A species-specific determinant on ß2-microglobulin required for Ly49A recognition of its MHC class I ligand
Motoaki Mitsuki1,2,
Naoki Matsumoto1 and
Kazuo Yamamoto1
1 Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan 2 Glyco-chain Functions Laboratory, Supra-biomolecular System Group, RIKEN Frontier Research System, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
Correspondence to: N. Matsumoto; E-mail: nmatsu{at}k.u-tokyo.ac.jp
Transmitting editor: W. M. Yokoyama
 |
Abstract
|
---|
The mouse inhibitory NK cell receptor Ly49A recognizes the mouse MHC class I molecule H-2Dk. The present study focuses on the species specificity of ß2-microglobulin (ß2m), an invariant component of MHC class I, in the interaction between Ly49A and H-2Dk. Transfection of the ß2m-defective mouse cell line R1E/TL8x.1 with human (h) ß2m induced cell-surface expression of H-2Dk, but failed to protect the cells from killing by Ly49A+ NK cells. In contrast, the cells transfected with mouse (m) ß2m were protected from killing by Ly49A+ NK cells. These data indicate that Ly49A distinguishes mß2m from hß2m when it recognizes the H-2Dk complexes. To identify the species-specific determinant of ß2m required for Ly49A recognition of H-2Dk, we prepared a panel of mß2m mutants and tested the H-2Dk that included each of the ß2m mutants for its capacity to engage Ly49A on NK cells. Ly49A failed to functionally recognize the H-2Dk that included the mß2m with K3R and Q29G mutations. Moreover, Ly49A was able to recognize the H-2Dk that included the hß2m with R3K and G29Q mutations. These data indicate that Lys3 and Gln29 consist of the central part of the species-specific determinant of ß2m required for Ly49A recognition of H-2Dk. The two residues are conserved in the mouse and the rat, in which NK cells use Ly49 family molecules as the receptors specific for MHC class I. These results suggest functional importance of ß2m in NK cell recognition of target cells.
Keywords: C-type lectin, cytotoxicity, inhibitory receptor, H-2 antigen, NK cell
 |
Introduction
|
---|
NK cell recognition of target cells involves MHC class I molecules on the target cells (1). NK cells express inhibitory receptors specific for MHC class I molecules. The receptors transmit inhibitory signals to prevent NK cell killing of target cells expressing appropriate MHC class I ligands (2). The MHC class I-specific inhibitory NK cell receptors are classified into two groups by their structural features. One group of the receptors that belong to the C-type lectin superfamily includes mouse and rat Ly49 and CD94/NKG2 receptors, which are used in various species including mouse, rat and human. Another group of the receptors that belongs to the Ig superfamily includes human killer cell Ig-like receptors (KIR) (3,4).
Mouse Ly49A, a member of Ly49 family, is a homodimer of type II transmembrane proteins (5,6), and recognizes the mouse MHC class I molecules H-2Dd, Dk (7,8) and Dp (9). A MHC class I molecule is a complex of the three non-covalently associating components: a heavy chain unique to each MHC class I molecule, an invariant ß2-microglobulin (ß2m) and a peptide of 89 amino acids. Ly49A recognition of the MHC class I molecules has been most extensively studied on that of H-2Dd. Ly49A recognizes a conformation of H-2Dd that depends on peptide binding (10,11), but does not depend on carbohydrate moieties of the H-2Dd heavy chain (12). The crystal structure of the Ly49A/H-2Dd complex revealed two Ly49A binding sites on a single molecule of H-2Dd, termed site 1 and site 2 (13). Site 1 includes the N-terminus of the
1
-helix and the C-terminus of the
2
-helix, while site 2 spans all the three structural domains of H-2Dd:
1/
2 and
3 domains, and ß2m. Of the two Ly49A binding sites on H-2Dd, site 2 is the functional binding site for Ly49A that leads to inhibition of NK cell cytotoxicity (14) and is also a major Ly49A binding site detectable in physical binding assays (14,15). Two research groups reported that efficient recognition of mouse MHC class I ligands by Ly49A (14,16) and Ly49C (16) requires association of the MHC class I heavy chains with mouse (m) ß2m, and that Ly49A and Ly49C are unable to bind MHC class I molecules that include human (h) ß2m as their subunits. Nevertheless, the molecular details of the species-specific determinant of ß2m required for recognition of the MHC class I ligands by Ly49 family receptors have not been experimentally addressed.
