International Immunology

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (10) Request Permissions Google Scholar Articles by Matsumoto, N. Articles by Yamamoto, K. Search for Related Content PubMed PubMed Citation Articles by Matsumoto, N. Articles by Yamamoto, K. Social Bookmarking          What's this?

International Immunology, Vol. 13, No. 5, 615-623, May 2001
© 2001 Japanese Society for Immunology

H-2 allele specificity of the NK cell C-type lectin-like MHC class I receptor Ly49A visualized by soluble Ly49A tetramer

Naoki Matsumoto, Kyoko Tajima, Motoaki Mitsuki and Kazuo Yamamoto

Laboratory of Molecular Medicine, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

Correspondence to: N. Matsumoto

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Ly49A is a C-type lectin-like receptor on NK cells that recognizes MHC class I ligands, H-2D^d and D^k. The engagement of Ly49A with the ligands inhibits activation of NK cells and protects target cells from lysis by NK cells. Here we express the extracellular region of Ly49A with an N-terminal biotinylation tag in Escherichia coli to obtain soluble Ly49A (sLy49A) after refolding. sLy49A is indistinguishable from native Ly49A expressed on NK cells serologically and in the ability to specifically bind H-2D^d after tetramerization with R-phycoerythrin-coupled streptavidin. The fluorescently labeled tetramer of sLy49A is applied to explore MHC class I haplotype specificity of Ly49A. We demonstrate the hierarchical reactivity of Ly49A with H-2 of various alleles in the order of d > k, r > p > v > q > s > z. Reactivity of sLy49A tetramer to spleen lymphocytes from B10.QBR mice (H-2K^b, I^b, D^q, Qa-1/Tla^b) but not from C57BL/10 mice (H-2^b) identifies H-2D^q and L^q as candidates for a Ly49A ligand. Binding of sLy49A tetramer to H-2D^q- or L^q-transfected cell lines demonstrates that the two highly related MHC class I molecules, H-2D^q and L^q, are ligands for Ly49A. sLy49A tetramer staining also demonstrates preferential expression of Ly49A ligand on a subset of B cells in P/J mice. These results provide the basis to examine the molecular mechanism by which Ly49A discriminates polymorphic MHC class I molecules.

Keywords: biotin ligase, biotinylation, BirA, flow cytometry, inhibitory receptor, NK cells, streptavidin, tetramer

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
NK cells express receptors that recognize MHC class I molecules (1,2). The recognition of specific ligands on target cells by these receptors leads to inhibition or activation of NK cells depending on the motif found in cytoplasmic or transmembrane regions of the receptors. The receptors mediate NK cell discrimination of self from non-self cells by recognition of specific ligands on target cells. The inhibitory MHC class I receptors expressed on NK cells are classified into two types by their structure. One type represented by human killer cell Ig-like receptors is a type I transmembrane protein with two or three Ig-like domains in the extracellular region (3). The second type of receptors, which includes mouse Ly49 and human and mouse CD94/NKG2, is a dimer of type II transmembrane proteins with extracellular C-type lectin-like domains (1,2,4–6).

Mouse Ly49 is a family of closely related receptors with >10 member (7,8). To date, binding to classical MHC class I molecules has been shown for Ly49A (9), C (10), D (11,12), G2 (13,14) and I (15), and the MHC class I reactivity and specificity for the rest of the molecules remains to be determined. Ly49A is a primary member of the mouse Ly49 family and recognizes the classical MHC class I molecules, H-2D^d and D^k (9,16,17). Engagement of Ly49A by MHC class I ligands on target cells inhibits the activation of NK cells and protects the target cells from lysis by Ly49A⁺ NK cells. The MHC class I specificity of Ly49A was first defined by functional target cell killing assays. Ly49A⁺ NK cells are unable to kill mouse tumor cells with H-2^d or H-2^k haplotypes (9). Introduction of H-2D^d but not of K^d and L^d into H-2^b tumor cells protects the tumor cells from killing by Ly49A⁺ NK cells. However, the findings that another inhibitory receptor Ly49G2 recognizes the same ligand H-2D^d (13,14) and that activating receptor Ly49D also recognizes H-2D^d (11,12) has complicated the interpretation of results from functional killing assays using NK cells with multiple receptors. The MHC class I specificity of Ly49 molecules has also been examined by physical cell–cell binding assay (18,19). However, even in a system where a single species of Ly49 is expressed on recipient cells, potential involvement of other cell adhesion molecules makes this result dependent on other factors. MHC class I molecules purified from tumor cell lines were used to demonstrate binding of EL4 tumor cells, which express Ly49A (17). More recently soluble MHC class I tetramers with single species of peptides were used to examine the specificity of Ly49 family molecules (15,20). However, the methods are not suitable to test a large number of highly polymorphic MHC class I molecules for binding to NK cell receptors.

In the present study we developed a system to examine the interaction of Ly49A with potential MHC class I ligands using a soluble form of Ly49A (sLy49A), which was expressed in Escherichia coli and refolded in vitro. We demonstrate that sLy49A upon tetramerization and fluorescence labeling can bind H-2D^d expressed on tumor or normal cells with indistinguishable specificity from native Ly49A on NK cells. By using sLy49A tetramer we demonstrate H-2 allele specificity of Ly49A in MHC class I recognition and heterogeneity of spleen B cells from P/J mice in expression of Ly49A ligands.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cells
C1498 cells obtained form ATCC (Rockville, MD) were maintained in RPMI 1640 medium (Sigma, St Louis, MO) supplemented with 10% heat-inactivated FCS (Intergen, New York, NY), 25 mM HEPES (Dojin, Kumamoto, Japan), 100 µg/ml penicillin, 100 U/ml streptomycin, 2 mM glutamine and 50 µM 2-mercaptoethanol. C1498 cells transfected with H-2D^d, K^d or L^d (9,21) were maintained in the same medium supplemented with 0.5 mg/ml of G418 sulfate (CalbiochemNovabiochem, San Diego, CA).

