Search

β-hexosaminidase A (HexA) protein is responsible for the degradation of GM2 gangliosides in the central and peripheral nervous systems. Tay-Sachs disease occurs when HexA within Hexosaminidase does not properly function and harmful GM2 gangliosides begin to build up within the neurons. In this study, in silico methods such as SIFT, PolyPhen-2, PhD-SNP, and MutPred were utilized to analyze the effects of nonsynonymous single nucleotide polymorphisms (nsSNPs) on HexA in order to identify possible pathogenetic and deleterious variants. Molecular dynamics (MD) simulations showed that two mutants, P25S and W485R, experienced an increase in structural flexibility compared to the native protein. Particularly, there was a decrease in the overall number and frequencies of hydrogen bonds for the mutants compared to the wildtype. MM/GBSA calculations were performed to help assess the change in binding affinity between the wildtype and mutant structures and a mechanism-based inhibitor, NGT, which is known to help increase the residual activity of HexA. Both of the mutants experienced a decrease in the binding affinity from -23.8 kcal/mol in wildtype to -20.9 and -18.7 kcal/mol for the P25S and W485R variants of HexA, respectively., (© 2021 Wiley Periodicals LLC.)

β-N-acetylhexosaminidase from the fungus Aspergillus oryzae is a secreted extracellular enzyme that cleaves chitobiose into constituent monosaccharides. It belongs to the GH 20 glycoside hydrolase family and consists of two N-glycosylated catalytic cores noncovalently associated with two 10-kDa O-glycosylated propeptides. We used X-ray diffraction and mass spectrometry to determine the structure of A. oryzae β-N-acetylhexosaminidase isolated from its natural source. The three-dimensional structure determined and refined to a resolution of 2.3 Å revealed that this enzyme is active as a uniquely tight dimeric assembly further stabilized by N- and O-glycosylation. The propeptide from one subunit forms extensive noncovalent interactions with the catalytic core of the second subunit in the dimer, and this chain swap suggests the distinctive structural mechanism of the enzyme's activation. Unique structural features of β-N-acetylhexosaminidase from A. oryzae define a very stable and robust framework suitable for biotechnological applications. The crystal structure reported here provides structural insights into the enzyme architecture as well as the detailed configuration of the active site. These insights can be applied to rational enzyme engineering., Database: Structural data are available in the PDB database under the accession number 5OAR., Enzyme: β-N-acetylhexosaminidase (EC 3.2.1.52)., (© 2017 Federation of European Biochemical Societies.)

GM 1 has generally been considered as the major receptor that binds to cholera toxin subunit B (CTB) due to its low dissociation constant. However, using a unique nanocube sensor technology, we have shown that CTB can also bind to other glycolipid receptors, fucosyl-GM 1 and GD 1 b. Additionally, we have demonstrated that GM 2 can contribute to CTB binding if present in a glycolipid mixture with a strongly binding receptor (GM 1 /fucosyl-GM 1 /GD 1 b). This hetero-multivalent binding result was unintuitive because the interaction between CTB and pure GM 2 is negligible. We hypothesized that the reduced dimensionality of CTB-GM 2 binding events is a major cause of the observed CTB binding enhancement. Once CTB has attached to a strong receptor, subsequent binding events are confined to a 2D membrane surface. Therefore, even a weak GM 2 receptor could now participate in second or higher binding events because its surface reaction rate can be up to 10 4 times higher than the bulk reaction rate. To test this hypothesis, we altered the surface reaction rate by modulating the fluidity and heterogeneity of the model membrane. Decreasing membrane fluidity reduced the binding cooperativity between GM 2 and a strong receptor. Our findings indicated a new protein-receptor binding assay, that can mimic complex cell membrane environment more accurately, is required to explore the inherent hetero-multivalency of the cell membrane. We have thus developed a new membrane perturbation protocol to efficiently screen receptor candidates involved in hetero-multivalent protein binding., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An efficient method was developed for the synthesis of a GM2 derivative suitable for the conjugation with various biomolecules. This GM2 derivative was covalently linked to keyhole limpet hemocyanin (KLH) and monophosphoryl lipid A (MPLA) to form novel therapeutic cancer vaccines. Immunological evaluations of the resultant conjugates in mice revealed that they elicited robust GM2-specific overall and IgG antibody responses. Moreover, the GM2-MPLA conjugate was disclosed to elicit strong immune responses without the use of an adjuvant, proving its self-adjuvant property. The antisera of both conjugates showed strong binding and mediated similarly effective complement-dependent cytotoxicity to GM2-expressing cancer cell line MCF-7. Based on these results, it was concluded that both GM2-MPLA and GM2-KLH are promising candidates as therapeutic cancer vaccines, whereas fully synthetic GM2-MPLA, which has homogeneous and well-defined structure and self-adjuvant property, deserves more attention and studies.

Gangliosides, glycosphingolipids containing one or more sialic acids in the glycan chain, are involved in various important biological processes in cell plasma membranes (PMs). However, the behaviors and functions of gangliosides are poorly understood, primarily because of the lack of fluorescent analogs that are equivalent to native gangliosides that can be used as chemical and physical probes. In this study, we developed entirely chemical methods to synthesize fluorescent gangliosides (GM3, GM2, GM1, and GD1b) in which the glycan components are site-specifically labeled with various fluorescent dyes. The functional evaluations of the synthesized fluorescent gangliosides demonstrated the great influence of fluorescent dye on the physical properties of gangliosides in PMs and revealed the fluorescent ganglioside analogs which show similar behaviors to the native gangliosides., (© 2017 Elsevier Inc. All rights reserved.)

Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM1 and GM1-like gangliosides binding to CTB. We confirmed the previously observed CTB-GM1 positive cooperativity. Surprisingly, we demonstrated fucosyl-GM1 has approximately 7 times higher CTB binding capacity than GM1. In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM1 caused the increased binding capacity. This was unintuitive, as GM1 exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM1 and GM2, we observed the mild binding avidity fucosyl-GM1 activated GM2 receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM2 receptors have negligible binding avidity in pure GM2 bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms.

The development of carbohydrate-based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor-associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus-like particle derived from bacteriophage Qβ. Although the copper-catalyzed azide-alkyne cycloaddition reaction efficiently introduced 237 copies of GM2 per Qβ, this construct failed to induce significant amounts of anti-GM2 antibodies compared to the Qβ control. In contrast, GM2 immobilized on Qβ through a thiourea linker elicited high titers of IgG antibodies that recognized GM2-positive tumor cells and effectively induced cell lysis through complement-mediated cytotoxicity. Thus, bacteriophage Qβ is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We successfully displayed a series of gangliosides on small bicelles with a uniform confined size, offering nanoscale standardized membrane mimics for spectroscopic characterization of weak encounter complexes formed between ganglioside clusters and amyloidogenic proteins. This enabled probing of initial membrane-landing processes of α-synuclein as a therapeutic target by NMR spectroscopy.

Functional analysis of carbohydrates is needed to understand the initial interface between membranes and the outer world. For this analysis we need individual protocols such as a method to modify the surfaces of nanoparticles with a variety of carbohydrates effectively and exhaustively, to synthesize an oligosaccharide on each particle's surface by chemical or enzymatic sugar elongation reaction, and to analyze the binding properties of carbohydrates. In this article, we describe the basic strategies for scooping up proteins from crude sample mixtures via interaction with carbohydrates. This approach was used to identify proteins that interacted with GM2, a ganglioside that is abundant on the surfaces of human lung cancer cells.

The development of analytical tools is important for understanding the infection mechanisms of pathogenic bacteria or viruses. In the present work, a functional carbohydrate microarray combined with a fluorescence immunoassay was developed to analyze the interactions of Vibrio cholerae toxin (ctx) proteins and GM1-related carbohydrates. Ctx proteins were loaded onto the surface-immobilized GM1 pentasaccharide and six related carbohydrates, and their binding affinities were detected immunologically. The analysis of the ctx-carbohydrate interactions revealed that the intrinsic selectivity of ctx was GM1 pentasaccharide ≫ GM2 tetrasaccharide > asialo GM1 tetrasaccharide ≥ GM3trisaccharide, indicating that a two-finger grip formation and the terminal monosaccharides play important roles in the ctx-GM1 interaction. In addition, whole cholera toxin (ctxAB(5)) had a stricter substrate specificity and a stronger binding affinity than only the cholera toxin B subunit (ctxB). On the basis of the quantitative analysis, the carbohydrate microarray showed the sensitivity of detection of the ctxAB(5)-GM1 interaction with a limit-of-detection (LOD) of 2 ng mL(-1) (23 pM), which is comparable to other reported high sensitivity assay tools. In addition, the carbohydrate microarray successfully detected the actual toxin directly secreted from V. cholerae, without showing cross-reactivity to other bacteria. Collectively, these results demonstrate that the functional carbohydrate microarray is suitable for analyzing toxin protein-carbohydrate interactions and can be applied as a biosensor for toxin detection.

Spermatogonia are a potential source of adult pluripotent stem cells and can be used for testis germ cell transplantation. Markers for the isolation of these cells are of great importance for biomedical applications. Primordial germ cells and prepubertal spermatogonia in many species can be identified by their binding of Dolichos biflorus agglutinin (DBA). This lectin binds to two different types of glycans, which are α-linked N-acetylgalactosamine (GalNac) and β-linked GalNac, if this is part of the Sda or GM2 glycotopes. We used the MAB CT1, which is specific for the trisaccharides motif NeuAcα2-3(GalNAcβ1-4)Galβ1-, which is common to both Sda and GM2 glycotopes, to further define the glycosylation of DBA binding germ cells. In porcine embryos, CT1 bound to migratory germ cells and gonocytes. CT1/DBA double staining showed that the mesonephros was CT1 negative but contained DBA-positive cells. Gonocytes in the female gonad became CT1 negative, while male gonocytes remained CT1 positive. In immunohistological double staining of cattle, pig, horse and llama testis, DBA and CT1 staining was generally colocalised in a subpopulation of spermatogonia. These spermatogonia were mainly single, sometimes paired or formed chains of up to four cells. Our data show that the Sda/GM2 glycotope is present in developing germ cells and spermatogonia in several species. Owing to the narrower specificity of the CT1 antibody, compared with DBA, the former is likely to be a useful tool for labelling and isolation of these cells.

