(Received for publication, July 9, 1996, and in revised form, September 17, 1996)
From the One of the predominant proteins in the bark of
elderberry (Sambucus nigra) has been identified as a novel
type 2 ribosome-inactivating protein that exhibits a normal RNA
N-glycosidase activity, but is devoid of carbohydrate
binding activity. Sequence analysis of the corresponding cDNA
clones revealed a striking homology to the previously cloned bark
lectins from elderberry, suggesting that the new protein is a
lectin-related protein. Molecular modeling of the protein confirmed
that its A chain is fully active, whereas its B chain contains two
functionally inactive carbohydrate-binding sites. These findings not
only demonstrate for the first time the occurrence of a type 2 ribosome-inactivating protein with an inactive B chain, but also offer
interesting perspectives for the synthesis of immunotoxins with an
improved selectivity.
Seasonally fluctuating bark proteins play an important role in the
nitrogen metabolism of deciduous trees of the temperate regions (1).
Although these presumed storage proteins have been found in several
tree and shrub species, only a few of them have been purified and
characterized in some detail. Surprisingly, all bark proteins
identified thus far are lectins or ribosome-inactivating proteins
(RIP)1 (1). Lectins have been defined as
proteins possessing at least one noncatalytic domain that binds
reversibly to specific mono- or oligosaccharides (2). They are
widespread in the plant kingdom and form a heterogeneous group of
proteins (3). Unlike lectins, RIP are a homologous group of proteins
that possess a highly specific rRNA N-glycosidase activity
and are capable of catalytically inactivating ribosomes (4). Type 1 and
2 RIP are distinguished according to their molecular structure (4).
Type 1 RIP consist of a single catalytically active subunit of ~30
kDa, whereas type 2 RIP are composed of A chains with
N-glycosidase activity and B chains with a carbohydrate
binding activity comparable to that of lectins. Since type 2 RIP are
fully capable of agglutinating cells and/or precipitating
glycocojugates, they are also considered as lectins.
Lectins have been identified as major proteins in the bark of the
legume trees Robinia pseudoacacia (black locust) (5, 6),
Sophora japonica (Japanese pagoda tree) (7), and
Cladrastis lutea (yellow wood) (8). Besides in legumes, bark
lectins have also been found in different species of the genus
Sambucus (elderberry; family Caprifoliaceae) (9-11).
Recently, molecular cloning of the cDNAs encoding the
Neu5Ac( A reinvestigation of the bark proteins of elderberry resulted in the
isolation of a novel type 2 RIP called SNLRP. Characterization of the
protein and molecular cloning of its corresponding cDNA demonstrated that this novel RIP has the same overall structure as the
classical type 2 RIP, but contains a B chain that is devoid of
carbohydrate binding activity. Our findings not only demonstrate for
the first time the occurrence of a type 2 RIP with an inactive B chain,
but also provide additional evidence that the bark of elderberry is
highly specialized in the accumulation of RIP and related proteins.
All experiments were carried out with bark
samples obtained from a single S. nigra tree. Samples were
collected as described previously (12). Radioisotopes were obtained
from ICN. A cDNA synthesis kit, the multifunctional phagemid
pT7T318U, restriction enzymes, and
DNA-modifying enzymes were obtained from Pharmacia Biotech Inc.
Escherichia coli XL1 Blue competent cells were purchased from Stratagene.