Mice
C57BL/6J mice were purchased from Clea Japan (Tokyo, Japan). BALB/cCr Slc, C3H/HeSlc, C57BL/10SnSlc, B10.A/SgSnSlc, B10.BR/SgSnSlc, B10.D2/nSnSlc and B10.QBR/SxSlc NZW/N Slc mice were purchased from Nippon SLC (Shizuoka, Japan). P/J, PL/J, RIIIS/J, SM/J and SWR/J mice were generous gifts from Dr Toshihiko Shiroishi (Genetic Stock Research Center, National Institute of Genetics, Mishima, Japan).

Antibodies and reagents
Anti-H-2D^d antibodies, 34-5-8S, 34-1-2S, 34-4-20S and 34-2-12S (22), and anti-H-2L^d antibodies, 30-5-8S and 28-14-8S (23), were purified from culture supernatants with Protein A-affinity column chromatography. Anti-H-2D^q antibody, 117 (24), was purchased from PharMingen (San Diego, CA). Anti-L^q, 248 (24), was a generous gift from Sherry Turner (McLaughlin Research Institute, Great Falls, MT) and was used as culture supernatant. Anti-Ly49A antibodies, A1 (25), YE1/32, YE1/48 (26) and JR9.318 (27) were purified from culture supernatants with Protein A- or Protein G-affinity column chromatography. YE1/48 antibody was chemically biotinylated with sulfo-NHC-LC-biotin (Pierce, Rockford, IL). Anti-CD16/32 antibody, 2.4G2 (28), was used as culture supernatant. FITC-conjugated goat anti-mouse IgG F(ab')₂ fragments were purchased from ICN Pharmaceuticals (Aurora, OH). Streptavidin–agarose was from Calbiochem-Novabiochem. All other reagents were purchased from Wako Chemicals (Osaka, Japan) unless otherwise indicated.

Construction of plasmids
A DNA fragment containing the 5' NdeI site followed by the sequence encoding biotinylation signal, SmaI and BamHI sites was created by PCR amplification of a synthesized template; 5'-GGAATTCCATATGTCCGGCCTGAACGACATC-TTCGAAGCTCAGAAAATCGAATGGCACGAAGGCGCGCC-CGGGTCGAGGATCCCG-3' with primers; 5'-GGAATTCCATATGTCCGGC-3' and 5'-CGGGATCCTCGACCCG-3'. NdeI and BamHI sites in the template and the primers are shown in italic. The product digested with NdeI and BamHI was cloned between NdeI and BamHI sites of pET3c (29). This construct, designated pET3Nbio, allowed the expression of a protein with the N-terminal biotinylation signal (30), MSGLNDIFEAQKIEWH, in E. coli and in vitro enzymatic biotinylation of the expressed protein.

cDNA fragment encoding the extracellular region of Ly49A was amplified by PCR with primers 5'-GATCCCGGGAAAATTTTTCAGTATGATCAAC-3' and 5'-CGTGGATCCTTTAACACTCATTGGAG-3' using Ly49A cDNA (clone pA1.3) (31) as a template. These primers contained SmaI and BamHI sites respectively, as indicated in italic. The amplified fragment was digested with SmaI and BamHI, then cloned between SmaI and BamHI sites of pET3Nbio to construct pET3NbioLy49A. All the PCR reactions were carried out with KOD Plus polymerase (Toyobo, Osaka, Japan). The sequences of DNA encoding biotinylation tag and Ly49A were confirmed by sequencing the constructs using automatic sequencer LS-2000 (LI-COR, Lincoln NE) or ABI310 (ABI).

Preparation of sLy49A
BL21 (DE3) pLysS strain of E. coli was transformed with pETNbioLy49A. Expression of sLy49A was induced by the addition of isopropyl ß-D-thiogalactopyranoside (0.5 mM), when the OD₆₀₀ of the cell culture reached 0.5–0.7. The cells were then cultured for 3 h before harvesting by centrifugation. The cell pellets from 1 l of culture were washed with 20 mM Tris–HCl buffer (pH 8.0) containing 150 mM NaCl (TBS) and then resuspended in 20 ml of the same buffer. After the addition of PMSF (1 mM), the cells were lysed by freezing and thawing. After addition of Triton X-100 and DTT to the final concentration of 0.5% (v/v) and 1 mM respectively, the lysates were sonicated on ice and then centrifuged at 8000 g for 15 min. The pellets were washed 4 times with TBS containing 0.5% (v/v) Triton X-100, 1 mM DTT and 0.5 mM EDTA, and then twice with TBS. The pellets were solubilized in 10 ml of 100 mM Tris–HCl buffer (pH 8.0) with 6 M guanidine–HCl, 1 mM DTT and 0.1 mM EDTA, and then used for refolding by dilution as described by Garboczi et al. (32) with minor modifications. Briefly, 90 mg of the solubilized protein was diluted into 500 ml of 100 mM Tris–HCl buffer (pH 8.2), containing 0.4 M arginine, 0.5 mM glutathione oxidized form, 5 mM glutathione reduced form, 0.1 mM PMSF and 0.1 mM EDTA, then incubated at 4°C for 72 h. The diluted protein was then dialyzed extensively against 10 mM Tris–HCl buffer (pH 8.5) containing 20 mM NaCl and 0.1 mM PMSF. The refolded sLy49A was concentrated by reverse dialysis against polyethylene glycol 20,000 and ultrafiltration using Centriplus10 (Millipore, Bedford, MA). The refolded sLy49A was purified by anion-exchange column chromatography on a UNO Q-6 column (BioRad, Hercules, CA) and gel-filtration chromatography. In-anion exchange chromatography with 10 mM Tris–HCl buffer (pH 8.5) as a mobile phase the correctly refolded sLy49A was recovered in the flow-through fraction. The gel-filtration column chromatography was performed on a Superdex-200 10/30 column (Amersham-Pharmacia Biotech, Little Chalfont, UK) equilibrated with 10 mM Tris–HCl buffer (pH 8.0) containing 50 mM NaCl at the flow rate of 0.5 ml/min. The column was calibrated with gel-filtration standards from BioRad. Refolding of sLy49A was monitored by sandwich ELISA using two anti-Ly49A antibodies, YE1/32 and YE1/48. YE1/32 antibody coated on 96-well plastic plates (high protein binding; Greiner Japan, Tokyo, Japan) was used to capture sLy49A, and bound sLy49A was detected by biotinylated YE1/48 antibody, horseradish peroxidase-conjugated streptavidin (SA; Sigma) and o-phenylenediamine substrate (Sigma FAST OPD; Sigma). The refolding of Ly49A was also monitored by four kinds of anti-Ly49A antibodies, A1, YE1/32, YE1/48 and JR9.318, with standard methods. Briefly, refolded Ly49A absorbed onto 96-well plastic plates (Greiner) was incubated with anti-Ly49A antibodies, and binding of antibodies was detected by alkaline phosphatase-conjugated goat anti-mouse IgG (H + L) (BioRad) and p-nitrophenylphosphate (Sigma FAST pNPP).

