Metabolism of exogenous gangliosides GM1 and chemically modified GM1 in mice

Ganglioside GM1(NeuAc), labeled at the C-3 position of sphingosine with tritium, was injected into C3H/He, C57BL/10, B10.AQR mice intraperitoneally. The incorporation and the distribution of the radioactivity in various organs were examined. The injected [3H]GM1(NeuAc) was mainly incorporated in the liver and hydrolyzed sequentially. Sialic acid of ganglioside GM1(NeuAc) and metabolites was converted to N-glycolyl type from N-acetyl type. An appreciable amount of the sphingosine moiety in the administered GM1(NeuAc), moreover, was reutilized, being converted to sphingomyelin, and incorporated into alkyl chain of the ether lipid in phosphatidylethanolamine. The distributions of radioactivity in the metabolites of GM1(NeuAc) administered to the three strains of mice were different from each other. In other organs, GM1(NeuAc) was incorporated and metabolized only slightly. The N-methylamide, at the carboxyl group of the sialic acid, of the labeled ganglioside GM1(GM1(NeuAc)-NMe) was injected into C3H/He mice. Most of the administered [3H]GM1(NeuAc)-NMe was incorporated in the liver, and was metabolized to GM3(NeuAc)-NMe, via GM2(NeuAc)-NMe, within 24 h. GM3(NeuAc)-NMe was the only radioactive compound in the subsequent 10 weeks, but disappeared from the liver gradually. N-Methylamide-modified gangliosides were resistant to hydrolysis by mouse hepatic sialidase, to elongation by glycosyltransferase and to N-glycolylation at N-acetylneuraminic acid by monooxygenase.     

A model of a gp120 V3 peptide in complex with an HIV-neutralizing antibody based on NMR and mutant cycle-derived constraints.

The 0.5beta monoclonal antibody is a very potent strain-specific HIV-neutralizing antibody raised against gp120, the envelope glycoprotein of HIV-1. This antibody recognizes the V3 loop of gp120, which is a major neutralizing determinant of the virus. The antibody-peptide interactions, involving aromatic and negatively charged residues of the antibody 0.5beta, were studied by NMR and double-mutant cycles. A deuterated V3 peptide and a Fab containing deuterated aromatic amino acids were used to assign these interactions to specific V3 residues and to the amino acid type and specific chain of the antibody by NOE difference spectroscopy. Electrostatic interactions between negatively charged residues of the antibody Fv and peptide residues were studied by mutagenesis of both antibody and peptide residues and double-mutant cycles. Several interactions could be assigned unambiguously: F96(L) of the antibody interacts with Pro13 of the peptide, H52(H) interacts with Ile7, Ile9 and Gln10 and D56(H) interacts with Arg11. The interactions of the light-chain tyrosines with Pro13 and Gly14 could be assigned to either Y30a(L) and Y32(L), respectively, or Y32(L) and Y49(L), respectively. Three heavy-chain tyrosines interact with Ile7, Ile20 and Phe17. Several combinations of assignments involving Y32(H), Y53(H), Y96(H) and Y100a(H) may satisfy the NMR and mutagenesis constraints, and therefore at this stage the interactions of the heavy-chain tyrosines were not taken into account. The unambiguous assignments [F96(L), H52(H) and D56(H)] and the two possible assignments of the light-chain tyrosines were used to dock the peptide into the antibody-combining site. The peptide converges to a unique position within the binding site, with the RGPG loop pointing into the center of the groove formed by the antibody complementary determining regions while retaining the beta-hairpin conformation and the type-VI RGPG turn [Tugarinov, V., Zvi, A., Levy, R. & Anglister, J. (1999) Nat. Struct. Biol. 6, 331-335].     

