The Functional Binding Site for the C-type Lectin–like Natural Killer Cell Receptor Ly49A Spans Three Domains of Its Major Histocompatibility Complex Class I Ligand

Naoki Matsumotoa, Motoaki Mitsukia, Kyoko Tajimaa, Wayne M. Yokoyamab, and Kazuo Yamamotoa
a Laboratory of Molecular Medicine, Department of Integrated Biosciences, The University of Tokyo Graduate School of Frontier Sciences, Tokyo 113-0033, Japan
b Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110

Correspondence to: Naoki Matsumoto, Laboratory of Molecular Medicine, Department of Integrated Biosciences, The University of Tokyo Graduate School of Frontier Sciences, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel:81-3-5841-8785 Fax:81-3-5841-8923 E-mail:nmatsu{at}k.u-tokyo.ac.jp.


  Abstract
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Natural killer (NK) cells express receptors that recognize major histocompatibility complex (MHC) class I molecules and regulate cytotoxicity of target cells. In this study, we demonstrate that Ly49A, a prototypical C-type lectin–like receptor expressed on mouse NK cells, requires species-specific determinants on ß2-microglobulin (ß2m) to recognize its mouse MHC class I ligand, H-2Dd. The involvement of ß2m in the interaction between Ly49A and H-2Dd is also demonstrated by the functional effects of a ß2m-specific antibody. We also define three residues in {alpha}1/{alpha}2 and {alpha}3 domains of H-2Dd that are critical for the recognition of H-2Dd on target cells by Ly49A. In the crystal structure of the Ly49A/H-2Dd complex, these residues are involved in hydrogen bonding to Ly49A in one of the two potential Ly49A binding sites on H-2Dd. These data unambiguously indicate that the functional effect of Ly49A as an MHC class I–specific NK cell receptor is mediated by binding to a concave region formed by three structural domains of H-2Dd, which partially overlaps the CD8 binding site.

Key Words: ß2-microglobulin, inhibitory receptor, cytotoxicity, mutation, H-2 antigens


  Introduction
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

NK cells are a population of lymphocytes with an ability to spontaneously kill tumor cells and infected cells (1). Target recognition by NK cells involves MHC class I molecules on target cells (2). NK cells express C-type lectin–like or Ig-like receptors for MHC class I molecules (3) (4). Engagement of these MHC class I receptors by its ligands inhibits or activates NK cells, depending on a motif found in the cytoplasmic region or a positively charged amino acid residue in the transmembrane segment of the receptors (3) (5) (6).

Mouse NK cells express receptors of the Ly49 family, comprised of >10 members. These molecules are homodimers of type II transmembrane proteins with C-type lectin–like domains in the extracellular region (7) (8) (9) (10) (11) (12). Ly49A, the prototype member of this family, is an inhibitory receptor specific for the mouse MHC class I molecules H-2Dd and H-2Dk (13). MHC class I is a ternary complex of a heavy chain, which consists of {alpha}1/{alpha}2 and {alpha}3 domains, ß2-microglobulin (ß2m),1 and a peptide bound to a groove in the {alpha}1/{alpha}2 domain (14). Several lines of evidence suggested the involvement of the {alpha}1/{alpha}2 domain in the recognition of H-2Dd by Ly49A. The 34-5-8S antibody, which recognizes the {alpha}1/{alpha}2 domain of H-2Dd, but not an antibody against the {alpha}3 domain (34-2-12S) inhibits functional and physical interaction between Ly49A and H-2Dd (13) (15). Ly49A recognizes the natural mutant MHC class I molecule dm-1, which has the {alpha}1 and NH2-terminal half of the {alpha}2 domain of H-2Dd with the rest of the molecule derived from H-2Ld, which is not a ligand for Ly49A (16). Ly49A recognizes only the peptide-bound form of H-2Dd molecules, but there is no apparent specificity for peptides as long as they have the anchoring residues required to bind H-2Dd (17) (18). Despite the homology of Ly49A to C-type lectins, the ability of Ly49A to bind certain carbohydrates like fucoidan or dextran sulfate (19), and the presence of two Asn-linked carbohydrates in H-2Dd, binding of Ly49A to H-2Dd does not depend on carbohydrates on H-2Dd (20). Our previous study using Dd/Kd chimeric molecules has shown that the polymorphic determinant of H-2Dd that restricts Ly49A reactivity lies in the NH2-terminal halves of {alpha}1 and {alpha}2 regions of H-2Dd that form the bottom of the {alpha}1/{alpha}2 domain (20). Recently, Tormo et al. (21) resolved the crystal structure of the Ly49A/H-2Dd complex, providing two possible Ly49A binding sites on H-2Dd, and suggested that site 1, which includes the NH2 terminus of {alpha}1 {alpha}-helix and COOH terminus of {alpha}2 {alpha}-helix, is the functional binding site for Ly49A rather than site 2, a concave region formed by {alpha}1/{alpha}2 and {alpha}3 domains, and ß2m. However, experimental data explaining which represents the functional Ly49A binding site on H-2Dd that leads to inhibition of NK cell cytotoxicity had been missing.

In this study, we first focused on ß2m, which had not been thought to be involved in the recognition of H-2Dd by Ly49A, and demonstrate the essential role of ß2m in recognition. This result led us to explore a panel of individually Ala-substituted mutants of H-2Dd, in which mutations were introduced into {alpha}1/{alpha}2 or {alpha}3 domains, to functionally interact with Ly49A. We found specific residues in the {alpha}1/{alpha}2 and {alpha}3 domains of H-2Dd that are critically important for Ly49A interaction, as determined by soluble Ly49A (sLy49A) binding and inhibition of cytotoxicity by Ly49A+ NK cells. Surprisingly, our results indicate that site 2 rather than site 1 is the functional binding site of Ly49A on H-2Dd that results in the inhibition of NK cell cytotoxicity.


  Materials and Methods
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Mice.
C57BL/6J mice were obtained from Clea. This study was approved by the Animal Experiment Review Board of the Faculty of Pharmaceutical Sciences at The University of Tokyo, Tokyo, Japan.

Cells and Antibodies.
C1498 cells, which have C57BL/6 origin with H-2b and ß2mb, and Daudi cells were obtained from American Type Culture Collection. Ly49A-transfected Chinese hamster ovary (CHO) cells and H-2Dd-, Kd-, or Ld-transfected C1498 cells were established as described (13) (19) (20). Ly49A+ IL-2–activated NK cells were prepared from C57BL/6 mouse splenocytes as described (13). S19.8 (anti–mouse ß2mb; reference (22)), BBM1 (anti–human ß2m; reference (23)), 34-5-8S (anti–H-2Dd {alpha}1/{alpha}2; reference (24)), 34-2-12S (anti–H-2Dd {alpha}3; reference (24)), and A1 (anti-Ly49A; reference (25)) were purified from culture supernatants. Fab and F(ab')2 fragment of antibodies were prepared with standard methods. Because both of the anti-ß2m antibodies are mouse IgG2b isotypes, it is difficult to make F(ab')2 fragments; Fab fragments of these antibodies were instead used in cell-mediated cytotoxicity assays to avoid antibody-dependent cellular cytotoxicity (ADCC). Daudi cells were transfected by electroporation with mouse ß2mb or human ß2m cDNA (a gift from Dr. R.K. Ribaudo, Molecular Applications Group, Silver Spring, MD) that was cloned into pApuro vector (26) together with wild-type H-2Dd cDNA cloned into pHßAPr-1neo vector, and stable transfectants were established as described (20).

