From the
Indirect immunofluorescence studies revealed that when fixed,
permeabilized cultured human cells were incubated with ricin A chain,
the toxin molecule localized in a staining pattern indicative of
binding to the endoplasmic reticulum and to nucleoli. Chemical
cross-linking experiments were performed to identify the cellular
components that mediated the binding of ricin A chain. Conjugates were
formed between
Ricin is a heterodimeric ribosome-inactivating protein, derived
from the seeds of Ricinus communis (castor beans), that
consists of a 30/32-kDa A chain linked by a disulfide bond to a 32-kDa
B chain. The A chain exists in two major species, the 30-kDa A
Early work by Hedblom et
al.(10) demonstrated that ricin A chain is capable of
binding to 60 S subunits of rat liver ribosomes with a dissociation
constant of 2.2
Single-chain
ribosome-inactivating proteins, such as gelonin, momordin I, and
saporin-S6, and the A chains of double chain toxins, such as ricin and
abrin c, have highly similar amino acid sequences at their putative
active sites (12 and references therein; 13). These
ribosome-inactivating proteins all have the same specificity for
adenine 4324 of naked 28 S rRNA and exhibit comparable levels of
inhibition in rabbit reticulocyte lysate translation systems. However,
these same ribosome-inactivating proteins show very different levels of
activity against ribosomes of other species, such as Homo sapiens (HeLa cells), E. coli, and various metazoan organisms (12
and references therein).
Taken together, these data suggest that the
variations in sensitivity of ribosomes of different species to
ribosome-inactivating proteins having identical rRNA substrate
specificities reflect differences in ribosomal protein content or
interactions. Ribosomal proteins may differentially bind various
ribosome-inactivating proteins. Alternatively, ribosomal proteins may
maintain the rRNA in a conformation susceptible to attack
(4, 14) by some ribosome-inactivating proteins but not by others.
Exactly which ribosomal proteins are involved and the nature of their
contribution to the increased efficiency of catalysis by
ribosome-inactivating proteins remains to be determined. This report is
a first step toward elucidating the roles of ribosomal proteins in the
inactivation of eukaryotic ribosomes by ricin A chain. We describe our
success in identifying the first two ricin A chain-associating proteins
to be isolated, mammalian ribosomal proteins L9 and L10e.
Unless stated otherwise,
reagents were purchased from Sigma. The protein cross-linking agents,
disuccinimidyl suberate (DSS)
In some experiments, cells were co-stained with a
monoclonal antibody specific for protein disulfide isomerase, a marker
protein of the endoplasmic reticulum. The HP13 mouse monoclonal
antibody
(17) , a generous gift of Dr. C. Kaetzel of Case Western
Reserve University, Cleveland, OH, was used at a final dilution of 1:20
and added to fixed, permeabilized cells simultaneously with ricin A
chain. Bound HP13 antibody was detected using Texas Red-conjugated goat
anti-mouse IgG (Catalog No. OB5000-TxRd, Fisher) at 1:100.
The
ribosomal pellet was resuspended by homogenization in 1 ml of 25
mM Tris-HCl, pH 7.5, containing 0.076 M NaCl, 5%
(w/v) SDS, 1 mM PMSF, 2 mM EDTA, 1 µM
leupeptin, 1 µM pepstatin, and 100 µg/ml RNase A Type
II-A, using 12 strokes of a tight-fitting borosilicate glass pestle
homogenizer. The resuspended sample was transferred to a screw-cap
Microfuge tube, heated for 5 min in a boiling water bath to enhance
solubilization, and subsequently microfuged for 10 min at 14,000 rpm.
The supernatant was removed from the residual insoluble material in the
pellet, transferred to a 50-ml screw cap tube, and diluted to 48 ml
with 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl,
1 mM PMSF, 2 mM EDTA, 1 µM leupeptin,
and 1 µM pepstatin. Avidin-agarose (ImmunoPure Immobilized
Avidin, Product No. 20225, Pierce) was treated with bovine serum
albumin (0.1 mg/ml for 30 min) to block any nonspecific binding sites.
1 ml of a 50% slurry of the bovine serum albumin-blocked avidin-agarose
was added to the solubilized, diluted ribosomal material, and the
mixture was rotated at 4 °C overnight. The avidin-agarose beads
were pelleted by centrifugation, and the supernatant was discarded. The
beads were washed 5 times by resuspension in 10 ml of 10 mM
Tris-HCl, pH 8, containing 0.1% SDS, 0.1% Triton X-100, and 2
mM EDTA, followed by centrifugation and aspiration of the
supernatant. The beads (approximately 500 µl) then were
transferred, by multiple washings, into a screw-cap Microfuge tube and
collected by centrifugation. The supernatant was aspirated.
The
avidin-agarose beads were resuspended in 1 ml of reducing
(2
The complex of ricin A chain
cross-linked to the protein later identified as ribosomal protein L10e
was isolated using a purification scheme similar to that described
above for the ricin A chain-L9 complex, with the following
modifications. A preparation of total SW2 cell membranes, derived from
1.4
In reaction mixtures containing proteins from the homogenate of SW2
cells in addition to
When the shorter cross-linking reagent, DFDNB, was used, a
second
Neither the indirect immunofluorescence experiments using whole
cells nor the cross-linking studies using cell membrane fractions could
resolve whether the ricin A chain-binding proteins were membrane
proteins of the endoplasmic reticulum or components of the ribosomes
that stud the endoplasmic reticulum and sediment with the membrane
fraction. To address this question, we isolated ribosomes from rat
liver and from E. coli and tested the purified ribosome
preparations in cross-linking experiments with
In addition to the major 55-kDa and 70-kDa
cross-linked species indicated by the large arrows in
Fig. 2
(a and b), several less prominent bands
can be seen on these autoradiographs; their positions are indicated by
the small arrowheads in Fig. 2, a and
b. These proteins, some of which can be generated more
efficiently using other cross-linking agents, are being investigated
currently and will be the subject of a future report.
