Int. 1. Exp. Path. (1992) 73, 43-49
Purification and amino-terminal sequence of the dengue virus-induced cytotoxic factor Madhu Khanna & U.C. Chaturvedi Postgraduate Department of Microbiology, K.G. Medical College, Lucknow -226 003, India Received for publication i 8 March I 99 I Accepted for publication 20 September I 99 I
Summary. The present study was undertaken to purify the dengue type 2 virus (DV)-induced cytotoxic factor (CF) and analyse its amino-terminal sequence. Spleens collected from DVinfected moribund mice were made into a single cell suspension and cultured for 24 h. The culture supernatant was purified using fast protein liquid chromatography (FPLC) and polyacrylamide gel electrophoresis (PAGE). CF could be purified in a single step by FPLC and native PAGE. The isoelectric point of CF was pH 6.5. Purified CF had a molecular weight of 45 kDa on native-PAGE while on SDS-PAGE it dissociated into two bands of 20 and 2 5 kDa. AntiCF-antisera reacted specifically with CF bands separated on native and SDS-PAGE in a Western blot assay. A sequence of I9 amino-acids of the N-terminus of CF was analysed which on comparison with that of other known cytotoxic proteins indicated that CF differs from them. Thus, CF appears to be a unique cytotoxic protein induced by a virus. Keywords: dengue virus, cytotoxic factor, amino-acid sequence, chromatography Dengue type 2 virus (DV) induces a subpopulation of mouse splenic T lymphocytes (ThyI.2+, LyI+2 -) to produce a cytotoxic factor (CF) which kills lymphoid cells of different animal species but has no effect on different tissue culture cells (Chaturvedi et al. i980a, 198ia; Shuklaetal. 1982). The cells that are killed by CF are mainly I-A negative macrophages and helper T cells (Chaturvedi et al. 198 7a; Khanna et al. I988). Intravenous inoculation of CF depresses foot-pad swelling reaction (DTH), leucocyte migration inhibition and the antibody plaque forming cell responses of mice to sheep erythrocytes (Chaturvedi et al. i98ib). The macrophages are reduced in number and
their ability to attach, migrate and phagocytose are depressed in such mice (Chaturvedi et al. I983; Nagar et al. I984). Similar effects are also observed in DV-infected mice and are considered to be mediated by CF (Gulati et al. I982; Nagar et al. I985). The subcellular changes seen in macrophages and lymphocytes of DV-infected mice (Nath et al. I983) can be reproduced by in-vivo or in-vitro treatment with CF (Chaturvedi et al. i98 7b). Recent studies have shown that CF increases capillary permeability and damages the blood-brain barrier in mice via release of histamine (Khanna et al. I990; Dhawan et al. iggoa; Chaturvedi et al. 199I). The adverse effects of DV infection on macro-
Correspondence: Professor U.C. Chaturvedi, Department of Microbiology, K.G. Medical College, Lucknow -226 003, India. 43
M. Khanna & U.C. Chaturvedi phages could be abrogated by pretreatment bund mice were teased out in chilled phosof mice with cycloheximide, which inhibits phate-buffered saline pH 7 (PBS) and a single CF production, or by surgical removal of the cell suspension was prepared. The cells were spleen, the site of CF production (Dalakoti et washed and cultured in PBS at 3 70C in the al. I982; Nagar et al. I985). Human blood presence of 5% CO2 (Khanna et al. I989, leucocytes treated in vitro with CF show I990). After 24 h the cultures were hardiminished phagocytic activity and E-rosette vested; cells were spun down at 3000 g for formation by T cells (Chaturvedi et al. I982; I 0 min at 40C and the clear supernatant was Gulati et al. I984). Such adverse effects of CF assayed for the cytotoxic activity. It was are inhibited by pretreatment of blood leuco- divided in small aliquots and stored at - 20°C or dried in a Speed Vac (Savant cytes with plasma membrane stabilizing substances (Gulati et al. I983). Similar effects Instruments Inc, Hicksville, USA). By culture are produced in monkeys inoculated with CF of cells in PBS the yield of CF was reduced as (Nagar et al. I986). Some of the changes about 30% of the cells died in 24 h, but the produced in human blood leucocytes by in- advantage was exclusion of extraneous provitro treatment with CF and those produced teins from the medium and the serum. A by inoculation of CF in monkeys are similar similar preparation obtained from normal mouse spleen cells (NH) was used as control. to those described in cases of DHF (Wells et al. NH had no cytotoxic activity. 1980). CF is a highly potent protein having a broad spectrum proteinase-like activity Preparation of anti-CF-antisera (Khanna et al. I989), but its precise chemical Antisera against CF was prepared in rabbits nature is not known. The present study, by giving weekly subcutaneous injections for undertaken to purify and analyse its amino6 months of FPLC-purified I00 uig CF protein terminal sequence, shows that CF appears to emulsified in complete Freund's adjuvant. be a unique cytotoxic protein. The antisera inhibited in-vitro cytotoxicity and other activities of CF (Khanna et al. Materials and methods 1990).
