Crystal structure of human CD1e reveals a groove suited for lipid-exchange processes

CD1e is the only human CD1 protein existing in soluble form in the late endosomes of dendritic cells, where it facilitates the processing of glycolipid antigens that are ultimately recognized by CD1b-restricted T cells. The precise function of CD1e remains undefined, thus impeding efforts to predict the participation of this protein in the presentation of other antigens. To gain insight into its function, we determined the crystal structure of recombinant CD1e expressed in human cells at 2.90-Å resolution. The structure revealed a groove less intricate than in other CD1 proteins, with a significantly wider portal characterized by a 2 Å-larger spacing between the α1 and α2 helices. No electron density corresponding to endogenous ligands was detected within the groove, despite the presence of ligands unequivocally established by native mass spectrometry in recombinant CD1e. Our structural data indicate that the water-exposed CD1e groove could ensure the establishment of loose contacts with lipids. In agreement with this possibility, lipid association and dissociation processes were found to be considerably faster with CD1e than with CD1b. Moreover, CD1e was found to mediate in vitro the transfer of lipids to CD1b and the displacement of lipids from stable CD1b–antigen complexes. Altogether, these data support that CD1e could have evolved to mediate lipid-exchange/editing processes with CD1b and point to a pathway whereby the repertoire of lipid antigens presented by human dendritic cells might be expanded.     

Membrane texture induced by specific protein binding and receptor clustering: active roles for lipids in cellular function

Biological membranes are complex, self-organized structures that define boundaries and compartmentalize space in living matter. Composed of a wide variety of lipid and protein molecules, these responsive surfaces mediate transmembrane signaling and material transport within the cell and with its environment. It is well known that lipid membrane properties change as a function of composition and phase state, and that protein-lipid interactions can induce changes in the membrane's properties and biochemical response. Here, molecular level changes in lipid organization induced by multivalent toxin binding were investigated using grazing incidence X-ray diffraction. Structural changes to lipid monolayers at the air-water interface and bilayers at the solid-water interface were studied before and after specific binding of cholera toxin to membrane embedded receptors. At biologically relevant surface pressures, protein binding perturbed lipid packing within monolayers and bilayers resulting in topological defects and the emergence of a new orientationally textured lipid phase. In bilayers this altered lipid order was transmitted from the receptor laden exterior membrane leaflet to the inner leaflet, representing a potential mechanism for lipid mediated outside-in signaling by multivalent protein binding. It is further hypothesized that cell-surface micro-domains exhibiting this type of lipid order may serve as nucleation sites for vesicle formation in clathrin independent endocytosis of cholera toxin.     

The 3-Deoxy Analogue of α-GalCer: Disclosing the Role of the 4-Hydroxyl Group for CD1d-Mediated NKT Cell Activation

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Interleukin (IL)-4 promotes T helper type 2-biased natural killer T (NKT) cell expansion, which is regulated by NKT cell-derived interferon-gamma and IL-4

CD1d-restricted natural killer T (NKT) cells can rapidly produce T helper type 1 (Th1) and Th2 cytokines and also play regulatory or pathological roles in immune responses. NKT cells are able to expand when cultured with alpha-galactosylceramide (alpha-GalCer) and interleukin (IL)-2 in a CD1d-restricted manner. However, the expansion ratio of human NKT cells is variable from sample to sample. In this study, we sought to determine what factor or factors are responsible for efficient in vitro expansion of NKT cells from various inbred mouse strains. Although the proportion of NKT cells in the spleen was nearly identical in each mouse strain, the growth rates of NKT cells cultured in vitro with alpha-GalCer and IL-2 were highly variable. NKT cells from the B6C3F1 and BDF1 mouse strains expanded more than 20-fold after 4 days in culture. In contrast, NKT cells from the strain C3H/HeN did not proliferate at all. We found that cell expansion efficiency correlated with the level of IL-4 detectable in the supernatant after culture. Furthermore, we found that exogenous IL-4 augmented NKT cell proliferation early in the culture period, whereas interferon (IFN)-gamma tended to inhibit NKT cell proliferation. Thus, the ratio of production of IL-4 and IFN-gamma was important for NKT cell expansion but the absolute levels of these cytokines did not affect expansion. This finding suggests that effective expansion of NKT cells requires Th2-biased culture conditions.     