Increased glycosphingolipid levels in serum and aortae of apolipoprotein E gene knockout mice

The apolipoprotein E gene knockout (apoE-/-) mouse develops atherosclerosis that shares many features of human atherosclerosis. Increased levels of glycosphingolipid (GSL) have been reported in human atherosclerotic lesions; however, GSL levels have not been studied in the apoE-/- mouse. Here we used HPLC methods to analyze serum and aortic GSL levels in apoE-/- and C57BL/6J control mice. The concentrations of glucosyl ceramide (GlcCer), lactosyl ceramide (LacCer), GalNAcbeta1-4Galbeta1-4Glc-Cer (GA2), and ceramide trihexoside (CTH) were increased by approximately 7-fold in the apoE-/- mouse serum compared with controls. The major serum ganglioside, N-glycolyl GalNAcbeta1-4[NeuNAcalpha2-3]Galbeta1-4Glc-Cer (N-glycolyl GM2), was increased in concentration by approximately 3-fold. A redistribution of GSLs from HDL to VLDL populations was also observed in the apoE-/- mice. These changes were accompanied by an increase in the levels of GSLs in the aortic sinus and arch of the apoE-/- mice. The spectrum of gangliosides present in the aortic tissues was more complex than that found in the lipoproteins, with the latter represented almost entirely by N-glycolyl GM2 and the former comprised of NeuNAcalpha2-3Galbeta1-4Glc-Cer (GM3), GM2, N-glycolyl GM2, GM1, GD3, and GD1a. In conclusion, neutral GSL and ganglioside levels were increased in the serum and aortae of apoE-/- mice compared with controls, and this was associated with a preferential redistribution of GSL to the proatherogenic lipoprotein populations. The apoE-/- mouse therefore represents a useful model to study the potential role of GSL metabolism in atherogenesis.     

N-Acetylneuraminyllactosylceramide, GM3-NeuAc, a new influenza A virus receptor which mediates the adsorption-fusion process of viral infection. Binding specificity of influenza virus A/Aichi/2/68 (H3N2) to membrane-associated GM3 with different molecular species of sialic acid

Agglutinates of native chicken erythrocytes caused by influenza virus A/Aichi/2/68 (H3N2) at 4 degrees C were potently fused and lysed at low pH (optimum pH 5.3) at 37 degrees C. Exogenous gangliosides GM3 (Sia alpha 2-3Gal beta 1-4Glc beta 1-ceramide) and GM2 (GalNAc beta 1-4(Sia alpha 2-3)-Gal beta 1-4Glc beta 1-ceramide) were integrated into the membranes of chicken asialoerythrocytes within 5-min incubation at 37 degrees C. We found that the incorporation of ganglioside GM3 containing N-acetylneuraminic acid into asialoerythrocytes restored the biological responsiveness to the virus as established by agglutination at 4 degrees C and fusion and hemolysis at 37 degrees C at pH 5.3. Biological responsiveness of GM3-NeuAc-erythrocytes to the virus was considerably higher than that of GM3-NeuGc-erythrocytes under the same experimental conditions. Treatment of the GM3-NeuAc-erythrocytes with neuraminidase again resulted in the complete abolishment of the response to the virus. Erythrocytes containing GM2-NeuAc showed no detectable biological responses toward the virus. The above results indicate that the hemagglutinin of influenza virus A/Aichi/2/68 (H3N2) recognizes the sialyloligosaccharide chain of ganglioside GM3 as its receptor which mediates the adsorption and fusion process on the virus entry into the host cells and has more preferential specificity for binding to N-acetylneuraminic acid-containing GM3 than that to N-glycolyl type in the target cell membranes.     

Preparation and specificity of avian anti-GM2(NeuGc) ganglioside antiserum

Antibodies to N-glycolyl neuraminic acid-containing GM2 ganglioside, GM2(NeuGc), were prepared by immunizing chickens. The specificity of the antibodies was examined by the double immunodiffusion test and solid-phase radioimmunoassay (RIA). One(C-4) of two antisera produced did not cross-react with GM3(NeuGc) but the other(C-3) did as assessed by the double immunodiffusion test. In RIA, the antibody activity of C-4 antiserum was detected only in the IgG fraction. Specificity of the serum was examined using authentic glycolipids which were structurally related to GM2(NeuGc). The antiserum showed a high specificity for the homologous ganglioside by either an RIA or an inhibition assay. This antiserum is a useful tool for the detection of GM2(NeuGc) in human and animal tissues under normal and/or disease condition.     

