Non-natural glycosphingolipids and structurally simpler analogues bind HIV-1 recombinant Gp120

Interactions of recombinant gp120 (rgp120) with non-natural glycosphingolipids (GSLs) and structurally simpler analogues have been studied using a competitive adhesion assay. Conjugates of cellobiosyl ceramide and melibiosyl ceramide were synthetically prepared as water-soluble GSL analogues. These ligands were screened against a panel of biologically relevant analogues, and the results show that their interactions with rgp120 are comparable to natural cellular receptors. Glycolipid interactions with rgp120 were probed further by the synthesis and testing of structurally simpler analogues that were obtained by reductive amination of lactose, cellobiose, and melibiose with a biotinylated amino ethylene glycol moiety. RGp120 did not recognize conjugates lacking a lipid component. However, palmitoylation of the secondary amino alditols yielded compounds with comparable rgp120 affinity to the natural cellular receptor, galactosyl ceramide (GalCer). Taken together, the SAR showed that both a hydrophobic and a hydrophilic component are required for rgp120 recognition. Moreover, structural variability in the carbohydrate headgroup did not significantly alter rgp120 recognition indicating that this interaction is not highly specific.     

Synthesis of biotinylated glycoconjugates and their use in a novel ELISA for direct comparison of HIV-1 Gp120 recognition of GalCer and related carbohydrate analogues

As part of our program directed toward the design and synthesis of high-affinity ligands for the GalCer-binding site on the HIV cell surface glycoprotein, gp120, we required a reliable method for qualitatively assessing relative binding affinities for related analogues. Due to the hydrophilic nature of these synthetic conjugates, difficulties were encountered with typical ELISA methods, which rely upon hydrophobic interactions to anchor the ligand to a microtiter plate. Other types of assays were also problematic due to nonspecific binding of gp120. Therefore, we developed a general method for plating water-soluble ligands on microtiter plates using biotin/NeutrAvidin recognition for adhesion. A water-soluble GalCer analogue was prepared by conjugating psychosine to biotin using a novel tetraethylene glycol linker. In a similar manner, LacCer and GlcCer analogues were prepared and these conjugates were plated into microtiter wells containing NeutrAvidin. Unoccupied sites were blocked using biotin functionalized as a primary amide. Gp120 binding to galactosyl sphingosine, GalSph (19), GlcSph (22), and LacSph (23) conjugates was assessed through incubation with recombinant HRP-gp120. It was determined that LacSph has the strongest interaction with gp120. The binding affinities of GalSph and GlcSph were similar to each other and less strong than LacSph. These data contradict earlier studies where HPTLC showed that LacCer and GlcCer do not significantly bind gp120. They also contradict liposome-based assays that reported psychosine is not recognized by gp120. The extent of plating for each biotinylated molecule was quantified using HRP-biotin, allowing direct comparison of ligand plating efficiencies for the first time. Several other synthetic biotin conjugates were prepared and tested, demonstrating the feasibility of performing ELISA on water-soluble ligands.     

Glycosphingolipid acyl chain orientational order in unsaturated phosphatidylcholine bilayers.

The glycosphingolipid, galactosyl ceramide (GalCer), was studied by 2H nuclear magnetic resonance (NMR) in fluid phospholipid bilayer membranes, with regard to arrangement of its acyl chain. For this purpose, species with perdeuterated 18-carbon fatty acid (18:0[d35]GalCer) or with perdeuterated 24-carbon fatty acid (24:0[d47] GalCer) were dispersed in bilayers of the 18-carbon phospholipid, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC). For 18:0[d35] GalCer, smoothed profiles of the order parameter, SCD, were found to be very similar to one another over the range of glycolipid concentration, 5-40 mol%. In addition, they were very similar to orientational order parameter profiles well known from the literature on phospholipid and glycolipid acyl chains (which deals in general with membranes of homogeneous chain length in the range 14-18 carbons). Corresponding order parameter profiles for the long-chain species, 24:0[d47] GalCer, were also similar to one another for glycolipid concentrations between 5 and 40 mol%. Their shapes, however, were distinctly different from those of the shorter chain analogues. SCD profiles for the two species were quantitatively similar to a membrane depth of C15. SCD values at C16 and C17 were approximately 20 and 30%, respectively, higher for the long-chain glycosphingolipid than for its short-chain analogue in SOPC. Nitroxide spin labels attached rigidly to C16 of the long-chain glycolipid in SOPC gave electron paramagnetic resonance (EPR) order parameters that were twice as high as for a spin label at C16 on the shorter chain glycolipid. Comparison was made between spectra of 24:0[d47] GalCer in SOPC and fully hydrated bilayers of the pure 24:0[d47] GalCer, a system that is considered to be partially interdigitated in fluid and gel phases. The resultant 2H NMR order parameter profiles displayed similar features, indicating that related organizational properties exist in these fluid systems. Effective chain length of 24:0[d47] GalCer within the SOPC membrane was calculated using the method of Schindler and Seelig (1975. Biochemistry, 14:2283-2287). The result suggested that the long-chain fatty acid should protrude roughly one third of the host matrix chain length across the bilayer midplane. However, a treatment of the same order parameters making very few assumptions about chain conformation indicated a high degree of orientational flexibility for the "extra" length of the long chain fatty acid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural determinants of miscibility in surface films of galactosylceramide and phosphatidylcholine: effect of unsaturation in the galactosylceramide acyl chain

