From the Cancer Prevention Division, National Cancer
Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, the ¶ Department of Biochemistry, University of Shizuoka School of
Pharmaceutical Sciences, 52-1 Yada, Shizuoka-shi 422-8526, and the
Instrumental Analysis Research Center for Life Science,
Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo
113-8510, Japan
Received for publication, November 27, 2002, and in revised form, January 2, 2003
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ABSTRACT |
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Pierisin-1, a cytotoxic protein found
naturally in the cabbage butterfly, induces apoptosis of mammalian
cells. Our recent studies suggest that pierisin-1 consists of an
N-terminal ADP-ribosyltransferase domain, and a C-terminal region that
binds to receptors on the surfaces of target cells and incorporates the
protein into cells. The present study was undertaken to identify
receptors for pierisin-1. The cross-linking and cloning experiments
suggested that the proteins on cell membrane had no binding ability to
pierisin-1. Inhibitory assays of fractionated lipids from human
cervical carcinoma HeLa cells, which are highly sensitive to
pierisin-1, indicated neutral glycosphingolipids on the cell surface to
show receptor activity. Inhibitory assays and TLC immunostaining using
anti-pierisin-1 antibodies demonstrated two neutral glycosphingolipids
as active components. Analysis of their structures with
glycosphingolipid-specific antibodies and negative secondary ion mass
spectrometry identified them as globotriaosylceramide (Gb3) and
globotetraosylceramide (Gb4). The receptor activities of Gb3 and Gb4
for pierisin-1 were also confirmed with these authentic compounds.
Pierisin-1-insensitive mouse melanoma MEB4 cells were found to lack
pierisin-1 receptors, including Gb3 and Gb4, but pretreatment of the
cells with glycosphingolipid Gb3 or Gb4 enhanced their sensitivity to
pierisin-1. Thus, Gb3 and Gb4 were proven to serve as pierisin-1
receptors. The C-terminal region of pierisin-1 consists of possible
lectin domains of a ricin B-chain, containing QXW
sequences, which are essential for its structural organization.
Alteration of QXW by site-directed mutagenesis caused
marked reduction of pierisin-1 cytotoxicity. Thus, our results suggest
that pierisin-1 binds to Gb3 and Gb4 receptors at the C-terminal
region, in a manner similar to ricin, and then exhibits cytotoxicity
after incorporation into the cell.
Pierisin-1 is a 98-kDa protein present in the cabbage
butterfly that has potent cytotoxic activity (1, 2). Among 13 mammalian cell lines so far tested, human cervical carcinoma HeLa cells
are the most sensitive to the cytotoxic effects of pierisin-1, whereas
mouse melanoma MEB4 cells are the least sensitive, with an
IC50 value ~5000 times higher (3-5). Pierisin-1 is a
potent inducer of apoptosis of mammalian cells, which is accompanied by
cleavage of DNA to nucleosome units and of poly(ADP-ribose) polymerase
(2, 3). Cloning of a complementary DNA (cDNA) of piersin-1 from
Pieris rapae revealed that pierisin-1 shares sequence
homology with the enzyme units of ADP-ribosylating toxins, including
the A-subunit of cholera toxin in its 27-kDa N-terminal region (6).
Furthermore, substitution of a glutamic acid residue at a presumed
NAD-binding site caused loss of cytotoxic activity, suggesting an
essential role for ADP-ribosylating activity in exerting the
cytotoxicity of piersin-1 (6). Similarly, pierisin-2 from Pieris
brassicae has been suggested to exert its action through ADP-ribosyltransferase (5). Recently, we reported that the target
molecule for mono(ADP-ribosyl)ation catalyzed by pierisin-1 is a DNA,
but not a protein, providing a contrast to bacteria-derived ADP-ribosylating toxins such as cholera toxin and pertussis toxin (7).
Pierisin-1 efficiently catalyzes the ADP-ribosylation of
double-stranded DNA. The ADP-ribose moiety of NAD is transferred by
pierisin-1 to the amino group at N2 of the
deoxyguanosine base (7).
An in vitro expressed peptide consisting of only the
N-terminal region exhibited cytotoxicity and apoptosis-inducing
activity when it was incorporated by electroporation (4). However, the N-terminal peptide alone could not be incorporated into the cells. The
remaining 71-kDa C-terminal region plays a role in binding and
internalization of the whole protein into the target mammalian cells
(4). The C-terminal region of pierisin-1 shares sequence similarity
with HA-33 (or HA1), a subcomponent of hemagglutinin of botulinum toxin
(8, 9). Recent reports of a requirement for sialic acid or galactose
moieties for binding of HA-33 suggest that glycolipids or glycoproteins
might similarly play a role in binding of pierisin-1 to cells (10, 11).
