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(Received for publication, August 29,
1994; and in revised form, November 14, 1994) From the
CEL-III is one of four Ca
Animal lectins have been of great interest recently, as their
various important functions in organisms are being suggested. Most
animal lectins have been classified into one of two groups, i.e. Ca Some C-type lectins have been isolated
from marine invertebrates, such as a sea urchin (Anthocidaris
crassispina)(7) , an acorn barnacle (Megabalanus
rosa)(8) , a tunicate (Polyandrocarpa
misakiensis)(9) , and a sea cucumber (Stichopus
japonicus)(10) . We have recently purified four
galactose/GalNAc-specific Ca Hemolytic and cytolytic proteins have
been isolated from various
origins(12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) .
Lytic actions of some proteins have been ascribed to enzymatic
activity(25, 26) , perturbation of the activities of
membrane-associated enzymes(27) , or pore formation in the
membranes (28) . In this study, we examined the interaction of
CEL-III with the erythrocyte membrane as well as with artificial lipid
vesicles, in order to elucidate the mechanism for hemolysis by CEL-III.
C. echinata body
fluid (350 ml) was taken, and CaCl
Figure 1:
Hemolytic activity (A) and
stability (B) of CEL-III at various temperatures. A,
reactions were performed with 5 µg/ml CEL-III and 5% (v/v) rabbit
erythrocytes in TBS containing 10 mM CaCl
Figure 2:
pH
dependence of the hemolytic activity (A) and pH stability (B) of CEL-III. The following buffers were used for both
experiments. pH 2, 0.01 M HCl; pH 3-5.5, 10 mM sodium acetate buffer; pH 6-7, 10 mM Bis-Tris-HCl
buffer; pH 7.5-8.5, 10 mM Tris-HCl buffer; pH
9-10, 10 mM sodium borate buffer. A, activity
measurements were performed, in duplicate, with 0.5 µg/ml (
Figure 3:
Immunoblotting of the erythrocyte
membranes treated with CEL-III (A) and aggregates of CEL-III
formed on SDS-PAGE (B). A, the membranes prepared
from the CEL-III-treated erythrocytes were solubilized with sample
buffer containing 1% SDS and subjected to 12.5% SDS-PAGE under
nonreducing conditions. After electrophoretic transfer to a
nitrocellulose membrane, CEL-III was detected with mouse anti-CEL-III
antiserum. Lane 1, chicken erythrocytes; lane 2,
rabbit erythrocytes; lane 3, human erythrocytes; lane
4, horse erythrocytes. B, SDS-PAGE (lane 1) and
its immunoblotting (lane 2) of native
CEL-III.
Figure 4:
Osmotic protection against hemolysis by
various carbohydrates. The hemolysis of rabbit erythrocytes was
measured in the presence of carbohydrates (15 mM) as
indicated. Measurements were performed in duplicate with 5% rabbit
erythrocytes and 5 µg/ml CEL-III in TBS containing 10 mM CaCl
Figure 5:
CEL-III-induced release of ATP from rabbit
erythrocytes osmotically protected by dextran 8, and hemolysis in the
absence of dextran 8. Rabbit erythrocytes (5%) were incubated with
CEL-III (5 µg/ml) in TBS containing 10 mM CaCl
Figure 6:
Interaction of CEL-III with the artificial
lipid vesicles. CEL-III was incubated with liposomes trapping
carboxyfluorescein in TBS containing 10 mM CaCl
Since galactose- or GalNAc-containing carbohydrates
effectively inhibited the hemolysis, it was suggested that CEL-III
exerted lytic action by damaging the erythrocyte membrane, after
binding to the specific carbohydrates on the cell surface(11) .
In order to elucidate this mechanism, the interactions of CEL-III with
the erythrocyte membrane and with artificial lipid vesicles were
investigated. When lysis of rabbit erythrocytes by CEL-III was measured
under different conditions, one of the characteristic features was
temperature dependence. The highest activity was exhibited at a low
temperature (10 °C), suggesting that hemolysis by CEL-III may be a
nonenzymatic reaction. Furthermore, the composition of the major lipids
in the rabbit erythrocyte membrane, e.g. phosphatidylcholine,
phosphatidylethanolamine, and sphingomyelin, did not change after
treatment with CEL-III, as confirmed by thin-layer chromatography (data
not shown). Therefore, we postulated that hemolysis by CEL-III was
caused by formation of transmembrane pores, as with some bacterial and
invertebrate
hemolysins(12, 13, 14, 15, 16, 17, 18, 19, 20) .