Cell-mediated Cytotoxicity Assay and Cell–Cell Adhesion Assay.
Cell-mediated cytotoxicity of Ly49A+ NK cells against H-2Dd–transfected C1498 cells was tested by a 4-h 51Cr-release assay as described (20). All cytotoxicity assays were done in triplicate. When indicated, target or Ly49A+ NK cells were preincubated for 15 min with anti–MHC class I or anti-Ly49A antibodies, respectively. To prevent ADCC, anti-ß2m and anti–H-2Dd antibodies were used as Fab and F(ab')2 fragments, respectively. Intact antibodies and F(ab')2 fragments were used at 10 µg/ml and Fab fragments were used at 40 µg/ml. Binding of H-2Dd–transfected cells to Ly49A-transfected CHO cells was examined as described previously (15) (20). When indicated, target cells or Ly49A+ NK cells were preincubated for 15 min with anti–MHC class I antibodies or anti-Ly49A antibody, respectively.

Preparation of sLy49A Tetramer.
sLy49A was prepared as described elsewhere (Matsumoto, N., K. Tajima, M. Mitsuki, and K. Yamamoto, manuscript submitted for publication). In brief, the extracellular domain of Ly49A with NH2-terminal biotinylation sequence tag (27) was expressed in Escherichia coli using an efficient T7 RNA polymerase-based system (28). The recombinant protein was in vitro refolded by dilution (29) and purified by cation exchange and gel filtration column chromatography. The sLy49A was biotinylated by biotin ligase BirA (Avidity). sLy49A tetramer was formed by incubating the biotinylated sLy49A with R-PE–conjugated streptavdin (BD PharMingen) at a molar ratio of 4:1.

ß2m Replacement Studies.
H-2Dd–transfected C1498 cells were cultured for 16 h in the presence or absence of 4 µM human ß2m (purified from plasma; Calbiochem) in RPMI 1640 free from FCS at 37°C. Then the cells were used for flow cytometry or cell-mediated cytotoxicity assay. After 16 h of culture under FCS-free condition, >99% of the cells were viable.

Site-directed Mutagenesis and Stable Transfection of Cells.
Point mutations were introduced by primer extension with T4 DNA polymerase using the Altered Sites® II system (Promega) or by sequential PCR steps as described by Cormack (30). To introduce point mutation by the PCR-based technique, we introduced individual mutations into the 5' fragment of H-2Dd cDNA, which encodes a signal sequence and {alpha}1/{alpha}2 domain, or 3' fragment, which encodes the rest of H-2Dd, by PCR using the following terminal primers: 5'-CCTGCAGGTCGACTCTAGAG-3' and 5'-GTTCTTAAGAGCGTAGCATTCCCGTTC-3' for the 5' fragment and 5'-CTCTTAAGAACAGATCCCCCAAAGGC-3' and 5'-GGATCCACACCAGGCAGCTG-3' for the 3' fragment. These primers contain SalI, AflII, or BamHI site (indicated in italics). The sequence of primers used in the first PCR step will be provided on request. All the PCR reactions were performed using KOD-Plus-DNA polymerase (Toyobo). Each of the 5' or 3' fragments was subcloned into the SmaI site of pBluescript® II SK+ (Stratagene), and the sequence was confirmed by reading both strands using an LS-2000 sequencer (LI-COR). Then, each of the mutant 5' fragments and wild-type 3' fragment or wild-type 5' fragment and each of the mutant 3' fragments was directionally cloned into pHßApr-1neo vector and the constructs were used for the transfection of C1498 cells by electroporation as described (20).

Flow Cytometry.
Cells were stained with 10 µg/ml of indicated primary antibodies and then with FITC-goat anti–mouse IgG F(ab')2. For sLy49A staining, cells were stained with PE-labeled sLy49A tetramer and then fixed with 0.5% paraformaldehyde in PBS. In both cases, the stained cells were analyzed using a FACScaliburTM with CELLQuestTM software. Binding of the sLy49A tetramer to each mutant H-2Dd was calibrated, with the expression of H-2Dd detected by 34-2-12S or 34-5-8S antibodies in the following formula: sLy49A tetramer binding index = (mean fluorescence intensity [MFI] of sLy49A tetramer stained cells - MFI of streptavidin-PE stained cells)/(MFI of 34-2-12S or 34-5-8S stained cells - MFI of control antibody stained cells). sLy49A tetramer binding of each H-2Dd mutant is expressed as the relative value of the binding index when wild-type H-2Dd is adjusted to 100. Because introduction of E227A mutation into H-2Dd abrogated the 34-2-12S epitope, we evaluated the expression of E227A mutant by reactivity with the 34-5-8S antibody.


  Results
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Anti–Mouse ß2mb Antibody Inhibits Recognition of H-2Dd by Ly49A.
To investigate the possible involvement of ß2m in the recognition of H-2Dd by Ly49A, we examined the effect of the S19.8 antibody, which reacts with the b allele of mouse ß2m, on the interaction of Ly49A with H-2Dd in target cell killing assays. In these experiments, antibody fragments lacking the Fc region were used to avoid the potential effect of target lysis by ADCC mediated by NK cells through their Fc receptors. F(ab')2, or Fab for IgG2b isotypes, fragments were used. As reported previously (13), Ly49A+ LAK cells were unable to kill H-2Dd–transfected C1498 (H-2b) lymphoma cells efficiently (Fig 1 A). Importantly, the addition of S19.8 Fab fragments to the killing assay, as well as the positive control anti–H-2Dd {alpha}1/{alpha}2 antibody (34-5-8S) F(ab')2 fragments or intact anti-Ly49A antibody (A1), reversed the H-2Dd–mediated inhibition of the target cell killing by Ly49A+ LAK cells (Fig 1 A). By contrast, negative control anti–H-2Dd {alpha}3 antibody (34-2-12S) F(ab')2 fragments had no effect. We also investigated the effect of S19.8 antibody on the physical binding of H-2Dd–transfected C1498 cells to Ly49A-transfected CHO cells (Fig 1 B). Anti–mouse ß2m antibody, as well as anti–H-2Dd {alpha}1/{alpha}2 or anti-Ly49A antibody, completely abrogated the binding of H-2Dd–transfected C1498 cells to Ly49A-transfected CHO cells (Fig 1 B). These results clearly demonstrate inhibition of the functional and physical interaction between H-2Dd and Ly49A by the anti-ß2mb antibody S19.8 and suggest the possible involvement of ß2m in Ly49A binding to H-2Dd.