Indeed, we found that addition of the lipid fraction prepared
from HeLa cells, being highly sensitive to pierisin-1, to the medium
inhibited the cytotoxic activity of pierisin-1 as did the membrane
fraction (4). Therefore, components of the glycolipids on the membrane in HeLa cells might act as receptors for pierisin-1.
The present study was designed to explore this possibility. Two
glycosphingolipids were thereby identified as receptor molecules. Immunostaining with antibodies against glycosphingolipids and their
structural analysis by mass spectrometry indicated them to be the
neutral glycosphingolipids
Gb31 and
Gb4.2 The possible structure
and function of the C-terminal region of pierisin-1 were also
investigated by site-directed mutagenesis.
Cell Lines--
Human cervical carcinoma HeLa cells and mouse
melanoma MEB4 cells were obtained from the Institute of Physical and
Chemical Research (RIKEN) cell bank (Tsukuba, Japan). The cells were
cultured in RPMI 1640 medium supplemented with 5% heat-inactivated
fetal bovine serum (Invitrogen, Carlsbad, CA) and 50 µg/ml kanamycin sulfate (Invitrogen) unless otherwise described. Cell cultures were
maintained at 37 °C in an atmosphere of 95% air and 5%
CO2.
Chemical Cross-linking of Pierisin-1 to HeLa Cells--
The
cross-linking studies of pierisin-1 to binding protein were carried out
as described previously (12). HeLa cells were preincubated for 30 min
at 37 °C in binding buffer (128 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 50 mM Hepes, pH 7.4, 5 mg/ml BSA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 10 µg/ml pepstatin A), and then appropriate
amounts of pierisin-1 (0-1.6 µg/ml) in the same buffer were added
followed by incubation for 1 h at 4 °C. After washing four
times with binding buffer, the cells were treated for an additional 15 min with 0.3 mM disuccinimidyl suberate. Cells were washed
once with sucrose buffer (0.25 M sucrose, 1 mM
EDTA, 10 mM Tris-HCl, pH 7.4, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin A) and solubilized in
solubilization buffer (1% Triton X-100, 1 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 mM Tris-HCl, pH 7.0) for 40 min at 4 °C. The solubilized
material was clarified by centrifugation at 10,000 × g
for 10 min and subjected to SDS-PAGE, followed by Western blotting with
the anti-pierisin-1 polyclonal antibody.
Expression Cloning of a cDNA Encoding the Pierisin-1
Receptor--
For the panning procedure, a modification of the
procedure of Almenoff et al. (13, 14) was employed. In the
first round of screening, COS cells were transfected with a human liver
cDNA library (Takara 9505). After 72 h, the cells were pooled
and resuspended in panning buffer (PBS containing 5 mM EDTA
and 0.02% NaN3 5% fetal calf serum), and the panning
plates were coated with pierisin-1 (44 µg per plate in 50 mM Tris-HCl buffer, pH 9.5) for 3 h and blocked with
PBS containing 0.1% BSA at 4 °C for overnight. The transfected COS
cells were then distributed into the coated plates, allowed to attach
for 10 min at room temperature, then washed three times gently with
panning buffer. Then cells remaining on the dishes were lysed, and
plasmid DNA was recovered by the alkaline Miniprep method and amplified
in Escherichia coli to obtain material for the next cycle of panning.
Purification of Glycosphingolipids--
Receptor activity was
monitored by inhibitory effects on cytotoxicity of pierisin-1 to HeLa
cells using a method described previously (4). Total lipids were
extracted from 5 × 108 harvested cells according to
the method of Murayama et al. (15). Briefly, packed cells
were extracted twice with 2-propanol/hexane/water (55:25:20, v/v) by
sonication for 5 min followed by centrifuging. The pellet was
re-extracted with chloroform/methanol (2:1, v/v) and subsequently with
chloroform/methanol (1:1, v/v). The combined extract was then
evaporated, and the residue was dissolved in 1 ml of solvent A
(chloroform/methanol/water, 30:60:8, v/v).