44
Mice
Assay of cytotoxic activity of CF
The study was carried out on inbred Swiss albino mice aged 3-4 months obtained from the mice colony maintained in this Department.
CF and its purified preparations were assayed for the cytotoxic activity using normal mouse spleen cells as target (Chaturvedi et al. ig80a). Briefly, I00 MI of the single cell suspension of the target cells (2 x j06 cells) was mixed with equal volumes of the CF preparation in a 96-well microtitre Perspex plate and incubated for i h at 4TC. Viability of the cells was then counted using the trypan blue dye exclusion method and the percentage of non-viable cells was expressed after deduction of the background values.
Virus Dengue type 2 virus (DV), strain P23085 was used in the form of infected mouse brain suspension as described in Chaturvedi et al. (1977).
Preparation of CF CF is secreted by the DV-primed spleen cells
Results
in tissue culture fluid or can be extracted from such cells (Chaturvedi et al. i98oa). The spleens collected from DV-infected mori-
Purification by FPLC CF was purified by Pharmacia fast protein
Amino-terminal sequence of dengue-induced cytotoxic factor
a-O 4-
0
0.
-0 U,
D0
45
100 90 80 70 60 50 40 30 20 10
I-
I U) CuQ a1) =
cu
0
z 15 20 Fraction number
10
25
30
Fig. i. Purification of CF on fast protein liquid chromatography using Superose-12 gel column. The , the fractions were monitored for - - -, protein at 280 nm with a UV-cord and were assayed for cytotoxic activity.
liquid chromatography (FPLC) using an HR 10/30 column prepacked with Superose-i2 gel. The elution was performed with PBS at a flow rate of ml/3 min and monitored at 280 nm with a UV-cord. The peaks were collected and assayed for cytotoxicity. The profile of different protein peaks eluted from crude CF is presented in Fig. The cytotoxic activity was found only in a small, sharp peak of protein eluted at Fraction 25. In different FPLC runs, the cytotoxic activity continued to appear in the same elution fraction. The proteins in the cytotoxic peak thus obtained were dried on Speed Vac, reconstituted, and re-run on the FPLC. In such preparations a single peak of protein having cytotoxic activity appeared. The purified CF was also assayed for amidolytic activity using chromogenic peptide substrates as described elsewhere (Khanna et al. I 989). The cytotoxic peak was not found in NH. i
i.
Polyacrylamide gel electrophoresis (PAGE) CF purified on FPLC was electrophoresed in 12% polyacrylamide native gels (Lammeli 1970) at a constant current of 30 mA in a vertical slab gel system. The gels were stained with Coomassie Brilliant Blue-R 250 stain or silver stain. For identifying the CF
band, one lane of the electrophoresed gel was cut and stained. The stained lane was aligned by the side of the main gel and portions were cut along the position of the stained protein band. The proteins were eluted from the gel and tested for cytotoxic activity to identify the CF-band in PAGE. Some of the gels were run with molecular weight markers (Weber & Osborn I969), which indicated that CF had a molecular weight of about 45 kDa (Fig. 2). No CF band was found in NH. Sodium dodecyl sulphate (SDS)-PAGE was carried out with 1 2% polyacrylamide slab gel (Lammeli 1970). The protein sample was denatured with I% SDS at go°C for 5 min under reducing conditions and then electrophoresed on SDS-PAGE. Molecular weight markers were included in some of the runs. The gels were stained with Coomassie Brilliant Blue-R 250 or silver nitrate as stated above. On SDS-PAGE the purified preparation of CF was split into two bands of smaller molecular weight occupying positions of 20 and 25 kDa (Fig. 3). Isoelectric focusing (IEF) Phast gel IEF-3-9 media was used for isoelectric focusing of purified CF using the Pharmacia Phast Gel electrophoresis system (Allen & Humphries I975). Marker proteins
46 Lane 1
2
M. Khanna & U.C. Chaturvedi of pH range 3.5-8.65 were included in the run. The gels were run for 500 V h at 50C and stained with Phast gel Blue R at 5o°C. i
The isoelectric point of CF was found to be at pH 6.5. kDa
-_94 66
Immunoblotting Purified CF and the NH proteins (for control) were resolved on 12% native or SDS-PAGE gels and then electroblotted on to nitrocellulose paper. It was blocked with 3% bovine serum albumin prepared in blocking buffer containing o. I M Tris HCI pH 7.5 and o.o5% Triton XThe blots were incubated with appropriately diluted anti-CF-antisera (Khanna et al, I990) at room temperature (2s5C) on a rocking table. After washing thrice, it was incubated with anti-rabbit IgG linked to horseradish peroxidase for 2.5 h at room temperature. After extensive washing, the blots were developed using diaminobenzidine and hydrogen peroxide. For control, the blots were treated with anti-DV-antisera in place of anti-CF-antisera. It was observed that the single band of CF obtained on native PAGE reacted with anti-CF antibody. No reaction was observed with anti-DV-antisera or normal rabbit sera. Similarly, on SDSPAGE, both the bands of CF reacted with the anti-CF-antisera. The blots obtained from NH did not react with any of the antisera used. i oo.
