Isolation and characterization of four type 2 ribosome
inactivating pulchellin isoforms from Abrus pulchellus
seeds
Priscila V. Castilho
1,2
, Leandro S. Goto
2
, Lynne M. Roberts
3
and Ana Paula U. Arau
´
jo
1,2
1 Programa de Po
´
s-graduac¸a˜o em Gene
´
tica e Evoluc¸a˜o, Universidade Federal de Sa˜o Carlos, Brazil
2 Instituto de Fı
´
sica de Sa˜o Carlos, Universidade de Sa˜o Paulo, Sa˜o Carlos, Brazil
3 Department of Biological Sciences, University of Warwick, Coventry, UK
Ribosome-inactivating proteins (RIPs; rRNA N-glyco-
sidases; EC 3.2.2.22) are found predominantly in
plants but they may also occur in fungi and bacteria
[1]. Collectively, unless mutated, they are all rRNA-
specific N-glycosidases capable of selectively cleaving a
glycosidic bond to release an adenine within the uni-
versally conserved sarcin ⁄ ricin loop of the large rRNA
in 60S ribosomal subunits [2]. This modification pre-
vents the binding of elongation factors and thereby

´
sica
Molecular, IFSC, PO Box 369,
Zip 13560-970, Sa˜o Carlos, Brazil
Fax: +55 16 33715381
Tel: +55 16 33739834
E-mail:
(Received 14 November 2007, revised 11
December 2007, accepted 20 December
2007)
doi:10.1111/j.1742-4658.2008.06258.x
Abrus pulchellus seeds contain at least seven closely related and highly toxic
type 2 ribosome-inactivating pulchellins, each consisting of a toxic A-chain
linked to a sugar binding B-chain. In the present study, four pulchellin
isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion
exchange and chromatofocusing chromatographies, and investigated with
respect to toxicity and sugar binding specificity. Half maximal inhibitory
concentration and median lethal dose values indicate that P I and P II have
similar toxicities and that both are more toxic to cultured HeLa cells and
mice than P III and P IV. Interestingly, the secondary structural character-
istics and sugar binding properties of the respective pairs of isoforms corre-
late well with the two toxicity levels, in that P I ⁄ P II and P III ⁄ P IV form
two specific subgroups. From the deduced amino acids sequences of the
four isoforms, it is clear that the highest similarity within each subgroup is
found to occur within domain 2 of the B-chains, suggesting that the
disparity in toxicity levels might be attributed to subtle differences in
B-chain-mediated cell surface interactions that precede and determine toxin
uptake pathways.
Abbreviations
GalNAc, N-acetylgalactosamine; IC

will contribute to an understanding of their role(s)
in vivo. Abrus pulchellus tenuiflorus (Leguminosae-Papi-
lionoideae) seeds contain a highly toxic type 2 RIP
named pulchellin. It exhibits specificity for galactose
and galactose-containing structures, can agglutinate
human and rabbit erythrocytes, and kills mice and the
microcrustacean Artemia salina at very low concentra-
tions [10]. Similar to the RIP in A. precatorius seeds
[14], this toxic activity is presented by a mixture of clo-
sely related isoforms. In the present study, four pulchel-
lin isoforms were isolated, and their amino acids
sequences deduced by cDNA cloning and verified by
MS. Half maximal inhibitory concentration (IC
50
) and
median lethal dose (LD
50
) values from HeLa cells and
mice divided them into two subgroups: the more toxic
forms (P I and P II) and the less toxic forms (P III and
P IV). In similar pairwise combinations, their interac-
tion with specific sugars was also shown to differ. From
a comparison of deduced amino acid sequences within
each subgroup, it is striking that the members of each
show closest identity in domain 2 of the B-chain. The
potential implications of this are discussed.
Results
Nomenclature of the toxic pulchellin lectins
The abbreviation P is followed by the Roman numer-
als I, II, III and IV and refers to each pulchellin

assays. Although samples from the asterisked peak in
Fig. 1A displayed hemagglutination and toxicity
toward mice (data not shown), additional efforts to
cleanly isolate the isoform were not successful and
further characterization was abandoned. A chromato-
focusing step was included to separate the P III and
P IV isoforms from the eluate P III ⁄ P IV (Fig. 1C).
Isoelectric focusing gave pI of 5.8, 5.7, 5.5 and 5.2 for
the four isoforms respectively.
Secondary structure of the pulchellin isoforms
and melting temperature
CD-spectral analyses were performed as described in
the Experimental procedures. As can be seen from
Fig. 2, the far-UV CD spectra of the pulchellin iso-
forms suggest only subtle differences in the content of
secondary structure, which was confirmed by the spec-
tral deconvolution using cdpro software. Thermal sta-
bility was also monitored by CD, following changes
in each spectrum with increasing temperature. The
P. V. Castilho et al. Characterization of four pulchellin isoforms
FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS 949
predicted content of secondary structure and melting
temperatures found for the four isoforms are given in
Table 1.
Sequence comparison of pulchellins from
A. pulchellus seeds
Using RT-PCR and a primer set, full length cDNA
clones were prepared and sequenced with primer walk-
ers as detailed in the Experimental procedures. Accord-
ing to the extent of similarity, seven different sequences