The Langmuir film balance technique has been used to define the surface structure and determine the mixing behavior of galactosylceramide (GalCer) and phosphatidylcholines in surface phases. To determine the effect of unsaturation on surface behavior, chain-pure GalCer species containing either oleoyl (18:1 delta 9), eicosenoyl (20:1 delta 11), or eicosadienoyl (20:2 delta 11,14) fatty acyl chains were synthesized. Using bovine brain GalCer as a reference, surface pressure versus molecular area (phi-A) isotherms of the pure lipids were measured and analyzed by determining their compressibilities and by using an equation of state for lipid monolayers. This information, when coupled with surface potential versus molecular area (delta V-A) analyses, provides insights into GalCer surface structure in terms of molecular packing and orientation. Lipid mixing behavior was determined by classical approaches which involve analyzing the average molecular area, the average surface dipole moment, and surface pressure as a function of film composition. The results indicate that, in contrast to the complex mixing behavior displayed by bovine brain GalCer and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), chain-pure GalCer species containing either oleoyl, eicosenoyl, or eicosadienoyl fatty acyl chains are miscible with POPC over the entire composition range. Moreover, increasing amounts of GalCer containing eicosenoyl acyl chains systematically elevate dipalmitoylphosphatidylcholine's (DPPC) liquid-expanded-to-liquid-condensed transition pressure. Such behavior is consistent with GalCer being miscible with the liquid-expanded phase of DPPC. Thus, fatty acyl unsaturation is a critical parameter governing the mixing behavior of GalCer with phosphatidylcholine.     

Divalent cation-mediated interaction between cerebroside sulfate and cerebrosides: an investigation of the effect of structural variations of lipids by electrospray ionization mass spectrometry.

Divalent cations mediate a carbohydrate-carbohydrate association between the two major glycolipids, galactosylceramide (GalCer) and its sulfated form, cerebroside sulfate (CBS), of the myelin sheath. We have suggested that interaction between these glycolipids on apposed extracellular surfaces of myelin may be involved in the stability or function of this multilayered structure. A mutant mouse lacking galactolipids because of a disruption in the gene that encodes a galactosyltransferase forms myelin that initially appears relatively normal but is unstable. This myelin contains glucosylceramide (GlcCer) instead of GalCer. To better understand the role of GlcCer in myelin in this mutant, we have compared the ability of divalent cations to complex CBS (galactosyl form) with GlcCer or GalCer in methanol solution by using positive ion electrospray ionization mass spectrometry. Because both the alpha-hydroxylated fatty acid species (HFA) and the nonhydroxylated fatty acid species (NFA) of these lipids occur in myelin, we have also compared the HFA and NFA species. In addition to monomeric Ca2+ complexes of all three lipids and oligomeric Ca2+ complexes of both GalCer and GlcCer, Ca2+ also caused heterotypic complexation of CBS to both GalCer and GlcCer. The heterotypic complexes had the greatest stability of all oligomers formed and survived better at high declustering potentials. Complexes of CBS with GlcCer were less stable than those with GalCer. This was confirmed by using the free sugars and glycosides making up the carbohydrate headgroups of these lipids. HFA species of CBS and GalCer formed more stable complexes than NFA species, but hydroxylation of the fatty acid of GlcCer had no effect. The ability of GlcCer to also complex with CBS, albeit with lower stability, may allow GlcCer to partially compensate for the absence of GalCer in the mouse mutant.     

