Purification and Partial Characterization of Antiviral Proteins from Chenopodium album L. Leaves

J Plant Physiol.

VtJI.

• JOURNAL OF • PLANT PHYSIOLOGY

156 pp. 808-810 (2000)

http://www.urbanfischer.de/journals/jpp

© 2000 URBAN &FISCHER Verlag

I

Short Communication

I

Purification and Partial Characterization of Antiviral Proteins from Chenopodium album L. Leaves Som Dutt, A. Balasubrahmanyam and M. L. Lodha Division of Biochemistry, Indian Agricultural Research Institute, New Delhi - 110012, India Received September 6, 1999 . Accepted December 10, 1999

Summary

Two antiviral proteins from leaves of Chenopodium album L. have been purified and their characteristics compared. Both of these impart resistance in hypersensitive hosts: against tobacco mosaic virus in Nicotiana tabacum cv. Samsun NN and N. glutinosa, and sunnhemp rosette virus in Cyamopsis tetragonoloba. One of them exhibited a single polypeptide with a molecular mass of 25 kO, and the other one is a complex showing polypeptide bands of 27, 25 and 18 kO on SOS-PAGE. The 25 kD protein and the complex protein inhibited more than 90% lesion formation at a concentration of20-22lJ,gmL -1. Both of the proteins are basic in nature and can tolerate high temperature (up to 90 ·C).

Key words: Chenopodium album, antiviral proteins, purification. Introduction

A large number of higher plants are known to contain potent inhibitors of plant viruses and a few have been purified and characterised (Verma et al., 1995 a, b). Most of these are basic proteins with molecular weights ranging from 24,000 to 32,000 and act against different plant viruses (Irvin et al., 1980; Kubo et al., 1990; Lodge et al., 1993). Several species of Chenopodium are known to possess antiviral activity viz., Chenopodium amaranticolor (Alberghina, 1976), C album (Yoshizaki and Murayama, 1966; Smookler, 1971), C quinoa (Saksena and Mink, 1969) and C ambrosoides (Verma and Baranwal, 1983). Here, we describe the purification to apparent homogeneity of virus inhibitors from leaves of C album which can impart a very high level of resistance to tobacco mosaic virus and sunnhemp rosette virus on their respective local lesion hosts. Materials and Methods

Bioassay The tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) were maintained on their respective systemic hosts, i.e. to-

bacco (Nicotiana tabacum) var. NP33 and sunnhemp (Crotalaria juncea). Nicotiana glutinosa and N tabacum cv. Samsun NN were used as hypersensitive hosts for TMV and Cyamopsis tetragonoloba

for SRV depending on availability of test plants and seasons. The procedures described by Balasubrahmanyam et al. (2000) were followed for the virus inoculum preparation and application of the test samples (leaf crude extract or antiviral principle at different stages of purification) on test plant leaves. The percentage inhibition was calculated using the formula: % inhibition = C-T/C x 100, where C and T are the average number of lesions in control plants and treated plants, respectively.

Purification and Characterization of Virus Inhibitor About 40 g of dried leaves of C. album were homogenised with 6-7 volumes of extraction buffer (0.1 mol/L sodium acetate buffer, pH 5.2, containing 12 mmol/L ~-mercaptoethanol) and 12 mg of polyvinyl polypyrollidone in a blender. The soluble protein of the crude extract was fractionated by slow addition of solid ammonium sulphate to 0-30, 30-60, 60-80, and 80-100 per cent saturation, followed by low speed centrifugation and dialysis. The clear dialysed samples were tested for their antiviral activity on the test hosts. Since activity was found in two protein fractions precipitating between 30 % and 60 %, and 60 % and 80 % ammonium sulphate saturation, all of the protein precipitating between 30 % and 80 % saturation was used for further purification. The active protein fraction 0176-16171001156/808 $ 12.0010

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Antiviral Proteins from Chenopodium Leaves obtained in the above step was loaded onto a column (20 x 1.5 em) of DEAE-Cellulose equilibrated with buffer A (20 mmollL sodium phosphate, pH 6.2, containing 10 mmol/L NaCl). Washing with several bed volumes of column buffer eluted the unbound protein. The bound protein was eluted with 0.5 mol/L NaCl prepared in the same buffer. Both fractions were tested for their antiviral activity. The active DEAE-Cellulose unadsorbed protein was fractionated on a CM-Sepharose column (35 x 1 em) equilibrated with buffer B (20 mmollL sodium acetate buffer, pH 5.2., containing 10 mmollL NaCl and 0.01 % sodium azide). The bound protein was eluted from the column with a discontinuous gradient of NaCi (0.1, 0.2, 0.3, 0.4 and 0.5 mollL) prepared in the equilibrating buffer. Fractions falling within the protein peak were pooled and tested for the antiviral activity. The pooled protein sample was chromarographed on a Sephadex G-75 column (61 x 1.5 em) equilibrated with buffer C (20 mmollL sodium acetate, pH 5.2, containing 0.01 % sodium azide). Fractions falling within the protein peak were pooled and tested for antiviral activity. Protein estimation was done by the Bradford method (Bradford, 1976) using bovine serum albumin (BSA) as a standard. The presence of carbohydrate was tested by the Molisch test (Vogel, 1971). The molecular weights of the purified proteins were determined under native conditions by calibrated gel-permeation chromatography in a Sephadex G-75 column using the MW-70 kit (Sigma, USA). The molecular weights of the purified proteins were also determined under denatured conditions by electrophoresis in a 12 % separating gel by SDS-PAGE (Laemmli, 1970) using the MW SDS-70 kit (Sigma, USA). The proteins were visualised with Coomassie blue.