In the present study, we constructed an experimental system that enabled us to evaluate the ability of various ß2m mutants to make complexes with heavy chains of the MHC class I H-2Dk and further evaluate the capacity of the H-2Dk complexes to engage Ly49A. Using this system, we demonstrate that the interaction between Ly49A and H-2Dk depends on the species from which ß2m that constitutes H-2Dk is derived. We further demonstrate that residues 3 and 29 of ß2m define the species specificity of ß2m in Ly49A recognition of H-2Dk using various mutants of mß2m and hß2m. The results help our understanding of the Ly49MHC class I interactions, as well as providing an insight into molecular evolution of ß2m.
 |
Methods
|
---|
Mice
C57BL/6J mice were purchased from Nippon CLEA (Tokyo, Japan); 8- to 17-week-old female mice were used for the experiments.
Cells
A mouse H-2k lymphoma R1.1 and its ß2-m-defective mutant R1E/TL8x.1 (R1.E), both of which are negative for Fc
receptors, were obtained from the ATCC (Manassas, VA), and were maintained in RPMI 1640 medium (Sigma, St Louis, MO) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-mercaptoethanol and 25 mM HEPES. Ly49A+ or Ly49A IL-2-activated NK cells were prepared from C57BL/6J mouse splenocytes as described (12).
Antibodies
All mAb were purified using Protein A- or Protein G-affinity chromatography from hybridoma culture supernatants. 15-5-5S (anti-H-2Dk), S19.8 (anti-mß2m), BBM1 (anti-hß2m) and MAR18.5 (anti-rat
light chain) were obtained from ATCC. A1 (anti-Ly49A) was a gift from Dr Wayne M. Yokoyama (Washington University School of Medicine, St Louis, MO). Fluorescein-conjugated goat anti-mouse IgG F(ab')2 was purchased from ICN Biomedicals (Irvine, CA).
Site-directed mutagenesis
Point mutations were introduced by sequential PCR steps as described by Cormack (17). All single mutations were generated with overlapping primers encoding mutations, external 5' primers (mß2m EcoRI; 5'-GGAATTCAGTCGCGGTCGCTT-3', mß2m BglII; 5'-TCAGATCTGGTCGCTTCAGTCGTC-3', hß2m SalI; 5'-CCAGTCGACGATAATACCATGAGTC-3') and external 3' primers (mß2m SalI 5'-CAGTCGACCATGATGCTTGA TCAC-3', hß2m SacII; 5'-CTGGAGCTCCACCGCGGT-3') using KOD-plus-DNA polymerase (Toyobo, Osaka, Japan) and mß2m (C57BL/6J strain) or hß2m cDNA (generous gifts from Dr R. K. Ribaudo, Molecular Applications Group, Silver Spring, MD) as templates. As external 5' primers, the mß2m EcoRI primer was used to generate mK3R, mQ6K and mP47E mutants, and the mß2m BglII primer was used to generate the other mouse mutant ß2m. The mutant ß2m cDNA fragments were subcloned into the SmaI site of pBluescript II SK+ (Stratagene, La Jolla, CA) and sequences were confirmed by a LS-2000 sequencer (LI-COR, Lincoln, NE). The mutant ß2m cDNA fragments were then cloned into the pApuro expression vector (18).
Stable transfection of cells
R1.E cells (4 x 106) were transfected in 0.5 ml of RPMI 1640 medium with 1020 µg of linearized plasmids harboring mutant ß2m by electroporation using a BTX-600 Electro Cell Manipulator (Harvard Apparatus, Holliston, MA) with the following setting: 0.4-cm gap, 1200 µF, 300 V and 720
. The transfectants were selected and maintained in the same medium as used for R1.E cells that was supplemented with 0.5 µg/ml of puromycin (Sigma).