Monoclonal antibody detects monosialogangliosides having a sialic acid alpha 2----3-galactosyl residue

A murine monoclonal antibody (mAb), designated mAb 202, was generated using a human melanoma cell line, UCLASO-M14 as the immunogen. mAb 202 reacted with two (GM2 and GM3) of the four (GM2, GM3, GD2, and GD3) gangliosides expressed by M14. Several authentic monosialogangliosides, including GM4, GM3, GM2, GM1, GM1b, and sialylparagloboside were then tested for their binding to 202 mAb by the immune adherence inhibition assay, TLC-enzyme immunostaining, and enzyme-linked immunosorbent assay. All showed positive binding but in varying degrees. GM4 showed the strongest affinity. No significant differences of reactivity were observed between the sialic acid derivatives, N-acetyl and N-glycolyl, in these gangliosides. Disialogangliosides such as GD3, GD2, GD1a, and GD1b, trisialoganglioside GT1b, and neutral glycolipids including GlcCer, GalCer, LacCer, GbOs3Cer, GbOs4Cer, GgOs3Cer, GgOs4Cer, and nLcOs4Cer were all negative. These results indicate that the 202 mAb detects sialyl alpha 2----3Gal residue in the monosialoganglioside, irrespective of the internal structure. Since GM4 is not expressed by M14 cells, the terminal disaccharide (sialyl alpha 2----3Gal) in GM3 and/or GM2 must have been the epitope responsible for the generation of the antibody.     

Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells

We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position. Pulse-chase analysis of the 3H/14C ratio showed that the N-acetyl groups of both GD3 and of the monosialoganglioside GM3 were turning over faster than the parent molecules. Selective periodate oxidation showed that both the inner and outer sialic acid residues of GD3 incorporated 3H-label in the in vitro reaction, and showed similar turnover of N-acetylation in the pulse-chase study. Taken together, these results indicate that both the O- and N-acetyl groups of the sialic acid residues of gangliosides turn over faster than the parent molecules. They also demonstrate a novel re-N-acetylation reaction that predicts the existence of de-N-acetyl gangliosides in melanoma cells.     

Structural rationale for the broad neutralization of HIV-1 by human monoclonal antibody 447-52D

447-52D is a human monoclonal antibody isolated from a heterohybridoma derived from an HIV-1-infected individual. This antibody recognizes the hypervariable gp120 V3 loop, and neutralizes both X4 and R5 primary isolates, making it one of the most effective anti-V3 antibodies characterized to date. The crystal structure of the 447-52D Fab in complex with a 16-mer V3 peptide at 2.5 A resolution reveals that the peptide beta hairpin forms a three-stranded mixed beta sheet with complementarity determining region (CDR) H3, with most of the V3 side chains exposed to solvent. Sequence specificity is conferred through interaction of the type-II turn (residues GPGR) at the apex of the V3 hairpin with the base of CDR H3. This novel mode of peptide-antibody recognition enables the antibody to bind to many different V3 sequences where only the GPxR core epitope is absolutely required.     

Neutralization of NGF-TrkA receptor interaction by the novel antagonistic anti-TrkA monoclonal antibody MNAC13: a structural insight

MNAC13, a mouse monoclonal antibody, recognizes with high affinity and specificity the neurotrophin receptor TrkA and displays a neutralizing activity toward the NGF/TrkA interaction. Detailed knowledge of the molecular basis determining the specificity of this antibody is of importance because of its potential use as a modulator of the TrkA-mediated NGF activity. Here, we report a full biochemical and structural characterization of the MNAC13 antibody. Epitope mapping studies, by serial deletion mutants and by phage display, reveal a conformational epitope that is localized on the carboxy-terminal region of the first immunoglobulin-like domain (d4) of TrkA. The X-ray crystal structure of the MNAC13 Fab fragment has been determined and refined to 1.8 A resolution. The antigen-binding site is characterized by a crevice, surrounded by hydrophilic-charged residues on either side, dipping deep toward three mainly hydrophobic subsites. Remarkably an isopropanol molecule has been found to bind in one of the hydrophobic crevices. Overall, the surface topology (shape and electrostatic potential) of the combining site is consistent with the binding data on TrkA ECD serial deletions mutants. The structure of the MNAC13 Fab fragment may assist in the rational structure-based design of high affinity humanized forms of MNAC13, appropriate for therapeutic approaches in neuropathy and inflammatory pain states.     