45
29
-14.2
polyacrylamide gel electrophoresis purified by FPLC (Lane i) with the molecular weight markers (Lane 2). The gels were silver
Fig.
2. Native
of CF
stained.
Lane
1
2
Amino-terminal
sequence
analysis of CF*
The crude CF purified on FPLC was run on 12% PAGE. The CF band with a molecular weight position of 43-45 kDa was electroeluted using mM phosphate buffer (pH 7) for I5 h at 4°C. The electroeluted protein solution was dialysed against 5% acetic acid with four changes. The concentrated protein solution (5 ug) was applied on a Polybrenecoated TFA-treated glass fibre disc and sequenced using a pulsed liquid phase sequencer model 477 A (Applied Biosystems io
Fig. 3. SDS-PAGE of CF purified on native PAGE (Lane ) and FPLC (Lane 2). The gels were stained with Coomasie Brilliant Blue. i
* Done with the help of Dr B.R. Srinivasa, ASTRA Research Centre, Bangalore.
Amino-terminal sequence of dengue-induced cytotoxic factor V A D D
D C L E P L Y N
E R
47
S C E L R E
Fig. 4. Amino-acid sequence of the N-terminus of CF.
Inc.). The amino acid analysis was done using a PTH amino acid analyser Model i20A. The N-terminal amino-acid sequence of CF is presented in Fig 4. The sequencing was done twice and identical patterns were obtained. It was observed that the sequence is hydrophilic in nature with two cysteines at positions 6 and i 6. Discussion The main point brought out in the present study is that DV-induced CF is a unique protein molecule which differs from other cytotoxic proteins, such as tumour necrosis factor (TNF) a, TNF-f,, and T-cell serine esterase in its amino-acid sequence as shown by a comparison at database. CF has two cysteines at position 6 and I 6 while murine TNF-f has one at 84, human TNF-,B has none and TNF-a has two cysteines, the first at 84 (reviewed by Ruddle et al. i987). Even at the time of discovery of CF in 1979, differences from mouse lymphotoxin (TNF-fJ) were realized (Chaturvedi et al. Ig80b) including their target cells (Chaturvedi et al. ig8ia, I987a; Khanna et al. i988). Further, the isoelectric point of 6.5 for CF, as reported here, differs from that of TNF-cx (5.6) and TNF-f (5.8) (reviewed by Aggarwal et al. I 9 8 7). Another point of difference is that the cytotoxic activity of TNF-ac, TNF-f,, serine esterases and perforins is Ca2 +-independent (reviewed by Clark I988). In contrast, the presence of Ca2+ is obligatory for the induction, secretion and the cytotoxic activity of CF (Dhawan et al. I990, I99I; Khanna et al.
tested on PAGE (Chaturvedi et al. ig80b) as was done in the present study. Another good method for a single step purification of CF was preparative PAGE using 12% native gels. The cytotoxic protein was localized in a single band at the 45 kDa position. But on SDS-PAGE, CF dissociated into two bands which migrated to 20 and 25 kDa positions. In the Western blot test, using native PAGE the single band of CF at 45 kDa position reacted specifically with anti-CF-antibodies, while in SDS-PAGE both the CF bands reacted with equal intensity. It has been shown that anti-CF-antibody inhibits the cytotoxic activity of CF in vitro and CFinduced increase in capillary permeability in vivo (Khanna et al. I 990). This confirmed the specificity of CF as located on native and SDSPAGE. Similar findings have been reported for the TNF-# which elutes in a single peak on gel filtration and gives a single band at 64 kDa (reviewed by Aggarwal et al. I987). The sequence of CF described here was the only one noted in repeated determinations, which indicated it to be the predominant sequence. The question which remains to be resolved is whether the two subunits of 20 and 25 kDa of CF noted on SDS-PAGE, have the same amino-terminus sequence. Thus, CF appears to be a unique dengue virus-induced cytotoxic protein which differs from other cytotoxic proteins in a number of properties including the amino-terminal sequence.
