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Isolation and Characterization of an Apoptosis-Inhibiting Component from the Hemolymph of Bombyx mori
Biochemical and Biophysical Research Communications 285, 224 –228 (2001) doi:10.1006/bbrc.2001.5148, available online at http://www.idealibrary.com on
Isolation and Characterization of an Apoptosis-Inhibiting Component from the Hemolymph of Bombyx mori Eun Jeong Kim, Won Jong Rhee, and Tai Hyun Park 1 School of Chemical Engineering, Seoul National University, Kwanak-Gu Shilim-Dong San 56-1, Seoul 151-744, Korea
Received June 8, 2001
Silkworm (Bombyx mori) hemolymph showed an apoptosis-inhibiting activity in insect cells (Sf 9) infected with baculovirus (AcNPV). The addition of silkworm hemolymph into the culture medium increased the host cell longevity due to its apoptosis-inhibition activity. Components with an apoptosis-inhibiting effect were purified from the silkworm hemolymph by heat treatment, gel-filtration chromatography, and ion-exchange chromatography. The component with highest activity was characterized by periodic acid– Schiff staining, isoelectric focusing, MALDI-TOF–mass spectrometry, and N-terminal sequencing and was found to be a nonglycosylated monomeric protein with a molecular weight of ca. 28,000 Da. © 2001 Academic Press
Apoptosis is programmed form of cell death that is morphologically distinguishable from necrosis (1, 2) and is accompanied by the condensation of nuclei and cytoplasm, loss of microvilli and the convolution of plasma membranes, and nuclear and cell segmentation (3), whereas necrosis is typified by cell swelling, mild clumping of chromatin, and cell lysis, which causes the release of the cell constituents. The programmed cell death occurs in most animal tissues to eliminate harmful cells or those that are produced in excess, and these are mediated by apoptosis (4, 5). The decision as to whether a cell undergoes apoptosis or necrosis is influenced by a variety of environmentally and internally regulatory stimuli. Recent evidence suggests that alterations in cell survival contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases, neurodegenerative disorders, and AIDS (acquired immunodeficiency syndrome) (6). Regulation of cell death may have therapeutic potential in these diseases, therefore, many research groups have been and continue to investigate the regulation of apoptosis. 1
To whom correspondence and reprint requests should be addressed. Fax: ⫹82-2-875-9348. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
The disruption of cell physiology as a result of viral infection can cause an infected cell to undergo apoptosis (6), and in particular, the infection of an insect cell with a baculovirus was reported to induce apoptosis (7), which involved the activation of host caspases (cysteine-dependent, aspartate-specific protease) (8). Spodoptera frugiperda and the Autographa californica nuclear polyhedrosis virus (AcMNPV) have been widely used as a model insect cell and baculovirus, respectively, for studying the baculovirus regulation of host apoptosis. A specific gene product, P35, which prevents an apoptotic response, was identified in AcMNPV and silkworm baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV) (7, 9). Various other genes are also known to regulate apoptosis in the cells of mammalian immune system (10 –12). Baculovirus also carries genes which produce inhibitors of apoptosis (IAPs), and IAP homologues have been found in a diversity of insect and mammal genomes (13, 14). Insect hemolymph was used as a culture medium in the early stages of insect tissue culture. On the basis of the chemical analysis of insect hemolymph, a synthetic medium was formulated for insect cell culture (15, 16), but it still had to be supplemented with insect hemolymph (17). However these days, fetal bovine serum (FBS) has replaced insect hemolymph as a supplement in insect cell medium. In a previous study (18), we reported that the production of recombinant protein in an insect cell– baculovirus system was increased by supplementing the medium with silkworm hemolymph. We also found that the silkworm hemolymph increased host cell longevity (19) by inhibiting baculovirus-induced insect cell apoptosis (20). This indicates that the component, which inhibits the apoptosis is contained in the silkworm hemolymph. In this article, the apoptosis-inhibiting component was isolated from silkworm hemolymph and characterized. MATERIALS AND METHODS Collection of silkworm hemolymph. Silkworm hemolymph was collected from the fifth-instar larvae by clipping the side of an abdominal leg. The collected hemolymph was heat-treated at 60°C for