To test this postulate, the binding of CEL-III to the erythrocyte
membrane was analyzed by immunoblotting the proteins bound to the
CEL-III-treated membranes (Fig. 3). Irreversibly bound proteins
that reacted with anti-CEL-III antiserum were detected in high
molecular mass form only in those erythrocytes susceptible to hemolysis
by CEL-III, such as human and rabbit cells. This result suggested that
CEL-III aggregated in the membrane after binding to the carbohydrates
on the erythrocyte surface by lectin activity and then formed
ion-permeable transmembrane pores so that the erythrocytes were
ruptured by colloid-osmotic shock. Since dextran 4 (4-6 kDa) and
dextran 8 (8-12 kDa) showed remarkable osmotic protection against
lysis, the pores formed by the aggregates of CEL-III may have a
functional radius smaller than 1.75 nm(32) . In this case the
carbohydrate binding activity of CEL-III is obviously not directly
responsible for lysis, since agglutination was observed instead of
lysis when the erythrocytes were protected by the dextrans. This result
also indicates that CEL-III has more than one carbohydrate-binding site
per single polypeptide chain, since CEL-III is a monomeric protein in
native form as confirmed by gel-permeation high performance liquid
chromatography (data not shown). Native CEL-III exhibited multiple
high molecular mass bands on SDS-PAGE (Fig. 3B),
although no aggregates were observed in this CEL-III preparation when
analyzed by gel-permeation high performance liquid chromatography (data
not shown). These aggregates seemed to be induced by interaction with
SDS and were different from those induced by interaction with the
erythrocyte membrane. In fact, aggregate formation was also observed
when CEL-III was incubated with other detergents, e.g. Triton
X-100, Tween 20, and sodium deoxycholate. ( The experiment shown in Fig. 6demonstrated that CEL-III
induced the release of carboxyfluorescein trapped in liposomes
consisting of egg yolk phosphatidylcholine and human globoside. Release
of carboxyfluorescein increased with time following a rapid
agglutination of the liposomes, which indicates that CEL-III binds to
the vesicles via the carbohydrate moiety of globoside containing GalNAc
at its nonreducing end. Although the release of carboxyfluorescein was
much slower than the hemolysis, this result demonstrated that CEL-III
could also form transmembrane pores in the artificial lipid vesicles.
The difference in the apparent rate of reactions between liposomes and
erythrocytes might be related to the composition of their lipid or
carbohydrate receptors. Of the C. echinata lectins, CEL-I
and CEL-IV were found to have apparent homology with C-type CRDs, like
the other invertebrate Ca There have been few reports of lectins with
cytolytic activity, whereas recently a hemolytic lectin from the
mushroom Laetiporus sulfureus and egg vitelline coat lysins
from Mytilus edulis sperm, the latter with homology with
C-type lectins, have been reported(35, 36) . Although
their lytic mechanisms are not clear, it would be interesting to
investigate their relationship with CEL-III. Many invertebrate lectins
are thought to be involved in the defense mechanisms of organisms, not
only neutralizing foreign substances by binding to their carbohydrate
moieties, but also by activating phagocytes(5, 6) .
CEL-III may be a novel type of invertebrate
Ca
Volume 270,
Number 8,
Issue of February 24, 1995 pp. 3560-3564
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-dependent
galactose/N-acetylgalactosamine (GalNAc)-binding lectins from
the marine invertebrate Cucumaria echinata which exhibits
hemolytic activity, especially toward rabbit and human erythrocytes.