Acknowledgements
1991).
FPLC was a useful method for purification as the CF could be purified in a single step. In earlier studies the cytotoxic activity was eluted in a single peak on agarose gel electrophoresis and on Sephadex G-ioo column but the purity of the CF protein was not
We sincerely thank Dr S. Anand Kumar the Director, Dr B.R. Srinivasa and Mr K.R. Swaminathan, ASTRA Research Centre, Bangalore for providing help during these studies including analysis of the amino-acid sequence. Thanks are due to the Distributed
48
M. Khanna & U.C. Chaturvedi
Information Centre, Indian Institute of Science, Bangalore for help in the Database analysis. The study was carried out with the financial assistance of the Council of Scientific and Industrial Research, New Delhi.
References AGGARWAL B.B., AIYER R.A., PENNICA D., GRAY P.W. & GOEDDEL D.V. (I987) Human tumour necrosis factors: structure and receptor interactions. In Tumour Necrosis Factor and Related Cytotoxins. Ciba Foundation Symposium, I 3I. Eds G. Bock & J. Marsh. John Wiley, Chichester, PP. 39-51. ALLEN J.C. & HUMPHRIES C. (1975) In Isoelectric focusing. Eds J.P. Arbuthnott & J.A. Beeley, Butterworths, London, pp. 347-354. CHATURVEDI U.C., BHARGAVA A. & MATHUR A. (ig8oa) Production of cytotoxic factor in the spleen of dengue virus-infected mice. Immunology 40, 665-671. CHATURVEDI U.C., DALAKOTI H. & MATHUR A. (ig80b) Characterization of the cytotoxic factor produced in the spleen of dengue virusinfected mice. Immunology 41, 387-392. CHATURVEDI U.C., DHAWAN R., KHANNA M. & MATHUR A. (i99I) Breakdown of the bloodbrain barrier during dengue virus infection of mice. J. Gen. Virol. 72, 859-866. CHATURVEDI U.C., GIJLATI L. & MATHUR A. (I982) Inhibition of E-rosette formation and phagocytosis by human blood leucocytes after treatment with the dengue virus-induced cytotoxic factor. Immunology 45, 679-685. CHATURVEDI U.C., MATHUR K.R., GIJLATI L. & MATHUR A. (i98ia) Target lymphoid cells for the cytotoxic factor produced in the spleen of dengue virus-infected mice. Immunol. Lett, 13i6. CHATURVEDI U.C., NAGAR R., GIJLATI L. & MATHUR A. (i987a) Variable effects of dengue virusinduced cytotoxic factors on different subpopulations of macrophages. Immunology 6i, 297301. CHATURVEDI U.C., NAGAR R. & MATHUR A. (I983) Effect of dengue virus infection on Fc-receptor functions of mouse macrophages. J. Gen. Virol. 64, 2399-2407. CHATURVEDI U.C., NATH P., GULATI L., JAIN A. & MATHUR A. (I98 7b) Subcellular changes in spleen cells of mice treated with the dengue virus-induced cytotoxic factor. Indian 1. Med. Res. 86, 284-289.
CHATURVEDI U.C., SHUKLA M.I., MATHUR K.R. & MATHUR A. (198 ib) Dengue virus-induced cytotoxic factor suppresses immune response of mice to sheep erythrocytes. Immunology 43,
311-3I6. CHATURVEDI U.C., TANDON P. & MATHUR A. (I9 77) Effect of immunosuppression on dengue virus infection in mice. 1. Gen. Virol. 