We have established that ricin A chain associates with
eukaryotic ribosomes in the proximity of two protein components,
forming covalent complexes of 55 kDa or 70 kDa when cross-linked to
ribosomes using the homobifunctional amine-reactive cross-linking
agents DSS or DFDNB, respectively. Through purification of the
complexes and internal peptide sequence analyses, we have identified
the polypeptides as L9 and L10e of the large ribosomal subunit. These
identifications are consistent with data generated by indirect
immunofluorescence, localizing ricin A chain binding in permeabilized
cells to the nucleoli and the endoplasmic reticulum, sites of
eukaryotic ribosome biogenesis (30) and protein synthesis,
respectively. The ability of ricin A chain to form a similar 55-kDa
protein complex with ribosomes prepared from three different mammalian
species (human, rabbit, and rat), but not with ribosomes from E.
coli, suggests that the L9 protein is highly conserved among
eukaryotes, but not between eukaryotes and prokaryotes. This conclusion
is supported by reports of eukaryotic homologs of rat L9 cloned from
H. sapiens(31) and S. cerevisiae(32) ,
having amino acid sequence identity over a 192-residue overlap to rat
ribosomal L9 of 99% and 49%, respectively. Significantly less homology,
26%, was found between rat ribosomal L9 and its E. coli homolog L6
(27, 33) .
The 70-kDa ricin A chain
complex was generated readily using ribosomes purified from either
cultured human cells or rat liver, less efficiently when rabbit
reticulocyte ribosomes were used, and not at all when E. coli ribosomes were used as the ricin A chain-binding substrate.
Deduced amino acid sequence identities between human L10e and
equivalent proteins from other species have been reported to be 95% for
Rattus norvegicus(34) , 90% for Rattus rattus (GenPept Updates) and Mus musculus (35), 84% for
Gallus gallus (GenBank Updates), 55% for Saccharomyces
cerevisiae(36, 37) , 25% for Halobacterium
cutirubrum(29) , and 15% for E. coli(29) .
Although the precise roles of L9 and L10e in the process of
eukaryotic protein synthesis are unknown, clues to the functions of
these proteins may be deduced by analogy to their prokaryotic homologs,
L6 and L10
(29, 33, 38) . Recent studies showing
that L10 homologs in yeast and rat are required for the assembly of a
multiprotein complex onto eukaryotic ribosomes that is similar to the
(L7/L12)
Our finding that ricin A chain can be cross-linked
to two ribosomal protein components of the translocation domain is
likely to be of physiological relevance, because EF-2-catalyzed GTP
hydrolysis and translocation are the steps of protein synthesis
inhibited by ricin (51). Indeed, EF-2 has been shown to protect
ribosomes from inactivation by ricin A chain
(52, 53) and to prevent binding of ricin A chain to ribosomes in the
presence of a nonhydrolyzable GTP analog (54). Likewise, ricin can
inhibit the binding of EF-2 to the ribosome (55, 56). These results
suggest that EF-2 and ricin A chain compete for identical or closely
located binding sites, such that prior binding of one protein precludes
access of the other to the ribosome.
We believe this to be the first
report identifying ribosomal proteins in the immediate vicinity of the
site of ricin A chain association with the ribosome. Gelonin, a protein
having precisely the same mechanism of action as ricin A
chain
(26) , was incapable of inhibiting the association of ricin
A chain with L9 or L10e in our cross-linking experiments. One
explanation for this result may be that gelonin associates with the
ribosome at the same binding site as ricin A chain but with much lower
affinity. Alternatively, gelonin may bind to the ribosome with a
different orientation than ricin A chain. The observed differential
sensitivity of protein translation systems from various sources to
inactivation by ricin A chain and by single chain ribosome-inactivating
proteins such as gelonin
(12) may reflect differences in the way
these distinct ribosome-inactivating proteins associate with ribosomal
proteins of various species. Indeed, even though ricin A chain and
gelonin catalyze the same reaction, there is only 35.5% amino acid
sequence identity between them
(13) . Interestingly, saporin-6, a
ribosome-inactivating protein extracted from the seeds of Saponaria
officinalis and having the identical site of rRNA depurination as
ricin A chain, has been shown by chemical cross-linking experiments to
form a complex of approximately 60 kDa with a component of the 60 S
subunit of yeast ribosomes
(57) . The identity of the yeast
ribosomal saporin-binding protein is as yet unreported, but it is
intriguing to speculate that it will turn out to be YL9, the yeast
homolog of mammalian ribosomal protein L9
(32) .
What is the
role of ribosomal proteins L9 and L10e in ricin A chain activity? Endo
and Tsurugi
(6) demonstrated that the context provided to
eukaryotic rRNA by ribosomal proteins is required for efficient
catalysis of rRNA depurination by ricin A chain. Although ricin A chain
has similar K
Regardless of the importance of the association of ricin A chain
with L9 or L10e for ricin A chain function, our data should be of
interest to those attempting to build a macromolecular model of the
mammalian ribosome. Although much progress has been made in solving the
structure of the E. coli organelle, much less is known about
the protein-protein and protein-rRNA contacts and functional
interactions in eukaryotic ribosomes. The observation that ricin A
chain binds to the ribosome in close proximity to ribosomal proteins L9
and L10e now can be combined with pre-existing knowledge that ricin A
chain depurinates the 28 S rRNA specifically at adenine 4324 to provide
new insight toward assembling a structural model of the 60 S ribosomal
subunit.