Elderberry bark was
lyophilized and powdered in a coffee mill. Fifty g of the finely ground
tissue were extracted in 500 ml of 0.2 M NaCl containing
0.2 g/liter ascorbic acid (adjusted to pH 6.5) by stirring at room
temperature for 1 h. The homogenate was centrifuged at 9000 × g for 15 min, and the resulting supernatant was decanted
and filtered through glass wool to remove the floating particles. SNAI
and the mixture of SNAII and SNAV were removed from the partially
purified extract by consecutive affinity chromatography on
fetuin-Sepharose 4B and GalNAc-Sepharose 4B, respectively, as described
previously (12, 15). The lectin-depleted extract (in 1.5 M
ammonium sulfate) was loaded onto a column (5 × 5 cm, 100-ml bed
volume) of phenyl-Sepharose 4B equilibrated with 1.5 M
ammonium sulfate in 50 mM sodium acetate (pH 6.5). After
washing the column with 1.5 M ammonium sulfate until the
A280 fell below 0.01, the proteins were eluted
with 50 mM Tris-HCl (pH 9.0). SDS-polyacrylamide gel
electrophoresis (PAGE) showed that the fraction retained on the
phenyl-Sepharose column contained almost exclusively a single polypeptide band. Further purification was achieved by ion exchange chromatography and gel filtration. The proteins were dialyzed against
20 mM sodium formate (pH 3.8) and loaded onto a column (15 × 2.6 cm, 75-ml bed volume) of S Fast Flow (Pharmacia,
Uppsala) equilibrated with the same buffer. After loading the proteins, the column was washed with 200 ml of buffer, and the proteins were
eluted with a linear gradient (500 ml) of increasing NaCl concentration
(from 0 to 1 M). The proteins eluting in the main peak were
dialyzed against water, lyophilized, and dissolved in 20 ml of
phosphate-buffered saline (1.5 mM
KH2PO4, 10 mM
Na2HPO4, 3 mM KCl, and 140 mM NaCl (pH 7.4)). Any insoluble material was precipitated
by centrifugation at 12,000 × g for 10 min, and the supernatant was chromatographed on a column (40 × 5 cm, 800-ml bed volume) of Sephacryl 100 equilibrated with phosphate-buffered saline. The protein eluting in the main peak was essentially pure SNLRP.
Analytical gel filtration of the purified
proteins was performed on a Pharmacia Superose 12 column using
phosphate-buffered saline containing 0.2 M galactose (to
avoid possible binding to the column) as running buffer. Molecular mass
reference markers were catalase (240 kDa), Ricinus communis
agglutinin (120 kDa), ricin (60 kDa), chymotrypsinogen (25 kDa), and
the elderberry lectins SNAI (240 kDa) and SNAV (120 kDa).
Total neutral sugar was determined by
the phenol/H2SO4 method (16), with
D-glucose as standard. Agglutination assays were conducted
using human, rabbit, and pigeon erythrocytes (15).
Lectin preparations were analyzed by SDS-PAGE using 12.5-25%
acrylamide gradient gels as described by Laemmli (17). Proteins separated by SDS-PAGE and electroblotted on a polyvinylidene difluoride membrane were sequenced on an Applied Biosystems Model 477A protein sequencer interfaced with an Applied Biosystems Model 120A on-line analyzer.
Cyanogen bromide cleavage of the proteins (2 mg) was done in 0.1 ml of
70% formic acid containing 10 mg of cyanogen bromide. After incubation
for 15 h at 37 °C (in the dark), peptides were recovered by
evaporation under vacuum.
A cDNA
library was constructed with poly(A)-rich mRNA isolated from
S. nigra bark using the cDNA synthesis kit from
Pharmacia (12). cDNA fragments were inserted into the
EcoRI site of the multifunctional phagemid
pT7T318U. The library was propagated in
E. coli XL1 Blue.
The cDNA library was screened using a 32P-labeled
synthetic oligonucleotide derived from the consensus sequence of the A
chain (5 The hydrophobic cluster
analysis (HCA) (21, 22) was performed to delineate the structurally
conserved regions along the amino acid sequences of the A and B chains
of LRPSN1, LRPSN2, and ricin, which was used as a
model. HCA plots were generated on a Macintosh LC using the program
HCA-Plot2 (Doriane, Paris).
