Báo cáo Y học: A complex fruit-specific type-2 ribosome-inactivating protein from elderberry (Sambucus nigra) is correctly processed and assembled in transgenic tobacco plants doc

A complex fruit-specific type-2 ribosome-inactivating protein from elderberry ( Sambucus nigra ) is correctly processed and assembled in transgenic tobacco plants Ying Chen 1, *, Frank Vandenbussche 1 , Pierre Rouge ´ 2 , Paul Proost 3 , Willy J. Peumans 1 and Els J. M. Van Damme 1 1 Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Belgium; 2 Institut de Pharmacologie et Biologie Structurale, UMR-CNRS 5089, Toulouse Cedex, France; 3 Rega Institute, Laboratory of Molecular Immunology, Katholieke Universiteit Leuven, Belgium Fruits of elderberry (Sambucus nigra) express small quanti- ties of a type-2 ribosome-inactivating protein with an exclusive specificity towards the NeuAc(a2,6)Gal/GalNAc disaccharide and a unique molecular structure typified by the occurrence of a disulfide bridge between the B-chains of two adjacent protomers. A cDNA clone encoding this so-called Sambucus nigra fruit specific agglutinin I (SNA-If) was iso- lated and expressed in tobacco (Samsun NN) under the control of the 35S cauliflower mosaic virus promoter. Characterization of the purified protein indicated that the recombinant SNA-If from tobacco leaves has the same molecular structure and biological activities as native SNA- If from elderberry fruits, demonstrating that transgenic tobacco plants are fully capable of expressing and correctly processing and assembling a type-2 ribosome-inactivating protein with a complex molecular structure. None of the transformants showed a phenotypic effect, indicating that the ectopically expressed SNA-If does not affect the viability of the tobacco cells. Bioassays further demonstrated that none of the transgenic lines exhibited a decreased sensitivity to infection with tobacco mosaic virus suggesting that the elderberry type-2 RIP SNA-If does not act as an antiviral agent in planta. Keywords: elderberry; ribosome-inactivating protein; Sam- bucus nigra, transgenic tobacco. Ribosome-inactivating proteins (RIPs) are an extended but heterogeneous group of plant proteins comprising an RNA N-glycosylase domain (EC 3.2.2.22) that catalyzes the endohydrolysis of the N-glycosylic bond at one specific adenine of the large ribosomal RNA [1–3]. As this de-adenylation has a detrimental effect on the ability to bind elongation factor 2, the ribosomes become inactive [4,5]. At present, type-1, type-2 and type-3 RIPs have been characterized [3]. In type-2 RIPs, the RNA N-glycosylase domain is tandemly arrayed to an unrelated lectin domain. Both domains are derived from a single precursor, which is post-translationally cleaved into an A- and B-chain har- boring the N-terminal RNA N-glycosylase and C-terminal lectin domain, respectively. All type-2 RIPs are built up of protomers consisting of an A- and B-chain linked by a disulfide bridge. Depending on the number of protomers (also referred to as [A-s-s-B] pairs), native type-2 RIPs are monomers, dimers or tetramers. In all dimeric and tetra- meric type-2 RIPs, the protomers are held together by noncovalent interactions except in the tetrameric Neu- Ac(a2,6)Gal/GalNAc-specific lectins from Sambucus spe- cies, which consist of four [A-s-s-B] pairs that are pair-wise linked through a disulfide bridge between the B-chains of two adjacent protomers into a [A-s-s-B-s-s-B-s-s-A] 2 struc- ture [4,6,7]. This implies that the assembly of SNA-I requires the formation of an intermolecular disulfide bridge. SNA-I also differs from all other type-2 RIPs in its carbohydrate- binding specificity. In contrast to most other type-2 RIPs that interact with Gal, GalNAc or Gal/GalNAc, the B-chain of SNA-I specifically recognizes terminal sialic acid linked a-2,6 to Gal/GalNAc residues. As SNA-I is the only known lectin that distinguishes NeuAc(a2,6)Gal/GalNAc from NeuAc(a2,3)Gal/GalNAc [8], it is an extremely useful tool for the analysis of sialylated N- and O-glycans [9]. SNA-I was originally isolated from elderberry bark where it represents  5% of the total soluble protein [10]. Later, a very similar lectin called Sambucus nigra fruit specific agglutinin I (SNA-If) was identified as a minor protein in ripe elderberry fruits [11]. To corroborate the relationship between SNA-If and its well-characterized homologue from elderberry bark, a Correspondence to E. J. M. Van Damme, Catholic University of Leuven, Laboratory for Phytopathology and Plant Protection, Willem de Croylaan 42, B-3001 Leuven, Belgium. Fax: + 32 16 322976, Tel.: + 32 16 322372, E-mail: Els.VanDamme@agr.kuleuven.ac.be Abbreviations: HCA, hydrophobic cluster analysis; LECSNA, cDNA encoding SNA; RIP, ribosome-inactivating protein; SNA, Sambucus nigra agglutinin; SNLRP, Sambucus nigra lectin-related protein; TMV, tobacco mosaic virus. Enzyme: ribosome-inactivating protein (RNA N-glycosylase) (EC 3.2.2.22). *Present address: China Import and Export Commodity Inspection Technology Institute, Gaobeidian North Road, Chaoyang District, Beijing, P. R. of China. Note: the nucleotide sequence reported in this paper has been sub- mitted to the GenBankTM/EMBL Data library under the accession number AF012899. (Received 17 January 2002, revised 22 April 2002, accepted 24 April 2002) Eur. J. Biochem. 