From the
Ly-49 represents a family of type II transmembrane proteins
containing C-type lectin domains. At least two members of the Ly-49
family, namely Ly-49A and Ly-49C, are expressed by distinct subsets of
natural killer cells and bind to class I major histocompatibility
complex antigens on the surface of target cells. In this report we have
established that Ly-49C mediates carbohydrate recognition. The sulfated
glycans fucoidan,
Natural killer cells are a class of lymphocytes that mediate
cytotoxicity against cells infected by virus or bacteria, as well as
many transformed cell types
(1) . NK
Recent
studies have implicated a group of molecules containing C-type lectin
domains as potential NK cell receptors. These include the genetically
linked Ly-49 (mouse)
(5, 6, 7, 8, 9, 10) ,
NKR-P1 (mouse, rat, and human)
(11, 12, 13, 14) , and NKG2 (human)
(15) multigene families. These genes encode type II
transmembrane proteins, with a distal extracellular region homologous
to the carbohydrate recognition domain (CRD) of C-type lectins.
Although members of each multigene family are highly related at both
the nucleotide and amino acid level, sequence comparisons between
multigene families show low but significant similarity only at the
amino acid level, and this is mainly limited to the lectin-like domain
(
The Ly-49
multigene family comprises at least eight distinct genes. These related
molecules have been termed Ly-49A-H and are between 49 and 91%
identical at the amino acid level
(8, 9, 10) .
Ly-49A and Ly-49C expression defines distinct single positive and
double positive subsets of NK cells
(10) , and both function as
receptors for class I MHC molecules
(10, 16, 17) . Ly-49A binds to purified,
immobilized H-2D
Ly-49C, recognized by
the 5E6 monoclonal antibody (mAb), binds to target cells of diverse
haplotypes
(10) , whereas Ly-49A has been shown only to bind
H-2
Neuraminidase- and sham-treated
GM979 cells were washed twice and stained with 100 ng/ml FITC-labeled
peanut lectin in a 100-µl volume for 30 min at 4 °C. This was
followed by two washes in phosphate-buffered saline, 2% fetal calf
serum, with propidium iodide added to the final wash at 1 µg/ml.
Cells were analyzed on a FACSortequipped with Lysis II software
(Becton Dickson & Co.), and dead cells were gated out.
COS cells expressing
Ly-49C were preincubated with each polysaccharide for 1 h, followed by
the 5E6 Ab for 30 min. Similar to what was observed in the cell-cell
assay, dextran sulfate ( M
Ly-49C, like all members of the Ly-49 multigene family,
contains a domain homologous to C-type animal lectins
(8) . Many
proteins have been shown to bind carbohydrates through this conserved
domain, including the hepatic lectins, mannose binding proteins, and
the selectins
(24, 25) . The present study provides
evidence that Ly-49C is also a carbohydrate-binding protein.
Interestingly, those polysaccharides that are recognized by Ly-49C also
inhibit selectin functions
(28) . Selectins and Ly-49 share
amino acid homology only in the putative CRD (
Our studies
have shown that
Previous studies have shown that many sugars inhibit NK
activity in a standard cytotoxicity assay. Both soluble and cell
surface carbohydrates appear to interfere with cytotoxicity by
inhibiting the activation of lysis at a post cell binding stage
(33) . Without having clarified the mechanisms of NK cell
recognition, these studies suggested that NK cells may possess lectin
receptors which recognize carbohydrate structures on the target cell
surface. The recent molecular characterization of lectin-like receptors
expressed by NK cells (Ly-49, NKR-P1, and NKG2)
(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) has provided a basis by which carbohydrate binding
properties of putative NK cell receptors can be evaluated.
Carbohydrate recognition has recently been reported for both rat
NKR-P1
(26, 34) and Ly-49A
(27) . Interestingly,
these molecules appear to behave differently than all other C-type
lectins
(24, 25) in that normal ligand binding occurs
in the absence of exogenous Ca
Carbohydrate
recognition by NKR-P1 appears to activate NK cell lytic functions
(34) , which follows from previous studies demonstrating that
cross-linking of the antigen activates NK cells
(36) . Ly-49C
and NKR-P1 are distantly related molecules, which suggests that they
have distinct ligand specificities. Indeed, both chondroitin sulfate
and sulfated heparin oligosaccharides inhibit NKR-P1 binding at very
low concentrations
(34) , but have no effect on Ly-49C.