Monolithic columns containing ganglioside GM2 and GM3 mimics were prepared for selective removal of serum anti-ganglioside antibodies from patients with acute and chronic immune-mediated neuropathies. ELISA results demonstrated that anti-GM2 IgM antibodies in human sera and a mouse monoclonal anti-GM2 antibody were specifically and selectively adsorbed by monolithic GM2 mimic columns and not by blank monolithic columns or monolithic GM3 mimic columns. In control studies, serum antibodies against the ganglioside GQ1b from another neuropathy patient were not depleted by monolithic GM2 mimic columns. Fluorescence microscopy with FITC-conjugated anti-human immunoglobulin antibodies showed that the immobilized ganglioside mimics were evenly distributed along the column. The columns were able to capture ∼95% of the anti-GM2 antibodies of patients after only 2 min of incubation. A monolithic column of 4.4 μL can deplete 28.2 μL of undiluted serum. These columns are potential diagnostic and therapeutic tools for neuropathies related to anti-ganglioside antibodies.

beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.

The GM2 ganglioside represents an important target for specific anticancer immunotherapy. We designed and synthesized a neoglycopeptide immunogen displaying one or two copies of the GM2 tetrasaccharidic moiety. These glycopeptides were prepared using the Huisgen cycloaddition, which enables the efficient ligation of the alkyne-functionalized biosynthesized GM2 with an azido CD4(+) T cell epitope peptide. It is worth noting that the GM2 can be produced on a gram scale in bacteria, which can be advantageous for a scale-up of the process. We show here for the first time that a fully synthetic glycopeptide, which is based on a ganglioside carbohydrate moiety, can induce human tumor cell-specific antibodies after immunization in mice. Interestingly, the monovalent, but not the divalent, form of GM2 peptide construct induced antimelanoma antibodies. Unlike traditional vaccines, this vaccine is a pure chemically-defined entity, a key quality for consistent studies and safe clinical evaluation. Therefore, such carbohydrate-peptide conjugate represents a promising cancer vaccine strategy for active immunotherapy targeting gangliosides.

Gal-PUGNAc (see picture), a highly selective inhibitor for beta-hexosaminidases HEXA and HEXB is cell-permeable and modulates the activity of HEXA and HEXB in tissue culture, increasing ganglioside GM2 levels. Gal-PUGNAc should allow the role of these enzymes to be studied at the cellular level without generating a complex chemical phenotype from concomitant inhibition of O-GlcNAcase.

To elucidate the mechanism underlying the hydrolysis of the GalNAcbeta1-->4Gal linkage in ganglioside GM2 [GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glcbeta1-->1' Cer] by beta-hexosaminidase A (Hex A) with GM2 activator protein, we designed and synthesized two kinds of GM2 linkage analogues-6'-NeuAc-GM2 and alpha-GalNAc-GM2. In this paper, the efficient and systematic synthesis of these GM2 analogues was described. The highlight of our synthesis process is that the key intermediates, newly developed sialyllactose derivatives, were efficiently prepared in sufficient quantities; these derivatives directly served as highly reactive glycosyl acceptors and coupled with GalNTroc donors to furnish the assembly of GM2 tetrasaccharides in large quantities.

Objectives: Gangliosides (GGs) are considered as diagnostic biomarkers and therapeutic targets and agents. The goal of this study was to develop a tandem mass spectrometry (MS/MS) method for the simultaneous measurement of both GM1 and GM2 gangliosides in human cerebrospinal fluid (CSF) samples in order to be able to determine their concentrations in patients with Tay-Sachs and Sandhoff disease and assess whether drugs or transplantation affect their concentrations., Design and Methods: An API-4000 tandem mass spectrometer equipped with TurboIonSpray source and Shimadzu HPLC system was employed to perform the analysis using isotope dilution with deuterium labeled internal standards. To a 1.5 mL conical plastic Eppendorf centrifuge tube, 40 microL of human CSF sample was added and mixed with 400 microL of internal standard solution for deproteinization. After centrifugation, 100 microL of supernatant was injected onto a C-18 column. After a 2.5 min wash, the switching valve was activated and the analytes were eluted from the column with a water/methanol gradient into the MS/MS system. Quantification by multiple reaction-monitoring (MRM) analysis was performed in the negative mode., Results: The within-day coefficients of variation were <3% for GM1 and <2% for GM2 and the between-day coefficients of variation were <5% for both GM1 and GM2 at all concentrations tested. Accuracy ranged between 98% and 102% for both analytes. Good linearity was also obtained within the concentration range of 10-200 ng/mL (6.5-129.3 nmol/L) for GM1 and 5-100 ng/mL (3.6-72.3 nmol/L) for GM2 (r> or =0.995)., Conclusions: A new simple, accurate, and fast isotope dilution tandem mass spectrometry method was developed for the simultaneous quantification of GM1 and GM2 gangliosides in a small amount of human CSF. Concentrations were measured in "normal" CSF and in CSF from patients with Tay-Sachs disease.

The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcbeta1-->4(Neu5Acalpha2-->3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to beta-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcbeta1-->4Gal linkage in GM2 was converted to the GalNAcbeta1-->6Gal, both the GalNAc and the Neu5Ac became susceptible to beta-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates.

Pathogenic organisms or oncogenically transformed cells often express complex carbohydrate structures at their cell surface, which are viable targets for active immunotherapy. We describe here a novel, immunologically neutral, linker methodology for the efficient preparation of highly defined vaccine conjugates that combine complex saccharide antigens with specific TH-cell peptide epitopes. This novel heterobifunctional approach was employed for the conjugation of a (1-->2)-beta-mannan trisaccharide from the pathogenic fungus Candida albicans as well as the carbohydrate portion of tumor-associated ganglioside GM2 to a TH-cell peptide epitope derived from the murine 60 kDa self heat-shock protein (hsp60). Moreover, the linkage chemistry has proven well suited for the synthesis of more complex target structures such as a biotinylated glycopeptide, a three component vaccine containing an immunostimulatory peptide epitope from interleukin-1 beta (IL-1 beta), and for the conjugation of complex carbohydrates to carrier proteins such as bovine serum albumin.

