Molecular Basis for E-cadherin Recognition by Killer Cell Lectin-like Receptor G1 (KLRG1)

The killer cell lectin-like receptor G1, KLRG1, is a cell surface receptor expressed on subsets of natural killer (NK) cells and T cells. KLRG1 was recently found to recognize E-cadherin and thus inhibit immune responses by regulating the effector function and the developmental processes of NK and T cells. E-cadherin is expressed on epithelial cells and exhibits Ca²⁺-dependent homophilic interactions that contribute to cell-cell junctions. However, the mechanism underlying the molecular recognition of KLRG1 by E-cadherin remains unclear. Here, we report structural, binding, and functional analyses of this interaction using multiple methods. Surface plasmon resonance demonstrated that KLRG1 binds the E-cadherin N-terminal domains 1 and 2 with low affinity (K_d ∼7–12 μm), typical of cell-cell recognition receptors. NMR binding studies showed that only a limited N-terminal region of E-cadherin, comprising the homodimer interface, exhibited spectrum perturbation upon KLRG1 complex formation. It was confirmed by binding studies using a series of E-cadherin mutants. Furthermore, killing assays using KLRG1⁺NK cells and reporter cell assays demonstrated the functional significance of the N-terminal region of E-cadherin. These results suggest that KLRG1 recognizes the N-terminal homodimeric interface of domain 1 of E-cadherin and binds only the monomeric form of E-cadherin to inhibit the immune response. This raises the possibility that KLRG1 detects monomeric E-cadherin at exposed cell surfaces to control the activation threshold of NK and T cells.Natural killer (NK)³ cells play a critical role in the innate immune system because of their ability to kill other cells. For example, NK cells can kill virus-infected cells and tumor cells without presensitization to a specific antigen, and they produce various cytokines, including interferon-γ and tumor necrosis factor-α (1). NK cells are controlled by both inhibitory and activating receptors that are expressed on their surfaces (2). The killer cell Ig-like receptor, Ly49, CD94/NKG2, and paired Ig-like type 2 receptor families include both inhibitory and activating members and thus are designated as paired receptor families. On the other hand, some inhibitory receptors, including KLRG1 (killer cell lectin-like receptor G1), and activating receptors, such as NKG2D, also exist. The integration of the signals from these receptors determines the final functional outcome of NK cells.These inhibitory and activating receptors can also be divided into two structurally different groups, the Ig-like receptors and the C-type lectin-like receptors, based on the structural aspects of their extracellular regions. The Ig-like receptors include killer cell Ig-like receptors and the leukocyte Ig-like receptors, and the C-type lectin-like receptors include CD94/NKG2(KLRD/KLRC), Ly49(KLRA), NKG2D(KLRK), NKR-P1(KLRB), and KLRG1. Many of these immune receptors recognize major histocompatibility complex class I molecules or their relatives (2–4), but there are still many orphan receptors expressed on NK cells. KLRG1 was one such orphan receptor; however, E-cadherin was recently found to be a ligand of KLRG1 (5, 6). Although major histocompatibility complex-receptor interactions have been extensively examined, the molecular basis of non-major histocompatibility complex ligand-receptor recognition is poorly understood.KLRG1 is a type II membrane protein, with one C-type lectin domain in the extracellular region, one transmembrane region, and one immunoreceptor tyrosine-based inhibitory motif. KLRG1 is expressed on a subset of mature NK cells in spleen, lungs, and peripheral blood during normal development. KLRG1 expression is induced on the surface of NK cells during viral responses (7, 8). NK cells expressing KLRG1 produce low levels of interferon-γ and cytokines and have a slow in vivo turnover rate and low proliferative responsiveness to interleukin-15 (9). Furthermore, KLRG1 is recognized as a marker of some T cell subsets, as follows. KLRG1 defines a subset of T cells, short lived effector CD8 T cells (SLECs), which are mature effector cells that express high levels of KLRG1 and cannot be differentiated into long lived memory CD8 T cells. In addition, memory precursor effector cells express low levels of KLRG1 and harbor the potential to become long lived memory CD8 T cells (10). Since SLECs exhibit stronger effector function than memory precursor effector cells, it is potentially beneficial, in terms of preventing harmful excess cytotoxicity, that SLECs express KLRG1 at a higher level to inhibit the immune response. Taken together, the expression of KLRG1 during the viral response and normal development might confer the inhibition of effector function and the regulation of NK and T cell proliferation (9).E-cadherin plays a pivotal role in Ca²⁺-dependent cell-cell adhesion and also contributes to tissue organization and development (11–14). E-cadherin is primarily expressed on epithelial cells, and its extracellular region consists of several domains that include cadherin motifs (15, 16). These domains mediate Ca²⁺-dependent homophilic interactions to facilitate cell adhesion. When E-cadherins form cis- or trans-homodimers, they utilize their N-terminal regions as an interface, which can dock with domain 1 of another E-cadherin to form strand exchange (17). Therefore, the N-terminal region plays important roles in homophilic binding and cell adhesion.KLRG1 recognizes E-cadherins (and other class I cadherins), which are widely expressed in tissues and form tight adhesive cell-cell junctions, and Ito et al. (5) demonstrated that E-cadherin binding by KLRG1 inhibits NK cytotoxicity. Further, Gründermann et al. (6) showed that the E-cadherin-KLRG1 interaction inhibits the antigen-induced proliferation and induction of the cytolytic activity of CD8 T cells. Therefore, it is plausible that E-cadherin recognition by KLRG1, expressed on the surfaces of NK cells and T cells, may raise their activation thresholds by transducing inhibitory signals. Such an inhibition would prevent the excess injury of normal cells, which might result in inflammatory autoimmune diseases. KLRG1 may also have an important role in monitoring and removing cancer cells that lose E-cadherin expression. A recent report demonstrated that N-terminal domains 1 and 2 of E-cadherin are critical for KLRG1 recognition (18); however, despite accumulating evidence supporting the functional importance of the E-cadherin-KLRG1 interaction, the molecular basis of this interaction is poorly understood. Here, we report that the N-terminal region of E-cadherin, comprising the dimer interface, is the binding site for KLRG1. This suggests that KLRG1 does not recognize the dimeric form of E-cadherin but rather recognizes the monomeric form, which is exposed on the cell surfaces of disrupted or infected cells. This may suppress excess immune responses.     