Flow cytometry analysis
Cells (23 x 105) were stained with 10 µg/ml of the indicated primary antibodies for 30 min at room temperature and then with fluorescein-conjugated goat anti-mouse IgG F(ab')2 for 15 min on ice. The stained cells were analyzed using a FACSCalibur with CellQuest software (BD Biosciences, San Jose, CA).
Cell cytotoxicity assays
Specific killing of targets by NK cells was determined by standard 4-h 51Cr-release assays as described (12). In brief, cells were labeled with 50 µCi of [51Cr]sodium chromate (Amersham Biosciences, Tokyo, Japan) for 90 min at 37°C. The cells were washed and then incubated with Ly49A+ or Ly49A NK cells at the indicated E:T ratios in 96-well U-bottom microplates (BD Biosciences) at 37°C for 4 h. Radioactivities released into supernatants were scintillation-counted using a TopCount scintillation counter (Perkin-Elmer, Boston, MA). Specific cytotoxicity was calculated as previously described (12).
 |
Results
|
---|
Failure of Ly49A to recognize H-2Dk that includes hß2m
To investigate whether Ly49A recognizes the H-2Dk that contains hß2m as a subunit, we transfected the R1.E cell line, which is a ß2m-deficient mutant of Fc
receptor-negative mouse lymphoma R1.1 (H-2k), with hß2m or mß2m cDNAs. The transfectants were assayed for expression of H-2Dk and hß2m or mß2m (Fig. 1A). R1.E cells transfected with either hß2m or mß2m cDNAs expressed H-2Dk on the cell surface, while untransfected R1.E cells did not express H-2Dk on the surface. These results indicate that hß2m forms a complex with the H-2Dk heavy chain and induces H-2Dk expression on the cell surface. We next tested the capacity of the H-2Dk that contained hß2m or mß2m as a subunit to engage Ly49A and to protect R1.E cells from killing by Ly49A+ IL-2-activated NK cells (Fig. 1B). Untransfected R1.E cells were efficiently killed by either Ly49A+ and Ly49A NK cells, and addition of an anti-Ly49A antibody did not affect NK cell cytotoxicity. R1.E cells transfected with mß2m cDNA were protected from killing by Ly49A+ NK cells and the protection was reversed by addition of an anti-Ly49A antibody, suggesting that Ly49A recognizes the H-2Dk that includes mß2m, in agreement with the previous study (7). Importantly, R1.E cells transfected with hß2m cDNA were killed efficiently by Ly49A+ NK cells despite the cell-surface expression of the H-2Dk that included hß2m. These data indicate that Ly49A recognizes the H-2Dk that includes mß2m and transmits negative signals to inhibit NK cell cytotoxicity, but is unable to recognize the H-2Dk that contains hß2m. These results also suggest that Ly49A recognition of H-2Dk involves a species-specific determinant of ß2m that also takes part in Ly49A recognition of H-2Dd (14,16).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1. Recognition of the H-2Dk molecules that contain mß2m or hß2m as subunits by Ly49A. (A) R1.1 cells, R1.E cells and R1.E transfectants (mß2m or hß2m) were stained with the anti-H-2Dk mAb 15-5-5S (bold lines), anti-hß2m or anti-mß2m mAb (filled histograms) or the isotype-matched control mAb MAR18.5 (dotted lines) and then analyzed by flow cytometry. For staining for ß2m, the R1.E transfectant with hß2m was stained with the anti-hß2m mAb BBM1, while the other cells were stained with the anti-mß2m mAb S19.8. Mean fluorescence intensities (MFI) for anti-H-2Dk staining are shown in the upper right corner of each panel. Of note, the S19.8 mAb is not reactive with the ß2m allele expressed in R1.1 cells. (B) R1.1 cells, R1.E cells and R1.E transfectants (mß2m or hß2m) were assayed for killing by Ly49A+ (left panels) or Ly49A (right panels) NK cells at the indicated E:T ratios. The NK cells were pre-incubated with medium alone (open squares), the anti-Ly49A mAb A1 (open circles) or the isotype-matched control mAb MAR18.5 (open triangles) at a final concentration of 10 µg/ml. The data shown are representative of at least three independent experiments with similar results.