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Figure 1. Anti-ß2m antibody inhibits Ly49A interaction with its MHC class I ligand H-2Dd. (A) Reversal of H-2Dd/Ly49A–mediated inhibition of target cell killing by anti-ß2m antibody. Killing of H-2Dd–transfected mouse lymphoma C1498 cells by Ly49A+ NK cells in the presence of various antibodies was examined by standard 51Cr-release assay at an E/T ratio of 20:1. Anti–mouse ß2m or anti–H-2Dd antibodies were used as Fab or F(ab')2 fragments to prevent ADCC. Means ± SD of triplicate studies are shown. (B) Inhibition of H-2Dd–transfected cells binding to Ly49A-transfected cells by anti-ß2m antibody. Binding of H-2Dd–transfected C1498 cells to Ly49A-transfected CHO cells was examined in the presence of various antibodies. The antibodies were used at 10 µg/ml. Means ± SD of triplicate studies are shown.

Failure of Ly49A To Recognize H-2Dd Complexed with Human ß2m.
ß2m bound to MHC class I molecules on the cells in culture can be replaced by exogenously added ß2m (31) (32). It is conceivable that some MHC class I molecules on C1498 cells and their transfectants were associated with bovine ß2m, because those cells were maintained in the presence of FCS. The observation that anti–mouse ß2m antibody completely abrogated the recognition of H-2Dd by Ly49A (Fig 1) suggested that H-2Dd complexed with ß2m from bovine or other species might not be recognized by Ly49A. To investigate the species-specific involvement of ß2m in Ly49A recognition of H-2Dd, we used human ß2m to which a serological reagent was available. Incubation of H-2Dd–transfected mouse C1498 cells with human ß2m induced expression of human ß2m epitope detected by BBM1 antibody (Fig 2 A) and decreased expression of mouse ß2m epitope by 34% in MFI compared with H-2Dd–transfected C1498 cells cultured in the absence of human ß2m (Fig 2A and Fig B). Incubation of H-2Dd–transfected C1498 cells with human ß2m also increased expression of H-2Dd by 12.4%, consistent with the reported ability of human ß2m to stabilize surface expression of mouse MHC class I molecules (33). These data indicate that a substantial number of H-2Dd molecules were complexed with human ß2m. These cells were then tested for killing by Ly49A+ NK cells in the presence or absence of various antibodies (Fig 2 C). To evaluate the interaction of Ly49A with H-2Dd complexed with human ß2m, H-2Dd complexed with mouse ß2m was masked by anti–mouse ß2m antibody. Addition of anti–mouse ß2m antibody Fab fragments as well as the anti–H-2Dd {alpha}1/{alpha}2 antibody F(ab')2 fragments reversed the inhibition of Ly49+ NK cell–mediated killing of H-2Dd–transfected C1498 cells cultured in the presence of human ß2m (Fig 2 C). Further addition of anti–human ß2m antibody Fab fragments did not show any effect. These data imply that H-2Dd complexed with human ß2m is unable to inhibit killing by Ly49A+ LAK cells; however, a firm conclusion could not be obtained due to the incomplete exchange of human ß2m for mouse ß2m.



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Figure 2. H-2Dd complexed with human ß2m cannot protect target cell killing by Ly49A+ NK cells. H-2Dd–transfected mouse lymphoma C1498 cells were cultured in the presence (A and C) or absence of human ß2m (B and D). Then the cells were tested for binding of anti–mouse ß2m antibody S19.8 (shaded area), anti–H-2Dd {alpha}3 antibody 34-2-12S (thin line), anti–human ß2m antibody BBM1 (bold line), or control antibody (dotted line) by flow cytometry (A and B). The cells were tested for killing by Ly49A+ NK cells in the presence of various antibodies (C and D) at an E/T ratio of 20:1. Anti-ß2m or anti–H-2Dd antibodies were used as Fab or F(ab')2 fragments, respectively.

To further address this issue, we transfected ß2m defective human cell line Daudi with human or mouse ß2m together with H-2Dd heavy chain (Fig 3A and Fig B). We recently prepared sLy49A tetramer, which specifically binds H-2Dd (Matsumoto, N., K. Tajima, M. Mitsuki, and K. Yamamoto, manuscript submitted for publication). sLy49A tetramer bound Daudi cells transfected with mouse ß2m and H-2Dd (Fig 3 C) but not those transfected with human ß2m and H-2Dd (Fig 3 D), despite the equivalent level of H-2Dd expression on those cells (Fig 3A and Fig B). These results clearly demonstrate the species-specific ability of mouse ß2m to support Ly49A binding to H-2Dd and suggest the direct involvement of ß2m in the recognition of H-2Dd by Ly49A.



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Figure 3. sLy49A tetramer binds H-2Dd complexed with mouse ß2m but not with human ß2m. ß2m-defective human Daudi cells were stably transfected with mouse ß2m (A and C) or human ß2m (B and D) together with H-2Dd heavy chain. The cells were assayed for binding of anti–H-2Dd antibody 34-2-12S (A and B, bold line), control antibody (A and B, thin line), PE-labeled sLy49A tetramer (C and D, bold line), or streptavidin-PE (C and D, thin line) by flow cytometry. Staining of these cells with 34-5-8S gave similar results to 34-2-12S (data not shown).

Disruption of Ly49A Recognition of H-2Dd by the Introduction of Three Individual Mutations in {alpha}1/{alpha}2 or {alpha}3 Domains of H-2Dd.
Previous observations indicate the importance of the {alpha}1/{alpha}2 domain, especially the NH2-terminal halves of the {alpha}1 and {alpha}2 regions of H-2Dd that form the bottom of the {alpha}1/{alpha}2 domain, in Ly49A recognition of H-2Dd (16) (34). Moreover, our analysis indicates that ß2m may be directly involved in the recognition. These observations prompted us to prepare a panel of Ala substituted mutants of H-2Dd and to explore their interaction with Ly49A. Residues substituted with Ala were chosen from solvent-exposed residues in {alpha}1/{alpha}2 or {alpha}3 domain based on the crystal structure of H-2Dd (35) (36). These sites include the residues involved in hydrogen bonding between H-2Dd and Ly49A at the two putative interaction sites in the crystal structure of the Ly49A/H-2Dd complex reported by Tormo et al. during the course of this study (21).

C1498 mouse lymphoma cells were stably transfected with the mutant H-2Dd cDNA constructs. The transfectants with a similar level of H-2Dd expression, as assayed by staining with 34-2-12S or 34-5-8S antibodies, were selected for further analysis (Fig 4). Most of the H-2Dd mutants tested here were equally reactive with both 34-2-12S and 34-5-8S antibodies (data not shown). However, substitution of Glu227 in the {alpha}3 domain of H-2Dd with Ala (E227A) disrupted the epitope recognized by 34-2-12S but not of 34-5-8S as reported by Connolly et al. (37). The panel of H-2Dd mutant transfectants was then assayed for binding of the sLy49A tetramer (Fig 5). Individual Ala substitution of the residues Arg6, Asp122 in the {alpha}1/{alpha}2 domain of H-2Dd, or Lys243 in the {alpha}3 domain completely abrogated the ability of H-2Dd to bind sLy49A tetramer, whereas substitution of Arg111 partially inhibited the binding. The rest of the mutants had similar capacities to bind the sLy49A tetramer as wild-type H-2Dd. The same panel of H-2Dd mutants was also tested for the ability to protect tumor cells from killing by Ly49A+ LAK cells (Fig 6). Introduction of R6A, D122A, or K243A mutation into H-2Dd completely abrogated the protective activity of H-2Dd against killing by Ly49A+ LAK cells. None of the other mutations tested here significantly impaired the ability of H-2Dd to protect C1498 cells from killing by Ly49A+ LAK cells (Fig 6). Neither one of the H-2Dd mutants tested here nor wild-type H-2Dd protected C1498 cells from killing by Ly49A- LAK cells (data not shown). These results indicate that Arg6, Asp122, and Lys243 are essential for the physical binding of Ly49A and also for the functional binding of Ly49A that leads to inhibition of NK cell cytotoxicity. Importantly, these residues are involved in hydrogen bonding to Ly49A in one out of two binding sites in the crystal structure of the Ly49A/H-2Dd complex (Fig 7; reference (21)).