Molecular modeling of A and B chains of LRPSN1 and
LRPSN2 was carried out on a Silicon Graphics Iris 4D25G
workstation using the programs InsightII, Homology, and Discover
(Biosym Technologies, San Diego, CA). The coordinates of ricin (code
2aai) were taken from the Brookhaven National Laboratory Protein Data
Bank (23) and used to build the three-dimensional models. Energy
minimization was performed by several cycles of steepest descent and
conjugate gradient using the cvff force field of Discover. The program
TurboFrodo (Bio-Graphics, Marseille, France) run on a Silicon Graphics
Indigo R3000 workstation was used to perform the superimposition of the models.
The amino acid sequence alignments were performed on a MicroVAX 3100 (Digital, Evry, France) using the ialign program of PIR/NBRF (Washington, D. C.). MacClade (24) was run on a Macintosh LC 630 to
build a parsimony phylogenetic tree relating the different RIP.
Elderberry bark contains at least four different proteins
that have been identified as type 2 RIP or lectins. To avoid confusion in the designation of the proteins and their genes, the following nomenclature has been used. (i) SNAI and LECSNAI refer to
the Neu5Ac( SNLRP was purified
from a lectin-depleted extract using a combination of hydrophobic
interaction chromatography, ion exchange chromatography, and gel
filtration. Starting from 50 g of lyophilized bark, 127 mg of
SNLRP were obtained. A comparable yield has been reported for SNAI (1 mg/g of wet bark) (9) and SNAII (2.6 mg/g of dry bark) (15), suggesting
that SNLRP is as abundant as the latter two bark proteins.
The molecular structure of SNLRP was determined by SDS-PAGE and gel
filtration. Unreduced SNLRP yielded two polypeptide bands of ~60 and
62 kDa, whereas the reduced protein yielded several bands with
molecular masses ranging between 30 and 34 kDa (Fig. 1).
It should be mentioned that the reduced samples also yielded the same
high molecular mass bands as the unreduced samples probably because of
the reassociation of the A and B chains during electrophoresis. Gel
filtration of the native protein on a Superose 12 column yielded a
single symmetrical peak eluting with an apparent molecular mass of 60 kDa (data not shown). According to these results, SNLRP is composed of
two disulfide bridge-linked subunits of ~32 kDa.
Determination of the carbohydrate content of SNLRP yielded a value of
3.0% (by mass). Assuming a molecular mass of 170 Da per
monosaccharide, the number of sugar residues amounts to ~11/native molecule of 60 kDa. In analogy to the ricin glycan chains, which consist of 6-8 monosaccharide residues, the native protein SNLRP contains on average 1.5 oligosaccharide side chains/molecule.
N-terminal sequencing of the blotted peptides yielded little conclusive
information because the signal was weak, and double peaks were obtained
at almost all positions of the sequence (data not shown). Therefore,
SNLRP was cleaved with cyanogen bromide, and the fragments were
sequenced. Several single and double sequences were obtained (Fig.
2) that exhibited ~50% sequence identity to the
deduced amino acid sequences of the A and B chains of SNAI and SNAV. On
the basis of these results, it was presumed that SNLRP is a type 2 RIP
composed of disulfide bridge-linked A and B chains.
The
possible ribosome inactivating activity of SNLRP was checked by
measuring its inhibitory effect on protein synthesis in a reticulocyte
lysate. Native (i.e. unreduced) SNLRP strongly reduced the
incorporation of labeled amino acids, with the concentration required
for 50% inhibition being ~0.5 µg/ml. These results leave no doubt
that SNLRP strongly inhibits mammalian ribosomes. Further details on
the ribosome inactivating activity, toxicity, and
N-glycosidase activity of SNLRP will be published
elsewhere.3
To assess the possible
lectin activity of SNLRP, the agglutination activity of the purified
protein was assayed with different types of red blood cells. SNLRP
failed to agglutinate untreated as well as trypsin-treated human,
rabbit, and pigeon erythrocytes, even at a final concentration of 10 mg/ml. Because this apparent lack of agglutination activity does not
necessarily imply that SNLRP has no carbohydrate binding activity, the
binding of purified SNLRP to various immobilized sugars (galactose,
GalNAc, lactose, mannose, fucose, GlcNAc, and GlcNAcn) and
glycoproteins (fetuin, asialofetuin, mucin, asialomucin, thyroglobulin,
and ovomucoid) was checked. Since the protein was not retained on any
of these affinity matrices, one can reasonably assume that SNLRP has no
carbohydrate binding activity.
