Van Damme1 1 Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Belgium;2Institut de Pharmacologie et Biologie Structurale, UMR-CNRS 5089, Toulouse Cedex
Trang 1A complex fruit-specific type-2 ribosome-inactivating protein
and assembled in transgenic tobacco plants
Ying Chen1,*, Frank Vandenbussche1, Pierre Rouge´2, Paul Proost3, Willy J Peumans1
and Els J M Van Damme1
1
Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Belgium;2Institut de Pharmacologie
et Biologie Structurale, UMR-CNRS 5089, Toulouse Cedex, France;3Rega 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 nigrafruit 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)
Trang 2cDNA 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
M A T E R I A L S A N D M E T H O D S
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 nigratree bearing yellow fruits
Tobacco (Nicotiana tabacum var Samsun NN) plants
were grown in a greenhouse under 16-h light cycles (55%
humidity and 20/18C 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 SacI and XbaI 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 SacI and XbaI 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 tumefaciensGV3101 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)16-benzylaminopurine, 100 mgÆL)1 timentin, 100 mgÆL)1cefotaxime, 100 mgÆL)1carbenicillin and 5 mgÆL)1 phosphinothricin Resistant shoots were transferred to Murashige and Skoog medium with 0.1 mgÆL)1a-naphthalene acetic acid, 100 mgÆL)1timentin,
100 mgÆL)1 cefotaxime, 100 mgÆL)1 carbenicillin and
5 mgÆL)1phosphinothricin 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 Nmembrane (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 0.1MTris/HCl (pH 8.7) contain-ing 0.2M NaCl and 0.2M 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
Trang 3excised 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
1 mL of 50 mM 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 [0.1M
Tris/HCl (pH 7.8), 4% SDS, 10% glycerol and 0.1M
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)1ascorbic 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 CaCl2(1 gÆL)1) was added to the supernatant and the
pH adjusted to 9.0 with 1MNaOH After standing for at
least 1 h in the cold room (2C), the extract was
centrifuged at 8000 g for 10 min and the supernatant
filtered through filter paper (Whatman 3MM) The cleared
extract was adjusted to pH 2.8 with 1MHCl and applied
on a column (2.6· 5 cm; 50 mL bed volume) of S Fast
Flow (Amersham Pharmacia, Uppsala, Sweden)
equili-brated with 20 mM acetic acid After loading the extract,
the column was washed with 500 mL 20 mMNa-formate
(pH 3.8) and the bound proteins eluted with 250 mL of
0.5MNaCl in 0.1MTris (pH 8.7) The eluate was adjusted
to pH 7.0 and loaded on a column (1.6 cm· 5 cm;
10 mL bed volume) of fetuin–Sepharose 4B After
passing the partially purified protein fraction, the column
was washed with 0.2M NaCl until A280< 0.01 and the
bound lectin desorbed with 20 mMacetic 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 lL of a 1%
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
RNAN-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 1.0Macidic aniline (pH 4.5) and analyzed in a 1.2% agarose-formamide gel to visualize the Endo fragment
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 theHOMOLOGYprogram [20] to maintain proper side-chain orientation Energy minimiza-tion and relaxaminimiza-tion 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 withMOLSCRIPT[22],BOBSCRIPT[23] and RASTER3D[24]
Docking of galactose in the carbohydrate-binding sites of the B-chain of SNA-If was performed with theHOMOLOGY program The lowest apparent binding energy (Ebind 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)] and weak [2.5 A˚ < dist(D-A) < 3.5 A˚ and 105 < ang (D-H-A) < 120] hydrogen bonds (D ¼ donor, A ¼ acceptor and H¼ hydrogen) found in the ricin–lactose complex, was calculated with the
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
Trang 4for 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 mM 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 usingSASsoftware [25]
R E S U L T S
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 2 kb
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.
Trang 5than 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 Natoms 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
EcoRI
HindIII
35S enh
XbaI SacI
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.
Trang 6Trp37 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]2structure 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-ted that SN A-If and SN A-I contain 6.7 and 4.9%
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 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
Trang 7that 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]
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 subunitsb(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
a
Molecular mass determined by gel filtrationbThe 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.
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 loaded as follows: Lanes 1 and 3, native SNA-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).
Trang 8of 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 pM
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
D I S C U S S I O N
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
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 tabacumvar 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
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 (+)
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 recombinant SNA-If.
Trang 9correctly process and assemble the typical
[A-s-s-B-s-s-B-s-s-A]2structure 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
expressed in tobacco [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) of the type-1 RIP from Phytolacca 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 plantaantiviral 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
A C K N O W L E D G E M E N T S 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.
R E F E R E N C E S
1 Nielsen, K & Boston, R.S (2001) Ribosome-inactivating pro-teins: a plant perspective Annu Rev Plant Physiol Plant Mol Biol 52, 785–816.