We wish to express our gratitude to Dr. Nancy Kedersha
and Gail McKenzie for assistance with fluorescence microscopy and to
Dr. C. Kaetzel of Case Western Reserve University, Cleveland, OH, for
the generous gift of the HP13 anti-protein disulfide isomerase
antibody. We thank Drs. Walter A. Blättler and Rajeeva Singh for
advice, Dr. Walter A. Blättler for critical reading of the
manuscript, and Rita Steeves and Carlos Acevedo for assistance in the
preparation of biotinylated ricin A chain.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
I-labeled ricin A chain and two proteins
present in preparations of total cell membranes and in samples of
purified mammalian ribosomes. Specificity of the ricin A chain-ribosome
interaction was demonstrated by inhibition of formation of the
complexes by excess unlabeled ricin A chain, but not by excess
unlabeled gelonin, another ribosome-inactivating protein. Complexes of
ricin A chain cross-linked to the ribosomal proteins were purified and
subjected to proteolytic digestion with trypsin. Amino acid sequencing
of internal tryptic peptides enabled identification of the ricin A
chain-binding proteins as L9 and L10e of the mammalian large ribosomal
subunit.
form which has a single complex oligosaccharide unit and the
32-kDa A
form which has a high mannose type oligosaccharide
in addition to the complex unit
(1) . The B chain mediates
binding to cell surface glycoproteins and thereby facilitates cellular
uptake of the toxin (for review, see Ref. 2). The A chain, once in the
cytoplasm and liberated from the B chain
(3) , acts as an
N-glycosidase that attacks the 60 S subunit of eukaryotic
ribosomes, removing a specific adenine in 28 S rRNA
(4) . While
the precise mechanism underlying the catalytic activity of ricin A
chain has been
determined
(5, 6, 7, 8, 9) , much
less is known about how ricin A chain gains access to ribosomes and the
roles, if any, that proteins play in the delivery and binding of ricin
A chain to its ribosomal target.
10
M and a ratio of
1 mol of ricin A chain/mol of subunits. That ribosomal proteins play an
important role in making rRNA highly susceptible to attack by ricin A
chain was established by Endo and Tsurugi
(6) , who showed that
rat rRNA in the context of the ribosome is depurinated at adenine 4324
by ricin A chain with a K
nearly
10
-fold greater than that measured using naked 28 S rRNA.
Naked 23 S rRNA of Escherichia coli is depurinated by ricin A
chain at the homologous N-glycosidic bond, adenine 2660, at
concentrations similar to those effective on mammalian naked rRNA, yet
ricin is ineffective against intact E. coli ribosomes
(4, 6, 11) .
Cell Lines and Materials
The human small cell
lung carcinoma line SW2
(15) was maintained as exponentially
growing cultures in RPMI 1640 medium supplemented with 10%
heat-inactivated iron-fortified bovine calf serum and 2 mML-glutamine. The human bladder carcinoma squamous cell line
SCaBER (ATCC HTB 3) was maintained in Dulbecco's modified
Eagle's medium containing 10% heat-inactivated fetal bovine serum
and 2 mML-glutamine.
(
)
and
1,5-difluoro-2,4-dinitrobenzene (DFDNB), and biotin-HPDP were purchased
from Pierce. Bovine calf serum and fetal bovine serum were from JRH
Biosciences (Lenexa, KS); media and other cell culture reagents were
from BioWhittaker, Inc. (Walkersville, MD). Plasticware was purchased
from Becton Dickinson and Co. Methanol was purchased from J. T. Baker
Inc. Ricin A chain was obtained from Inland Laboratories (Austin, TX).
Gelonin (Catalog No. G-2394) and the rabbit polyclonal anti-ricin
antibody (Catalog No. R-1254) were purchased from Sigma.
Adsorption of Rabbit Anti-ricin Antibody
Prior to
use in immunofluorescence experiments, the rabbit anti-ricin antibody
was presorbed with fixed permeabilized SCaBER cells to remove any cell
cross-reacting antibodies. SCaBER cells (10 cells) were
removed from tissue culture plates by trypsinization, washed with 30 ml
of PBS, and pelleted by centrifugation. The cell pellet was resuspended
in 1 ml of -20 °C methanol for 5 min and then washed three
times in PBS containing 5 mM NaN
by resuspension
and centrifugation. The buffer was aspirated, and the fixed cell pellet
was resuspended in 50 µl of rabbit anti-ricin antibody at 17 mg/ml
in PBS containing 5 mM NaN
. Adsorption was allowed
to proceed for 2 h at room temperature, after which time the cells were
removed by centrifugation. The adsorbed antibody was transferred to a
fresh tube and stored at 4 °C.
Indirect Immunofluorescence Microscopy
The
adherent cell line, SCaBER, was chosen for immunofluorescence
experiments based on its good morphological characteristics, our
previous experience in fixing and labeling these cells, and
availability. Cells were plated at 2 10
cells per
well onto sterile glass coverslips in 24-well tissue culture plates.
Three days later, the medium was aspirated from the wells, and the cell
monolayers were fixed and permeabilized by the addition of 1 ml of
methanol at -20 °C for 5 min. The cells then were rinsed
three times in PBS and blocked for 30 min with PBS containing 5% normal
goat serum and 5 mM NaN
. All subsequent reagent
dilutions were made in PBS containing 5% normal goat serum and 5
mM NaN
. Antibodies routinely were microfuged for 5
min at 14,000 rpm to remove any grossly aggregated material prior to
use. A minimum reagent volume of 100 µl was added per well and
incubated at room temperature with gentle rocking to ensure an even
distribution of the sample over the monolayer. Ricin A chain at
10
M was added to the fixed cell monolayers
for 75 min; alternatively, PBS containing 5% normal goat serum and 5
mM NaN
alone was added to control wells. Between
incubations with reagents, three washes with 1 ml of PBS, for 5 min
each with gentle rocking, were performed. Adsorbed rabbit anti-ricin
antibody was diluted 1:130 and added for 1 h, followed by three washes.