The cDNA clones encoding the putative novel RIP were
isolated following a strategy whereby only cDNA clones differing
from LECSNAI and LECSNAV were recovered. A total
cDNA library was screened with an oligonucleotide corresponding to
the consensus sequence of the A chain
(5
LRPSN1 contains a 1734-base pair open reading frame encoding
a polypeptide of 578 amino acids with one possible initiation codon at
position 13 of the deduced amino acid sequence. Assuming that this
methionine is used as the translation initiation site, the primary
translation product is a polypeptide of 566 amino acids (62,733 Da)
that contains the N-terminal amino acid sequences of both the A and B
chains of SNLRP. According to the rules of von Heijne (25), a signal
peptide is cleaved between residues 22 and 23 of the RIP precursor. The
resulting polypeptide of 60,333 Da is further processed by one or more
proteolytic cleavages to yield the A and B chains of mature SNLRP.
Since the B chain starts with the sequence DDEKCTVVDV, a cleavage must
take place between residues 305 and 306 of the primary translation
product, resulting in an A chain of 31,592 Da and a B chain of 28,759 Da. Taking into consideration that the A chain of the SNAI homologue of
S. sieboldiana is processed after the sequence VTS (13), it
is possible that also the A chain of SNLRP1 is post-translationally processed at this position. The deduced amino acid sequence of LRPSN1 contains five putative
N-glycosylation sites at positions 114, 127, 259, 472, and 522 of the precursor (Fig. 3).
LRPSN2 strongly resembles LRPSN1. It encodes a
polypeptide of 573 amino acids with one possible initiation codon at
position 9 of the deduced amino acid sequence. Translation starting
with this methionine results in a polypeptide of 565 amino acids
(62,324 Da). A possible cleavage site for the processing of the signal peptide was identified between residues 25 and 26 of the primary translation product, which is in good agreement with the N-terminal amino acid sequence of the A chain. Cleavage of the signal peptide at
this site will result in a lectin polypeptide of 59,769 Da. Further
processing of this polypeptide to yield the A and B chains of mature
SNLRP implies a cleavage between residues 304 and 305, resulting in an
A chain of 30,996 Da and a B chain of 28,791 Da. The deduced amino acid
sequence of LRPSN2 contains two putative N-glycosylation sites at positions 258 and 521 of the
precursor (Fig. 3).
To corroborate the possible occurrence of two different isoforms, a
total preparation of SNLRP was analyzed by ion exchange chromatography
on a Mono-S column. The protein eluted in two peaks (data not shown),
which are referred to as SNLRP1 and SNLRP2. SDS-PAGE revealed that
SNLRP1 and SNLRP2 are composed of polypeptides with a slightly
different size. As shown in Fig. 1, unreduced and reduced isoform 1 yielded a single polypeptide band of 62 and 34 kDa, respectively.
Unreduced isoform 2 migrated in a doublet of 60 and 62 kDa, whereas the
reduced protein yielded several polypeptides with molecular masses
ranging between 30 and 34 kDa. N-terminal sequencing of the 34-kDa
polypeptide of isoform 1 yielded a double sequence that corresponds to
the N terminus of the A and B chains of SNLRP1. The upper and lower
bands of isoform 2 yielded unique sequences corresponding to the
N-terminal sequences of the B and A chains, respectively, of SNLRP2
(Fig. 2).
SNLRP was modeled using the
coordinates of ricin, the three-dimensional structure of which has been
resolved by x-ray crystallography (23, 26). Although it must be
emphasized that the results of these modeling studies have to be
interpreted with care, they can give interesting information about
structural homologies between related proteins.