269, 2897–2906 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02962.x cDNA encoding SNA-If was isolated and analyzed. In addition, the complete coding sequence of SNA-If was introduced into Nicotiana tabacum Samsun NN using Agrobacterium mediated transformation and transgenic plants expressing SNA-If were generated. Analyses of the recombinant SNA-If demonstrated that the transgenic tobacco plants correctly process and assemble this complex type-2 RIP including the formation of the intermolecular disulfide bond. None of the transformants was affected in its viability or growth indicating that the host ribosomes are not susceptible to the ectopically expressed SNA-If. Bio- assays further showed that the transgenic plants were as sensitive as control plants towards infection with tobacco mosaic virus (TMV), indicating that SNA-If does not act as an antiviral protein in planta. MATERIALS AND METHODS Plant materials Immature fruits from elderberry destined for the extraction of RNA were collected around mid-July and processed immediately. Mature fruits used for the isolation of SNA-If were harvested around mid-September and stored at )20 °C until use. All berries were collected from a single S. nigra tree bearing yellow fruits. Tobacco (Nicotiana tabacum var. Samsun NN) plants were grown in a greenhouse under 16-h light cycles (55% humidity and 20/18 °C temperature day/night). Transformation vector The plant transformation vector pGB19 was constructed by transfer of the EcoRI–HindIII fragment of the plasmid pFF19 (containing the cauliflower mosaic virus enhancer (duplicated), promoter and polyadenylation signal) [12] into pGPTV-BAR [13] from which the b-glucuronidase gene was removed by EcoRI/HindIII digestion. The vector pGB19 contained the phosphinothricin acetyltransferase (bar) gene, conferring phosphinothricin resistance. RNA isolation, construction and screening of cDNA library Immature fruits were gently homogenized with a mortar and pestle, taking care not to damage the seeds, and the total cellular RNA was then prepared as described by Van Damme & Peumans [14]. A cDNA library was constructed with total RNA using the CapFinder cDNA synthesis kit from Clontech (Palo Alto, USA). cDNA fragments were inserted into the EcoRI site of pUC18 (Amersham Phar- macia Biotech, Uppsala, Sweden) and the library propaga- ted in Escherichia coli XL1 Blue (Stratagene, La Jolla, CA, USA). The cDNA library was screened with a random- primer-labelled cDNA clone encoding SNA-I from S. nigra bark. Positively reacting colonies were selected and used for the isolation and sequencing of the inserts, as described previously [14]. Plasmid construction All plasmids were constructed by standard cloning tech- niques. An SacI–XbaI cassette containing the complete coding sequence of SNA-If was amplified by PCR using LECSNA-If as a template with the primers 5¢-GCGCGAG CTC/ATGAGAGTGGTAACAAAATTA-3¢ (5¢ primer containing SacI site for cloning) and 5¢-GCGCTCTAGA/ CTATGCTGGTTGGGTGGTAGT-3¢ (3¢ primer with added XbaI site). The restricted cassette (1.8 kb) was subcloned into the SacIandXbaI sites of the plasmid pFF19. Sequencing reactions on this plasmid were carried out to confirm the sequence of the SNA-If coding region. After confirmation of the sequence, the plasmid was digested with SacIandXbaI and the insert cloned into the plant transformation vector pGB19. The resulting plasmid, pGB19-SNA-If, contained the SNA-If transgene under the control of the 35S promoter from cauliflower mosaic virus and the selectable marker phosphinothricin acetyltransf- erase (bar) under the control of the nopaline synthase promoter. Transformation of tobacco Agrobacterium tumefaciens GV3101 was transformed with the plasmid pGB19-SNA-If by electroporation. The Agro- bacterium strain containing the construct was used for transformation of tobacco (Samsun NN) leaf discs, as described by Rogers et al. [15]. Shoots were selected on Murashige and Skoog medium with 0.1 mgÆL )1 a-naphtha- lene acetic acid, 1 mgÆL )1 6-benzylaminopurine, 100 mgÆL )1 timentin, 100 mgÆL )1 cefotaxime, 100 mgÆL )1 carbenicillin and 5 mgÆL )1 phosphinothricin. Resistant shoots were transferred to Murashige and Skoog medium with 0.1 mgÆL )1 a-naphthalene acetic acid, 100 mgÆL )1 timentin, 100 mgÆL )1 cefotaxime, 100 mgÆL )1 carbenicillin and 5mgÆL )1 phosphinothricin for rooting. Northern blot analysis RNA was prepared from transgenic tobacco leaves as described by Chen et al. [16], dissolved in RNase-free water and quantitated spectrofotometrically. Approximately 30 lg of total RNA was denatured in glyoxal and dimeth- ylsulfoxide, and separated in a 1.2% (w/v) agarose gel. Following electrophoresis the RNA was transferred to an Immobilon N membrane (Millipore, Bedford MA, USA) and the blot hybridized using a random-primer-labelled cDNA insert or a specific oligonucleotide probe for SNA-If. Analytical methods Crude extracts and purified proteins were analyzed by SDS/PAGE using 15% (w/v) acrylamide gradient gels. Analytical gel filtration was performed on a Pharmacia Superose 12 column (Amersham Pharmacia Biotech, Uppsala, Sweden) using 0.1 M Tris/HCl (pH 8.7) contain- ing 0.2 M NaCl and 0.2 M galactose as running buffer. About 0.3 mg of protein was loaded on the column. The well-characterized elderberry bark type-2 RIPs SNA-I (240 kDa), SNA-V (120 kDa) and SNLRP (60 kDa) were used as molecular mass markers. Protein concentration and total neutral sugar were determined as described previously [14,16]. For N-terminal amino-acid sequencing, purified proteins were separated by SDS/PAGE and electroblotted on a poly(vinylidene difluoride) membrane. Polypeptides were 2898 Y. Chen et al. (Eur. J. Biochem. 269) Ó FEBS 2002 excised from the blots and sequenced on a model 477 A/ 120 A or Procise 491 cLC protein sequencer (Applied Biosystems, Foster City CA, USA). Extraction of proteins and Western blot analysis Samples (200 mg) of tobacco leaves were homogenized in 1mL of 50m M acetic acid using a Fastprep system (Bio101, Vista CA, USA) and centrifuged at 13 000 g for 5 min. The supernatants were taken off and used for protein analysis. A 200-lL aliquot of each extract was lyophilized and dissolved in 20 lL loading buffer [0.1 M Tris/HCl (pH 7.8), 4% SDS, 10% glycerol and 0.1 M 2-mercaptoethanol]. Fifteen microliters of each sample were analyzed by SDS/PAGE on a 1% acrylamide gel. After electrophoresis, proteins were transferred to an Immobilon P membrane (Millipore, Bedford MA, USA) using a semidry blotting system. Immunodetection of SNA- If was done as described by Chen et al. [16] using an affinity-purified polyclonal rabbit antibody raised against SNA-I from elderberry bark as primary antibody. Purification