FITC-labeled goat anti-rabbit IgG (Catalog No. OB1420-FITC, Fisher) at
1:100 was used to probe for bound anti-ricin antibody; the incubation
with the secondary antibody was for 1 h in the dark. After the final
incubation, the coverslips were washed three times in PBS and mounted
onto microscope slides using a polyvinyl-based mounting
medium
(16) . Fluorescence was viewed through a Nikon FXA
microscope equipped with epifluorescence optics and the appropriate
filter cube for the detection of fluorescein. Photographs were taken on
Fujichrome film.
Ricin A chain and gelonin were labeled with
[I-Labeling of Ricin A Chain and
Gelonin
I]NaI by the IODO-GEN method
(18) using
IODO-GEN Iodination Reagent purchased from Pierce (Product No. 28600)
and [
I]NaI purchased from DuPont NEN (Catalog
No. NEZ-033H). Twenty-five microliters of IODO-GEN Iodination Reagent
at 50 µg/ml in chloroform was dried to form a film on 12-
75-mm glass test tubes. Ricin A chain (120 µg in 40 µl of
PBS/glycerol, 1:1) or gelonin (100 µg in 100 µl of PBS) was
added to the IODO-GEN-coated tubes together with 10 µl of 1
M sodium phosphate, pH 7.0, and 0.33 mCi of
[
I]NaI, and the mixtures were incubated for 30
min on ice. The reactions were stopped by transferring the protein
solutions to fresh tubes that did not contain IODO-GEN. Non-radioactive
NaI (10 µl of a 1 M solution in water) was added and the
mixtures were incubated for 10 min at room temperature. Ricin A chain
and gelonin then were separated from free
[
I]NaI on NAP-5 (Pharmacia Biotech Inc.)
columns of Sephadex G-25 equilibrated in PBS. Pooled fractions of the
I-labeled ribosome-inactivating proteins were diluted
with an equal volume of glycerol and stored at -20 °C.
Preparation of SW2 Cell Homogenate, Membrane, and Cytosol
Fractions
SW2 cells were pelleted by centrifugation, washed
twice in 0.145 M NaCl, and stored at -80 °C. The
pellet from 5 10
cells was resuspended in 4 ml of
25 mM Hepes-KOH buffer, pH 7.4, containing 0.25 M
sucrose and 1 mM PMSF, and homogenized using 20 strokes of a
tight-fitting borosilicate glass pestle homogenizer. The cell lysate
was centrifuged for 10 min at 6,000 rpm in a Microfuge, and the
supernatant was removed to a fresh tube. The pellet was resuspended in
another 4 ml of 25 mM Hepes-KOH buffer, pH 7.4, containing
0.25 M sucrose and 1 mM PMSF, and the homogenization
and centrifugation steps were repeated. The homogenate supernatants
were pooled and, in some cases, subjected to two rounds of freezing in
liquid nitrogen and quick-thawing at 37 °C. A portion of the
homogenate was separated into membrane and cytosol fractions by
centrifugation at 4 °C for 2 h at 160,000
g
(42,000 rpm) in a Type 50.2 Ti rotor (Beckman Instruments). The
resulting supernatant, taken free of any contaminating pellet material,
was designated the SW2 cell cytosol. The pellet, which is referred to
as the SW2 total membrane fraction, was resuspended by gentle
homogenization in 800 µl of 25 mM Hepes-KOH buffer, pH
7.4, containing 0.25 M sucrose and 1 mM PMSF. The
homogenate, membrane, and cytosol preparations were aliquoted, frozen
in liquid nitrogen, and stored at -80 °C.
Isolation of Ribosomes from Rabbit Reticulocytes, Rat
Liver, and E. coli
Rabbit reticulocyte blood was purchased from
Pel-Freeze Biologicals, and ribosomes were purified from the rabbit
reticulocytes according to standard
procedures
(19, 20, 21) . Ribosomes were isolated
from fresh rat livers (purchased from Taconic, Germantown, NY) and from
the E. coli strain DHFF` according to protocols described
by Spedding
(22) , with the exception that the reducing agent was
omitted. The final pelleting step for the ribosomes was made through a
sucrose cushion prepared in 20 mM Hepes-KOH, pH 7.4, a buffer
compatible with the cross-linking agents used, rather than Tris-HCl.
The ribosomal pellets were rinsed gently with 25 mM Hepes-KOH,
pH 7.4, containing 0.1 M KCl, and resuspended in the same
buffer at 53 A
units/ml, 580 A
units/ml, and 410 A
units/ml for the
rabbit reticulocyte, rat liver, and E. coli preparations,
respectively. The concentrations of the ribosome preparations were
estimated by adsorbance at 260 nm, where 1 A
unit is equal to 18 pmol of 80 S ribosomes or to 23 pmol of 70 S
ribosomes
(22) .
Cell fractionation samples were tested for the
ability to be cross-linked to ricin A chain. SW2 cell membrane
fractions, SW2 cytosol fractions, or purified rabbit, rat, or E.
coli ribosome preparations were diluted into 25 mM
Hepes-KOH buffer, pH 7.4, containing 0.25 M sucrose, and mixed
with 1 µl of I-Labeled Ricin A Chain Cross-linking
Protocol
I-labeled ricin A chain (approximately
0.25 µg) in a total volume of 20 µl. The mixtures were
incubated for 1 h at room temperature to allow binding to occur. Then,
one of two different amine-reactive cross-linking agents, DSS or DFDNB,
was added to the reaction mixtures at a final concentration of 0.3
mg/ml from freshly prepared stock solutions in dimethyl sulfoxide (3.3
mg/ml). Cross-linking was allowed to proceed for 2 h at room
temperature, after which time the reaction was quenched with the
addition of glycine, pH 7.4, to 0.1 M and further incubation
for 30 min at room temperature. Portions of the reaction mixtures (10
to 20 µl) were subjected to SDS-PAGE and autoradiography.