Antioxidant activity of gangliosides GM1 and GT1b in the Fenton type of reaction was investigated by EPR spectroscopy using DMPO as a spin trap. Hydroxyl radical spin adduct signal intensity was significantly reduced in the presence of gangliosides at their micellar concentrations. Mean micellar hydrodynamic diameter was not changed, whereas significant changes in negative Zeta potential values were observed as evidenced by Zetasizer Nano ZS. This study showed that the primary mode of ganglioside action was not due to direct scavenging of OH., but rather to the inhibition of hydroxyl radical formation. This phenomenon is related to the ability of ganglioside micelles to bind oppositely charged ferrous ions, thus reducing their concentration and consequently inhibiting OH. formation.

Ganglioside GM1 and its seven potential catabolic products: asialo-GM1, GM2, asialo-GM2, GM3, Lac-Cer, Glc-Cer and Cer, were labeled with tetramethylrhodamine (TMR) to permit ultra-sensitive analysis using laser-induced fluorescence (LIF) detection. The preparation involved acylation of the homogenous C(18)lyso-forms of GM1, Lac-Cer, Glc-Cer and Cer with the N-hydroxysuccinimide ester of a beta-alanine-tethered 6-TMR derivative, followed by conversion of these labeled products using galactosidase, sialidase, and sialyltransferase enzymes. The TMR-glycolipid analogs produced are detectable on TLC down to the 1 ng level by the naked eye. All eight compounds could be separated within 4 min in capillary electrophoresis where they could be detected at the zeptomole (ca. 1000 molecule) level using LIF.

The aggregation of alpha-synuclein is believed to be a key step in the etiology of Parkinson's disease. Alpha-synuclein is found in the cytosol and is associated with membranes in the presynaptic region of neurons and has recently been reported to be associated with lipid rafts and caveolae. We examined the interactions between several brain sphingolipids and alpha-synuclein and found that alpha-synuclein specifically binds to ganglioside GM1-containing small unilamellar vesicles (SUVs). This results in the induction of substantial alpha-helical structure and inhibition or elimination of alpha-synuclein fibril formation, depending on the amount of GM1 present. SUVs containing total brain gangliosides, gangliosides GM2 or GM3, or asialo-GM1 had weak inhibitory effects on alpha-synuclein fibrillation and induced some alpha-helical structure, while all other sphingolipids studied showed negligible interaction with alpha-synuclein. alpha-Synuclein binding to GM1-containing SUVs was accompanied by formation of oligomers of alpha-synuclein. The familial mutant A53T alpha-synuclein interacted with GM1-containing SUVs in an analogous manner to wild type, whereas the A30P mutant showed minimal interaction. This is the first detailed report showing a direct association between GM1 and alpha-synuclein, which is attributed to specific interaction between helical alpha-synuclein and both the sialic acid and carbohydrate moieties of GM1. The recruitment of alpha-synuclein by GM1 to caveolae and lipid raft regions in membranes could explain alpha-synuclein's localization to presynaptic membranes and raises the possibility that perturbation of GM1/raft association could induce changes in alpha-synuclein that contribute to the pathogenesis of PD.

A series of GM2 analogs in which GM2 epitope was coupled to a variety of glycosyl lipids were designed and synthesized to investigate the mechanism of enzymatic hydrolysis of GM2 ganglioside. The coupling of N-Troc-protected sialic acid and p-methoxyphenyl galactoside acceptor gave the crystalline disaccharide, which was further coupled with galactosamine donor to give the desired GM2 epitope trisaccharide. After conversion into the corresponding glycosyl donor, the trisaccharide was coupled with galactose, glucose and artificial ceramide (B30) to give the final compounds. The result on hydrolysis of GM2 analogs indicates that GM2 activator protein requires one spacer sugar between GM2 epitope and the lipid moiety to assist the hydrolysis of the terminal GalNAc residue.

Glycolipids are a group of compounds with a broad range of applications. Two types of glycolipids (alkylpolyglycosides and gangliosides) were examined with regard to their physicochemical properties. Despite their structural differences, they have in common that they are amphiphilic molecules and able to aggregate to form monolayers, bilayers, micelles, lyothropic mesophases or vesicles. The structures of glycolipid micelles were investigated by different experimental techniques in addition to molecular dynamic simulations. The knowledge of the physicochemical properties of gangliosides enables a better understanding of their biological functions. Structural features were obtained for the monosialogangliosides GM1, GM2 and GT1b from bovine brain by means of mass spectrometry. Further the aggregation behaviour was determined by small-angle neutron and dynamic light scattering experiments. Interaction studies of these compounds were carried out by means of surface plasmon resonance using gangliosides incorporated liposomes. They were used as model membranes that interact with the lectins WGA, RCA and HPA. The interaction of lectins immobilized to a modified silicon surface was investigated by in-situ ellipsometry.