|
|
Previously, Wang et al. (15) examined the physical interaction between the recombinant soluble forms of Ly49A and H-2Dd with BIAcore to demonstrate a pivotal role of Lys58 of mß2m in the interaction between Ly49A and H-2Dd. To investigate the role of Lys58 of mß2m in the functional interaction between Ly49A and H-2Dk, we established R1.E cells stably transfected with a K58A mutant of mß2m. Transfection of R1.E cells with the mutant mß2m induced cell-surface expression of H-2Dk as in the case of the cells transfected with the wild-type mß2m (Fig. 2A). However, H-2Dk that contained the K58A mutant of mß2m failed to protect the cells from killing by Ly49A+ NK cells. Even though R1.E transfected with the K58A mutant of mß2m expressed low levels of H-2Dk (MFI = 9.3), R1.E cells transfected with the wild-type mß2m that expressed lower levels of H-2Dk (MFI = 7.4) could be protected from killing by Ly49A+ NK cells (data not shown), indicating that low-level expression of H-2Dk with MFI values as low as 7.4 is sufficient to protect the cells from killing by Ly49A+ NK cells, if the H-2Dk contained wild-type mß2m. These results suggest that the functional interaction between Ly49A and H-2Dk requires the side-chain of mß2m Lys58.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 2. Ly49A recognition of H-2Dk complexed with various mutants of mß2m. (A) R1.E cells and R1.E transfectants (wild-type ß2m or various mß2m mutants) were stained with the anti-H-2Dk mAb (bold lines) or the control mAb (dotted line). MFI for anti-H-2Dk staining are shown in the upper right corner of each panel. (B) R1.E cells and R1.E transfectants (wild-type ß2m or various mß2m mutants) were assayed for killing by Ly49A+ NK cells at the indicated E:T ratios. NK cells were pre-incubated with medium alone (open squares), the anti-Ly49A mAb (open circles) or the control mAb (open triangles) at a final concentration of 10 µg/ml. The data shown are representative of at least three independent experiments with similar results.
|
|
Expression of the H-2Dk that contained mß2m mutants with the human type residues
In order to elucidate the mß2m residues that determine the species specificity of ß2m required for Ly49A recognition of H-2Dk, we prepared a panel of mß2m mutants, in which individual residues were substituted with the corresponding residues found in hß2m. mß2m and hß2m both consist of 99 amino acid residues, but their amino acid sequences differ from each other by
30% (Fig. 3). Ly49A recognizes the complexes of the H-2Dd heavy chains and mß2m or rat ß2m, but fails to recognize those of the H-2Dd heavy chains and hß2m or bovine ß2m (12,19). Considering the similar involvement of ß2m in Ly49A recognition of H-2Dd and Dk, we chose residues of mß2m that satisfied the following two criteria to introduce mutations: (i) the residues that are conserved in mouse and rat, but not in human and bovine ß2m, and (ii) the residues that are exposed to the molecular surface of H-2Dd (20,21) (Fig. 3). We prepared mutants of mß2m in which mß2m residues that satisfied the above criteria were replaced with the corresponding human residues.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 3. Alignment of amino acid sequences of ß2m from various species. Alignment of amino acid sequences of ß2m from mouse (PIR unique ID: MGMSB2), rat (PIR unique ID: A26842), human (PIR unique ID: MGHUB2) and bovine (PIR unique ID: MGBOB2) are shown. mß2m residues indicated by reverse triangles with numbers were mutated into the corresponding residues found in hß2m.
|
|
We transfected R1.E cells with the mß2m mutant cDNAs and assayed for cell-surface expression of H-2Dk (Fig. 2A). R1.E cells transfected with most of the mß2m mutant cDNAs expressed H-2Dk on the cell surface at levels comparable to that of H-2Dk on the R1.E cells transfected with wild-type mß2m. However, two mutants of mß2m (K3R and Q6K) did not induce cell-surface expression of H-2Dk (unpublished data). The mß2m mutant-transfected clones with expression of H-2Dk comparable to the wild-type mß2m transfectant were chosen and used in the following assays.