SDS-PAGE and Autoradiography
SDS-PAGE was
performed under reducing conditions using 9% or 10% polyacrylamide gels
(analytical gels, 12 cm 14 cm
0.75 mm; preparative
gels, 12 cm
14 cm
1.5 mm or 3 mm) according to the
method of Laemmli
(23) . Samples were prepared in 62.5
mM Tris-HCl buffer, pH 6.8, containing 2% (w/v) SDS, 10% (v/v)
glycerol, 0.001% bromphenol blue (w/v), and 5% (v/v)
-mercaptoethanol and heated for 5 min in a boiling water bath.
When mini-gels (8 cm
8 cm
1 mm) were used, the
acrylamide concentration was 10% or 12%, and the gels were purchased
from Novex (San Diego, CA). Estimates of relative molecular mass were
made using Rainbow Markers (Amersham). Gels containing samples of
I-labeled ricin A chain were dried and exposed to Fuji
x-ray film at -80 °C for 6 to 16 h using a Cronex Lightning
Plus intensifying screen (DuPont NEN).
Reduction and Biotinylation of Ricin A Chain
Ricin
A chain at 3 mg/ml in PBS/glycerol (1:1) was reduced by reaction with
30 mM dithiothreitol for 50 min at 30 °C. Excess
dithiothreitol was removed by passage of the mixture through a PD-10
column (Pharmacia Biotech Inc.) using PBS containing 1 mM EDTA
as eluent. The reduced ricin A chain was reacted with biotin-HPDP
(N-(6-[biotinamido]hexyl)-3`-(2`pyridyldithio)propionamide)
according to the manufacturer's instructions in order to link
biotin to the thiol of cysteine 259 near the C terminus of ricin A
chain
(24) .
Purification of the Cross-linked Ricin A Chain-Ribosomal
Protein Species
The complex of ricin A chain cross-linked to the
protein subsequently identified as ribosomal protein L9 was purified as
follows. Rat liver ribosomes (9 mg in 350 µl of 25 mM
Hepes-KOH, pH 7.4, containing 0.1 M KCl) and biotinylated
ricin A chain (280 µg in 500 µl of PBS) were mixed together in
8 ml of 25 mM Hepes-KOH, pH 7.4, containing 50 mM
KCl, 0.125 M sucrose, and 1 mM PMSF, and incubated
for 1 h at room temperature. The final concentrations of ribosomes and
ricin A chain during the binding step were approximately 4
10
M and 1
10
M, respectively. Control samples, in which either the
ribosomes or the biotinylated ricin A chain were omitted, were
performed in parallel and resulted in the absence of any protein
comigrating with the cross-linked species at the end of the experiment
(data not shown). DSS was added to 0.3 mg/ml from a 10 mg/ml stock
solution freshly prepared in dimethyl sulfoxide. Cross-linking was
allowed to proceed for 2 h at room temperature, after which time the
reaction was quenched with the addition of 0.1 M glycine, pH
7.4, and incubation for an additional 30 min. The reaction mixture was
layered over a 5-ml cushion of 50 mM Tris-HCl, pH 7.8,
containing 0.5 M sucrose, 12.5 mM MgCl
,
and 80 mM KCl, and centrifuged for 2 h at 160,000
g
(42,000 rpm) in a Beckman 50.2 Ti rotor.
)SDS-PAGE sample buffer and heated for 5 min in a boiling
water bath. The beads were removed by centrifugation and the
solubilized proteins that were released into the supernatant were
electrophoresed on 10% polyacrylamide preparative gels. A second
extraction of the avidin-agarose beads using 300 µl of reducing
(1
)SDS-PAGE sample buffer was performed to yield additional
material. Biotinylated ricin A chain (10 µg/well) was run on
several other lanes of the same gels. Proteins on the gels were
electroblotted onto Immobilon-P (Millipore Corp.) for 90 min at 400 mA
in 10 mM CAPS, pH 11, containing 10% methanol, at 4 °C.
The blots were rinsed with water, then with 100% methanol, and stained
for several minutes with Amido Black (0.1% Amido Black, 1% acetic acid,
40% methanol) to visualize the protein bands. Following destaining in
several changes of water, bands of the 55-kDa cross-linked species were
excised and pooled. In all, bands excised from 8 lanes, in which
150-µl samples had been loaded per well, were sufficient to
generate enough material for internal peptide sequencing. We estimate
that this corresponded to approximately 8 µg of total protein.
Control bands of non-cross-linked biotinylated ricin A chain also were
excised and used to generate an HPLC profile of the trypsin fragments
derived from ricin A chain alone.
10
cells and resuspended in 43 ml, was used as
the source of crude protein to be cross-linked to ricin A chain. DFDNB,
rather than DSS, was used as the cross-linking agent and was added to a
final concentration of 0.6 mg/ml from a stock solution of 20 mg/ml in
dimethyl sulfoxide. Following quenching and ultracentrifugation of the
sample, the pellet of biotinylated ricin A chain-bound ribosomes and
membranes was resuspended in 8 ml of solubilization buffer and
incubated for 30 min at 37 °C, rather than for 5 min at 100 °C.
Insoluble material was removed by centrifugation. The solubilized
sample was diluted to 0.1% (w/v) SDS and purified using avidin-agarose,
SDS-PAGE, and electroblotting, as described above for ribosomal protein
L9.