B-chain
A-chain
0 5 10 15 20 25
0
20
40
60
80
100
0
20
40
60
80
100
P IV
P III
% of buffer B
Absorbance at 280 nm (a.u.)
Elution volume (mL)
C
B
A
Fig. 1. Mono Q elution profile of pulchellin isoforms. (A) The four
isoforms were eluted with a linear gradient of 0–20% 1
M NaCl
in 20 m
M Tris–HCl, pH 8, for 45 min (dashed line) at a flow rate
of 1 mLÆmin
)1

L·Mol
–1
·cm
–1
)
Wavelen
g
th (nm)
Fig. 2. Circular dichroism spectra of P I, P II, P III and P IV. CD
spectra of P I (solid), P II (dash), P III (dot) and P IV (dash dot) were
measured in the far-UV range (195–250 nm) in 1 mm path length
quartz cuvettes and recorded as an average of 16 scans. CD spec-
tra were measured in protein solution of 0.125 mgÆmL
)1
(Tris
20 m
M,pH8,10mM NaCl added).
Table 1. Secondary structure content (expressed as %) and melt-
ing temperatures found for P I, P II, P III and P IV. Secondary struc-
ture values were obtained by the spectral deconvolution using
CDPRO software. For all deconvolutions, rmsd values were less than
1. The melting temperatures were calculated based on CD thermal
scans (at 232 nm) of the proteins.
Secondary structure
content (%) P I P II P III P IV
Helix 13 12 10 16
b sheet 32 32 30 31
Turn 22 23 24 20
Unordered 33 33 36 33
Melting

P. V. Castilho et al. Characterization of four pulchellin isoforms
FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS 951
fragment containing these changes (underlined at the
N-terminus of P II).
Overall, the four isoforms precursors have 562 (P I),
563 (P II) and 561 (P III and P IV) aminoacyl resi-
dues. The protein outside this family to which they
showed the highest amino acid identity was abrin, at
approximately 94%. Besides abrin, the pulchellin iso-
forms were also compared with ricin and mistletoe lec-
tin I (Fig. 3), with which they showed approximately
47% amino acid identity to both sequences.
The respective A-chains contain 251 (P II) and 250
(P I, P III and P IV) amino acids. P I and P IV
A-chains have two N-glycosylation sites, whereas P II
and P III only one. In the four pulchellin A-chains,
the residues involved in the active site cleft are the
same as in abrin and ricin A-chains. This suggests that
the catalytic reaction is exactly the same. The sugar
binding pulchellin B-chains are 264 (P I and P II) or
263 (P III and P IV) amino acids in length and contain
two N-glycosylation sites. Soler et al. [17] defined two
homologous carbohydrate binding sites that were
shared in mistletoe lectin I, ricin-d and abrin-a
B-chains. Based on these previously published observa-
tions, we predict residues comprising the two sugar
binding pockets in the pulchellins (Fig. 3).
In order to compare the similarity of the A- and
B-chains of the four isoforms, a pairwise alignment
was performed and the values of identity expressed in

(1.7 nm) for P IV]. LD
50
experiments also showed vari-
ability in the toxicity to mice, with the most potent
toxin being P II (15 lgÆkg
)1
), followed by P I
(25 lgÆkg
)1
), P IV (60 lgÆkg
)1
) and P III (70 lgÆkg
)1
).
These results indicate that the pulchellin isoforms are
highly toxic, but not as much as mistletoe lectin
(LD
50
5–10 lgÆkg
)1
) [19], ricin (IC
50
0.001 nm
and LD
50
2.6 lgÆkg
)1
) [20] and abrin (IC
50
0.0037 nm