of SNA-If from tobacco Leaves of transgenic plants (500 g) were homogenized in 2.5 L of a solution of 1 gÆL )1 ascorbic acid using a Waring blender. The homogenate was poured through a sieve (pore size: 1.5 mm) and centrifuged at 3000 g for 10 min. Solid CaCl 2 (1 gÆL )1 ) was added to the supernatant and the pH adjusted to 9.0 with 1 M NaOH. After standing for at least 1 h in the cold room (2 °C), the extract was centrifuged at 8000 g for 10 min and the supernatant filtered through filter paper (Whatman 3 MM ). The cleared extract was adjusted to pH 2.8 with 1 M HCl and applied on a column (2.6 · 5cm; 50 mL bed volume) of S Fast Flow (Amersham Pharmacia, Uppsala, Sweden) equili- brated with 20 m M acetic acid. After loading the extract, the column was washed with 500 mL 20 m M Na-formate (pH 3.8) and the bound proteins eluted with 250 mL of 0.5 M NaCl in 0.1 M Tris (pH 8.7). The eluate was adjusted to pH 7.0 and loaded on a column (1.6 cm · 5cm;  10 mL bed volume) of fetuin–Sepharose 4B. After passing the partially purified protein fraction, the column waswashedwith0.2 M NaCl until A 280 <0.01and the bound lectin desorbed with 20 m M acetic acid. The affinity- purified lectin was dialyzed against appropriate buffers and stored at )20 °C until use. Agglutination assays Agglutination assays were performed in 96-well microtiter plates in a final volume of 50 lL containing 40 lLofa1% suspension of red blood cells and 10 lL of extracts or lectin solutions. To determine the specific agglutination activity, the lectin was serially diluted with twofold increments. Agglutination was assessed visually after 1 h at room temperature. Rabbit erythrocytes were treated with trypsin as described previously [6]. The carbohydrate-binding specificity of the type-2 RIPs was checked by inhibition of agglutination of trypsin- treated rabbit erythrocytes with galactose and fetuin. Therefore 10 lL of serial dilutions of the inhibitor stock solution were incubated with 10 lL of lectin solution. After 30 min, 30 lL of a 2% suspension of trypsinized rabbit erythrocytes were added and the agglutination examined after 1 h. RNA N -glycosylase activity assay The RNA N-glycosylase activity of the RIPs was deter- mined by the method of Endo et al. [5] with minor modifications as described by Chen et al.[16].Rabbit reticulocyte lysate and wheat germ ribosomes were used as a substrate. RNA was extracted from the reaction mixtures, treated with freshly prepared 1.0 M acidic aniline (pH 4.5) and analyzed in a 1.2% agarose-formamide gel to visualize the ÔEndo fragmentÕ. Molecular modelling Hydrophobic cluster analysis (HCA) [17] of SNA-If was carried out using ricin as a model. Molecular modelling of the A- and B-chains of SNA-If was carried out on a Silicon Graphics O2 R10000 workstation with the programs INSIGHT II , HOMOLOGY and DISCOVER (MSI, San Diego CA, USA) using the atomic coordinates of ricin (RCSB Protein Data Bank code 2AAI) [18]. Steric conflicts resulting from the replacement or the deletion of some residues in SNA-If were corrected during the model building procedure using the rotamer library [19] and the search algorithm implemented in the HOMOLOGY program [20] to maintain proper side-chain orientation. Energy minimiza- tion and relaxation of the loop regions was carried out by several cycles of steepest descent and conjugate gradient using the consistent valence force field (CVFF) forcefield of DISCOVER. The program TURBOFRODO (Bio-Graphics, Marseille, France) was run on the O2 workstation to draw the Ramachandran plot and to perform the superposition of the models. PROCHECK [21] was used to assess the geometric quality of the three-dimensional models. Molecular dia- grams were drawn with MOLSCRIPT [22], BOBSCRIPT [23] and RASTER 3 D [24]. Docking of galactose in the carbohydrate-binding sites of the B-chain of SNA-If was performed with the HOMOLOGY program. The lowest apparent binding energy (E bind expressed in kcalÆmol )1 ) compatible with the hydrogen bonds (considering Van der Waals interactions and strong [2.5 A ˚ < dist(D-A) < 3.1 A ˚ and 120° < ang(D-H-A)] andweak[2.5A ˚ < dist(D-A) < 3.5 A ˚ and 105° <ang (D-H-A) < 120°] hydrogen bonds (D ¼ donor, A ¼ acceptor and H ¼ hydrogen) found in the ricin–lactose complex, was calculated with the 1 cvff forcefield and used to anchor the pyranose ring of Gal into the binding sites of SNA-If. Bioassay with tobacco mosaic virus Seeds of transformed tobacco were sterilized by successive soaking in 70% ethanol and a solution of 5% NaOCl containing 0.05% Tween 20 before selection on Murashige and Skoog medium containing phosphinothricin (5 mgÆL )1 ). Seedlings, which were phenotypically healthy after the appearance of the first two true leaves, were transferred to soil. A further selection was made by a simple agglutination test on a small piece of leaf. Only plants giving a strong lectin activity with rabbit erythrocytes were used Ó FEBS 2002 Expression of a type-2 RIP in tobacco (Eur. J. Biochem. 269) 2899 for the experiments. When plants reached the six-leaf stage the upper two fully expanded leaves were mechanically infected with TMV (strain TMV vulgare) by rubbing the virus suspension in 100 m M phosphate buffer (pH 7.2) containing 2% poly(vinylpyrrolidone) in the presence of Carborundum powder. Inoculated plants were maintained in a greenhouse for 1 week. After 4 days, the number of local lesions on the infected leaves was counted. The size of the lesions (10 per plant) was measured under a microscope seven days post infection. Data obtained from each experiment were analyzed separately for statistical signifi- cance using SAS software [25]. RESULTS Nomenclature of lectins/RIPs and corresponding cDNAs The first lectin to be isolated from elderberry was the Neu5Ac(a2,6)Gal/GalNAc-specific bark agglutinin, which according to its origin was called S. nigra agglutinin (SNA) [10]. Though already discovered in 1984, SNA was recog- nized as a type-2 RIP only upon cloning of the correspond- ing gene in 1996 [6]. After the discovery of additional lectins with a different molecular structure and specificity [26], SNA was renamed SNA-I. Further research on the lectins/ RIPs from elderberry revealed that fruits also contain small quantities of a type-2 RIP resembling SNA-I from the bark. To distinguish this presumed fruit-specific homologue from SNA-I it is referred to as SNA-If [11]. cDNA clones encoding SNA-I and SNA-If are indicated by LECSNA-I and LECSNA-If, respectively. Recombinant SNA-If expressed in transgenic tobacco will be referred to as rSNA-If. Isolation and characterization of a cDNA clone encoding SNA-If Previous work indicated that elderberry fruits express, in addition to an abundant Gal/GalNAc-specific lectin, small quantities of a RIP that resembles SNA-I in terms of molecular structure and specificity. To check whether this minor fruit lectin is identical to SNA-I from the bark or is a fruit-specific homologue the corresponding cDNA was cloned and analyzed. Screening of a cDNA library constructed with RNA from elderberry fruits yielded a few cDNA clones of  2kb encoding SNA-If. Sequencing revealed that the clone LECSNA-If contains an ORF of 1806 bp encoding a polypeptide of 602 amino acids with a possible initiation codon at position 33 of the deduced amino-acid sequence (Fig. 1). Translation starting with this codon yields a primary translation product of 570 amino acid residues (with a calculated m of 62.7 kDa). Cleavage of the signal peptide between residues 28 and 29 gives a polypeptide of 542 amino-acid residues (59.7 kDa) with an N-terminal sequence identical to that of the A-chain of SNA-If. Conversion of this propeptide into the mature protomer of SNA-If requires the excision of the linker between the A- and B-chain. As the mature B-chain of SNA-If starts with the sequence GGGYEKV, a proteolytic cleavage must take place between amino-acid residues 308 and 309 (of the primary translation product). The exact position of the cleavage site between the C-terminus of the A-chain and the N-terminus of the linker peptide has not been determined. However, due to the analogy of the processing of the closely related type-2 RIP from Sambucus sieboldiana [7], the linker most probably comprises residues N290–G309 of the primary translation product. As a result, the mature A- and B-chains each comprise 261 residues. Molecular modelling of SNA-If As could be expected on the basis of the high degree of similarity between the amino-acid sequences of both the A- and B-chain (58 and 68%, respectively) of SNA-If and ricin, the modelled SNA-If closely resembles ricin (Fig. 2, upper part). As with ricin, the A-chain of SNA-If contains eight a helices (labeled A–H) and six strands of b sheet (labeled a–f) exhibiting a left-handed twist of about 110° when observed along the hydrogen bonds [27]. However, due to a deletion of three residues just preceding the second a helix (labeled B), this a helix of SNA-If is slightly shorter Fig. 1. Alignment of the deduced amino-acid sequences of cDNA clones encoding SNA-If (LECSNA-If), SNA-I (LECSNA-I) and ricin (RICI_RICCO). The N-terminal sequences of the A- and B-chains of SNA-If are underlined. Putative N-glycosylation sites are shown in grey. Residues forming the carbohydrate-binding sites are boxed in black. Amino acids that are conserved among SNA-If, SNA-I and ricin are indicated by asterisks. 2900 Y. Chen et al. (Eur. J. Biochem. 269) Ó FEBS 2002 than the corresponding a helix in the ricin A-chain. The A-chain of SNA-If possesses five putative N-glycosylation sites (Asn12-Leu-Thr, Asn34-His-Thr, Asn62-Pro-Ser, Asn112-Phe-Thr and Asn204-Trp-Ser). Three of these sites (namely Asn12-Leu-Thr, Asn34-His-Thr and Asn112-Phe- Thr) are located in well-exposed and flexible loops, and are therefore presumably glycosylated (as confirmed by the carbohydrate content of SNA-If; see below). Moreover, the presence of a glycosylated Asn12 in the A-chain explains the blank signal during N-terminal protein sequence analysis (due to the poor extraction yields for glycosylated amino acid) (data not shown). The active site responsible for the RNA N-glycosylase activity of the ricin A-chain comprises five essential residues (Tyr80, Tyr123, Glu177, Arg180 and Trp211) [27]. In addition, other residues located in the vicinity of the active site (i.e. Asn78, Arg134, Gln173, Ala178, Glu208 and Asn209) are probably necessary to maintain the catalytic conformation of the active site. All these residues are strictly conserved in SNA-If (Tyr78, Tyr117, Glu171, Arg174, Trp205, and Asn76, Arg128, Gln167, Ala172, Glu202, Asn203). The Ca–Ca distance between Cys256 of the A-chain and Cys8 of the B-chain of SNA-If (4.82 A ˚ )is virtually identical to that between Cys259 of the A-chain and Cys4 of the B-chain of ricin (4.81 A ˚ in ricin), which form the disulfide bridge connecting both chains. One can reasonably assume therefore that the A- and B-chain of SNA-If are covalently linked by a disulfide bridge between these two Cys residues. The B-chain of SNA-If consists mainly of short strands of b sheet interconnected by loops and arranged in two structurally equivalent domains 1 and 2 (Fig. 2, upper part). The same is true for the B-chain of ricin. Each domain comprises three homologous subdomains (1a,1b and 1c for domain 1; 2a,2b and 2c for domain 2) of approximately 40 residues. Domain 2 of SNA-If possesses three putative N-glycosylation sites (Asn184-Arg-Ser, Asn218-Gly-Thr and Asn236-Val-Ser) which are all accessible for glycosyla- tion because they are located in well-exposed loops. The structure of the B-chain of SNA-If is stabilized by four intrachain disulfide bonds. Two of these disulfide bonds (linking Cys24-Cys43 and Cys65-Cys77, respectively) are located in domain 1, and two others (linking Cys147- Cys162 and Cys188-Cys205, respectively) are located in domain 2. The sugar-binding activity of SNA-If relies on two carbohydrate-binding sites located at the N- and C-terminus of the B-chain (more precisely in subdomains 1a and 