Murugesapillai Mylvaganam

New aspects of the regulation of glycosphingolipid receptor function.

We propose that the fatty acid heterogeneity of glycosphingolipids may compensate for the relative few and simple glycosphingolipid structures found in mammalian cells. Variation in GSL fatty acid composition may mediate aglycone regulation of GSL membrane receptor function by a differential interaction with cholesterol and other membrane components which may be differentially organized within plasma membrane lipid domains. These concepts are specifically illustrated in model membrane studies and in relation to the role of the glycolipid, globotriaosyl ceramide (Gb(3)) in verotoxin-induced renal pathology and gp120 binding in HIV infection.

A Convenient Oxidation of Natural Glycosphingolipids to Their “Ceramide Acids” for Neoglycoconjugation BOVINE SERUM ALBUMIN-GLYCOSYLCERAMIDE ACID CONJUGATES AS INVESTIGATIVE PROBES FOR HIV gp120 COAT PROTEIN-GLYCOSPHINGOLIPID INTERACTIONS

A new method to cleave the double bond of sphingolipids has been developed. Using limited concentrations of KMnO4 and an excess of NaIO4, in a neutral aqueous tert-butanol solvent system gave nearly quantitative yields of the oxidized product. A variety of natural glycosphingolipids (GSLs): GlcC, GalC, SGC, LC, Gb3C, Gb4C, Gg4C, Gb5C, and GM1C, gave the corresponding acids: 2-hydroxy-3-(N-acyl)-4-(O-glycosyl)-oxybutyric acids, i.e. “glycosyl ceramide acids” (GSL·CCOOH) in excellent yields (80–90%). Deacyl GSLs (dGSLs) were oxidized to acids containing the oligosaccharides devoid of hydrocarbon chains, i.e. “ceramide oligosaccharides” (dGSL·NRR1 CCOOH, where R = R1 = H; R = H, R1 = CH3CO; or R = R1 = Me). The efficacy of this method was demonstrated by transforming natural GSLs: GlcC, GalC, GalS, SGC, LC, Gb3C, and Gb4C into neoglycoproteins via coupling glycosyl ceramide acids (except GalS, which was coupled directly) to bovine serum albumin (BSA). Mass spectroscopic analysis of GalC-BSA conjugates, (GalC·CONH) n BSA and (GalS·NHCO) n BSA gave a value of 9 ± 1 and 16 ± 2 for n. Neoglycoconjugates derived from GlcC, GalC (type I and II and the behenic analog), SGC, LC, and Gb3C were recognized by the recombinant human immunodeficiency virus coat protein gp120 (rgp120). The GalS conjugate showed significantly reduced binding, and the Gb4C conjugate showed no binding. Thus, rgp120/GSL-BSA interaction requires a terminal galactose and/or glucose residue. TerminalN-acetylgalactosamine containing GSLs are not bound. The ceramide acid conjugates provide a more effective scaffold for presentation of glycone for rgp120 binding than those derived from dGSLs. The retention of receptor specificity of the glycoconjugates was validated by retention of the expected binding specificity of VT1 and VT2e for Gb3C and Gb4C conjugates, respectively. These studies open a new vista in the generation of glycoconjugates from GSLs and further emphasize the role of aglycone in glycolipid recognition.

Adamantyl Glycosphingolipids Provide a New Approach to the Selective Regulation of Cellular Glycosphingolipid Metabolism

Mammalian glycosphingolipid (GSL) precursor monohexosylceramides are either glucosyl- or galactosylceramide (GlcCer or GalCer). Most GSLs derive from GlcCer. Substitution of the GSL fatty acid with adamantane generates amphipathic mimics of increased water solubility, retaining receptor function. We have synthesized adamantyl GlcCer (adaGlcCer) and adamantyl GalCer (adaGalCer). AdaGlcCer and adaGalCer partition into cells to alter GSL metabolism. At low dose, adaGlcCer increased cellular GSLs by inhibition of glucocerebrosidase (GCC). Recombinant GCC was inhibited at pH 7 but not pH 5. In contrast, adaGalCer stimulated GCC at pH 5 but not pH 7 and, like adaGlcCer, corrected N370S mutant GCC traffic from the endoplasmic reticulum to lysosomes. AdaGalCer reduced GlcCer levels in normal and lysosomal storage disease (LSD) cells. At 40 μm adaGlcCer, lactosylceramide (LacCer) synthase inhibition depleted LacCer (and more complex GSLs), such that only GlcCer remained. In Vero cell microsomes, 40 μm adaGlcCer was converted to adaLacCer, and LacCer synthesis was inhibited. AdaGlcCer is the first cell LacCer synthase inhibitor. At 40 μm adaGalCer, cell synthesis of only Gb3 and Gb4 was significantly reduced, and a novel product, adamantyl digalactosylceramide (adaGb2), was generated, indicating substrate competition for Gb3 synthase. AdaGalCer also inhibited cell sulfatide synthesis. Microsomal Gb3 synthesis was inhibited by adaGalCer. Metabolic labeling of Gb3 in Fabry LSD cells was selectively reduced by adaGalCer, and adaGb2 was produced. AdaGb2 in cells was 10-fold more effectively shed into the medium than the more polar Gb3, providing an easily eliminated “safety valve” alternative to Gb3 accumulation. Adamantyl monohexosyl ceramides thus provide new tools to selectively manipulate normal cellular GSL metabolism and reduce GSL accumulation in cells from LSD patients.