We
have found that fucosidase treatment of GM979 significantly reduced its
adhesion to Ly-49C (Fig. 3). This observation, along with the
inhibition by fucoidan, suggests that fucose may serve as a ligand for
Ly-49C. Daniels et al. (27) have shown that
Ly-49A-mediated adhesion is also inhibited by fucoidan, at
concentrations similar to the Ly-49C ID
Daniels et al. (27) also reported
(as data not shown) that sialidase treatment of D
Ly-49A and C have both been shown to
bind to class I MHC molecules
(10, 16, 17) . The
polymorphic
All class I MHC molecules contain a conserved
N-linked glycosylation site (Asn-86) which lies near the
junction of the
Class I MHC oligosaccharides are known
to be heterogeneous, as displayed by the distinct carbohydrate
structures of K
Ly-49A appears to act as a
negative regulator
(18) , but it is unknown if Ly-49C
recognition is inhibitory or activating to the NK cell. It has been
proposed that NK cell target recognition may be a complex mixture of
positive and negative signals and these signals may be delivered by
molecules such as NKR-P1 and Ly-49
(43) . The characterization
of Ly-49C as a carbohydrate-binding protein suggests that lectin
interactions may be central to this process.
We thank Dr. Graeme Dougherty for the 3c12 antibody
and the CD44H cDNA and Dr. Robert Kay for the vector PAX142.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-carrageenan, and dextran sulfate were found to
be potent inhibitors of Ly-49C-mediated cell adhesion, whereas other
polysaccharides of similar size, charge, or sulfate content were
noninhibitory. All of the polysaccharides which inhibited Ly-49C
adhesion also blocked the binding of the antibody 5E6 to
Ly-49C-expressing COS cells, confirming the direct protein-carbohydrate
interaction. The enzymatic removal of specific carbohydrates from the
target cell surface has shown that Ly-49C-mediated adhesion is not
sialic acid-dependent, but is significantly decreased following
fucosidase treatment. These results suggest an important role for
carbohydrate recognition by natural killer cell receptors.
(
)
cells produce cytokines following stimulation
(1) ,
and in the mouse, are capable of rejecting allogeneic hemopoietic cells
(2) . Although the manner in which NK cells recognize target
cells is poorly understood, it is clear that the mechanism is distinct
from that utilized by T cells. NK cells neither rearrange nor express
the T cell receptor
(3) and undergo normal development in mice
deficient for the recombinase-activating gene
(4) .
25% identity)
(8, 12, 15) .
and H-2D
(16) , and the
recognition of these antigens on the target cell appears to deliver a
negative signal to the NK cell which prevents lysis
(18) . This
observation is concordant with the prediction that NK cells possess
receptors that deliver negative signals upon interaction with class I
MHC on the target cell surface
(19) .
(10, 16, 17) . Although
the functional consequences of Ly-49C recognition are unknown, the
molecule has been implicated in the H-2-directed (and NK cell-mediated)
phenomenon of hybrid resistance. 5E6
(Ly-49C
) NK cells reject H-2
, but
not H-2
, bone marrow cells when transplanted into
irradiated hosts
(20) . In this study, we have shown that cell
adhesion mediated by Ly-49C is inhibited by several sulfated glycans,
each of which blocks the binding of the mAb 5E6, and that treatment of
target cells with fucosidase drastically reduces their adhesion to
Ly-49C expressing COS cells.
Cell Lines and Reagents
The cell lines COS-1 and
GM979 were obtained from American Type Culture Collection (Rockville,
MD) and were maintained in Dulbecco's modified minimum essential
medium, supplemented with 5% fetal calf serum. Dextran sulfate
( M500,000), fucoidan,
-carrageenan,
chondroitin sulfates A, B, and C, heparan sulfate, hyaluronan, dextran
( M
500,000), neuraminidase (from Vibrio
cholerae, type II),
-L-fucosidase (from bovine
kidney), and FITClabeled peanut lectin (from Arachis hypogaea) were
purchased from Sigma.