Protein assay
Protein was assayed using the BCA protein assay kit (Pierce) with BSA as standard.

Biotinylation of sLy49A and formation of fluorescent sLy49A tetramer
Purified Ly49A was biotinylated with biotin ligase BirA (Avidity, Denver, CO) following the manufacturer's protocol and free biotin was removed by gel-filtration chromatography using a Superdex-200 10/30 column. The biotinylation was monitored with a sandwich ELISA system using YE1/32 as capturing antibody and horseradish peroxidase-conjugated SA (Sigma) as detecting reagent. Soluble Ly49A tetramer was formed by incubating the biotinylated sLy49A with R-phycoerythrin–coupled SA (PE–SA) (PharMingen).

Flow cytometry
For staining with antibody, cells were incubated with 10 µg/ml of purified antibodies for 30 min. Then, cells were washed 3 times with HBSS (Gibco BRL) containing 0.1% BSA and 0.1% sodium azide (FACS buffer), and were stained with 10 µg/ml of FITC–goat anti-mouse IgG F(ab')₂ for 20 min. The cells were washed 2 times with the same buffer and analyzed on a FACSCalibur with CellQuest software (Becton Dickinson Immunocytometry Systems, Mountain View, CA). In total, 10,000 events of live cells gated by forward and side scattering and exclusion of propidium iodide were acquired for analysis.

For staining with sLy49A tetramer, cells were incubated on ice for 60 min with the indicated concentration of sLy49A tetramer and then washed 2 times with FACS buffer. Then cells were fixed by adding equal volumes of 1% paraformaldehyde in PBS. The stained cells were analyzed as described above, and events from the cells gated by forward and side scattering were collected. For inhibition experiments, cells or Ly49A tetramer were preincubated with the indicated concentration of antibodies for 20 min on ice, before incubating cells with sLy49A tetramer.

Viable spleen lymphocytes from 6- to 16-week-old female mice were prepared by density-gradient centrifugation over Lympholyte M (Cedarlane, Ontario, Canada) at 200 g for 20 min. The spleen lymphocytes were preincubated with anti-CD16/32 antibody, 2.4G2, then stained with sLy49A tetramer as described above.

For two-color analysis, spleen lymphocytes were incubated with FITC–goat anti-mouse IgG F(ab')₂ fragment and sLy49A tetramer for 1 h, and then washed twice with FACS buffer. The cells were fixed and analyzed as described above.

Transfection
For expression of H-2D^q and L^q, C1498 cells were independently transfected with linearized cosmid by electroporation, then selected in the presence of 1 mg/ml of G418 sulfate and stable clones were established by limiting dilution as described (21). The cosmid clones 5.1 and 33.3, which harbor H-2D^q and L^q respectively (33), were generous gifts from Dr Ted Hansen (Washington University School of Medicine). The established clones were assayed for expression of H-2D^q and L^q by staining with 117 and 248 antibodies respectively.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Preparation of sLy49A
The extracellular region of Ly49A was tagged with the N-terminal biotinylation signal (30), expressed in E. coli and refolded. The refolded sLy49A was purified to homogeneity by column chromatography. On SDS–PAGE, purified sLy49A migrated as a 22.4-kDa protein under non-reducing conditions and as 25.7-kDa mass under reducing conditions, indicating that sLy49A exists as a monomer with intramolecular disulfide bonds (Fig. 1A). Because native Ly49A expressed on NK cells is a disulfide-linked homodimer, we examined the behavior of sLy49A in gel-permeation chromatography. When 250 µl of 67.2 µM sLy49A protein was injected, sLy49A was eluted as a single asymmetrical peak at 32.05 min with an apparent mass of 24 kDa (Fig. 1B). In the course of purifying refolded sLy49A, we observed that when sLy49A was applied to gel-filtration column immediately after concentration by ultrafiltration, a shoulder peak was eluted on the earlier side of the main sLy49A peak. However, this shoulder peak, most likely sLy49A dimer, diminished when the same sample was incubated for 3 h on ice (Fig. 2A). SDS–PAGE analysis of the eluted fractions under non-reducing condition showed only the presence of sLy49A monomer (data not shown). These data suggest that a non-covalently bonded dimer of sLy49A is reversibly formed under high concentrations, in this case, achieved locally by ultrafiltration.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Preparation of sLy49A. (A) Refolded sLy49A (1.5 µg) was analyzed on 12.5% T SDS–PAGE under reducing or non-reducing conditions. The gel was stained with Coomassie brilliant blue. Mol. wt markers were phosphorylase b (97.4 kDa), BSA (66.3 kDa), aldolase (42.4 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and hen egg lysozyme (14.4 kDa). Each lane is as follows: R, reducing condition; NR, non-reducing condition; M, mol. wt markers. Estimated mol. wt values of sLy49A under reducing and non-reducing conditions are indicated with arrows. (B) Refolded sLy49A was analyzed by Superdex 200 column chromatography at the flow rate of 0.5 ml/min. Elution of mol. wt standards is shown by arrows on top.

