(Received for publication, August 29, 1994; and in revised form, November 14, 1994)
From the
CEL-III is one of four Ca-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.
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-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.
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-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.
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 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.
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. 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%.
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 ()
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%.
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 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%.
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 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%.
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. ()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.
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-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) .
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-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.