A comparison of the HCA plots of the A chains of ricin,
LRPSN1, and LRPSN2 indicates that the secondary
structural features (
The 6 residues that are believed to constitute the active RNA
N-glycosidase site of ricin (Tyr80,
Tyr123, Glu177, Als178,
Arg180, and Trp211) (27-29) are fully
conserved in the A chains of LRPSN1 (Tyr78,
Tyr117, Glu172, Ala173,
Arg175, and Trp206) and LRPSN2
(Tyr77, Tyr116, Glu168,
Ala169, Arg171, and Trp202).
Similarly, most of the residues that are located in the vicinity of the
active site of the A chain of ricin and hence are probably necessary
for the catalytic conformation of the site, i.e.
Asn78, Arg134, Gln173,
Glu208, and Asn209, are conserved in the A
chains of LRPSN1 (Asn76, Arg129,
Gln168, Glu203, and Asn204) and
LRPSN2 (Asn75, Arg127,
Gln164, Glu199, and Asn200).
Therefore, the results of these modeling experiments fully confirm that
the A chain of SNLRP possesses RNA N-glycosidase activity.
The B chains of ricin and SNLRP share a common three-dimensional
structure. Sequence comparisons revealed that the four subdomains designated 1
Since SNLRP has no carbohydrate binding activity, a more detailed
comparison was made between the carbohydrate-binding sites of the B
chain of ricin and the corresponding parts of the B chain of the
elderberry protein. Several amino acid residues that constitute the
binding sites of the ricin B chain are apparently changed in the
corresponding sites of LRPSN1 and LRPSN2.
Asp22, Gln35, Trp37,
Asn46, and Gln47 of the binding site of domain
1 of the ricin B chain are replaced by Asp23,
Gln36, Leu38, Ser45, and
Gln46 in both LRPSN1 and LRPSN2
(i.e. two changes). Similarly, Asp234,
Ile246, Tyr248, Asn255, and
Gln256, forming the binding site of domain 2 of the ricin B
chain, are replaced by Glu230,
Ile242, Tyr244, Asn251, and
Gln252 in the B chains of LRPSN1 and
LRPSN2 (i.e. one change).
Taking into consideration the amino acid replacements occurring in the
B chains of SNLRP and assuming that the orientation of the galactose
moiety of lactose bound to the binding sites of the ricin B chain is
conserved in the B chains of LRPSN1 and LRPSN2,
docking experiments were performed with Gal and GalNAc. The results
indicate that, in SNLRP, a hydrogen bond is lacking between
Ser45 (which replaces Asn46 of the ricin B
chain) and O-3 (site of domain 1) and a that steric hindrance occurs
between Glu230 (which replaces Asp234 of the
ricin B chain) and O-4 (site of domain 2). In addition, the hydrogen
bond interconnecting O- A comparison of
the deduced amino acid sequences of LRPSN1 and
LRPSN2 and those of LECSNAI and
LECSNAV indicates that nonagglutinating SNLRP is more
closely related to SNAI than to SNAV. The dendrogram in Fig.
7 clearly shows that LECSNAI on the one hand
and LRPSN1 and LRPSN2 on the other form separate
subgroups.
All type 2 RIP from elderberry share a reasonable sequence homology
with the toxin and agglutinin of castor bean and abrin, respectively. A
closer examination of the sequence homologies between the two domains
of elderberry RIP and their homologues from other plants indicates that
the B chains of the respective proteins share more homology than the A
chains. However, the sequence that is believed to be essential for RIP
activity (SEAAR) is well conserved in all type 2 RIP.
