Alteration of lectin pattern in potato tuber by virus X

Plant Science, 58 (1988) 9 - 1 4 Elsevier Scientific Publishers Ireland Ltd.

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ALTERATION OF LECTIN P A T T E R N IN POTATO TUBER BY VIRUS X

CECILIA SCHEGGIA', ANTONIO E. PRISCO°, PRAKASH M. DEYb, GUSTAVO R. DALEO" and RAFAEL PONT LEZICA** •Instituto de lnvestigadones Bioldgicas and Departmnento de Biologla, Facultad de Cie~ias Ezactas y Naturals,, Universidad Nacienal de Mar del Plata, C.C. 13/,8, 7600 Mar del Plata (Argentiz~ 'Departmcitt of Biocltemistry, Royal HoUotuay and Bedford New College, University of London, Egham HiK Egham, Surrey TW~O OEX (U..K.) (Received April 27th, 1988) (Revision Received June 8th, 1988) (Accepted June 13th, 1988)

Solanum tuberosum agglutinin (STA) isolated from healthy or virus X-infected tubers show quantitative and qualitative differences. Infected tubers contain on the average 63% more STA than noninfeeted ones based on equal fresh weight. However, lectin from beth origins have the same speeifie activity on hemagglutination assays and the same subcollular distribution. The main differences between lectias from beth sources were: the immuneeleetrophoreti¢ pattern against antiSTA, the electrophoretic pattern on SDS gel and the isoelectrie point of isoleetinz, the hydroxyprline and carbohydrate content. These changes suggest that virus infection could induce the expression of different genes encoding potato isoleetins. Key words: carbohydrate-binding proteins; potato lectin; Solanum tuberosum agglutinin; potato virus X

Introduction Lectins are proteins or glycoproteins that have the capacity of specific recognition and reversible binding to carbohydrate, without altering the structure of any of the recognized glycosyl ligands [1]. STA, a lectin which can bind oligomers of N-acetylglucosamine [2,3], is the best-studied lectin of the Solanaceae family. It has been described as a hydroxyproline-rich glycoprotein containing about 50% carbohydrate of which over 90% is arabinose. All of the carbohydrate is attached to a hydroxyprolinerich domain which is also rich in serine; the *To whom all correspondence should be addressed at: Department of Biology, Washington University, Campus Box 1137, St. Louis, MO 63130, U.S.A. Abbreviations: ELISA, enzyme-linked immunosorbent assay; PBS, phosphate buffer saline; PVX, potato virus X; SDS-PAGE, sodium dedecylanlphate-polyacrylamide gel electrofphoresis; STA, Solanum tuberosum agglutinin; STA ÷pvx, Solanum tuberosum agglutinin from tubers infected with potato virus X; STAvr, Solanum tuberosum agglutinin from virus-free tubers.

other domain of the glycoprotien has a high cysteine and glyeine content [4]. The linkages of galactose and arabinose to the protein are the same as the linkages of these sugars in extensin [5--7]. Despite the vast amount of information on lectin structure and distribution, their biological function in the plant is not yet known [8]. Many hypotheses have been postulated based on the carbohydrate-binding properties of leetins, and one of these is the possible role of lectins in the recognition process between host and pathogens. Previous work of our laboratory on STA have shown that it is an extracellular glycoprotein associated with the cell wall [9]. On the other hand, preliminary evidence has indicated that some differences could be found in lectins isolated from healthy or virusinfected tubers [10]. It is becoming clear now that gene expression could be altered by environmental stress. It is believed that these changes allow plants to make biochemical and structural adjustments that enable them to cope with the stress conditions [11]. Most of the

0168-9452/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

10 studied effects were elicited by physical or chemical agents, namely anaerobiosis, high temperature, UV light, heavy metals, etc. In this report we present evidences indicating that virus infection can also induce changes in STA content and structure and that these changes could be due to the expression of different genes. Materials and methods