Several novel, fully synthetic, carbohydrate-based antitumor vaccines have been assembled. Each construct consists of multiple cancer-related antigens displayed on a single polypeptide backbone. Recent advances in synthetic methodology have allowed for the incorporation of a complex oligosaccharide terminating in a sialic acid residue (i.e., GM2) as one of the carbohydrate antigens. Details of the vaccine synthesis as well as the results of preliminary immunological investigations are described herein.

The ganglioside-activator protein is an essential cofactor for the lysosomal degradation of ganglioside GM2 (GM2) by beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interphase. Mutations in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. In order to efficiently and sensitively probe the glycolipid binding and membrane activity of this cofactor, we synthesized two new fluorescent glycosphingolipid (GSL) probes, 2-NBD-GM1 and 2-NBD-GM2. Both compounds were synthesized in a convergent and multistep synthesis starting from the respective gangliosides isolated from natural sources. The added functionality of 2-aminogangliosides allowed us to introduce the chromophore into the region between the polar head group and the hydrophobic anchor of the lipid. Both fluorescent glycolipids exhibited an extremely low off-rate in model membranes and displayed very efficient resonance energy transfer to rhodamine-dioleoyl phosphoglycerol ethanolamine (rhodamine-PE) as acceptor. The binding to GM2-activator protein (GM2AP) and the degrading enzyme was shown to be unaltered compared to their natural analogues. A novel fluorescence-resonance energy transfer (FRET) assay was developed to monitor in real time the protein-mediated intervesicular transfer of these lipids from donor to acceptor liposomes. The data obtained indicate that this rapid and robust system presented here should serve as a valuable tool to probe quantitatively and comprehensively the membrane activity of GM2AP and other sphingolipid activator proteins and facilitate further structure-function studies aimed at delineating independently the lipid- and the enzyme-binding mode of these essential cofactors.

Oligosaccharidic moieties of GM(2) and GM(3) gangliosides bearing an allyl or a propargyl aglycon, are efficiently biosynthesized on the gram scale by growing metabolically engineered Escherichia coli cells in the presence of the corresponding lactoside acceptors and sialic acid.

The human MHC class I-like molecule CD1b is distinctive among CD1 alleles in that it is capable of presenting a set of glycolipid species that show a very broad range of variation in the lengths of their acyl chains. A structure of CD1b complexed with relatively short acyl chain glycolipids plus detergent suggested how an interlinked network of channels within the Ag-binding groove could accommodate acyl chain lengths of up to 80 carbons. The structure of CD1b complexed with glucose monomycolate, reported in this study, confirms this hypothesis and illustrates how the distinctive substituents of intracellular bacterial glycolipids can be accommodated. The Ag-binding groove of CD1b is, uniquely among CD1 alleles, partitioned into channels suitable for the compact accommodation of lengthy acyl chains. The current crystal structure illustrates for the first time the binding of a natural bacterial lipid Ag to CD1b and shows how its novel structural features fit this molecule for its role in the immune response to intracellular bacteria.

GM2 ganglioside, beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)-]beta-Gal-(1-4)-beta-Glc-(1-1)-Cer, is the main ganglioside in the brain of Tay-Sachs patients. In this work, GM2 ganglioside was extracted from a Variant B Tay-Sachs human brain, purified to homogeneity of the oligosaccharide moiety by silica gel chromatography. It was further fractionated for the first time into the molecular species differing in the ceramide structures by reverse-phase flash chromatography. The GM2 ganglioside species were characterized by gas-chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. The major GM2 species contained the ceramides with d18:1-18:0 (40.5% of the total GM2 species), d20:1-18:0 (31%) and d18:1-20:0 (12%). We also found minor GM2 species with the ceramides with d18:1-24:1 (4%), d18:1-22:0 (2%) and d18:2-24:1 (1%), which have not been reported previously.

Tay-Sachs disease (TSD) is a classical glycosphingolipid (GSL) storage disease. Although the genetic and biochemical bases for a massive cerebral accumulation of ganglioside GM2 in TSD have been well established, the mechanism for the neural dysfunction in TSD remains elusive. Upon analysis of GSLs from a variant B TS brain, we have detected a novel GSL that has not been previously revealed. We have isolated this GSL in pure form. Using NMR spectroscopy, mass spectrometry, and chemical synthesis, the structure of this unusual GSL was established to be a taurine-conjugated GM2 (tauro-GM2) in which the carboxyl group of N-acetylneuraminic acid was amidated by taurine. Using a rabbit anti-tauro-GM2 serum, we also detected the presence of tauro-GM2 in three other small brain samples from one variant B and two variant O TSD patients. On the other hand, tauro-GM2 was not found in three normal human brain samples. The presence of tauro-GM2 in TS brains, but not in normal brains, indicates the possible association of this unusual GM2 derivative with the pathogenesis of TSD. Our findings point to taurine conjugation as a heretofore unelucidated mechanism for TS brain to cope with water-insoluble GM2.

Two metabolically engineered Escherichia coli strains have been constructed to produce the carbohydrate moieties of gangliosides GM2 (GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc; Gal = galactose, Glc = glucose, Ac = acetyl) and GM1 (Galbeta-3GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc. The GM2 oligosaccharide-producing strain TA02 was devoid of both beta-galactosidase and sialic acid aldolase activities and overexpressed the genes for CMP-NeuAc synthase (CMP = cytidine monophosphate), alpha-2,3-sialyltransferase, UDP-GlcNAc (UDP = uridine diphosphate) C4 epimerase, and beta-1,4-GalNAc transferase. When this strain was cultivated on glycerol, exogenously added lactose and sialic acid were shown to be actively internalized into the cytoplasm and converted into GM2 oligosaccharide. The in vivo synthesis of GM1 oligosaccharide was achieved by taking a similar approach but using strain TA05, which additionally overexpressed the gene for beta-1,3-galactosyltransferase. In high-cell-density cultures, the production yields for the GM2 and GM1 oligosaccharides were 1.25 g L(-1) and 0.89 g L(-1), respectively.