A reinvestigation of the bark proteins from elderberry revealed
the occurrence of SNLRP, a predominant RIP that is structurally related
to type 2 RIP, but possesses a B chain that is apparently devoid of
carbohydrate binding activity. Molecular cloning confirmed that SNLRP
is structurally and evolutionary closely related to the previously
cloned elderberry type 2 RIP SNAI and, to a lesser extent, also to
SNAV. However, despite its high sequence homology to SNAI, SNLRP is
apparently devoid of carbohydrate binding activity. Molecular modeling
of SNLRP confirmed the results of the protein synthesis inhibition
experiments and agglutination/carbohydrate binding assays. According to
the model, the A chain of SNLRP contains a catalytically active
N-glycosidase site, whereas both sugar-binding sites of the
B chain are functionally inactive because of a few amino acid
substitutions that prevent proper binding of carbohydrates to either
one of the two sites.
SNLRP is the first documented example of a type 2 RIP with a
functionally inactive B chain. Since it is clearly devoid of carbohydrate binding activity, SNLRP cannot be considered as a lectin,
but as a lectin-related protein. In the past, several other
lectin-related proteins have been isolated and characterized. Well
known examples are the Phaseolus vulgaris arcelins and
The isolation and identification of SNLRP demonstrate that elderberry
bark accumulates large amounts of at least three different type 2 RIP.
Two of these RIP, namely SNAI and SNAV, contain B chains that recognize
structurally unrelated sugars (Neu5Ac( The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U58357[GenBank] and U58358[GenBank].
Laboratory for Phytopathology and Plant
Protection,
Center for Human Genetics,
2-6)Gal/GalNAc2-specific agglutinins
from Sambucus nigra and Sambucus sieboldiana revealed that both lectins are type 2 RIP with an unusual specificity and molecular structure (12, 13). Similarly, molecular cloning of
Nigrin b (now called SNAV) confirmed that this protein is a typical
GalNAc-specific type 2 RIP (14, 15).
Materials
CTT GCC GCT TCC GAG ACC ATT TGA AT-3
) of
type 2 RIP and oligonucleotides that react exclusively with
LECSNAI (5
-GGG GGC GAG TAC GAA AAA-3
) and
LECSNAV (5
-GAC GGG GAA ACG TGT ACG-3
)
cDNA clones. Hybridization was carried out overnight as
described previously (18). After washing, filters were blotted
dry, wrapped in Saran Wrap, and exposed to Fuji film overnight at
70 °C. All colonies that reacted positively with the consensus
sequence but were negative with the two other oligonucleotides were
selected and rescreened at low density using the same conditions.
Plasmids were isolated from purified single colonies on a miniprep
scale using the alkaline lysis method as described by Mierendorf and
Pfeffer (19) and sequenced by the dideoxy method (20). DNA sequences
were analyzed using programs from PC Gene and Genepro.
Nomenclature of the S. nigra Bark Lectins/RIP and Their
Genes
2-6)Gal/GalNAc2-specific type 2 RIP and the
corresponding cDNA clone, respectively (12). (ii) SNAII refers to
the GalNAc-specific lectin composed of two subunits that are homologous
to the B chains of SNAV (10). (iii) SNAV and LECSNAV refer
to the GalNAc-specific type 2 RIP and the corresponding cDNA clone,
respectively (15). (iv) SNLRP and LRPSN refer to the novel
nonagglutinating type 2 RIP and its corresponding cDNA clone.
Fig. 1.
SDS-PAGE of purified SNLRP. Samples (25 µg each) of reduced and unreduced total SNLRP were run in lanes
1 and 4, respectively. Lanes 2 and
3 were loaded with 25 µg of reduced isoforms 1 and 2, respectively. Unreduced isoforms were loaded in lanes 5 and 6, respectively. Molecular mass reference proteins
(lane R) were lysozyme (14 kDa), soybean trypsin inhibitor
(20 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), bovine serum
albumin (67 kDa), and phosphorylase b (94 kDa).
[View Larger Version of this Image (112K GIF file)]
Fig. 2.
N-terminal amino acid sequences of SNLRP and
cyanogen bromide cleavage fragments.