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Figure 4. Expression of H-2Dd mutants on transfected C1498 cells. Individual Ala substitution of each residue was introduced into the indicated residues of H-2Dd. Each mutant is described as original amino acid residue in one letter code followed by residue number and by A, the one letter code for Ala. C1498 cells that were untransfected or transfected with wild-type H-2Dd or individual H-2Dd mutants other than E227A mutant were stained with 34-2-12S antibody (bold lines) or control antibody (broken lines). C1498 cells transfected with H-2Dd E227A mutant were stained with 34-5-8S (bold lines) or control antibody (broken lines), because E227A mutation disrupted the 34-2-12S epitope.



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Figure 5. Binding of sLy49A tetramer to H-2Dd mutant transfectants. Each of the H-2Dd mutant transfectants was stained with sLy49A tetramer or streptavidin-PE alone. Binding of sLy49A tetramer to each H-2Dd mutant transfectant is expressed as relative value to wild-type H-2Dd transfectants as described in Materials and Methods. The mutants in site 1 and site 2 are represented by white and black bars, respectively. The other mutants and wild-type are represented by gray bars; R6A, D122A, and K243A mutations, which virtually disrupted sLy49A tetramer binding, are located in site 2. Definition for site 1 and site 2 followed that of Tormo et al. (21) and is shown in Fig 7.



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Figure 6. Inhibition of Ly49A+ LAK cell–mediated killing of tumor cells by H-2Dd mutants. Each H-2Dd mutant transfectant was assayed for killing by Ly49A+ LAK cells in the absence or presence of anti-Ly49A antibody or control antibody at an E/T ratio of 20:1. Inhibitory activity of each H-2Dd mutant is expressed as percent inhibition of target cell killing mediated by Ly49A in the formula: % inhibition of killing = (% specific cytotoxicity in the presence of the anti-Ly49A antibody A1 - % specific cytotoxicity in the absence of the antibody)/(% specific cytotoxicity in the presence of antibody 100x). Killing assay at an E/T ratio of 4:1 gave similar results (data not shown). Control antibody did not show any significant effect on killing by Ly49A+ LAK cells (data not shown). The mutants in site 1 and site 2 are represented by white and black bars, respectively. The other mutants and wild-type are represented by gray bars.



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Figure 7. H-2Dd mutations and antibody epitopes in the Ly49A/H-2Dd complex. An MHC class I molecule and Ly49A molecules are depicted as molecular surface and ribbon diagrams, respectively (the coordinates were provided by Dr. D.H. Margulies, National Institutes of Health, Bethesda, MD). The bottom view is orthogonal to the top view. The molecular surface of H-2Dd heavy chain and ß2m are yellow and pink, respectively. The residues of H-2Dd that affected Ly49A binding upon Ala substitution are red or orange and are labeled, whereas those that did not affect Ly49A binding upon Ala substitution are dark green (those in site 1) or light green (others). The residues involved in antigenic epitopes recognized by the anti–mouse ß2m antibody S19.8 and the anti–H-2Dd {alpha}1/{alpha}2 antibody 34-5-8S are blue. The glycosylation site Asn86 is magenta. The graphics image was prepared with Swiss-PdbViewer (reference (59)).


  Discussion
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Our data clearly indicate the functional Ly49A binding site on H-2Dd that is associated with inhibition of NK cell cytotoxicity. The crystal structure of the Ly49A/H-2Dd complex defined two possible binding sites for Ly49A on a single H-2Dd molecule (site 1 and site 2 in Fig 7; reference (21)). Site 2 spans the three structural domains that constitute the MHC class I molecule, {alpha}1/{alpha}2, {alpha}3, and ß2m. Several new lines of evidence indicate that site 2, rather than site 1, is the functional binding site for Ly49A. (a) 34-5-8S antibody, which recognizes the {alpha}1/{alpha}2 domain, inhibits the interaction between Ly49A and H-2Dd (Fig 1 and Fig 2; reference (13)). We recently mapped the epitope of 34-5-8S to a region containing Glu104 and Gly107 of the H-2Dd heavy chain (Matsumoto, N., W. Yokoyama, S. Kojima, and K. Yamamoto, manuscript submitted for publication), which is located in the neighborhood of site 2 (Fig 7). (b) The anti-ß2mb antibody S19.8 completely inhibited the functional and physical interaction between Ly49A and H-2Dd (Fig 1). S19.8 recognizes an epitope containing Ala85 and His34 of mouse ß2m of b allele (38) (39), which is also juxtaposed to site 2 (Fig 7). (c) To bind H-2Dd, Ly49A required a complex of H-2Dd with mouse ß2m, but not with human or bovine ß2m (Fig 1 Fig 2 Fig 3). On the other hand, H-2Dd complexed with rat ß2m can bind Ly49A as shown by Sundback et al. (40). ß2m contributes 25% of the binding surface in site 2 of the Ly49A/H-2Dd complex structure (21). Importantly, the surface of ß2m has the species-specific residues found in mouse and rat ß2m but not in human or bovine ß2m (data not shown). In particular, in the crystal structure of the Ly49A/H-2Dd complex, site 2 encompasses the side chains of Lys3, Gln29, and Lys58 of mouse ß2m. Of these, Lys3 and Gln29 are replaced with Arg and Gly, respectively, in human and bovine ß2m but are conserved in rat ß2m, suggesting significance of Lys3 and Gln29 in species-specific contribution of ß2m to the interaction between Ly49A and H-2Dd. However, we cannot exclude the possibility that the replacement in other residues in human or bovine ß2m forces H-2Dd heavy chain to have a different conformation from H-2Dd complexed with mouse or rat ß2m. Mutation analysis is in progress to identify ß2m residues that account for the species specificity. (d) Ala substituted mutation into any one of the residues of H-2Dd heavy chain, Arg6 and Asp122 in {alpha}1/{alpha}2 and Lys243 in {alpha}3, completely abrogated the physical and the functional interaction between H-2Dd and Ly49A (Fig 5 and Fig 6). Introduction of R111A mutation into {alpha}1/{alpha}2 of H-2Dd partially inhibited the ability of H-2Dd to bind Ly49A to such an extent that the effect was not evident in the functional protection assay (Fig 5 and Fig 6). Importantly, these four residues are involved in hydrogen bonding to Ly49A in site 2 in the crystal structure of the Ly49A/H-2Dd complex (Fig 7; reference (21)). Interestingly, not all of the Ala substitution of the residues that putatively disrupt hydrogen bonds between Ly49A and H-2Dd in site 2 resulted in loss in binding and function: E227A and E232A mutants of H-2Dd were fully functional in binding and protection from killing (Fig 5 and Fig 6). However, detailed examination of the crystal structure of the Ly49A/H-2Dd complex revealed that multiple hydrogen bonds are provided by each side chain of the residues of which Ala substitution completely (R6, D122, K243) or partially (R111) abolished the interaction between Ly49A and H-2Dd. By contrast, each side chain of the residues E227 and E232 forms only single hydrogen bond (data not shown). These differences could account for the observed absence in binding and functional effects of the E227A and E232A mutants. (e) Another possible Ly49A binding site on H-2Dd (site 1) was also revealed by the crystal structure of the Ly49A/H-2Dd complex (21). None of the Ala substitutions that were expected to disrupt hydrogen bonds between Ly49A and site 1 in the crystal structure showed any effect on sLy49A tetramer binding, or on the functional interaction of H-2Dd with Ly49A (Fig 5 Fig 6 Fig 7). However, our data do not exclude the possibility that Ly49A interacts with H-2Dd through site 1 so weakly that the interaction was not detectable by sLy49A tetramer staining. The weak interaction through site 1 might be associated with cis interaction between Ly49A and H-2Dd on NK cells that leads to modulation of the Ly49A receptor as observed by Kåse et al. (43).