[39] - Oxidation of Aglycone of Glycosphingolipids: Serine and Ceramide Acid Precursors for Soluble Glycoconjugates

A new oxidation protocol for the cleavage of sphingosine double bonds is described. The procedure is applicable to both natural and deacyl glycolipids and can be applied to microgram quantities of precursors. Under neutral conditions, glycosyl ceramide acids are obtained and under basic conditions glycosyl serine acids are obtained. The glycosyl ceramide acid-based glycoconjugates--BSA-neoglycoprotein and adamantyl-neohydrocarbon--demonstrate the importance that an aglycone can play in carbohydrate-protein interaction. Studies with HIV coat protein gp120 and BSA-neoglycoprotein conjugates derived from galactosylceramide (GalC) showed that binding affinities of the conjugates depend on the manner in which the glycosyl unit is coupled to the protein. Deacyl-GalC conjugates, in which the glycosyl unit is coupled via the amine of the sphingosine, showed significantly lower affinity as compared to glycosylceramide acid conjugates. In the case of Gb3-VT1 binding, it was found that ceramide acid conjugates bound to VT1 better than the serine acid conjugates. These studies show that the aglycone organization, particularly the region adjacent to the carbohydrate region (or in a membrane environment, the aglycone-glycone interface) modulate carbohydrate presentation. It is possible that in each of the conjugates described above, the interface region could have different hydrogen-bonding networks (see Scheme 4.) This, in turn, could influence the solvation and/or conformation of this region and thereby influence ligand binding.

Synthesis of a novel photoactivatable glucosylceramide cross-linker

The biosynthesis of glucosylceramide (GlcCer) is a key rate-limiting step in complex glycosphingolipid (GSL) biosynthesis. To further define interacting partners of GlcCer, we have made a cleavable, biotinylated, photoreactive GlcCer analog in which the reactive nitrene is closely apposed to the GlcCer head group, by substituting the native fatty acid with d, l-2-aminohexadecanoic acid. Two amino-GlcCer diastereomer cross-linkers (XLA and XLB) were generated. XLB proved an effective lactosylceramide (LacCer) synthase substrate while XLA was inhibitory. Both probes specifically bound and cross-linked the GlcCer binding protein, glycolipid transfer protein (GLTP), but not other GSL binding proteins (Shiga toxin and cholera toxin). GlcCer inhibited GLTP cross-linking. Both GlcCer cross-linkers competed with microsomal nitrobenzoxadiazole (NBD)-GlcCer anabolism to NBD-LacCer. GLTP showed marked, ATP-dependent enhancement of cell-free intact microsomal LacCer synthesis from endogenous or exogenous liposomal GlcCer, supporting a role in the transport/membrane translocation of cytosolic and extra-Golgi GlcCer. GLTP was specifically labeled by either XLA or XLB GlcCer cross-linker during this process, together with a (the same) small subset of microsomal proteins. These cross-linkers will serve to probe physiologically relevant GlcCer-interacting cellular proteins.

Oxidation of the primary hydroxyl group of galactose of galactaosyl ceramide analogue by chemical method-precursors for the synthesis of labeled conjugates

Isotopic labeling of the C-6 of a model glycosphingolipid (2S, 3R, 4E)-2-(1-adamantanacetamido)-3-hydroxy-4-octadecenyl-beta-D-galactopyranoside, GalCAda, is described. Oxidation of (2S, 3R, 4E)-2-(1-adamantanacetamido)-3-(benzoyloxy)-4-octadecenyl-2,3,4-tri-O-benzoyl-beta-D-galactopyranoside with o-iodoxybenzoic acid gave the dialdoside derivative in good yield. Reduction of the dialdoside with sodium borodeuteride gave the deuterium labeled D-GalCAda, with a cumulative yield of 35%.