electrophoresis. Fifty-seven spots were newly expressed compared to the image of the first day in fifth instar. The results implied that these proteins are related to biosynthesis of silk protein and metamorphosis preparation from larva to pupa. In total, 19 protein spots including 6 special spots expressed in seventh day were analyzed through MALDI-TOF-MS. The relations between proteins and growth and development of silkworm were discussed. Keywords: silkworm (Bombyx mori L.) • hemolymph • proteomic analysis • 2D electrophoresis • MALDI-TOF-MS
Introduction The mulberry silkworm, Bombyx mori, has been domesticated for silk production for more than 5000 years. Currently, it is the major economic resource for more than 30 million families in countries such as China, India, Vietnam, and Thailand. With the development of biotechnology, B. mori has been used as an important bioreactor for the production of recombinant proteins.1-3 In addition, B. mori is the model organism for Lepidoptera, the second most numerous order of insects, including many species important for agriculture and forestry. Advances in silkworm research not only have a great impact in improving sericulture, but also may facilitate the development of new strategies for pest control. The economic and scientific significance of silkworms have made them the subject of intensive genetic studies since the last century and, thus, the most important insect genetic model after Drosophila melanogaster. More than 400 mutations have been identified, and more than 1000 silkworm strains are maintained as genetic resources.4-6 These mutations affect many fundamental aspects of the insect life cycle, including egg and egg-shell formation, early embryonic pattern formation, development and diapause, larval feeding behavior, and molting, among others.7 Moreover, the genetic physical map and draft sequence for the genome of the domesticated silkworm has been reported,8 covering 90.9% of all known silkworm genes. The gene count is 18 510, which exceeds the
13 379 genes reported for D. melanogaster. Comparative analyses to fruitfly, mosquito, spider, and butterfly reveal both similarities and differences in gene content. Silkworm larva is the solely feeding stage in which larvae take in their nutrition (food) for all of the life process. Generally speaking, larvae before the fourth instar are called young, and those after the fourth instar are called grown silkworm. At the end of final instar (fifth instar), larvae cease feeding, and their bodies become shorter, stouter, and transparent. These larvae are called mature larvae, which begin to coon, and later to pupate in the cocoons. The fifth instar is a transition period for biosynthesizing and spinning silk proteins, and for metamorphosis from larva to pupa. Although the contents of hemolymph and the activities of enzymes such as amino transferase, phosphatase, carbohydrate, and lipid metabolism will be certainly changing during this period, the molecular mechanism of this short period is still not well-understood. Proteomics is a large-scale study of the gene expression at the protein level, which ultimately provides direct measurement of protein expression levels and insight into the activity state of all relevant proteins.9 In this report, we utilized the proteomic approach to investigate the proteome of the fifth instar hemolymph during growth and development, and to improve the understanding of this important bioprocess and gene expression situation.
Materials and Methods * To whom correspondence should be addressed. College of Animal Sciences, Zhejiang University, Hangzhou 310029, P. R. China. Tel: +86 571 86971659. E-mail:
[email protected]. 10.1021/pr0603093 CCC: $33.50
2006 American Chemical Society
Experimental Animal. Disease-free eggs of the silkworm strain P50 (Dazhao) were maintained in silkworm germplasm Journal of Proteome Research 2006, 5, 2809-2814
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Figure 1. Comparison of protein expression of the first day and the seventh day in silkworm hemolymph. (A) The first day of hemolymph 2D analysis results in detection of approximately 241 discrete spots, while (B) the seventh day consists of approximately 298 discrete protein spots.