Ly49A failed to recognize the H-2Dk that contained mß2m with K3R and Q29G mutations
To investigate the functional recognition of the H-2Dk that included each of the mß2m mutants by Ly49A, we tested the R1.E cells transfected with a panel of mß2m mutants for their protection from killing by Ly49A+ NK cells (Fig. 2B). Expression of the single mß2m mutants T75K, E89K and T92I as well as that of the wild-type mß2m protected R1.E cells from killing by Ly49A+ NK cells and the protection was reversed in the presence of an anti-Ly49A antibody. In contrast, Q29G mutation partially impaired the protective activity of H-2Dk. Gln29 of mß2m is in the close proximity of Lys3, of which substitution by Arg ablated ability of mß2m to induce cell-surface expression of H-2Dk. Considering the possibility that Arg3 is incompatible with Gln29 in the milieu of mß2m, but is compatible with Gly29, which is found in hß2m, we produced a Q29G/K3R double mutant of mß2m and transfected R1.E cells with the mutant. As we expected, the Q29G/K3R mutant of mß2m was able to induce cell-surface expression of H-2Dk (Fig. 2A). The Q29G/K3R transfectants of R1.E cells were also assayed for killing by Ly49A+ NK cells (Fig. 2B). Importantly, the H-2Dk that included mß2m with K3R and Q29G mutations failed to protect the cells from killing by Ly49A+ NK cells. These data suggest that Lys3 and Gln29 of mß2m are required for Ly49A recognition of H-2Dk.
Ly49A recognizes the H-2Dk that includes hß2m with R3K and G29Q mutations
To further validate the above results, we designed an inverse experiment in which residues of hß2m were substituted by the corresponding residues found in mß2m and the H-2Dk that contained each of the hß2m mutants were tested for functional recognition by Ly49A. We introduced either one or both of R3K and G29Q mutations into hß2m, and stably transfected R1.E cells with these hß2m mutant cDNAs. Transfection of R1.E cells with any of the hß2m mutant cDNAs induced cell-surface expression of H-2Dk comparable to the wild-type hß2m transfectant (Fig.4A). The wild-type as well as mutant hß2m transfectants were tested for killing by Ly49A+ NK cells (Fig. 4B). Expression of the R3K/G29Q double mutant of hß2m protected R1.E cells from killing by Ly49A+ NK cells, while that of the R3K and G29Q single mutants of hß2m failed to protect the cells. These data clearly indicate that simultaneous introduction of the R3K and G29Q mutations is sufficient to give hß2m a capacity to constitute the H-2Dk complex competent to functionally engage Ly49A.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 4. Ly49A recognition of H-2Dk complexed with hß2m mutants. (A) Expression of H-2Dk induced by introduction of wild-type or various mutants of hß2m on the surface of R1.E cells. The cells were stained with an anti-H-2Dk mAb (bold lines) or a control mAb (dotted lines). MFI values for anti-H-2Dk staining are shown in the upper right corner of each panel. (B) R1.E cells that were untransfected or transfected with wild-type or mutants of hß2m were assayed for killing by Ly49A+ NK cells at the indicated E:T ratios. NK cells were pre-incubated with medium alone (open squares), anti-Ly49A mAb (open circles) or isotype-matched control mAb (open triangles) at the final concentration of 10 µg/ml. The data shown are representative of at least three independent experiments with similar results.