2 Peumans, W.J., Hao, Q & Van Damme, E.J.M (2001) Ribo-some-inactivating proteins from plants: more than RNA N-gly-cosylases? FASEB J 15, 1493–1505.
3 Van Damme, E.J.M., Hao, Q., Chen, Y., Barre, A., Vandenbus-sche, F., Desmyter, S., Rouge´, P & Peumans, W.J (2001) Ribo-some-inactivating proteins: a family of plant proteins that do more than inactivate ribosomes Crit Rev Plant Sci 20, 395–465.
4 Endo, Y & Tsurugi, K (1987) RNA N-glycosidase activity of ricin A-chain Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes J Biol Chem 262, 8128–8130.
5 Endo, Y., Mitsui, K., Motizuki, M & Tsurugi, K (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes The site and the characteristics of the modification in 28S ribosomal RNA caused by the toxins J Biol Chem 262, 5908–5912.
6 Van Damme, E.J.M., Barre, A., Rouge´, P., Van Leuven, F & Peumans, W.J (1996) The NeuAc (a-2,6)-Gal/GalNAc binding lectin from elderberry (Sambucus nigra) bark, a type 2 ribosome inactivating protein with an unusual specificity and structure Eur.
J Biochem 235, 128–137.
7 Kaku, H., Tanaka, K., Tazaki, K., Minami, E., Mizuno, H & Shibuya, N (1996) Sialylated oligosaccharide-specific plant lectin from Japanese elderberry (Sambucus sieboldiana) bark tissue has a homologous structure to type-II ribosome-inactivating proteins, ricin and abrin cDNA cloning and molecular modeling study.
J Biol Chem 271, 1480–1485.
8 Shibuya, N., Goldstein, I.J., Broekaert, W.F., Nsimba-Lubaki, M., Peeters, B & Peumans, W.J (1987) The elderberry (Sambucus nigra L.) bark lectin recognizes the N eu5Ac (a2–6) Gal/GalNAc sequence J Biol Chem 262, 1596–1601.
9 Peumans, W.J & Van Damme, E.J.M (1998) Plant lectins: Spe-cific tools for the identification, isolation, and characterization of O-linked glycans Crit R ev Biochem Mol Biol 33, 209–258.
10 Broekaert, W.F., Nsimba-Lubaki, M., Peeters, B & Peumans, W.J (1984) A lectin from elder (Sambucus nigra L.) bark Biochem J 221, 163–169.
Trang 1011 Peumans, W.J., Roy, S., Barre, A., Rouge´, P., Van Leuven, F &
Van Damme, E.J.M (1998) Elderberry (Sambucus nigra) contains
truncated Neu5Ac (a-2,6)Gal/GalNac-binding type 2
ribosome-inactivating proteins FEBS Lett 425, 35–39.
12 Timmermans, M., Maliga, P., Vieira, J & Messing, J (1990) The
pFF plasmids: cassettes utilising CaMV sequences for expression
of foreign genes in plants J Biotechnol 14, 333–334.
13 Becker, D., Kemper, E., Schell, J & Masterson, R (1992) New
plant binary vectors with selectable markers located proximal to
the left T-DNA border Plant Mol Biol 20, 1195–1197.
14 Van Damme, E.J.M., Roy, S., Barre, A., Rouge´, P., Van Leuven,
F & Peumans, W.J (1997) The major elderberry (Sambucus nigra)
fruit protein is a lectin derived from a truncated type 2
ribosome-inactivating protein Plant J 12, 1251–1260.
15 Rogers, S.G., Horsch, R.B & Fraley, R.T (1985) Gene transfer in
plants: Production of transformed plants using Ti-plasmid vectors.
Methods Enzymol 118, 627–640.
16 Chen, Y., Peumans, W.J & Van Damme, E.J.M (2002) The
Sambucus nigra type-2 ribosome-inactivating protein SNA-I’
exhibits in planta antiviral activity in transgenic tobacco FEBS
Lett 516, 27–30.
17 Lemesle-Varloot, L., Henrissat, B., Gaboriaud, C., Bissery, V.,
Morgat, A & Mornon, J.P (1990) Hydrophobic cluster analysis:
procedure to derive structural and functional information from
2D-representation of protein sequences Biochimie 72, 555–574.
18 Rutenber, E., Katzin, B.J., Collins, E.J., Mlsna, D., Ready, M.P.
& Robertus, J.D (1991) Crystallographic refinement of ricin to
2.5 A˚ Proteins 10, 240–250.
19 Ponder, J.W & Richards, F.M (1987) Tertiary templates for
proteins Use of packing criteria in the enumeration of allowed
sequences for different structural classes J Mol Biol 193, 775–
791.
20 Mas, M.T., Smith, K.C., Yarmush, D.L., Aisaka, K & Fine,
R.M (1992) Modeling the anti-CEA antibody combining site by
homology and conformational search Proteins Struct Func.