Trypsin Digestion, HPLC Separation of Peptides, and Amino
Acid Sequencing
In situ trypsin digestions, HPLC
separation of the peptides, and microsequence analyses were performed
at the W. M. Keck Foundation Protein Chemistry Facility of the
Worcester Foundation for Experimental Biology in Shrewsbury, MA.
Electroblotted, excised bands of ricin A chain and the ricin A
chain-ribosomal protein cross-linked species were digested in situ with trypsin, as described by Fernandez et
al.(25) . The tryptic fragments were separated using a
Brownlee Aquapore C8 microbore column (1 250 mM) on a
Hewlett Packard 1090M HPLC system equipped with a UV diode array
detector. With 0.1% trifluoroacetic acid as solvent A and 0.08%
trifluoroacetic acid in acetonitrile/water (70:30 v/v) as solvent B, a
linear gradient from 0 to 55% in solvent B was developed over 90 min at
a flow rate of 50 µl/min. Peptide peaks were collected manually in
1.5-ml Microfuge tubes. Comparison of the HPLC peptide profiles derived
from the ricin A chain-ribosomal protein cross-linked species with that
derived from ricin A chain alone enabled the selection of peptides
unique to the putative ribosomal proteins. The most prominent non-ricin
A chain peptide from each complex was subjected to automated Edman
degradation on an Applied Biosystems 477A Sequencer equipped with a
Model 120A in-line phenylthiohydantoin analyzer or a Model 492 Procise
Sequencer.
Localization of Ricin A Chain Binding by Indirect
Immunofluorescence
Indirect immunofluorescence microscopy was
used as an initial approach toward determining what specific ricin A
chain binding sites existed, if any, in permeabilized human cells. As
shown in Fig. 1a, the indirect immunofluorescence
staining pattern obtained with ricin A chain was indicative of ricin A
chain binding to the endoplasmic reticulum, where many ribosomes are
localized, and to nucleoli, where ribosomal particles are assembled.
Only a low level of background staining was observed in a control
sample, which was not treated with ricin A chain but received both the
primary and secondary antibodies (Fig. 1b). Double
labeling experiments (data not shown), performed as described under
``Experimental Procedures,'' confirmed the association of
ricin A chain with the endoplasmic reticulum, by demonstrating that
ricin A chain co-localized with protein disulfide isomerase, a marker
enzyme of that organelle.
Figure 1:
Localization of ricin A chain binding
by indirect immunofluorescence microscopy. Fixed, permeabilized SCaBER
cells were incubated for 1 h with 10M
ricin A chain (a) or buffer alone (b). The cell
monolayers then were washed, and bound ricin A chain was detected by
processing with a polyclonal rabbit anti-ricin antibody followed by a
FITC-labeled goat anti-rabbit IgG secondary
antibody.
Chemical cross-linking
experiments were performed to identify cellular components to which
ricin A chain can bind. Because these studies required bulk cell
culture, the nonadherent human small cell lung carcinoma line SW2 was
chosen, rather than the adherent SCaBER cell line which was used for
immunofluorescence. Cells were homogenized and fractionated into
cytosol and membrane fractions, and these preparations were tested for
the presence of putative ricin A chain-binding proteins by incubating
them for 1 h with I-Labeled Ricin A Chain Cross-linking
to Proteins from Fractionated Cells
I-labeled ricin A chain. Either DSS or
DFDNB then was added and the cross-linking reactions were allowed to
proceed for 2 h at room temperature. The reaction mixtures were
analyzed by SDS-PAGE and autoradiography; results using DSS and DFDNB
are shown in Fig. 2, a and b, respectively.
Figure 2:
Cross-linking of I-labeled
ricin A chain to cell fractions.
I-labeled ricin A chain
at a final concentration of 4
10
M
was cross-linked to proteins in various cell fractions using either DSS
(a) or DFDNB (b). Lane 1, buffer alone;
lane 2, SW2 cell homogenate from 4.8
10
cells; lane 3, SW2 cell cytosol from 4.8
10
cells; lane 4, SW2 cell membranes from 4.8
10
cells; lane 5, 10 pmol of rat liver
ribosomes; lane 6, 10 pmol of E. coli ribosomes.
Following quenching of the cross-linking reactions, the mixtures were
analyzed by SDS-PAGE and autoradiography. Only half of the rat liver
ribosome sample cross-linked with DSS was loaded (a, lane
5) to avoid overexposure by the prominent 55-kDa band generated in
this reaction mixture. The arrows point to the major ricin A
chain-cross-linked species generated with each cross-linking agent. The
arrowheads indicate minor cross-linked species. The major
doublet running at 30-32 kDa is monomeric
I-labeled
ricin A chain. Positions of the molecular weight standard markers are
indicated as M
10
.
In control samples in which I-labeled ricin A chain
was incubated with buffer alone (Fig. 2, a and
b, lane 1), the only radiolabeled species observed
were the prominent
I-labeled doublet of monomeric ricin A
chain at 30 kDa/32 kDa, a fainter diffuse band representing
cross-linked
I-labeled ricin A chain-ricin A chain dimers
running between 60 kDa and 64 kDa and heavily cross-linked large
aggregates of
I-labeled ricin A chain that failed to
enter the separating gel. We have noted that ricin A chain tends to
aggregate more in the absence of other protein, leading to higher
levels of cross-linked ricin A chain dimers and multimers in buffer
control samples than in samples containing various cell fractions.
I-labeled ricin A chain and DSS
(Fig. 2a, lane 2), a new band was observed
running as a tight doublet at approximately 55 kDa (indicated by the
large arrow). Since monomeric ricin A chain itself runs as a
doublet, the 55-kDa doublet likely represents a single SW2 cell protein
cross-linked to both of the differentially glycosylated forms of ricin
A chain. The 55-kDa species was enriched in samples containing the SW2
total cell membrane fraction as the source of binding proteins
(Fig. 2a, lane 4). Under optimal binding and
cross-linking conditions, the amount of
I-labeled ricin A
chain present in this cross-linked product represented 25% of that
present in the monomer bands of
I-labeled ricin A chain,
as determined by excision from the gel and counting of the respective
bands in a
counter. The 55-kDa species was not generated in the
SW2 cell cytosol reaction mixture (Fig. 2a, lane
3).