36, 449-458. CLARK W.R. (I988) Perforin-a primary or auxillary lytic mechanism? Immunol. Today 9, IOI104. DALAKOTI H., CHATURVEDI U.C. & MATHUR A. (I982) Inhibition of production of dengue virus-induced cytotoxic factor by treatment with cycloheximide and mitomycin C. Indian J. Exp. Biol. 20, I-3. DHAWAN R., CHATIJRVEDI U.C., KHANNA M., MATHUR A., TEKWANI B.L. & PANDEY V.C. (199 I) Obligatory role of Ca2+ in the cytotoxic activity of dengue virus-induced cytotoxin. Int. J. Exp. Path. 72, 31-39. DHAWAN R., KHANNA M., CHATURVEDI U.C. & MATHUR A. (199oa) Effect of dengue virusinduced cytotoxin on capillary permeability. Int. J. Exp. Path. 71, 83-88. DHAWAN R., KHANNA M., CHATURVEDI U.C. & MATHUR A. (I99ob) Role of Ca2+ in induction and secretion of dengue virus-induced cytokines. |. Biosci. 15, 409-4I6. GULATI L., CHATURVEDI U.C. & MATHUR A. (I982) Depressed macrophage functions in dengue virus-infected mice. Role of the cytotoxic factor. Br. J. Exp. Path. 63, 194-202. GULATI L., CHATURVEDI U.C. & MATHUR A. (I983) Plasma membrane-acting drugs inhibit the
effect of dengue virus-induced cytotoxic factor. Ann. Immunol. (Inst. Pasteur) 134C, 227-235. GULATI L., CHATURVEDI, U.C. & MATHUR A. (I984) Effect of dengue virus-induced macrophage cytotoxin on functions of human blood leucocytes. Indian 1. Med. Res. 79, 709-7I5. KHANNA M., CHATURVEDI U.C. & MATHUR A. (I988) Abrogation of helper T cells by dengue virus-induced cytotoxic factor. Curr. Sci. 57, 411-414. KHANNA M., CHATURVEDI U.C., SRINIVASA B.R., SWAMINATHAN K.R. & MATHUR A. (I989) Proteinase-like activity in the cytotoxic factor produced by T cells during dengue virus infection. Immunology, 67, 32-37. KHANNA M., CHATURVEDI U.C., SHARMA M.C., PANDEY V.C. & MATHUR A. (I990) Increased capillary permeability mediated by a dengue
virus-induced lymphokine. Immunology 69,
449-453.
KHANNA M., CHATURVEDI U.C., DHAWAN R., TEK-
Amino-terminal sequence of dengue-induced cytotoxic factor WANI B.L. & PANDEY V.C. (I 99I) Presence of Ca2+ is obligatory for the cytotoxic activity of dengue virus-induced cytotoxic factor. Imimunology 72, 73-78. AMMELI U.K. ( 19 70) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685. 4AGAR R., CHATURVEDI U.C. & MATHUR A. (I984) Effect of dengue virus-induced cytotoxic factor on Fc-receptor functions of mouse macrophages. Br. I. Exp. Path. 65, 11-17. 4AGAR R., CHATURVEDI U.C. & MATHUR A. (I985)
Abrogation of dengue virus-induced alterations of Fc-receptor functions of macrophages by splenectomy or drug treatment. Indian J. Med. Res. 8i, 537-546. 4AGAR R., CHATURVEDI, U.C., MATHUR A. & KUMAR
A. (1986) Effect of dengue virus-induced cytotoxic factor on blood leucocytes of monkeys. Indian J. Med. Res. 84, 339-347. 4ATH P., TANDON P., GIJLATI L. & CHATURVEDI U.C. (i983) Histological and ultrastructural study
49
of spleen during dengue virus infection of mice. Indian J. Med. Res. 78, 83-90. RIJDDLE N.H., Li C-B., TANG W., GRAY P.W. & MCGRATH K.M. (I987) Lymphotoxin: Cloning, regulation and mechanism of killing. In Tumour Necrosis Factor and Related Cytotoxins. Ciba Foundation Symposium I3I. Eds G. Bock & J. Marsh. John Wiley, Chichester, pp. 52-82. SHIJKLA M.I., DALAKOTI H. & CHATURVEDI U.C. (19 82) Ly phenotype of T lymphocytes producing dengue virus-induced immunosuppressive factors. Indian J. Exp. Biol. 20, 525-528. WELLS R.A., ScoTT R.Mc.N., PAVANAND K., SATHITSATHEIN V., CHEAMUDON U. & MCDERMOTT R.P. (ig80) Kinetics of peripheral blood leucocyte alterations in Thai children with dengue haemorrhagic fever. Infect. Immun. 28, 428433. WEBER K. & OSBORN M. (I:969) The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. 1. Biol. Chem. 244, 4406-4412.