Materials Potato (Solanum tuberosum cv. Huinkul) tubers were provided by S. Mackrey and Co, Balcarce. This cultivar was grown in the area of Balcarce (Buenos Aires province) which is chronically infected with virus-X. These tubers have been infected through more than ten generations. Virus-free tubers were obtained by cell culture and were a generous gift from Dr. R. Tizio, Univ. Nac. de Cuyo, Mendoza and from the INTA Experimental Station, Balcarce. Preparation of antigen was performed as described [9] and the anti-STA serum was obtained with the collaboration of the Laboratorio de Inmunologla, Produccibn Animal, EERA-INTA Balcarce. Sepharose-N, N', N"-triacetychitotriose was prepared as described [12]. Ampholines were purchased from LKB, Sweden, all other chemicals were obtained from Fluka, Switzerland. Methods Determination of virus infection in tubers was performed by Diagnbsticos Vegetales SRL using an ELISA test. All infected tubers were selected for the presence of virus X only, tubers containing other viruses were discarded. STA was extracted with PBS and isolated according to Desai and Allen [12], but using a chitin column for affinity chromatography. The lectin was eluted from chitin with 0.2 M NH4OH [13] and freeze
concentration was measured with the Biuret reagent [16] using bovine serum albumin as standard. Total carbohydrates were determined by the phenol sulfuric acid method [17]. SDS-PAGE was performed in 12% acrylamide slab gels as described by Lugtenberg et al. [18]. Gel isoelectrofocusing was performed in 15% acrylamide slab gels (pH range 5 " 11.5), 0.01 M glutamic acid and 0.01 M ethanolamine were used as anode and cathode buffers respectively. The gels were prefocused at 200, 300, 400 and 500 V (15 rain each), and then the samples were applied, the gel was run for 5 h at 400 V. The proteins were stained with the silver procedure previously described [19]. The gel was electrofocused for 3 h at 4 °C and 500 V.

Amino acid anlysis Duplicate, weighed samples of lyophylized protein, corrected for moisture content, were hydrolyzed in vacuo for 24 h in I ml 6 M HC1 at 110°C. Amino acids were analysed in a Beckman model 119 analyser. Values for serine and threonine were determined by extrapolation to zero time, and isoleucine was determined after 48 h of hydrolysis. Tryptophan was estimated spectrophotometrically in 0.1 M-NaOH [20]. Cysteine was estimated by measuring cysteic acid residues after performic acid oxidation [21] or by reduction of the protein followed by carboxymethylation with iodoacetic acid [22] and amino acid analysis. While the oxidation method results in destruction of tryptophan, this limitation does not apply to the latter procedure. Results Virus-free tubers and tubers infected with P V X were extracted with P B S and S T A was isolated by affinity chromatography on chitin columns. As shown in Table I, PVX-infected tubers had 2 2 % more protein than healthy ones based on equal fresh weight. On the other hand, the S T A content measured by radial immunodiffusion, or as the protein retained by the affinity column, was 64 or 580/0 higher respectively, in infected tubers than in controls

11 T ~ l e I. Protein and STA content of virus-free and PVXinfected tubers. Protein content was measured with the Biuret reagent. STA content was estimmted by (A) radial immunodifftmion in 1% agarose gel; (B) protein retained by affinity chromatography column. Hemaglutinating activity was determined with rabbit red blood cells using serially diluted STA. Each value is the average of four determinations ± S~D. Determinations

Virus-free

Total protein (mg/g fresh wt.)

4.02 ± 0.20 4.92 ± 0.23

STA content (tJ/gfresh wt.) Method A MethodB Hemagglutinatingactivity (units/mgprotein)

Fig. 2. Immunoelectrophoresis of STA extracted from virus-free (1) and PVX-infected tubers (2).

PVX-infected

111 ± 8 69 ± 4

169 ± 11 113 ± 5

181 ± 1

195 ± 11

on a fresh weight basis. No significant difference was found in hemagglutinatination specific activity against rabbit red blood cells in both samples.

1 2 O'-~

66 K -~'-

45 K,-~

Fig. 1. SDS gel eleetrophoresis on 12% acrylamide of STA purified from infected tubers (lane 1) and non-infected (lane 2). O, origin; F, front; 66 K, bovine albumin marker (Mr 66 000);45 K, ovalbuminmarker (Mr 45 000).