2c, respectively). Both sites consist of five amino-acid residues (Asp26, Gln39, Gly41, Asn48 and Gln49 for the site of subdomain 1a; Asp231, Ile243, Tyr245, Asn252 and Gln253 for the site of subdomain 2c), which are identical to those found in the corresponding carbohydrate-binding sites of ricin except for Gly41 which replaces the more bulky Fig. 2. Three-dimensional model of SNA-If and its carbohydrate-bind- ing site. 4 Upper panel: ribbon diagram of the three-dimensional model of SNA-If. The A- (light grey) and B- (dark grey) chains are linked by a disulfide bridge (*) between two Cys residues located at the C-terminal and N-terminal end of the A- and B-chain, respectively. The B-chain consists of two domains each of which contains one carbohydrate- binding site (w for domain 1, q for domain 2). The N- and C-terminal end of both chains are indicated. Lower panels: docking of galactose (Gal) in the monosaccharide-binding sites of subdomain 1a (indicated by w on the three-dimensional model) and 2c (indicated by q on the three-dimensional model). Dashed lines correspond to the hydrogen bonds connecting the oxhydryls O3, O4 and O6 of the sugar (dark grey) to the amino acid residues (Asp26, Gln39, Gly41, Asn48 and Gln49 for 1a, and Asp231, Ile243, Tyr245, Asn252 and Gln253 for 2 c) of the binding sites. O and N atoms of the amino acids are coloured white and black, respectively. pGB19-SNA-If (13.9 kb) bar pAg7 Pnos 35S prom SNA-If 1.8kb 35S polyA RB LB Eco RI Hin dIII 35S enh XbaI Sac I Fig. 3. 5 Schematic representation of vector pGB19-SNA-If. The plasmid is derived from pGPTV-BAR [14]. 35S prom, CaMV35S promoter; 35S enh, CaMV35S enhancer (duplicated); 35S polyA, CaMV35S polyadenylation signal; RB, right border, LB, left border; Pnos, nop- aline synthase promoter, bar, phosphinothricin acetyltransferase gene; pAg7, T-DNA gene7 polyA signal. Ó FEBS 2002 Expression of a type-2 RIP in tobacco (Eur. J. Biochem. 269) 2901 Trp37 residue of site 1 of ricin. Docking experiments showed that Gal anchors into the binding sites of sub- domains 1a and 2c by a network of five and four hydrogen bonds, respectively (Fig. 2, lower part). The network of hydrogen bonds is very similar to that occurring in the corresponding sites of ricin. However, site 1 of SNA-If is most probably less reactive than site 1 of ricin due to the replacement of the bulky Trp37 residue by Gly41. SNA-If is a fruit-specific homologue of SNA-I A comparison of the deduced sequences revealed that the primary translation products of LECSNA-If and LEC- SNA-I share 94% identity at the amino-acid level. For the mature A- and B-chains, the sequence identity is 97 and 94%, respectively. Two important conclusions can be drawn from the sequence data. First, the mature protomers of both SNA-If and SNA-I proteins contain 11 Cys residues at identical positions. This implies that SNA-If contains the same extra Cys-residue (Cys47 of the mature B-chain) that allows the formation of an intermolecular disulfide bridge between the B-chains of two adjacent protomers of SNA-I [6]. Accordingly, one can reasonably expect that native SNA-If adopts the same [A-s-s-B-s-s-B-s-s-A] 2 structure as SNA-I. Secondly, there is a difference in the distribution of putative glycosylation sites along the sequences. In SNA-I, the A- and B-chain contain six and two putative glycosy- lation sites, respectively, whereas in SNA-If only five putative glycosylation sites occur in the A-chain but three sites are present in the B-chain. As will discussed below, this difference in the distribution of the glycosylation sites results in a different glycosylation pattern of the A- and B-chains of SNA-If and SNA-I. Comparison of the molecular structure and biological activities of SNA-I and SNA-If To check whether the differences in sequence affect the structure and/or activity of the proteins the molecular structure and biological activities of SNA-If and SNA-I were compared. In a first approach, the molecular structure of the native protein and the composing polypeptides was analyzed by gel filtration and SDS/PAGE. Both proteins eluted with an apparent m  240 kDa upon gel filtration on a Superose 12 column indicating that the native lectins are tetrameric type-2 RIPs. SDS/PAGE under nonreducing conditions yielded the same typical banding pattern (show- ing several high molecular mass bands, which, as was previously demonstrated, are due to the formation of interchain disulfide bonds [6]) for both lectins. In contrast, SDS/PAGE of the reduced proteins yielded different patterns for the fruit and bark lectin. SNA-If migrated as a single band of 35 kDa (Fig. 5) whereas SNA-I behaves as a typical type-2 RIP consisting of two different polypeptide bands of 33 and 35 kDa, respectively [6,10]. N-Terminal sequencing of the 35 kDa polypeptide of SNA-If yielded a double sequence in which the N-terminal sequences of both the A- and B-chain of SNA-If could be recognized. These results suggested that both SNA-I and SNA-If are tetra- meric type-2 RIPs with a similar [A-s-s-B-s-s-B-s-s-A] 2 structure. Determination of the total carbohydrate content indica- tedthatSNA-IfandSNA-Icontain6.7and4.9% covalently bound sugars, respectively. Assuming a molecu- lar mass of 180 Da per monosaccharide, the number of sugar residues would be 26 and 19, respectively, which implies that SNA-If and SNA-I contain four and three N-glycan chains (consisting of 6–7 monosaccharide units), respectively. In other words, SNA-If contains one extra N-glycan as compared to SNA-I. Taking into consideration that the A-chain of SNA-I (33 kDa) is  2 kDa smaller than that of SNA-If (whereas the calculated m of the naked mature polypeptides is virtually identical), one can reason- ably assume that this extra N-glycan is located in the A-chain of SNA-If. To assess the possible effect of the differences in sequence on the biological activities of both the A- and B-chains the agglutination activity and carbohydrate- binding specificity, and RNA N-glycosylase activity of SNA-If and