|
|
 |
Discussion
|
---|
In the present study, we demonstrated that Ly49A was unable to recognize the H-2Dk that included hß2m as a subunit, but was able to recognize the H-2Dk that included mß2m in functional assays. Furthermore, we found that the introduction of a K58A mutation into mß2m, which significantly impairs physical interaction between Ly49A and H-2Dd (15), also impaired the capacity of H-2Dk to engage Ly49A in functional assays. These results clearly indicate that Ly49A recognizes the H-2Dk surface that includes ß2m and the recognition depends on the species of ß2m that constitute H-2Dk, as was shown for Ly49A recognition of another MHC class I ligand H-2Dd (14,16). Similarly, the physical interaction between Ly49C and H-2Kb also depends on the source of the ß2m subunit (16). Once established that Ly49A recognition of H-2Dk depends on the species of ß2m, we next sought to identify the species-specific determinant on mß2m required for Ly49A recognition of H-2Dk. Our findings that Q29G single mutation of mß2m partially impaired Ly49A recognition of H-2Dk, and simultaneous introduction of the K3R and Q29G mutations into mß2m completely abrogated Ly49A recognition of H-2Dk, indicate that the two residues are essential for efficient Ly49A recognition of H-2Dk in the context of other residues of mß2m. In the inverse experiment, in which residues of hß2m were substituted by those found in mß2m, Ly49A functionally recognized the H-2Dk that contained the hß2m with R3K and G29Q mutations, demonstrating that the two mutations are sufficient to endow hß2m an ability to substitute mß2m in Ly49A recognition of H-2Dk. These results clearly indicate that Lys3 and Gln29 determine the species specificity of ß2m in Ly49A recognition of its ligand H-2Dk. These results also illustrate for the first time that the mß2m residues Lys3, Gln29 and Lys58, the last two of which have been shown to contribute to the physical interaction between Ly49A and H-2Dd (15), play a critical role in the functional interaction between Ly49A and its MHC class I ligand.
The current findings can be interpreted in light of the crystal structure of the Ly49A/H-2Dd complex (13). Ly49A appears to contact similar residues of ß2m when it binds H-2Dd and H-2Dk, since both bindings were sensitive to replacement of the mß2m subunits with hß2m, and also to K58A mutation on the mß2m subunits as shown in this and the previous studies (1416). In the Ly49A/H-2Dd complex the side-chains of Lys3, Gln29 and Lys58 of ß2m form hydrogen bonds with the residues of Ly49A (Fig. 5). The structure of the Ly49A/H-2Dd complex can nicely explain our conclusion that Lys3 and Gln29 determine the species specificity of ß2m in the interaction between Ly49A and H-2Dk, suggesting that our current findings can be extended to the interaction between Ly49A and H-2Dd.
Comparison of amino acid sequences of ß2m from various species revealed that Lys3 and Gln29 are conserved in some animals of the family Muridae such as the house mouse, Mus musculus, and the Norway rat, Rattus norvegicus. The genomes of these animals bear multiple Ly49 genes (2224). In particular, mouse NK cells are known to use Ly49 family receptors to monitor MHC class I expression. In contrast, in other species such as primates and bovine, each of their genomes bears multiple KIR genes, but has a unique gene homologous to Ly49 (2,4,2528), which ultimately turned into a pseudogene in higher primates including the human. Moreover, human NK cells use KIR to monitor MHC class I expression, instead of Ly49. Thus, there appears to be a correlation between the conservation of Lys3 and Gln29 of ß2m and the expression of multiple Ly49 receptors specific for MHC class I molecules, at least among the animals listed above. We propose that the selective pressure to maintain the effective interactions between Ly49 family molecules and MHC class I molecules had conserved Lys3 and Gln29 of ß2m in the house mouse and the Norway rat. Conversely, acquisition of Lys3 and Gln29 by ancestral ß2m in a common ancestor of the house mouse and the Norway rat might have enabled ancestral Ly49 to interact with MHC class I with an affinity high enough to monitor MHC class I expression; subsequently, Ly49 family molecules might have rapidly expanded and evolved to those seen currently in the house mouse and the Norway rat. ß2m of the Chinese hamster, Cricetulus griseus (Swiss-Prot accession no. Q9WV24), and the hispid cotton rat, Sigmodon hispidus (Swiss-Prot accession no. Q8CIQ3), both of which also belong to the family Muridae, have sequences of intermediate type with Arg3 and Gln29. Whether NK cells in these animals use Ly49 family receptors or KIR to monitor MHC class I expression is an interesting question that remains to be answered.