Computational 3-D modeling and site-directed mutation of an antibody that binds Neu2en5Ac, a transition state analogue of a sialic acid.

An antibody against a transition state analog (TSA) may share some common features with an enzyme that produces such a transition state. SIC172 antibody binds specifically to Neu2en5Ac, a TSA of Neu5Ac in the sialidase reaction, but has no catalytic activity. To understand how the antibody recognizes Neu2en5Ac and to find out if it is possible to convert it to a catalytic antibody, we made and sequenced the SIC172 ScFv, and constructed a 3-D model of it. The VH-CDR3 contains a unique sequence with Cys at H95. The 3-D model showed that Cys-H95 is exposed inside the antigen-binding cavity. After affinity docking, 4 types emerged. In type I, the carboxyl group of Neu2en5Ac is located near the Cys-H95 and neighboring positively charged residues. The change of Cys-H95 to Asp by site-directed mutation decreased the binding activity, while a change to Arg did not. These and other mutation experiments, and further modeling of mutant Fv, support the 3-D model and docking type I. A comparison with sialidase indicates that SIC172 antibody appears to have some groups of residues that are conserved at the active site of the enzyme. The possibility of Neu2en5Ac-binding antibody being converted to a catalytic antibody is discussed.     

Analysis of structure and function of the B subunit of cholera toxin by the use of site-directed mutagenesis

Oligonucleotide-directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT-B) altered at residues Cys-9, Gly-33, Lys-34, Arg-35, Cys-86 and Trp-88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT-B polypeptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT-B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly-33 and negatively or positively charged residues for Trp-88. Substitution of lysine or arginine for Gly-33 did not affect immunoreactivity or GM1-binding activity of CT-B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg-35 interfered with formation of holotoxin, but none of the observed substitutions for Lys-34 or Arg-35 affected binding of CT-B to GM1. The Cys-9, Cys-86 and Trp-88 residues were important for establishing or maintaining the native conformation of CT-B or protecting the CT-B polypeptide from rapid degradation in vivo.     

Incorporation of ganglioside analogues into fibroblast cell membranes. A spin-label study

The uptake of ganglioside analogues by a permanent mouse fibroblast cell line has been studied by radio-tracer techniques and ESR spectroscopy with 3H- and nitroxide-labeled compounds. Analogues of GM1, GM2, and GM3 monosialogangliosides and of GD1a and GD3 disialogangliosides were synthesized. The spin-label group was situated on the 5-, 9-, or 13-carbon atom of the C18 fatty acid chain, and the 3H label was in the carbohydrate moiety. Part of the ganglioside associated with the cells could be removed by trypsin treatment and was shown to consist of ganglioside micelles attached to the cell surface. The trypsin-resistant component displayed characteristic anisotropic ESR spectra which closely resembled those of the same spin-labeled analogues at low dilution in liposomes prepared from the extracted cell lipids. The flexibility gradient, polarity profile, and temperature dependence displayed by the spectra were similar to those found for fluid phospholipid bilayer model membranes, and the high effective order parameters suggested a location in the cell plasma membrane. Similar results were obtained for all the different ganglioside analogues, indicating a common anchoring region in the hydrophobic interior of the membrane. Under the incubation conditions used the amount of trypsin-resistant ganglioside analogue taken up by the cells was about 15 nmol/mg of cellular protein, irrespective of the nature of the oligosaccharide moiety. By use of the natural ganglioside [3H]GM3, the trypsin-resistant uptake was about 19 nmol/mg of cellular protein. Although these amounts are quite similar, the uptake kinetics differed between the true ganglioside GM3 and the ganglioside analogues.     

Structural insights into steroid hormone binding: the crystal structure of a recombinant anti-testosterone Fab fragment in free and testosterone-bound forms.