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Protein purification. To facilitate purification, the hemolymph was heat-treated at various temperatures for 30 min. After chilling and centrifuging, the precipitated proteins were removed. Heattreated and centrifuged supernatants were loaded on a Superdex 200 HR (1 ⫻ 30 cm, Amersham–Pharmacia Biotech) and eluted with 50 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl at a flow rate of 0.5 ml/min. The eluant was monitored at 280 nm and 0.5 ml fractions were collected. Active fractions from the Superdex 200 HR column were desalted into the starting buffer (120 mM Tris and 80 mM Bis–Tris, pH 9.0) using a HiTrap desalting column (1.6 ⫻ 2.5 cm, Amersham–Pharmacia Biotech). The desalted fractions were injected onto a Mono Q HR (0.5 ⫻ 5 cm, Amersham–Pharmacia Biotech) preequilibrated with same starting buffer and eluted with a linear gradient of 20 column volumes of elution buffer (120 mM Tris and 80 mM Bis–Tris, pH 5.0) at a flow rate of 0.5 ml/min. The eluant was monitored at 280 nm and 1-ml fractions were collected. The purified fractions were concentrated using lyophilizer. Electrophoresis. SDS–PAGE was carried out using 15% polyacrylamide separating gels, and 5% polyacrylamide stacking gels, using the method described by Laemmli (23). A low-range protein molecular weight standard (Amersham–Pharmacia Biotech) was used and gels were stained using Coomassie blue. Gels were stained for glycoproteins using a periodic acid–Schiff (PAS)-based stain (Sigma). Isoelectric focusing was carried out on PhastGel IEF 3-9 (Amersham–Pharmacia Biotech) using PhastSystem (Amersham– Pharmacia Biotech). The isoelectric points (pI) of the purified proteins were measured using a pI calibration kit (pI 3.5–9.3, Amersham–Pharmacia Biotech). Mass spectrometry. Matrix-assisted laser desorption ionization time of flight (MALDI-TOF)–mass spectrometry was carried out using a Voyager-DE (PerSeptive Biosystem). CHCA (␣-cyano-4hydroxy-cinnamic acid) (Sigma) and myoglobin were used as a matrix and as an internal standard, respectively.
FIG. 1. (A) Fractionation of silkworm hemolymph by Superdex 200 column chromatography. (B) SDS–PAGE analysis of F1 and FII.
30 min. then chilled and centrifuged (21). The supernatant was filtered through a 0.2-m membrane filter and stored 4°C for the later use. Insect cell culture and baculovirus infection. S. frugiperda (Sf 9) cells were cultivated in Grace medium (Gibco) supplemented with 5% fetal bovine serum (FBS, Gibco), 0.35 g/liter NaHCO 3, and antibiotic–antimicotic (Gibco) at 28°C. Whole silkworm hemolymph, used as a control, or the isolated component from each purification step was added to the medium, and their effects were investigated on baculovirus-induced insect cell apoptosis. Cells were infected in the late exponential growth phase (5 days in culture) with the recombinant baculovirus, which in this study was A. californica nuclear polyhedrous virus (AcNPV) that produces -galactosidase under the control of a polyhedrin promoter. For the infection, the medium was aspirated and a virus stock solution added. A multiplicity of infection (m.o.i.) of 13 was used for all experiments. After incubating for 1 h, the virus solution was replaced with the medium used before the infection. Apoptosis assay. Cell concentrations were measured using a hemocytometer and viable cells were detected using the trypan blue exclusion test. Since dead cells absorb trypan blue (Sigma), they can be identified under the optical microscope. The cell viability was defined by the ratio of the viable cell number to the total cell number. Cellular DNA was extracted using the previously reported method (22), and the DNA fragmentation pattern was examined by 2% agarose gel electrophoresis.
N-terminal sequencing. SDS–PAGE gel was transferred to a PVDF membrane by electroblotting. After the transfer the membrane was stained with Ponseau S (0.2% Ponseau S in 1% acetic acid) and destained with deionized water. The stained band was then cut out and air-dried. Amino acid sequencing was carried out using a Precise Protein sequencing system (Applied Biosystems).
RESULTS AND DISCUSSION In our previous study (20), the host insect cell viability remained at a high level for 6 days after baculo-
FIG. 2. Effect of each fraction on cell viability. Medium was supplemented with 5% FBS and each fraction was obtained from Superdex 200 chromatography.