Genet 14, 483–498.
21 Laskowski, R.A., MacArthur, M.W., Moss, D.S & Thornton,
J.M (1993) PROCHECK: a program to check the
stereo-chemistry of protein structures J Appl Cryst 26, 283–291.
22 Kraulis, P.J (1991) Molscript: a program to produce both detailed
and schematic plots of protein structures J Appl Cryst 24, 946–
950.
23 Esnouf, R.M (1997) An extensively modified version of Molscript
that includes greatly enhanced coloring capabilities J Mol.
Graphics 15, 132–134.
24 Merritt, E.A & Bacon, D.J (1997) Raster3D photorealistic
molecular graphics Methods Enzymol 277, 505–524.
25 Cody, R.P & Smith, J.K (1991) Applied Statistics and the SAS
Programming Language Elsevier Science Publishers, New York,
NY.
26 Kaku, H., Peumans, W.J & Goldstein, I.J (1990) Isolation and
characterization of a second lectin (SNA-II) present in elderberry
(Sambucus nigra L.) bark Arch Biochem Biophys 277, 255–262.
27 Katzin, B.J., Collins, E.J & Robertus, J.D (1991) Structure of
ricin A-chain at 2.5 A˚ Proteins 10, 251–259.
28 Van Damme, E.J.M., Peumans, W.J., Barre, A & Rouge´, P (1998) Plant lectins: a composite of several distinct families of structurally and evolutionary related proteins with diverse biolo-gical roles Crit Rev Plant Sci 17, 575–692.
29 Van Damme, E.J.M., Barre, A., Rouge´, P., Van Leuven, F & Peumans, W.J (1996) Characterization and molecular cloning of Sambucus nigra agglutinin V (nigrin b), a GalNAc-specific type 2 ribosome-inactivating protein from the bark of elderberry (Sam-bucus nigra) Eur J Biochem 237, 505–513.
30 Van Damme, E.J.M., Barre, A., Rouge´, P., Van Leuven, F & Peumans, W.J (1997) Isolation and molecular cloning of a novel type 2 ribosome-inactivating protein with an inactive B chain from elderberry (Sambucus nigra) bark J Biol Chem 272, 8353–8360.
31 Sehnke, P.C., Pedrosa, L., Paul, A.L., Frankel, A.E & Ferl, R.J (1994) Expression of active, processed ricin in transgenic tobacco.
J Biol Chem 269, 22473–22476.
32 Tagge, E.P., Chandler, J., Harris, B., Czako, M., Marton, L., Willingham, M.C., Burbage, C., Afrin, L & Frankel, A.E (1996) Preproricin expressed in Nicotiana tabacum cells in vitro is fully processed and biologically active Prot Exp Purif 8, 109–118.
33 Sehnke, P.C & Ferl, R.J (1999) Processing of preproricin in transgenic tobacco Prot Exp Purif 15, 188–195.
34 Hussain, K., Bowler, C., Roberts, L & Lord, J.M (1989) Expression of ricin B chain in Escherichia coli FEBS Lett 244, 383–387.
35 Richardson, P.T., Gilmartin, P., Colman, A., Roberts, L.M & Lord, J.M (1988) Expression of functional ricin B chain in Xenopus oocytes Bio/Technology 6, 565–570.
36 Richardson, P.T., Roberts, L.M., Gould, J.H & Lord, J.M (1988) The expression of functional ricin B-chain in Sacchar-omyces cerevisiae Biochim Biophys Acta 950, 385–394.
37 Frankel, A., Roberts, H., Gulick, H., Afrin, L., Vesely, J & Willingham, M (1994) Expression of ricin B chain in Spodoptera frugiperda Biochem J 303, 787–794.
38 Lodge, J.K., Kaniewshi, W.K & Tumer, N.E (1993) Broad spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein Proc Natl Acad Sci USA 90, 7089–7093.
39 Go¨rschen, E., Dunaeva, M., Hause, B., Reeh, I., Wasternack, C.
& Parthier, B (1997) Expression of the ribosome-inactivating protein JIP60 from barley in transgenic tobacco leads to an abnormal phenotype and alterations on the level of translation Planta 202, 470–478.
40 Stevens, W.A., Spurdon, C., Onyon, L.J & Stirpe, F (1981) Effect
of inhibitors of protein synthesis from plants on tobacco mosaic virus infection Experientia 37, 257–259.
41 Taylor, S., Massiah, A., Lomonossoff, S., Roberts, L.M., Lord, J.M & Hartley, M (1994) Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection Plant
J 5, 827–835.
42 Kumar, M.A., Timm, D.E., Neet, K.E., Owen, W.G., Peumans, W.J & Rao, A.G (1993) Characterization of the lectin from the bulbs of Eranthis hyemalis (winter aconite) as an inhibitor of protein synthesis J Biol Chem 268, 25176–25183.