Purification and amino-terminal sequence of the dengue virus-induced cytotoxic factor Madhu Khanna & U.C. Chaturvedi Postgraduate Department of Microbiology, K.G. Medical College, Lucknow -226 003, India Received for publication i 8 March I 99 I Accepted for publication 20 September I 99 I
Summary. The present study was undertaken to purify the dengue type 2 virus (DV)-induced cytotoxic factor (CF) and analyse its amino-terminal sequence. Spleens collected from DVinfected moribund mice were made into a single cell suspension and cultured for 24 h. The culture supernatant was purified using fast protein liquid chromatography (FPLC) and polyacrylamide gel electrophoresis (PAGE). CF could be purified in a single step by FPLC and native PAGE. The isoelectric point of CF was pH 6.5. Purified CF had a molecular weight of 45 kDa on native-PAGE while on SDS-PAGE it dissociated into two bands of 20 and 2 5 kDa. AntiCF-antisera reacted specifically with CF bands separated on native and SDS-PAGE in a Western blot assay. A sequence of I9 amino-acids of the N-terminus of CF was analysed which on comparison with that of other known cytotoxic proteins indicated that CF differs from them. Thus, CF appears to be a unique cytotoxic protein induced by a virus. Keywords: dengue virus, cytotoxic factor, amino-acid sequence, chromatography Dengue type 2 virus (DV) induces a subpopulation of mouse splenic T lymphocytes (ThyI.2+, LyI+2 -) to produce a cytotoxic factor (CF) which kills lymphoid cells of different animal species but has no effect on different tissue culture cells (Chaturvedi et al. i980a, 198ia; Shuklaetal. 1982). The cells that are killed by CF are mainly I-A negative macrophages and helper T cells (Chaturvedi et al. 198 7a; Khanna et al. I988). Intravenous inoculation of CF depresses foot-pad swelling reaction (DTH), leucocyte migration inhibition and the antibody plaque forming cell responses of mice to sheep erythrocytes (Chaturvedi et al. i98ib). The macrophages are reduced in number and
their ability to attach, migrate and phagocytose are depressed in such mice (Chaturvedi et al. I983; Nagar et al. I984). Similar effects are also observed in DV-infected mice and are considered to be mediated by CF (Gulati et al. I982; Nagar et al. I985). The subcellular changes seen in macrophages and lymphocytes of DV-infected mice (Nath et al. I983) can be reproduced by in-vivo or in-vitro treatment with CF (Chaturvedi et al. i98 7b). Recent studies have shown that CF increases capillary permeability and damages the blood-brain barrier in mice via release of histamine (Khanna et al. I990; Dhawan et al. iggoa; Chaturvedi et al. 199I). The adverse effects of DV infection on macro-
Correspondence: Professor U.C. Chaturvedi, Department of Microbiology, K.G. Medical College, Lucknow -226 003, India. 43
M. Khanna & U.C. Chaturvedi phages could be abrogated by pretreatment bund mice were teased out in chilled phosof mice with cycloheximide, which inhibits phate-buffered saline pH 7 (PBS) and a single CF production, or by surgical removal of the cell suspension was prepared. The cells were spleen, the site of CF production (Dalakoti et washed and cultured in PBS at 3 70C in the al. I982; Nagar et al. I985). Human blood presence of 5% CO2 (Khanna et al. I989, leucocytes treated in vitro with CF show I990). After 24 h the cultures were hardiminished phagocytic activity and E-rosette vested; cells were spun down at 3000 g for formation by T cells (Chaturvedi et al. I982; I 0 min at 40C and the clear supernatant was Gulati et al. I984). Such adverse effects of CF assayed for the cytotoxic activity. It was are inhibited by pretreatment of blood leuco- divided in small aliquots and stored at - 20°C or dried in a Speed Vac (Savant cytes with plasma membrane stabilizing substances (Gulati et al. I983). Similar effects Instruments Inc, Hicksville, USA). By culture are produced in monkeys inoculated with CF of cells in PBS the yield of CF was reduced as (Nagar et al. I986). Some of the changes about 30% of the cells died in 24 h, but the produced in human blood leucocytes by in- advantage was exclusion of extraneous provitro treatment with CF and those produced teins from the medium and the serum. A by inoculation of CF in monkeys are similar similar preparation obtained from normal mouse spleen cells (NH) was used as control. to those described in cases of DHF (Wells et al. NH had no cytotoxic activity. 1980). CF is a highly potent protein having a broad spectrum proteinase-like activity Preparation of anti-CF-antisera (Khanna et al. I989), but its precise chemical Antisera against CF was prepared in rabbits nature is not known. The present study, by giving weekly subcutaneous injections for undertaken to purify and analyse its amino6 months of FPLC-purified I00 uig CF protein terminal sequence, shows that CF appears to emulsified in complete Freund's adjuvant. be a unique cytotoxic protein. The antisera inhibited in-vitro cytotoxicity and other activities of CF (Khanna et al. Materials and methods 1990).
44
Mice
Assay of cytotoxic activity of CF
The study was carried out on inbred Swiss albino mice aged 3-4 months obtained from the mice colony maintained in this Department.
CF and its purified preparations were assayed for the cytotoxic activity using normal mouse spleen cells as target (Chaturvedi et al. ig80a). Briefly, I00 MI of the single cell suspension of the target cells (2 x j06 cells) was mixed with equal volumes of the CF preparation in a 96-well microtitre Perspex plate and incubated for i h at 4TC. Viability of the cells was then counted using the trypan blue dye exclusion method and the percentage of non-viable cells was expressed after deduction of the background values.