Macro-ripple phase formation in bilayers composed of galactosylceramide and phosphatidylcholine.

As determined by freeze fracture electron microscopy, increasing levels of bovine brain galactosylceramide (GalCer) altered the surface structure of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers by inducing a striking "macro-ripple" phase in the larger, multilamellar lipid vesicles at GalCer mole fractions between 0.4 and 0.8. The term "macro-ripple" phase was used to distinguish it from the P beta' ripple phase observed in saturated, symmetric-chain length phosphatidylcholines. Whereas the P beta' ripple phase displays two types of corrugations, one with a wavelength of 12-15 nm and the other with a wavelength of 25-35 nm, the macro-ripple phase occurring in GalCer/POPC dispersions was of one type with a wavelength of 100-110 nm. Also, in contrast to the extended linear arrays of adjacent ripples observed in the P beta' ripple phase, the macro-ripple phase of GalCer/POPC dispersions was interrupted frequently by packing defects resulting from double dislocations and various disclinations and, thus, appeared to be continuously twisting and turning. Control experiments verified that the macro-ripple phase was not an artifact of incomplete lipid mixing or demixing during preparation. Three different methods of lipid mixing were compared: a spray method of rapid solvent evaporation, a sublimation method of solvent removal, and solvent removal using a rotary evaporation apparatus. Control experiments also revealed that the macro-ripple phase was observed regardless of whether lipid specimens were prepared by either ultra-rapid or manual plunge freezing methods as well as either in the presence or absence of the cryo-protectant glycerol. The macro-ripple phase was always observed in mixtures that were fully annealed by incubation above the main thermal transition of both POPC and bovine brain GalCer before rapid freezing. If the GalCer mixed with POPC contained only nonhydroxy acyl chains or only 2-hydroxy acyl chains, then the occurrence of macro-ripple phase decreased dramatically.     

Molecular dynamics simulations and 2H NMR study of the GalCer/DPPG lipid bilayer

Molecular dynamics simulations were performed on a two-component lipid bilayer system in the liquid crystalline phase at constant pressure and constant temperature. The lipid bilayers were composed of a mixture of neutral galactosylceramide (GalCer) and charged dipalmitoylphosphatidylglycerol (DPPG) lipid molecules. Two lipid bilayer systems were prepared with GalCer:DPPG ratio 9:1 (10%-DPPG system) and 3:1 (25%-DPPG system). The 10%-DPPG system represents a collapsed state lipid bilayer, with a narrow water space between the bilayers, and the 25%-DPPG system represents an expanded state with a fluid space of approximately 10 nm. The number of lipid molecules used in each simulation was 1024, and the length of the production run simulation was 10 ns. The simulations were validated by comparing the results with experimental data for several important aspects of the bilayer structure and dynamics. Deuterium order parameters obtained from (2)H NMR experiments for DPPG chains are in a very good agreement with those obtained from molecular dynamics simulations. The surface area per GalCer lipid molecule was estimated to be 0.608 +/- 0.011 nm(2). From the simulated electron density profiles, the bilayer thickness defined as the distance between the phosphorus peaks across the bilayer was calculated to be 4.21 nm. Both simulation systems revealed a tendency for cooperative bilayer undulations, as expected in the liquid crystalline phase. The interaction of water with the GalCer and DPPG oxygen atoms results in a strong water ordering in a spherical hydration shell and the formation of hydrogen bonds (H-bonds). Each GalCer lipid molecule makes 8.6 +/- 0.1 H-bonds with the surrounding water, whereas each DPPG lipid molecule makes 8.3 +/- 0.1 H-bonds. The number of water molecules per GalCer or DPPG in the hydration shell was estimated to be 10-11 from an analysis of the radial distribution functions. The formation of the intermolecular hydrogen bonds was observed between hydroxyl groups from the opposing GalCer sugar headgroups, giving an energy of adhesion in the range between -1.0 and -3.4 erg/cm(2). We suggest that this value is the contribution of the hydrogen-bond component to the net adhesion energy between GalCer bilayers in the liquid crystalline phase.     

