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Receptor Glycosylation Regulates Ly-49 Binding to MHC Class I ¹

Llewellyn H. Mason^2,*, Jami Willette-Brown^*, Stephen K. Anderson, W. Gregory Alvord, Richard L. Klabansky, Howard A. Young^* and John R. Ortaldo^*

^* Laboratory of Experimental Immunology, National Cancer Institute-Clinical Cancer Research, and Basic Research Program, Science Applications International Corporation-Frederick, Frederick, MD 21702; and Data Management Services, National Cancer Institute, Frederick, MD 21702

	Abstract

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Murine NK cells express the Ly-49 family of class I MHC-binding receptors that control their ability to lyse tumor or virally infected host target cells. X-ray crystallography studies have identified two predominant contact sites (sites 1 and 2) that are involved in the binding of the inhibitory receptor, Ly-49A, to H-2D^d. Ly-49G2 (inhibitory) and Ly-49D (activating) are highly homologous to Ly-49A and also recognize H-2D^d. However, the binding of Ly-49D and G₂ to H-2D^d is of lower affinity than Ly-49A. All Ly-49s contain N-glycosylation motifs; however, the importance of receptor glycosylation in Ly-49-class I interactions has not been determined. Ly-49D and G₂ contain a glycosylation motif (NTT (221–223)), absent in Ly-49A, adjacent to one of the proposed binding sites for H-2D^d (site 2). The presence of a complex carbohydrate group at this critical site could interfere with class I binding. In this study, we are able to demonstrate for the first time that Ly-49D binds H-2D^d in the presence of mouse ₂-microglobulin. We also demonstrate that glycosylation of the NTT (221–23) motif of Ly-49D inteferes with recognition of H-2D^d. Alteration of the Ly-49D-NTT (221–23) motif to abolish glycosylation at this site resulted in enhanced H-2D^d binding and receptor activation. Furthermore, glycosylation of Ly-49G2 at NTT (221–23) also reduces receptor binding to H-2D^d tetramers. Therefore, the addition of complex carbohydrates to the Ly-49 family of receptors may represent a mechanism by which NK cells regulate affinity for host class I ligands.

	Introduction

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The recognition of self MHC class I by NK cell inhibitory receptors has been proposed as a regulatory mechanism for preventing autoreactivity of NK cells (1). Down-regulation of class I on host cells following infection or transformation leads to enhanced susceptibility to lysis by activating receptors on NK cells. Inhibitory receptors function through the tyrosine phosphorylation of their immunoreceptor tyrosine-based inhibitory motifs and the subsequent recruitment of phosphatases that can terminate activation signals (2). Although the human killer Ig-related receptor and mouse Ly-49 families primarily consist of inhibitory receptors, they also contain receptors that lack immunoreceptor tyrosine-based inhibitory motifs and can activate NK cells through their association with immunoreceptor tyrosine-based activation motif-bearing adaptor molecules such as DAP12 (3, 4, 5, 6, 7, 8, 9).

Members of the Ly-49 gene family have been shown to recognize class I as ligand in several different assay systems. NK cells expressing inhibitory receptors such as Ly-49A, C/I, and G₂ do not lyse target cells bearing specific class I molecules, unless Ab to either the receptor or class I is added to block this interaction (10, 11, 12). Cells expressing Ly-49 proteins can bind to target cells expressing the appropriate class I molecule (13), and NK cells can lyse Con A blasts from mice expressing certain H-2 haplotypes (11). Cells transfected with specific Ly-49 receptors can bind to tetrameric proteins consisting of class I, ₂-microglobulin (₂m), ³ and peptide (14). The rejection of bone marrow allografts is also mediated by NK cells, and controlled by host Ly-49 phenotype and class I expression of the donor marrow (15, 16). Studies using class I tetramers demonstrate that the ability of Ly-49 receptors to bind class I does not depend on the presence of carbohydrate on the class I molecules (14). The crystal structure of Ly-49A bound to H-2D^d has identified two important binding sites involved in this interaction, sites 1 and 2 (17). However, other studies suggest that ₂m plays an important role in the interaction of these molecules (18, 19).