To remove phospholipids, mild alkaline degradation was performed in 0.5 N sodium hydroxide in methanol at 37 °C for 1 h,
followed by dialysis against water overnight and lyophilization. The
lyophilized sample was dissolved in solvent A and applied to
DEAE-Sephadex A-25 column (bed volume 16 ml, acetate form) (Amersham
Biosciences, Buckinghamshire, UK). Neutral glycosphingolipids were
recovered in the flowthrough fractions, while acidic glycosphingolipids were eluted with solvent A containing 0.8 M sodium acetate.
For further purification of individual glycosphingolipids, the
flowthrough fractions were developed on TLC plates of Silica Gel 60 (E. Merck, Darmstadt, Germany) with solvent B (chloroform/methanol/water, 65:25:4, v/v). Spots on TLC plates corresponding to each
glycosphingolipid were scraped off, and the lipids were extracted with
solvent B. The extracts were then evaporated, and the residue was
suspended with water, dialyzed overnight against 10 mM
EDTA, and then lyophilized. EDTA was removed by passage through a
Sephadex LH-20 column (Amersham Biosciences) equilibrated with solvent
A. The eluates were used for further experiments.
TLC Immunostaining--
TLC immunostaining for detection of
molecules bound with pierisin-1 was performed as follows.
Glycosphingolipids were developed on a plastic TLC plate (Polygram SIL
G, Macherey-Nagel, Germany) using solvent B. Two chromatograms were
developed in parallel on the same sheet. One was visualized with
orcinol reagent for chemical detection of glycosphingolipids. The other
was soaked overnight in phosphate-buffered saline containing 1% bovine
serum albumin (PBS/BSA) at 4 °C to block nonspecific antibody
binding. The plate was incubated for 1 h with pierisin-1 (2 µg/ml) in PBS/BSA. After rinsing with PBS, the plate was soaked for
10 min at room temperature in PBS containing 4% formaldehyde. After
washing four times with PBS, the plate was incubated for 2 h at
room temperature with 1:1000 diluted rabbit anti-pierisin-1 antisera
(4) in PBS/BSA containing 5% goat serum. The plate was then washed
five times with PBS containing 0.05% Tween 20 and washed once with PBS/BSA. Pierisin-1, bound with anti-pierisin-1 antibody, was incubated
for 1 h at room temperature in 1:2000 diluted horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (#NA9340, Amersham
Biosciences) in PBS/BSA. After washing five times with PBS, the bound
antibody was visualized with an ECL chemiluminescence kit (Amersham Biosciences).
TLC immunostaining with anti-Gb3 and anti-Gb4 antibodies was performed
using the procedure of Miyamoto et al. (16) with a slight
modification. Mouse anti-Gb3 IgM monoclonal antibody (TU-1) (16) and
mouse anti-Gb4 IgM monoclonal antibody, which was kindly provided by
Dr. Sen-itiroh Hakomori, Pacific Northwest Research Institute, Seattle,
WA (9G7) (17), were used for Gb3 and Gb4 detection, respectively. The
horseradish peroxidase-conjugated goat anti-mouse IgG/M antibody
(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used as
the secondary antibody. The plate for Gb3 detection was stained in
substrate solution (2 mM of 4-chloro-1-naphthol, 1.2 mM
N,N'-diethyl-p-phenylenediamine, 0.04% hydrogen peroxide in 0.1 M citrate buffer, pH 6.0).
The bound anti-Gb4 antibody was visualized by using an ECL
chemiluminescence kit.
For conversion of Gb4 to Gb3, the isolated glycosphingolipids were
incubated with TLC Blotting/Secondary Ion Mass
Spectrometry--
Glycosphingolipids that were separated on the TLC
plate were transferred to a polyvinylidene difluoride membrane by the
TLC blotting method (18). The appropriate position, sized at about 2 mm
in diameter, was cut out and placed on the SIMS target tip with 0.5 µl of triethanolamine as the SIMS matrix. The SIMS spectra of the
glycosphingolipids in negative ion mode were obtained using a TSQ70
triple-quadrupole mass spectrometer (ThermoFinnigan, San Jose, CA)
equipped with a 20-KeV cesium ion gun.