Galactosylceramide domain microstructure: impact of cholesterol and nucleation/growth conditions

Galactosylceramide (GalCer), a glycosphingolipid, is believed to exist in the extracellular leaflet of cell membranes in nanometer-sized domains or rafts. The local clustering of GalCer within rafts is thought to facilitate the initial adhesion of certain viruses, including HIV-1, and bacteria to cells through multivalent interactions between receptor proteins (gp120 for HIV-1) and GalCer. Here we use atomic force microscopy (AFM) to study the effects of cholesterol on solid-phase GalCer domain microstructure and miscibility with a fluid lipid 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) in supported lipid bilayers. Using "slow-cooled vesicle fusion" to prepare the supported lipid bilayers, we were able to overcome the nonequilibrium effects of the substrate (verified by comparison to results for giant unilamellar vesicles) and accurately quantify the dramatic effect of cholesterol on the GalCer domain surface area/perimeter ratio (A(D)/P) and DLPC-GalCer miscibility. We compare these results to a supported lipid bilayer system in which the bilayer is rapidly cooled (nonequilibrium conditions), "quenched vesicle fusion", and find that the microstructures are remarkably similar above a cholesterol mol fraction of approximately 0.06. We determined that GalCer domains were contained in one leaflet distal to the mica substrate through qualitative binding experiments with Trichosanthes kirilowii agglutinin (TKA), a galactose-specific lectin, and AFM of Langmuir-Blodgett deposited GalCer/DLPC supported lipid bilayers. In addition, GalCer domains in bilayers containing cholesterol rearranged upon tip-sample contact. Our results further serve to clarify why discrepancies exist between different model membrane systems and between model membranes and cell membranes. In addition, these results offer new insight into the effect of cholesterol and surrounding lipid on domain microstructure and behavior. Finally, our observations may be pertinent to cell membrane structure, dynamics, and HIV infection.     

HIV-1 gp41 envelope residues 650-685 exposed on native virus act as a lectin to bind epithelial cell galactosyl ceramide

The initial step in the interaction between human immunodeficiency virus (HIV-1) and epithelial cells is the binding of HIV-1 envelope glycoproteins to the epithelial cell galactosyl ceramide (GalCer). Here we show that HIV-1 envelope gp41 residues 650-685 bind GalCer in a galactose-specific manner. The gp41 residues that display this lectin activity are highly conserved among HIV-1 isolates and constitute three regions: residues 650-661, which encompass a charged helix; residues 662-667, referred to as the conserved epitope ELDKWA, the epitope recognized by antibodies that neutralize HIV-1 entry in epithelial and CD4(+)-mononucleated cells; and residues 668-685, a hydrophobic Trp-rich sequence that stabilizes the structure of the galactose binding site. Similar to other galactose-specific lectins, the gp41 lectin site is active only as an oligomer. Finally the orientation of the galactose toward the gp41 lectin site appears to be controlled by the lipid microenvironment of the epithelial membrane. From the experimental data we construct a theoretical model of the interaction between gp41 and GalCer based on thermodynamic considerations. This model integrates the dynamics and the spatial organization of the viral envelope glycoproteins, GalCer organized in raft microdomains in the apical region of the epithelial cell membrane and the interfacial water. Characterization of the minimal sequence and structure of gp41 in direct interaction with GalCer may help unravel the still unknown immunogenic determinant able to elicit antibodies against ELDKWA and target of one of the rare neutralizing antibodies against gp41.     

Glycosphingolipid fatty acid arrangement in phospholipid bilayers: cholesterol effects.

Deuterium wide line NMR spectroscopy was used to study cholesterol effects on the ceramide portions of two glycosphingolipids (GSLs) distributed as minor components in fluid membranes. The common existence of very long fatty acids on GSLs was taken into account by including one glycolipid species with fatty acid chain length matching that of the host matrix, and one longer by 6 carbons. N-stearoyl and N-lignoceroyl galactosyl ceramide with perdeuterated fatty acid (18:0[d35] GalCer and 24:0[d47] GalCer) were prepared by partial synthesis. They were dispersed in bilayer membranes having the 18-carbon-fatty-acid phospholipid, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), as major component. Glycolipid fatty acid chain behavior and arrangement were analyzed using order profiles derived from their 2H-NMR spectra. Cholesterol effects on order parameter profiles for 18:0[d35] GalCer, with chain length equal to that of the host matrix, followed the pattern known for acyl chains of phospholipids. The presence of sterol led to restriction of trans/gauche isomerization along the length of the chain, with the largest absolute increase in order parameters being toward the surface, but somewhat greater relative effect just below the "plateau" region. In cholesterol-containing membranes, order parameter profiles for the long chain species, 24:0[d47] GalCer, showed a characteristic secondary "plateau" associated with carbon atoms C14 to C23, a feature also present in SOPC bilayers without cholesterol and in pure hydrated 24:0[d47] GalCer. Cholesterol-induced ordering effects on the long chain glycolipid were similar to those described for the shorter chain species, but were minimal at the methyl terminus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adhesion between cerebroside bilayers