The hemolytic activity of CEL-III was also
Ca-dependent and was found to be inhibited by
galactose or GalNAc-containing carbohydrates, suggesting that the
hemolysis was caused by CEL-III binding to specific carbohydrates on
the erythrocyte membrane by Ca-dependent lectin
activity, followed by partial destruction of the membrane. The activity
of CEL-III was highest at 10 °C and decreased markedly with
increasing temperature, unlike usual enzymatic reactions. The hemolytic
activity of CEL-III increased with increasing pH from neutral to 10,
but almost no hemolysis was observed below pH 6.5. Immunoblotting
analysis of proteins from the erythrocyte membrane after treatment with
CEL-III indicated that CEL-III aggregates were irreversibly bound to
the membrane. When erythrocytes were incubated with CEL-III in the
presence of dextran with molecular masses greater than 4 kDa, lysis was
impeded considerably, while a concomitant release of ATP was detected
from these osmotically protected cells. It was found that CEL-III
released carboxyfluorescein from artificial globoside-containing lipid
vesicles, and it is suggested that CEL-III is a novel pore-forming
protein with the characteristics of a Ca-dependent
lectin, which may act as a toxic protein to foreign microorganisms.
-dependent (C-type) and independent (S-type or
galectin)(1, 2) . In vertebrates, C-type lectins are
classified into six groups, which include proteoglycan core protein,
hepatic lectin, mannose-binding protein, and selectin(3) .
These proteins are composed of C-type CRDs, (
)which exhibit
some degree of homology with each other, and additional domains or
regions with the characteristic functions of individual proteins.
C-type lectins purified from invertebrates, on the other hand, are
generally smaller than those from vertebrates, and many of them consist
of only a single CRD. One of the most probable roles of invertebrate
C-type lectins is to act as humoral factors in the defense mechanism,
as do immunoglobulins in vertebrates; enhanced fly lectin production
after injury to the body wall and its activation of phagocytes in
vitro have been demonstrated(4, 5, 6) ,
and it has also been suggested that this lectin plays an important role
during developmental stages.-dependent lectins
(CEL-I-CEL-IV) from the marine invertebrate Cucumaria
echinata(11) , and we have determined the complete and
partial amino acid sequences of CEL-IV and CEL-I, respectively. (
)It was found from the determined sequences that these two
lectins have apparent structural homology with other C-type lectins,
especially with those from marine invertebrates. Interestingly, CEL-III
exhibited strong hemolytic activity when incubated with human and
rabbit erythrocytes, while only weak agglutination was observed with
chicken and horse erythrocytes. The hemolytic activity of CEL-III was
inhibited by several galactose or GalNAc-containing carbohydrates and
was prevented in the presence of EDTA, suggesting that the hemolysis
was caused by CEL-III binding to galactose or GalNAc-containing
carbohydrates on the erythrocyte membrane by
Ca-dependent lectin activity, followed by partial
destruction of the membrane.
Materials
The C. echinata samples were
a generous gift from N. Ikeda (Fukuoka Fisheries and Marine Technology
Research Center). The samples were stored at -30 °C until
use. Chicken, horse, and rabbit blood samples were obtained from Nippon
Bio-Test Laboratories (Tokyo). Inulin and egg yolk phosphatidylcholine
were obtained from Nacalai Tesque (Kyoto). Dextran 4 and dextran 8 were
from Serva. Luciferase-luciferin and human globoside were products of
Sigma. Cellulofine GCL-2000-c was from Seikagaku Kogyo (Tokyo).
Peroxidase-conjugated goat anti-mouse IgG was from Organon Teknika
(West Chester, PA).Preparation of Lactosyl-Cellulofine and
GalNAc-Cellulofine Columns
The columns were prepared by
cross-linking lactose or GalNAc with Cellulofine GCL-2000-c using
divinyl sulfone as described by Teichberg et al.(29) . Purification of CEL-III
In a previous paper we
reported the purification of the four C. echinata lectins by
column chromatography using lactosyl-Sepharose 4B, Sephacryl S-200, and
Q-Sepharose(11) . In the present study we also used a newly
developed procedure, described below, which involved two affinity
columns, i.e. lactosyl- and GalNAc-Cellulofine columns, to
facilitate the separation of CEL-III. was added to 10
mM. This solution was applied to a lactosyl-Cellulofine column
(3.6 
9 cm) equilibrated with TBS (10 mM Tris-HCl
buffer, pH 7.5, containing 0.15 M NaCl) and 10 mM CaCl. After washing the column with the same buffer,
adsorbed proteins were eluted with 20 mM EDTA followed by 0.1 M lactose in TBS. CEL-I and CEL-III were eluted from the
column with 20 mM EDTA, and CEL-IV with 0.1 M lactose, whereas CEL-II was not retained in this column. The
fraction containing a mixture of CEL-I and CEL-III was further
separated using the GalNAc-Cellulofine column (1.3 2 cm) with
the same buffer after dialysis against TBS and addition of CaCl to 10 mM. Since CEL-I has an extremely high affinity for
GalNAc in addition to a moderate affinity for galactose-containing
carbohydrates(11) , CEL-I was bound to the column more strongly
than CEL-III, so the former was eluted with 0.1 M lactose,
while the latter had to be eluted with 20 mM EDTA. A small
amount of CEL-III aggregate was sometimes observed with this
preparation, which could be removed by gel filtration on Sephacryl
S-200. The CEL-III purified by this method exhibited the same specific
activity as that purified by the previous method(11) .
