Analysis of the membrane-interacting domains of myelin basic protein by hydrophobic photolabeling.
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Myelin basic protein is a water soluble membrane protein which interacts with acidic lipids through some type of hydrophobic interaction in addition to electrostatic interactions. Here we show that it can be labeled from within the lipid bilayer when bound to acidic lipids with the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID) and by two lipid photolabels. The latter included one with the reactive group near the apolar/polar interface and one with the reactive group linked to an acyl chain to position it deeper in the bilayer. The regions of the protein which interact hydrophobically with lipid to the greatest extent were determined by cleaving the TID-labeled myelin basic protein (MBP) with cathepsin D into peptides 1-43, 44-89, and 90-170. All three peptides from lipid-bound protein were labeled much more than peptides from the protein labeled in solution. However, the peptide labeling pattern was similar for both environments. The two peptides in the N-terminal half were labeled similarly and about twice as much as the C-terminal peptide indicating that the N-terminal half interacts hydrophobically with lipid more than the C-terminal half. MBP can be modified post-translationally in vivo, including by deamidation, which may alter its interactions with lipid. However, deamidation had no effect on the TID labeling of MBP or on the labeling pattern of the cathepsin D peptides. The site of deamidation has been reported to be in the C-terminal half, and its lack of effect on hydrophobic interactions of MBP with lipid are consistent with the conclusion that the N-terminal half interacts hydrophobically more than the C-terminal half. Since other studies of the interaction of isolated N-terminal and C-terminal peptides with lipid also indicate that the N-terminal half interacts hydrophobically with lipid more than the C-terminal half, these results from photolabeling of the intact protein suggest that the N-terminal half of the intact protein interacts with lipid in a similar way as the isolated peptide. The similar behavior of the intact protein to that of its isolated peptides suggests that when the purified protein binds to acidic lipids, it is in a conformation which allows both halves of the protein to interact independently with the lipid bilayer. That is, it does not form a hydrophobic domain made up from different parts of the protein. (+info)
The natural killer T (NKT) cell ligand alpha-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells.
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The natural killer T (NKT) cell ligand alpha-galactosylceramide (alpha-GalCer) exhibits profound antitumor activities in vivo that resemble interleukin (IL)-12-mediated antitumor activities. Because of these similarities between the activities of alpha-GalCer and IL-12, we investigated the involvement of IL-12 in the activation of NKT cells by alpha-GalCer. We first established, using purified subsets of various lymphocyte populations, that alpha-GalCer selectively activates NKT cells for production of interferon (IFN)-gamma. Production of IFN-gamma by NKT cells in response to alpha-GalCer required IL-12 produced by dendritic cells (DCs) and direct contact between NKT cells and DCs through CD40/CD40 ligand interactions. Moreover, alpha-GalCer strongly induced the expression of IL-12 receptor on NKT cells from wild-type but not CD1(-/-) or Valpha14(-/-) mice. This effect of alpha-GalCer required the production of IFN-gamma by NKT cells and production of IL-12 by DCs. Finally, we showed that treatment of mice with suboptimal doses of alpha-GalCer together with suboptimal doses of IL-12 resulted in strongly enhanced natural killing activity and IFN-gamma production. Collectively, these findings indicate an important role for DC-produced IL-12 in the activation of NKT cells by alpha-GalCer and suggest that NKT cells may be able to condition DCs for subsequent immune responses. Our results also suggest a novel approach for immunotherapy of cancer. (+info)
Structure and properties of totally synthetic galacto- and gluco-cerebrosides.