Of the Ly-49 receptors studied to date, there is strong evidence suggesting that Ly-49A, G₂, and D recognize H-2D^d, and that the rank order of binding appears to be A>G₂>D (10, 12, 13, 14). Functional, cell-cell binding and tetramer-binding assays are all available to support this hierarchy of binding. It must be noted, however, that Ly-49D has only been shown to recognize H-2D^d in functional assays, and is proposed to have a very low affinity for this class I protein (14). There is also a high degree of homology among Ly-49A, G₂, and D in their extracellular domains (>85%) that suggest these receptors share similar contact sites with H-2D^d. Margulies and colleagues (20) have suggested that Ly-49G₂ is likely to have the same binding sites for H-2D^d as Ly-49A. Most of the well-characterized Ly-49 receptors in B6 mice share common glycosylation motifs in the extracellular stalk region at amino acids NCS (86–88) and NKS (103–105). However, Ly-49D and G₂ share an additional glycosylation motif at NTT (221–23) that would be juxtaposed to a region of the Ly-49A binding site 2 (RKYNIRD (223–29)), as demonstrated by Tormo et al. (17). We hypothesized that if the NTT (221–23) glycosylation motifs of Ly-49D and G₂ were indeed glycosylated, the presence of a large carbohydrate group may account for the lower affinity of binding that these receptors display for H-2D^d, primarily due to steric hindrance. We systematically determined which of the various glycosylation motifs of Ly-49D were used, and generated point mutations of Ly-49D, G₂, and A to determine the ability of receptor glycosylation to affect recognition of the class I ligand, H-2D^d.

	Materials and Methods

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Plasmids/constructs

Point mutations were introduced into Ly-49 cDNAs using the Stratagene (La Jolla, CA) Quick Change site-directed mutagenesis kit to remove the carboxyl-terminal N-glycosylation site (NTT^221–223) from Ly-49D and Ly-49G2 and introduce one into Ly-49A (NTR^221–223). A variety of different cDNA constructs was made for Ly-49D with the following substitutions: Ly-49D^N221R, Ly-49D^T223R, Ly-49D^N221A, Ly-49D^N86A, Ly-49D^N103A, Ly-49D^N169A, and Ly-49D^N221Q. The following Ly-49G2 constructs were also made: NTT^T223R and NTT^N221Q. Ly-49G2^T223R confers complete homology to the Ly-49A^208–241 sequence in addition to disrupting the glycosylation site. We also constructed the following cDNAs for Ly-49A: NTR^TR222–23ST, NTR^TR222–23RT, NTR^R223M, and NTR^{NTR221–23QTT}. Each mutation generated was sequenced and cloned into mammalian expression vectors using the EF1 promoter.

Murine cDNA for DAP12 was obtained from the American Type Culture Collection (Manassas, VA), GenBank accession AA098506. The chimeric molecule of murine DAP12/ (DZ) was made by fusion of the extracellular and transmembrane regions of murine DAP12 (aa 1–67) to the intracellular region of murine TCR- (aa 52–143) in pSVL2 (a gift from J.-P. Kinet (Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA)). The amino acid sequence of the junction is LAVYSLGRAKFSRS. A truncated DAP12 was engineered by introduction of an XbaI site at the 3' end of the transmembrane region to which a PCR-generated tail was ligated. The -tail fragment was amplified from murine cDNA with the forward primer 5'-CTCTAGAGCAAAATTCAGCAGGAG-3' and the reverse primer 5'-GCTCTAGACTTTGTCCTCCTGTCCTTCAC-3', both containing an XbaI restriction site. The complete chimera was cloned into the pEF-Bos expression vector (a kind gift of G. Koretzky, University of Pennsylvania, Philadelphia, PA).

Transfection and receptor analysis

The 293T cells were transfected using Fugene 6 (Roche Biochemicals, Indianapolis, IN) with either 1 µg of cDNA for Ly-49A, Ly-49G2, or 1 µg of the combined cDNAs for Ly-49D and DAP12. Green fluorescence protein expression plasmid was cotransfected to monitor transfection efficiency. After overnight incubation at 37°C, cells were analyzed by flow cytometry for receptor level using the following mAb: 4D11/Ly-49G2, 4E5/Ly-49D, YE148, or 12A8/Ly-49A. Surface proteins were biotinylated with biotin-7-normal human serum, according to manufacturer’s protocol (Boehringer Mannheim, Indianapolis, IN), subjected to SDS-PAGE, and blotted using HRP-labeled streptavidin. Biotinylated receptors were visualized using chemiluminescence.