A Glycolipid α-GalCer Derivative, 7DW8-5 as a Novel Mucosal Adjuvant for the Split Inactivated Influenza Vaccine

Influenza virus infects the host and transmits through the respiratory tract (i.e., the mouth and nose); therefore, the development of intranasal influenza vaccines that mimic the natural infection, coupled with an efficient mucosal adjuvant, is an attractive alternative to current parenteral vaccines. However, with the withdrawal of cholera toxin and Escherichia coli heat-labile endotoxin from clinical use due to side effects, there are no approved adjuvants for intranasal vaccines. Therefore, safe and effective mucosal adjuvants are urgently needed. Previously, we reported that one derivative of α-Galactosylceramide (α-GalCer), 7DW8-5, could enhance the protective efficacy of split influenza vaccine by injection administration. However, the mucosal adjuvanticity of 7DW8-5 is still unclear. In this study, we found that 7DW8-5 promotes the production of secret IgA antibodies and IgG antibodies and enhances the protective efficacy of the split influenza vaccine by intranasal administration. Furthermore, co-administration of 7DW8-5 with the split influenza vaccine significantly reduces the virus shedding in the upper and lower respiratory tract after lethal challenge. Our results demonstrate that 7DW8-5 is a novel mucosal adjuvant for the split influenza vaccine.     

基于网络药理学及分子对接探讨山楂改善代谢性高血压的作用机制

目的 运用网络药理学和分子对接技术探讨山楂改善代谢性高血压（metabolic hypertension,MH）的物质基础及作用机制。方法 利用HERB、ETCM数据库并结合文献调研收集山楂成分;通过SwissTargetPrediction、GeneCards等数据库筛选山楂的全部成分改善MH的靶点;借助Cytoscape软件构建山楂“活性成分-靶点-疾病”网络;利用DAVID进行GO富集及KEGG通路分析;并借助Autodock软件对核心成分和核心靶点进行分子对接验证。结果 从山楂中筛选出89个活性成分,作用于84个靶点,其中山楂改善MH的核心活性成分为山楂酸、阿里红酸B、亚麻酸、亚油酸、甲基正壬酮、芹菜素、熊果酸等;核心靶点为CYP19A1、PPARA、ESR1、PTGS2、PPARG、NR3C1、MMP9、TNF等;作用机制主要涉及炎症、糖脂代谢、血管内皮功能等多条信号通路;分子对接显示山楂核心活性成分与核心靶点具有较高亲和力。结论 山楂可能通过多成分调节TNF、IL-17、AGE-RAGE、HIF-1、cGMP-PKG等多条信号通路,进而抑制炎症反应、改善糖脂代谢异常、改善血管内皮功能等综合发挥改善MH的作用。     