I-labeled ricin A chain cross-linked product was
observed running at approximately 70 kDa, indicated by the large
arrow in Fig. 2b. As with the 55-kDa species, the
70-kDa product was prominent in reaction mixtures containing SW2 cell
membranes (Fig. 2b, lane 4), but not in those
containing SW2 cell cytosol (Fig. 2b, lane 3).
I-labeled
ricin A chain. The 55-kDa and 70-kDa
I-labeled ricin A
chain-cross-linked species were enriched in samples containing rat
liver ribosomes (Fig. 2, a and b, lane
5), suggesting that these ricin A chain-binding proteins were
ribosomal or ribosome-associated proteins. The E. coli ribosome preparation did not support the generation of either of
these ricin A chain-cross-linked species, although several high
molecular weight products were observed in the sample of E. coli ribosomes cross-linked with DFDNB (Fig. 2b,
lane 6).
Formation of the
Specificity of
the ricin A chain-ribosome interaction was demonstrated by competition
experiments using either ricin A chain or gelonin, another
ribosome-inactivating protein having the identical mechanism of
ribosome inactivation
(26) in a broad spectrum of species
including rat and rabbit
(12, 26) . A 50-fold molar
excess of unlabeled ricin A chain or a 70-fold molar excess of gelonin
was added to the reaction mixtures during the time in which
I-Labeled Ricin A
Chain Cross-linked Species Is Inhibited by Excess Unlabeled Ricin A
Chain but Not by Excess Unlabeled Gelonin
I-labeled ricin A chain was preincubated with the
ribosomes, before the addition of the cross-linking reagent. Whereas
unlabeled ricin A chain produced nearly complete inhibition of
formation of the
I-labeled ricin A chain-cross-linked
products (Fig. 3, a and b, lane 3),
unlabeled gelonin was unable to prevent
I-labeled ricin A
chain cross-linking to either of the ribosomal proteins (Fig. 3,
a and b, lane 5). The samples run in
lane 4 of Fig. 3, a and b, represent
an additional buffer control since the stock solution of ricin A chain
was in PBS containing 50% glycerol. Note that in the experiment shown
in Fig. 3a we used ribosomes purified from rabbit
reticulocytes as the substrate for generation of the 55-kDa
cross-linked species; similar results were obtained with rat liver
ribosomes. Additional evidence that gelonin exhibits binding
characteristics different from those of ricin A chain, even though they
inactivate ribosomes by acting on the rRNA in a similar manner, was
provided by experiments in which gelonin was labeled with
[
I]NaI and substituted for
I-labeled ricin A chain in the standard cross-linking
protocol using rat ribosomes and DSS. No
I-labeled
gelonin-cross-linked products were observed (data not shown).
Figure 3:
Cross-linking of I-labeled
ricin A chain to ribosomes: competition by ricin A chain but not by
gelonin.
I-labeled ricin A chain was cross-linked to
ribosomes in reaction mixtures containing buffer or an excess of an
unlabeled competing ribosome-inactivating protein. In a,
ribosomes were purified from rabbit reticulocytes and cross-linked to
I-labeled ricin A chain using DSS. In b,
ribosomes were isolated from rat liver and cross-linked to
I-labeled ricin A chain using DFDNB. The reaction
mixtures contained the following components. Lane 1,
I-labeled ricin A chain; lane 2, ribosomes
+
I-labeled ricin A chain; lane 3,
ribosomes + a 50-fold molar excess of unlabeled ricin A chain
+
I-labeled ricin A chain; lane 4,
ribosomes + glycerol-containing buffer +
I-labeled ricin A chain; lane 5, ribosomes
+ a 70-fold molar excess of unlabeled gelonin +
I-labeled ricin A chain. Arrows mark the
positions of the 55-kDa and 70-kDa cross-linked species in a and b, respectively. The prominent doublet running at
30-32 kDa is monomeric
I-labeled ricin A chain. The
positions of the molecular weight standard markers are indicated as
M
10
.
Purification of the Ricin A Chain-Ribosomal Protein
Complexes
We performed large-scale cross-linking experiments to
generate sufficient amounts of the ricin A chain-ribosomal protein
complexes for purification and internal peptide sequence analyses. SW2
cell membranes were used as the source of ribosomal proteins to produce
the DFDNB-cross-linked 70-kDa complex, and rat liver ribosomes were
used to produce the DSS-cross-linked 55-kDa complex. As described in
detail under ``Experimental Procedures,'' the use of
biotinylated ricin A chain enabled us to purify the complexes on
avidin-agarose. The ricin A chain-ribosomal protein complexes were
recovered from the resin by heating the avidin-agarose samples in
SDS-containing buffer. The solubilized proteins then were subjected to
SDS-PAGE and electroblotted onto Immobilon-P. A blot of the purified
55-kDa complex stained with Amido Black in shown in Fig. 4.
Biotinylated ricin A chain alone was run in lanes 1 and
4, and the purified complex was run in lanes 2 and
3. The highly purified 55-kDa complex ran as a doublet, as
does ricin A chain, and was easily resolved from contaminating traces
of non-cross-linked ricin A chain and monomeric avidin, which ran at
the dye front and which presumably leached off the agarose beads during
boiling in SDS. Similar results were obtained for the 70-kDa complex,
purified using the same strategy.