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FIG. 3. (A) Fractionation of FII by Mono Q column chromatography. (B) SDS–PAGE analysis of FII-1 and FII-2 obtained by Mono Q chromatography.
virus infection in the medium supplemented with 5% silkworm hemolymph, whereas its viability began to decrease 3 days after infection in a medium without silkworm hemolymph. It was also shown that DNA fragmentation was not observed during the first 3 days, while it occurred afterward in the medium without silkworm hemolymph. Whereas the DNA fragmentation was not observed until the sixth day in the medium supplemented with 5% silkworm hemolymph. Significantly, the time when the DNA fragmentation began corresponded to the time when the cell viability started to decrease (19). This also indicates that cell viability measurements can be used as an assay method for isolating the apoptosis-inhibiting component from silkworm hemolymph. Therefore, cell viability was measured 7 days after infection in media supplemented with each fraction to determine the apoptosis-inhibiting activity of the fractionated silkworm hemolymph. Silkworm hemolymph must be heat-treated for use as a medium supplement, since the intact hemolymph darkens visibly due to the activity of tyrosinase, which
synthesizes melanin via intermediary quinones (15). The production of toxic quinones in the medium consequently inhibits cell growth. Heat treatment and centrifugation facilitate the purification step by removing many proteins, as well as tyrosinase from the silkworm hemolymph. More proteins were removed by higher temperature heat treatments; however, these reduced the apoptosis-inhibiting activity. It was shown that 60°C is the most appropriate heat-treatment temperature and the apoptosis-inhibiting component is stable at 60°C (20). After heat treatment and centrifugation at 60°C for 30 min, the supernatant was loaded onto a gelfiltration column to purify the effective component. Five fractions were obtained, as is shown in Fig. 1A. Among these fractions, FI and FII showed high apoptosis-inhibitory activity as shown in Fig. 2, despite the lower protein concentration of FII, as is shown in Fig. 1B, cell viability was higher in FII than in FI. The molecular mass of the major protein contained in FI was approximately 70,000 Da, and this protein accounted for about 60% of the hemolymph proteins. The major protein contained in FII was found to have a molecular mass of ca. 30,000 Da, and accounted for 30% of the protein present. That is, these two proteins accounted for almost all of the hemolymph protein. FII, which had the higher apoptosis-inhibiting activity, was further purified by anion-exchange chromatography with a linear gradient elution of 20 column volumes (pH 9.0 –5.0), and four fractions were obtained as shown in Fig. 3A. Among these fractions, the major fractions FII-1 and FII-2 showed apoptosis-inhibitory activity, as shown in Fig. 4. The SDS–PAGE results of FII-1 and FII-2 are shown in Fig. 3B. The fraction FI obtained from gel-filtration chromatography was also further purified using the same anion-exchange chromatographic method, and major
FIG. 4. Effect of each fraction on cell viability. Medium was supplemented with 5% FBS and each fraction was obtained by Mono Q column chromatography.
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three fractions (FI-1, FI-2, and FI-3) were obtained (data not shown). Table 1 summarizes the protein concentrations of each fraction. Fractions were concentrated by lyophilization and added to the culture medium at the same concentration (0.2 mg protein/ medium ml) to determine the specific activity of apoptosis inhibition. The last column in Table 1 is the relative apoptosis-inhibiting activity of each fraction. It represents the relative cell viability (7 days after infection) in the medium supplemented with the same amount (0.2 mg/ml) of each fraction. The percentage is the relative cell viability to that in 5% silkworm hemolymph medium. Many of these fractions resulted in the inhibition of apoptosis; however, their effects were not additive and FII-2 showed the highest value (92%). In the periodic acid–Schiff reagent staining, detection of glycoproteins FI-1, FI-3, FII-1, and FII-2 was not indicated (data not shown), whereas FI-2 was stained, indicating that only FI-2 is a glycoprotein. The isoelectric points (pI) of FI-1, FI-2, FII-1, and FII-2 were approximately 6.62, 5.76, 6.93, and 5.96, respectively. FII-2, which had the highest activity, was further analyzed by MALDI-TOF–mass spectrometry and found to be a monomeric protein with the molecular weight of ca. 28,000 Da. The N-terminal amino acid sequence of the first 24 residues of FII-2 are as follows: NH 2 -Ala-Asp-Ser-Asp-Val-Pro-Asn-Asp-Ile-Leu-GluGlu-Gln-Leu-Tyr-Asn-Ser-Val-X-Val-Ala-Asp-X-Tyr. A database search using this amino acid sequence as a template for -BLAST resulted in a 95% homology with a low molecular weight lipoprotein, the so called “30K protein” of unknown function (24). The amino acid sequence of FII-2 is compared with that of 30K protein in Table 2.