)1
) and horse
(41.7 ngÆmL
)1
) erythrocytes. P III and P IV aggluti-
nated only rabbit blood (18.5 ngÆmL
)1
and
12.3 ngÆmL
)1
, respectively).
To determine their carbohydrate binding specificity,
a series of hemagglutination inhibition assays were
carried out using 14 sugars of three classes. Whereas
agglutination was inhibited by galactose and its deriva-
tives [such as N-acetylgalactosamine (GalNAc),
methyl-a-d-galactopyranoside], it was evident that, at
doses up to 100 mm, glucose, mannose, a-methylman-
noside, fucose, maltose, xylose and saccharose did not
inhibit agglutination (Table 3).
All four pulchellins were shown to interact with ga-
lactosides, although the minimum sugar concentration
that promoted inhibition of hemagglutination varied.
The failure to bind glucose, mannose, a-methylmanno-
side, fucose, maltose, xylose and saccharose shows that
an axial hydroxyl group at C4 is not only an impor-
tant binding group for the lectin, but also that a
reversed configuration at this position might prevent
sugar recognition. P I and P II were able to inhibit
hemagglutination in the presence of GalNAc whereas

Fig. 4. Inhibition of protein synthesis in HeLa cells. Each isoform
was diluted serially in DMEM ⁄ fetal bovine serum and added to
HeLa cells at the concentrations shown. The incorporation of
[
35
S]methionine into new cellular proteins was subsequently deter-
mined as described in the Experimental procedures. Each value is
the mean for triplicate samples. h,PI;
, P II; s, P III; d, P IV.
P. V. Castilho et al. Characterization of four pulchellin isoforms
FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS 953
P III and P IV did not. The hemagglutination inhibi-
tion caused by methyl- a -d-galactopyranoside suggests
that the -OH on C2, C3 and C4, which have the same
configuration as those in galactose and lactose, are
responsible for the strong interaction with the iso-
forms. Interestingly, P II was the only isoform with
affinity for rhamnose. As a result, P II lacked the
galactose and ⁄ or N-acetyl galactosamine specificity
that is a characteristic feature of the archetypal type 2
RIP (with few exceptions).
The most striking difference in sugar binding prefer-
ence was observed with GalNAc (Table 3). We there-
fore performed cytotoxicity assays in which the
various pulchellins were pre-incubated or not with free
GalNAc to determine whether this sugar can prevent
surface binding of toxin in a manner that might indi-
cate a possible basis for the distinctive subgroup
potencies (Fig. 4). For P I and P II, we observed
improved levels of cellular protein synthesis as the

peptide joining the A- and B-chains, that must be
removed during protein maturation upon their biosyn-
thesis. The pre-sequence resembles a true endoplasmic
signal peptide to direct the proteins into the secretory
pathway. The additional N-terminal sequence may
function in a manner akin to the N-terminal propeptide
found in preproricin [24]. It is most likely cleaved after
an Asn residue once the protein is deposited in vacu-
oles. The intervening linker peptides are also extremely
similar and, by analogy to that of preproricin, may well
contain a vacuolar targeting signal [25].
Alignment of the immature polypeptide sequences
(Fig. 3) shows that some residues are conserved only
amongst the pulchellin isoforms (Fig. 3). Although
Table 3. Carbohydrate-binding specifity of P I, P II, P III and P IV.
In the first well, 100 lL of each sugar at 100 m
M was placed and
50 lL was taken and serially two-fold diluted in wells containing
50 lL of NaCl ⁄ P
i
. Then, 50 lL of each isoform solution
(112 lgÆmL
)1
) was added to the wells. Following incubation, 50 lL
of a 1% erythrocyte solution was added. Numbers indicate the
minimal concentration that inhibits agglutination.
Sugar
Minimum concentration for
inhibition (m
M)

P III and P IV), previously shown capable of inhibiting 90% protein
synthesis within 4 h, was used in all preincubations. Each
toxin was mixed with increasing concentrations of GalNAc in
DMEM ⁄ FCS for 30 min. at 37 °C. The mixtures were added to
cells for 4 h and remaining protein synthesis determined as detailed
in the Experimental procedures. Each value is the mean for tripli-
cate samples. h,PI;
, P II; s, P III; d, P IV.
Characterization of four pulchellin isoforms P. V. Castilho et al.
954 FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS
three isoforms (P I, P II and P IV) have the nine con-
served cysteines in the B-chains, P III has only eight of
these and lacks Cys506, indicating that it must lack
one of the usual four intra-chain disulfide bridges.
The primary sequences of the catalytic A-chains
were found to be only slightly different (Fig. 3) but
not in the pairwise manner indicated from cytotoxities
(Fig. 4). Indeed, virtually all of the changes within the
pulchellin A-chains revealed pairwise identity of P II ⁄
P III and P I ⁄ P IV (Table 2). However, these differ-
ences lie outside the residues that are known, from
other ribosome inactivating proteins, to determine the
major folds and the catalytic site (Asn71, Tyr73,
Tyr112, Arg123, Gln159, Glu163, Arg166, Glu194,
Asn195, Trp197, P I numbering) [26]. Indeed, these
residues are retained in positions corresponding exactly
to those in the A-chains of ricin and abrin [26]. Over-
all, it is therefore unlikely that the A-chains differ sig-
nificantly in catalytic activity.
The toxicity values found for the pulchellin isolectins