Table 1: Purification of antiviral principle from leaves of Chenopodium album*. Fraction

Total protein (mg)

Crude extract 262 30-80% Ammonium sulphate 127.4 DEAE-Cellulose unadsorbed 16.2 CM-Sepharose purified (i) 0.1 mollL NaCI eluted Peak I 3.37 Peak II 0.06 Peak III 2.83 (ii) 0.2mo1lL NaCl eluted Peak IV ·7.69 Peak V 0.2 Sephadex G-75 chromatography Peak A 0.35 Peak B 1.00

Protein in sample (l1g/mL)

% Inhibition

2620 12,740 108

93.1 93.5 94.3

136 20 152

4.0 21.6 93.2

225 32

85.1 Not tested

20 22

97.8 90.7

* Data refers to 40 g of dried leaf material. Note: The antiviral activity was tested against sunnhemp rosette virus (SRV) using C. tetragonoloba as test plant.

a

b

c

d

e

Results

Upon stepwise ammonium sulphate precipitation of soluble protein from crude extract, most of the antiviral activity was recovered in the protein fraction precipitating berween 30 % and 80 % ammonium sulphate. The active fraction was subjected to DEAE-Cellulose chromatography. The antiviral activity that came through in the void volume was further fractionated by CM-Sepharose cation exchange chromatography using a discontinuous linear NaCl gradient to elute bound proteins. The elution at 0.1 mollL NaCI resulted in three protein peaks, peaks I, II and III. The elution with 0.2 mollL NaCI gave rise to rwo peaks, peaks IV and V. Of these, the peak III and IV fractions exhibited very high activity (Table 1). The relevant pooled fractions were subjected to size exclusion chromatography on Sephadex G-75. This resulted in a single protein peak for each of the rwo pooled fractions, designated as peak A and peak B (corresponding to peak III and peak IV of CM-sepharose chromatography, respectively). These were subsequently found to possess high antiviral activity (Table 1). The purified antiviral principles from rwo active peaks of the Sephadex G-75 column, i.e. peaks A and B, tested positive for the Bradford reagent, indicatingtheir proteinaceous nature. However, they tested negative with the Molisch test, suggesting the absence of carbohydrate moiety in the antiviral proteins. Electrophoresis of the protein fraction of peak A under denaturing conditions revealed a single prominent band with a molecular weight of 25 kD. The protein fraction of peak B exhibited rwo prominent polypeptide bands with molecular weights of 25 and 18 kD, and a minor polypeptide band of 27 kD (Fig. 1). Molecular weights of purified antiviral pro-

Fig. 1: SDS-PAGE (12 %) of antiviral proteins purified from leaves of Chenopodium album. Lanes a & b: show 4 and 811g of Sephdex G-75 purified peak A protein; lane c: molecular weight markers (from top to bottom, BSA, 66,000; egg albumin, 45,000; trypsinogen, 24,000; lactoglobulin, 18,400 and lysozyme, 14,300), and lanes d & e: 4 and 811g of Sephadex G-75 purified peak B protein. The purified protein fractions obtained from the Sephadex G-75 chromatography were lyophilised and suspended in Laemmli buffer before loading onto the gel.

teins in their native state were determined by calibrated gelpermeation chromatography using a Sephadex G-75 column. The protein eluting under peak A with elution volume of 59.5 mL exhibited a molecular weight of 50.5 kD, whereas the protein eluting under peak B with elution volume of 49.0 mL exhibited a molecular weight of 72 kD (data not shown).

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Som Dutt, A. Balasubrahmanyam and M. L. Lodha

Various concentrations of these two proteins, ranging from 10 -150 Ilg mL -\, were applied on tobacco cv. Samsun NN plants to test for the resistance response against TMV. The protein fraction showing 25 kD molecular weight on SOSPAGE inhibited lesion formation bi more than 90 % at a concentration as low as 20 Ilg mL - , whereas the complex protein exhibited the same response at a concentration of 221lg mL -\ (results not shown). Both of the proteins displayed tolerance to temperature and remained entirely unaffected even after 10 min of incubation at 90·C. However, at around 95 ·C, they began to lose the ability to impart resistance (results not shown).