[View Larger Version of this Image (22K GIF file)]
-CTTGCC GCT TCC GAG ACC ATT TGA AT-3
) and oligonucleotides corresponding to the N-terminal sequences of the B chain of SNAI (5
-GGG GGC GAG TAC GAA AAA-3
) and SNAV
(5
-GAC GGG GAA ACG TGT ACG-3
), which react exclusively with
cDNA clones encoding LECSNAV and LECSNAI,
respectively. All colonies that reacted positively with the consensus
sequence but were negative with the two other oligonucleotides were
selected and purified. Subsequent sequence analysis revealed the
occurrence of two groups of cDNA clones (LRPSN1 and
LRPSN2) encoding proteins containing the N-terminal amino
acid sequences of SNLRP as well as the sequences of all the cyanogen
bromide fragments (Fig. 3).
Fig. 3.
Comparison of the deduced amino acid
sequences of the cDNA clones encoding SNLRP1, SNLRP2, SNAI, and
SNAV. The arrowhead indicates the processing site for
the cleavage of the signal peptide. Dashes denote gaps
introduced to obtain maximal homology. The determined N-terminal amino
acid sequences of the A and B chains of SNLRP1 and SNLRP2 and their
cyanogen bromide cleavage fragments are underlined. Putative
glycosylation sites are shown in boldface. Since the first
ATG codon is probably used as the translation initiation site, the
deduced amino acids preceding this methionine are shown in
lower-case letters. The numbers above the
sequences refer to the positions of the residues along the A and B
chains of SNLRP1.
[View Larger Version of this Image (46K GIF file)]
-helices and
-sheets) are readily conserved
(Fig. 4). Despite some discrepancies due to the
occurrence of a few deletions or insertions, the three-dimensional
models of the A chains of LRPSN1 and LRPSN2 built
from the coordinates of the A chain of ricin are very similar. The
model contains eight
-helices and a six-stranded
-sheet with a
left-handed twist similar to that found in the ricin A chain (Fig.
5).
Fig. 4.
Comparison of the HCA plots of the A chains
of ricin (A), LRPSN2 (B), and
LRPSN1 (C). The -helices and strands of the
-sheet delineated on the HCA plot of the ricin A chain are indicated on the HCA plots of the A chains of LRPSN1 and
LRPSN2. These delineations were used to recognize the
structurally conserved regions between the A chains of ricin and
LRPSN proteins.
[View Larger Version of this Image (70K GIF file)]
Fig. 5.
Stereoviews of the three-dimensional models
of the A chain (A), B chain (B), and
heterodimer (C) of LRPSN2. The helices (medium thick lines) and left-handed twisted six-stranded
-sheet (heavy lines) of the A chain are indicated. It is
assumed that, like ricin, LRPSN2 results from the
association of the A and B chains by a disulfide bridge between
Cys253 and Cys5.
[View Larger Version of this Image (47K GIF file)]
, 1
, 1
, and 1
and 2
, 2
, 2
, and 2
,
which compose the two respective domains of the ricin B chain (26), are
easily recognized along the B chains of LRPSN1 and
LRPSN2 on the basis of both sequence alignments and
structural features. All cysteine residues involved in the folding of
the ricin B chain appear at very conserved positions in
LRPSN1 and LRPSN2. HCA plot analysis clearly
illustrates the structural similarities between the B chains of ricin
and the two isoforms of SNLRP, suggesting that the three-dimensional
models of the B chains of LRPSN1 and LRPSN2 are
closely related to that of the ricin B chain (Fig. 6).
The B chains do not contain any secondary structures such as helices or
sheets. As for ricin (23), it is assumed that the A and B chains of
both LRPSN1 and LRPSN2 are linked by a disulfide
bridge occurring between Cys257 of the A chain and
Cys5 of the B chain of LRPSN1 and between
Cys253 of the A chain and Cys5 of the B chain
of LRPSN2, which are conserved in the amino acid sequences
of both chains (Fig. 5).
Fig. 6.