 

View larger version (13K): [in this window] [in a new window]   Fig. 2. Gel-filtration chromatography of reversibly formed sLy49A dimer and sLy49A–SA complex. (A) Concentrated sLy49A was analyzed by gel-filtration column chromatography immediately after concentration (0 hr) or after 3 h of incubation (3 hr). During 3 h of incubation most of the sLy49A dimer dissociated into monomer. (B) Excess amount of sLy49A was mixed with SA and the mixture was analyzed by gel-filtration chromatography. sLy49A tetramer complexed with SA and sLy49A monomer were detected.

 
Specific recognition of H-2D^d by sLy49A tetramer
To multimerize sLy49A, we enzymatically biotinylated sLy49A using BirA biotin ligase. Absorption analysis with SA-coupled agarose indicated that at least 95% of sLy49A was biotinylated (data not shown). Biotinylated sLy49A was used to form tetramers with SA. In gel-filtration chromatography, the complex of sLy49A and SA was eluted at the position of 160 kDa, as expected from the molecular mass of sLy49A tetramer (100 kDa) and SA (60 kDa) (Fig. 2B).

To use sLy49A tetramer for flow cytometry, we tetramerized sLy49A using PE–SA. The fluorescent sLy49A tetramer was tested for binding to C1498 cells untransfected or transfected with H-2D^d, K^d or L^d. sLy49A tetramer bound to H-2D^d-transfected C1498 cells, although not significantly to H-2K^d-, L^d-transfected or untransfected C1498 cells (Fig. 3A). The binding was concentration dependent (Fig. 3B), while it was not saturated even at the highest concentration used here. The binding of fluorescently labeled sLy49A tetramer was competed partially by 10-fold amount of unlabeled sLy49A tetramer, indicating the specificity of the binding. To further test the specificity, we examined the effect of various antibodies that recognize H-2D^d or Ly49A. 34-5-8S antibody, which recognizes a conformational epitope in the ₁/₂ domain of H-2D^d (34), completely abrogated the binding of sLy49A tetramer to H-2D^d, while 34-1-2S and 34-4-20S antibodies, both of which recognize unidentified epitopes in ₁/₂, only partially inhibited the binding of sLy49A tetramer (Fig 3C). 34-2-12S antibody, which recognizes the ₃ domain of H-2D^d, enhanced the binding of sLy49A tetramer to H-2D^d. Each of four different antibodies against Ly49A, A1, YE1/32, YE1/48 and JR9-318, completely inhibited the binding of Ly49A tetramer to H-2D^d. These results indicate that sLy49A tetramer has an ability to bind H-2D^d with similar specificity to native Ly49A expressed on NK cells.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Binding of sLy49A tetramer to H-2Dd-transfected C1498 cells. (A) H-2Dd-specific binding of Ly49A tetramer. C1498 cells transfected with H-2Dd, Kd or Ld or untransfected were incubated with 20 µg/ml of sLy49A tetramer (bold line) or PE–SA alone (dotted line). Data are representative of three independent experiments with similar results. (B) Dose-dependent binding of Ly49A to H-2Dd-transfected cells. H-2Dd-transfected C1498 cells were incubated with the indicated concentration of sLy49A tetramer and analyzed by flow cytometry. Data are representative of three independent experiments with similar results. (C) Inhibition of sLy49A tetramer binding to H-2Dd by antibodies against H-2Dd or Ly49A. H-2Dd-transfected cells were incubated with 20 µg/ml of sLy49A tetramer in the presence or absence of 50 µg/ml of indicated antibodies. Data are representative of three independent experiments with similar results.

 
H-2 allele specific binding of sLy49A tetramer to spleen lymphocytes
We applied sLy49A tetramer to explore the reactivity of Ly49A with MHC class I from various haplotypes. Spleen cells from a panel of inbred or H-2 congenic mice (Table 1) were tested for sLy49A tetramer staining by flow cytometry (Fig. 4). Very bright staining was obtained for spleen cells from strains of mice with H-2^{d, k, a, u and r} haplotypes, and moderate staining was observed for spleen cells from mice with H-2^{q and v}. Spleen cells from mice of H-2^{s and z} haplotypes were weakly stained but those from H-2^b were negligibly stained. Hierarchy of sLy49A tetramer staining of spleen lymphocytes from mice of various haplotypes was in the order of d, a, u > k, r > v > q > s > z. Complete abrogation of Ly49A tetramer binding to spleen lymphocytes by anti-Ly49A antibody A1 F(ab')₂ fragments indicates the specificity of staining (data not shown). Of these haplotypes, Ly49A staining of spleen cells from mice of H-2^{d, a and u} haplotypes is due to the binding of Ly49A to H-2D^d and the binding was completely inhibited by 34-5-8S antibody (data not shown), because these haplotypes have the H-2D^d allele (Table 1). Spleen cells from SWR/J mice (H-2^q) and B10.QBR mice, of which H-2 region has the K^b, I^b, D^q, Qa-1/Tla^b genotype (35), were stained moderately by sLy49A tetramer, while spleen cells from C57BL/10 mice were not visibly stained. These results indicate that the MHC class I located in the H-2D region, H-2D^q and (or) L^q, is responsible for the reactivity of Ly49A against spleen cells from SWR/J and B10.QBR mice.

View this table: [in this window] [in a new window]   Table 1.

 

View larger version (45K): [in this window] [in a new window]   Fig. 4. MHC class I specificity of Ly49A visualized by sLy49A tetramer. Spleen lymphocytes from the indicated strains of mice were stained with sLy49A tetramer (bold line), PE–SA alone (thin line) or unstained (dotted line). The name of each mouse strain is noted in each panel. Data are representative of three independent experiments with similar results.