Effects of Glycosphingolipid on Cytotoxicity of Pierisin-1 in
MEB4 Cells--
MEB4 cells, maintained in Opti-MEM
(Invitrogen)-reduced serum (0.5% FBS (HyClone)) medium for at least 1 week, were trypsinized and suspended in a reduced serum medium at a
density of 5 × 104 cells/ml. In the next step, a
100-µl aliquot of the cell suspension was dispensed in each well of
the 96-well plate and cultured for 18 h. The cells in each well
were then incubated for 6 h with 100 µl of the reduced serum
medium containing 100 µg/ml (Gb4, Gb5, or GM3) or 10 µg/ml (Gb3) of
glycosphingolipid (Sigma Chemical Co., St. Louis, MO). After removal of
the glycosphingolipid-containing medium, the cells in each well were
washed with the medium and incubated for 1 h with 100 µl of the
medium containing pierisin-1. After washing they were further cultured
in the same medium for 48 h, and the effects of the incorporated
glycosphingolipid on the cytotoxicity of pierisin-1 were examined by
phase-contrast microscopy and WST-1 cell proliferation assay described
previously (2).
Site-directed Mutagenesis--
A DNA fragment containing the
altered sequence at desired position was amplified from an intact
pierisin-1 cDNA subclone (6) by overlap extension PCR technique
(19, 20). To obtain overlapped 5'- and 3'-fragments, two separate PCR
reactions were carried out, using a 5' primer
(5'-TTCCCAGTCACGACGTTGTA-3') and a 3' primer (primer A, shown in Table
I) for the 5'-fragment and a 5' primer (primer B, shown in Table I) and a 3' primer
(5'-ATAACAATAACAACAACCTCGG-3') for the 3'-fragment. Primers A and B are
complementary to each other and contain mutations of interest.
Following the amplifications, these PCR products were subjected to a
second round of PCR to obtain full-length mutated DNA fragment. The
25-µl reaction mixture contained 5 µl each of PCR solution of 5'-
and 3'-fragments. The thermocycle conditions were 5 cycles of
denaturation at 98 °C for 15 s, re-annealing at 60 °C for
30 s, and extension at 72 °C for 2 min. Using this protocol,
more than half the 5'- and 3'-fragments could be converted to
full-length DNA fragments. The resultant DNA was used as the template
for the in vitro expression system described previously (6)
using MEGAscript and rabbit reticulocyte lysate (Ambion, Austin, TX).
The PCR primer for attachment of T7 promoter sequence was
5'-TAATACGACTCACTATAGGGCGAATTGCCACCATGGCTGACCGTCAACCTTA-3'. The
cytotoxicity of each translated protein in HeLa cells was assessed by
the WST-1 cell proliferation assay (2).
Isolation of Pierisin-1 Receptors from HeLa Cells--
Pierisin-1
exhibits cytotoxicity against mammalian cells after being incorporated
into the cells by interaction of its C-terminal region with the
receptor on the cell membrane. To identify the possible receptor
protein, we performed cross-linking experiments. HeLa cells were
incubated with pierisin-1, cross-linked at each amino group by
disuccinimidyl suberate, and then subjected to Western blotting.
However, no cross-reacting bands were observed. Specific binding of
pierisin-1 to a membrane protein was further examined by expression
cloning of a cDNA encoding a possible receptor from a human liver
cDNA library through the affinity panning system. The selectivity
for pierisin-1 receptor expression was analyzed using COS cells
transiently transfected with the human liver cDNA library. To
select COS clones expressing receptor cDNA, the transfected cells
were placed on panning plates coated with pierisin-1. However, positive
cells were not detected. These results suggested that the protein
fraction has no ability bind to pierisin-1.
It is plausible that receptor molecules competitively inhibit the
binding of pierisin-1 to the cells, thereby inhibiting cytotoxicity. We
have reported that membrane fractions from HeLa cells and total lipid
fraction of HeLa cells inhibited cytotoxic activity of pierisin-1 (4).