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The structural and thermal properties of aqueous dispersions of the totally synthetic cerebrosides, D-erythro-N-palmitoyl galactosyl- and glucosyl-C18-sphingosine (C16:0-GalCer and C16:0-GluCer, respectively) have been studied using differential scanning calorimetry (DSC) and X-ray diffraction. Over the temperature range 0-100 degrees C, both C16:0-GalCer and C16:0-GluCer show complex thermal transitions characteristic of polymorphic behavior of exclusively bilayer phases. On heating, hydrated C16:0-GalCer undergoes an exothermic bilayer-bilayer transition at 59 degrees C to produce a stable bilayer crystal form. X-ray diffraction at 70 degrees C reveals a bilayer structure with an ordered hydrocarbon chain-packing arrangement. This ordered bilayer phase undergoes an endothermic chain-melting transition at 85 degrees C to the bilayer liquid crystalline state. Similar behavior is exhibited by hydrated C16:0-GluCer which undergoes the exothermic transition at 49 degrees C and a chain-melting transition at 87 degrees C. The exothermic transitions observed on heating hydrated C16:0-GalCer and C16:0-GluCer are irreversible and dependent upon previous chain melting, prior cooling rate, and time of incubation at low temperatures. Thus, the structure and properties of totally synthetic C16:0-GalCer and C16:0-GluCer with identical sphingosine (C18:1) and fatty acid (C16:0) chains are quite similar, suggesting that the precise isomeric structure of the linked sugar plays only a minor role in regulating the properties of hydrated cerebrosides. Further, these studies indicate that the complex thermal behavior and bilayer phase formation exhibited by these single-sugar cerebrosides are intrinsic properties and not due to the heterogeneity of the sphingosine base found in natural and partially synthetic cerebrosides. (+info)
A novel recognition motif of human NKT antigen receptor for a glycolipid ligand.
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Murine NKT cells can recognize alpha-galactosylceramide (alpha-GalCer) in the context of a class Ib CD1d molecule. Here we show that alpha-GalCer can selectively activate freshly isolated human Valpha24(+)Vbeta11(+) cells, functionally defining the human NKT cells. The naive human NKT cell repertoire consisted of cells expressing an invariant Valpha24JalphaQ chain and a diverse array of beta chains derived from a single Vbeta11 gene segment. Stimulation with alpha-GalCer expanded a polyclonal subset of the human NKT cell repertoire carrying a novel complementarity-determining region (CDR) 3beta consensus motif that may directly interact with the sugar moiety of alpha-GalCer. Our data suggest that certain redundancy is allowed for CDR3beta of NKT antigen receptor to interact with the ligand and provide a first clue to understand the novel protein-carbohydrate interaction mechanisms. (+info)
A novel function of Valpha14+CD4+NKT cells: stimulation of IL-12 production by antigen-presenting cells in the innate immune system.
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The balance between Th1 and Th2 development is determined by IL-4 and IL-12. While the role for CD4+ NK1.1+ T (NKT) cells in influencing this balance has been recognized based on their capacity to produce IL-4, it is unknown how IL-12 is produced in the innate immune system in which they participate. This study demonstrates that Ag-activated CD4+ NKT cells express CD40 ligand (CD40L) (CD154), which engages CD40 on APC and stimulates them to produce IL-12. Culture of B cell-depleted spleen cells from C57BL/6 mice with alpha-galactosylceramide (alpha-GalCer) capable of selectively stimulating Valpha14/Jalpha281+ NKT cells resulted in the production of IL-12 together with IFN-gamma and IL-4. alpha-GalCer-induced IL-12 production occurred in I-Abbeta-deficient mice, but not in beta2-microglobulin-deficient and Valpha14/Jalpha281 TCR-deficient mice, and was inhibited by anti-CD40L mAb. Of CD4+ and CD4- NKT cells, the capacity to express CD40L/CD154 and trigger IL-12 production following alpha-GalCer stimulation was exhibited preferentially by the CD4+ NKT subset. IL-12 production was also observed in alpha-GalCer-treated mice. Production of IL-12 preceded IFN-gamma production, and IL-12 was required for IFN-gamma, but not IL-4, production. A stimulatory/inhibitory relationship existed between IL-12 and IL-4 production. These results illustrate a novel function of CD4+ NKT cells that could be involved in the regulation of Th1 vs Th2 development. (+info)
Modulation of nanotube formation by structural modifications of sphingolipids.