STA extracted from virus-free ( S T A vF) and infected tubers (STA ÷Pvx) and eluted from the chitin column were submitted to a new affinity chromatography step on a Sepharose-N, N ' N " triacetylchitotriose column [12]. SDS-PAGE consistently showed one band having an apparent Mr of 42 000 for STA w. However, STA ÷Pvx presented variations in the electrophoretic pattern from one preparation to the other. In this case two, or sometimes three bands can be found, the major one having identical electrophoretic mobility as the non-infected sample, and 40 000 and 36 000 for the new polypeptides (Fig. 1). It should be noted that most preparations only showed the 42 and 40 K bands. Western blot anlaysis using anti-STA serum indicated that all the polypeptides were recognized by the antibody. In order to study the possible effect of virus infection on subcellular distribution infected and non-infected tubers were ground in PBS, and separated into different subcellular fractions (cell wall, starch grain, membrane-bound and soluble fractions) [9]. Each fraction was extracted with ammonia, with the exception of the soluble fraction, and STA content was measured by radial immunodiffusion. No differences were found in subcellular distribution, 63% (healthy) and 650/0 (infected) of STA was found associated with the cell walls. Immunoelectrophoresis showed that STA w presents a slight double precipitation band running in the direction of the anode that is absent in STA ÷vvx (Fig. 2). These differences are consistent with those obtained by isoelectrofocusing on polyacrylamide gels (Fig. 3). Healthy tubers present three isolectins with pI of 6.6, 6.9 and 7.3. On the other hand, the PVX-

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1 2

pH -11 -10

Table II. Aminoacid and carbohydrate composition of STA obtained from virus-free and PVX-infected tubers. Results are expressed as molar ratios relative to hydroxyproline ( = 50). The values are to the nearest whole residue. ND, not detected. Amino acid

STAvF

STA°Pvx A l l e n et al. [4]

Matsumoto et al. [13]

Hyp Asp" Thrb Ser b Glu" Pro Gly Ala 1/2 Cys~ Val Met Ile Leu

50 20 24 30 13 24 32 13 15 ND 3 4 7 4 6 ND 4 ND ND 2

23 17 27 30 16 25 39 13 17 ND 3 7 4 4 16 ND 5 ND ND 3

50 12 14 31 17 17 30 10 26 1 1 4 3 8 1 ND 9 1 3 8

8 ND 4 4

251

249

246

201

140

55

182

184

-9 -8

-6 Fig. 3. Gel isoelectrofocusing from STA extracted from PVX-infected (1) and virus-free tubers (2).

Tyr d

infected t u b e r s contained four isolectins (pI 7.0, 7.3, 7.8 and 7.9). A m i n o acid analysis of both S T A samples w e r e p e r f o r m e d and the r e s u l t s are s h o w n in Table II. The amino acid and c a r b o h y d r a t e composition of S T A published b y o t h e r a u t h o r s [4,13] had been included in t h e table for comparison. Slight differences can be o b s e r v e d in the amino acid composition from S T A vF and t h o s e obtained from o t h e r a u t h o r s , t h a t could be due to differences b e t w e e n cultivars. T h e main changes w e r e o b s e r v e d in S T A vF and S T A ÷evx, with the l a t t e r displaying a r e m a r k a b l y lower c o n t e n t of h y d r o x y p r o l i n e (54°/0 less in S T A ÷evx) and a h i g h e r c o n t e n t of glycine and phenylalanine. F u r t h e r m o r e , i m p o r t a n t differences w e r e obained in the glycosylation pattern. S T A ÷vvx contains only 390/0 c a r b o h y d r a t e s as c o m p a r e d with S T A vv.

Discussion P o t a t o lectin c o n t e n t was found to be 5 8 64% h i g h e r in t u b e r s chronically infected with P V X t h a n in non-infected ones w h e n m e a s u r e d on a fresh w e i g h t basis. H o w e v e r , w h e n comparison was m a d e on p r o t e i n basis, t h e infected

Phe His Lys Orn Arg Trp Total residues Total carbc~ hydrates"

50 9 11 19 16 20 22 8 14 1 1 4 3 5 1 1

"As aspartic acid/asparagine or glutamic acid/glutamine. aObtained by extrapolation to zero time of hydrolysis. °Determined after performic acid treatment. dValues determined after 48 h hydrolysis. •Normalized for arabinose.

