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

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

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

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

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

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,

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],

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

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

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

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

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

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

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

.
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

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

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

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

Research-Flanders. P. P. is a postdoctoral fellow of this fund.
REFERENCES
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.&VanDamme,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.,VanLeuven,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.&

,P.,VanLeuven,
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-

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.,VanLeuven,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.