Collectively, our current results combined with the crystal structure of the Ly49A/H-2Dd complex (21) unambiguously indicate that the functional binding site of Ly49A lies in a concave region formed by the bottom of the {alpha}1/{alpha}2 and {alpha}3 domains, and ß2m. These results also explain the previous observation that a single chain H-2Dd molecule, in which ß2m is covalently linked to H-2Dd heavy chain through a peptide spacer, fails to interact with Ly49A (41). The peptide spacer is expected to cross in the middle of site 2, thereby interfering with the binding of Ly49A to site 2. Also, the current data provide a basis for the observation that the Ly49A binding site on MHC class I is distinct from the binding site for T cell receptor, which binds the top of the {alpha}1/{alpha}2 domain (42). Similarly, the identification of the binding site for Ly49A provides an understanding of other observations.

Functional binding of Ly49A to site 2 of H-2Dd well explains the observation that binding of Ly49A to H-2Dd requires the presence of peptide in the groove of H-2Dd {alpha}1/{alpha}2 domain (17) (18). In the crystal structure of the Ly49A/H-2Dd complex, the side chains of Thr238 and Arg239 of Ly49A-1, which has a smaller contact area with H-2Dd than the other Ly49A subunit Ly49A-2 (Fig 7), are hydrogen bonded to main chain carbonyl oxygens of Tyr85 and Asn86, respectively, both of which are located in the COOH-terminal end of {alpha}1 {alpha}-helix (21). The side chain of Ser192 of Ly49A-1 is hydrogen bonded to the amide groups of Met138 and Ala139, both of which are in the NH2-terminal end of {alpha}2 {alpha}-helix. Binding of a peptide to the peptide-binding groove of H-2Dd would bring two {alpha}-helices of H-2Dd to a position where Tyr85, Asn86, Met138, and Ala139 are available for hydrogen bonding to the residues in Ly49A-1. The notion that mutations in the peptide-binding groove of H-2Dd deteriorate the interaction between H-2Dd and Ly49A is remarkable in this context (Matsumoto, N., W. Yokoyama, S. Kojima, and K. Yamamoto, manuscript submitted for publication; references (45), (46)). It is also noteworthy that the binding of Ly49C and Ly49I to H-2Kb and Kd, respectively, is restricted by the peptide bound to MHC class I (44) (47). Interestingly, position 7, which is proximal to the end of the groove, of H-2Kb–bound peptide is critically involved in the peptide specificity of Ly49C binding to H-2Kb (47). Provided that Ly49C binds to a similar site on H-2Kb as Ly49A functionally binds H-2Dd (site 2), the peptide bound to H-2Kb might affect the conformation of the end regions of the {alpha}-helices and thereby influence the binding of Ly49C.

The functional Ly49A binding site on H-2Dd is located beneath the {alpha}1/{alpha}2 domain (Fig 7), and it partially overlaps the CD8 binding site (48) (49). CD8 has a stalk region of 30–50 residues, which is highly O-glycosylated (50), and its extended structure enables the unique Ig-like domain to reach MHC class I on the opposing target cell (49). Similarly, Ly49A has a stalk region of 68 residues, which connects the C-type lectin–like domain to the transmembrane domain and has three potential N-glycosylation sites (7) (8). N-glycosylation on these sites might keep the stalk region of polypeptide in an extended conformation to enable the C-type lectin–like domain of Ly49A to reach the recognition surface beneath the {alpha}1/{alpha}2 domain of H-2Dd. It is of note that two of the three N-glycosylation sites were highly conserved among other Ly49 members (data not shown).

H-2Dd has two N-glycosylation sites at Asn86 and Asn176. Results from investigations on the role of carbohydrate moieties in recognition of H-2Dd by Ly49A were controversial (20) (51) (52). However, it was established that Ly49A does not require carbohydrates on H-2Dd to interact with H-2Dd (20) (42). Because site 2 is located in the neighborhood of the N-glycosylation site at Asn86, the carbohydrate attached to this site might influence the Ly49A binding. We modeled H-2Dd with a high mannose-type glycochain on Asn86 by transplanting that from human CD2, of which dynamic structure including the carbohydrate moiety was determined by nuclear magnetic resonance (53) (Matsumoto, N., H. Iijima, and K. Yamamoto, unpublished data). The carbohydrate in any conformation found in CD2 is well accommodated in the interface of Ly49A at site 2. Moreover, the model raises the possibility that the carbohydrate might interact with the surface of Ly49A that corresponds to the carbohydrate-recognition surface found in typical carbohydrate-binding C-type lectins (54). This could account for the finding that optimal binding of H-2Dd–expressing cells to immobilized Ly49A is compromised by a sulfation inhibitor (51). The modeling of the H-2Dd with carbohydrate moieties on Asn86 also provides an insight into the stoichiometry of Ly49A binding to H-2Dd. From the model provided by the crystal structure of the Ly49A/H-2Dd complex, one Ly49A dimer could associate with two H-2Dd molecules. However, the carbohydrate modeled on Asn86 of H-2Dd occupied the space where the H-2Dd molecule that interacts through site 1 is supposed to fill. Therefore, the stoichiometry of binding of the Ly49A dimer to the N-glycosylated H-2Dd is postulated to be one to one; when a single Ly49A dimer on NK cells binds H-2Dd on target cells via site 2, the same Ly49A molecule would not be able to interact with MHC class I on NK cells via site 1.

