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*
Laboratory of Molecular Medicine, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Tokyo, Japan;
Department of Parasitology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and
Howard Hughes Medical Institute, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1/2 domain of H-2Dd.
Here we have substituted polymorphic as well as conserved residues of
H-2Dd 1/2 domain (when compared with
H-2Kd, which does not interact with Ly49A). We then tested
the ability of the H-2Dd mutants to interact with Ly49A by
soluble Ly49A tetramer binding and NK cell cytotoxicity inhibition
assays. Individual introduction of mutations converting the
H-2Dd residue into the corresponding H-2Kd
residue (N30D, D77S, or A99F) in H-2Dd partially abrogated
the interaction between Ly49A and H-2Dd. Introduction of
the three mutations into H-2Dd completely abolished Ly49A
recognition. Individual introduction of D29N or R35A mutation into the
residues of H-2Dd that are conserved among murine MHC class
I severely impaired the interaction. The crystal structure of
H-2Dd reveals that D77 and A99 are located in the peptide
binding groove and that N30, D29, and R35 are in the interface of the
three structural domains of MHC class I: 1/2, 3, and
2-microglobulin. These data suggest that Ly49A can
monitor mutations in MHC class I inside and outside of the peptide
binding groove and imply that inhibitory MHC class I-specific receptors
are sensitive to mutations in MHC class I as well as global loss of MHC
class I. Our results also provide insight into the molecular basis of
Ly49A to distinguish MHC class I polymorphism. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inhibitory receptors of both groups have one or two immune receptor tyrosine-based inhibitory motifs (ITIM)3 characterized by an IXYXXL sequence in the cytoplasmic region (17). Upon ligand recognition, the Tyr residue in the ITIM is phosphorylated and recruits the Src homology domain-containing protein tyrosine phosphatase 1 with Src homology 2 domains. The recruited Src homology domain-containing protein tyrosine phosphatase 1 is believed to dephosphorylate unknown critical substrates to shut down positive signaling. There are activating type of MHC class I receptors of C-type lectin and Ig-type with extracellular domains similar to those of the inhibitory receptors. Instead of cytoplasmic ITIM, the activating receptors have characteristic charged amino acid residues in the transmembrane region, and by that associate with DAP12, an adapter component with cytoplasmic immune receptor tyrosine-based activating motif (18). Engagement of such receptors initiates an activation signaling cascade analogous to T or B cell receptor signaling.
Mouse NK cells express the Ly49 family of receptors, which includes
>10 members (9, 10, 11, 12, 19). Several lines of evidence
demonstrate that Ly49A, a primary member of Ly49 family, is an
inhibitory receptor that specifically recognizes a conformational
epitope on H-2Dd. Introduction of
H-2Dd, but not H-2Kd or
Ld, functionally protects C1498 lymphoma cells
from lysis by Ly49A+ NK cells (20).
Also, Ly49A expression on NK cells is modulated in mice expressing
H-2Dd (21). Physical
interaction is supported by studies of Chinese hamster ovary
cells transfected with Ly49A that bind
H-2Dd-transfected C1498 cells (22).
The recognition of 1/2 domain of H-2Dd is
supported by two lines of indirect evidence. First, recognition of
H-2Dd by Ly49A is inhibited by the 1/2
domain-specific Ab 34-5-8S, but not by 34-2-12S Ab, which recognizes
3 domain of H-2Dd (20). Second,
Ly49A recognizes the natural mutant MHC class I molecule dm1,
which has 1 and the N-terminal half of 2 domain of
H-2Dd and the rest of the regions from
H-2Ld, which is not a ligand for Ly49A
(23). 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 (24, 25), in
contrast with peptide-specific recognition of MHC class I molecules by
TCRs. Ly49A has a functional C-type lectin domain that can bind the
polyanionic carbohydrate, fucoidan (26). However, Ly49A
can recognize the H-2Dd that lacks carbohydrate
moiety (23). Recently, Tormo et al. reported the crystal
structure of the Ly49A/H-2Dd complex
(27), providing firm evidence for specific interaction of
Ly49A with H-2Dd. The structure reveals the
presence of two possible binding sites on H-2Dd
for Ly49A. The authors suggested that one of the binding sites (site 1)
that is formed by the N-terminal end of 1 helix and the
C-terminal end of 2 helix of H-2Dd might
be the functional binding site involved in the inhibitory recognition
of H-2Dd by Ly49A expressed on NK cells.