The structure, hydration properties, and adhesion energy of the membrane glycolipid galactosylceramide (GalCer) were studied by osmotic stress/X-ray diffraction analysis.(1) Fully hydrated GalCer gave a repeat period of 67 A, which decreased less than 2 A with application of applied osmotic pressures as large as 1.6 x 10(9) dyn/cm(2). These results, along with the invariance of GalCer structure obtained by a Fourier analysis of the X-ray data, indicated that there was an extremely narrow fluid space (less than the diameter of a single water molecule) between fully hydrated cerebroside bilayers. Electron density profiles showed that the hydrocarbon chains from apposing GalCer monolayers partially interdigitated in the center of the bilayer. To obtain information on the adhesive properties of GalCer bilayers, we incorporated into the bilayer various mole ratios of the negatively charged lipid dipalmitoylphosphatidylglycerol (DPPG) to provide known electrostatic repulsion between the bilayers. Although 17 and 20 mol % DPPG swelled (disjoined) the GalCer bilayers by an amount predictable from electrostatic double-layer theory, 5, 10, 13, and 15 mol % DPPG did not disjoin the bilayers. By calculating the magnitude of the electrostatic pressure necessary to disjoin the bilayers, we estimated the adhesion energy for GalCer bilayers to be about -1.5 erg/cm(2), a much larger value than that previously measured for phosphatidylcholine bilayers. The observed discontinuous disjoining with increased electrostatic pressure and this relatively large value for adhesion energy indicated the presence of an attractive interaction, in addition to van der Waals attraction, between cerebroside bilayers. Possible attractive interactions are hydrogen bond formation and hydrophobic interactions between the galactose headgroups of apposing GalCer bilayers.     

Specificity of the glycolipid transfer protein from pig brain

Lipid specificity has been studied in the lipid transfer reaction facilitated by the glycolipid transfer protein from pig brain. The lipid transfer was measured by determining the transfer of a radioisotopically labeled lipid from donor liposomes to either acceptor liposomes or mitochondria. Whenever possible, the liposomes contained 1 mol % of the lipid whose transfer was under study. The transfer protein accelerates the transfer of glucosylceramide, galactosylceramide (GalCer), lactosylceramide (LacCer), galactosylceramide 3-sulfate, globotriaosylceramide, LacCer sulfate, sialosyl-LacCer, globotetraosylceramide, and globopentaosylceramide. An inverse relationship is found between the length of sugar chains in glycosphingolipids and the transfer rates. In addition to the glycosphingolipids, the transfer protein facilitates the transfer of galactosyldiacylglycerol, digalactosyldiacylglycerol, glucosyldiacylglycerol, and diglucosyldiacylglycerol. The protein does not facilitate the transfer of dimannosyldiacylglycerol. The transfer of periodate-oxidized and subsequently reduced derivatives of GalCer and LacCer is facilitated by the transfer protein. The derivatives of GalCer are transferred at lower rates than GalCer, whereas the derivatives of LacCer are transferred at higher rates than LacCer. The transfer protein does not facilitate the transfer of phosphatidylcholine, phosphatidylinositol, cholesterol, or cholesteryloleate. These results suggest that the glycolipid transfer protein from pig brain has specificity to hydroxyl groups present in the sugar residue directly linked to either ceramide or diacylglycerol. The presence of glucose or galactose linked to these hydrophobic moieties makes the glycolipid transferable by the protein.     