Human beta-hexosaminidase A (Hex A) (alphabeta) is composed of two subunits whose primary structures are approximately 60% identical. Deficiency of either subunit results in severe neurological disease due to the storage of GM2 ganglioside; Tay-Sachs disease, alpha deficiency, and Sandhoff disease, beta deficiency. Whereas both subunits contain active sites only the alpha-site can efficiently bind negatively charged 6-sulfated hexosamine substrates and GM2 ganglioside. We have recently identified the alphaArg(424) as playing a critical role in the binding of 6-sulfate-containing substrates, and betaAsp(452) as actively inhibiting their binding. To determine if these same residues affect the binding of the sialic acid moiety of GM2 ganglioside, an alphaArg(424)Gln form of Hex A was expressed and its kinetics analyzed using the GM2 activator protein:[3H]-GM2 ganglioside complex as a substrate. The mutant showed a approximately 3-fold increase in its K(m) for the complex. Next a form of Hex B (betabeta) containing a double mutation, betaAspLeu(453)AsnArg (duplicating the alpha-aligning sequences), was expressed. As compared to the wild type (WT), the mutant exhibited a >30-fold increase in its ability to hydrolyze a 6-sulfated substrate and was now able to hydrolyze GM2 ganglioside when the GM2 activator protein was replaced by sodium taurocholate. Thus, this alpha-site is critical for binding both types of negatively charge substrates.

The human genome encodes five nonpolymorphic major histocompatibility complex class I-like glycoproteins, CD1a to CD1e, that present lipid antigens for specific recognition by T lymphocytes. Using single alkyl chain detergents, we developed a protocol to generate recombinant human CD1b-lipid complexes. We present here the crystal structures of CD1b in complex with either phosphatidylinositol or ganglioside GM2 at 2.3 A and 2.8 A resolutions, respectively. The antigen-binding groove houses four interlinked hydrophobic channels that are occupied by the alkyl chains of the glycolipid plus two detergent molecules. A distinct exit beneath the alpha 2 helix further contributes to the plasticity of the binding groove. These structures reveal the mechanism by which two alkyl chain lipids bind to CD1b, and how CD1b can adapt to ligands of different alkyl chain length. They also suggest how very long alkyl chains, such as those of mycolic acid, could be fully contained within the binding groove. These results extend the spectrum of potential CD1b ligands by revealing that, in addition to two alkyl chain lipids, mono-alkyl and triple-alkyl chain lipids can be accommodated in the binding groove.

The outer cores of the lipooligosaccharides (LOS) of many strains of Campylobacter jejuni mimic human gangliosides in structure. A population of cells of C. jejuni strain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM(2) and GM(3) gangliosides, with minor amounts of structures mimicking GD(1b) and GD(2). Genetic analyses of genes involved in the biosynthesis of the outer core of C. jejuni 81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, an N-acetylgalactosaminyltransferase. Variation in the number of G residues within cgtA affected the length of the open reading frame, and these changes in cgtA corresponded to a change in LOS structure from GM(2) to GM(3) ganglioside mimicry. Site-specific mutation of cgtA in 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM(3) ganglioside. Compared to wild-type 81-176, the cgtA mutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of the neuC1 gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.

Lysosphingolipids, which lack the fatty acid moiety of sphingolipids, are known to be accumulated in some variants of sphingolipid storage diseases. Here, we report that lysosphingolipids with naturally occurring stereochemical configurations induce apoptosis in mouse neuroblastoma Neuro2a cells. The intracellular dehydrogenase activity and [3H]thymidine incorporation of Neuro2a cells were strongly suppressed by the addition of lysosphingolipids in a dose-dependent manner, whereas the parental sphingolipids had no effect. Intranucleosomal DNA fragmentation, chromatin condensation, and phosphatidylserine externalization, which are typical features of apoptosis, were observed when the cells were cultured with 40-80 microM of lysosphingolipids for 24-48 h in the presence of 5% fetal calf serum. Activation of caspase-3-like enzyme occurred after addition of lysosphingolipids followed by incubation at 37 degrees C for 24 h. The addition of an inhibitor of caspases, ZVAD-fmk, to the Neuro2a cell culture completely inhibited the elevation of caspase-3 activity but not the DNA fragmentation. These results may indicate that a caspase-3 independent signaling pathway is involved in the lysosphingolipid-induced apoptosis and suggest that accumulation of lysosphingolipids, but not parental sphingolipids, triggers the apoptotic cascade in neuronal cells of patients with sphingolipidoses.