at Zhejiang University, and hatched and reared under standard condition at 26 ( 2 °C, 70-85% relative humidity, and 12 h light:12 h dark photoperiod with mulberry leaves. The hemolymph was collected daily from 15 worms from the first day to the matured fifth instar and stored at -80 °C until ready to use. 2D Electrophoresis and Gel Visualization. Isoelectric focusing (IEF) was perfomed following the manufacturer’s instructions (Amersham Biosciences) using IPGphor pH3-10, Linear) isoelectric focusing system (Amersham Biosciences). Briefly, 25 µL of hemolymph was mixed with 125 µL of lysis solution containing 8 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris base, 2% IPG buffer (pH 3-10), and 30 mM DTE. The mixture was fully lysed for 1 h in an ice bath and sonicated for 3 min, then centrifuged twice at 15 000g for 15 min at 4 °C. The supernatant was mixed further with 300 µL of rehydration solution containing 8 M urea, 2% CHAPS, 0.5% IPG buffer (pH 3-10), 0.4% DTT, and 0.002% bromophenol blue to a total volume of about 450 µL and applied to a 24 cm IPG strip (pH 3-10, Linear). Rehydration and IEF were carried out on the IPGphor platform automatically at 20 °C, and the total Vh of IEF was about 87 000 Vh for 27 h. After IEF separation, the gels were equilibrated for 15 min in an equilibration buffer (50 mM Tris base, 6 M urea, 30% glycerol, 2% SDS, and 0.002% bromophenol blue) containing 1% DTT and subsequently in the same buffer except DTT was replaced with 2.5% iodoacetamide for another 15 min. The equilibrated gel strip was subjected to 12.5% SDS-PAGE and sealed with 0.5% agarose. SDS-PAGE was performed at constant power of 5 W/gel for 45 min, and switched to 15 W/gel until the bromophenol blue frontier reached the bottom of the gel. The separated proteins were visualized by silver staining as described by Amersham Biosciences. 2D Protein Image Acquisition and Analysis. All the 2D images were scanned at an optical resolution of 300 dpi by high-resolution image scanner (Amersham Biosciences). The images were analyzed using the ImageMaster 2D software supplied by the manufacturer. In-Gel Digestion. The protein spots of interest were excised manually from the silver-stained gels with a clean scalpel blade. Each sample was washed twice in milli-Q water, then destained 2810
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by washing with a 1:1 solution of 30 mM potassium ferrocyanide and 100 mM sodium thiosulfate, and equilibrated in 100 mM ammonium bicarbonate for 20 min to pH 8.0. After washing twice in milli-Q water, the gels were dehydrated by addition of acetonitrile, then dried in a SpeedVac (Thermo Savant) for 15 min. Subsequently, the spots’ gel were rehydrated in 10-20 µL of trypsin (Sigma, St. Louis, MO) solution (20 ng/ µL in 40 mM NH4HCO3 in 9% acetonitrile) and incubated at 37 °C overnight. Peptides were extracted twice by adding 50 µL of the solution including 50% acetonitrile and 5% trifluoroacetic acid for 15 min, respectively. After they were dried in a SpeedVac, peptides extracted were dissolved in 10 µL of 0.1% trifluoroacetic acid prior to applying to the sample plate. Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Analysis. Peptide mixture (2 µL) was mixed with an equal volume of 10 mg/mL R-cyano-4-hydroxycinnamic acid (Sigma) saturated with 50% acetonitrile in 0.1% trifluoroacetic acid, and analyzed by a Voyager-DE STR MALDI-TOF MS using a delayed ion extraction and ion mirror reflector mass spectrometer (Applied Biosystems, Foster City, CA). The instrument setting was reflector mode with 160-ns delay extraction time, positive, 60-65% grid voltage, and 20-kV accelerating voltage. Laser shots at 200 per spectrum were used to acquire the spectra with mass range from 1000 to 4000 Da. External calibration was performed with Peptide Mass Standard kit (Perspective Biosystems). The matrix and the autolytic peaks of trypsin served as internal standards for mass calibration. Protein Identification and Database Searching. Protein identification using peptide mass fingerprinting (PMF) was performed by the MASCOT search engine (http://www.matrixscience.com) against the NCBI protein database.
Results The Standard Protein Expression of Silkworm Hemolymph. The silkworm hemolymph is an opening system, which flows through all inside organs and takes on the metabolism and regulation functions. The fifth instar is a transition period for biosynthesizing and spinning silk proteins, and for metamorphosis from larva to pupa. It can be assumed that the hemolymph of the fifth instar will focus on the metabolism of
Proteomic Analysis of the Silkworm Hemolymph
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Figure 2. Special 2D protein spots of silkworm hemolymph during development of the fifth instar. (A) P50blood5-1; (B) P50blood5-7.
the activities of enzymes such as amino transferase, phosphatase, carbohydrate, lipid, and so forth. In total, 25 µL of hemolymph was used for 2D analysis, and the separated proteins were visualized by silver staining and analyzed using the ImageMaster 2D software. Figure 1 showed as many as 241 protein spots were expressed in the beginning of the fifth instar. Among them, most proteins were concentrated in pI 3.5-6.5; their number was 139, which reached 76% to the total protein spots. As for the protein molecular sizes, 182 protein spots concentrated between 35 and 90 kDa, which comes to 75% of the total spots. When the larvae grow to the seventh day (total fifth instar duration was 9 days), 298 protein spots were visualized through 2D electrophoresis. Fifty-seven spots were newly expressed compared to the image of the first day in fifth instar. The results implied that these proteins are related to biosynthesis of silk protein and metamorphosis preparation from larva to pupa. Special 2D Protein Spots of Silkworm Hemolymph during Development of the Fifth Instar. The 2D image was selected with frame according to the calculation of pI and molecular sizes (Figure 2). As shown in Figure 2, spots 1-9 presented the same molecular sizes and different isoelectric point (pI), and so were spots 10-13. It inferred they are the same proteins, which have different decoration.