Our recent studies have indicated that another site on
H-2Dd, termed site 2, forms the functional
interaction site with Ly49A (28). However, other studies,
including investigations by our laboratory, also indicate that there
may be functional consequences of H-2Dd residues
that are not directly contacted by Ly49A. Specifically, we previously
localized regions of H-2Dd that are important for
recognition by Ly49A by using a series of recombinant MHC class I
molecules between H-2Dd and
Kd (23). The important areas include
-sheet regions that form the bottom of 1/2 domain rather than
helices of 1/2 domains, which form the functional binding
site for Ly49A by the crystal structure data and our more recent
mutagenesis data (28). Furthermore, other investigators
have reported that mutations in residues that affect the peptide
binding cleft may also affect Ly49A interaction (29, 30).
Therefore, the contribution of H-2Dd residues
that do not directly contact Ly49A requires further investigation.
To address this issue in more detail, we produced a panel of
H-2Dd mutants with a single or multiple mutations
to examine the role of each residue in the interaction with Ly49A. The
H-2Dd mutants expressed on C1498 lymphoma cells
were tested for their ability to interact with Ly49A by binding of
soluble Ly49A (sLy49A) complex and by functional inhibition of killing.
We found that simultaneous introduction of three specific mutations
inside and outside of the peptide binding groove totally disrupted the
epitope on H-2Dd that is recognized by Ly49A. We
also demonstrate that mutations in conserved residues also impair the
epitope of H-2Dd required for recognition by
Ly49A. Taken together, our results suggest that Ly49A recognizes a
conformational epitope that is sensitive to substitution in residues in
1/2 domains, including both polymorphic and conserved residues.
We discuss the molecular mechanism by which Ly49A detects mutations and
polymorphism in MHC class I in light of the recently identified
functional binding site of Ly49A on H-2Dd
(28).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were obtained from CLEA Japan (Tokyo, Japan).
Cells
C1498 cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS (Sigma, St. Louis, MO), 25 mM HEPES (Dojindo, Kumamoto, Japan), 100 µg/ml penicillin, 100 U/ml streptomycin, 2 mM glutamine, and 5 µM 2-ME. Dd5 was established by transfecting C1498 cells with wild-type Dd expression vector as described previously (23). Dd5 and C1498 cells transfected with Dd mutant cDNA were maintained in the same medium supplemented with 0.5 mg/ml G418 sulfate (Life Technologies, Rockville, MD). L cells, generous gifts from Dr. Ted H. Hansen (Washington University, St. Louis, MO), were maintained in DMEM supplemented with 10% heat-inactivated FCS, 25 mM HEPES, 100 µg/ml penicillin, 100 U/ml streptomycin, 2 mM glutamine, nonessential amino acids (Life Technologies), and 1 mM sodium pyruvate (Life Technologies).
Ly49A+ or Ly49A- lymphokine-activated killer (LAK) cells were prepared from splenocytes from 8- to 16-wk-old C57BL/6J mice as described previously (20). Recombinant human IL-2, a generous gift form Ajinomoto Corporation (Tokyo, Japan), was used for culture. Both populations were >98% positive for NK1.1 by FACS analysis. Ly49A+ cell preparations were >97% positive for anti-Ly49A Ab (A1) and Ly49A- preparations contained <2% Ly49A+ cells.