Class I binding of glycosylation mutants (Ly-49D/A/G₂ at 37°C)

MHC tetramer-binding assays were performed by incubating Ly-49-transfected 293T cells with MHC class I PE tetramers at 37°C for 30 min in FACS buffer (0.5% BSA and 0.2% sodium azide in PBS). Cells were washed and analyzed by flow cytometry on a FACScan (BD PharMingen, San Diego, CA). MHC tetramers were prepared containing the following peptides: H-2D^d (RGPGRAFVTI) and H-2D^b (KAVYNFATC), and complexed with murine ₂m by the National Institute of Allergy and Infectious Diseases MHC Tetramer Core Facility at Emory University (Atlanta, GA). In experiments with swainsonine (SW)-treated cells, SW was added to cultures (0.5 µg/ml) 24 h before transfection and maintained after the transfection.

IL-2 production from Jurkat

Jurkat cells were cotransfected with 20 µg of Ly-49D cDNA and accessory chain DAP12/ by electroporation, incubated overnight, then washed and assayed for receptor expression by flow cytometry. Transfected cells were plated in triplicate at 10⁶ cells/ml in 48-well plates with PMA (10 ng/ml) for 24 h before analyzing supernatants for human IL-2 by ELISA (R&D Systems, Minneapolis, MN). Ly-49-expressing Jurkat cells were cocultured alone or with YB/H-2D^b (YBD^b) or YB/H-2D^d (YBD^d) stably transfected target cells at a 3:1 ratio. Blocking experiments were performed by inclusion of 2 µg/ml anti-H-2D^d mAb clone 34.5.8S or mAb clone 24.2.12. Ab cross-linking was performed by culturing cells with 2 µg/ml of anti-Ly-49 Ab (4E5) or rat IgG control Ab in 48-well plates that had been precoated with F(ab')₂ anti-rat.

RNase protection assay

The Multiprobe RNase protection assay was performed according to the manufacturer’s directions (BD PharMingen, San Diego, CA) with the following modifications: 1) Hybridization. Probes were synthesized with ³³P UTP (70–80 µC/full reaction) using the BD PharMingen In Vitro Transcription Kit. Following incubation, yeast tRNA and EDTA were added, as described by the manufacturer (BD PharMingen); the reaction was placed on G25 Microspin columns; and the probe was purified by centrifugation for 2 min at 3000 rpm. A total of 0.5–1.0 x 10⁶ cpm was added to each RNA in a final hybridization volume of 10–20 µl (at least 50% BD PharMingen hybridization buffer). 2) RNase inactivation A master cocktail, containing 200 µl Ambion RNase inactivation/precipitation reagent III (Ambion, Austin, TX), 50 µl ethanol, 5 µg yeast tRNA, and 1 µl Ambion GycoBlue coprecipitate per RNA sample, was used to precipitate the protected RNA. After adding the individual RNase-treated samples to 250 µl of the inactivation/precipitation cocktail, the samples were mixed well, placed at -70°C for 15 min, and subjected to centrifugation at 14,000 rpm for 15 min in a room temperature microcentrifuge. The supernatants were decanted; a sterile cotton swab was used to remove excess liquid; and the pellet was resuspended in 3 µl of BD PharMingen sample buffer.

Statistical analysis

Tetramer-binding data were normalized according to the level of receptor expressed by each transfectant, and the data were presented as a ratio of the tetramer binding (mean fluorescence intensity (MFI)) relative to receptor expression (MFI) for each transfectant. To control for experiment-to-experiment variation, Ly-49A, D, and G mutant ratios were compared with their respective wild-type (WT) ratios performed under the same experimental conditions. We computed paired analyses, (parametric) t tests, and (nonparametric) Wilcoxon Signed Ranks tests. For Ly-49D receptors, we hypothesized, a priori, that D^NTR and D^QTT binding would be higher than those for the WT receptor, D^NTT. Additionally, for Ly-49G receptors, we hypothesized, a priori, that G^NTR and G^QTT responses would be higher than those for the WT receptor, G^NTT. For the Ly-49A receptors, we predicted that ratio measures for the WT receptor, A^NTR, would be higher than those for the mutant receptors A^NTM, A^NRT, A^NST, and A^QTT. As all of our hypotheses were one-directional and because interpretations as to statistical significance were identical for parametric and nonparametric tests, we report probabilities from one-tailed, paired t tests.