Another interesting issue comes from the increased 17 protein spots of the seventh day in the fifth instar hemolymph compared to the first day of fifth instar. It inferred that these proteins are related to biosynthesis of silk protein and metamorphosis preparation from larva to pupa. Analysis of Peptide Mass Fingerprinting (PMF) Patterns and Homology Searching. The protein spots of interest were excised manually from the silver-stained gels with a clean scalpel blade. The spots’ gel was rehydrated in 10-20 µL of trypsin solution (20 ng/µL in 40 mM NH4HCO3 in 9% acetonitrile) and incubated at 37 °C overnight for digestion. The digested peptide mixture was analyzed by a Voyager- DE STR MALDI-TOF MS using a delayed ion extraction and ion mirror reflector mass spectrometer. A total of 19 spots including 6 special spots expressed in seventh day was analyzed through MALDI-TOF-MS. Among them, spots 1-9 presented the same molecular sizes and different isoelectric point (pI) and were identified as methyltransferase (EC 2.1.1.51) (Figure 3 and Table 1). The matched peptides for spot 5 are shown in bold red font as follows: Similarly, spots 10-13 were identified as Ribosomal protein. Figure 4 and Table 2 showed the peptide mass fingerprinting and its database searching results of protein spot 11. Journal of Proteome Research • Vol. 5, No. 10, 2006 2811
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Figure 3. PMF of spot 5 extracted from 2-DE gel. Table 1. PMF Database Searching Results of Protein Spot 5a Homolohous protein name RRNA(Guanine-N(1)-)methyltransferase (EC 2.1.1.51) Start-End
24-42 114-134 172-199 178-199 227-251 a
NCBI accession No. ABE55566
pI 3.51
Sequence Coverage
MW (Delta) 31833
32%
Observed
Mr (expt)
Mr (calc)
Delta
Sequence
2145.8398 2273.9056 2940.9376 2272.9049 2938.9808
2144.8326 2272.8983 2939.9303 2271.8976 2937.9735
2145.0676 2273.1143 2940.6006 2271.2561 2937.4040
-0.2351 -0.2160 -0.6703 0.6415 0.5695
K.GHQFDCAKEGYVNLLPVQK.K R.NLDEGPCCQLQGLDISKTAIR.Y K.AEELARVIAGNGILITVSPGPMHHFAIK.E R.VIAGNGILITVSPGPMHHFAIK.E R.LESQMSLDNAVDIVHFLNMTPYSWK.L
No match to: 1335.6239, 1446.6349, 2104.8475, 2163.8424, 3211.1333, 3215.9011.
Figure 4. PMF of spot 11 extracted from 2-DE gel. Table 2. PMF Database Searching Results of Protein Spot 11a NCBI accession No. EAN27451
pI 3.84
MW (Delta) 20834
Sequence Coverage 37%
Observed
Mr (expt)
Mr (calc)
Delta
Sequence
1654.6615 1666.6694 3213.9038 1123.5173
1653.6542 1665.6621 3212.8965 1122.5100
1652.8885 1665.8660 3212.6862 1121.7033
0.7656 -0.2039 0.2104 0.8068
K.QVYHDKVVPELQAK.F K.FGYSNVMQVPRLQK.I K.IVVNMGVGEAIADKNFLNNAIGNLEAITGQK.A R.LINAALPRVR.D
Homolohous protein name Ribosomal protein L5 Start-End
6-19 20-33 34-64 103-112 a
No match to: 1020.4888, 1271.6032, 1655.6523, 2018.7746, 2163.7624, 2272.8997, 2273.8501, 2317.7908, 3212.0155.
The matched peptides for spot 11 are shown in bold red font as follows:
Six special spots expressed in seventh day of fifth instar were 2812
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analyzed through MALDI-TOF-MS. They are presented herein
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Proteomic Analysis of the Silkworm Hemolymph
Table 3. Mass Spectrometric Details of Six Special Spots Proteins Expressed in the Seventh Day of Fifth Instar spot no.