TABLE 1
Protein Concentrations and Relative Apoptosis-Inhibiting Activity of Each Fraction
Heat-treated SH FI FI-1 FI-2 FI-3 FII FII-1 FII-2 FII ⬃ FV
Protein (mg)/ SH (ml)
Protein (mg)/ medium (ml)
Ratio % (each fraction/ total)
Relative apoptosisinhibiting activity (%)
35.43 20.62 5.49 5.93 5.82 10.80 4.43 4.17 4.02
1.77 1.03 0.27 0.29 0.29 0.54 0.22 0.21 0.20
100 58.19 15.50 16.75 16.43 30.47 12.49 11.78 11.34
100
TABLE 2
Comparison of N-Terminal Amino Acid Sequences of FII-2 and 30K Protein 30K protein FII-1 30K protein FII-2
1 MKPAIVILCL 21 DVPNDILEEQ DVPNDILEEQ
11 FVASLYA AD AD 31 LYNSVVVAD LYNSV –VAD
S S Y –
The 30K protein is a specific type of plasma proteins and is one of the so-called “storage proteins” in the silkworm hemolymph. These are synthesized in the fat body of the feeding larvae and released into the hemolymph. During the larval-pupal transformation, they are transported from the silkworm hemolymph to the fat body cells and stored as protein granules (25). Deductions based upon the nucleotide sequence of 30K cDNA suggest that the nascent molecule has a signal sequence (24). The 1⬃17 region of its amino-terminal sequence, as shown in Table 2, is a signal sequence. The mechanism of baculovirus-induced insect cell apoptosis is not fully understood; however, it is surmised that the apoptosis in insect cells involves a cascade of caspase activation and Sf-caspase-1 is the principal effector caspase in Sf 9 cells. In the insect cellbaculovirus system, silkworm hemolymph may work directly on the baculovirus-induced apoptosis cascade mechanism or increase the expression of the antiapoptotic baculoviral gene such as p35. Recently, we found that silkworm hemolymph inhibits not only the baculovirus-induced apoptosis but also the apoptosis induced by various chemicals such as actinomycin D, camptothecin, and staurosporine. Moreover, it also inhibits the apoptosis of mammalian cells including human cells (unpublished). These results indicate that silkworm hemolymph itself contains anti-apoptotic components itself and 30K protein is one of them. Further study is required to elucidate the mechanism for the inhibition of apoptosis by 30K protein. ACKNOWLEDGMENTS
48.25 ⫾ 4.51 57.13 ⫾ 1.75 61.53 ⫾ 0.70 73.40 ⫾ 1.74 92.16 ⫾ 3.26 Negligible
The authors acknowledge the financial support of the Korea Science & Engineering Foundation through the Nano Bio-Electronic & System Center. This work was supported in part by the Brain Korea 21 Program from the Ministry of Education. Silkworms were kindly provided by Dr. Sam-Eun Kim, Department of Sericulture and Entomology, National Institute of Agricultural Science and Technology, Suwon, Korea.
REFERENCES
Note. The relative apoptosis-inhibiting activity represents the relative cell viability (7 days after infection) in the medium supplemented with the same amount (0.2 mg/ml) of each fraction. The percentage is the relative cell viability to that in 5% silkworm hemolymph medium. 227
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3. Cotter, T. G., Lennons, S. V., Glynn, J. M., and Green, D. R. (1992) Cancer Res. 52, 997–1005. 4. Ellis, R. E., Yuan, J., and Horvitz, H. R. (1991) Annu. Rev. Cell Biol. 7, 663– 698. 5. Raff, M. C., Barres, B. A., Burne, J. F., Coles, H. S., Ishizaki, Y., and Jacobson, M. D. (1993) Science 262, 695–700. 6. Thompson, C. B. (1995) Science 267, 1456 –1462. 7. Clem, R. J., Fecheimer, M., and Miller, L. K. (1991) Science 254, 1388 –1390. 8. Miller, L. K., Kaiser, W. J., and Seshagiri, S. (1998) Semin. Virol. 8, 445– 452. 9. Kamito, S. G., Majima, K., and Maeda, S. (1993) J. Virol. 67, 455– 463. 10. Henderson, S., Rowe, M., Gregory, C., Croom-Carter, D., Wang, F., Longnecker, R., Kieff, E., and Rickinson, A. (1991) Cell 65, 1107–1115. 11. Hockenbery, D., Nunez, D., Milliman, C., Schreiber, R. D., and Korsmeyer, S. J. (1990) Nature 348, 334 –336. 12. Yonish-Rauach, E., Resnitzky, D., Lotem, J., Sachs, L., Kimchi, A., and Oren, M. (1991) Nature 352, 345–336. 13. Liston, P., Roy, N., Tamai, K., Lefebvre, C., Baird, S., ChertonHorvat, G., Farahani, R., McLean, M., Ikeda, J. E., MacKenzie, A., and Koneluk, R. G. (1996) Nature 379, 349 –353.