Ly49A distinguishes polymorphic MHC class I molecules (Matsumoto, N., K. Tajima, M. Mitsuki, and K. Yamamoto, manuscript submitted for publication; reference (44)). However, the critical residues identified in this study are conserved among mouse MHC class I molecules, including H-2Dd and Dk, which are ligands for Ly49A, and H-2Db, Kb, and Kd, which are not ligands for Ly49A (data not shown). Some of the polymorphic residues exposed on the surface of non-Ly49A ligand MHC class I that correspond to site 2 (data not shown) might determine the reactivity with Ly49A. In this context, we previously reported that NH2-terminal halves of {alpha}1 and {alpha}2 regions of H-2Dd are critically important for the recognition of H-2Dd by Ly49A by analyzing H-2Dd/Kd chimeric molecules (20). Sundback et al. (40) also reported the inability of the H-2Db {alpha}2 domain to support recognition of H-2Dd by Ly49A by exon shuffling between H-2Dd and Db. Mutational studies on H-2Dd revealed that polymorphic residues inside and outside of the peptide-binding groove affect the recognition of H-2Dd by Ly49A (45) (46) (Matsumoto, N., W. Yokoyama, S. Kojima, and K. Yamamoto, manuscript submitted for publication). Of particular importance, we recently found that the substitution of polymorphic Asn30 of H-2Dd with Asp, which is found in non-Ly49A ligands Kb and Kd, partially abolished the functional as well as physical recognition of H-2Dd by Ly49A (Matsumoto, N., W. Yokoyama, S. Kojima, and K. Yamamoto, manuscript submitted for publication). The effect of the mutations in these sites may be conformational, since these residues identified by mutational studies are not found in the interface between Ly49A and H-2Dd that was functionally identified in this study (site 2). It should also be noted that a comparison of the crystal structure of MHC class I molecules revealed variation of the relative orientation of {alpha}3 domain or ß2m to {alpha}1/{alpha}2 domain among mouse and human MHC class I molecules (36). Configuration of these domains might be critical for the interaction with Ly49A because Ly49A interacts with the surface of H-2Dd that spans the {alpha}1/{alpha}2 and {alpha}3 domains, and ß2m in site 2 (Fig 7). Therefore, the structural basis for MHC class I allele specificity of Ly49A remains to be examined in detail by site-directed mutagenesis.

The structure of the human Ig-like NK cell receptor KIR2DL2 in complex with its MHC class I ligand, HLA-Cw3, has been determined (55). Our analysis clearly demonstrated that Ly49A recognizes the surface of MHC class I that is distinct from the surface recognized by KIR2DL2. The interaction of KIR2DL2 with MHC class I is abrogated by individual amino acid substitutions of the residues in KIR2DL2 that disrupt hydrogen bonds between KIR2DLD and MHC class I (55). Similarly, the presence of hydrogen bonds critical for Ly49A/H-2Dd association was shown in our assays (Fig 5 and Fig 6). Thus, despite the structural difference and the difference in the binding sites on MHC class I, binding of the functionally similar receptors Ly49A and KIR2DL2 to MHC class I is critically mediated by hydrogen bonds and is very sensitive to individual disruption of hydrogen bonds.

The identification of the functional Ly49A binding site on H-2Dd provides a molecular basis for understanding the recognition of the MHC class I or related molecules not only by other members of the Ly49 family but also by other C-type lectin–like NK cell receptors, including HLA-E or Qa-1 recognition by CD94/NKG2A (56) and MIC-A, B, and RAE recognition by human and mouse NKG2D, respectively (57) (58). While our studies predict that other C-type lectins, such as CD94/NKG2A, may interact with MHC class I–related molecules in the same way, several findings suggest that there may be differences. CD94/NKG2A is a heterodimer of two related chains with unique C-type lectin–like domains, whereas Ly49A is a homodimer. CD94/NKG2A recognizes the nonclassical MHC class I molecule HLA-E (Qa-1 in mouse), whereas Ly49A recognizes the classical MHC class I molecules like H-2Dd and Dk. Recognition of the MHC class I ligand by CD94/NKG2A is dependent on the sequence of the MHC class I–bound peptide (59) (60), whereas Ly49A has no apparent specificity for MHC class I–bound peptide (17) (18). CD94 and NKG2A have relatively short stalk regions, 28 residues in human CD94 and 24 residues in human NKG2A, compared with Ly49A, which has a stalk region of 68 residues. One might argue that the short stalk region is not compatible with the idea that CD94/NKG2A interacts with the similar site on HLA-E as Ly49A functionally interacts with H-2Dd. However, the stalk regions with 24–28 residues are able to stretch for at least 8 nm in an extended conformation and would be capable of placing C-type lectin–like domains for CD94/NKG2A to bind the similar site on HLA-E. Biochemical as well as structural studies on the interaction between other members of the C-type lectin–like NK cell receptors and their ligands are needed to show whether the similar sites on MHC class I or its related molecules are used as receptor binding interface.

Structurally based studies such as this work together with the recently resolved crystal structures of the Ly49A/H-2Dd and the KIR2DLD/HLA-Cw3 complexes (21) (55) have unveiled the mode of recognition of MHC class I molecules by MHC class I–specific NK cell receptors of the two structurally different families. These studies are also important with respect to NK cell biology in general. The missing-self hypothesis predicts that NK cells monitor the expression of MHC class I molecules and kill the cells with aberrant expression of MHC class I associated with such events as tumorigenesis or infection (2). The structural studies suggest how NK cell receptors can sense the aberrant expression of MHC class I molecules, in addition to global loss of expression.


  Footnotes

1 Abbreviations used in this paper: ADCC, antibody-dependent cellular cytotoxicity; ß2m, ß2-microglobulin; CHO, Chinese hamster ovary; MFI, mean fluorescence intensity; sLy49A, soluble Ly49A.
N. Matsumoto and M. Mitsuki contributed equally to this work.


  Acknowledgements
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

We thank Drs. R.K. Ribaudo and M.J. Shields for their helpful discussions and reagents, Dr. D.H. Margulies for the coordinates for the Ly49A/H-2Dd complex, and Dr. H. Iijima for his critical reading of the manuscript and modeling of the Ly49A complexed with N-glycosylated H-2Dd.

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (12672107), by a grant for Research on Health Sciences focusing on Drug Innovation from the Japan Health Science Foundation (12259), and by grants from the National Institutes of Health to W.M. Yokoyama, who is an Investigator for the Howard Hughes Medical Institute.

Submitted: 11 September 2000
Revised: 28 November 2000
Accepted: 4 December 2000


  References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

  1. Trinchieri, G. 1989. Biology of natural killer cells. Adv. Immunol. 47:187-376[Medline].

  2. Karre, K., Ljunggren, H.G., Piontek, G., and Kiessling, R. 1986. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature. 319:675-678[Medline].

  3. Lanier, L.L. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359-393[Medline].

  4. Yokoyama, W.M. 1999. Natural killer cells. In Paul W.E., ed. Fundamental Immunology. Philadelphia, Lippincott-Raven Publishers, 575-603.

  5. Moretta, A., Biassoni, R., Bottino, C., Pende, D., Vitale, M., Poggi, A., Mingari, M.C., and Moretta, L. 1997. Major histocompatibility complex class I-specific receptors on human natural killer and T lymphocytes. Immunol. Rev. 155:105-117[Medline].

  6. Long, E.O., Burshtyn, D.N., Clark, W.P., Peruzzi, M., Rajagopalan, S., Rojo, S., Wagtmann, N., and Winter, C.C. 1997. Killer cell inhibitory receptors: diversity, specificity, and function. Immunol. Rev. 155:135-144[Medline].

  7. Yokoyama, W.M., Jacobs, L.B., Kanagawa, O., Shevach, E.M., and Cohen, D.I. 1989. A murine T lymphocyte antigen belongs to a supergene family of type II integral membrane proteins. J. Immunol. 143:1379-1386[Abstract/Free Full Text].