Treatment of HeLa cells with 2 ng/ml pierisin-1 at a pulse duration of
15 min induced cell death in about 50% of the cells. Contrary to this,
preincubation of 2 ng/ml pierisin-1 with about 50 µg of total lipid
fraction from HeLa cells before the treatment caused cell death in only
20%. Furthermore, an inhibitory assay of fractionated lipid from HeLa
cells suggested that a polar lipid fraction, which contains glycolipids
and phospholipids, has a binding ability to pierisin-1 (data not
shown). To determine which of the polar lipids on HeLa cells is the
receptor candidate, we first obtained glycosphingolipids from the total
lipids of HeLa cells by mild alkaline degradation and dialysis and then
analyzed the inhibitory effects on cytotoxicity. The inhibitory effects of the glycosphingolipids on cytotoxicity were stronger than for the
parent material, the total lipid fraction, suggesting that the
glycosphingolipids are the major source of pierisin-1 receptor. When
the glycosphingolipids were further separated into neutral and acidic
glycosphingolipids by DEAE-Sephadex A-25 column chromatography, about
75% of the inhibitory activity was recovered in flowthrough fractions,
whereas no such activity was observed in any of the eluted fractions,
which contain acidic glycosphingolipids. These findings indicate that
neutral glycosphingolipids are pierisin-1 receptor candidates. None of
the samples isolated from pierisin-1-insensitive MEB4 cells with the
same methods exhibited any inhibitory effects.
The neutral glycosphingolipids from HeLa cells were developed on a TLC
plate using solvent B (Fig. 1). Of the
various glycosphingolipids in the fraction stained with orcinol
reagent, two major doublet bands, each doublet probably representing
different sugar residues with two different classes of ceramide, were
clearly detected by TLC immunostaining with anti-pierisin-1 antibodies
(Fig. 1). The mobility of these two sets of bands was similar to those
of authentic Gb3 and Gb4, which also exhibited binding ability with pierisin-1.
In the next step, we purified both positive doublet bands, designated
as fractions I and II (Fig. 1), by preparative TLC and analyzed their
effects on pierisin-1 cytotoxicity. As expected, a decrease was noted
following addition of either of the two fractions (data not shown),
suggesting Gb3 and Gb4 to be receptor glycolipids, present in HeLa cells.
Structural Analyses of Receptor Glycosphingolipids--
Fractions
I and II showed positive bands on TLC immunostaining with anti-Gb3 and
anti-Gb4 antibodies, respectively (Fig.
2). Moreover, degradation products of
both fraction II and authentic Gb4 with
For further confirmation of the structures of the isolated
glycosphingolipids, each glycosphingolipid from fractions I and II was
analyzed by negative ion SIMS (Fig. 3).
Deprotonated molecules ([M-H] Inhibitory Potential of Various Glycolipids on Cytotoxic Activity
of Pierisin-1--
We then examined the inhibitory effects of a series
of glycolipids on the cytotoxicity of pierisin-1 to HeLa cells. As
shown in Table II, authentic Gb3 and Gb4
exhibited clear inhibition. Furthermore, authentic Gb5 exhibited
similar activity as well as binding to pierisin-1 on TLC immunostaining
with anti-pierisin-1 antibody (data not shown). However, GalCer,
GlcCer, and LacCer had no inhibitory activity. An oligonucleotide
sugar, Gal Effects of Glycosphingolipids on Cytotoxicity of Pierisin-1 in MEB4
Cells--
In pierisin-1-insensitive MEB4 cells, no glycolipids
binding to pierisin-1, including Gb3 and Gb4, were detected by TLC
immunostaining (data not shown). Authentic Gb4 was added to MEB4 cells
in reduced serum medium and incubated. After removal of Gb4, the cells
were treated with pierisin-1. Cytotoxic assay demonstrated that
sensitivity to pierisin-1 was clearly enhanced (Fig.
4A). Similar effects were
observed with one-tenth the concentration of authentic Gb3 (Fig.
4A). Similarly, Gb5 enhanced cell sensitivity, although GM3
did not show any effect. No morphological changes were observed on
treatment of cells with any glycosphingolipids in the absence of
pierisin-1. Fig. 4B illustrates pierisin-1-induced
morphological changes of MEB4 cells, with or without Gb4 pretreatment.
Concentrations of pierisin-1 that exhibited little effect on control
MEB4 cells caused shrinkage detachment after pretreatment with Gb4. The
results thus suggest that authentic glycosphingolipids, Gb3 and Gb4,
also serve as pierisin-1 receptors.
Role of the QXW Sequence in the C-terminal Region of
Pierisin-1--
Although the C-terminal region of pierisin-1 is known
to share sequence homology with HA-33, the structure of HA-33 remains unclear. Recent data base searches indicated pierisin-1 and HA-33 to
exhibit sequence similarity with the lectin domain of ricin B-chain.
The QXW sequence pattern is present in each subdomain of the
lectin domain of ricin B-chain and is important for its structural
organization and function (21, 22). This QXW sequence was
found in the C-terminal region of pierisin-1 (Fig.