SNA-I were compared. As shown in Table 1, no difference could be detected between SNA-If and SNA- I for what concerns their specific agglutination activity and the inhibition of agglutination by lactose and fetuin. In addition, both type-2 RIPs exhibited the same RNA N-glycosylase activity. It can be concluded therefore that SNA-If and SNA-I exhibit very similar if not identical sugar-binding properties and catalytic activities. These findings are in agreement with the results of the molecular modelling, which showed that all amino-acid residues involved in the catalytic activity of the A-chain and the sugar-binding activity in the B-chain are identical in both type-2 RIPs. Expression of SNA-If in transgenic tobacco plants The unique molecular structure of SNA-I/SNA-If and their homologues from other Sambucus species raises the question whether the formation of the characteristic intermolecular disulfide bridge occurs exclusively in the parent plant or can also be performed by unrelated species. To address this question, SNA-If was expressed in transgenic tobacco plants (Fig. 3). Fifteen independent phosphinothricin resistant tobacco lines were obtained after transformation of leaf discs with the SNA-If construct, from which seven lines (designated 25101– 25107) were selected for further analysis. PCR amplifica- tion using genomic DNA and primers corresponding to the N- and C-terminus of the coding sequence of SNA-If yielded the expected fragment of approximately 1.8 kb for all seven lines (data not shown). The presence of the mRNA encoding SNA-If was checked by Northern blot analysis. As shown in Fig. 4A, four of the seven transgenic lines yielded a clear signal upon hybridization with a probe specific for SNA-If. No bands could be detected in the untransformed line under the same hybridization condi- tions. Western blot analysis of crude leaf extracts con- firmed that the four lines that reacted positively upon Northern blot analysis contained polypeptides of  35 kDa reacting with anti-(SNA-I) Ig. No signal was detected in the three other lines and in the untransformed tobacco. Agglutination assays further revealed that only extracts from the four lines that reacted positively in the Northern and Western blot analysis exhibited lectin activity, indica- ting that these lines express an active form of SNA-If. Semi-quantitative agglutination assays with the crude extracts (using purified SNA-I as a standard) indicated 2902 Y. Chen et al. (Eur. J. Biochem. 269) Ó FEBS 2002 that the expression level of SNA-If in the transgenic plants varied between 1 and 5 lgÆmg )1 protein. No visible phenotype was observed in any of the transformants expressing SNA-If. Recombinant SNA-If is correctly processed and assembled but differently glycosylated in transgenic tobacco To check whether the lectin expressed in transgenic tobacco plants corresponds to recombinant SNA-If (rSNA-If), and if so, whether this rSNA-If is identical to native SNA-If, the ectopically expressed lectin was purified from leaves of the transgenic tobacco plants and compared to the genuine fruit protein. Both SNA-If and rSNA-If eluted with an apparent m of  240 kDa upon gel filtration on a Superose 12 column, indicating that the recombinant lectin also is a tetrameric type-2 RIP. SDS/PAGE under nonreducing conditions further demonstrated that rSNA-If yielded the same typical banding pattern characterized by the occur- rence of several high molecular mass bands as SNA-If. This implies that rSNA-If has the same [A-s-s-B-s-s-B-s-s-A] 2 structure as SNA-If, which demonstrates that the tobacco cells are capable of forming the intermolecular disulfide bridge between the B-chains of two adjacent protomers. SDS/PAGE of the reduced proteins showed a different pattern for rSNA-If and SNA-If. Whereas both the A- and B-chains of SNA-If migrated with an apparent molecular mass of 35 kDa, rSNA-If yielded two polypeptides of 33 and 35 kDa, respectively (Fig. 5). N-Terminal sequencing showed that the A- and B-chains of rSNA-If start with the sequences VTPPVYPSVSFNLT and YEKVCSSVVEVTR RIS, respectively, indicating that the observed differences in molecular mass are not due to a different proteolytic processing in tobacco even though the first three amino- acid residues of the B-chain are cleaved from rSNA-If in tobacco. Determination of the total sugar content showed that rSNA-If contains only 3.4% covalently bound carbo- hydrate whereas SNA-If contains 6.7% sugar. Assuming a molecular mass of 180 Da per monosaccharide, the number Table 1. Comparison of the molecular structure and biological activities of SNA-I, SNA-If and rSNA-If. Type-2 RIP m native type-2 RIP a (kDa) m subunits b (kDa) Specific agglutination activity c (lgÆmL )1 ) IC 50 lactose d (m M ) IC 50 fetuin d (lgÆmL )1 ) Specific RNA N-glycosylase activity (p M ) A-chain B-chain SNA-I 240 33 (1) 35 (2) 7.5 10 60 50 SNA-If 240 35 (2) 35 (2) 7.5 10 60 50 rSNA-If 240 33 (1) 35 (1) 7.5 10 60 50 a Molecular mass determined by gel filtration b The number between brackets refers to the number of N-glycan chains per polypeptide c Lowest lectin concentration that still gives agglutination d Concentration required for 50% inhibition of the agglutination of trypsin- treated rabbit erythrocytes at a lectin concentration of 20 lgÆmL )1 . Fig. 4. Northern and Western blot analysis of tobacco transformed with pGB19-SNA-If. 6 (A) Northern blot analysis of transformed tobacco. The blot was hybridized using a random-primer-labelled oligonucle- otide probe specific for SNA-If. RNA samples were loaded as follows: Lane WT, untransformed tobacco; lanes 1–7, transformed tobacco lines 25101, 25102, 25103, 25104, 25105, 25106 and 25017, respectively. (B) Western blot analysis of transformed tobacco. Approximately 50 lg of total soluble leaf protein was loaded in each slot. Specific antibodies were used for the detection of SNA-If after blotting of the proteins. Samples were loaded as follows: Lane P, pure SNA-If from elderberry; lane WT, untransformed tobacco plant; lanes 1–7, trans- formed tobacco lines 25101, 25102, 25103, 25104, 25105, 25106 and 25017, respectively. 