Chromatography was performed at 7 °C. The proteins thus purified
were dialyzed against TBS and stored frozen at -30 °C.Protein Determination
Protein concentrations were
determined with bicinchoninic acid by the method given by Smith et
al.(30) , using bovine serum albumin as a standard.Determination of Hemolytic Activity
Hemolytic
activity of CEL-III was determined by the absorbance at 540 nm due to
hemoglobin released from the erythrocytes. CEL-III in TBS containing 10
mM CaCl (100 µl) was mixed with the same
volume of erythrocyte suspension (10%, v/v) in the same buffer. After
incubation, the suspension was centrifuged, and lysis was determined by
the supernatant's absorbance at 540 nm.Antiserum
Anti-CEL-III antiserum was produced in
mice using CEL-III purified by the previous method (11) as the
antigen. Initially, 20 µg of protein in complete Freund's
adjuvant was injected intraperitoneally. Booster injections of the same
amount of the protein in incomplete Freund's adjuvant were
administered twice, at 3-week intervals. Blood was taken 1 week after
the final injection, and the antiserum was prepared.Immunoblotting
A 5% (v/v) erythrocyte suspension
was incubated with CEL-III (10 µg/ml) in 1 ml of TBS containing 10
mM CaCl for 15 min at 20 °C. After lysis had
been completed, the membrane was pelleted by centrifugation at 13,000
g for 5 min and serially washed twice with 10 mM Tris-HCl, pH 8.0, once with the same buffer containing 0.1 M lactose, and once with the same buffer containing 10 mM EDTA. One-fifth of the membrane pellet was solubilized with the
sample buffer containing 1% SDS and then subjected to 12.5% SDS-PAGE.
The proteins separated in the polyacrylamide gel were transferred to a
nitrocellulose membrane in the transfer buffer (20% ethanol, 25 mM Tris, 192 mM glycine, and 0.1% SDS) for 3 h at 180 mA.
The membrane was blocked with 5% nonfat dry milk in TBS (blocking
buffer) for 60 min at room temperature, then incubated with mouse
anti-CEL-III antiserum (1000-fold dilution), followed by
peroxidase-conjugated goat anti-mouse IgG (2000-fold dilution), in the
blocking buffer at room temperature for 60 min. Proteins were detected
by incubating the membrane with 0.1 M Tris-HCl, pH 7.6,
containing 0.08% 3,3`-diaminobenzidine tetrahydrochloride and 0.05%
HO.Measurement of ATP Release from Erythrocytes
ATP
released from rabbit erythrocytes was measured by the firefly
assay(31) . A 10% (v/v) suspension of rabbit erythrocytes was
incubated with CEL-III (16 µg/ml) in 0.8 ml of TBS containing 10
mM CaCl and 15 mM dextran 8 at 20 °C.
After centrifugation, 0.2 ml of supernatant was mixed with 0.1 ml of
luciferase-luciferin solution (10 mg/ml), and its luminescence was
measured at 560 nm (band path 10 nm) using a Hitachi 650-10SC
fluorescence spectrophotometer without an excitation light.Preparation of Liposomes Containing
Carboxyfluorescein
Egg yolk phosphatidylcholine (20 mg) and
human globoside (0.4 mg) were dissolved in 0.5 ml of chloroform and
dried under reduced pressure in a conical glass tube. After addition of
1 ml of TBS containing 0.1 M carboxyfluorescein, the lipids
were hydrated by vortex mixing for 15 min at room temperature. The
suspension was then sonicated for 10 min at room temperature using a
Tomy Seiko UR-200P ultrasonic disruptor, and subjected to gel
filtration on a column of Cellulofine GCL-2000-c in TBS. The fractions
containing small unilamellar vesicles were collected.Measurement of the Release of Carboxyfluorescein from the
Liposomes
The liposome trapping 0.1 M carboxyfluorescein in 0.5 ml of TBS containing 10 mM CaCl was mixed with CEL-III (0.45 µg/ml) in 0.1 ml
of the same buffer at 20 °C. The fluorescence of carboxyfluorescein
at 523 nm excited at 470 nm was measured at appropriate intervals using
a Hitachi 650-10SC fluorescence spectrophotometer.