In this study we analyzed by immunofluorescence, laser confocal microscopy, immunoelectron microscopy and label fracture technique the ganglioside distribution on the plasma membrane of several different cell types: human peripheral blood lymphocytes (PBL), Molt-4 lymphoid cells, and NIH 3T3 fibroblasts, which mainly express monosialoganglioside GM3, and murine NS20Y neuroblastoma cells, which have been shown to express a high amount of monosialoganglioside GM2. Our observations showed an uneven distribution of both GM3 and GM2 on the plasma membrane of all cells, confirming the existence of ganglioside-enriched microdomains on the cell surface. Interestingly, in lymphoid cells the clustered immunolabeling appeared localized over both the microvillous and the nonvillous portions of the membrane. Similarly, in cells growing in monolayer, the clusters were distributed on both central and peripheral regions of the cell surface. Therefore, glycosphingolipid clusters do not appear confined to specific areas of the plasma membrane, implying general functions of these domains, which, as structural components of a cell membrane multimolecular signaling complex, may be involved in cell activation and adhesion, signal transduction and, when associated to caveolae, in endocytosis of specific molecules.

To understand the reason why, in the absence of GM2 activator protein, the GalNAc and the NeuAc in GM2 (GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer) are refractory to beta-hexosaminidase A and sialidase, respectively, we have recently synthesized a linkage analogue of GM2 named 6'GM2 (GalNAcbeta1-->6(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer). While GM2 has GalNAcbeta1-->4Gal linkage, 6'-GM2 has GalNAcbeta1-->6Gal linkage (Ishida, H., Ito, Y., Tanahashi, E., Li, Y.-T., Kiso, M., and Hasegawa, A. (1997) Carbohydr. Res. 302, 223-227). We have studied the enzymatic susceptibilities of GM2 and 6'GM2, as well as that of the oligosaccharides derived from GM2, asialo-GM2 (GalNAcbeta1-->4Galbeta1--> 4Glcbeta1-1'Cer) and 6'GM2. In addition, the conformational properties of both GM2 and 6'GM2 were analyzed using NMR spectroscopy and molecular mechanics computation. In sharp contrast to GM2, the GalNAc and the Neu5Ac of 6'GM2 were readily hydrolyzed by beta-hexosaminidase A and sialidase, respectively, without GM2 activator. Among the oligosaccharides derived from GM2, asialo-GM2, and 6'GM2, only the oligosaccharide from GM2 was resistant to beta-hexosaminidase A. Conformational analyses revealed that while GM2 has a compact and rigid oligosaccharide head group, 6'GM2 has an open spatial arrangement of the sugar units, with the GalNAc and the Neu5Ac freely accessible to external interactions. These results strongly indicate that the resistance of GM2 to enzymatic hydrolysis is because of the specific rigid conformation of the GM2 oligosaccharide.

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti-ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M-1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.

Monosialogangliosides (GM1, GM2, GM3 and GM4) were reconstituted in lipid monolayers at the air-water interface. The binding amounts and the initial binding rates of wheat germ agglutinin (WGA) to the monosialoganglioside monolayers were quantitatively studied by use of a quartz-crystal microbalance (QCM). A QCM was horizontally attached to the monolayer from the air phase, and the binding behavior (mass increase) was followed by the frequency decrease of the QCM. WGA binding affinities for the ganglioside monolayers were influenced by hydrophilic head groups of lipid matrices, densities of gangliosides, and sequences of oligosaccharide in gangliosides. Binding of WGA to the gangliosides reconstituted in a phosphatidylcholine (sphingomyelin and distearoylphosphatidylcholine) matrix was strongly suppressed, but not in a neutral glycolipids (GlcCer, GalCer, and LacCer), dipalmitoylphosphatidylethanolamine, and dipalmitoylphosphatidylethanolamine matrix. WGA showed high affinity for monolayers containing 20 mol% gangliosides, but only low affinity for 100% ganglioside monolayers. WGA preferably binds to gangliosides in the following sequence: GM3 > GM4 >> GM2 = GM1. No affinities of WGA for GM2 and GM1 were observed. The combined techniques of monolayer and QCM have the advantages of investigating recognition properties of gangliosides.

To establish a model system for the study of ganglioside metabolism of the human brain tumor, medulloblastoma, we have chemically characterized the gangliosides of the Daoy cell line. These cells contain a high concentration of gangliosides (143 +/- 13 nmol LBSA/10(8) cells). The major species have been structurally confirmed to be GM2 (65.9%), GM3 (13.0%), and GD1a (10.3%). Isolation of individual gangliosides homogeneous in both carbohydrate and ceramide moieties by reversed-phase HPLC and analysis by negative-ion fast atom bombardment collisionally activated dissociation tandem mass spectrometry have allowed us to unequivocally characterize ceramide structures. In the case of GM2, 10 major ceramide subspecies were identified: d18:1-hC16:0, d18:1-C16:0, d18:0-C16:0, d18:1-C18:0, d18:1-C20:0, d18:1-C22:0, d18:2-C24:1, d18:1-C23:1, d18:1-C24:1, and d18:1-C24:0. Taken together with previous studies, these findings in buman medulloblastoma cells support the view that high expression and marked heterogeneity of ceramide structure are general characteristics of tumor gangliosides, molecules which are shed by the tumor cells and which are biologically active in vivo.