Mass spectrometric details protein name
39
Dehydrogenase
43
zinc-finger protein zpr1
44
Hypothetical protein
45
Alcohol dehydrogenase II (Fragment)
46
Transcriptional regulator
47
HAD-type hydrolase/phosphatase
theoretical Mr
observed Mr
peptide sequence
1138.3324 3210.5286 2704.4544 2937.7361 1020.3084 2164.4950 2939.8018 1992.8234 3150.1300 3212.5236 1445.4925 1019.3490 2256.5715 1637.5093 3210.8298 1019.3475 3209.2181 2143.4839 1137.4080 2987.0280 2500.2739 1069.1478 2162.0417 1019.1088 1253.1283 2269.4491 1306.2366 2704.8862 1019.2810 2938.5314 2271.4629
1139.3397 3211.5359 2705.4617 2938.7434 1019.3012 2163.4878 2940.8091 1993.8307 3151.1373 3213.5309 1446.4998 1020.3563 2257.5788 1638.5166 3211.8371 1020.3548 3210.2254 2143.4839 1138.4152 2988.0353 2501.2811 1070.1551 2163.0490 1020.1161 1254.1355 2270.4563 1307.2439 2705.8934 1020.2883 2939.5386 2272.4702
ICENIEYQK ICENIEYQKSIEAITIQNSTVYAACSYR MVANSSGLIGIYYLGTIGAYKLDGTK YKLYDFTLDGDYAYLATGDVFSGESK VNLTEMASR AVVGTGYGESGHVYIIDSQGR LLLTVIPYFREVVLMSFECPHCGFK KESAPQLYDQINAFIEK SGSVPFTITVDDITGNSWIEMKPGRDGDR VFSQETDSMTPEQVANWQQFLCNLTAAR ALLKMDCQGLVVR MDCQGLVVR LIQDFVLLTTAVEVAQRWR DVIQELHLGLDKMK HLTGTLILVNSLDVLRAAAFSPADQDDFVI GVMIGDGQSR INPEAPLDKVCIMSCGFCTGFGATVNVAKPK RGQTVAIFGLGAVGLAAMEGAR FGLTDFVNPK DLLLQTASNIMREGDVVDISLSELSLR EGDVVDISLSELSLRSGLNSALVK AGLLKALLDR HISKCIDTYYDYPYLNR DSDEAEAKR AYNRFIGEGVK LLVTDIDGTITHQSHHLDKK LFFLTGRYYK FSPTPIAQDLHEYVDPRYFPNAK VVNILYDGK GDFKIVMSSAPEEMHVHADFLAPPADK NGILSAWEAGVRYYDDLMSL
with protein names, observed masses, and matched peptide sequences (Table 3).
Discussion The silkworm, B. mori, is a large-size domestic insect that has been continuously selected to improve silk production in sericulture for several thousand years. Since the silkworm can be easily mass-cultured in a laboratory by using advanced artificial diets, B. mori is also a good experimental insect and is now considered to be one of the most thoroughly studied lepidopteran insects in the field of physiology, biochemistry, pathology, and genetics.10 In addition, recent advances in the expressed sequence tag (EST) project in B. mori have catalogued more than 8500 independent EST clones from various cDNA libraries prepared from more than 20 organs or tissues in different developmental stages.11 Xia et al. reported a draft sequence for the genome of the domesticated silkworm (B. mori), covering 90.9% of all known silkworm genes and estimated the gene count to be 18 510, which exceeds the 13 379 genes reported for D. melanogaster. Comparative analyses to fruitfly, mosquito, spider, and butterfly reveal both similarities and differences in gene content.8 These advances are now urging the elucidation of cloned gene function and will further facilitate the elucidation of molecular mechanisms underlying a wide variety of biological events in this insect species. Such a task can be accomplished, however, only with appropriate and efficient methodological tools to elucidate gene function and regulation. Proteomics is a large-scale study of the gene expression at the protein level, which ultimately provides direct measurement of protein expression levels and insight into the activity state of all relevant proteins. Key elements of classical proteomics
are the separation of proteins in a sample using twodimensional gel electrophoresis (2-DE) and their subsequent identification by biological mass spectrometry (MS).9,12 Silkworm, B. mori, as an ideal animal model has its scientific importance for elucidating developmental mechanisms, especially the metamorphosis mechanism, from egg, larval, pupa, and adult in its life cycle. Another interesting and unique event is its capability of high biosynthesis level for silk proteins in the late stage of worm. The fifth instar, especially the middle (usually the fourth and fifth day) of fifth instar, is the highly efficient stage for synthesizing silk proteins. After the seventh day of