Temperature and pH Dependence of the Hemolytic Activity
of CEL-III
Our previous results suggested that hemolysis by
CEL-III was caused by the interaction of CEL-III with the erythrocyte
membrane after it has bound to the Gal/GalNAc-containing carbohydrates
on the erythrocyte surface(11) . To obtain information on the
nature of the interaction between CEL-III and the erythrocyte membrane,
we examined the hemolytic activity under different conditions. As shown
in Fig. 1A, when the hemolytic activity of CEL-III was
measured at various temperatures using rabbit erythrocytes, the highest
activity was observed at 10 °C, but activity decreased with
increasing temperature. At 30 °C, the hemolysis had decreased to
25% of that at 10 °C. It is evident that such a decrease in
hemolytic activity at higher temperatures is not due to the instability
of the protein, since the hemolytic activity of CEL-III was retained
after incubation at up to 50 °C for 30 min (Fig. 1B). This suggests that the hemolysis is not an
enzymatic reaction and might depend largely on the binding of CEL-III
to the specific carbohydrates on the surface of the erythrocyte. On the
other hand, the remarkable decrease in the activity below 10 °C
might be related to reduced membrane fluidity at low temperatures. Fig. 2shows the pH dependence profile of the hemolytic activity
and stability of CEL-III. The hemolytic activity of CEL-III increased
with increasing pH up to pH 10, while almost no hemolysis was observed
below pH 6.5 (Fig. 2A). However, after pretreatment at
different pH values for 15 h, a decrease in hemolytic activity was
observed only in the acidic region below pH 5 (Fig. 2B), indicating that the low activity in the
acidic region shown in Fig. 2A was not due to
irreversible inactivation of the protein. This might reflect the
presence of some ionizable group of amino acid residue with a
pK
around 9-10, which is involved in the
hemolytic activity.
. B, hemolysis was measured at 20 °C in TBS containing 10
mM CaCl, after pretreatment at indicated
temperatures for 30 min. The pH of the buffer was adjusted to 7.5 at
each temperature prior to measurement. The measurements were performed
in duplicate. The highest values were taken as
100%.
)
or 5 µg/ml (
) CEL-III and 5% rabbit erythrocytes in the
presence of 0.15 M NaCl and 10 mM CaCl. B, pH stability was measured after treating CEL-III in the
buffers at the pH values indicated for 15 h at room temperature,
followed by dialysis against TBS containing 10 mM CaCl. The protein concentration was adjusted to 5
µg/ml. The value for the erythrocytes lysed with 0.1% Triton X-100 (A) or the highest value indicated (B) was taken as
100%.
Immunoblotting Analysis
Since there was a
possibility that the erythrocyte membrane had been damaged by the
irreversible binding of CEL-III, the interaction between CEL-III and
the erythrocytes was examined by immunoblotting analysis of the
proteins solubilized from the erythrocyte membranes treated with
CEL-III (Fig. 3). In this experiment, the erythrocyte membranes
treated with CEL-III were washed with 10 mM Tris-HCl buffer,
pH 8.0, containing 0.1 M lactose, followed by the same buffer
containing 10 mM EDTA, to remove any CEL-III bound to the
membrane by its carbohydrate binding ability. The proteins solubilized
from the membranes were then subjected to SDS-PAGE and immunoblotting
analysis. The results clearly showed high molecular mass protein
reacting with anti-CEL-III antiserum in the membranes from rabbit and
human erythrocytes (Fig. 3A, lanes2 and 3) but not in those from horse and chicken
erythrocytes. This suggests that CEL-III was tightly bound to the
membranes of rabbit and human erythrocytes in an aggregated form, in
accordance with the fact that these erythrocytes are susceptible to the
hemolytic action of CEL-III. It is apparent that these aggregates were
derived from the CEL-III monomer, since the antiserum used in this
experiment was raised against the monomer protein purified by gel
filtration on Sephacryl S-200(11) . On the other hand, when
native CEL-III was subjected to SDS-PAGE, high molecular mass bands
were often observed in addition to the monomer band. These high
molecular mass species were also confirmed to be aggregates of CEL-III
by the reaction with the antiserum (Fig. 3B),
indicating that even native CEL-III has a tendency to form aggregates
under the conditions for SDS-PAGE. The aggregates formed during
SDS-PAGE are apparently different from those induced by interaction
with the erythrocyte membranes, since the former always appear as
multiple bands of smaller molecular size than the latter.