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Galactosylceramides (GalCers) containing nervonoyl (24:1(Delta15(cis))) acyl chains have the capacity to assemble into nanotubular microstructures in excess water (. Biophys. J. 69:1976-1986). To define the structural parameters that modulate nanotube formation, GalCer derivatives were synthesized that contained cis monounsaturated acyl chains with the formula X:1((X-9)). X indicates the total acyl carbon number (24, 22, 20, or 18), and 1 indicates a single cis double bond, the location of which is designated by the superscript (X-9). Deep etching of freeze-fractured 24:1(Delta15(cis)) GalCer dispersions followed by replica production and transmission electron microscopic analysis confirmed nanotube morphology (25-30-nm diameter). Control experiments revealed that tubule formation was promoted by cooling through the main enthalpic phase transition coupled with repetitive freeze-thaw cycling. Imparting a negative charge to the sugar headgroup of 24:1(Delta15)GalCer via sulfate dramatically altered mesomorpholgy and resulted in myelinic-like, multilamellar structures. Removal of the sugar headgroup (24:1(Delta15)Cer) resulted in flattened cylindrical structures with a cochleate appearance. Compared to these large-scale changes in morphology, more subtle changes were induced by structural changes in the acyl chain of 24:1(Delta15)GalCer. 22:1(Delta13)GalCer dispersions consisted of long, smooth tubules (35-40-nm diameters) with a strong tendency to self-align into bundle-like aggregates. In contrast, the microstructures formed by 20:1(Delta11)GalCer resembled helical ribbons with a right-handed twist. Ribbon widths averaged 30-35 nm, with helical pitches of 80-90 nm. 18:1(Delta9)GalCer displayed a variety of morphologies, including large-diameter multilamellar cylinders and liposome-like structures, as well as stacked, plate-like arrays. The results are discussed within the context of current theories of lipid tubule formation. (+info)
In vitro production of cytokines is influenced by sulfatide and its precursor galactosylceramide.
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Effects of sulfatide and its precursor galactosylceramide (gal-cer) on the kinetics of production of cytokines were studied. In human mononuclear leucocytes, gal-cer but not sulfatide induced significantly increased amounts of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF) mRNA. In phytohemagglutinin-stimulated cultures, gal-cer increased the levels of IL-1beta and IL-6 mRNA and secreted IL-1beta and IL-6, while sulfatide decreased the amounts of IL-6 mRNA and secreted IL-6. Gal-cer also increased TNF secretion. In lipopolysaccharide-stimulated cells, sulfatide but not gal-cer decreased the secretion of IL-1beta and IL-10, a potent suppressor of production of many cytokines. Thus, sulfatide and gal-cer affect cytokine production differently, most likely at the level of gene expression. This may have implications in diseases where inflammatory cytokines play a pathogenic role. (+info)
Cutting edge: activation of NK T cells by CD1d and alpha-galactosylceramide directs conventional T cells to the acquisition of a Th2 phenotype.
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NK T cells recognize glycolipid Ags such as alpha-galactosylceramide (alpha-GalCer) presented by the MHC class I-like molecule CD1d. In this paper we have studied the in vivo effects of alpha-GalCer on the generation of adaptive immune responses. Treatment of mice with alpha-GalCer resulted in rapid activation of NK T cells and production of the cytokines IL-4 and IFN-gamma. However, after this initial stimulation, NK T cells became polarized for the production of IL-4. Further, as soon as 6 days after alpha-GalCer injection, a marked increase in serum IgE levels was observed. Administration of alpha-GalCer at the time of priming of mice with protein Ag resulted in the generation of Ag-specific Th2 cells and a profound increase in the production of IgE. Collectively, these findings indicate that alpha-GalCer may be useful for modulating immune responses toward a Th2 phenotype during prophylaxis and therapy. (+info)