The aglycone of sulfogalactolipids can alter the sulfate ester substitution position required for hsc70 recognition.

3'-Sulfogalactolipids(SGLs), sulfogalactosyl ceramide (SGC), and sulfogalactoglycerolipid (SGG) bind to the N-terminal ATPase-containing domain of members of the heat shock protein 70 family. We have probed this binding specificity using a series of synthetic positional sulfated or phosphorylated glycolipid analogues, containing either a long-chain bisalkyl hydrocarbon-2-(tetradecyl)hexadecane (B30) or C(18) ceramide (SGC(18)) backbone. By TLC overlay and receptor ELISA, recombinant hsc70 bound ceramide-based glycoconjugates having 3'- or 4'-sulfogalactose glycone moieties and the 4'-sulfogalactose positional isomer conjugated to B30. Hsc70 binding was significantly decreased to the 3'-sulfogalactose conjugated to the long-chain branched alkane. 3'-Sulfoglucose conjugated to B30 was not bound, nor were similarly conjugated di-, tri-, and tetra-sulfated or phosphorylated galactolipids. These results highlight the importance of the position, rather than the number of sulfate esters within the galactose ring. This binding selectivity was shared by the sea urchin hsp70-related sperm receptor. A 3'-SGC-based soluble inhibitor, in which the acyl chain was replaced with an adamantyl group, inhibited binding of hsc70 to both 3'- and 4'-SGC species with an IC(50) of 50 and 75 microM, respectively, indicating a shared sulfogalactose binding site. These studies demonstrate the highly specific nature of hsc70/SGL binding and show, for the first time, that the lipid aglycone can alter the substitution position requirement for glycolipid recognition.

Hsp70s contain a specific sulfogalactolipid binding site. Differential aglycone influence on sulfogalactosyl ceramide binding by recombinant prokaryotic and eukaryotic hsp70 family members.

Specific 3'-sulfogalactolipid [SGL-sulfogalactosyl ceramide (SGCer) and sulfogalactosylglycerolipid (SGG)] binding is compared for hsp70s cloned from Helicobacter pylori, Haemophilus influenzae, Chlamydia trachomatis serovar E, Escherichia coli, murine male germ cells, and the hsp70-like extracellular domain within the sperm receptor from Strongylocentrotus purpuratus. This lectin activity, conserved among the different hsp70 family members, is modulated by the SGL aglycone. This is shown by differential binding to both SGC fatty acid homologues and 3'-sulfogalactolipid neoglycoproteins generated by coupling bovine serum albumin (BSA) and glycosyl ceramide acids synthesized by oxidation of the double bond of sphingosine. Eukaryotic hsp70s preferentially bound the SGCer fatty acid homologues SG(24)Cer, SG(18)Cer, and SG(20:OH)Cer, while prokaryotic hsp70s bound SG(18:1)Cer and SG(20:OH)Cer. Eukaryotic hsp70s bound SGCer-BSA and SG(24)Cer-BSA conjugates where the latter is the main constituent in SGCer-BSA, while prokaryotic hsp70s bound SG(20:OH)Cer-BSA. None of the hsp70s bound sulfogalactosyl sphingosine (SGSph) or SGSph-BSA, further demonstrating the important role of the aglycone. Although the primary SGL recognition domain of all hsp70s is conserved, we propose that aglycone organization differentially influences the interaction with the sub-site. Heterogeneous SGCer aglycone isoforms in cells and the differential in vitro binding of eukaryotic and prokaryotic hsp70s may relate to their different adhesin roles in vivo as mediators of germ cell and bacterial/host interactions, respectively.

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.

Oxidation of glycosphingolipids under basic conditions: synthesis of glycosyl "serine acids" as opposed to "ceramide acids". Precursors for neoglycoconjugates with increased ligand binding affinity.

Two types of oxidative cleavage of the double bond of glycosphingolipids (GSLs) are described. Oxidation of peracetylated GSL precursors with stoichiometric proportions of KMnO4 and an excess of NaIO4, in a neutral aqueous tert-butanol solvent system, gave nearly quantitative yields of the glycosyl ceramide acid, 2-hydroxy-3-(N-acyl)-4-(O-glycosyl)oxybutyric acid [Mylvaganam, M., and Lingwood, C. A. (1999) J. Biol. Chem. 274, 20725-20732]. However, if the reaction medium was made alkaline, the hydroxyallylic function of the sphingolipid, as a whole, was oxidized and the glycosyl serine acid, 2-(N-acyl)-3-(O-glycosyl)oxypropionic acid, was obtained in good yield. This represents a new type of oxidation reaction. Optimized conditions gave glycosyl ceramide or serine acids with greater than 90% selectivity and in good yields (90%). Oxidation of dGSLs gave serine and ceramide oligosaccharides, devoid of hydrocarbon chains. An intriguing glycosyl species containing 5-hydroxy-4-oxo-3-hydroxy-2-(N-acyl)sphingosine (hydroxy-acyl intermediate) was identified via ESMS analyses. We propose that further oxidation of this intermediate is pH-dependent and will be oxidized to either serine or ceramide acids. On the basis of MS-MS analysis of specific homologues of serine and ceramide acids, two types of collision-induced dissociation (CID) patterns have been established. These CID patterns were then used in the identification of serine and ceramide acids synthesized from natural GSL samples. Also, on a qualitative basis, this oxidation protocol, in conjunction with ESMS, provides a novel method for characterizing the aglycone composition (acyl chain length, unsaturation position, dihydrosphingosine content, etc.) of natural GSLs. A novel class of neohydrocarbon conjugates were synthesized by coupling the acids to rigid hydrocarbon frames such as 2-aminoadamantane. Preliminary studies with conjugates derived from globotriaosyl ceramide (Gb3C), lactosyl ceramide (LC), and galactosyl ceramide (GalC) bound verotoxin with the expected specificity but with affinities much greater than that of the natural glycolipid. Also, the ceramide acid-based conjugates were better ligands than serine acid conjugates.

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. NRR1CCOOH, 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)nBSA and (GalS.NHCO)nBSA 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. Terminal N-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 globotriaosyl ceramide: a monovalent soluble mimic which inhibits verotoxin binding to its glycolipid receptor.

The globotriaosylceramide (Gb3) verotoxin (VT) interaction is one of several examples of glycolipid receptors where the ceramide (or lipid) free oligosaccharides fail to show the expected binding parameters. We present a novel, yet simple strategy to synthesize monovalent, water soluble glycosphingolipid mimics which retain receptor function. Replacing the fatty acid chain with rigid, three dimensional hydrocarbon frames, such as adamantane, gives a novel class of neohydrocarbon glycoconjugates. Such adamantyl conjugates derived from Gb3 showed significantly enhanced solubility in water compared to natural Gb3. Adamantyl-Gb3 showed a thousand fold enhanced inhibitory activity (IC50 = 1 microM) for VT-Gb3 binding as compared to a lipid free Gb3 oligosaccharide derivative, alphaGal1-4betaGal1-4betaGlc1-O-CH2CH(CH2SO2C 4H9)2 (IC50 > 2 mM). This represents a new approach to the generation of antagonists of glycolipid receptors.