	Results

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Glycosylation motifs of Ly-49D, A, and G₂

Fig. 1A is a schematic display of Ly-49D, A, and G₂ and depicts the cytoplasmic, transmembrane, stalk, and CRD (carbohydrate recognition domain) domains, along with possible N-linked glycosylation motifs (NXS/T). Most Ly-49 receptors contain two conserved glycosylation motifs that are in the stalk region, NCS^86–88 and NKS^103–05. It is evident from this diagram that Ly-49D contains two additional glycosylation motifs (NSS^169–71 and NTT^221–23), while Ly-49G2 shares one additional glycosylation motif (NTT^221–23) with Ly-49D in the CRD. The CRD domain has been shown to contain the binding sites for interactions with class I ligands (17). Our attention was drawn to the fact that both Ly-49D and G₂ contained a similar glycosylation motif NTT^221–23, not shared by Ly-49A, and which is immediately adjacent to the proposed binding site 2 of Ly-49A for H-2D^d (17). Addition of a carbohydrate to a single N-linked glycosylation site can add substantially to the molecular mass of a protein (up to 5 kDa). We speculated that if these motifs were indeed glycosylated, they could physically interfere with the binding of Ly-49D and G₂ to H-2D^d.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. A, Ly-49A, D, and G2 glycosylation motifs. This figure presents a schematic diagram of the potential glycosylation sites (NXS/T) found in the extracellular domains of Ly-49A, D, and G2. The consensus sequence is taken from Tormo et al. (17 ). B, 293T cells were either untreated or treated with 0.5 µg/ml SW for 24 h before transfection with 1 µg Ly-49D plus DAP12 cDNA. H-2Dd tetramer-binding assays were performed on the transfectants after incubation for an additional 18 h (see Fig. 5). Ly-49A binding to H-2Dd is presented as a positive control. One of two representative experiments is shown.

Site-specific mutations of Ly-49D glycosylation motifs

We next prepared individual Ly-49D mutants at each glycosylation motif (NCS^86–89, NKS^103–05, NSS^169–71, and NTT^221–23) in which asparagine was replaced with alanine. After transfection of 293T cells, surface biotinylation, and immunoprecipitation, we compared the molecular mass of each mutant with Ly-49D^wt. As can be seen in Fig. 2, site-directed mutagenesis of the four sites suggested that three of the four motifs are glycosylated. The most amino-terminal NCS^86–89 motif common to all Ly-49 receptors does not change in molecular mass upon abolishing this motif and is probably not glycosylated. This motif contains a cysteine and may therefore be involved in disulfide bond formation. However, NKS^103–05, NSS^169–71, and NTT^221–23 appear to be glycosylated as mutation of these motifs results in reduction in the molecular mass of Ly-49D. Because glycosylation of the NTT^221–23 motif of Ly-49D was evident, we prepared additional mutants to address the importance of glycosylation in Ly-49 binding to class I.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Molecular mass of Ly-49D glycosylation mutants. Ly-49Dwt and the following mutants, NCSN86A, NKSN103A, NSSN169A, and NTTN221A, were transfected into 293T cells, surface biotinylated, and immunoprecipitated with mAb 4E5. SDS-PAGE, transfer to Immobilon, and blotting with streptavidin-HRP were performed. The relative molecule mass of each mutant was compared with Ly-49Dwt receptor.

To specifically address the role of the NTT^221–223 glycosylation motif of Ly-49D and G₂, as well as the nonglycosylated Ly-49A motif NTR^221–23 in their binding to H-2D^d, we created a series of point mutations to disrupt these motifs. Several site-specific mutations were made that abolished the glycosylation motif NTT^221–223 of Ly-49D, while a single mutant was prepared for Ly-49G2. We also generated a mutant that added a glycosylation motif to Ly-49A (NST^221–23) and examined binding to H-2D^d. Fig. 3 depicts the Ly-49A, D, and G₂ WT sequences for aa 200–255 and highlights the point mutations made for each of the receptors.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Site-directed mutagenesis of Ly-49 glycosylation motifs. The consensus sequences of Ly-49A, D, and G2 are shown for aa 200–255 of the CRD. The bold bars denote site 2 contacts of Ly-49A for H-2Dd, as identified by Tormo et al. (17 ). The Ly-49 mutants generated for this study are listed, and the individual amino acid substitutions made are in boldface.