The monoclonal anti-testosterone antibody (3-C(4)F(5)) has a relatively high affinity (3 x 10(8) m(-1)) with an overall good specificity profile. However, the earlier characterized binding properties have shown that both the affinity and specificity of this antibody must be improved if it is intended for use in clinical immunoassays. In this paper, the crystal structures of the recombinant anti-testosterone (3-C(4)F(5)) Fab fragment have been determined in the testosterone-bound and free form at resolutions of 2.60 and 2.72 A, respectively. The high affinity binding of the (3-C(4)F(5)) Fab is mainly determined by shape complementarity between the protein and testosterone. Only one direct hydrogen bond is formed between the hydroxyl group of the testosterone D-ring and the main-chain oxygen of Gly100(J)H. The testosterone is deeply bound in a hydrophobic pocket, and the close shape complementarity is mainly formed by the third complementarity-determining regions (CDR) of the heavy and light chain. Comparison of the bound structure with the free structure indicates conformational changes in the protein upon testosterone binding. The conformational changes of the side chains of two residues Glu95H and Tyr99H in the CDR-H3 are particularly essential for the binding. Interesting similarities in the binding of different steroids were also observed upon comparison of the available structures of anti-steroid antibodies.     

Ganglioside expression in macrophages from endotoxin responder and nonresponder mice

Peritoneal macrophage ganglioside patterns and ganglioside sialic acid content were compared for two congenic strains of mice having differing responses to bacterial lipopolysaccharide. Resident macrophage ganglioside patterns from C3H/HeJ mice (endotoxin hyporesponsive) and C3H/HeN mice (endotoxin responsive) were similar. Macrophages elicited with phenol-extracted or butanol-extracted endotoxin showed distinctly more complex ganglioside patterns in C3H/HeN mice. C3H/HeJ macrophages showed distinct, but less complex changes when elicited with butanol-extracted endotoxin. As expected, there were minimal alterations induced by phenol-extracted endotoxin in the C3H/HeJ patterns. When injected with whole killed E. coli, both strains of mice exhibited complex ganglioside patterns; however, there were relative differences in the quantities of multiple gangliosides. Differences in ganglioside patterns were mirrored in the relative ratios of N-acetyl- to N-glycolylneuraminic acid. When macrophages were activated by administration of either endotoxin preparation, macrophage gangliosides from C3H/HeN mice always contained a higher proportion of N-acetylneuraminic acid compared with C3H/HeJ macrophage gangliosides. Oxidative metabolism of the macrophage populations was assessed by PMA-induced H2O2 release. This indicated that endotoxin activation produced an increase in PMA-induced H2O2 release as well as a shift of sialic acid class from the N-glycolyl type to the N-acetyl type. However, no direct correlation could be made between ganglioside composition, sialic acid content, and macrophage function. These data indicate that both ganglioside composition and sialic acid composition of macrophages are profoundly altered with endotoxin activation. The data further indicate that under conditions which C3H/HeJ mice respond to Gram-negative bacteria, their macrophage ganglioside patterns still differ from normal mice.     

Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.

Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.     

Computational study of bindings of HK20 Fab and D5 Fab to HIV-1 gp41

Antibodies HK20 and D5 have been shown to target HIV-1 gp41, thereby inhibiting membrane fusion that facilitates viral entry. The binding picture is static, based on the X-ray crystal structures of the Fab regions and gp41 mimetic five-helix bundle. In this study, we carried out molecular dynamics simulation to provide the dynamic binding picture. Calculated binding free energies are within reasonable range of and follow the trend of the experimental values: -15.28 kcal/mol for HK20 Fab (expt. -11.60 kcal/mol) and -17.90 kcal/mol for D5 Fab (expt. -11.70 kcal/mol). Alanine scanning at protein-protein interface reveals that the highest contributors to binding for HK20 Fab are F54 and I56, both of V(H) region, as well as R30' of V(L) region; whereas for D5 Fab, F54 of V(H) region, as well as W32' and Y94' of V(L) region. HK20 F54 and I56, as well as D5 I52, F54, and T56, bind to the gp41 hydrophobic binding pocket, an important region targeted by many other fusion inhibitors. Hydrogen bonding analysis also identifies high-occupancy hydrogen bonds at the periphery of gp41 hydrophobic pocket. Considering that almost all interface residues are turn residues, further work may be directed to turn mimics. Pre-orientation by the hydrogen bonds to poise this particular turn towards the binding pocket may also be a point worth pursuing.     

