The activating Ly49W and inhibitory Ly49G NK cell receptors display similar affinities for identical MHC class I ligands.

The Ly49 receptor family plays an important role in the regulation of murine natural killer (NK) cell effector function. They recognize cell surface-expressed class I MHC (MHC-I) and are functionally equivalent to the killer Ig-related receptors (KIRs) in human NK cells. Ly49s exist in activating and inhibitory forms with highly homologous extracellular domains, displaying greater variability in the stalk regions. Inhibitory Ly49s can recognize self-MHC-I and therefore mediate tolerance to self. The role of activating Ly49 receptors is less clear. Some activating Ly49 receptors have been shown to recognize MHC-I molecules. The binding affinity of activating Ly49 receptors with MHC-I is currently unknown, and we sought to examine the affinities of two highly related receptors, an activating and an inhibitory Ly49 receptor, for their shared MHC-I ligands. The ectodomain of inhibitory Ly49G of the BALB/c mouse strain is highly similar to the Ly49W activating receptor in the nonobese diabetic (NOD) mouse. Recombinant soluble Ly49G and W were expressed, refolded, and analyzed for binding affinity with MHC-I by surface plasmon resonance. We found that Ly49G and Ly49W bound with similar affinity to the same MHC-I molecules. These results are a first determination of an activating Ly49 receptor affinity for MHC-I and show that, unlike prior results obtained with activating and inhibitory KIR receptors, functional homologues to Ly49 receptors, activating and inhibitory Ly49, can recognize common MHC-I ligands, with similar affinities.

Recognition of class I MHC by a rat Ly49 NK cell receptor is dependent on the identity of the P2 anchor amino acid of bound peptide.

Members of the rodent Ly49 receptor family control NK cell responsiveness and demonstrate allele specificity for MHC class I (MHC-I) ligands. For example, the rat Ly49i2 inhibitory NK cell receptor binds RT1-A1(c) but not other rat MHC class Ia or Ib molecules. RT1-A1(c) preferentially binds peptides with proline at the second, or P2, position, which defines it as an HLA-B7 supertype MHC-I molecule. Previously, our laboratory showed that mutations within the MHC-I supertype-defining B-pocket of RT1-A1(c) could lead to alterations in P2 anchor residues of the peptide repertoire bound by RT1-A1(c) and loss of recognition by Ly49i2. Although suggestive of peptide involvement, it was unclear whether the peptide P2 anchor residue or alteration of the RT1-A1(c) primary sequence influenced Ly49i2 recognition. Therefore, we directly investigated the role of the P2 anchor residue of RT1-A1(c)-bound peptides in Ly49i2 recognition. First, fluorescent multimers generated by refolding soluble recombinant RT1-A1(c) with individual synthetic peptides differing only at the P2 anchor residue were examined for binding to Ly49i2 NK cell transfectants. Second, cytotoxicity by Ly49i2-expressing NK cells toward RMA-S target cells expressing RT1-A1(c) bound with peptides that only differ at the P2 anchor residue was evaluated. Our results demonstrate that Ly49i2 recognizes RT1-A1(c) bound with peptides that have Pro or Val at P2, whereas little or no recognition is observed when RT1-A1(c) is complexed with peptide bearing Gln at P2. Thus, the identity of the P2 peptide anchor residue is an integral component of MHC-I recognition by Ly49i2.

The rat RT1-A1c MHC molecule is a xenogeneic ligand recognized by the mouse activating Ly-49W and inhibitory Ly-49G receptors.

Mouse Ly-49 receptors are known to recognize xenogeneic ligands from hamster and rat. However, until now, there has been no description of a specific rat xenogeneic ligand for any mouse Ly-49 receptor. In this report, we identify RT1-A1c, a rat classical class I MHC molecule, as a ligand for the Ly-49G(BALB/c) inhibitory receptor and the closely related activating receptor, Ly-49W. Xenogeneic class I recognition of targets from PVG but not DA strain rats was mapped to the classical region of the RT1c haplotype by using Con A blasts from RT1c/RT1av1 intra-MHC recombinant rats as targets for RNK-16 cells expressing either Ly-49W or Ly-49G(BALB/c) receptors. Individual expression of class I molecules from PVG and DA rat strains in YB2/0 target cells demonstrate the xenogeneic recognition to be allele specific, because other class I molecules of the RT1c haplotype, RT1-A2c and RT1-U2c, and a classical class I molecule encoded by the RT1av1 haplotype, RT1-Aa, are not recognized by Ly-49W and -G(BALB/c). Furthermore, specificity for RT1-Ac can be transferred from Ly-49W to Ly-49P, which is normally unable to recognize RT1-Ac, by substitution of three residues shared by Ly-49W and -G(BALB/c) but not Ly-49P. These residues are located in the Ly-49 beta4-beta5 loop, which can determine class I allele specificity in mouse Ly-49 receptor interactions with mouse class I ligands, suggesting that mouse Ly-49 recognition of rat class I molecules follows similar principles of interaction. These findings have implications for xenotransplantation studies and for discerning Ly-49 recognition motifs present in MHC molecules.