Preparation of sLy49A tetramer
sLy49A was prepared as described elsewhere (48). Briefly, the extracellular domain of Ly49A with N-terminal biotinylation sequence tag (31) was expressed in Eschericia coli using an efficient T7 RNA polymerase-based system (32). The recombinant protein was refolded in vitro by dilution (33) and purified by cation exchange and gel filtration column chromatography. The sLy49A was biotinylated by biotin ligase BirA (Avidity, Denver, CO). sLy49A tetramer was formed by incubating the biotinylated sLy49A with R-PE-conjugated streptavidin (BD PharMingen, San Diego, CA) at the molar ratio of 4:1.
Monoclonal Abs
34-5-8S (anti-H-2Dd 1/2),
34-2-12S (anti-H-2Dd 3) (34),
A1 (anti-Ly49A) (35), PK136 (anti-NK1.1)
(36), 53-6.7 (anti-mouse CD8) (37),
and H57-597 (anti-mouse TCR ) (38) were purified
from culture supernatants by protein A or protein G affinity column
chromatography.
Site-directed mutagenesis
Mutations were introduced using the Altered Sites II
site-directed mutagenesis kit (Promega, Madison, WI) according to the
manufacturer’s protocol. Multiple mutations were introduced by several
rounds of mutations using two selectable markers, ampicillin and
tetracycline resistance. Sequences of the primers used for
site-directed mutagenesis will be provided on request. Sequences of all
mutants were verified by sequencing both strands using ABI373A, ABI377
sequencers, or LS2000 DNA sequencer (Shimadzu, Kyoto, Japan).
H-2Dd cDNAs with mutations were directionally
subcloned into an expression vector pHAPr-neo driven by human
-actin promoter as described previously (23).
Transfection
C1498 cells were transfected with electroporation using ECM600 (BTX, San Diego, CA) under the following conditions: 0.4 cm gap, 300 V, 1,000 µF, and 720 ohm resistance. After electroporation, the cells were cultured for 2 days and then selected in the presence of 1 mg/ml G418 sulfate and cloned by limiting dilution as described previously (23). The clones were assayed by flow cytometry, and clones expressing an equivalent level of H-2Dd to wild-type Dd transfectant were selected for further analysis. L cells were transiently transfected with 1 µg of plasmid and 8 µl of Transfectamine (Life Technologies) in OPTI-MEM (Life Technologies) and were analyzed by flow cytometry after 48 h of culture.
Flow cytometry
Cells were stained with 10 µg/ml purified Abs for 30 min. Then cells were washed three times with HBSS containing 0.1% BSA and 0.1% sodium azide and were stained with 10 µg/ml FITC-goat anti-mouse IgG F(ab')2 (ICN Pharmaceuticals, Costa Mesa, CA) for 15 min. The cells were washed two times with the same buffer and analyzed on FACSCalibur with CellQuest software (BD Biosciences, San Jose, CA). For staining with sLy49A tetramer, cells were incubated on ice for 30 min with the 20 µg/ml sLy49A tetramer, then washed three times with the buffer. The cells were fixed with 1% paraformaldehyde in PBS and then analyzed as described above. Ten thousand events of cells gated by forward and side scattering were acquired for analysis. Binding of sLy49A tetramer to each mutant H-2Dd was calibrated with the expression of H-2Dd detected by 34-2-12S Ab 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-stained cells) - (MFI of control Ab-stained cells)]. sLy49A tetramer binding of each H-2Dd mutant is expressed as the relative value of the binding index as compared with that of wild-type H-2Dd, which was adjusted to 100.