Osmotic Protection of Erythrocytes by
Carbohydrates
Since CEL-III was irreversibly bound to the
membranes of the erythrocytes susceptible to its hemolytic action, it
was postulated that the hemolytic activity of CEL-III was due to the
formation of ion-permeable pores in the membrane, as with some
cytolytic
proteins(17, 18, 19, 20, 21, 22) .
Therefore, we examined this possibility by performing the osmotic
protection experiment using several carbohydrates as protectants. As
shown in Fig. 4, when rabbit erythrocytes were incubated with
CEL-III in the presence of several carbohydrates with varying molecular
masses, lysis was inhibited increasingly as the size of the
carbohydrates increased; sucrose and melezitose gave only slight
protection against lysis of rabbit erythrocytes, whereas inulin,
dextran 4 (4-6 kDa), and dextran 8 (8-12 kDa) gave 62%,
98%, and 99% protection against lysis, respectively. Moreover, washing
the erythrocytes treated with CEL-III in the presence of 15 mM dextran 8 with TBS resulted in immediate lysis of the cells. These
results suggest that the erythrocytes were ruptured by colloid-osmotic
shock after CEL-III had formed ion-permeable pores in the membrane.
.
ATP Release from Erythrocytes Treated with
CEL-III
In order to confirm the formation of the transmembrane
pores by CEL-III, the release of ATP from the erythrocytes treated with
CEL-III in the presence of dextran 8 was examined by the firefly
luciferase assay (31) . As shown in Fig. 5, ATP was
released from rabbit erythrocytes protected with 15 mM dextran
8 when the cells were incubated with CEL-III, supporting the above
idea. The increase in ATP release paralleled the hemolysis measured in
the absence of dextran 8 with a slight delay, showing that the
formation of ion-permeable pores preceded the hemolysis. In addition to
rabbit erythrocytes, ATP release was also detected with human
erythrocytes, but not with chicken and horse cells, in agreement with
the susceptibility of these cells to the hemolytic action of CEL-III.
in the presence () or absence () of 15 mM dextran 8. The ATP released from the erythrocytes was measured by
the firefly luciferase assay(31) . The highest values were
taken as 100%.
Interaction of CEL-III with Liposomes
The
interaction of CEL-III with the membrane was further examined by using
artificial lipid vesicles as shown in Fig. 6. When CEL-III was
added to a solution of carboxyfluorescein-trapping liposomes prepared
with egg yolk phosphatidylcholine and a small amount of human globoside
(2%, w/w) as a receptor, the fluorescence intensity at 523 nm was
enhanced, due to the carboxyfluorescein released from the liposomes. At
the same time, a slight turbidity was seen in the solution, due to
cross-linking of the liposomes by CEL-III. In contrast, the increase in
fluorescence intensity was much smaller both in the solution without
the protein and in the presence of 0.1 M lactose. The release
of carboxyfluorescein from the liposomes treated with CEL-III was quite
slow compared with the hemolysis and ATP release form osmotically
protected erythrocytes (Fig. 5).
at
20 °C in the absence () and presence () of 0.1 M lactose, and the increase in fluorescence at 523 nm was recorded
with excitation at 470 nm. 
, blank solution without CEL-III.
The fluorescence intensity of the solution after treatment with 0.1%
Triton X-100 was taken as 100%.