14. Duckett, C. S., Nava, V. E., Gedrich, R. W., Clem, R. J., Van Dongen, J. L., Gilfillan, M. C., Shiels, H., Hardwick, J. M., and Thompson, C. B. (1996) EMBO J. 15, 2685–2694. 15. Wyatt, S. S. (1956) J. Gen. Physiol. 39, 841– 852. 16. Wyatt, G. R., Loughheed, T. C., and Wyatt, S. S. (1956) J. Gen. Physiol. 39, 853– 868. 17. Grace, T. C. D. (1962) Nature 195, 788 –789. 18. Ha, S. H., and Park, T. H. (1997) Biotechnol. Lett. 19, 1087–1091. 19. Rhee, W. J., Kim, E. J., and Park, T. H. (1999) Biotechnol. Prog. 15, 1028 –1032. 20. Rhee, W. J., and Park, T. H. (2000) Biochem. Biophys. Res. Commun. 271, 186 –190. 21. Ha, S. H., Park, T. H., and Kim, S.-E. (1996) Biotechnol. Tech. 10, 401– 406. 22. Enari, M., Hase, A., and Nagata, S. (1995) EMBO J. 14, 5201– 5208. 23. Laemmli, U. K. (1970) Nature 227, 680 – 685. 24. Sakai, N., Mori, S., Izumi, S., Haino-Fukushima, K., Ogura, T., Maekawa, H., and., Tomino, S. (1988) Biochim. Biophys. Acta 949, 224 –232. 25. Mine, E., Izumi, S., Katsuki, M., and Tomino, S. (1983) Dev. Biol. 97, 329 –337.
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Isolation and Characterization of an Apoptosis-Inhibiting Component from the Hemolymph of Bombyx mori Eun Jeong Kim, Won Jong Rhee, and Tai Hyun Park 1 School of Chemical Engineering, Seoul National University, Kwanak-Gu Shilim-Dong San 56-1, Seoul 151-744, Korea
Received June 8, 2001
Silkworm (Bombyx mori) hemolymph showed an apoptosis-inhibiting activity in insect cells (Sf 9) infected with baculovirus (AcNPV). The addition of silkworm hemolymph into the culture medium increased the host cell longevity due to its apoptosis-inhibition activity. Components with an apoptosis-inhibiting effect were purified from the silkworm hemolymph by heat treatment, gel-filtration chromatography, and ion-exchange chromatography. The component with highest activity was characterized by periodic acid– Schiff staining, isoelectric focusing, MALDI-TOF–mass spectrometry, and N-terminal sequencing and was found to be a nonglycosylated monomeric protein with a molecular weight of ca. 28,000 Da. © 2001 Academic Press
Apoptosis is programmed form of cell death that is morphologically distinguishable from necrosis (1, 2) and is accompanied by the condensation of nuclei and cytoplasm, loss of microvilli and the convolution of plasma membranes, and nuclear and cell segmentation (3), whereas necrosis is typified by cell swelling, mild clumping of chromatin, and cell lysis, which causes the release of the cell constituents. The programmed cell death occurs in most animal tissues to eliminate harmful cells or those that are produced in excess, and these are mediated by apoptosis (4, 5). The decision as to whether a cell undergoes apoptosis or necrosis is influenced by a variety of environmentally and internally regulatory stimuli. Recent evidence suggests that alterations in cell survival contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases, neurodegenerative disorders, and AIDS (acquired immunodeficiency syndrome) (6). Regulation of cell death may have therapeutic potential in these diseases, therefore, many research groups have been and continue to investigate the regulation of apoptosis. 1
To whom correspondence and reprint requests should be addressed. Fax: ⫹82-2-875-9348. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
The disruption of cell physiology as a result of viral infection can cause an infected cell to undergo apoptosis (6), and in particular, the infection of an insect cell with a baculovirus was reported to induce apoptosis (7), which involved the activation of host caspases (cysteine-dependent, aspartate-specific protease) (8). Spodoptera frugiperda and the Autographa californica nuclear polyhedrosis virus (AcMNPV) have been widely used as a model insect cell and baculovirus, respectively, for studying the baculovirus regulation of host apoptosis. A specific gene product, P35, which prevents an apoptotic response, was identified in AcMNPV and silkworm baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV) (7, 9). Various other genes are also known to regulate apoptosis in the cells of mammalian immune system (10 –12). Baculovirus also carries genes which produce inhibitors of apoptosis (IAPs), and IAP homologues have been found in a diversity of insect and mammal genomes (13, 14). Insect hemolymph was used as a culture medium in the early stages of insect tissue culture. On the basis of the chemical analysis of insect hemolymph, a synthetic medium was formulated for insect cell culture (15, 16), but it still had to be supplemented with insect hemolymph (17). However these