 
Recognition of H-2D^q and L^q by Ly49A
To identify the MHC class I molecules that sLy49A tetramer binds in the q haplotype, we established H-2D^q or L^q transfectants and examined binding of sLy49A tetramer to them. C1498 (H-2^b) cells were stably transfected with the cosmid clones 5.1 or 33.3 that have H-2D^q or L^q genes respectively (33). The established transfected clones were confirmed for expression of H-2D^q or L^q, using specific antibodies (Fig. 5B and C). Then the transfected clones were assayed for binding of sLy49A tetramer. The H-2D^q- or L^q-transfected cells were weakly but significantly stained by sLy49A tetramer, while untransfected C1498 cells were negligibly stained (Fig. 5E–G). The sLy49A tetramer staining of H-2D^q- or L^q-transfected cells was far weaker than that of H-2D^d transfectants, suggesting a weak interaction between Ly49A and each of H-2D^q and L^q.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Binding of sLy49A tetramer to H-2Dq and Lq. C1498 cells were stably transfected with H-2Dq (B and F), H-2Lq (C and G) or H-2Dd (D and H) or untransfected (A and E). (A–D) The cells were stained with anti-MHC class I antibodies (bold line) or control antibody (dotted line) and then with FITC–goat anti-mouse IgG F(ab')2. Anti-MHC class I antibodies anti-Dd (34-2-12S), anti-Dq (117) and anti-Lq (248) were used. (E–H) The cells were stained with sLy49A tetramer (bold line) or SA–PE alone (thin line) or unstained (dotted line). The data are representative of three independent experiments.

 
Heterogeneous staining of P/J spleen lymphocytes by Ly49A tetramer
When spleen lymphocytes from P/J mice were analyzed for sLy49A tetramer binding, we observed heterogeneous staining unlike other strains (Fig. 4). One population of lymphocytes was stained moderately to the similar level as spleen cells from SM/J mice with H-2^v. Another population of lymphocytes was stained weakly to the similar level as spleen cells from B10.S mice with H-2^s. To further characterize the heterogeneous staining of P/J spleen cells with sLy49A, the spleen cells were analyzed by two-color staining for sIg expression as well as sLy49A binding (Fig. 5). The sIg⁺ population contained both moderately and weakly sLy49A-stained cells (Fig. 5E), while all of the sIg^– population of P/J lymphocytes was stained weakly with sLy49A (Fig. 5F). Thus, a subset of B cells in P/J mice is moderately stained with Ly49A tetramer, while P/J mouse T cells are weakly stained with Ly49A tetramer.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We demonstrated that sLy49A expressed in E. coli could bind its ligand H-2D^d. Combined with the result that sLy49A is serologically reactive with four different antibodies against Ly49A, this indicates that sLy49A expressed in bacteria has retained the conformational epitope required for interaction with H-2D^d and specific antibodies despite the absence of disulfide-linked homodimerization. The behavior of sLy49A in gel-filtration chromatography (Figs 2B and 3) suggests that sLy49A is in equilibrium between the monomer and dimer. Recently Natarajan et al. have shown that the extracellular region of Ly49A similarly expressed in E. coli forms non-covalent dimers at high concentration by analytical ultracentrifugation (36). Furthermore, the recently resolved crystal structure of the Ly49A–H-2D^d complex indicates the ability of Ly49A C-type lectin-like domain to dimerize in the absence of interchain disulfide bonds (37). As tetramerization with SA juxtaposes two sLy49A monomers, we speculate that sLy49A would form a dimer homologous to native Ly49A in the sLy49A–SA complex. Indistinguishable ability of sLy49A tetramer to bind H-2D^d from that of the native Ly49A strongly suggests this idea; however, we cannot exclude the possibility that the artificially formed Ly49A dimer in the sLy49A tetramer might be structurally different from the native Ly49A dimer.

The binding of the sLy49A tetramer to H-2D^d-transfected C1498 cells is specifically mediated by Ly49A, shown by the observation that any of four different antibodies against Ly49A inhibited the binding of the sLy49A tetramer to H-2D^d-transfected cells and that PE–SA minimally stained the H-2D^d-transfected cells (Fig. 2A and C). H-2D^d dependency of the staining of H-2D^d-C1498 by sLy49A tetramer was confirmed by the absence of the staining in untransfected C1498 cells and C1498 cells transfected with H-2K^d or L^d and inhibition of the staining by 34-5-8S antibody recognizing the peptide-dependent epitope in the ₁/₂ domain of H-2D^d (34). Interestingly, 34-2-12S antibody, which recognizes an epitope in the ₃ domain of H-2D^d (38), enhanced the binding of sLy49A tetramer (Fig. 3C). In the cell–cell binding assay, we observed the similar enhancement on binding of H-2D^d-transfected C1498 cells to Ly49A-transfected Chinese hamster ovary cells by 34-2-12S antibody (N. Matsumoto, unpublished observation). The enhancement of binding might be mediated by dimerization of H-2D^d on the cell surface by 34-2-12S, arranging two H-2D^d molecules in a position suitable for recognition by a Ly49A dimer. Otherwise, binding of 34-2-12S antibody to H-2D^d might induce a conformational change of H-2D^d that is favorable to Ly49A binding. Of three mAb that recognize the ₁/₂ domain of H-2D^d only 34-5-8S antibody completely inhibited binding of sLy49A tetramer to H-2D^d cells, while 34-4-20S or 34-1-2S antibodies partially inhibited the binding. Interestingly, epitopes recognized by 34-5-8S and 34-4-20S antibodies are mapped to the same region of H-2D^d (39). Further mapping of epitopes recognized by these antibodies using site-directed mutagenesis would help to understand the site of H-2D^d interacting with Ly49A.