Ly-49 receptors and their functions.

Ly-49 receptors are lectin-like type II transmembrane disulfide-bonded homodimers expressed on natural killer (NK) cells and some T-cell subsets. Cell-mediated cytotoxicity and release of cytokines/chemokines are functions regulated by Ly-49 recognition of class I major histocompatibility complex proteins (MHC-I) or virus-encoded MHC-like product(s). Here we examine diversity and conservation found within the Ly-49 gene family and explore the importance of polymorphism in Ly-49 receptor expression, specificity, and function. Several parallels are evident between Ly-49 receptors in rodents and killer Ig-related (KIR) receptors in humans, including receptor gene amplification and diversification, expression patterns, MHC-I specificity, shared signaling, and ultimate effects on NK-cell functions. These similarities suggest that insights gained in defining Ly-49 receptor functions in small animal models could have direct relevance to existing clinical challenges where there may be opportunities to manipulate human NK cells and KIR receptors for therapeutic benefit.

Reciprocal transfer of class I MHC allele specificity between activating Ly-49P and Ly-49W receptors by exchange of beta 4-beta 5 loop residues.

Receptors of the Ly-49 multigene family regulate rodent NK cell functions. Ly-49Rs are highly polymorphic and exist in either activating or inhibitory forms. Examples of both Ly-49 receptor types have been shown to recognize class I MHC ligands. Ly-49Rs can distinguish between class I alleles, but the molecular basis of this discrimination is unknown. Two activating receptors, Ly-49P and Ly-49W, differ in class I recognition, recognizing H-2D(d), or H-2D(d) and D(k), respectively. In this report, we demonstrate that specificity for H-2D(k) can be transferred from Ly-49W to Ly-49P by substituting 3 aa predicted to reside in the beta4-beta5 loop of Ly-49W into Ly-49P. Replacement of these same residues of Ly-49W with corresponding residues in Ly-49P eliminates H-2D(k) recognition while still preserving H-2D(d) recognition. Further mutagenesis indicates that all 3 aa facilitate optimal class I specificity exchange. These results provide the first evidence for a specific site on Ly-49Rs, the beta4-beta5 loop, in determining class I MHC allele specificity.

Expression of monolysocardiolipin acyltransferase activity is regulated in concert with the level of cardiolipin and cardiolipin biosynthesis in the mammalian heart.

BACKGROUND: Monolysocardiolipin acyltransferase (MLCL AT) catalyzes the acylation of monolysocardiolipin to cardiolipin in mammalian tissues. We previously reported that cardiac cardiolipin levels, MLCL AT and cardiolipin synthase activities were all elevated in rats made hyperthyroid by thyroxine treatment. In this study, we examined if cardiac mitochondrial MLCL AT activity was dependent upon the biosynthesis and level of cardiolipin in the heart. Rat heart mitochondrial MLCL AT activity was determined under conditions in which the levels of cardiac cardiolipin and cardiolipin synthase activity were either reduced or unaltered using four different disease models in the rat. In addition, these parameters were examined in a murine model of cardiac cell differentiation. RESULTS: In rats made hypothyroid by treatment with 6-n-propyl-2-thiouracil in the drinking water for 34 days, cardiac cardiolipin content was decreased 29% (p < 0.025) and this was associated with a 32% decrease (p < 0.025) in cardiolipin synthase and a 35% reduction (p < 0.025) in MLCL AT activities. Streptozotocin-induced diabetes or hyperinsulinemia in rats did not affect cardiac cardiolipin content nor MLCL AT and cardiolipin synthase activities. Finally, cardiolipin content, MLCL AT and cardiolipin synthase activities were unaltered during murine P19 teratocarcinoma cell differentiation into cardiac myocytes. In all models, phospholipase A2 activities were unaltered compared with controls. CONCLUSION: We propose a general model in which the expression of MLCL AT activity is regulated in concert with the biosynthesis and level of cardiolipin in the heart.