5). Tryptophan residue at the
QXW sequence pattern was conserved at 11 of 12 subdomains.
To clarify whether the QXW sequence of pierisin-1 is
essential as in ricin, site-directed mutagenesis was conducted to alter
the conserved tryptophan residue (Table
III). Replacement of the residue by
glycine in each In the present study, two active neutral glycosphingolipids
exerting inhibitory effects on the cytotoxicity of pierisin-1 were
isolated. The binding abilities of the glycosphingolipids to pierisin-1
were demonstrated by TLC immunostaining. Their identities as Gb3 and
Gb4 were revealed using anti-Gb3 and -Gb4 antibodies and confirmed by
negative ion SIMS. As noted above, Gb3 and Gb4 may serve as pierisin-1
receptors, and this appears to be the case for Gb5, whereas several
structurally related molecules, including LacCer, GM3, asialo-GM2, and
globotriaose, were found to be without activity. Accordingly, neutral
glycosphingolipids with terminally or internally located saccharide
chains of Gal Our results provided strong evidence that the lack of pierisin-1
receptors such as Gb3 and Gb4 in MEB4 cells is the reason for poor
incorporation of pierisin-1 and hence their insensitive phenotype. In
addition, among seven mammalian cell lines with different sensitivities
to pierisin-1, binding and incorporation correlated with the
sensitivity of the cells to the toxic effects of pierisin-1. In fact,
we have also confirmed the presence of abundant amounts of Gb3 and Gb4
receptors in pierisin-1-sensitive human gastric carcinoma TMK-1 cells
and human breast carcinoma MCF-7 cells, in addition to HeLa cells (data
not shown). Thus, the presence of pierisin-1 receptor on the cells is
an important factor affecting pierisin-1 sensitivity.
Ricin, a toxic protein found in the castor bean Ricinus
communis, is composed of a sugar-binding subunit B, which attaches to receptors on the surfaces of target cells, and a subunit A, which
acts as an N-glycosidase inactivating cellular ribosomes (23, 24). Ricin binds to both glycoproteins and glycolipids with
terminal galactose units and can therefore interact with a large number
of different molecules on cell surfaces. The ricin B-chain has two
sugar-binding domains, each of which is composed of three copies ( Pierisin-1 is present naturally in the cabbage butterfly. Because
levels of the protein increases in the fifth instar larvae (6),
elimination of cells in larval tissues by expression of a receptor is
plausible. However, the glycolipid composition in insects, including
the cabbage butterfly, has not yet been fully elucidated. Analyses of
several Dipteran insect species, such as the fruit fly, and
other invertebrates such as the Biwa pearly mussel, suggest that
invertebrates possess characteristic mannose-containing glycolipids as
the core sequence of Man
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-N-acetylhexosaminidase from jack beans (Seikagaku Kogyo, Tokyo, Japan). The resulting products were also assayed by TLC immunostaining.
List of primers used for overlap extention PCR
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
TLC immunostaining of neutral
glycosphingolipid isolated from HeLa cells. Total lipids were
extracted from 5 × 108 harvested cells. After mild
alkaline degradation and dialysis against water, the lyophilized
glycosphingolipid sample was dissolved in an appropriate volume of
solvent A (chloroform/methanol/water, 30:60:8, v/v) and applied to a
DEAE-Sephadex A-25 column. Neutral glycosphingolipids, which were
recovered in the flowthrough fractions, were dissolved in 1 ml of
solvent A. Samples (5 µl) and authentic Gb3 (0.5 µg) and Gb4 (0.5 µg) were resolved on TLC plates with solvent B
(chloroform/methanol/water, 65:25:4). Lane F is the
flowthrough fraction. A, staining with orcinol reagent.
B, immunostaining with anti-pierisin-1 antibody. The two
doublet bands appearing on immunostaining are assigned as
"I" and "II." We have prepared the
neutral glycosphingolipid fraction three times, independently, and the
similar results were reproducibly obtained.
-N-acetylhexosaminidase were confirmed to be Gb3 by TLC
immunostaining using anti-Gb3 antibody (data not shown). These data
strongly suggested that sugar residues of the glycosphingolipids isolated from HeLa cells, exhibiting receptor activity, are identical to those of Gb3 and Gb4, respectively.
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Fig. 2.
