H-2 allele specificity of the NK cell C-type lectin-like MHC class I receptor Ly49A visualized by soluble Ly49A tetramer.
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.
Matsumoto
N
N
Laboratory of Molecular Medicine, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Tajima
K
K
Mitsuki
M
M
Yamamoto
K
K
D000483:Alleles; D000818:Animals; D000950:Antigens, Ly; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006183:H-2 Antigens / Q000235:genetics*; D066298:In Vitro Techniques; D007694:Killer Cells, Natural / Q000276:immunology*; D037102:Lectins / Q000378:metabolism; D037181:Lectins, C-Type; D008024:Ligands; D008214:Lymphocytes / Q000276:immunology; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D055650:NK Cell Lectin-Like Receptor Subfamily A; D020836:Protein Structure, Quaternary; D055630:Receptors, NK Cell Lectin-Like; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013154:Spleen / Q000276:immunology
D000950:Antigens, Ly; D002352:Carrier Proteins; D017931:DNA Primers; D006183:H-2 Antigens; C527088:Klra1 protein, mouse; D037102:Lectins; D037181:Lectins, C-Type; D008024:Ligands; D008565:Membrane Proteins; D055650:NK Cell Lectin-Like Receptor Subfamily A; D055630:Receptors, NK Cell Lectin-Like; D011994:Recombinant Proteins