Specificity of Ly49 to polymorphic MHC class I molecules has been examined by various methods: functional killing assay by sorted NK cells (9), cell–cell adhesion assay (18,19), cell adhesion assay to solid-phase MHC class I (17), binding of MHC class I tetramer to cells expressing Ly49 (15,20) or surface plasmon resonance analysis using sLy49 and MHC class I molecules (36). Our method has advantages over these methods in some points. Primarily, the use of flow cytometry makes our method suitable to estimate the relative avidity of interaction quantitatively. Secondly, use of soluble receptors excludes involvement of cell adhesion receptors and activation receptors, found in cell adhesion assays and killing assays respectively. Finally, use of soluble receptors rather than soluble ligands enables us to examine the MHC class I ligands in a natural setting: MHC class I molecules in glycosylated forms complexed with naturally occurring peptides. It also enables the examination of Ly49A reactivity of individual cells as shown in Fig. 5. Recently, a similar technique has been used to demonstrate binding of the C-type lectin-like receptor CD94/NKG2A to the human non-classical MHC class I HLA-E (40). A chemical biotinylated form of Ly49A has also been used to detect the interaction of Ly49A with its cellular ligands (36). However, inability of chemical biotinylation to control sites and extents hinders the preparation of the Ly49A tetramer used in this study, and obliged researchers to perform the staining in two steps. This constraint appears to limit the sensitivity to detect interaction between Ly49A and its ligands; Chung et al. recently reported the absence of Ly49A reactivity in B10.S (H-2^s) and B10.Q (H-2^q) mice (41), in contrast with our observations (Fig. 4 and Table 1).

Our results demonstrated that Ly49A interacts with many allelic forms of MHC class I other than the previously described H-2D^d, H-2^k and H-2D^p (9,42). Hanke et al. recently examined MHC allele specificity of Ly49A by cell–cell adhesion assay and functional recognition assay using T cells from Ly49A transgenic mice (15). Their results of the functional recognition assay gave the following order to H-2 alleles for Ly49A recognition: d > k, r > v > f > q, s. Our result that sLy49A tetramer bound H-2 in the order of d > k, r > p > v > q > s > z is consistent with the functional data by Hanke et al. These findings suggest that functional inhibitory activity of each MHC class I in recognition by Ly49A correlates with the physical binding ability of each MHC class I.

The reactivity of sLy49A tetramer with spleen lymphocytes from B10.QBR mice with H-2K^b, I^b, D^q, Qa-1/Tla^b and SWR mice with H-2^q but not with C57BL/6 mice with H-2^b indicated that H-2D^q and (or) L^q is responsible for the binding of Ly49A. Individual expression of H-2D^q and L^q enabled us to demonstrate that both H-2D^q and L^q are recognized by Ly49A even though the binding of sLy49A tetramer to H-2D^q- or L^q-transfected cells was far weaker than that to H-2D^d transfectants (Fig. 5). The weak reactivity of H-2D^q- or L^q-transfected cells could be the result of the lower (10-fold) expression of H-2D^q or L^q on the transfectants compared to H-2D^d (Fig. 5B and C), and of the weaker interaction between sLy49A tetramer and H-D^q and L^q: ~50-fold less binding to H-2D^q and L^q than that to H-2D^d as estimated from sLy49A tetramer staining of B10.QBR and B10.D2 spleen lymphocytes (Fig. 4). The current finding that Ly49A recognizes both of H-2D^q and L^q is consistent with the high homology between D^q and L^q (43). So far, accumulating data suggest that Ly49A is a specific receptor for allelic forms of MHC class I encoded in the H-2D region. However, our data clearly demonstrate that H-2D in b, s and z and H-2L^d are not good ligands for Ly49A. Structural comparison of H2-D capable of binding Ly49A with H-2D unable to bind Ly49A will help to reveal the structural basis of allele specific recognition of H2-D by Ly49A

Lack of significant staining of tumor or spleen cells from mouse with H-2^b haplotypes (Figs 2 and 3) is in contrast with the recent observation by Michaelsson et al. that soluble H-2D^b tetramers with LCMV gp33 peptides bind Ly49A (20). We speculate that the lack of glycosylation in the H-2D^b tetramers or the binding of LCMV gp33 peptides could account for the unexpected binding behavior. Functional recognition of H-2D^b by Ly49A has never been demonstrated. However, reduction in Ly49A⁺ NK cell frequency in wild-type compared to ß₂-microglobulin-deficient C57BL/6 mice (44,45) suggests the presence of Ly49A ligand(s) complexed with ß₂-microglobulin in C57BL/6 mice. Small numbers of glycosylation-defective H-2D^b molecules or H-2D^b molecules complexed with special peptides present in wild-type C57BL/6 mice, which are not detectable in our or previously described methods, may account for the modulation of Ly49A frequency.

Interestingly, sLy49A tetramer differentially stained a subset of B cells from P/J mice (Fig. 5). Olsson-Alheim et al. demonstrated functional recognition of H-2D^p by Ly49A (42). The preferential binding of sLy49A tetramer to a subset of B cells might result from differential expression of H-D^p, differential modification of H-2D^p including glycosylation, difference in H-2D^p-bound peptides or otherwise preferential expression of a novel ligand other than H-2D^p. It should be noted that recent studies demonstrated that recognition of H-2K^b and K^d by Ly49C and Ly49I respectively is influenced by bound peptides (15,20,46). Recognition of H-2D^d by Ly49A is not influenced by bound peptide (47,48), but it is possible that the peptide(s) that is selectively expressed in a subset of B cells favors high-affinity binding of H-2D^p by Ly49A. It is also noteworthy that recognition of H-2D^d by Ly49A is influenced by sulfation of H-2D^d-bound carbohydrates (49). Further analysis is required to elucidate a molecular mechanism that explains the preferential expression of Ly49A ligand on a subset of B cells in P/J mice. It is also important to characterize the B cell subset defined by intermediate binding of sLy49A tetramer and its function. Future studies are under planning, focusing on these subjects.