Our current results together with the previous findings (1416) demonstrated the inhibitory Ly49 receptors Ly49A and Ly49C use ß2m as a part of interface when they bind their MHC class I ligands. Interestingly, the activating receptor mouse Ly49D recognizes mouse H-2Dd and also a xenogeneic MHC class I molecule from the Chinese hamster (29), of which ß2m has K3R substitution. The inhibitory receptor Ly49G2 also recognizes a structure(s) expressed on Chinese hamster ovary cells (30), even though the nature of the structure, including whether the structure is Chinese hamster MHC class I or not, remains elusive. Moreover, the other activating receptor mouse Ly49H recognizes the virally encoded xenogeneic MHC class I-like molecule m157, which does not associate with the ß2m subunit (31). Thus, activating members of the Ly49 family and possibly an inhibitory member of the Ly49 family may not use ß2m as a part of the interface when they recognize their xenogeneic ligands. Whether Ly49D uses ß2m as a part of the interface when it recognizes H-2Dd, also, remains elusive.
The present study together with the previous studies (1416) has also shown that ß2m constitutes a part of the interface between MHC class I molecules and the NK cell receptors Ly49A and Ly49C. During the preparation of this manuscript, it was reported that the binding site for LIR-1, an inhibitory receptor of the Ig superfamily expressed on human leukocytes, on its MHC class I ligand also include the surface of ß2m (32). Moreover, human (33) and mouse (34) CD8
homodimers contact ß2m residues when they bind classical MHC class I molecules. In particular, Lys58 of ß2m plays a critical role in the interaction between human MHC class I and human CD8 (35). These studies, including ours, highlight the importance of ß2m as a critical part of the interfaces between the MHC class I molecules and the immune receptors.
In conclusion, Ly49A recognition of H-2Dk includes species-specific residues of ß2m, and the specificity is determined by Lys3 and Gln29 of ß2m. These results contribute to our understanding of the molecular mechanism underlying Ly49A recognition of its MHC class I ligands, but also suggest the functional importance of ß2m in NK cell recognition of target cells.
 |
Acknowledgements
|
---|
We thank Drs W. M. Yokoyama and R. K. Ribaudo for reagents. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (12672107 and 15590057) (N. M.), and a grant from the Kato Memorial Bioscience Foundation (N. M.).
 |
Abbreviations
|
---|
ß2mß2-microglobulin
hhuman
KIRkiller cell Ig-like receptor
mmouse
MFImean fluorescence intensity
R1.ER1E/TL8x.1
 |
References
|
---|
- Ljunggren, H. G. and Karre, K. 1990. In search of the missing self: MHC molecules and NK cell recognition. Immunol. Today 11:237.[CrossRef][ISI][Medline]
- Long, E. O. 1999. Regulation of immune responses through inhibitory receptors. Annu. Rev. Immunol. 17:875.[CrossRef][ISI][Medline]
- Yokoyama, W. M. 1999. Natural killer cells. In Paul, W. E., ed., Fundamental Immunology, 4th edn, p. 575. Lippincott-Raven, Philadelphia, PA.
- Lanier, L. L. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359.[CrossRef][ISI][Medline]
- 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.[Abstract/Free Full Text]
- 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.[Abstract/Free Full Text]
- 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.[CrossRef][ISI][Medline]
- Kane, K. P. 1994. Ly-49 mediates EL4 lymphoma adhesion to isolated class I major histocompatibility complex molecules. J. Exp. Med. 179:1011.[Abstract]
- Olsson-Alheim, M. Y., Sundback, J., Karre, K. and Sentman, C. L. 1999. The MHC class I molecule H-2Dp inhibits murine NK cells via the inhibitory receptor Ly49A. J. Immunol. 162:7010.[Abstract/Free Full Text]
- 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.[ISI][Medline]
- 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.[Abstract/Free Full Text]
- 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.[ISI][Medline]
- 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.[CrossRef][ISI][Medline]
- Matsumoto, N., Mitsuki, M., Tajima, K., Yokoyama, W. M. and Yamamoto, K. 2001. 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. J. Exp. Med. 193:147.[Abstract/Free Full Text]
- Wang, J., Whitman, M. C., Natarajan, K., Tormo, J., Mariuzza, R. A. and Margulies, D. H. 2002. Binding of the natural killer cell inhibitory receptor Ly49A to its major histocompatibility complex class I ligand. Crucial contacts include both H-2Dd and ß2-microglobulin. J. Biol. Chem. 277:1433.[Abstract/Free Full Text]
- Michaelsson, J., Achour, A., Rolle, A. and Karre, K. 2001. MHC class I recognition by NK receptors in the Ly49 family is strongly influenced by the ß2-microglobulin subunit. J. Immunol. 166:7327.[Abstract/Free Full Text]
- 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. and Struhl, K., eds, Current Protocols in Molecular Biology, p. 8.5.1. Wiley, New York.