View larger version (65K): [in this window] [in a new window]   FIGURE 4. Alteration of glycosylation site changes molecular mass of the Ly-49 receptors. The 293T cells were transfected with the Ly-49A, D, or G2wt, or the indicated mutant; incubated for 18 h at 37°C; surface biotinylated; immunoprecipitated; applied to SDS-PAGE; and transferred to Imobilon. Observed molecular masses are indicated in kDa at the bottom of each lane.

The resulting glycosylation-deficient mutants of Ly-49D and G₂, as well as the glycosylated mutant of Ly-49A were initially tested for their ability to bind to class I tetramers consisting of class I protein, human ₂m, and peptide. However, in light of recent reports that there are differences observed between using human ₂m and mouse ₂m in the binding of Ly-49 receptors (18, 19), the data presented in this work used tetramers prepared with mouse ₂m. In our experiments, the differences between the human and mouse forms of ₂m in the tetramers were primarily quantitative when assaying Ly-49A-, D-, and G₂-transfected 293T cells (data not shown).

Fig. 5 demonstrates that all Ly-49D mutants were expressed at similar levels upon transfection of 293T cells. Recognition by mAb 4E5 also suggests that there were no major conformational changes of Ly-49D upon mutation of the receptor. The transfected 293T cells were tested for their ability to bind H-2D^b (control) and H-2D^d tetramers composed of mouse ₂m and peptide at 37°C. Our H-2D^d tetramer-binding results demonstrated that a significant percentage of Ly-49D^wt-transfected cells (32%) bound H-2D^d, while there was no binding to H-2D^b. This is the first report demonstrating that Ly-49D can directly bind to the class I ligand H-2D^d. In addition, we observed an increased binding of Ly-49D to H-2D^d with all of our glycosylation-deficient mutants. Our results demonstrated the order of Ly-49D binding to H-2D^d tetramers was as follows: Ly-49D^T223R>Ly-49D^N221R>Ly-49D^N221A>Ly-49D^wt. These results support our hypothesis that glycosylation of the Ly-49D receptor interferes with binding to H-2D^d, because even the most conserved glycosylation mutant, Ly-49D^N221A, increased binding to H-2D^d. Upon close examination of the tetramer-binding profiles of these Ly-49D mutants, it was apparent that the arginine at position NTT^T223R may be important in conferring Ly-49D binding to H-2D^d, as this is the identical residue found in Ly-49A^wt at this location. Even when arginine is transfered to position 221 of Ly-49D (RTT²²¹), binding to H-2D^d is increased over that of the more conservative mutation, Ly-49D^N221A. Not only did the Ly-49D^N221R and Ly-49D^T223R arginine mutants increase Ly-49D binding to H-2D^d, we also observed an increase in binding to H-2D^b. Therefore, the arginine at position 223 of Ly-49A may be important in binding class I molecules other than H-2D^d, because overexpression of Ly-49A in the presence of mouse ₂m results in strong binding to H-2D^b, as has been previously described (19, 21).

View larger version (49K): [in this window] [in a new window]   FIGURE 5. Increased H-2Dd tetramer binding of Ly-49D glycosylation mutants. The 293T cells were transfected with either Ly-49Awt as a positive control, Ly-49Dwt, or the glycosylation mutants of Ly-49D. After overnight incubation, the transfected cells were examined for receptor expression (mAb4E5/Ly-49D or YE148/Ly-49A) and binding to the indicated class I tetramers (filled histograms) as compared with mock-transfected controls (gray histograms). Tetramer binding was performed at 37°C and used mouse 2m. Numbers in histograms represent percentage of postive cells. One representative experiment of five performed is shown.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Altered tetramer binding of Ly-49A, G2, and D glycosylation mutants. Ly-49A, G2, and D WT and mutant receptors were transfected into 293T cells and, after 18 h at 37°C, analyzed for binding to H-2Dd tetramers (WT, thick lines; mutant receptor, thin lines) as compared with mock-transfected controls (gray histograms). The percentage of positive cells binding to the tetramers and mean channel intensity (MCI) are presented. One of six representative experiments is shown.