COS Cell Expression and Cell Adhesion
Assays
Ly-49C, CD44H, and the vector PAX142 were transfected
into COS cells as described previously
(10) . Cells to be
analyzed by FACSwere allowed to grow uninterrupted for 3 days
following transfection. Cells tested in adhesion assays were split to
Falcon 3002 plates 24 h post-transfection (2 10
cells/plate). 48 h later, these plates were used in a
quantitative cell-cell adhesion assay. 5
10
labeled
GM979 cells (1 µCi of Na
CrO
/10
cells) were overlaid on adherent transfected COS cells for 2 h,
after which the plates were washed three times, bound cells were lysed
with 10% Triton X-100, and radioactivity was determined. Each condition
was tested in at least three independent experiments. Carbohydrates
were added to COS cells simultaneously with the labeled GM979 cells. In
experiments involving EGTA, assays were performed in
Ca
-free Hanks' balanced salt solution (HBSS)
containing 1% BSA, 2 mM MgCl
. 2 mM
CaCl
was added to control plates, whereas 5 mM
EGTA was added to experimental plates. Both COS cells and GM979 were
washed twice in the appropriate buffers before the start of the assay.
Glycosidase Treatment of GM979 and Lectin Binding
Assay
GM979 cells were labeled with Cr, washed
twice in a 150 mM sodium chloride, 4 mM calcium
chloride, pH 5.5, buffer, and then resuspended. Neuraminidase treatment
was for 1 h at 37 °C in a 50-µl reaction volume with 0.40 or
0.22 unit/ml of enzyme. Fucosidase treatment was for 1 h at room
temperature with 0.28 or 0.19 unit/ml in a 100-µl reaction volume.
Immediately following enzymatic treatment, cells were tested in the
cell adhesion assay described above.
Polysaccharides, Antibodies, and Flow
Cytometry
R-Phycoerythrin-conjugated 5E6 was purchased from
PharMingen, and the mAb 3c12 (anti-CD44H)
(21) was provided by
Dr. Graeme Dougherty (Terry Fox Laboratory, Vancouver, Canada). All
stainings were carried out at cell concentrations of 1
10
/ml. COS cells were incubated with carbohydrate for 1 h
at room temperature (control samples were treated with an equal volume
of HBSS), followed by a 30-min incubation with either 5E6 or 3c12. A
secondary goat anti-mouse IgG-R-phycoerythrin staining step was
required with the antibody (Ab) 3c12. Cells were washed twice following
Ab staining, and dead cells were gated out with propidium iodide. Mean
fluorescence values were determined for each condition and compared
with the control which did not receive carbohydrate. Each
polysaccharide was tested in at least three independent experiments
(transfections and FACSanalysis).
Sulfated Polysaccharides Inhibit Ly-49C-mediated
Adhesion
Previous work has demonstrated that Ly-49C expressed on
COS cells binds to cell lines of various haplotypes
(H-2) and that this interaction is inhibited by an
anti-class I MHC Ab
(10) . In this study, we have used this
system to test the potential carbohydrate recognition properties of
Ly-49C, which are suggested by its amino acid similarity to known
lectins
(6, 8) . Several polysaccharides were tested for
their ability to inhibit the adhesion of GM979 to COS cells transfected
with the Ly-49C cDNA (Fig. 1). Of those tested, dextran sulfate
( M
500,000), fucoidan, and
-carrageenan were
found to inhibit at concentrations ranging from 5
10
to 2
10
M
().
Figure 1:
Polysaccharide dose-response inhibition
of Ly-49C-mediated cell adhesion. 0% inhibition corresponds to adhesion
of the cell line GM979 (Cr-labeled) to Ly-49C-transfected
COS cells in the absence of carbohydrate, and 100% inhibition is
equivalent to GM979 binding to COS cells transfected with the vector
PAX142. COS cells were incubated simultaneously with GM979 and dextran
sulfate ( M
500,000),
-carrageenan, fucoidan,
dextran ( a), heparan sulfate ( b), hyaluronan
( c), chondroitin sulfate B ( d), chondroitin sulfate A
( e), or chondroitin sulfate C ( f). Each point
represents the mean ± S.E. of at least three independent
experiments.
Those polysaccharides which inhibited adhesion are
all negatively charged sulfated glycans
(22, 23) . An
important role for sulfate is indicated by the failure of nonsulfated
dextran to inhibit at a concentration 400 times higher than the
IDvalue determined for dextran sulfate ().
Chondroitin sulfates A, B, and C and heparan sulfate represent a panel
of sulfated anionic polysaccharides which do not affect Ly-49C-mediated
adhesion. Hyaluronan is of intermediate size and charge compared to
fucoidan and
-carrageenan
(22) . Its lack of inhibitory
activity in this system rules out the possibility of a nonspecific
charge effect.
Inhibition of 5E6 Binding to Ly-49C
The mAb 5E6
appears to recognize a functional domain of Ly-49C, as demonstrated by
its ability to completely inhibit Ly-49C-mediated cell adhesion
(10) . The polysaccharide inhibition seen in the cell binding
assays suggests that certain carbohydrates may specifically interact
with Ly-49C. We therefore tested the series of carbohydrates which
strongly inhibit Ly-49C mediated adhesion for their ability to compete
with the Ab 5E6 for binding to the molecule.
500,000), fucoidan, and
-carrageenan all significantly inhibited the binding of 5E6 to
Ly-49C, whereas hyaluronan had no effect (Fig. 2). As a control,
we have expressed the common form of CD44 (CD44H) on COS cells and
performed the same set of experiments using the anti-CD44 Ab 3c12. All
polysaccharides tested had no effect on the interaction of 3c12 with
CD44, indicating that the effect is both Ly-49C- and
carbohydrate-specific. These results suggest that Ly-49C binds to
dextran sulfate, fucoidan, and
-carrageenan.
Figure 2:
Competition of antibody binding by
polysaccharides. COS cells expressing Ly-49C or CD44H were exposed to
fucoidan, -carrageenan, dextran sulfate ( M
500,000), or hyaluronan for 1 h at room temperature, followed by
the Ab 5E6 (Ly-49C) or 3c12 (CD44H) for 30 min. Cells were washed
twice, analyzed by FACS, and mean fluorescence was determined for
each condition. Ab binding is shown as the percentage relative to
control levels (in the absence of carbohydrate). Values shown are the
mean ± S.E. of at least three independent transfections,
stainings, and FACSanalysis.
Glycosidase Treatment of GM979
To directly
evaluate the role of cell surface carbohydrates in Ly-49C-mediated
adhesion, GM979 cells were treated with fucosidase or neuraminidase and
tested in the cell-cell adhesion assay. Treatment of cells with
fucosidase resulted in a dose-dependent decrease in cell adhesion
(Fig. 3). Cell surface class I MHC levels were unaltered by
fucosidase treatment, as evaluated by staining with the Ab 34-1-2S
(data not shown). Removal of sialic acid resulted in an enhanced
adhesion to Ly-49C expressing COS cells (Fig. 3). The increased
binding of desialylated cells was Ly-49C-specific, because
neuraminidase-treated GM979 showed no altered binding to control COS
cells (data not shown). The effectiveness of the sialidase was verified
by the binding of peanut lectin only to enzymatically treated cells
(Fig. 4). This lectin binds to the terminal galactose of
glycoproteins following neuraminidase treatment.
Figure 3:
Effects of glycosidase treatment and
calcium depletion on Ly-49C-mediated adhesion. GM979 cells were treated
with neuraminidase or fucosidase as described under ``Materials
and Methods.'' In the calcium depletion experiments, adhesion was
tested in Ca-free HBSS containing 1% BSA, 2
mM MgCl
, 5 mM EGTA. 100% control adhesion
is equal to binding of GM979 in the absence of treatments, and 0%
adhesion is the level of binding detected to COS cells transfected with
the vector PAX142. Values shown are the mean ± S.E. of at least
three independent experiments.
Figure 4:
Peanut lectin binds only to
neuraminidase-treated GM979. Cells were treated with neuraminidase,
washed, stained with FITC-labeled peanut lectin, washed, and then
analyzed by flow cytometry. A, no enzyme, unstained;
B, no enzyme, FITC-peanut lectin; C, 0.28 unit/ml
neuraminidase, FITC-peanut lectin; D, 0.19 unit/ml
neuraminidase, FITC-peanut lectin.
One of the defining
features of C-type lectins is that ligand binding is calcium-dependent
(24, 25) . Upon testing Ly-49C-mediated adhesion for
this property, we have found that there is no requirement for exogenous
Ca, as demonstrated by the persistent binding in 5
mM EGTA (Fig. 3). Although this is in sharp contrast to
nearly all C-type lectins studied, it appears to be a property common
to the NK cell lectin branch of this superfamily
(26, 27) .