Synthesis of novel, multivalent glycodendrimers as ligands for HIV-1 gp120

Multivalent neoglycoconjugates are valuable tools for studying carbohydrate-protein interactions. To study the interaction of HIV-1 gp120 with its reported alternate glycolipid receptors, galactosyl ceramide (GalCer) and sulfatide, galactose- and sulfated galactose-derivatized dendrimers were synthesized, analyzed as ligands for rgp120 by surface plasmon resonance, and tested for their ability to inhibit HIV-1 infection of CXCR4- and CCR5-expressing indicator cells. Four different series of glycodendrimers were made by amine coupling spacer-arm derivatized galactose residues, either sulfated or nonsulfated, to poly(propylenimine) dendrimers, generations 1-5. One series of glycodendrimers was prepared from the ceramide saccharide derivative of purified natural GalCer, and another was from chemically synthesized 3-(beta-D-galactopyranosylthio)propionic acid. Synthesis of 3-sulfogalactopyranosyl-derivatized dendrimers was accomplished using the novel compound, 3-(beta-D-3-sulfogalactopyranosylthio)propionic acid. The fourth series was made by random sulfation of the 3-(beta-D-galactopyranosylthio)propionic acid functionalized dendrimers. Structures of the carbohydrate moieties were confirmed by NMR, and the average molecular weights and polydispersities of the different glycodendrimers were determined using MALDI-TOF MS. Surface plasmon resonance studies found that rgp120 IIIB bound to the derivatized dendrimers tested with nanomolar affinity, and to dextran sulfate with picomolar affinity. In vitro studies of the effectiveness of these compounds at inhibiting infection of U373-MAGI-CCR5 cells by HIV-1 Ba-L indicated that the sulfated glycodendrimers were better inhibitors than the nonsulfated glycodendrimers, but not as effective as dextran sulfate.     

Human immunodeficiency virus type 1 infection of SK-N-MC cells: domains of gp120 involved in entry into a CD4-negative, galactosyl ceramide/3' sulfo-galactosyl ceramide-positive cell line.

The primary receptor for human immunodeficiency virus (HIV) is the CD4 molecule; however, in vitro evidence suggests that a neutral glycolipid, galactosyl ceramide (GalCer) or a derivative molecule, 3' sulfogalactosyl ceramide (GalS), may serve as an alternative receptor for HIV type 1 (HIV-1) in cells of neural and colonic origin. Biochemical studies have demonstrated that recombinant gp120 envelope protein binds to GalCer/GalS in both solid-phase enzyme-linked immunosorbent assay and high-performance thin-layer chromatography overlays. We have used the SK-N-MC cell line, a CD4-negative, GalCer/GalS-positive cell line previously characterized as susceptible to HIV-1 infection, to identify virus isolates with either a positive infection phenotype, HIVHxB2, or a negative infection phenotype, HIV-1(89.6). Using a solid-phase virus binding assay, we determined the level of restriction in HIV-1(89.6) infection to be at the level of virus-glycolipid binding. Furthermore, using HIV-1HxB2-HIV-1(89.6) chimeras, we have identified a 193-amino-acid fragment from the envelope region of HIV-1HxB2 containing the V3, V4, and V5 regions which confers a positive infection phenotype on the HIV-1(89.6) background. Recombinant viruses which separate this 193-amino-acid fragment into two distinct chimeras are each able to confer a positive infection phenotype on the background of HIV89.6, suggesting that a stable GalCer/GalS-envelope interaction is dependent on the conformation of the envelope protein in the context of the viral membrane. Alternatively, the GalCer/GalS-gp120 bond may involve multiple sites on the oligomeric envelope protein.     

C-Glycoside analogues of beta-galactosylceramide with a simple ceramide substitute: synthesis and binding to HIV-1 gp120

The synthesis and HIV-1 gp120 binding of C- and aza-C-glycoside analogues of beta-galactosylceramide (GalCer) that contain a simple C-17 hydrocarbon chain as a ceramide substitute are described. Both compounds originate from stearic acid, and a carbohydrate-derived thioacetal-alcohol, and their syntheses are potentially general for beta-C-galactosides and their aza-C-partners. They showed potent and specific affinity for gp120 in an assay based on the change of surface pressure when the glycolipid monolayers were exposed to solutions of gp120. Interestingly, the aza-C-glycoside exhibited a significantly higher affinity than GalCer, whereas the C-glycoside was as active as GalCer.     

Post-translational Modification of κ-Casein in the Golgi Apparatus of Mid-lactating Mammary Glands from Rat and Cow

Bovine κ-casein, a phosphoglycoprotein, has mucin-type carbohydrate chains. Subcellular distribution of enzymes that take part in the post-translational modification of κ-casein was examined. In lactating mammary glands from rats and cows, N-acetyl-galactosaminyl transferase, galactosyl transferase, sialyl transferase, and casein kinase were localized specifically in the Golgi apparatus.