Virus Dengue type 2 virus (DV), strain P23085 was used in the form of infected mouse brain suspension as described in Chaturvedi et al. (1977).
Preparation of CF CF is secreted by the DV-primed spleen cells
Results
in tissue culture fluid or can be extracted from such cells (Chaturvedi et al. i98oa). The spleens collected from DV-infected mori-
Purification by FPLC CF was purified by Pharmacia fast protein
Amino-terminal sequence of dengue-induced cytotoxic factor
a-O 4-
0
0.
-0 U,
D0
45
100 90 80 70 60 50 40 30 20 10
I-
I U) CuQ a1) =
cu
0
z 15 20 Fraction number
10
25
30
Fig. i. Purification of CF on fast protein liquid chromatography using Superose-12 gel column. The , the fractions were monitored for - - -, protein at 280 nm with a UV-cord and were assayed for cytotoxic activity.
liquid chromatography (FPLC) using an HR 10/30 column prepacked with Superose-i2 gel. The elution was performed with PBS at a flow rate of ml/3 min and monitored at 280 nm with a UV-cord. The peaks were collected and assayed for cytotoxicity. The profile of different protein peaks eluted from crude CF is presented in Fig. The cytotoxic activity was found only in a small, sharp peak of protein eluted at Fraction 25. In different FPLC runs, the cytotoxic activity continued to appear in the same elution fraction. The proteins in the cytotoxic peak thus obtained were dried on Speed Vac, reconstituted, and re-run on the FPLC. In such preparations a single peak of protein having cytotoxic activity appeared. The purified CF was also assayed for amidolytic activity using chromogenic peptide substrates as described elsewhere (Khanna et al. I 989). The cytotoxic peak was not found in NH. i
i.
Polyacrylamide gel electrophoresis (PAGE) CF purified on FPLC was electrophoresed in 12% polyacrylamide native gels (Lammeli 1970) at a constant current of 30 mA in a vertical slab gel system. The gels were stained with Coomassie Brilliant Blue-R 250 stain or silver stain. For identifying the CF
band, one lane of the electrophoresed gel was cut and stained. The stained lane was aligned by the side of the main gel and portions were cut along the position of the stained protein band. The proteins were eluted from the gel and tested for cytotoxic activity to identify the CF-band in PAGE. Some of the gels were run with molecular weight markers (Weber & Osborn I969), which indicated that CF had a molecular weight of about 45 kDa (Fig. 2). No CF band was found in NH. Sodium dodecyl sulphate (SDS)-PAGE was carried out with 1 2% polyacrylamide slab gel (Lammeli 1970). The protein sample was denatured with I% SDS at go°C for 5 min under reducing conditions and then electrophoresed on SDS-PAGE. Molecular weight markers were included in some of the runs. The gels were stained with Coomassie Brilliant Blue-R 250 or silver nitrate as stated above. On SDS-PAGE the purified preparation of CF was split into two bands of smaller molecular weight occupying positions of 20 and 25 kDa (Fig. 3). Isoelectric focusing (IEF) Phast gel IEF-3-9 media was used for isoelectric focusing of purified CF using the Pharmacia Phast Gel electrophoresis system (Allen & Humphries I975). Marker proteins
46 Lane 1
2
M. Khanna & U.C. Chaturvedi of pH range 3.5-8.65 were included in the run. The gels were run for 500 V h at 50C and stained with Phast gel Blue R at 5o°C. i
The isoelectric point of CF was found to be at pH 6.5. kDa
-_94 66
Immunoblotting Purified CF and the NH proteins (for control) were resolved on 12% native or SDS-PAGE gels and then electroblotted on to nitrocellulose paper. It was blocked with 3% bovine serum albumin prepared in blocking buffer containing o. I M Tris HCI pH 7.5 and o.o5% Triton XThe blots were incubated with appropriately diluted anti-CF-antisera (Khanna et al, I990) at room temperature (2s5C) on a rocking table. After washing thrice, it was incubated with anti-rabbit IgG linked to horseradish peroxidase for 2.5 h at room temperature. After extensive washing, the blots were developed using diaminobenzidine and hydrogen peroxide. For control, the blots were treated with anti-DV-antisera in place of anti-CF-antisera. It was observed that the single band of CF obtained on native PAGE reacted with anti-CF antibody. No reaction was observed with anti-DV-antisera or normal rabbit sera. Similarly, on SDSPAGE, both the bands of CF reacted with the anti-CF-antisera. The blots obtained from NH did not react with any of the antisera used. i oo.