Our original observation that glycosylation of the NTT^221–23 motif of Ly-49G2 and D might lower the affinity of these receptors for H-2D^d prompted us to change this motif to the corresponding sequence of the Ly-49A receptor, NTR^221–23. However, we were concerned that the arginine at position 223 of Ly-49A^wt might play a role in binding H-2D^d because of its positive charge. Furthermore, the presence of an arginine was maintained in most of our glycosylation-deficient mutants. Therefore, we generated additional Ly-49 mutations, somewhat more conservative in nature, eliminating the arginine from this motif. Additional tetramer-binding experiments were performed with these Ly-49 mutations.

Fig. 7 displays the tetramer-binding data, expressed as a ratio of the MFI of tetramer binding to the MFI of receptor binding, from nine individual experiments comparing various mutations of the Ly-49A, G₂, and D receptors. Interestingly, the data shown in Fig. 7A demonstrate that the arginine at Ly-49A^R223 may be important for binding to H-2D^d, depending upon the amino acid substitution. The Ly-49A^NTM mutant (no arginine) demonstrated significantly less binding than WT receptor (p = 0.034). However, statistical analysis of the Ly-49A^QTT mutant, also arginine deficient, suggested no significant difference between the mutant and the WT receptor (p = 0.081). Upon examination of the Ly-49A glycosylation mutants NST^221–23 and NRT^221–23, binding to H-2D^d was significantly, although not dramatically, decreased in each case (p = 0.044 and p = 0.034, respectively). These results suggest that introduction of glycosylation into the 221–23 motif of Ly-49A interferes with binding to H-2D^d.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. Effects of arginine (223) and glycosylation on Ly-49 binding to H-2Dd tetramers. A, Mutations of Ly-49ANTR221–23 (NST, NRT, NTM, and QTT), and B, Ly-49G2 and DNTT221–23 (NTR and QTT) were generated, confirmed by sequencing, and transfected into 293T cells, as described above. Both Ly-49 WT and transfectants were tested for binding to H-2Dd tetramers at 37°C in the presence of mouse 2m, as described. Data are presented for nine individual experiments by calculating the ratio of tetramer binding (MFI) to the amount of receptor (MFI) expressed by each transfectant. Statistical analysis of one-tailed tests demonstrating that Ly-49Awt-binding ratios were higher than mutant ratios was as follows: ANTR vs ANTM p = 0.034, ANTR vs ANRT p = 0.034, ANTR vs ANST p = 0.044, and ANTR vs AQTT p = 0.081. Statistical analysis of one-tailed tests demonstrating that Ly-49G and Ly-49D mutant ratios were higher than Ly-49Gwt and Ly-49Dwt was as follows: GNTT vs GNTR p = 0.0012, GNTT vs GQTT p = 0.0186, DNTT vs DNTR p = 0.0001, and DNTT vs DQTT p = 0.0002.

RNase protection assay of IL-2 message in Ly-49D-transfected Jurkat cells

Although Ly-49 receptor expression in transfected 293T cells conferred specific binding of class I tetramers, it did not prove that these molecules maintain signal-transducing functions. Therefore, Jurkat cells were transfected with Ly-49D^wt or the glycosylation-deficient mutants; incubated with the class I-transfected YB20 cells, YBD^d or YBD^b, for 5 h; and IL-2 mRNA analyzed by RNase protection. Fig. 8 shows that when Jurkat cells are transfected with the Ly-49D^N221A and Ly-49D^T223R glycosylation mutants, IL-2 mRNA is induced following incubation with YBD^d targets as compared with YBD^b targets. Jurkat cells transfected with Ly-49D^wt displayed little, if any, IL-2 mRNA in the presence of H-2D^d targets after adjusting for control GAPDH mRNA. Interestingly, the Ly-49D^N221A mutant showed greater induction of IL-2 mRNA than the Ly-49D^T223R mutant (17- and 7-fold, respectively). These results suggest that our glycosylation-deficient Ly-49D mutants not only recognized H-2D^d with higher affinity than Ly-49D^wt, but that this recognition could be translated into up-regulation of effector cell function, in this case induction of IL-2 mRNA.