基于SIRT1/UCP2信号通路观察二陈汤对2型糖尿病合并非酒精性脂肪性肝病大鼠的影响

目的 探讨二陈汤对2型糖尿病（type 2 diabetes mellitus,T2DM）合并非酒精性脂肪性肝病（nonalcoholic fatty liver disease,NAFLD）大鼠糖脂代谢及氧化应激的作用,并探讨其可能作用机制。方法 40只♂ SD大鼠随机选取10只为正常对照组,其余大鼠造模成功后,随机选取20只分为模型组和二陈汤组,每组10只。正常对照组给予常规饲料喂养,模型组结合高脂高糖饲料喂养及单次腹腔注射链脲佐菌素30 mg·kg^-1以制备T2DM合并NAFLD大鼠模型,二陈汤组灌胃给予二陈汤（每天1.0 g·mL^-1生药量）。12周后检测各组大鼠生化指标、氧化应激指标;HE染色法观察肝脏组织病理变化;免疫组化法、q-PCR及Western blotting检测肝脏组织SIRT1/UCP2信号通路的表达。结果 与模型组比,二陈汤显著改善T2DM合并NAFLD大鼠体质量及代谢功能,降低空腹血糖、谷丙转氨酶、天门冬氨酸氨基转移酶、甘油三酯、总胆固醇、低密度脂蛋白胆固醇、极低密度脂蛋白、游离脂肪酸、空腹胰岛素、胰岛素抵抗指数、丙二醛水平,且高密度脂蛋白、超氧化物歧化酶、谷胱甘肽过氧化物酶、过氧化氢酶均明显升高。HE染色结果显示,模型组肝组织出现重度的脂肪变性,二陈汤显著减轻肝细胞脂肪性变。免疫组化、q-PCR与Western blotting结果显示,与模型组比较,二陈汤组SIRT1 mRNA和蛋白的表达显著上调,UCP2 mRNA与蛋白的表达显著降低。结论 二陈汤在一定程度上对T2DM合并NAFLD大鼠具有改善作用,其机制可能与调节SIRT1/UCP2信号通路有关。     

From the Cover: A nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant Alaskan beetle Upis ceramboides

Thermal hysteresis (TH), a difference between the melting and freezing points of a solution that is indicative of the presence of large-molecular-mass antifreezes (e.g., antifreeze proteins), has been described in animals, plants, bacteria, and fungi. Although all previously described TH-producing biomolecules are proteins, most thermal hysteresis factors (THFs) have not yet been structurally characterized, and none have been characterized from a freeze-tolerant animal. We isolated a highly active THF from the freeze-tolerant beetle, Upis ceramboides, by means of ice affinity. Amino acid chromatographic analysis, polyacrylamide gel electrophoresis, UV-Vis spectrophotometry, and NMR spectroscopy indicated that the THF contained little or no protein, yet it produced 3.7 ± 0.3 °C of TH at 5 mg/ml, comparable to that of the most active insect antifreeze proteins. Compositional and structural analyses indicated that this antifreeze contains a β-mannopyranosyl-(1→4) β-xylopyranose backbone and a fatty acid component, although the lipid may not be covalently linked to the saccharide. Consistent with the proposed structure, treatment with endo-β-(1→4)xylanase ablated TH activity. This xylomannan is the first TH-producing antifreeze isolated from a freeze-tolerant animal and the first in a new class of highly active THFs that contain little or no protein.     

Preliminary studies on sensitization of Lewis rats with sulfated glucuronyl paragloboside

A large number of patients with peripheral neuropathy and IgM paraproteinemia have IgM monoclonal antibodies which recognize a carbohydrate determinant shared by myelin-associated glycoprotein (MAG) and sulfated glucuronyl glycolipids (SGGLs). There is considerable evidence that these IgM monoclonal antibodies are responsible for demyelination in this disorder. To study the pathogenic role of SGGLs in this type of neuropathy, we sensitized Lewis rats with sulfated glucuronyl paragloboside (SGPG), a major SGGL. Fifty percent of the animals (8/16) developed neurological symptoms such as mild to moderate distal tail tone loss, with or without abnormal posture, along with development of anti-SGPG antibodies. These antibodies reacted with SGGLs, but not with rat MAG. Morphological studies showed: (1) axonal change in the lateral aspects of the dorsal columns in the spinal cord; and (2) damage to the endothelial cells in the spinal cord which suggested a breakdown of the blood-brain barrier. There was no obvious change in the peripheral nerve. Since no marked cellular infiltration was detected in these lesions, the clinicopathological findings observed could be induced by humoral mechanism, most likely anti-SGPG antibodies.     