)This is similar
to the case of Staphylococcus aureus 
-toxin, which forms
hexamers on treatment with deoxycholate. After binding to the
erythrocyte membrane as a monomer, S. aureus -toxin forms
transmembrane pores composed of toxin hexamers induced by interaction
with the membrane (33) . Likewise, detergents such as SDS and
deoxycholate might also induce a conformational change in CEL-III,
promoting aggregation. This appears to be closely related to the fact
that CEL-III forms aggregates irreversibly bound to the membrane when
incubated with erythrocytes susceptible to lysis by CEL-III. It is
probable that CEL-III forms aggregates in partially hydrophobic
environments like detergent solutions. CEL-III pore formation may also
be triggered by its conformational change in the partially hydrophobic
environment at the surface of the membrane following binding to the
galactose or GalNAc-containing carbohydrates of the erythrocytes. -dependent lectins whose
primary structures have been determined. Therefore, CEL-III can also be
expected to belong to the C-type lectin family because of the
Ca-dependent nature of its hemolytic and
hemagglutinating activity. Since C-type CRDs usually consist of
120-130 amino acid residues, the extra polypeptide portion of
CEL-III (45 kDa) might be responsible for other function(s), e.g. for membrane binding or aggregate formation. Alternatively, it
also seems possible that CEL-III has a structure consisting of multiple
CRDs like the structure of the macrophage mannose
receptor(34) .-dependent lectin that can act directly as a toxic
protein to foreign microorganisms. In fact, CEL-III was found to
exhibit cytotoxicity to some cultured cells.
Pore-forming
proteins have been purified from some invertebrates (12, 13, 14, 15, 16, 17, 18) ,
although they are not lectins. It is probable that the binding ability
of CEL-III to galactose or GalNAc-containing carbohydrates makes this
protein more specific to its target organisms.
))))
We thank N. Ikeda (Fukuoka Fisheries and Marine
Technology Research Center) for providing the C. echinata samples, Dr. T. Utsumi (Yamaguchi University) for helpful advice
on the preparation of liposomes, and S. Nishinohara and M. Furukawa for
aid in protein preparation and activity measurements.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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T. Uchida, T. Yamasaki, S. Eto, H. Sugawara, G. Kurisu, A. Nakagawa, M. Kusunoki, and T. Hatakeyama Crystal Structure of the Hemolytic Lectin CEL-III Isolated from the Marine Invertebrate Cucumaria echinata: IMPLICATIONS OF DOMAIN STRUCTURE FOR ITS MEMBRANE PORE-FORMATION MECHANISM J. Biol. Chem., August 27, 2004; 279(35): 37133 - 37141. [Abstract] [Full Text] [PDF]
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T. Hatakeyama, T. Suenaga, S. Eto, T. Niidome, and H. Aoyagi Antibacterial Activity of Peptides Derived from the C-Terminal Region of a Hemolytic Lectin, CEL-III, from the Marine Invertebrate Cucumaria echinata J. Biochem., January 1, 2004; 135(1): 65 - 70. [Abstract] [Full Text] [PDF]
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T. Hatakeyama, K. Shiba, N. Matsuo, T. Fujimoto, T. Oda, H. Sugawara, and H. Aoyagi Characterization of Recombinant CEL-I, a GalNAc-Specific C-Type Lectin, Expressed in Escherichia coli Using an Artificial Synthetic Gene J. Biochem., January 1, 2004; 135(1): 101 - 107. [Abstract] [Full Text] [PDF]
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H. Tateno and I. J. Goldstein Molecular Cloning, Expression, and Characterization of Novel Hemolytic Lectins from the Mushroom Laetiporus sulphureus, Which Show Homology to Bacterial Toxins J. Biol. Chem., October 17, 2003; 278(42): 40455 - 40463. [Abstract] [Full Text] [PDF]
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Y. Kouzuma, Y. Suzuki, M. Nakano, K. Matsuyama, S. Tojo, M. Kimura, T. Yamasaki, H. Aoyagi, and T. Hatakeyama Characterization of Functional Domains of the Hemolytic Lectin CEL-III from the Marine Invertebrate Cucumaria echinata J. Biochem., September 1, 2003; 134(3): 395 - 402. [Abstract] [Full Text] [PDF]
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T. Hatakeyama, M. Furukawa, H. Nagatomo, N. Yamasaki, and T. Mori Oligomerization of the Hemolytic Lectin CEL-III from the Marine Invertebrate Cucumaria echinata Induced by the Binding of Carbohydrate Ligands J. Biol. Chem., July 12, 1996; 271(28): 16915 - 16920. [Abstract] [Full Text] [PDF]
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