fifth instar, the worm is getting ready for metamorphosis from larva to pupa, although there still is protein synthesis. In this term, the enzyme activity for silk proteins synthesis is gradually reducing, and the carbohydrate and fatty acid synthesis are enhancing to store up the energy for use in pupa and adult stage. In this report, results showed 57 spots were newly expressed in the seventh day compared to the image of the first day in fifth instar. Generally speaking, large quantity of differentially decorated methyltransferase and Ribosomal protein were presented in the hemolymph of fifth instar. The results implied that these proteins are related to biosynthesis of silk protein. On the other hand, dehydrogenase, alcohol dehydrogenase, and hypothetical protein specially were expressed in the late of fifth instar. We suggested this is related to the fat synthesis in this stage in preparation for pupation. Zinc Finger Protein is a sequence-specific transcriptional repressor, which is involved in special intracellular nucleic acids binding, control of gene expression, and plays important role in cell division and differentiation, embryo development, and individual’s growth.13,14 The expressed zinc-finger protein and Journal of Proteome Research • Vol. 5, No. 10, 2006 2813
research articles hydrolase/phosphatase in the late fifth instar implied the synthetic activity of silk protein is gradually reducing, and the larva is tending toward metamorphosis preparation from the larval to pupal stage.
Concluding Remarks The proteomics research will not only provide a method for direct measurement of protein expression levels and insight into the activity state of all relevant proteins, but also the elucidation of gene function and regulation. The proteins, especially the enzymes, are differentially expressed during the growth and development of silkworm. This has implications for the physiological and biochemical proceedings and the gene regulation in the silkworm.
Acknowledgment. We thank Dr. Wu Xiao Qiong and Miss Shen Jing, Medical College of Zhejiang University, for their kind help through the experiment. The works were supported by the National basic Research Program of China under Grant No. 2005CB121003. References (1) Xiang, Z. H. Genetics and Breeding of the Silkworm; Chinese Agriculture Press: Beijing, China, 1995; pp 273-289.
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Li et al. (2) Tamura, T.; Thibert, C.; Royer, C.; Kanda, T.; Abraham, E.; Kamba, M.; Komoto, N.; Thomas, J. L.; Mauchamp, B.; Chavancy, G. Nat. Biotechnol. 2000, 18, 81-84. (3) Tomita, M.; Munetsuna, H.; Sato, T.; Adachi, T.; Hino, R.; Hayashi, M.; Shimizu, K.; Nakamura, N.; Tamura, T.; Yoshizato, K. Nat. Biotechnol. 2003, 21, 52-56. (4) Doira, H. Linkage Maps and List of Genetical Stocks Maintained in Kyushu University; Institute of Genetic Resources, Kyushu University: Fukuoka, Japan, 1992. (5) Fujii, H.; Banno, Y.; Doira, H.; Kihara, H.; Kawaguchi, Y. Important Genetic Resources, 2nd ed.; Institute of Genetic Resources, Kyushu University: Fukuoka, Japan, 1998. (6) Lu, C.; Dai, F.; Xiang, Z. Sci. Agric. Sin. 2003, 36, 968-975. (7) Nagaraju, J.; Goldsmith, M. R. Curr. Sci. 2002, 83, 415-425. (8) Xia, Q.; Zhou, Z.; Lu, C.; et al. Science 2004, 306, 1937-1940. (9) Nabby-Hansen, S.; Waterfield, M. D.; Cramer, R. Trends. Pharmacol. Sci. 2001, 22, 376-384. (10) Moto, K.; Kojima, H.; Kurihara, M.; Iwami, M.; Matsumoto, S. Insect Biochem. Mol. Biol. 2003, 33, 1-12. (11) Yoshiga, T.; Okano, K.; Mita, K.; Shimada, T.; Matsumoto, S. Gene 2000, 246, 339-345. (12) Berkelman, T.; Stenstelt, T.; Bjellquist, B.; Laird, N.; McDowell, M.; Olsson, I.; Westermeier, R. Amersham Biosciences; Sweden, 1998; p 87. (13) Xu, Y. S.; Wang, S. S.; Wang, H. B.; Lou, C. F. Acta Sericol. Sin. 2004, 30, 343-347. (14) Evans, R. M.; Holenberg, S. M. Cell 1988, 52, 1-3.
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