nation and cytotoxicities between the two subgroups
(Table 3, Figs 4 and 5), is that flanking residues may
be critical in preventing a P III ⁄ P IV interaction with
GalNAc. This hypothesis was also raised for the
mistletoe lectin I [29]. The pairwise alignment of the
isoforms reveals that, although the highest primary
sequence similarity of each subgroup is found in the
C-terminal half of the B-chains (domain 2; Table 2),
and that there is only a single conserved aromatic sub-
stitution in the residues that make up the putative
sugar binding pockets, there is some interesting varia-
tion in the flanking regions around the second sugar
binding pocket that could influence the P III ⁄ PIV
specificity and binding properties. Of particular interest
is the substitution G488R presented by P III ⁄ P IV.
In summary, our data describe a preliminary charac-
terization of a family of pulchellins and reveal a num-
ber of clear differences in B-chain behaviour. We
speculate that variations within domain 2 (C-terminal
half) of these lectins may be relevant for the different
patterns of cell surface binding that are likely to influ-
ence receptor clustering, entry of these toxins into cells
and ultimately their toxicities. Further studies aim to
investigate the proposed structure–function relation-
ships experimentally.
Experimental procedures
Abrus pulchellus seeds were obtained from a plant culti-
vated in the garden of our laboratory, in Sa
˜
o Carlos-SP,

linear gradient of buffer B (20 mm Tris–HCl, pH 8, con-
taining 1 m NaCl) from 0% to 20% for 40 min followed by
20–100% in 5 min. The corresponding peaks of P I and
P I) were collected, dialyzed and freezed. The peaks related
to the P III and P IV were dialyzed against 10 mm sodium
phosphate, pH 7 (buffer A from Mono P) and submitted to
a second chromatographic step in a Mono P 5 ⁄ 50 chro-
matofocusing column previously equilibrated with the same
buffer. One milliliter samples containing P III and P IV
(approximately 0.5 mg) were isolated by an elution gradient
of 0–100% of buffer B (10 mm sodium phosphate, pH 5.7)
for 20 min, holding for 5 min in 100% buffer B. The corre-
sponding peaks of P III and P IV were collected, dialyzed
and freezed. In the two last chromatographic steps (ion
exchange and chromatofocusing), the flow rate was main-
tained at 1 mLÆmin
)1
, the protein level was monitored at
280 nm and the pressure was maintained under 5.5 MPa.
SDS ⁄ PAGE was used to monitor the isolation as well as
the estimation of the apparent molecular weights and struc-
tural properties of the pulchellin isoforms.
Isoeletric focusing
Isoelectric focusing of the proteins was carried out on Phast
System (Pharmacia, Uppsala, Sweden). Samples reconstitu-
ted in MilliQ water were applied to Phast Gel IEF, pH 3–9,
and run according to the standard program. Gels were
stained with Comassie brilliant blue. The range of pI values
of each protein was estimated by using standard markers.
CD measurements

Hitachi, Vienna, Austria). RT-PCR (Super Script Choice
System for DNA Synthesis, Gibco BRL., Paisley, UK) was
performed in two steps. In the first step, for cDNA single
strand synthesis, 600 ng of RNA, 0.5 lg of oligo(dT) primer
and 10 mm of dNTPs were incubated for 5 min at 65 °C.
Subsequently, 4 lL of the first strand buffer 5 X and 2 lL
of dithiothreitol (0.1 m) was added and the reaction was
incubated for 2 min at 42 °C. Finally 1 lL of Superscript II
was added and the reaction was incubated for an additional
1 h at 65 °C. After the cDNA synthesis, the reaction was
precipitated with ethanol [31]. In the second RT-PCR step,
in order to isolate and amplify the cDNAs of the pulchellin
isoforms, the whole amount of the cDNA obtained in the
reaction described above was used. Several primer designs,
based on the N-terminal amino acid sequence of the iso-
forms and on the DNA sequence of pulchellin A- [15] and
B- [16] chains, were tested. These included: pair 1: primer
sense PulcA (5¢-GTC CAG TTT CAA ATG GAC AAA
AC-3¢) and primer anti-sense Oligo (dT)12–18 (Invitrogen,
Carlsbad, CA, USA) and pair 2: primer sense Nterm
(5¢-ATG GAC AAA ACT TTG AAR CTA CTG ATT TTA
TG-3¢) and anti-sense Cterm (5¢-TTA AAA CAA AGT
AAG CCA TAT TTG RTT NGG YTT-3¢). The reaction
mixtures [75 mm of MgSO
4
, 100 pmol of each primer,
10 mm of dNTPs (Promega), 5 lL of buffer HiFi 10 X
(Invitrogen), 2 U of Taq Platinum (Invitrogen), and MilliQ
water to a final volume of 50 lL] were submitted to PCR.
The conditions were initial denaturation of 2 min at 94 °C