View larger version (26K): [in this window] [in a new window]   FIGURE 8. Increased IL-2 mRNA induced by Ly-49D glycosylation mutants in response to H-2Dd. Jurkat cells were transfected with Ly-49Dwt cDNA (lanes 1 and 2), Ly-49DN221A (lanes 3 and 4), or Ly-49DT223R (lanes 5 and 6) plus DAP12/, and incubated at 37°C for 18 h. The transfectants were cocultured with YBDb targets (lanes 1, 3, and 5) or YBDd (lanes 2, 4, and 6) for an additional 5 h in the presence of 10 ng/ml PMA. RNase protection assay extracts were prepared and analyzed by RPA for human IL-2. Values were normalized for GAPDH expression and expressed as fold increase over stimulation in response to YBDb targets. Ly-49D expression on transfected Jurkat cells averaged between 20 and 27% for WT and mutant receptors. A representative experiment is shown of two such experiments performed.

To determine whether induction of IL-2 mRNA in our Ly-49D mutants could lead to secretion of IL-2 protein, Jurkat cells were transiently transfected with Ly-49D^wt and the glycosylation mutants, cross-linked with mAb 4E5 or control mAb, and incubated for 24 h in the presence of 10 ng/ml PMA to act as a costimulus. As can be seen in Fig. 9, cross-linking of surface Ly-49D on the mutants as well as the Ly-49D^wt resulted in the secretion of high levels of IL-2. Transfection of Jurkat with either Ly-49D^wt or mutant receptors resulted in 20–25% receptor expression (data not shown). These results confirmed that all of the Ly-49D constructs were capable of triggering the Jurkat cells to secrete IL-2 upon receptor cross-linking. We next determined whether these receptors could trigger IL-2 secretion in the presence of target cells expressing the specific class I ligand, H-2D^d.

View larger version (41K): [in this window] [in a new window]   FIGURE 9. IL-2 secretion by Ly-49D glycosylation mutants upon receptor cross-linking. Jurkat cells were transfected with cDNAs of Ly-49Dwt or the indicated glycosylation mutants along with DAP12/. After 18 h, the transfectants were cross-linked with either mAb 4E5 or control rat IgG2a in plates coated with F(ab')2 anti-rat Ab in the presence of 10 ng/ml PMA. After 24 h, supernatants were harvested and assayed for human IL-2 by ELISA. Ly-49D receptor expression was 20–25% for all transfectants. Data from one of six comparable experiments are shown.

Assay conditions were used to mimic effector-target cell interactions, and test the ability of our Ly-49D mutants to recognize the appropriate class I ligand in a functionally relevant model. Jurkat cells transfected with the Ly-49D^wt receptor or the glycosylation mutants were combined with the target cells YBD^b or YBD^d and incubated for 24 h, and supernatants were assayed for IL-2. Fig. 10A demonstrates that Ly-49D-transfected Jurkat cells can be stimulated to secrete IL-2 in the presence of H-2D^d targets, but not H-2D^b targets, confirming the ability of Ly-49D to recognize H-2D^d. These results confirm previous functional data indicating that Ly-49D recognizes H-2D^d as a ligand (22, 23, 24). Furthermore, these results demonstrate that both the conservative Ly-49D^N221A and Ly-49D^T223R glycosylation mutants secrete significantly higher levels of IL-2 than Ly-49D^wt in response to YBD^d, but not YBD^b, targets. This enhanced IL-2 secretion can be blocked by addition of mAb to H-2D^d, as can be seen in Fig. 10B. IL-2 secretion from WT Ly-49D-transfected Jurkat cells and all three glycosylation-deficient Ly-49D mutants was greatly reduced in the presence of mAb 34.5.8S (1–2 specific), but not mAb 24.2.12 (3 specific; data not shown). These results demonstrate the specificity of the Ly-49D/H-2D^d interactions. The results of our data confirm that Ly-49D recognizes H-2D^d, and that glycosylation of the NTT^221–23 motif of this receptor lowers the affinity of this recognition.