  8. Chan, P.Y., and Takei, F. 1989. Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48. J. Immunol. 142:1727-1736[Abstract/Free Full Text].

  9. Yokoyama, W.M., Kehn, P.J., Cohen, D.I., and Shevach, E.M. 1990. Chromosomal location of the Ly-49 (A1, YE1/48) multigene family. Genetic association with the NK 1.1 antigen. J. Immunol. 145:2353-2358[Abstract/Free Full Text].

  10. Wong, S., Freeman, J.D., Kelleher, C., Mager, D., and Takei, F. 1991. Ly-49 multigene family. New members of a superfamily of type II membrane proteins with lectin-like domains. J. Immunol. 147:1417-1423[Abstract/Free Full Text].

  11. Smith, H.R., Karlhofer, F.M., and Yokoyama, W.M. 1994. Ly-49 multigene family expressed by IL-2-activated NK cells. J. Immunol. 153:1068-1079[Abstract/Free Full Text].

  12. Brennan, J., Mager, D., Jefferies, W., and Takei, F. 1994. Expression of different members of the Ly-49 gene family defines distinct natural killer cell subsets and cell adhesion properties. J. Exp. Med. 180:2287-2295[Abstract].

  13. Karlhofer, F.M., Ribaudo, R.K., and Yokoyama, W.M. 1992. MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature. 358:66-70[Medline].

  14. Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L., and Wiley, D.C. 1987. Structure of the human class I histocompatibility antigen, HLA-A2. Nature. 329:506-512[Medline].

  15. Daniels, B.F., Karlhofer, F.M., Seaman, W.E., and Yokoyama, W.M. 1994. A natural killer cell receptor specific for a major histocompatibility complex class I molecule. J. Exp. Med. 180:687-692[Abstract].

  16. Karlhofer, F.M., Hunziker, R., Reichlin, A., Margulies, D.H., and Yokoyama, W.M. 1994. Host MHC class I molecules modulate in vivo expression of a NK cell receptor. J. Immunol. 153:2407-2416[Abstract/Free Full Text].

  17. Correa, I., and Raulet, D.H. 1995. Binding of diverse peptides to MHC class I molecules inhibits target cell lysis by activated natural killer cells. Immunity. 2:61-71[Medline].

  18. Orihuela, M., Margulies, D.H., and Yokoyama, W.M. 1996. The natural killer cell receptor Ly-49A recognizes a peptide-induced conformational determinant on its major histocompatibility complex class I ligand. Proc. Natl. Acad. Sci. USA. 93:11792-11797[Abstract/Free Full Text].

  19. Daniels, B.F., Nakamura, M.C., Rosen, S.D., Yokoyama, W.M., and Seaman, W.E. 1994. Ly-49A, a receptor for H-2Dd, has a functional carbohydrate recognition domain. Immunity. 1:785-792[Medline].

  20. Matsumoto, N., Ribaudo, R.K., Abastado, J.P., Margulies, D.H., and Yokoyama, W.M. 1998. The lectin-like NK cell receptor Ly-49A recognizes a carbohydrate-independent epitope on its MHC class I ligand. Immunity. 8:245-254[Medline].

  21. Tormo, J., Natarajan, K., Margulies, D.H., and Mariuzza, R.A. 1999. Crystal structure of a lectin-like natural killer cell receptor bound to its MHC class I ligand. Nature. 402:623-631[Medline].

  22. Tada, N., Kimura, S., Hatzfeld, A., and Hammerling, U. 1980. Ly-m11: the H-3 region of mouse chromosome 2 controls a new surface alloantigen. Immunogenetics. 11:441-449[Medline].

  23. Brodsky, F.M., Bodmer, W.F., and Parham, P. 1979. Characterization of a monoclonal anti-ß2-microglobulin antibody and its use in the genetic and biochemical analysis of major histocompatibility antigens. Eur. J. Immunol. 9:536-545[Medline].

  24. Ozato, K., Mayer, N.M., and Sachs, D.H. 1982. Monoclonal antibodies to mouse major histocompatibility complex antigens. Transplantation. 34:113-120[Medline].

  25. Nagasawa, R., Gross, J., Kanagawa, O., Townsend, K., Lanier, L.L., Chiller, J., and Allison, J.P. 1987. Identification of a novel T cell surface disulfide-bonded dimer distinct from the {alpha}/ß antigen receptor. J. Immunol. 138:815-824[Abstract/Free Full Text].

  26. Takata, M., Sabe, H., Hata, A., Inazu, T., Homma, Y., Nukada, T., Yamamura, H., and Kurosaki, T. 1994. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways. EMBO (Eur. Mol. Biol. Organ.) J. 13:1341-1349[Abstract].

  27. Schatz, P.J. 1993. Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Biotechnology. 11:1138-1143[Medline].

  28. Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185:60-89[Medline].

  29. Garboczi, D.N., Hung, D.T., and Wiley, D.C. 1992. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc. Natl. Acad. Sci. USA 89:3429-3433[Abstract].

  30. Cormack, B. 1997. Directed mutagenesis using the polymerase chain reaction. In Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A., Struhl K., eds. Current Protocols in Molecular Biology. New York, John Wiley & Sons, Inc, 1:8. 5.1–10.

  31. Schmidt, W., Festenstein, H., Ward, P.J., and Sanderson, A.R. 1981. Interspecies exchange of ß2-microglobulin and associated MHC and differentiation antigens. Immunogenetics. 13:483-491[Medline].

  32. Bernabeu, C., van de Rijn, M., Lerch, P.G., and Terhorst, C.P. 1984. ß2-microglobulin from serum associates with MHC class I antigens on the surface of cultured cells. Nature. 308:642-645[Medline].

  33. Pedersen, L.O., Stryhn, A., Holter, T.L., Etzerodt, M., Gerwien, J., Nissen, M.H., Thogersen, H.C., and Buus, S. 1995. The interaction of ß2-microglobulin (ß2m) with mouse class I major histocompatibility antigens and its ability to support peptide binding: a comparison of human and mouse ß2m. Eur. J. Immunol. 25:1609-1616[Medline].

  34. Karlhofer, F.M., Ribaudo, R.K., and Yokoyama, W.M. 1992. The interaction of Ly-49 with H-2Dd globally inactivates natural killer cell cytolytic activity. Trans. Assoc. Am. Physicians. 105:72-85[Medline].

  35. Achour, A., Persson, K., Harris, R.A., Sundback, J., Sentman, C.L., Lindqvist, Y., Schneider, G., and Karre, K. 1998. The crystal structure of H-2Dd MHC class I complexed with the HIV-1-derived peptide P18-I10 at 2.4 Å resolution: implications for T cell and NK cell recognition. Immunity. 9:199-208[Medline].

  36. Li, H., Natarajan, K., Malchiodi, E.L., Margulies, D.H., and Mariuzza, R.A. 1998. Three-dimensional structure of H-2Dd complexed with an immunodominant peptide from human immunodeficiency virus envelope glycoprotein 120. J. Mol. Biol. 283:179-191[Medline].