days, fetal bovine serum (FBS) has replaced insect hemolymph as a supplement in insect cell medium. In a previous study (18), we reported that the production of recombinant protein in an insect cell– baculovirus system was increased by supplementing the medium with silkworm hemolymph. We also found that the silkworm hemolymph increased host cell longevity (19) by inhibiting baculovirus-induced insect cell apoptosis (20). This indicates that the component, which inhibits the apoptosis is contained in the silkworm hemolymph. In this article, the apoptosis-inhibiting component was isolated from silkworm hemolymph and characterized. MATERIALS AND METHODS Collection of silkworm hemolymph. Silkworm hemolymph was collected from the fifth-instar larvae by clipping the side of an abdominal leg. The collected hemolymph was heat-treated at 60°C for
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Protein purification. To facilitate purification, the hemolymph was heat-treated at various temperatures for 30 min. After chilling and centrifuging, the precipitated proteins were removed. Heattreated and centrifuged supernatants were loaded on a Superdex 200 HR (1 ⫻ 30 cm, Amersham–Pharmacia Biotech) and eluted with 50 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl at a flow rate of 0.5 ml/min. The eluant was monitored at 280 nm and 0.5 ml fractions were collected. Active fractions from the Superdex 200 HR column were desalted into the starting buffer (120 mM Tris and 80 mM Bis–Tris, pH 9.0) using a HiTrap desalting column (1.6 ⫻ 2.5 cm, Amersham–Pharmacia Biotech). The desalted fractions were injected onto a Mono Q HR (0.5 ⫻ 5 cm, Amersham–Pharmacia Biotech) preequilibrated with same starting buffer and eluted with a linear gradient of 20 column volumes of elution buffer (120 mM Tris and 80 mM Bis–Tris, pH 5.0) at a flow rate of 0.5 ml/min. The eluant was monitored at 280 nm and 1-ml fractions were collected. The purified fractions were concentrated using lyophilizer. Electrophoresis. SDS–PAGE was carried out using 15% polyacrylamide separating gels, and 5% polyacrylamide stacking gels, using the method described by Laemmli (23). A low-range protein molecular weight standard (Amersham–Pharmacia Biotech) was used and gels were stained using Coomassie blue. Gels were stained for glycoproteins using a periodic acid–Schiff (PAS)-based stain (Sigma). Isoelectric focusing was carried out on PhastGel IEF 3-9 (Amersham–Pharmacia Biotech) using PhastSystem (Amersham– Pharmacia Biotech). The isoelectric points (pI) of the purified proteins were measured using a pI calibration kit (pI 3.5–9.3, Amersham–Pharmacia Biotech). Mass spectrometry. Matrix-assisted laser desorption ionization time of flight (MALDI-TOF)–mass spectrometry was carried out using a Voyager-DE (PerSeptive Biosystem). CHCA (␣-cyano-4hydroxy-cinnamic acid) (Sigma) and myoglobin were used as a matrix and as an internal standard, respectively.
FIG. 1. (A) Fractionation of silkworm hemolymph by Superdex 200 column chromatography. (B) SDS–PAGE analysis of F1 and FII.
30 min. then chilled and centrifuged (21). The supernatant was filtered through a 0.2-m membrane filter and stored 4°C for the later use. Insect cell culture and baculovirus infection. S. frugiperda (Sf 9) cells were cultivated in Grace medium (Gibco) supplemented with 5% fetal bovine serum (FBS, Gibco), 0.35 g/liter NaHCO 3, and antibiotic–antimicotic (Gibco) at 28°C. Whole silkworm hemolymph, used as a control, or the isolated component from each purification step was added to the medium, and their effects were investigated on baculovirus-induced insect cell apoptosis. Cells were infected in the late exponential growth phase (5 days in culture) with the recombinant baculovirus, which in this study was A. californica nuclear polyhedrous virus (AcNPV) that produces -galactosidase under the control of a polyhedrin promoter. For the infection, the medium was aspirated and a virus stock solution added. A multiplicity of infection (m.o.i.) of 13 was used for all experiments. After incubating for 1 h, the virus solution was replaced with the medium used before the infection. Apoptosis assay. Cell concentrations were measured using a hemocytometer and viable cells were detected using the trypan blue exclusion test. Since dead cells absorb trypan blue (Sigma), they can be identified under the optical microscope. The cell viability was defined by the ratio of the viable cell number to the total cell number. Cellular DNA was extracted using the previously reported method (22), and the DNA fragmentation pattern was examined by 2% agarose gel electrophoresis.