View larger version (9K): [in this window] [in a new window]   FIGURE 10. IL-2 secretion by Ly-49D glycosylation mutants in response to H-2Dd targets. Jurkat cells were transfected with Ly-49Dwt or the glycosylation mutants plus DAP12/ and cultured alone or with YBDb or YBDd targets for 24 h. Supernatants were collected and assayed for human IL-2 by ELISA. A, Ly-49D-transfected Jurkat cells plus YB targets in the absence of blocking Ab. B, Ly-49D-transfected Jurkat cells plus YBDd targets with or without blocking mAb to H-2Dd (34.5.8S-1,2 specific). Ly-49D expression was equivalent for both WT and mutant transfectants. The data are representative of nine such experiments performed.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We prepared a series of Ly-49D constructs disrupting each of the four possible glycosylation motifs of this receptor. These constructs were analyzed for their ability to bind class I tetramers in transfected 293T cells and trigger IL-2 secretion from transfected Jurkat cells in the presence of H-2D^d target cells. Our results led us to the following conclusions: 1) Three of four glycosylation motifs of Ly-49D are used, with only the N-terminal NCS^86–89 motif apparently nonglycosylated. 2) For the first time, we demonstrate Ly-49D^wt binding to H-2D^d tetramers at 37°C in the presence of mouse ₂m. 3) Glycosylation of Ly-49D at NTT^221–23 decreases the affinity of this receptor for H-2D^d. Disruption of this Ly-49D glycosylation motif increases both H-D^d binding and Ly-49D-mediated activation in the presence of H-2D^d targets. 4) Glycosylation of Ly-49G2 at the NTT^221–23 motif also reduces receptor binding for H-2D^d. 5) Together these results suggest that the proposed binding site 2 between Ly-49A and H-2D^d may also represent a contact site for Ly-49G₂ and D with this class I molecule.

Our findings that glycosylation of Ly-49 receptors can regulate the affinity of receptor/ligand binding are novel for NK cells and their cognate class I ligands, but have been implicated in the regulation of T cells. Carbohydrate residues on the TCRs from MgatV null mice are deficient in complex N-glycans, resulting in hyperproliferation in response to CD3 stimulation and enhanced TCR clustering (28). Moody et al. (29) have demonstrated that increased glycosylation of the CD8 coreceptors modulates ligand binding as thymocytes develop from the immature CD4⁺8⁺ stage to mature CD4^-8⁺ cells, with a reduced affinity for MHC I. Furthermore, Lowe (30) has suggested that galectin binding to the lactosamine chain of B1–6-branched N-glycans of the TCR restrains the lateral mobility of these receptors, reducing clustering and raising the threshold required for TCR complex clustering.

NK cells may therefore share similar characteristics with T cells regarding carbohydrate modification of their receptors. Although glycosylation of NK cell receptors may not account for all of the ligand specificity displayed by these receptors, they may function to control the affinity, and therefore the threshold levels required for activation. Considering that the complexity and levels of receptor glycosylation are considered to be cell type, tissue, and activation dependent, NK cell receptors may be differentially glycosylated depending on their state of activation. NK cells are activated by a variety of cytokines, including IL-2, IL-12, IL-15, and IL-18, along with IFN-. It is not known how these individual cytokines, or combinations thereof, affect NK cell receptor glycosylation in vivo. Receptor glycosylation may be of particular importance in regulating activating Ly-49 receptors that have the potential to interact with host class I ligands such as Ly-49D, L, P, R, U, H, and W (31, 32, 33, 34). All of these receptors contain additional N-glycosylation motifs outside of the conserved NKS^103–05 motif common to most Ly-49 proteins. It would be logical for these activating receptors to have a lower affinity for class I than their Ly-49 inhibitory counterparts, because the consequence of self recognition would be more severe. The results of our data suggest that Ly-49 class I recognition may not only be controlled by the level of expression of these receptors, but by inclusion of glycosylation sites that can modify receptor/ligand interactions.

	Footnotes

 
¹ This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract N01-CO-12400. The publisher or recipient acknowledges right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. The content of this publication does not necessarily reflect the views or policies of Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

² Address correspondence and reprint requests to Dr. Llewellyn H. Mason, Laboratory of Expriemental Immunology, Division of Basic Sciences, National Cancer Institute-Clinical Cancer Research, National Instutites of Health, Building 560, Room 31-93, Frederick, MD 21702-1201. E-mail address: MasonL{at}mail.ncifcrf.gov

³ Abbreviations used in this paper: ₂m, ₂-microglobulin; CRD, carbohydrate recognition domain; MFI, mean fluorescence intensity; SW, swainsonine; WT, wild type.

Received for publication May 9, 2002. Accepted for publication August 18, 2003.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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