Design,synthesis and biological evaluations of novel 7-[3-(1-aminocycloalkyl)pyrrolidin-1-yl]-6-desfluoro-8-methoxyquinolones with potent antibacterial activity against multi-drug resistant Gram-positive bacteria

A series of novel 6-desfluoro [des-F(6)] and 6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-8-methoxyquinolones bearing 3-(1-aminocycloalkyl)pyrrolidin-1-yl substituents at the C-7 position (1–6) was synthesized to obtain potent drugs for nosocomial infections caused by Gram-positive pathogens. The des-F(6) compounds 4–6 exhibited at least four times more potent activity against representative Gram-positive bacteria than ciprofloxacin or moxifloxacin. Among the derivatives, 7-[(3R)-3-(1-aminocyclopropan-1-yl)pyrrolidin-1-yl] derivative 4, which showed favorable profiles in preliminary toxicological and non-clinical pharmacokinetic studies, exhibited potent antibacterial activity against clinically isolated Gram-positive pathogens that had become resistant to one or more antibiotics.     

Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa

Background  

While the larval-juvenile transition (metamorphosis) in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose), and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE), and they were then compared to those of the barnacle.     

Seasonal variation of phlorotannin in sargassacean species from the coast of the Sea of Japan

Variations in phlorotannin concentrations among the developmental stages of brown algae have been reported; however, the phlorotannin concentration plasticity associated with fluctuations in environmental factors make it difficult to determine the essential ontogenetic variation. The phlorotannin concentrations in five perennial sargassacean species where newly sprouted branches appear in summer and become fertile the following spring were examined every month during a year; and correlation with the developmental or seasonal environmental factors was determined. Although the phlorotannin fluctuated greatly throughout the year, the fluctuation patterns were relatively similar among the five species: phlorotannin showed a peak during July and August; gradually decreased in the winter; and increased in April. Performing a multiple regression analysis, the phlorotannin concentration did not correlate with thallus size in all species; and phlorotannin amounts were significantly affected by ambient abiotic factors in some species. The phlorotannin contents in newly sprouted branches were always higher than those in the long main branches during all seasons. When the phlorotannin contents were determined monthly for S. fulvellum (Turner) C. Agardh where the thalli were cultured from embryos in outdoor tanks, the phlorotannin concentrations were 3–4% of the dry matter (DM) in the juveniles and decreased to less than 1% of the DM in thalli >7.5 cm in length. However, the phlorotannin in these cultured thalli suddenly increased to 5.3% DM after being transplanted to the inshore coast; and then the concentration gradually decreased. The data show higher phlorotannin concentrations in younger sargassacean algae thalli and fluctuation of the phlorotannin amounts with extrinsic environmental factors.     