45
29
-14.2
polyacrylamide gel electrophoresis purified by FPLC (Lane i) with the molecular weight markers (Lane 2). The gels were silver
Fig.
2. Native
of CF
stained.
Lane
1
2
Amino-terminal
sequence
analysis of CF*
The crude CF purified on FPLC was run on 12% PAGE. The CF band with a molecular weight position of 43-45 kDa was electroeluted using mM phosphate buffer (pH 7) for I5 h at 4°C. The electroeluted protein solution was dialysed against 5% acetic acid with four changes. The concentrated protein solution (5 ug) was applied on a Polybrenecoated TFA-treated glass fibre disc and sequenced using a pulsed liquid phase sequencer model 477 A (Applied Biosystems io
Fig. 3. SDS-PAGE of CF purified on native PAGE (Lane ) and FPLC (Lane 2). The gels were stained with Coomasie Brilliant Blue. i
* Done with the help of Dr B.R. Srinivasa, ASTRA Research Centre, Bangalore.
Amino-terminal sequence of dengue-induced cytotoxic factor V A D D
D C L E P L Y N
E R
47
S C E L R E
Fig. 4. Amino-acid sequence of the N-terminus of CF.
Inc.). The amino acid analysis was done using a PTH amino acid analyser Model i20A. The N-terminal amino-acid sequence of CF is presented in Fig 4. The sequencing was done twice and identical patterns were obtained. It was observed that the sequence is hydrophilic in nature with two cysteines at positions 6 and i 6. Discussion The main point brought out in the present study is that DV-induced CF is a unique protein molecule which differs from other cytotoxic proteins, such as tumour necrosis factor (TNF) a, TNF-f,, and T-cell serine esterase in its amino-acid sequence as shown by a comparison at database. CF has two cysteines at position 6 and I 6 while murine TNF-f has one at 84, human TNF-,B has none and TNF-a has two cysteines, the first at 84 (reviewed by Ruddle et al. i987). Even at the time of discovery of CF in 1979, differences from mouse lymphotoxin (TNF-fJ) were realized (Chaturvedi et al. Ig80b) including their target cells (Chaturvedi et al. ig8ia, I987a; Khanna et al. i988). Further, the isoelectric point of 6.5 for CF, as reported here, differs from that of TNF-cx (5.6) and TNF-f (5.8) (reviewed by Aggarwal et al. I 9 8 7). Another point of difference is that the cytotoxic activity of TNF-ac, TNF-f,, serine esterases and perforins is Ca2 +-independent (reviewed by Clark I988). In contrast, the presence of Ca2+ is obligatory for the induction, secretion and the cytotoxic activity of CF (Dhawan et al. I990, I99I; Khanna et al.
tested on PAGE (Chaturvedi et al. ig80b) as was done in the present study. Another good method for a single step purification of CF was preparative PAGE using 12% native gels. The cytotoxic protein was localized in a single band at the 45 kDa position. But on SDS-PAGE, CF dissociated into two bands which migrated to 20 and 25 kDa positions. In the Western blot test, using native PAGE the single band of CF at 45 kDa position reacted specifically with anti-CF-antibodies, while in SDS-PAGE both the CF bands reacted with equal intensity. It has been shown that anti-CF-antibody inhibits the cytotoxic activity of CF in vitro and CFinduced increase in capillary permeability in vivo (Khanna et al. I 990). This confirmed the specificity of CF as located on native and SDSPAGE. Similar findings have been reported for the TNF-# which elutes in a single peak on gel filtration and gives a single band at 64 kDa (reviewed by Aggarwal et al. I987). The sequence of CF described here was the only one noted in repeated determinations, which indicated it to be the predominant sequence. The question which remains to be resolved is whether the two subunits of 20 and 25 kDa of CF noted on SDS-PAGE, have the same amino-terminus sequence. Thus, CF appears to be a unique dengue virus-induced cytotoxic protein which differs from other cytotoxic proteins in a number of properties including the amino-terminal sequence.
Acknowledgements
1991).
FPLC was a useful method for purification as the CF could be purified in a single step. In earlier studies the cytotoxic activity was eluted in a single peak on agarose gel electrophoresis and on Sephadex G-ioo column but the purity of the CF protein was not
We sincerely thank Dr S. Anand Kumar the Director, Dr B.R. Srinivasa and Mr K.R. Swaminathan, ASTRA Research Centre, Bangalore for providing help during these studies including analysis of the amino-acid sequence. Thanks are due to the Distributed
48
M. Khanna & U.C. Chaturvedi
Information Centre, Indian Institute of Science, Bangalore for help in the Database analysis. The study was carried out with the financial assistance of the Council of Scientific and Industrial Research, New Delhi.
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