TLC immunostaining of pierisin-1 receptor
using anti-Gb3 and anti-Gb4 antibodies. Fractions I and II were
prepared from 5 × 108 harvested HeLa cells and
dissolved in 500 µl of solvent A. Samples (5 µl) and authentic Gb3
(0.5 µg) and Gb4 (0.5 µg) were resolved on TLC plates with solvent
B (chloroform/methanol/water, 65:25:4). A, staining with
orcinol reagent; B, immunostaining with anti-Gb3 antibody;
C, immunostaining with anti-Gb4 antibody.
) were observed at
m/z 1106.9 and 1132.9 in the spectrum for that from fraction I, as shown in Fig. 3A. The ion of
m/z 1106.9 corresponded to Gb3, consisting of
sphingenine (d18:1) and docosanoic acid (C22:0) as ceramide. The
ion of m/z 1132.9 corresponded to Gb3 consisting
of d18:1 and tetracosenoic acid (C24:1) as ceramide. The fragment ions
were weakly observed at m/z 808.7 and 970.7, corresponding to GlcCer and LacCer (d18:1/C24:1), respectively. Thus, the structure for both compounds was determined to be Gb3 (Gal-Gal-Glc-Cer). A deprotonated molecule was observed at
m/z 1336.1 in the spectrum of the
glycosphingolipid from fraction II, as shown in Fig. 3B,
thus corresponding to Gb4, consisting of d18:1 and C24:1 as ceramide.
Fragment ions observed at m/z 646.6, 808.8, 970.8, and 1133.0 corresponded to ceramide, GlcCer, LacCer, and Gb3
(d18:1/C24:1), respectively. Accordingly, the structure of the compound
was concluded to be Gb4 (GalNac-Gal-Gal-Glc-Cer).
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Fig. 3.
Negative SIMS spectra of the two neutral
glycosphingolipids with binding activity to pierisin-1. Fractions
I and II were prepared from 5 × 108 harvested HeLa
cells and dissolved in 500 µl of solvent A. Samples (5 µl) that
were separated on the TLC plate were transferred to a polyvinylidene
difluoride membrane by the TLC blotting method (18). SIMS spectra for
glycosphingolipids from fractions I and II are shown in A
and B, respectively, with interpretations of the fragment
ions. Those at m/z 595, 744, 893, and 1042 are
cluster ions of the triethanolamine used as a matrix. Although an
additional ion at m/z 1024.8 is apparent in
spectrum A, its presence may be due to impurity.
1-4Gal
1-4Glc, corresponding to the terminal sugar
sequence of Gb3, also showed no such inhibitory effect. Other
glycolipids such as gangliosides GM1, GM2, and GM3, as well as
asialo-GM1 and asialo-GM2 did not affect the cytotoxicity of
pierisin-1. Thus, Gb3 might be the minimal requirement structure for
the pierisin-1 receptor.
Inhibitory effects of various glycolipids on cytotoxicity of pierisin-1
, no suppression was observed.
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Fig. 4.
Effects of glycosphingolipids on the
cytotoxicity of pierisin-1 in MEB4 cells. A, MEB4 cells
treated with Gb3 (10 µg/ml) or Gb4 (100 µg/ml) for 6 h were
incubated with various doses of pierisin-1 for 1 h at 37 °C.
After further incubation for 48 h, pierisin-1 cytotoxicity was
examined with a WST-1 cell proliferation assay (2). Gb3 (closed
triangle), Gb4 (closed circle), control (open
circle). Each experiment was carried out in triplicate.
B, MEB4 cells with Gb4 (lower part) or without
Gb4 (upper part) were incubated for 1 h with 250 ng/ml
pierisin-1, and 48 h later, changes in cell morphology were
assessed by phase-contrast microscopy.
subdomain, the most conserved among the
,
,
and
subdomains (see Fig. 5), in domains 1, 2, 3, and 4 (W354G,
W505G, W656G, and W801G, respectively) showed a loss of at least 95%
of the cytotoxic activity against HeLa cells. This implies necessity of
all four domains for the exertion of cytotoxicity of pierisin-1. Next,
the conserved tryptophan in subdomains 3
and 3
was substituted
with glycine. The cytotoxic activities of the resultant mutant proteins
(W607G and W704G) decreased to less than 5% of the parent level,
similar to the 3
mutant, W656G. Furthermore, replacement of
tryptophan 656 in the 3
subdomain by other than glycine,
phenylalanine (W656F) or histidine (W656H), also resulted in a marked
reduction, by 82 and 89%, respectively, of cytotoxic activity against
HeLa cells. These results suggest that the QXW sequence in
each subdomain of every domain in the C-terminal region of pierisin-1
plays a structural role in the cytotoxicity.