Discussion

In the present study, the proteins from the leaves of Chenopodium album precipitating between 30 % and 80 % ammonium sulphate saturation showed the maximum antiviral activity, whereas the earlier reports indicated that maximum precipitation of virus inhibitor occurred at 70-90 % saturated ammonium sulphate (Smookler, 1971). In the case of C amaranticolor, the virus inhibitor was reported to be precipitating at 50 % saturation (Taniguchi and Goto, 1979).The antiviral activity was found in the OEAE-Cellulose unadsorbed fraction in contrast with the earlier report about the C amaranticolor antiviral inhibitor (Taniguchi and Goto, 1979). Smookler (1971) observed that the inhibitors from C amaranticolorand C album strongly bound to the CM-Sephadex column and required a high concentration of buffer to remove them. In a similar fashion, the virus inhibitors from C album eluted at 0.1 mollL and 0.2 mollL NaCl concentration, respectively, and they might belong to two different categories. Subsequent analysis by gel filtration on Sephadex G-75 revealed that both were eluting at two different elution volumes (59.5 mL, peak A, and 49.0 mL, peak B, respectively), thus favouring the above possibility. The size of one of the isolated proteins (peak A protein fraction) appeared to be 25 kD on SOS-PAGE, while on gel filtration it exhibited a size of 50.5 kO. This suggests that this protein might exist as a dimer in native state. However, the second protein fraction exhibited more than one polypeptide on SOS-PAGE (25 kO, 27 kD minor band and 18 kD) in spite of its elution as a single peak with a size of72kD from the Sephadex G-75 column. This indicates that the second active principle of C album exists as a complex protein in native condition. Such complex proteins were also observed in the case of Clerodendrum inerme (Prasad et al., 1995). Smookler (1971) suggested

that the molecular weights of antiviral proteins from Chenopodiales may range from 25 to 38 kD and possibly occur in extracts as dimers or trimers. The absence of sugar moiety in both of the purified fractions as indicated by the negative result with the Molisch test suggests that these proteins are similar to pokeweed antiviral proteins (PAP) (Irvin, 1995) in this aspect. Further studies are in progress to understand the mechanism of action of these antiviral proteins. Acknowledgements

Authors are thankful to ICAR Centre of Advanced Studies in Biochemistry for supporting the work. The senior author is grateful to the Indian Council of Agricultural Research for awarding a Junior Research Fellowship. References

Alberghina, A.: Phytopathol. 87, 17-27 (1976). Balasubrahmanyam, A., V. K Baranwal, M. L. Lodha, A. Varma and H. C. Kapoor: Plant Sci. (in press). Bradford, M. M.: Anal. Biochem. 72,248-254 (1976). Irvin, J. D., T. Kelly and J. D. Robertus: Arch. Biochem. Biophys. 200,418-428 (1980).

Irvin, J. D.: In: M. Chessin, D. Deborde and A. Zipf (eds.): Antiviral Proteins in Higher Plants, pp. 65-91, CRC Press, London (1995). Kubo, S., T. Ikeda, S. Imaizumi, Y. Takanami and Y. Mikami: Ann. Phytopathol. Soc. Japan 56, 481-482 (1990). Laemmli, U. K: Nature 227, 680-685 (1970). Lodge, J. K, W. K Kaniewski and N. E. Turner: Proc. Natl. Acad. Sci., USA 90, 7089-7093 (1993). Prasad, v., S. Srivatsava, Varsha and H. N. Verma: Plant Sci. 110, 73-82 (1995). Saksena, K N., and G. I. Mink: Phytopathol. 59, 61-63 (1969). Smookler, M. M.: Ann. Appl. BioI. 69, 157-168 (1971). Taniguchi, T., and T. Goto: Ann. Phytopath. Soc. Japan 45, 135141 (1979). . Verma, H. N., and V. K Baranwal: Proc. Indian Acad. Sci. (Plant Sci.) 92, 461-465 (1983). Verma, H. N., Varsha and V. K Baranwal: In: M. Chessin, D. Deborde and A. Zipf (eds.): Antiviral Proteins in Higher Plants, pp. 1-21, CRC Press, London (1995 a). Verma, H. N., Varsha and V. K Baranwal: In: M. Chessin, D. Deborde and A. Zipf (eds.): Antiviral Proteins in Higher Plants, pp. 23-27, CRC Press, London (1995 b). Vogel, A. I.: A text book of practical chemistry including quantitative organic analysis, 3rd ed., English Language Book Society and Longman Group Ltd, London, 1971. Yoshizaki, T., and D. Murayama: Ann. Phytopath. Soc. Japan 32, 267-274 (1966).