The substrate specificities indicate that these enzymes are actually responsible for the processing of κ-casein.

The presence of a phosphate group attached to κ-casein did not affect the rate of glycosylation by N-acetyl-galactosaminyl transferase, while the presence of carbohydrate chains attached to κ- casein strongly reduced the rate of phosphorylation by casein kinase. These results suggest that in the Golgi apparatus, phosphorylation of κ-casein precedes glycosylation.     

A new immunosensing method by galactose oxidase-mediated electrocatalysis using a virtual beaker array

We report a novel and convenient method for the determination of glycoproteins, especially antibodies, using galactose oxidase (GAO) on the basis of the contents of galactosyl and N-acetylgalactosaminyl residues in carbohydrate chains of glycoproteins. GAO converts galactose residues to their corresponding aldehyde and H₂O₂, the latter being electroactive and quantifiable by DC amperometry. The method does not require processes such as antibody labeling or the use of enzyme-tagged secondary antibodies. For an array-type immunosensing, the platform surface for antigen immobilization was specially designed by using differentiated surface wetting property of hydrophobic and hydrophilic patterns. We patterned the hydrophobic surface of the poly(dimethylsiloxane) substrate by microcontact printing with the poly(amidoamine) dendrimer ink, providing hydrophilic patterns on a hydrophobic base substrate. By applying aqueous solution on the patterned surface, an array of free-standing water droplets was made. With the prepared virtual beaker array, electrochemical immunosensing was performed by using anti-dinitrophenyl-IgG as a model target protein. From immunoassay with GAO-mediated electrocatalysis, a good correlation in amperometric signal with the target IgG was registered. The total assay time was about 20 min, including antibody recognition and signal registration.     

Composition and distribution of carbohydrate chains in glycoproteins of human erythrocyte membrane

The M-, N-, and MN-glycoproteins obtained from human erythrocytes by phenol-water extraction were purified by gel filtration and digested with Pronase and trypsin. The products of degradation were fractionated by gel filtration on Sephadex G-25 and DEAE-Sephadex A-50 and the fractions were examined by poly(acrylamide)-gel electrophoresis in the presence of dodecyl sodium sulfate, analyzed for carbohydrate and amino acid contents, and tested for M and N blood-group activity. From the results, it is suggested that the glycoprotein chains are composed of a hydrophobic moiety devoid of carbohydrate chains and a hydrophilic moiety containing carbohydrate chains of different compositions, irregularly distributed along the protein chains and linked to L-asparagine, L-serine, or L-threonine residues. The M and N activity typical for the undegraded glycoproteins, and the “basic” or “precursor-type” N activity, were found in different glycopeptide fractions.     

Fluid-phase chain unsaturation controlling domain microstructure and phase in ternary lipid bilayers containing GalCer and cholesterol

We report the microstructure and phase behavior of three ternary mixtures each containing a long-chain saturated glycosphingolipid, galactosylceramide (GalCer), and cholesterol at room temperature. The unsaturation level of the fluid-phase component was varied by lipid choice, i.e., saturated 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), singly unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or doubly unsaturated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). GalCer was used because of its biological significance, for example, as a ligand in the sexual transmission of HIV and stimulator of natural killer T-cells. Supported lipid bilayers of the ternary mixtures were imaged by atomic force microscopy and GalCer-rich domains were characterized by area/perimeter ratios (A/P). GalCer domain phase transitions from solid (S) to liquid (L) phase were verified by domain behavior in giant unilamellar vesicles, which displayed two-dimensional microstructure similar to that of supported lipid bilayers. As cholesterol concentration was increased, we observed approximately 2.5, approximately 10, and approximately 20-fold decreases in GalCer domain A/P for bilayers in L-S phase coexistence containing DOPC, POPC, and DLPC, respectively. The transition to L-L phase coexistence occurred at approximately 10 mol % cholesterol for bilayers containing DOPC or POPC and was accompanied by maintenance of a constant A/P. L-L phase coexistence did not occur for bilayers containing DLPC. We systematically relate our results to the impact of chain unsaturation on the interaction of the fluid-phase lipid and cholesterol. Physiologically, these observations may give insight into the interplay of fatty acid chain unsaturation, sterol concentration, and lipid hydrophobic mismatch in membrane phenomena.