- 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 J. 13:1341.[Abstract]
- Sundback, J., Nakamura, M. C., Waldenstrom, M., Niemi, E. C., Seaman, W. E., Ryan, J. C. and Karre, K. 1998. The
2 domain of H-2Dd restricts the allelic specificity of the murine NK cell inhibitory receptor Ly-49A. J. Immunol. 160:5971.[Abstract/Free Full Text]
- 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 P18I10 at 2.4 Å resolution: implications for T cell and NK cell recognition. Immunity 9:199.[ISI][Medline]
- 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.[CrossRef][ISI][Medline]
- 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.[Abstract/Free Full Text]
- 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.[Abstract]
- Rolstad, B., Naper, C., Lovik, G., Vaage, J. T., Ryan, J. C., Backman-Petersson, E., Kirsch, R. D. and Butcher, G. W. 2001. Rat natural killer cell receptor systems and recognition of MHC class I molecules. Immunol. Rev. 181:149.[CrossRef][ISI][Medline]
- Barten, R. and Trowsdale, J. 1999. The human Ly-49L gene. Immunogenetics 49:731.[CrossRef][ISI][Medline]
- Westgaard, I. H., Berg, S. F., Orstavik, S., Fossum, S. and Dissen, E. 1998. Identification of a human member of the Ly-49 multigene family. Eur. J. Immunol. 28:1839.[CrossRef][ISI][Medline]
- Mager, D. L., McQueen, K. L., Wee, V. and Freeman, J. D. 2001. Evolution of natural killer cell receptors: coexistence of functional Ly49 and KIR genes in baboons. Curr. Biol. 11:626.[CrossRef][ISI][Medline]
- McQueen, K. L., Wilhelm, B. T., Harden, K. D. and Mager, D. L. 2002. Evolution of NK receptors: a single Ly49 and multiple KIR genes in the cow. Eur. J. Immunol. 32:810.[CrossRef][ISI][Medline]
- Furukawa, H., Iizuka, K., Poursine-Laurent, J., Shastri, N. and Yokoyama, W. M. 2002. A ligand for the murine NK activation receptor Ly-49D: activation of tolerized NK cells from ß2-microglobulin-deficient mice. J. Immunol. 169:126.[Abstract/Free Full Text]
- Mason, L. H. 2000. Recognition of CHO cells by inhibitory and activating Ly-49 receptors. J. Leukoc. Biol. 68:583.[Abstract/Free Full Text]
- Arase, H., Mocarski, E. S., Campbell, A. E., Hill, A. B. and Lanier, L. L. 2002. Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science 296:1323.[Abstract/Free Full Text]
- Willcox, B. E., Thomas, L. M. and Bjorkman, P. J. 2003. Crystal structure of HLA-A2 bound to LIR-1, a host and viral major histocompatibility complex receptor. Nat. Immunol. 4:913.[CrossRef][ISI][Medline]
- 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

and HLA-A2. Nature 387:630.[CrossRef][ISI][Medline]
- 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

ectodomain fragment in complex with H-2Kb. Immunity 9:519.[ISI][Medline]
- Glick, M., Price, D. A., Vuidepot, A. L., Andersen, T. B., Hutchinson, S. L., Laugel, B., Sewell, A. K., Boulter, J. M., Dunbar, P. R., Cerundolo, V., Oxenius, A., Bell, J. I., Richards, W. G. and Jakobsen, B. K. 2002. Novel CD8+ T cell antagonists based on ß2-microglobulin. J. Biol. Chem. 277:20840.[Abstract/Free Full Text]
- Guex, N. and Peitsch, M. C. 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714.[ISI][Medline]