roabrin, proricin and mistletoe lectin I for identity analysis.
The nucleotide sequences of the four isoforms were
deposited in Genbank, with the accession numbers
(EU008735, EU008736, EU008737 and EU008738, for P I,
P II, P III and P IV respectively).
Amino acid sequence analysis
Samples of each pulchellin isoform were submitted to
SDS ⁄ PAGE and electroblotted on a poly(vinylidene difluo-
ride) membrane. Polypeptides were excised from the blots
and the N-terminal region was sequenced on an Applied
Biosystems model 477A protein sequencer interfaced with
an Applied Biosystems model 120A online analyzer
(Applied Biosystems, Weiterstadt, Germany). The standard
Edman degradation procedure was used [34].
LC-MS
⁄
MS analysis of tryptic peptides
Pulchellin isoforms (P I, P II, P III and P IV) (100 lg) were
desalted and dried in a SpeedVac SPD12P concentrator
(Thermo Savant, Holbrook, NY, USA). The samples were
solved in 25 lL of 50% (v ⁄ v) acetonitrile and 50 mm
NH
4
HCO
3
; subsequently 5 lLof45mm dithiothreitol were
added to each sample. After incubation for 1 h at 56 °C,
5 lL of 100 mm iodoacetamide were added followed by 2 h
of incubation in the dark at room temperature. After five-
fold dilution with 100 mm NH

teine carbamidomethylation and the variable methionine
oxidation as modifications. The PKL file generated was
used to perform a database search using the deduced pep-
tide sequences provided by the sequences previously cloned.
Cytotoxicity assays
HeLa cells were maintained in DMEM ⁄ fetal bovine serum
(10%). Cells were seeded at 1.5 · 10
4
⁄ well in a 96-well tis-
sue culture plate, allowed to grow overnight and incubated
for 4 h with 100 mL DMEM ⁄ fetal bovine serum containing
graded concentrations of pulchellin isoforms. Subsequently,
cells were washed twice with NaCl ⁄ P
i
and incubated in
NaCl ⁄ P
i
containing 10 lCiÆmL
)1
[
35
S] methionine for
30 min. Labelled proteins were precipitated with three
washes in 5% (w ⁄ v) trichloroacetic acid and the amount of
radiolabel incorporated was determined after the addition
of 100 mL ⁄ well of scintillation fluid, by scintillation count-
ing in a Micro-Beta 1450 Trilux counter (Perkin Elmer,
Waltham, MA, USA). For each value, the level of protein
synthesis was taken as a percentage of toxin-free control
cells, and the mean from four replicate samples was calcu-

FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS 957
96-well microtiter plates. All solutions and dilutions were
made in NaCl ⁄ P
i
(150 mm NaCl containing 5 mm sodium
phosphate buffer, pH 7.4). In each well, 25 lL of NaCl ⁄ P
i
was added and 50 lL of a solution containing each isoform
(112 lgÆmL
)1
) was placed in the first well and serially
diluted (two-fold dilution into successive wells). Next,
25 lL of 1% erythrocytes of each blood type suspension
was added and, after incubating the plates for 30 min at
37 °C and 30 min at 24 °C, the hemagglutination titer was
scored visually.
Hemagglutination-inhibition assays were performed
according to the following procedure. In the first well,
100 lL of each sugar solution (0.1 m) was placed and
50 lL was taken and serially two-fold diluted in wells con-
taining 50 lL of NaCl ⁄ P
i
. Next, 50 lL of each isoform
solution (112 lgÆmL
)1
) was added to each well. After incu-
bating for 30 min at 37 °C, 50 lL of a 1% erythrocyte
solution was added from the animal that showed the high-
est hemagglutination titer for each isoform, human
erythrocytes for P I and P II, and rabbit for P III and IV.

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