Figure 4:
Western blot of the purified 55-kDa ricin
A chain-ribosomal protein cross-linked complex. Biotinylated ricin A
chain was cross-linked to ribosomes, and the ricin A chain-ribosomal
protein complex was purified on avidin-agarose. The complex was eluted
from the avidin-agarose beads by boiling in SDS-containing buffer and
purified by SDS-PAGE and Western blotting onto a PVDF membrane. The
membrane was stained with Amido Black; positions of the molecular
weight standard markers are indicated as M
10
. Biotinylated ricin A chain alone was run in
lanes 1 and 4, 10 µg and 5 µg, respectively.
The ricin A chain-ribosomal protein complex was run in lanes 2 and 3. The arrow points to the ricin A
chain-cross-linked doublet species.
Internal Peptide Sequence Analyses Identify the Ribosomal
Proteins as L9 and L10e
Bands of the purified ricin A
chain-ribosomal protein complexes and of ricin A chain alone were
excised from the blots and subjected to in situ digestion with
trypsin. The peptides generated from each sample were separated by
HPLC, and the resulting chromatographs, shown in Fig. 5, were
compared in order to identify peptides of non-ricin A chain origin. The
peptide digest derived from the 55-kDa ricin A chain-ribosomal protein
complex, shown in the middle tracing (Fig. 5b),
and the peptide digest derived from the 70-kDa complex, shown in the
bottom tracing (Fig. 5c), both contained peaks
that were not present in the peptide digest derived from the sample of
ricin A chain alone, shown in the upper tracing (Fig. 5a). The unique peptides marked with
arrows in Fig. 5, b and c, were chosen
for amino acid sequencing.
Figure 5:
HPLC separation of tryptic peptides
derived from ricin A chain and the ricin A chain-ribosomal protein
complexes. Bands of ricin A chain and the ricin A chain-ribosomal
protein complexes were excised from Western blots and subjected to
in situ proteolytic digestion with trypsin. The peptides were
separated by HPLC and the resulting chromatograms are shown.
a, ricin A chain alone; b, the 55-kDa ricin A
chain-ribosomal protein complex; c, the 70-kDa ricin A
chain-ribosomal protein complex. Tracings b and c,
therefore, reflect peptides derived from both ricin A chain and the
cross-linked ribosomal protein. The peptides eluting in the peaks
designated with arrows in b and c were
chosen for amino acid sequencing.
Residues 2 through 14 of the peptide
derived from the 55-kDa complex were unambiguously identified as
FNHINVELSLLGK. The 13-amino acid sequence of the peptide was used to
perform a BLAST data base search and found to be a perfect match to
residues 38 to 50 of rat ribosomal protein L9, as deduced from the
nucleotide sequence obtained by Suzuki et al.(27) . Rat
ribosomal protein L9 contains 192 amino acids and has an molecular
weight of 21,879
(27) , making it an appropriately sized protein
to form a 55-kDa complex with ricin A chain. The sequence of 20
residues of the peptide derived from the 70-kDa complex was determined
to be AFLADPSAFVAAAPVAAATT. A BLAST data base search revealed a perfect
match to residues 267 to 286 of human ribosomal protein L10e, as
deduced from the cDNA sequence obtained by Rich and Steitz for the
human ribosomal phosphoprotein originally termed
P0
(28, 29) . Human ribosomal protein L10e contains 317
amino acids and has a molecular weight of 34,273
(28) .
L10 complex of E. coli ribosomes indicate
that analogies between the eukaryotic and bacterial ribosomal systems
are likely to be valid
(39, 40) . In E. coli,
both L6 and L10 have been ascertained by chemical cross-linking and
immunoelectron microscopy studies to be located in close proximity to
each other on the large ribosomal subunit, in the translocation domain
positioned at the interface between the large and small subunits
(41-44). Furthermore, both L6 and L10 are believed to be
functionally involved in the GTP hydrolysis step mediated by elongation
factor G during translocation. Both L10 and L6 have been cross-linked
to L7/12 dimers
(44, 45) , and both proteins have been
shown to stimulate the elongation factor G-dependent GTP hydrolysis
associated with L7/L12
(46, 47, 48) . L6 has been
identified as one of several proteins to which elongation factor G can
be cross-linked upon binding to the ribosome
(42, 49) .
In eukaryotic ribosomes, L9 has been cross-linked to elongation factor
EF-2
(50) .
values measured with rat
ribosomes, naked 28 S RNA, and a synthetic 19-mer oligonucleotide, the
k
measured for ricin A chain using intact
ribosomes as substrate is 10
-fold greater than that using
naked 28 S RNA or the synthetic oligonucleotide
(6, 8) .
This major difference in turnover number has prompted investigators to
propose a role for eukaryotic ribosomal proteins in inducing or
maintaining the proper rRNA secondary structure required to make the
specific adenine residue highly susceptible to attack by ricin A
chain
(14) . However, although the ribosomes of E. coli contain GAGA loop structures similar to that present in the ricin
A chain-susceptible site of eukaryotic rRNA, and although naked 23 S
RNA is depurinated by ricin A chain with a k
comparable to that of naked 28 S RNA, intact E. coli ribosomes are not inactivated by ricin A
chain
(6, 11) . On the basis of our observation that
ricin A chain can be cross-linked to ribosomal proteins L9 and L10e, we
propose that ricin A chain becomes an efficient catalyst of eukaryotic
ribosome depurination only upon binding to these ribosomal proteins,
and that ricin A chain is inactive against E. coli ribosomes
due to its inability to associate with the more divergent L6 and L10
proteins. Our proposal is supported by the observation that ricin A
chain cannot be cross-linked to these E. coli ribosomal
proteins under conditions used successfully to cross-link ricin A chain
to the homologous proteins from human, rabbit, and rat ribosomes.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.