Phenylpiperidine-type γ-secretase modulators target the transmembrane domain 1 of presenilin 1

Amyloid-β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.     

De novo design of foldable proteins with smooth folding funnel: automated negative design and experimental verification

De novo sequence design of foldable proteins provides a way of investigating principles of protein architecture. We performed fully automated sequence design for a target structure having a three-helix bundle topology and synthesized the designed sequences. Our design principle is different from the conventional approach, in that instead of optimizing interactions within the target structure, we design the global shape of the protein folding funnel. This includes automated implementation of negative design by explicitly requiring higher free energy of the denatured state. The designed sequences do not have significant similarity to those of any natural proteins. The NMR and CD spectroscopic data indicated that one designed sequence has a well-defined three-dimensional structure as well as alpha-helical content consistent with the target.     

Synthesis and mechanism of action of novel pyrimidinyl pyrazole derivatives possessing antiproliferative activity

Pyrimidinyl pyrazole derivatives 1-4, prepared as a new scaffold of an anti-tumor agent, showed antiproliferative activity against human lung cancer cell lines and inhibited tubulin polymerization. Furthermore, it was found that compound 2 bound at the colchicine site on tubulin, but the tubulin binding pattern was different from that of colchicine. Here, we describe the synthesis of the derivatives and the differences of the action mechanism on tubulin polymerization inhibition between compound 2 and colchicine.     

Effects of Silicon-Limitation on Growth and Morphology of Triparma laevis NIES-2565 (Parmales,Heterokontophyta)

The order Parmales (Heterokontophyta) is a group of small-sized unicellular marine phytoplankton, which is distributed widely from tropical to polar waters. The cells of Parmales are surrounded by a distinctive cell wall, which consists of several siliceous plates fitting edge to edge. Phylogenetic and morphological analyses suggest that Parmales is one of the key organisms for elucidating the evolutionary origin of Bacillariophyceae (diatoms), the most successful heterokontophyta. The effects of silicon-limitation on growth and morphogenesis of plates were studied using a strain of Triparma laevis NIES-2565, which was cultured for the first time in artificial sea water. The cells of T. laevis were surrounded by eight plates when grown with sufficient silicon. However, plate formation became incomplete when cells were cultured in a medium containing low silicate (ca. <10 µM). Cells finally lost almost all plates in a medium containing silicate concentrations lower than ca. 1 µM. However, silicon-limitation did not affect growth rate; cells continued to divide without changing their growth rate, even after all plates were lost. Loss of plates was reversible; when cells without plates were transferred to a medium containing sufficient silicate, regeneration of shield and ventral plates was followed by the formation of girdle and triradiate plates. The results indicate that the response to silicon-limitation of T. laevis is different from that of diatoms, where cell division becomes inhibited under such conditions.     

Sarcomere length nanometry in rat neonatal cardiomyocytes expressed with α-actinin–AcGFP in Z discs

Nanometry is widely used in biological sciences to analyze the movement of molecules or molecular assemblies in cells and in vivo. In cardiac muscle, a change in sarcomere length (SL) by a mere ∼100 nm causes a substantial change in contractility, indicating the need for the simultaneous measurement of SL and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiomyocytes at high spatial and temporal resolution. To accurately analyze the motion of individual sarcomeres with nanometer precision during excitation–contraction coupling, we applied nanometry techniques to primary-cultured rat neonatal cardiomyocytes. First, we developed an experimental system for simultaneous nanoscale analysis of single sarcomere dynamics and [Ca²⁺]_i changes via the expression of AcGFP in Z discs. We found that the averaging of the lengths of sarcomeres along the myocyte, a method generally used in today’s myocardial research, caused marked underestimation of sarcomere lengthening speed because of the superpositioning of different timings for lengthening between sequentially connected sarcomeres. Then, we found that after treatment with ionomycin, neonatal myocytes exhibited spontaneous sarcomeric oscillations (cell-SPOCs) at partial activation with blockage of sarcoplasmic reticulum functions, and the waveform properties were indistinguishable from those obtained in electric field stimulation. The myosin activator omecamtiv mecarbil markedly enhanced Z-disc displacement during cell-SPOC. Finally, we interpreted the present experimental findings in the framework of our mathematical model of SPOCs. The present experimental system has a broad range of application possibilities for unveiling single sarcomere dynamics during excitation–contraction coupling in cardiomyocytes under various settings.