Comparison of the HCA plots of the B chains
of ricin (A), LRPSN2 (B), and
LRPSN1 (C). Subdomains 1, 1
, 1
,
2
, 2
, and 2
delineated on the HCA plot of the ricin B chain
are indicated on the HCA plots of the B chains of LRPSN1 and
LRPSN2. These delineations were used to recognize the
structurally conserved regions between the B chains of ricin and
LRPSN proteins.
[View Larger Version of this Image (69K GIF file)]
1 of Glu230 to N-
2 of
Gln252 is lacking. This should prevent the B chains of
LRPSN1 and LRPSN2 from interacting with both
sugars and could explain why both proteins do not bind to
carbohydrates.
Fig. 7.
Phylogeny of amino acid sequences encoding
SNLRP (LRPSN1 and LRPSN2), SNAI
(LECSNAI), SNAV (LECSNAV), the lectin from
R. communis (AGGL_RICCO), and the RIP from
R. communis (RICI-RICCO) and Abrus
precatorius (ABRC_ABRPR). The dendrogram was
constructed using the simultaneous alignment and phylogeny program
CLUSTAL from the PC Gene Software package (IntelliGenetics).
[View Larger Version of this Image (11K GIF file)]
-amylase inhibitor, which are structurally and evolutionary closely
related to the E- and L-type agglutinins from the same species, but
exhibit no carbohydrate binding activity (30). Molecular cloning and modeling of an agglutinin from rhizomes of Polygonatum
multiflorum (Solomon's seal) revealed that this tissue contains,
besides a typical monocot mannose-binding lectin, also a protein that
is clearly related to the lectin, but exhibits no carbohydrate binding activity because its sugar-binding sites are not functional (31). Evidently, the isolation of SNLRP demonstrates that the occurrence of
lectin-related proteins is not restricted to the legume and monocot
mannose-binding lectins, but has to be extended to type 2 RIP. Although
no general conclusions can be drawn as yet, the occurrence of at least
three different types of lectin-related proteins suggests that several
lectin genes evolved into lectin-related genes or vice
versa. It should be emphasized, however, that the absence of
functionally active carbohydrate-binding chains does not necessarily
result in a biologically inactive protein. On the contrary, both the
bean
-amylase inhibitor and SNLRP exhibit a well defined inhibitory
or catalytic activity.
2-6)Gal/GalNAc2 and
GalNAc, respectively), whereas SNLRP possesses a functionally inactive
B chain. Evidently, the simultaneous occurrence of large amounts of
three different type 2 RIP raises the question of the physiological
meaning of the apparent specialization of elderberry bark toward the
accumulation of type 2 RIP. At present, one can only speculate about
the evolutionary advantage of possessing such a mixture of toxic
proteins. Most likely, a synergistic effect between the different RIP
eventually results in an increased resistance to phytophagous insects
and/or herbivorous animals.
*
This work was supported in part by Grant OT/94/17 from the
Catholic University of Leuven, by grants from CNRS and the Conseil Régional de Midi-Pyrénées, by Grant G.0223.97 from
the National Fund for Scientific Research (Belgium), and by Grant
7.0047.90 from the Nationaal Fonds voor Wetenschappelijk
Onderzoek-Levenslijn.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Senior Research Assistant of the National Fund for Scientific
Research (Belgium). To whom correspondence should be addressed. Tel.:
32-16-322379; Fax: 32-16-322976.
**
Research Director of the National Fund for Scientific Research
(Belgium).
1
The abbreviations used are: RIP,
ribosome-inactivating protein(s); SNA, S. nigra agglutinin;
SNLRP, S. nigra lectin-related protein; LECSNA,
cDNA encoding SNA; LRPSN, cDNA encoding SNLRP; PAGE,
polyacrylamide gel electrophoresis; HCA, hydrophobic cluster analysis.
2
Neuraminic acid is acetylated at the hydroxyl
group at C5.
3
M. G. Batelli, L. Barbieri, A. Bolognesi, L. Buonamici, P. Valbonesi, L. Polito, E. J. M. Van Damme, W. J. Peumans,
F. Stirpe, unpublished data.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.