  37. Connolly, J.M., Hansen, T.H., Ingold, A.L., and Potter, T.A. 1990. Recognition by CD8 on cytotoxic T lymphocytes is ablated by several substitutions in the class I {alpha}3 domain: CD8 and the T-cell receptor recognize the same class I molecule. Proc. Natl. Acad. Sci. USA. 87:2137-2141[Abstract].

  38. Margulies, D.H., Parnes, J.R., Johnson, N.A., and Seidman, J.G. 1983. Linkage of ß2-microglobulin and ly-m11 by molecular cloning and DNA-mediated gene transfer. Proc. Natl. Acad. Sci. USA. 80:2328-2331[Abstract].

  39. Hermel, E., Robinson, P.J., She, J.X., and Lindahl, K.F. 1993. Sequence divergence of ß2m alleles of wild Mus musculus and Mus spretus implies positive selection. Immunogenetics. 38:106-116[Medline].

  40. Sundback, J., Nakamura, M.C., Waldenstrom, M., Niemi, E.C., Seaman, W.E., Ryan, J.C., and Karre, K. 1998. The {alpha}2 domain of H-2Dd restricts the allelic specificity of the murine NK cell inhibitory receptor Ly-49A. J. Immunol. 160:5971-5978[Abstract/Free Full Text].

  41. Chung, D.H., Dorfman, J., Plaksin, D., Natarajan, K., Belyakov, I.M., Hunziker, R., Berzofsky, J.A., Yokoyama, W.M., Mage, M.G., and Margulies, D.H. 1999. NK and CTL recognition of a single chain H-2Dd molecule: distinct sites of H-2Dd interact with NK and TCR. J. Immunol. 163:3699-3708[Abstract/Free Full Text].

  42. Natarajan, K., Boyd, L.F., Schuck, P., Yokoyama, W.M., Eliat, D., and Margulies, D.H. 1999. Interaction of the NK cell inhibitory receptor Ly49A with H-2Dd: identification of a site distinct from the TCR site. Immunity. 11:591-601[Medline].

  43. Kåse, A., Johansson, M.H., Olsson-Alheim, M.Y., Karre, K., and Hoglund, P. 1998. External and internal calibration of the MHC class I-specific receptor Ly49A on murine natural killer cells. J. Immunol. 161:6133-6138[Abstract/Free Full Text].

  44. Hanke, T., Takizawa, H., McMahon, C.W., Busch, D.H., Pamer, E.G., Miller, J.D., Altman, J.D., Liu, Y., Cado, D., and Lemonnier, F.A. et al. 1999. Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors. Immunity. 11:67-77[Medline].

  45. Waldenstrom, M., Sundback, J., Olsson-Alheim, M.Y., Achour, A., and Karre, K. 1998. Impaired MHC class I (H-2Dd)-mediated protection against Ly-49A+ NK cells after amino acid substitutions in the antigen binding cleft. Eur. J. Immunol. 28:2872-2881[Medline].

  46. Nakamura, M.C., Hayashi, S., Niemi, E.C., Ryan, J.C., and Seaman, W.E. 2000. Activating Ly-49D and inhibitory Ly-49A natural killer cell receptors demonstrate distinct requirements for interaction with H2-Dd. J. Exp. Med 192:447-454[Abstract/Free Full Text].

  47. Franksson, L., Sundback, J., Achour, A., Bernlind, J., Glas, R., and Karre, K. 1999. Peptide dependency and selectivity of the NK cell inhibitory receptor Ly-49C. Eur. J. Immunol. 29:2748-2758[Medline].

  48. Gao, G.F., Tormo, J., Gerth, U.C., Wyer, J.R., McMichael, A.J., Stuart, D.I., Bell, J.I., Jones, E.Y., and Jakobsen, B.K. 1997. Crystal structure of the complex between human CD8{alpha}{alpha} and HLA-A2. Nature. 387:630-634[Medline].

  49. Kern, P.S., Teng, M.K., Smolyar, A., Liu, J.H., Liu, J., Hussey, R.E., Spoerl, R., Chang, H.C., Reinherz, E.L., and Wang, J.H. 1998. Structural basis of CD8 coreceptor function revealed by crystallographic analysis of a murine CD8{alpha}{alpha} ectodomain fragment in complex with H-2Kb. Immunity. 9:519-530[Medline].

  50. Classon, B.J., Brown, M.H., Garnett, D., Somoza, C., Barclay, A.N., Willis, A.C., and Williams, A.F. 1992. The hinge region of the CD8 {alpha} chain: structure, antigenicity, and utility in expression of immunoglobulin superfamily domains. Int. Immunol. 4:215-225[Abstract].

  51. Chang, C.S., and Kane, K.P. 1998. Evidence for sulfate modification of H-2Dd on N-linked carbohydrate(s): possible involvement in Ly-49A interaction. J. Immunol. 160:4367-4374[Abstract/Free Full Text].

  52. Lian, R.H., Freeman, J.D., Mager, D.L., and Takei, F. 1998. Role of conserved glycosylation site unique to murine class I MHC in recognition by Ly-49 NK cell receptor. J. Immunol. 161:2301-2306[Abstract/Free Full Text].

  53. Wyss, D.F., Choi, J.S., Li, J., Knoppers, M.H., Willis, K.J., Arulanandam, A.R., Smolyar, A., Reinherz, E.L., and Wagner, G. 1995. Conformation and function of the N-linked glycan in the adhesion domain of human CD2. Science. 269:1273-1278[Medline].

  54. Weis, W.I., Taylor, M.E., and Drickamer, K. 1998. The C-type lectin superfamily in the immune system. Immunol. Rev. 163:19-34[Medline].

  55. Boyington, J.C., Motyka, S.A., Schuck, P., Brooks, A.G., and Sun, P.D. 2000. Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand. Nature. 405:537-543[Medline].

  56. Braud, V.M., and McMichael, A.J. 1999. Regulation of NK cell functions through interaction of the CD94/NKG2 receptors with the nonclassical class I molecule HLA-E. Curr. Top. Microbiol. Immunol. 244:85-95[Medline].

  57. Bauer, S., Groh, V., Wu, J., Steinle, A., Phillips, J.H., Lanier, L.L., and Spies, T. 1999. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science. 285:727-729[Abstract/Free Full Text].

  58. Cerwenka, A., Bakker, A.B., McClanahan, T., Wagner, J., Wu, J., Phillips, J.H., and Lanier, L.L. 2000. Retinoic acid early inducible genes define a ligand family for the activating NKG2D receptor in mice. Immunity. 12:721-727[Medline].

  59. Kraft, J.R., Vance, R.E., Pohl, J., Martin, A.M., Raulet, D.H., and Jensen, P.E. 2000. Analysis of Qa-1b peptide binding specificity and the capacity of CD94/NKG2A to discriminate between Qa-1–peptide complexes. J. Exp. Med. 192:613-624[Abstract/Free Full Text].

  60. Llano, M., Lee, N., Navarro, F., Garcia, P., Albar, J.P., Geraghty, D.E., and Lopez-Botet, M. 1998. HLA-E-bound peptides influence recognition by inhibitory and triggering CD94/NKG2 receptors: preferential response to an HLA-G-derived nonamer. Eur. J. Immunol. 28:2854-2863[Medline].