N-terminal sequencing. SDS–PAGE gel was transferred to a PVDF membrane by electroblotting. After the transfer the membrane was stained with Ponseau S (0.2% Ponseau S in 1% acetic acid) and destained with deionized water. The stained band was then cut out and air-dried. Amino acid sequencing was carried out using a Precise Protein sequencing system (Applied Biosystems).
RESULTS AND DISCUSSION In our previous study (20), the host insect cell viability remained at a high level for 6 days after baculo-
FIG. 2. Effect of each fraction on cell viability. Medium was supplemented with 5% FBS and each fraction was obtained from Superdex 200 chromatography.
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FIG. 3. (A) Fractionation of FII by Mono Q column chromatography. (B) SDS–PAGE analysis of FII-1 and FII-2 obtained by Mono Q chromatography.
virus infection in the medium supplemented with 5% silkworm hemolymph, whereas its viability began to decrease 3 days after infection in a medium without silkworm hemolymph. It was also shown that DNA fragmentation was not observed during the first 3 days, while it occurred afterward in the medium without silkworm hemolymph. Whereas the DNA fragmentation was not observed until the sixth day in the medium supplemented with 5% silkworm hemolymph. Significantly, the time when the DNA fragmentation began corresponded to the time when the cell viability started to decrease (19). This also indicates that cell viability measurements can be used as an assay method for isolating the apoptosis-inhibiting component from silkworm hemolymph. Therefore, cell viability was measured 7 days after infection in media supplemented with each fraction to determine the apoptosis-inhibiting activity of the fractionated silkworm hemolymph. Silkworm hemolymph must be heat-treated for use as a medium supplement, since the intact hemolymph darkens visibly due to the activity of tyrosinase, which
synthesizes melanin via intermediary quinones (15). The production of toxic quinones in the medium consequently inhibits cell growth. Heat treatment and centrifugation facilitate the purification step by removing many proteins, as well as tyrosinase from the silkworm hemolymph. More proteins were removed by higher temperature heat treatments; however, these reduced the apoptosis-inhibiting activity. It was shown that 60°C is the most appropriate heat-treatment temperature and the apoptosis-inhibiting component is stable at 60°C (20). After heat treatment and centrifugation at 60°C for 30 min, the supernatant was loaded onto a gelfiltration column to purify the effective component. Five fractions were obtained, as is shown in Fig. 1A. Among these fractions, FI and FII showed high apoptosis-inhibitory activity as shown in Fig. 2, despite the lower protein concentration of FII, as is shown in Fig. 1B, cell viability was higher in FII than in FI. The molecular mass of the major protein contained in FI was approximately 70,000 Da, and this protein accounted for about 60% of the hemolymph proteins. The major protein contained in FII was found to have a molecular mass of ca. 30,000 Da, and accounted for 30% of the protein present. That is, these two proteins accounted for almost all of the hemolymph protein. FII, which had the higher apoptosis-inhibiting activity, was further purified by anion-exchange chromatography with a linear gradient elution of 20 column volumes (pH 9.0 –5.0), and four fractions were obtained as shown in Fig. 3A. Among these fractions, the major fractions FII-1 and FII-2 showed apoptosis-inhibitory activity, as shown in Fig. 4. The SDS–PAGE results of FII-1 and FII-2 are shown in Fig. 3B. The fraction FI obtained from gel-filtration chromatography was also further purified using the same anion-exchange chromatographic method, and major
FIG. 4. Effect of each fraction on cell viability. Medium was supplemented with 5% FBS and each fraction was obtained by Mono Q column chromatography.
