Natural killer (NK) cells express receptors that recognize major histocompatibility complex (MHC) class I molecules and regulate cytotoxicity of target cells. In this study, we demonstrate that Ly49A, a prototypical C-type lectin–like receptor expressed on mouse NK cells, requires species-specific determinants on β2-microglobulin (β2m) to recognize its mouse MHC class I ligand, H-2Dd. The involvement of β2m in the interaction between Ly49A and H-2Dd is also demonstrated by the functional effects of a β2m-specific antibody. We also define three residues in α1/α2 and α3 domains of H-2Dd that are critical for the recognition of H-2Dd on target cells by Ly49A. In the crystal structure of the Ly49A/H-2Dd complex, these residues are involved in hydrogen bonding to Ly49A in one of the two potential Ly49A binding sites on H-2Dd. These data unambiguously indicate that the functional effect of Ly49A as an MHC class I–specific NK cell receptor is mediated by binding to a concave region formed by three structural domains of H-2Dd, which partially overlaps the CD8 binding site.
Immune cells are known to express specific recognition molecules for cell surface glycans. However, mechanisms involved in glycan-mediated cell–cell interactions in mucosal immunity have largely been left unaccounted for. We found that several glycans preferentially expressed in nonmalignant colonic epithelial cells serve as ligands for sialic acid-binding Ig-like lectins (siglecs), the immunosuppressive carbohydrate-recognition receptors carried by immune cells. The siglec ligand glycans in normal colonic epithelial cells included disialyl Lewisa, which was found to have binding activity to both siglec-7 and -9, and sialyl 6-sulfo Lewisx, which exhibited significant binding to siglec-7. Expression of these siglec-7/-9 ligands was impaired upon carcinogenesis, and they were replaced by cancer-associated glycans sialyl Lewisa and sialyl Lewisx, which have no siglec ligand activity. When we characterized immune cells expressing siglecs in colonic lamina propriae by flow cytometry and confocal microscopy, the majority of colonic stromal immune cells expressing siglec-7/-9 turned out to be resident macrophages characterized by low expression of CD14/CD89 and high expression of CD68/CD163. A minor subpopulation of CD8+ T lymphocytes also expressed siglec-7/-9. Siglec-7/-9 ligation suppressed LPS-induced cyclooxygenase-2 expression and PGE2 production by macrophages. These results suggest that normal glycans of epithelial cells exert a suppressive effect on cyclooxygenase-2 expression by resident macrophages, thus maintaining immunological homeostasis in colonic mucosal membranes. Our results also imply that loss of immunosuppressive glycans by impaired glycosylation during colonic carcinogenesis enhances inflammatory mediator production.
Siglec-7 (p75/AIRM1) is an inhibitory receptor on human natural killer cells (NK cells) and monocytes. The cytoplasmic domain of Siglec-7 contains two signaling motifs: a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) (Ile435-Gln-Tyr-Ala-Pro-Leu440) and a membrane-distal motif (Asn458-Glu-Tyr-Ser-Glu-Ile463). We report here that, upon pervanadate (PV) treatment, Siglec-7 recruited the protein tyrosine phosphatases Src homology-2 (SH2) domain-containing protein-tyrosine phosphatase-1 (SHP-1) and SHP-2 less efficiently than did other inhibitory receptors such as Siglec-9 and leukocyte-associated Ig-like receptor (LAIR-1). Alignment of the amino acid sequences of the two Siglecs revealed only three amino acids difference in these motifs. To identify the amino acid(s) critical to recruitment efficiency, we prepared a series of Siglec-7-based mutants in which each of the three amino acids were replaced with the corresponding one of Siglec-9 (I435L, P439S, and N458T mutants). P439S and N458T mutants showed pronounced enhancement of SHP recruitment, but I435L mutant had little effect. A double mutant (P439S, N458T) or triple mutant (I435L, P439S, N458T) recruited SHPs as much as did Siglec-9, indicating that Pro439 in the proximal motif and Asn458 in the distal motif of Siglec-7 attenuate its ability to recruit phosphatases. These amino acids appeared to affect not only phosphatase recruitment but also the subsequent attenuation of Syk phosphorylation.
The mouse inhibitory NK cell receptor Ly49A recognizes the mouse MHC class I molecule H-2D(k). The present study focuses on the species specificity of beta(2)-microglobulin (beta(2)m), an invariant component of MHC class I, in the interaction between Ly49A and H-2D(k). Transfection of the beta(2)m-defective mouse cell line R1E/TL8x.1 with human (h) beta(2)m induced cell-surface expression of H-2D(k), but failed to protect the cells from killing by Ly49A(+) NK cells. In contrast, the cells transfected with mouse (m) beta(2)m were protected from killing by Ly49A(+) NK cells. These data indicate that Ly49A distinguishes mbeta(2)m from hbeta(2)m when it recognizes the H-2D(k) complexes. To identify the species-specific determinant of beta(2)m required for Ly49A recognition of H-2D(k), we prepared a panel of mbeta(2)m mutants and tested the H-2D(k) that included each of the beta(2)m mutants for its capacity to engage Ly49A on NK cells. Ly49A failed to functionally recognize the H-2D(k) that included the mbeta(2)m with K3R and Q29G mutations. Moreover, Ly49A was able to recognize the H-2D(k) that included the hbeta(2)m with R3K and G29Q mutations. These data indicate that Lys3 and Gln29 consist of the central part of the species-specific determinant of beta(2)m required for Ly49A recognition of H-2D(k). 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 beta(2)m in NK cell recognition of target cells.
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