In conclusion, Ly49A can discriminate alleles of H-2 with a hierarchy and bind H-2D^q or L^q in mice with the H-2^q allele. Reactivity with Ly49A defines a novel subset of B cells in P/J mice.

View larger version (45K): [in this window] [in a new window]   Fig. 6. sLy49A tetramer staining distinguishes a subset of B cells in P/J mice. Spleen lymphocytes from P/J (A), BALB/c (B) or C57BL/6 (C) mice were stained for sIg and Ly49A tetramer binding. sLy49A tetramer staining histograms of total (D), sIg+ (E) and sIg– (F) population of P/J mice lymphocytes is shown. Data are representative of similar data obtained from three mice of each strain that were individually analyzed.

 

	Acknowledgments

 
We thank Dr Toshihiko Shiroishi for providing mice, Dr Ted Hansen and Sherry Turner for reagents, and Dr Wayne M. Yokoyama for critical reading of the manuscript. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (12672107), and by a grant for Research on Health Sciences focusing on Drug Innovation from the Japan Health Science Foundation (12259).

	Abbreviations

 

PE phycoerythrin

SA streptavidin

sLy49A soluble Ly49A

	Notes

 
Transmitting editor: K. Okumura

Received 31 July 2000, accepted 24 January 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Lanier, L. L. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359.[Web of Science][Medline]
2. Yokoyama, W. M. 1999. Natural killer cells. In Paul, W. E., ed., Fundamental Immunology, 4th edn, p. 575. Lippincott-Raven, Philadelphia, PA.
3. 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.[Web of Science][Medline]
4. 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.[Web of Science][Medline]
5. Ryan, J. C. and Seaman, W. E. 1997. Divergent functions of lectin-like receptors on NK cells. Immunol. Rev. 155:79.[Web of Science][Medline]
6. Takei, F., Brennan, J. and Mager, D. L. 1997. The Ly-49 family: genes, proteins and recognition of class I MHC. Immunol. Rev. 155:67.[Web of Science][Medline]
7. 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.[Abstract]
8. 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]
9. 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.[Medline]
10. Brennan, J., Mahon, G., Mager, D. L., Jefferies, W. A. and Takei, F. 1996. Recognition of class I major histocompatibility complex molecules by Ly-49: specificities and domain interactions. J. Exp. Med. 183:1553.[Abstract/Free Full Text]
11. Nakamura, M. C., Linnemeyer, P. A., Niemi, E. C., Mason, L. H., Ortaldo, J. R., Ryan, J. C. and Seaman, W. E. 1999. Mouse Ly-49D recognizes H-2D^d and activates natural killer cell cytotoxicity. J. Exp. Med. 189:493.[Abstract/Free Full Text]
12. George, T. C., Mason, L. H., Ortaldo, J. R., Kumar, V. and Bennett, M. 1999. Positive recognition of MHC class I molecules by the Ly49D receptor of murine NK cells. J. Immunol. 162:2035.[Abstract/Free Full Text]
13. Mason, L. H., Ortaldo, J. R., Young, H. A., Kumar, V., Bennett, M. and Anderson, S. K. 1995. Cloning and functional characteristics of murine large granular lymphocyte-1: a member of the Ly-49 gene family (Ly-49G2). J. Exp. Med. 182:293.[Abstract/Free Full Text]
14. Johansson, M. H., Hoglund, E., Nakamura, M. C., Ryan, J. C. and Hoglund, P. 1998. Alpha1/alpha2 domains of H-2D^d, but not H-2L^d, induce 'missing self' reactivity in vivo—no effect of H-2L^d on protection against NK cells expressing the inhibitory receptor Ly49G2. Eur. J. Immunol. 28:4198.[Web of Science][Medline]
15. Hanke, T., Takizawa, H., McMahon, C. W., Busch, D. H., Pamer, E. G., Miller, J. D., Altman, J. D., Liu, Y., Cado, D., Lemonnier, F. A., Bjorkman, P. J. and Raulet, D. H. 1999. Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors. Immunity 11:67.[Web of Science][Medline]
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.[Abstract]
17. Kane, K. P. 1994. Ly-49 mediates EL4 lymphoma adhesion to isolated class I major histocompatibility complex molecules. J. Exp. Med. 179:1011.[Abstract/Free Full Text]
18. 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.[Abstract/Free Full Text]
19. 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/Free Full Text]
20. Michaelsson, J., Achour, A., Salcedo, M., Kase-Sjostrom, A., Sundback, J., Harris, R. A. and Karre, K. 2000. Visualization of inhibitory Ly49 receptor specificity with soluble major histocompatibility complex class I tetramers. Eur. J. Immunol. 30:300.[Web of Science][Medline]
21. 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.[Web of Science][Medline]
22. Ozato, K., Mayer, N. M. and Sachs, D. H. 1982. Monoclonal antibodies to mouse major histocompatibility complex antigens. Transplantation 34:113.[Web of Science][Medline]
23. Ozato, K., Hansen, T. H. and Sachs, D. H. 1980. Monoclonal antibodies to mouse MHC antigens. II. Antibodies to the H- 2Ld antigen, the products of a third polymorphic locus of the mouse major histocompatibility complex. J. Immunol. 125:2473.[Abstract]
24. Hasenkrug, K. J., Cory, J. M. and Stimpfling, J. H. 1987. Monoclonal antibodies defining mouse tissue antigens encoded by the H-2 region. Immunogenetics 25:136.[Web of Science][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/beta antigen receptor. J. Immunol. 138:815.[Abstract]
26. Chan, P. Y. and Takei, F. 1988. Characterization of a murine T cell surface disulfide-linked dimer of 45-kDa glycopeptides (YE1/48 antigen). Comparison with T cell receptor, purification, and partial amino acid sequences. J. Immunol. 140:161.[Abstract]
27. Roland, J. and Cazenave, P. A. 1992. Ly-49 antigen defines an alpha beta TCR population in i-IEL with an extrathymic maturation. Int. Immunol. 4:699.[Abstract/Free Full Text]
28. Unkeless, J. C. 1979. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J. Exp. Med. 150:580.[Abstract/Free Full Text]
29. 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.[Medline]
30. 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.
31. 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]
32. 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.[Abstract/Free Full Text]
33. Lee, D. R., Rubocki, R. J., Lie, W. R. and Hansen, T. H. 1988. The murine MHC class I genes, H-2D^q and H-2L^q, are strikingly homologous to each other, H-2L^d, and two genes reported to encode tumor-specific antigens. J. Exp. Med. 168:1719.[Abstract/Free Full Text]
34. Otten, G. R., Bikoff, E., Ribaudo, R. K., Kozlowski, S., Margulies, D. H. and Germain, R. N. 1992. Peptide and beta 2-microglobulin regulation of cell surface MHC class I conformation and expression. J. Immunol. 148:3723.[Abstract]
35. Germana, S. and Shinohara, N. 1991. Qa-1/Tla region alloantigen-specific CTL with alpha beta receptor. Immunology 74:578.[Web of Science][Medline]
36. 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.[Web of Science][Medline]
37. 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.[Medline]
38. 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.[Abstract/Free Full Text]
39. Abastado, J. P., Jaulin, C., Schutze, M. P., Langlade-Demoyen, P., Plata, F., Ozato, K. and Kourilsky, P. 1987. Fine mapping of epitopes by intradomain K^d/D^d recombinants. J. Exp. Med. 166:327.[Abstract/Free Full Text]
40. Brooks, A. G., Borrego, F., Posch, P. E., Patamawenu, A., Scorzelli, C. J., Ulbrecht, M., Weiss, E. H. and Coligan, J. E. 1999. Specific recognition of HLA-E, but not classical, HLA class I molecules by soluble CD94/NKG2A and NK cells. J. Immunol. 162:305.[Abstract/Free Full Text]
41. Chung, D. H., Natarajan, K., Boyd, L. F., Tormo, J., Mariuzza, R. A., Yokoyama, W. M. and Margulies, D. H. 2000. Mapping the ligand of the NK inhibitory receptor Ly49A on living cells. J. Immunol. 165:692.
42. Olsson-Alheim, M. Y., Sundback, J., Karre, K. and Sentman, C. L. 1999. The MHC class I molecule H-2D^p inhibits murine NK cells via the inhibitory receptor Ly49A. J. Immunol. 162:7010.[Abstract/Free Full Text]
43. Rubocki, R. J., Lee, D. R., Lie, W. R., Myers, N. B. and Hansen, T. H. 1990. Molecular evidence that the H-2D and H-2L genes arose by duplication. Differences between the evolution of the class I genes in mice and humans. J. Exp. Med. 171:2043.[Abstract/Free Full Text]
44. Held, W., Dorfman, J. R., Wu, M. F. and Raulet, D. H. 1996. Major histocompatibility complex class I-dependent skewing of the natural killer cell Ly49 receptor repertoire. Eur. J. Immunol. 26:2286.[Web of Science][Medline]
45. Salcedo, M., Diehl, A. D., Olsson-Alheim, M. Y., Sundback, J., Van Kaer, L., Karre, K. and Ljunggren, H. G. 1997. Altered expression of Ly49 inhibitory receptors on natural killer cells from MHC class I-deficient mice. J. Immunol. 158:3174.[Abstract]
46. 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.[Web of Science][Medline]
47. 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.[Web of Science][Medline]
48. 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]
49. Chang, C. S. and Kane, K. P. 1998. Evidence for sulfate modification of H-2D^d on N-linked carbohydrate(s): possible involvement in Ly-49A interaction. J. Immunol. 160:4367.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				N. Kawasaki, Y. Ichikawa, I. Matsuo, K. Totani, N. Matsumoto, Y. Ito, and K. Yamamoto The sugar-binding ability of ERGIC-53 is enhanced by its interaction with MCFD2 Blood, February 15, 2008; 111(4): 1972 - 1979. [Abstract] [Full Text] [PDF]