123 4R Fig. 5. SDS/PAGE of purified SNA-If from elderberry and transgenic tobacco. Samples (10 lg each) of the unreduced (lane 1–2) and reduced (lane 3–4) proteins were loadedas follows: Lanes 1and 3, nativeSNA-If; lanes 2 and 4, rSNA-If. Molecular mass reference proteins (lane R) were lysozyme (14 kDa), soybean trypsin inhibitor (20 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), BSA (67 kDa) and phos- phorylase b (94 kDa). Ó FEBS 2002 Expression of a type-2 RIP in tobacco (Eur. J. Biochem. 269) 2903 of sugar residues is 13 and 26, respectively, which implies that rSNA-If and SNA-If contain two and four N-glycan chains, respectively. This obvious difference in glycosylation not only accounts for the lower molecular mass of the A-chain of rSNA-If but also demonstrates that the primary translation product of SNA-If is differently glycosylated in elderberry and tobacco. Comparison of the biological activities of native and recombinant SNA-If To check whether the type-2 RIP expressed in tobacco possesses a fully active A- and B-chain, the agglutination activity and carbohydrate-binding specificity, and RNA N-glycosylase activity of SNA-If and rSNA-If were com- pared. As shown in Table 1, rSNA-If exhibits the same specific agglutination activity and sensitivity towards lactose and fetuin as SNA-If, suggesting that the B-chain of the recombinant lectin exhibits the same activity and specificity as that of the elderberry fruit protein. Assays of the RNA N-glycosylase activity using ribosomes from both animal and plant origin as a substrate yielded identical results for rSNA-If and SNA-If. Both proteins de-adenylated rabbit reticulocyte ribosomes (Fig. 6) but failed to depurinate wheat germ ribosomes (data not shown). Moreover, the minimal concentration required for RNA N-glycosylase activity on rabbit reticulocyte lysate ribosomes was 50 p M for both rSNA-If and SNA-If. It can be concluded therefore that the A-chains of the recombinant and the native SNA-If are equally active. Expression of SNA-If offers the transformants no protection against infection with tobacco mosaic virus Transgenic tobacco plants expressing SNA-If were mechan- ically infected with TMV and the development of symptoms of viral infection were compared to that occurring in untransformed plants. Four days post-infection, the number of lesions on the two infected leaves of each plant was determined and after 7 days the lesion size was measured. Untransformed plants developed 36 lesions per leaf while the transgenic lines 25103, 25104, 25106 and 25107 showed 28, 33, 38 and 30 lesions, respectively. There were no apparent differences in the size of the lesions on untrans- formed and transgenic plants indicating that the expression of SNA-If offers the transformants no resistance against infection with TMV. To assess the possible in vitro antiviral activity of SNA-If, tobacco leaves were infected with a suspension of TMV both in the absence and the presence of purified SNA-If. As neither the number nor the size of the lesions was significantly reduced, it can be concluded that SNA-If does not act as an antiviral protein in vitro against tobacco mosaic virus. DISCUSSION Biochemical analysis and molecular cloning provided ample evidence that S. nigra and other Sambucus species express a great variety of type-2 RIPs and related lectins with different molecular structures and carbohydrate-binding specificity [28]. Detailed studies demonstrated that virtually all tissues of the elderberry tree contain multiple type-2 RIPs/lectins. All elderberry type-2 RIPs/lectins can be classified into four groups. A first group are the tetrameric Neu5Ac(a2,6)Gal/ GalNAc-specific type-2 RIPs similar to the bark type-2 RIP SNA-I [6]. Dimeric galactose-specific type-2 RIP resembling SNA-V from the bark [29] form a second group, whereas the third group comprises the monomeric type-2 RIPs with an inactive B-chain, similar to SNLRP 2 from the bark [30]. A fourth group comprising the Gal/GalNAc-specific lectins similar to SNA-IVf are related to the dimeric galactose- specific type-2 RIPs, but are not RIPs because they are encoded by genes from which the complete A-chain is deleted [14]. At present, it is not clear whether the homologues from different tissues are identical proteins or represent individual tissue-specific proteins encoded by separate genes. To answer this question, we isolated and cloned the fruit-specific homologue of the tetrameric Neu5Ac(a2,6)Gal/GalNAc-specific SNA-I from bark. Our results clearly demonstrate that SNA-I and SNA-If are encoded by highly similar, but different genes, indicating that the expression of closely related homologues of a given type-2 RIP in different tissues of the elderberry tree is controlled by different genes. Despite the obvious differ- ences in sequence, native SNA-I and SNA-If have the same basic [A-s-s-B-s-s-B-s-s-A] 2 structure. However, due to a different distribution of glycosylation sites both homologues slightly differ for what concerns the glycosylation of the A- and B-chains. All amino-acid residues involved in the catalytic activity of the A-chain and the carbohydrate- binding activity of the B-chain are identical in SNA-I and SNA-If. This explains why no difference could be observed between the catalytic activities and sugar-binding properties of both homologues. To check whether transgenic plants are capable of expressing and correctly processing and assembling SNA- If, the coding sequence of LECSNA-If was introduced into Nicotiana tabacum var. Samsun NN using Agrobacterium- mediated transformation. Several lines were obtained, which expressed the RIP. Analysis of the recombinant protein indicated that rSNA-If has the same molecular structure and biological activities as SNA-If from elderberry fruits. This implies that the tobacco cells synthesize, and 12 34 56 -+ - + - + 28S rRNA 18S rRNA Fig. 6. RNA N-glycosylase activity of native and recombinant SNA-If towards rabbit reticulocyte lysate ribosomes. RNA bands were visual- ized by ethidium bromide staining. (–) and (+) 7 indicate no treatment and aniline treatment, respectively. The arrow indicates the position of the Endo’s fragment released from the rRNA. Samples were loaded as follows: Lanes 1–2, 1 m M native SNA-If; lanes 3–4, crude protein ex- tract of untransformed tobacco; lanes 5–6, 1 m M recombinant SNA-If. 2904 Y. Chen et al. (Eur. J. Biochem. 