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three fractions (FI-1, FI-2, and FI-3) were obtained (data not shown). Table 1 summarizes the protein concentrations of each fraction. Fractions were concentrated by lyophilization and added to the culture medium at the same concentration (0.2 mg protein/ medium ml) to determine the specific activity of apoptosis inhibition. The last column in Table 1 is the relative apoptosis-inhibiting activity of each fraction. It represents the relative cell viability (7 days after infection) in the medium supplemented with the same amount (0.2 mg/ml) of each fraction. The percentage is the relative cell viability to that in 5% silkworm hemolymph medium. Many of these fractions resulted in the inhibition of apoptosis; however, their effects were not additive and FII-2 showed the highest value (92%). In the periodic acid–Schiff reagent staining, detection of glycoproteins FI-1, FI-3, FII-1, and FII-2 was not indicated (data not shown), whereas FI-2 was stained, indicating that only FI-2 is a glycoprotein. The isoelectric points (pI) of FI-1, FI-2, FII-1, and FII-2 were approximately 6.62, 5.76, 6.93, and 5.96, respectively. FII-2, which had the highest activity, was further analyzed by MALDI-TOF–mass spectrometry and found to be a monomeric protein with the molecular weight of ca. 28,000 Da. The N-terminal amino acid sequence of the first 24 residues of FII-2 are as follows: NH 2 -Ala-Asp-Ser-Asp-Val-Pro-Asn-Asp-Ile-Leu-GluGlu-Gln-Leu-Tyr-Asn-Ser-Val-X-Val-Ala-Asp-X-Tyr. A database search using this amino acid sequence as a template for -BLAST resulted in a 95% homology with a low molecular weight lipoprotein, the so called “30K protein” of unknown function (24). The amino acid sequence of FII-2 is compared with that of 30K protein in Table 2.
TABLE 1
Protein Concentrations and Relative Apoptosis-Inhibiting Activity of Each Fraction
Heat-treated SH FI FI-1 FI-2 FI-3 FII FII-1 FII-2 FII ⬃ FV
Protein (mg)/ SH (ml)
Protein (mg)/ medium (ml)
Ratio % (each fraction/ total)
Relative apoptosisinhibiting activity (%)
35.43 20.62 5.49 5.93 5.82 10.80 4.43 4.17 4.02
1.77 1.03 0.27 0.29 0.29 0.54 0.22 0.21 0.20
100 58.19 15.50 16.75 16.43 30.47 12.49 11.78 11.34
100
TABLE 2
Comparison of N-Terminal Amino Acid Sequences of FII-2 and 30K Protein 30K protein FII-1 30K protein FII-2
1 MKPAIVILCL 21 DVPNDILEEQ DVPNDILEEQ
11 FVASLYA AD AD 31 LYNSVVVAD LYNSV –VAD
S S Y –
The 30K protein is a specific type of plasma proteins and is one of the so-called “storage proteins” in the silkworm hemolymph. These are synthesized in the fat body of the feeding larvae and released into the hemolymph. During the larval-pupal transformation, they are transported from the silkworm hemolymph to the fat body cells and stored as protein granules (25). Deductions based upon the nucleotide sequence of 30K cDNA suggest that the nascent molecule has a signal sequence (24). The 1⬃17 region of its amino-terminal sequence, as shown in Table 2, is a signal sequence. The mechanism of baculovirus-induced insect cell apoptosis is not fully understood; however, it is surmised that the apoptosis in insect cells involves a cascade of caspase activation and Sf-caspase-1 is the principal effector caspase in Sf 9 cells. In the insect cellbaculovirus system, silkworm hemolymph may work directly on the baculovirus-induced apoptosis cascade mechanism or increase the expression of the antiapoptotic baculoviral gene such as p35. Recently, we found that silkworm hemolymph inhibits not only the baculovirus-induced apoptosis but also the apoptosis induced by various chemicals such as actinomycin D, camptothecin, and staurosporine. Moreover, it also inhibits the apoptosis of mammalian cells including human cells (unpublished). These results indicate that silkworm hemolymph itself contains anti-apoptotic components itself and 30K protein is one of them. Further study is required to elucidate the mechanism for the inhibition of apoptosis by 30K protein. ACKNOWLEDGMENTS
48.25 ⫾ 4.51 57.13 ⫾ 1.75 61.53 ⫾ 0.70 73.40 ⫾ 1.74 92.16 ⫾ 3.26 Negligible
The authors acknowledge the financial support of the Korea Science & Engineering Foundation through the Nano Bio-Electronic & System Center. This work was supported in part by the Brain Korea 21 Program from the Ministry of Education. Silkworms were kindly provided by Dr. Sam-Eun Kim, Department of Sericulture and Entomology, National Institute of Agricultural Science and Technology, Suwon, Korea.
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Note. The relative apoptosis-inhibiting activity represents the relative cell viability (7 days after infection) in the medium supplemented with the same amount (0.2 mg/ml) of each fraction. The percentage is the relative cell viability to that in 5% silkworm hemolymph medium. 227
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