				D. Nawa, O. Shimada, N. Kawasaki, N. Matsumoto, and K. Yamamoto Stable interaction of the cargo receptor VIP36 with molecular chaperone BiP Glycobiology, September 1, 2007; 17(9): 913 - 921. [Abstract] [Full Text] [PDF]

				N. Kawasaki, I. Matsuo, K. Totani, D. Nawa, N. Suzuki, D. Yamaguchi, N. Matsumoto, Y. Ito, and K. Yamamoto Detection of Weak Sugar Binding Activity of VIP36 using VIP36-streptavidin Complex and Membrane-based Sugar Chains J. Biochem., February 1, 2007; 141(2): 221 - 229. [Abstract] [Full Text] [PDF]

				K. Tajima, N. Matsumoto, K. Ohmori, H. Wada, M. Ito, K. Suzuki, and K. Yamamoto Augmentation of NK cell-mediated cytotoxicity to tumor cells by inhibitory NK cell receptor blockers Int. Immunol., March 1, 2004; 16(3): 385 - 393. [Abstract] [Full Text] [PDF]

				J. Zimmer, V. Ioannidis, and W. Held H-2D Ligand Expression by Ly49A+ Natural Killer (NK) Cells Precludes Ligand Uptake from Environmental Cells: Implications for NK Cell Function J. Exp. Med., November 19, 2001; 194(10): 1531 - 1539. [Abstract] [Full Text] [PDF]

				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]

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (10) Request Permissions Google Scholar Articles by Matsumoto, N. Articles by Yamamoto, K. Search for Related Content PubMed PubMed Citation Articles by Matsumoto, N. Articles by Yamamoto, K. Social Bookmarking          What's this?

Online ISSN 1460-2377 - Print ISSN 0953-8178

Copyright © 2009 Japanese Society for Immunology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics