©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Carbohydrate Recognition by a Natural Killer Cell Receptor, Ly-49C (*)

Jack Brennan (1) (2), Fumio Takei (1) (3), Simon Wong (4)(§), Dixie L. Mager (1) (2)(¶)

From the (1) Terry Fox Laboratory, British Columbia Cancer Agency and the Departments of (2) Medical Genetics and (3) Pathology, the University of British Columbia, Vancouver, British Columbia V5Z 1L3, Canada and the (4) Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, -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.


INTRODUCTION

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() 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) .

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 (25% identity) (8, 12, 15) .

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-2Dand 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) .

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(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.


MATERIALS AND METHODS

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 ( M500,000), 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 10cells/plate). 48 h later, these plates were used in a quantitative cell-cell adhesion assay. 5 10labeled GM979 cells (1 µCi of NaCrO/10cells) 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 CaClwas 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.

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.

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).


RESULTS

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 ( M500,000), fucoidan, and -carrageenan were found to inhibit at concentrations ranging from 5 10to 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 ( M500,000), -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.

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 ( M500,000), 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 ( M500,000), 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) .


DISCUSSION

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 (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 IDvalues 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.

Our studies have shown that -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).

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(26) . Decalcification of the purified proteins by high pH dialysis has shown that carbohydrate binding is indeed Ca-dependent, but that Cais 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.

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 IDvalue. Additionally, tunicamycin-treated target cells showed reduced adhesion to Ly-49A and increased sensitivity to Ly-49ANK 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).

Daniels et al. (27) also reported (as data not shown) that sialidase treatment of Dtarget 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) .

Ly-49A and C have both been shown to bind to class I MHC molecules (10, 16, 17) . The polymorphic 1/2 domain provides target cells with protection from lysis by NK cells (37) and is likely the region of H-2Drecognized 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.

All class I MHC molecules contain a conserved N-linked glycosylation site (Asn-86) which lies near the junction of the 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.

Class I MHC oligosaccharides are known to be heterogeneous, as displayed by the distinct carbohydrate structures of Kand Dat 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) .

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.

  
Table: Polysaccharide inhibition of Ly-49C-mediated adhesion



FOOTNOTES

*
This work was supported by a joint grant from the Cancer Research Society of Canada (to F. T. and D. M.), with core support provided by the British Columbia Cancer Agency. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: The British Columbia Institute for Child and Family Health, Division of Infectious and Immunological Diseases, 950 West 28th Ave., Vancouver, British Columbia V5Z 4H4, Canada.

To whom correspondence should be addressed: Terry Fox Laboratory, British Columbia Cancer Agency, 601 West 10th Ave., Vancouver, British Columbia V5Z 1L3, Canada. Tel.: 604-877-6070; Fax: 604-877-0712.

The abbreviations used are: NK, natural killer; CRD, carbohydrate recognition domain; MHC, major histocompatibility complex; Ab, antibody; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; HBSS, Hanks' balanced salt solution.


ACKNOWLEDGEMENTS

We thank Dr. Graeme Dougherty for the 3c12 antibody and the CD44H cDNA and Dr. Robert Kay for the vector PAX142.


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