Sites of protein-protein interaction on the mitochondrial F1-ATPase inhibitor protein.

We have investigated the structure of the mitochondrial F1-ATPase inhibitor protein from ox heart by using a differential trace-labelling method. This method has also been used to determine sites on the inhibitor protein involved in binding to both the isolated mitochondrial ATPase (F1) and to a specific anti-inhibitor antibody. Native, free inhibitor was trace-labelled on its lysine and serine residues with [14C]acetic anhydride, and inhibitor protein unfolded in guanidinium chloride or specifically bound to another protein, with [3H]acetic anhydride. Exposure/concealment of residues was deduced from the 14C/3H ratios of the peptides in a proteolytic digest of the inhibitor, after separation by h.p.l.c. None of the lysine or serine residues in the native inhibitor are as exposed as in the unfolded form. There is a gradient of reactivity, with residues 54-58 being most concealed and exposure increasing towards either end of the protein. A slight decrease in reactivity is noted in residues 1-3, suggesting that the N-terminus may be in a fairly restricted environment. These findings are discussed in the light of the predicted structure of the inhibitor protein. All but one of the labelled residues increases in reactivity when inhibitor protein binds to F1. The exception, Lys-24, is only slightly concealed. Hence, F1 binding appears not to involve the lysine or serine residues directly. This finding is consistent with the view that the F1-inhibitor interaction is hydrophobic in nature. Complementary information was provided using an anti-inhibitor antibody that binds to a site on the inhibitor different from that at which F1 binds. Binding of this antibody conceals residues 54, 58, and 65 considerably. This confirms that F1 does not interact with these hydrophilic residues on the inhibitor protein.     

De-N-acetyllactotriaosylceramide as a novel cationic glycosphingolipid of bovine brain white matter: isolation and characterization

A novel cationic lipid was separated from bovine brain white matter by a series of chromatographies on carboxymethyl-Sephadex and silica gel in chloroform and methanol. Its structure was identified unambiguously as de-N-acetyllactotriaosylceramide (deNAcLc(3)Cer) by mass spectrometry and (1)H NMR. The natural occurrence of this glycolipid in white matter extract was detected by immunostaining of thin-layer chromatography with monoclonal antibody 5F5, which is directed to deNAcLc(3)Cer and recognizes the terminal beta-glucosaminyl (GlcNH(2)) residue, having a free NH(2) group. A de-N-acetylase capable of hydrolyzing the N-acetyl group of Lc(3)Cer was detected in bovine brain extract using N-[(14)C]acetyl-labeled Lc(3)Cer as a substrate. The biogenesis and possible functional significance of deNAcLc(3)Cer are discussed.     

Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.

Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].     

Characterization of the binding epitope of the monoclonal antibody DMAb-1 to ganglioside GM2.

Several derivatives of ganglioside GM2 were synthesized for mapping of the binding epitope of a monoclonal antibody raised against this ganglioside. The GM2 ganglioside was modified in both the hydrophobic and the hydrophobilic part of the molecule. The synthesized derivatives were characterized with fast atom bombardment mass spectrometry (FAB-MS). Affinity of the monoclonal antibody for the GM2 derivatives was determined by enzyme-linked immunosorbent assay (ELISA) on microtitre plates or by TLC immunostaining. Modifying the GM2 sialic acid by deacetylation or blocking of the carboxyl moiety abolished the binding to the monoclonal antibody while the cleaving of the glycol group on the sialic acid tail led to a 70% reduced binding affinity. Removal of the fatty acid (lyso-GM2) eliminated the binding to the antibody. GM2 derivatives with fatty acid moieties of 8 carbon atoms or less showed almost no reactivity. GM2 with saturated fatty acids 16:0, 18:0 and 20:0 had binding affinity similar to natural GM2, while the 24:0 fatty acid had only half the binding affinity. The results demonstrate the importance of ganglioside fatty acid composition with regard to ligand binding between the monoclonal antibody and its specific ganglioside antigen. Thus, caution must be shown in the application of immunaffinity methods with monoclonal antibodies for the quantitative determination of glycosphingolipids from different tissues.