20% identity) and no
nucleotide similarity
(8) . Both fucoidan and dextran sulfate
inhibit adhesion mediated by L- and P-selectin at concentrations nearly
identical to the ID
values of Ly-49C
(28, 29, 30) . Fucoidan also potently blocks the
binding of the Ab Mel-14 to L-selectin
(31) . In this study,
those polysaccharides that inhibit cell adhesion were also found to
inhibit the binding of 5E6 to Ly-49C, suggesting that 5E6, like Mel-14
binding to L-selectin, recognizes the CRD of Ly-49C.
-carrageenan is an equally potent inhibitor of
Ly-49C-mediated adhesion. The monosaccharide cores of the inhibitory
polysaccharides are fucose, galactose, and glucose (fucoidan,
-carrageenan, and dextran sulfate, respectively)
(22, 32) . These polysaccharides are obviously not the
natural ligands for Ly-49C, as they are not found in mammalian cells.
It remains to be seen if the natural ligands consist of these
monosaccharides or if they are structurally analogous to the true
ligand(s).
(26) .
Decalcification of the purified proteins by high pH dialysis has shown
that carbohydrate binding is indeed Ca
-dependent, but
that Ca
is tightly bound at pH 8 and is not released
in the presence of EGTA or EDTA
(26, 27) . We have also
observed that Ly-49C-mediated adhesion is not inhibited by the removal
of exogenous Ca
. NK cell lectin-like receptors define
one of at least five distinct subgroups of the C-type lectin
superfamily
(35) . It appears that one property of this class
may be a high affinity for Ca
, which expresses itself
as exogenous calcium-independent ligand binding.
value.
Additionally, tunicamycin-treated target cells showed reduced adhesion
to Ly-49A and increased sensitivity to Ly-49A
NK cells
(27) . We have seen that tunicamycin also reduces
Ly-49C-mediated adhesion, but consider this observation difficult to
interpret due to the associated decrease in class I MHC expression
(data not shown).
target cells had no effect on their adhesion to Ly-49A. Contrary
to this, Ly-49C adhesion is increased following sialic acid removal
(Fig. 3), suggesting that a precursor structure may be masked by
sialic acid, the exposure of which allows for a stronger binding. This
is reminiscent of another C-type lectin, the asialoglycoprotein
receptor, which binds to the terminal galactose of glycoproteins
following the loss of sialic acid
(24, 25) .
Alternatively, the enhanced adhesion may simply be charge related,
resulting from the removal of negatively charged sialic acid moieties.
Control COS cells (not expressing Ly-49C), however, showed no increased
binding to sialidase-treated cells. Nonetheless, Ly-49C recognition is
clearly distinct from the sialic acid-dependent adhesion mediated by
the selectins
(28) .
1/
2 domain provides target cells with protection
from lysis by NK cells
(37) and is likely the region of
H-2D
recognized by Ly-49A
(17, 18) . A
recent report by Correa et al. (38) has carefully
examined the possibility that Ly-49A may recognize specific peptides
bound by D
. This work has established that Ly-49A
recognizes all class I-peptide complexes, rather than any particular
subset of them. It is clear that although recognition is not peptide
specific, it is peptide-dependent, suggesting that a conformation
consisting of an
/
chain plus peptide is recognized by
Ly-49A.
1/
2 domain. Murine class I molecules have an
additional conserved N-linked glycan at Asn-176 and no
O-linked sugars
(39) . Although the oligosaccharide
makeup of class I molecules is not well characterized, they are known
to be complex sialylated, fucosylated structures
(39, 40, 41, 42) . If Ly-49 molecules
are indeed recognizing cell surface carbohydrates, it is almost certain
that such structures are class I MHC-associated and are likely located
in the
1/
2 domain.
and D
at conserved
N-linked sites
(40) . It is therefore possible that
these oligosaccharide differences (which presumably result from
different amino acid sequences) are the basis of Ly-49 recognition. It
is also possible that Ly-49 recognizes the combination of a specific
carbohydrate and protein scaffolding, as appears to be the case for
both L- and P-selectin
(28) .
Table:
Polysaccharide inhibition of Ly-49C-mediated
adhesion
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.