Onion-like glycodendrimersomes from sequence-defined Janus glycodendrimers and influence of architecture on reactivity to a lectin

A library of eight amphiphilic Janus glycodendrimers (GDs) with d-mannose (Man) headgroups, a known routing signal for lectin-mediated transport processes, was constructed via an iterative modular methodology. Sequence-defined variations of the Janus GD modulate the surface density and sequence of Man after self-assembly into multilamellar glycodendrimersomes (GDSs). The spatial mode of Man presentation is decisive for formation of either unilamellar or onion-like GDS vesicles. Man presentation and Janus GD concentration determine GDS size and number of bilayers. Beyond vesicle architecture, Man topological display affects kinetics and plateau level of GDS aggregation by a tetravalent model lectin: the leguminous agglutinin Con A, which is structurally related to endogenous cargo transporters. The agglutination process was rapid, efficient, and readily reversible for onion-like GDSs, demonstrating their value as versatile tools to explore the nature of physiologically relevant glycan/lectin pairing.Supramolecular chemistry has enormous potential to help resolve fundamental questions in the realm of cell biology. One of the key challenges is the design of programmable models for vesicles and cells and their surfaces as a means of establishing a chemical platform that mimics natural features in size and shape, and also allows customized implementation of bioactive epitopes, in structural and topological terms. Natural complexity can conveniently be reduced to simple systems, whose degree of diversity can then be rationally reconstituted in a stepwise process. Focusing on surface properties, the recently gained access to uniform populations of stable glycodendrimersomes (GDSs) by a simple injection method using a solution of amphiphilic Janus glycodendrimers (GDs) as building blocks for self-assembly in a water-soluble aprotic solvent (1–3), has afforded a promising opportunity to realize this concept. Interestingly, the resulting GDSs, which have tunable surface features, can cover the size range of naturally occurring vesicles such as endo- and exosomes.When considering the choice of surface determinants, a natural class of molecules to study comes from the third alphabet of life, carbohydrates. In fact, glycan structures, unsurpassed in coding capacity relative to proteins and nucleic acids, serve as signals in many bioprocesses, from cell adhesion and growth regulation to diverse transport processes (4–9). Consequently, a wide array of synthetic products for the many roles of glycans must be prepared by diversity-oriented approaches (10–15). The self-assembly of Janus GDs into GDSs enables analysis of trans (bridging) contacts. Initial experiments with unilamellar GDSs have proven their reactivity with sugar receptors (lectins) (16). Thus, model studies of the impact of glycans on vesicle properties and on glycan–lectin recognition are now possible. The studies will be valuable in clarifying how glycan structure and topology of presentation on cell surfaces team up to achieve the physiological level of selectivity and specificity of lectin binding to distinct counter receptors (17).In this article, generation of onion-like GDSs is demonstrated for some d-mannose (Man)-presenting Janus GDs. Of note, they are structurally related to physiological multilayered vesicles (18–20). The carbohydrate headgroup Man was selected because this sugar is relevant for cellular transport. A group of intracellular cargo transporters are mannose-specific lectins, i.e., ERGIC-53, ERGL, VIP36, and VIPL, and they share the β-sandwich fold and Ca²⁺ dependence (21–24). However, their nature as type-1 membrane proteins hampers studies on GDS aggregation (agglutination). Close similarities between the tissue and legume lectins, resulting in their classification as leguminous-like (L type) (25, 26), provide the motivation to run the assays with the tetrameric leguminous lectin Con A (ConA), a popular model often used in glycocluster research (7, 9, 10, 12). The distance between two lectin sites for ConA binding to the surface of the same Janus GDS is about 64 Å or 70 Å, and the cations are bound to ConA to hold amino acid side chains in place for ligand binding (25). These aggregation studies unravel the impact of sequence-defined surface display of Man on lectin-dependent bridging between vesicles (rate and extent) and on stability of the aggregates under conditions of impaired protein–carbohydrate interactions.