269) Ó FEBS 2002 correctly process and assemble the typical [A-s-s-B-s-s-B-s-s- A] 2 structure including the formation of the intermolecular disulfide bridge between the B-chains of two different protomers. Apart from the expression of ricin and SNA-I¢ in tobacco plants [16,31,32], no reports have been published on the expression of other type-2 RIPs in a transgenic plant. Therefore, our results are straightforward because they demonstrate for the first time that transgenic tobacco is capable of expressing not only a simple type-2 RIP like ricin and SNA-I¢ but also a tetrameric type-2 RIP with a complex structure. In addition, our finding that SNA-If is less efficiently glycosylated in the tobacco cells than in the parent tissue confirms a similar observation made for ricin expressedintobacco[31]. Expression of SNA-If (at a level of 1–5 lgÆmg protein )1 ) does not cause any visible phenotype. This implies that the type-2 RIP is either nontoxic for the ribosomes of the host cells or has no access to its substrate because it is rigorously sequestered from the cytoplasmic compartment. A similar conclusion was drawn for ricin because this highly toxic type-2 RIP also caused no phenotype in transgenic tobacco [33]. Although the B-chain of ricin has been expressed in Escherichia coli [34], Xenopus oocytes [35], yeast systems [36] and insect cells [37], there are no reports of a successful expression of the whole ricin molecule in these systems due to host cell death as a result of ribosome inactivation by the A-chain [33]. This implies that whenever the production of a complete recombinant type-2 RIP is envisaged, plant systems are the only valuable candidates. The obvious absence of a phenotype due to the ectopic expression of type-2 RIPs contrasts with the detrimental effects of ectopically expressed type-1 and type-3 RIPs. For example, tobacco plants expressing high levels (>10 ngÆmg pro- tein )1 )ofthetype-1RIPfromPhytolacca americana exhibited a stunted, mottled phenotype, and the plants with the highest expression level of pokeweed antiviral protein (PAP) 3 were sterile [38]. Similarly, the expression of the type- 3 RIP JIP60 in transgenic tobacco under the control of a constitutive promoter led to an abnormal phenotype characterized by slower growth, shorter internodes, lanceo- late leaves, reduced root development and premature leaf senescence [39]. At present, it is not clear why ectopically expressed type-1 and type-3 but not type-2 RIP are cytotoxic for the plant host cell. Possibly plant cells succeed better in sequestering type-2 RIP from the cytoplasmic/ nuclear compartment than type-1 and type-3 RIP. This tight sequestration may be facilitated by the extensive glycosyla- tion of type-2 RIPs and the fact that a specific post- translational proteolytic processing in the vacuole is required to render the A-chain enzymatically active [3]. At present, the antiviral activity of type-2 RIP is far less documented than that of type-1 RIPs. Though there are several reports on the in vitro antiviral activity of abrin, ricin and moddecin [40,41] and a type-2 RIP from Eranthis hyemalis [42] conclusive evidence for in planta antiviral activity of a type-2 RIP has been obtained only for a type-2 RIP from S. nigra. According to a recent report, S. nigra agglutinin I¢ (SNA-I¢) clearly enhanced the resistance of transgenic tobacco plants against infection with TMV when expressed at a level of 1–10 lgÆmg protein )1 [16]. To check whether the closely related elderberry type-2 RIP SNA-If possesses a comparable antiviral activity, the sensitivity of transgenic tobacco plants expressing SNA-If to infection with TMV was compared to that of untransformed plants. As neither the number nor the size of the lesions was reduced, it can be concluded that ectopically expressed SNA-If offers no protection in planta against infection with TMV. It appears therefore that the previously demonstrated in planta antiviral activity of SNA-I¢ can not be extrapolated to SNA-If. Evidently, this observation raises the question why two closely related type-2 RIPs with a high degree of sequence identity and an identical carbohydrate-binding specificity behave so differently with respect to their protective activity against viruses. As SNA-If can be considered a variant of SNA-I¢ consisting of two SNA-I¢ molecules linked by a disulfide bridge, it is tempting to speculate that the lack of antiviral activity is somehow related to the higher degree of oligomerization (and hence greater size) of SNA-If. ACKNOWLEDGEMENTS This work was supported in part by grants from the Katholieke Universiteit Leuven, DG6 Ministerie voor Middenstand en Landbouw – Bestuur voor Onderzoek en Ontwikkeling, the Flemish Ministry for Science and Technology (BIL98/10) and the Fund for Scientific Research-Flanders. P. P. is a postdoctoral fellow of this fund. 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Biochem. 269) Ó FEBS 2002 . the type-2 RIP expressed in tobacco possesses a fully active A- and B-chain, the agglutination activity and carbohydrate-binding specificity, and RNA N-glycosylase activity of SNA-If and rSNA-If. A complex fruit-specific type-2 ribosome-inactivating protein from elderberry ( Sambucus nigra ) is correctly processed and assembled in transgenic tobacco plants Ying Chen 1, *, Frank Vandenbussche 1 ,. distinguishes NeuAc (a2 ,6)Gal/GalNAc from NeuAc (a2 ,3)Gal/GalNAc [8], it is an extremely useful tool for the analysis of sialylated N- and O-glycans [9]. SNA-I was originally isolated from elderberry