Cellular cytotoxicity assay
Cytotoxicity of Ly49A+ or Ly49A- LAK cells against C1498 transfectants was determined with a standard 51Cr release assay at the E:T ratios of 4 and 20 as described (23). Radioactivity released into supernatant was measured by liquid scintillation counting with Microbeta (Wallac, Turku, Finland). The percentage of specific cytotoxicity was calculated as described (23). Where Ab was included in the assay, LAK cells were incubated with Abs for 15 min at room temperature before addition of target cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The previous study examining a series of recombinant MHC class I
molecules between H-2Dd, which is a ligand for
Ly49A, and H-2Kd, which is not a ligand for
Ly49A, demonstrated the importance of the two regions, 1–52 and
90–107 of H-2Dd, for the recognition of
H-2Dd by Ly49A (23). These regions
contained multiple residues that are different between
Dd and Kd (Fig. 1
). To evaluate these findings further,
we compared the sequences of 1/2 domains of
H-2Dd and Dk (which bind
Ly49A) and those of H-2Kd and
Kb (which do not bind Ly49A) for residues that
might be informative in mutagenesis experiments (Fig. 1). In the region
of H-2Dd from 1 to 52, the N30D mutation was
generated because Asp30 is uniquely found in
Kd and Kb, whereas
Dd and Dk have
Asn30. The region from 90 to 107 involves the
antigenic epitope of the anti-H-2Dd Ab
34-5-8S (39), which can block the interaction between
Ly49A and H-2Dd (20). To determine
the epitope of 34-5-8S Ab as well as the critical residues for Ly49A
binding, we chose L95F, A99F, E104G, and G107W in this region, even
though these residues are not conserved in another Ly49A ligand,
H-2Dk. We also included D77S, which potentially
affects peptide binding.
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3 domain
(40) where we did not introduce any mutation. Clones with
levels of expression similar to the wild-type
H-2Dd transfectant were chosen for further
analysis (Fig. 2). Because we only
obtained W97R mutant transfectants with very low expression, W97R was
omitted from further analysis. The panel of the transfectants was then
examined for reactivity with 34-5-8S Ab, which recognizes a
conformational epitope of H-2Dd in 1/2
domain that is sensitive to peptide binding (41). Most of
the H-2Dd mutant transfectants were stained
equally with the two mAbs, 34-2-12S and 34-5-8S, whereas the
transfectants with E104G or G107W mutant showed diminished staining
with 34-5-8S Ab (Fig. 2). Transient transfection of L cells with these
mutant constructs showed similar results (data not shown). These
results suggest that the epitope of the 34-5-8S Ab contains the
hairpin loop that includes Glu104 and
Gly107 and are consistent with the previous
finding that the epitope recognized by 34-5-8S Ab is sensitive to the
replacement of the region from 90 to 107 of H-2Dd
with the similar region of H-2Kd
(39). Because Ly49A recognition of
H-2Dd is affected by 34-5-8S Ab, these data
suggest that Glu104 and
Gly107 are part or are in the proximity of the
Ly49A epitope.
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The panel of H-2Dd mutant transfectants was
assayed for binding of sLy49A tetramer and for killing by
Ly49A+ LAK cells. sLy49A tetramer binds
H-2Dd with similar specificity as Ly49A expressed
on NK cells (48). Surprisingly, neither E104G nor G107W mutation, which
affected 34-5-8S binding (Fig. 2
), showed any significant effect on the
binding of sLy49A tetramer to H-2Dd (Fig. 3). The L95F mutation also showed no
significant effect on sLy49A tetramer binding. In contrast,
introduction of N30D, D77S, or A99F mutation into
H-2Dd significantly reduced the binding of sLy49A
tetramer to H-2Dd (Fig. 3). In killing assays,
the protective activity of the N30D mutant against killing by
Ly49A+ LAK cells was partially abrogated, whereas
the other single mutants in the polymorphic residues showed similar
protective activity to wild-type H-2Dd (Fig. 4).
