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
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
Ly49A is a C-type lectin-like receptor on NK cells that recognizes MHC class I ligands, H-2Dd and Dk. 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-2Dd 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-2Kb, Ib, Dq, Qa-1/Tlab) but not from C57BL/10 mice (H-2b) identifies H-2Dq and Lq as candidates for a Ly49A ligand. Binding of sLy49A tetramer to H-2Dq- or Lq-transfected cell lines demonstrates that the two highly related MHC class I molecules, H-2Dq and Lq, 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
Transmitting editor: K. Okumura
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