t u b e r s contain only 2 6 - 3 5 % m o r e S T A t h a n h e a l t h y ones. The immunological m e t h o d d e t e c t e d 5 0 - 6 0 % m o r e S T A t h a n t h e affinity c h r o m a t o g r a p h y m e t h o d (Table I).This can be explained by the low yields obtained w h e n chitin is used as affinity m a t r i x [12]. The variations in t h e e l e c t r o p h o r e t i c p a t t e r n o b s e r v e d in the infected m a t e r i a l is not well u n d e r s t o o d . P r o t e o l y s i s by e n d o g e n o u s prc~ t e a s e s d u r i n g e x t r a c t i o n is unlikely since smaller p o l y p e p t i d e s are not d e t e c t e d in noninfected t u b e r s . F u r t h e r m o r e , w h e n homogeni-

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zation and extraction were made with 0.5 N NH4OH, when proteolytic activity was inhibited, the same pattern was observed (only one major band for STA vF and two or more for STA÷PVX).Some legume lectins also present this particular electrophoretic pattern on SDS gels, with a major band corresponding to the mature lectin, and several fragments in different proportion corresponding to non mature polypeptides [23- 26]. The subcellular distribution of the lectin and the specific agglutinating activity against rabbit red blood cells were found to be the same for both STA samples. This indicates that infected tubers had around 600/0 higher total activity than healthy ones. However, it is difficult to evaluate the importance of this parameter since little is known of the physiological role of agglutinating activity, if any. It has been reported that potato lectin is present in tubers of cv. King Edward VII as isc~ lectins having isoelectric points ranging from 7.7 to 9.6 [4]. The isolectins present in this cultivar show a shift toward more acidic pH (6.6-7.3). This could be explained by the lower content of lysine and absence or arginine in our cultivar as compared with that previously described. PVX infection of tubers slightly changes the isoelectric point of isolectins to a more basic region (7.0-7.9) (Figs. 1 and 2). Those differences could be attributed to changes in the aspartic/asparagine or glutamic/ glutamine ratios (Table II) between STA vF and STA ÷Pvx, since other amino acids affecting the isoeloctric point do not change. In spite of the presence of isolectins, only one monomer has been described for potato lectin. Its molecular mass ranges from 46 000 to 54 000 in previous reports [4,13,15], and 42 000 for STA w in this o n e . S T A ÷Pvx presents multiple bands but results differ from one preparation to another. Nevertheless, the 42 K band is always present, plus one or two extra bands, frequently having a higher electrophoretic mobility in SDS gels. SDS ÷Pvx is remarkably undergiycosylated compared to STA vF in our hands and in other reports. This decrease in carbohydrates is pre-

sumed to be directly related to the decreased content in hydroxyproline (Table II). Since most of the carbohydrates in STA consist of tri- or tetraarabinosides linked by O-giycosidyl bonds to the 44)H group of hydroxyproline [27], the absence of this hydroxylated amino acid leads to a lesser sugar content. One possibility is that virus infection alters post-translational processing by blocking the prolyl hydroxylase or by interfering with the giycosyl transferases. The consequence of this blockage should be a rise in proline content following he loss of hydroxyproline for the non hydroxylated polypeptide, or to maintain the level of hydroxyproline but not the amount of sugars for the under glycosylated protein. This does not happen because although STA ÷Pvxcontains half the hydroxyproline of non-infected STA, it maintains the proline content. A more likely possibility is that a different polypeptide is expressed in virus-infected tubers. This is consistent with the lower hydroxyproline levels and with the 1.6-fold increase in phenylalanine. The change in isolectin pattern, the rise in activity and total amount of lectin per gram fresh weight, and the lower content of hydroxyproline and sugars in the lectins extracted from PVX-infected tubers points to a change in gene expression associated with the infection. However, the possibility of interference with a posttranslational processing of the protein could not be ruled out. At present we have no direct experimental evidence supporting this assumption. However, it is becoming clear now that multiple lectin genes are present in plants displaying isolectin patterns [8]. Acknowledgements This work was partially supported by the ComisiSn de Investigaciones Cientificas de la Provincia de Buenos Aires (CIC) and the Consejo Nacional de Investigaciones Cientificas y T~cnicas (CONICET). We acknowledge the Fundacibn para Investigaciones Biolbgicas Aplicadas (FIBA) for the use of laboratory facilities. We are indebted to T-H. David Ho and J.E. Varner from the Department of Biology,

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Washington University, St. Louis MO. for critical revision of the manuscript and to our colleagues in the Instituto de Investigaciones Biolbgicas for continuous help and criticism. G.R.D. and R.P.L. are Career Investigators of CIC.

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