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). Introduction of either one
of D77S and A99F mutation together with N30D into
H-2Dd decreased the sLy49A binding to
H-2Dd compared with N30D single mutant
H-2Dd. Introduction of all three mutations into
H-2Dd at the same time completely abrogated the
sLy49A binding (Fig. 3). Simultaneous introduction of both D77S and
A99F into H-2Dd did not have significant effects
that differed from either D77S or A99F single mutation. In the killing
assay, a similar cooperative effect among N30D, D77S, and A99F
mutations in H-2Dd on the abrogation of
Ly49A-mediated protection of the tumor cell killing was observed
(Fig. 4). These results well explain the previous observation that exchange of the region 1–52 of H-2Dd with that of H-2Kd partially inhibits H-2Dd recognition by Ly49A and that exchange of the region 1–107 of H-2Dd with that of H-2Kd completely abrogated the H-2Dd recognition by Ly49A (23). The results also indicate that Ly49A binding to H-2Dd does not depend on the 34-5-8S epitope, even though 34-5-8S Ab blocks the Ly49A binding to H-2Dd.
Effect of mutation in conserved residues in 1 domain of
H-2Dd on Ly49A interaction with H-2Dd
In addition to these polymorphic residues, we introduced mutations
into conserved charged residues in 1 domain in the neighborhood of
Asn30 to evaluate their contribution to Ly49A
binding. These mutants include D29N, R35A, E41A, E46A, and R48A (Fig. 1). The H-2Dd mutant molecules were expressed on
C1498 cells (Fig. 2) and were tested for the binding of sLy49A tetramer
and the ability to inhibit killing by Ly49A+ LAK
cells. Introduction of D29N mutation completely abolished the ability
of H-2Dd to interact with Ly49A in the sLy49A
binding and the functional protection assay (Figs. 3 and 4).
Introduction of R35A mutation severely impaired the ability of
H-2Dd to interact with Ly49A in both of the
assays. Mutations in Glu41,
Glu46, or Arg48 did not
show any significant effect on the Ly49A-H-2Dd
interaction. Thus, our results show the ability of Ly49A to detect
mutations in various regions of H-2Dd to monitor
expression of normal MHC class I.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N30D, D29N, and R35A mutations located outside of the peptide binding
groove affected the recognition of H-2Dd by Ly49A
(Figs. 3 and 4). These mutations can be interpreted in light of the
crystal structure of the Ly49A/H-2Dd complex
reported by Tormo et al. (27) and recent identification of
the functional Ly49A-binding site on H-2Dd
(28). The crystal structure provided the two possible
binding sites on H-2Dd for Ly49A dimer. One site
that consists of the NH2-teminal end of 1
helix and the COOH-terminal of 2 helix has been suggested to be
a functional binding site for Ly49A because of the polymorphism and
geometry (27). In contrast with this possibility, we
recently established that Ly49A functionally binds the other site on
H-2Dd to inhibit NK cell cytotoxicity
(28). The functional binding surface is contributed by the
three structural domains that constitute H-2Dd,
1/2 and 3 domains and 2-microglobulin
(2m), and was referred to as site 2 by Tormo
et al. (27). Importantly, Asn30,
Asp29, and Arg25 are at the junction of these
three domains and are also in the neighborhood of the functional
binding site for Ly49A (Fig. 5).
Especially, the side chain of Arg35
(H-2Dd heavy chain) is involved in hydrogen
bonding with 2m in the crystal structure of
H-2Dd (42, 43). Disruption of this
hydrogen bond by R35A mutation might change the orientation of
2m against 1/2 domain and thereby
severely impair the Ly49A binding to H-2Dd. In
the crystal structure, Asp29 of
H-2Dd heavy chain makes a salt bridge with
Arg228 of Ly49A in the functional binding site.
Therefore, complete loss of the binding by introduction of D29N
mutation into H-2Dd heavy chain is also
consistent with this structure model.
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). The effect of D77S and A99F mutations
on Ly49A interaction with H-2Dd was not evident
in the killing assay when introduced alone (Fig. 4). However,
cointroduction of these mutations together with N30D mutation
significantly impaired the inhibitory recognition by Ly49A as well as
the binding of Ly49A tetramer. This observation might reflect a
threshold of ligand receptor interaction required for inhibition of
killing: the effect of D77S and/or A99F single or double mutations was
small so that the effect was insufficient to be detected by functional
inhibition assay unless the N30D mutation was also present.
The crystal structure of H-2Dd revealed that
Ala99 and Asp77 are located
in a critical position to bind two anchoring residues of the peptide,
Pro at position 3 and Arg at position 5, respectively (42, 43). Consequently, H-2Dd with these
mutations might not bind peptide or acquire new peptide specificities.
Two lines of evidence, albeit indirect, suggest that the mutants
H-2Dd with D77S and/or A99F mutation on the cell
surface are expressed with bound peptides. First,
H-2Dd molecules with one or both of D77S and A99F
mutations were expressed on the cell surface at levels comparable to
wild-type H-2Dd (Fig. 2). The notion that
expression of classical MHC class I molecules requires complex
formation with peptides (44) suggests that the
H-2Dd mutants are peptide-bound. Secondly, the
H-2Dd mutants were stained by 34-5-8S Ab, which
recognizes an epitope sensitive to peptide binding (41),
to the same extent as staining by mAb 34-2-12S (Fig. 2), which
recognizes 3 domain of H-2Dd
(40). In contrast with D77S and/or A99F mutants, the
inefficient expression of the W97R mutant (Fig. 2) likely results from
the impaired ability of the mutant to bind peptide. This is supported
by our observation that expression of the mutant was rescued by
incubation of the cells at 28°C (N. Matsumoto, unpublished
observation) that induce peptide-independent expression of MHC class I
(24, 25). Moreover, the W97R mutant was least reactive
with 34-5-8S when expressed on L cells (data not shown).
Recently, Waldenström et al. reported that simultaneous
introduction of S73W and D156Y mutation into
H-2Dd partially impairs
H-2Dd-mediated protection against killing by
Ly49A+ NK cells (29). These
substitutions putatively introduce a ridge inside the peptide binding
groove that is found in H-2Db. Their
interpretation is consistent with our current finding that substitution
in peptide binding groove influenced the recognition of
H-2Dd by Ly49A. However, we could not detect any
decrease in protection by the S73W, D156Y double mutant in our system
using C1498 cells (N. Matsumoto, unpublished data). The discrepancy
might be due to the difference in level of expression of mutant
H-2Dd or the difference in balance of activating
and inhibitory ligands on the target cells used in these experimental
systems. Correa et al. have shown that a stronger inhibitory signal is
required to overcome a stronger activating signal by comparing natural
killing and Ab-dependent cellular cytotoxicity (45).
During the preparation of this manuscript, Nakamura et al. reported
that introduction of triple mutations, W97Q, A99S, and W114L, into the
peptide binding groove of H-2Dd completely
abrogates recognition by Ly49A (30). This also underscores
the importance of the peptide binding groove for Ly49A recognition of
H-2Dd. In the crystal structure, the
COOH-terminal end of 1 helix and the
NH2-terminal end of 2 helix are engaged by
Ly49A with hydrogen bonds at the functional binding site. This
structure explains the peptide dependency of Ly49A binding to
H-2Dd and the sensitivity of the Ly49A binding to
the mutations in the peptide binding groove, observed in this and
previous studies (29, 30).
Our finding that E104G or G107W mutations impaired staining with
34-5-8S Ab suggests that 34-5-8S recognizes the hairpin loop,
including Glu104 and
Gly107, and indicates the essential role of these
residues for efficient binding of the Ab. This is consistent with the
previous finding that the 34-5-8S epitope is located in the region
between 90 and 107 (39). The current finding that
H-2Dd with E104G or G107W mutation was fully
recognized by Ly49A (Figs. 3 and 4) clearly segregates the Ly49A
epitope on H-2Dd from the 34-5-8S epitope,
despite the previous result that 34-5-8S Ab inhibits
H-2Dd recognition by Ly49A (20).