View larger version (49K):
[in a new window]
Fig. 5.
Sequence similarity between the C-terminal
region of pierisin-1 and the lectin domain of ricin B-chain.
A, horizontal bars indicate polypeptides of
pierisin-1. The C-terminal region of pierisin-1 is composed of four
presumed lectin domains, shown in the thick-lined box, and
each includes three peptides ,
, and
. B, alignment
of each pierisin-1 subdomain with that of the consensus sequence of
lectin domain of the ricin B-chain (smart00458). The conserved
QXW or similar sequences suggested by this alignment are
boxed.
Cytotoxic activity of pierisin-1 mutated in the conserved QxW sequence
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-4Gal or Gal
1-4Gal
1-4Glc might be required
for binding of pierisin-1. Because oligosaccharides sugars
themselves showed no receptor activity, a ceramide moiety
appears essential for receptor function.
,
, and
) of a galactose-binding subdomain of about 40 amino acid
residues (21). The most characteristic sequence feature is the presence
of a Gln-X-Trp pattern, where X is any amino acid
residue. These Trp residues constitute the hydrophobic core of the
sugar-binding domains and stabilize the C-terminal hook of each
subdomain (21, 25-27). Abrogation of lectin activity is observed with
substitution of tryptophan in the QXW sequence of the ricin
B chain (22). The C-terminal region of pierisin-1 is composed of four
presumed lectin-like domains, and each includes three subdomains
,
, and
. The QXW pattern is conserved partially or
completely conserved in all of the 12 subdomains of pierisin-1.
Site-directed mutation of C-terminal pierisin-1 by replacement of
tryptophan at any conserved QXW sequence in the present
study resulted in markedly reduced cytotoxic activity to HeLa cells.
These results suggest that the conserved QXW sequence in all
of
,
, and
subdomains in each domain of C-terminal region of
pierisin-1 might have an important structural role, and all four bind
to receptors. This would ensure efficient incorporation of pierisin-1
into cells. Thus, structure of the C-terminal region of pierisin-1 and
its binding to receptors may resemble those of the ricin
B-chain.
1-4Glc
1-1'Cer (28-31). Any receptor
activity of these particular glycolipids for pierisin-1 and whether
they are present in the cabbage butterfly are unknown. It should be
noted, however, that globo- series glycosphingolipids, such as Gb3, are
relatively primitive glycolipids. Hence, it is possible that
they might be found in insects. Alternatively, invading organisms with
pierisin-1 receptors might be the natural targets of pierisin-1. It is
also interesting to know the origin of pierisin-1, a unique guanine
ADP-ribosyltransferase combined with lectin domain of the ricin
B-chain. A full understanding of the evolutional aspect of pierisin-1
as well as determination of the presence and distribution of its
receptors in insects and possible target bio-organisms are important
issues for the characterization of the biological roles of
pierisin-1.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Sen-itiroh Hakomori for providing anti-Gb4 antibody.
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FOOTNOTES |
---|
* This study was supported by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science, a grant-in-aid for cancer research from the Ministry of Health, Labor and Welfare, Japan, and a research grant from the Princess Takamatsu Cancer Research Fund.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.
§ To whom correspondence should be addressed. Tel.: 81-3-3542-2511; Fax: 81-3-3543-9305; E-mail: yhibiya@gan2.ncc.go.jp.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M212114200
2 The assignment of gangliosides is based on Svennerholm's designation.
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ABBREVIATIONS |
---|
The abbreviations used are:
Gb3, globotriaosylceramide;
Gb4, globotetraosylceramide;
Gb5, globopentaosylceramide;
GalCer, galactosylceramide;
GlcCer, glucosylceramide;
LacCer, lactosylceramide;
Cer, ceramide;
PMSF, phenylmethylsulfonyl fluoride;
BSA, bovine serum albumin;
PBS, phosphate-buffered saline;
SIMS, secondary ion mass spectrometry;
GM1, Gal1-3GalNac
1-4(Neu5Aca2-3)Gal
1-4Glc
1-1'Cer;
GM2, GalNac
1-4(Neu5Aca2-3)Gal
1-4Glc
1-1'Cer;
GM3, Neu5Aca2-3Gal
1-4Glc
1-1'Cer..
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REFERENCES |
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