However, the hairpin loop, which contains the 34-5-8S epitope, is
located at the edge of the functional binding site of Ly49A on
H-2Dd, and the Ab is therefore in good proximity
to sterically hinder Ly49A binding (Fig. 5). Therefore, the
identification of the 34-5-8S epitope and the inhibition of the
Ly49A-H-2Dd interaction by 34-5-8S strongly
support the recent identification of the functional Ly49A binding site
as site 2 (28).
Ly49A is able to distinguish polymorphic MHC class I molecules. Ly49A
can recognize H-2Dd, Dk,
and Dp, but not H-2Db,
Kb, Kd, and
Ld (20, 46). Introduction of N30D,
D77S, and A99F, substitution of three Dd residues
to Kd-type completely abolished
Dd interaction with Ly49A (Figs. 3 and 4). This
observation explains the previous finding that the R6 recombinant MHC
class I molecule, which has residues 1–107 from
Kd, is not able to protect target cell killing by
Ly49A+ NK cells and that the R1-R5 recombinant
MHC class I molecules, all of which have residues 1–52 from
Kd and the residues 90–107 from
H-2Dd, partially protect target cells from
killing by Ly49A+ NK cells (23).
Our results well explain the inability of Ly49A to interact with H-2Kd and provide insight into how Ly49A distinguishes polymorphic MHC class I molecules. However, the residues found in H-2Kd are not always associated with other H-2 molecules that are not ligands for Ly49A. Additional residues that determine the reactivity of each polymorphic MHC class I molecule with Ly49A remain to be explored by further site-directed mutagenesis on H-2Dd to fully understand the basis of MHC class I specificity of Ly49A.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Naoki Matsumoto, Laboratory of Molecular Medicine, Department of Integrated Biosciences, University of Tokyo Graduate School of Frontier Sciences, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
3 Abbreviations used in this paper: ITIM, immuno-receptor tyrosine-based inhibitory motifs; LAK, lymphokine-activated killer; sLy49A, soluble Ly49A; 2m, 2-microglobulin; MFI, mean fluorescence intensity.
Received for publication September 18, 2000. Accepted for publication January 19, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ antigen receptor. J. Immunol. 138:815.[Abstract]
T cell receptors. J. Immunol. 142:2736.[Abstract]
2-microglobulin regulation of cell surface MHC class I conformation and expression. J. Immunol. 148:3723.[Abstract]
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M. A. Morris, J. Liu, V. Arora, T. C. George, J. Klem, J. D. Schatzle, V. Kumar, and M. Bennett B6 Strain Ly49I Inhibitory Gene Expression on T Cells in FVB.Ly49IB6 Transgenic Mice Fails to Prevent Normal T Cell Functions J. Immunol., October 1, 2002; 169(7): 3661 - 3666. [Abstract] [Full Text] [PDF]
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J. Sundback, A. Achour, J. Michaelsson, H. Lindstrom, and K. Karre NK Cell Inhibitory Receptor Ly-49C Residues Involved in MHC Class I Binding J. Immunol., January 15, 2002; 168(2): 793 - 800. [Abstract] [Full Text] [PDF]
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 J. Wang, M. C. Whitman, K. Natarajan, J. Tormo, R. A. Mariuzza, and D. H. Margulies Binding of the Natural Killer Cell Inhibitory Receptor Ly49A to Its Major Histocompatibility Complex Class I Ligand. CRUCIAL CONTACTS INCLUDE BOTH H-2Dd AND beta 2-MICROGLOBULIN J. Biol. Chem., January 4, 2002; 277(2): 1433 - 1442. [Abstract] [Full Text] [PDF]
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(polymorphic residues) or 
(conserved residues) on top of the sequence. B,
Structure of H-2Dd (PDB accession: 1bii) 
) or presence of
anti-Ly49A (•) or control Ab (