Our rapid method of microwave-mediated saponification for preparing lysoglycosphingolipids from their parent glycosphingolipids was also able to prepare lysogangliosides or modified lysogangliosides, which were identified by delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometric (DE MALDI-TOF MS) analysis. When GM3, GM2, and GM1 isolated from adult human brain gangliosides were subjected to the saponification, GM3 was found to give rise to only lyso-GM3 containing de-N-acetylneuraminic acid (de-N-acetyl lyso-GM3), whereas the GM2 produced both lyso-GM2 and the de-N-acetyl compound, and GM1 also gave both lyso-GM1 and the de-N-acetyl compound. In the saponification of GM1 and GDla, isolated from rat brain gangliosides, GM1 similarly produced both lyso-GM1 and the de-N-acetyl compound, but GDla was found to give rise to both dehydrated de-N-monoacetyl and dehydrated de-N-diacetyl lyso-GDla. However, the saponification of the GM1 fraction isolated from porcine brain gangliosides gave rise not only to both lyso-GM1 and the de-N-acetyl compound, but also unexpectedly to both lyso-fucosyl GM1 and its de-N-acetyl compound. The untreated GM1 fraction was examined by TLC and DE MALDI-TOF mass spectrometry, and proved to contain fucosyl-GM1. The DE MALDI-TOF MS analysis of the prepared lyso-gangliosides showed that their long chain bases consisted of d18:1 and d20:1 sphingosines in various ratios reflecting those of the different mammalian brain gangliosides.

The lysosomal beta-hexosaminidases are dimers composed of alpha and beta subunits. beta-Hexosaminidase A (alphabeta) is a heterodimer, whereas hexosaminidase B (betabeta) and S (alphaalpha) are homodimers. Although containing a high degree of amino acid identity, each subunit expresses a unique active site that can be distinguished by a differential ability to hydrolyze charged substrates. The site on the beta-subunit primarily degrades neutral substrates, whereas the alpha-subunit site is, in addition, active against sulfated substrates. Isozyme specificity is also exhibited with glycolipid substrates. Among human isozymes, only beta-hexosaminidase A together with the GM2 activator protein can degrade the natural substrate, GM2 ganglioside, at physiologically significant rates. To identify the domains of the human beta-hexosaminidase subunits that determine substrate specificity, we have generated chimeric subunits containing both alpha- and beta-subunit sequences. The chimeric constructs were expressed in HeLa cells to screen for activity and then selected constructs were produced in the baculovirus expression system to assess their ability to degrade GM2 ganglioside in the presence of GM2 activator protein. Generation of activity against the sulfated substrate required the substitution of two noncontinuous alpha-subunit sequences (amino acids 1-191 and 403-529) into analogous positions of the beta-subunit. Chimeric constructs containing only one of these regions linked to the beta-subunit sequence showed either neutral substrate activity only (amino acids 1-191) or lacked enzyme activity entirely (amino acids 403-529). Neither the chimeras nor the wild-type subunits displayed activator-dependent GM2-hydrolyzing activity when expressed alone. However, one chimeric subunit containing alpha amino acids 1-191 fused with beta amino acids 225 to 556, when co-expressed with the wild-type alpha-subunit, showed activity comparable with that of recombinant beta-hexosaminidase A formed by the co-expression of the alpha- and beta-subunits. This result indicates that the beta-subunit amino acids 225-556 contribute an essential function in the GM2-hydrolyzing activity of beta-hexosaminidase A.

The human glioma D-54MG cell line grown in vitro primarily expresses ganglio series gangliosides, particularly GM2. Subcutaneous injection of these cells into nude mice produced xenografts with an increased content of the human glioma-associated lacto series gangliosides, primarily 3'-isoLM1, an alteration that was dose dependent, with the highest dose (1 x 10(8)) resulting in a phenotype that was most like that of the inoculum. After one passage in vivo, the lacto series dominated and reached a proportional level that was kept throughout the 10 passages. The mRNA levels of the GM2-synthase clearly coincided with GM2 expression and was 20 times higher in cells grown in vitro than in those grown in vivo. These results support the view that ganglioside expression in human gliomas is strongly influenced by environmental factors.

GM2 ganglioside, although scarce in normal adult brain, is the predominant ganglioside accumulating in several types of lysosomal disorders, most notably Tay-Sachs disease. Pyramidal neurons of cerebral cortex in Tay-Sachs, as well as many other types of neuronal storage disorders, are known to exhibit a phenomenon believed unique to storage disorders: growth of ectopic dendrites. Recent studies have shown that a common metabolic abnormality shared by storage diseases with ectopic dendrite growth is the abnormal accumulation of GM2 ganglioside. The correlation between increased levels of GM2 and the presence of ectopic dendrites has been found in both ganglioside and nonganglioside storage disorders, the latter including sphingomyelin-cholesterol lipidosis, mucopolysaccharidosis, and alpha-mannosidosis. Quantitative HPTLC analysis has shown that increases in GM2 occur in proportion to the incidence of ectopic dendrite growth, whereas other gangliosides, including GM1, lack similar increases. Immunocytochemical studies of all nonganglioside storage diseases which exhibit ectopic dendritogenesis have revealed heightened GM2 ganglioside-immunoreactivity in the cortical pyramidal cell population, whereas nerurons in normal adult brain exhibit little or no staining for this ganglioside. Further, studies examining disease development have consistently shown that accumulation of GM2 ganglioside precedes growth of ectopic dendrites, indicating that it is not simply occurring secondary to new membrane production. These findings have prompted an examination for a similar relationship between GM2 ganglioside and dendritogenesis in cortical neurons of normal developing brain. Results show that GM2 ganglioside-immunoreactivity is consistently elevated in immature neurons during the period when they are undergoing active dendritic initiation, but this staining diminishes dramatically as the dendritic trees of these cells mature. Collectively, these studies on diseased and normal brain offer compelling evidence that GM2 ganglioside plays a pivotal role in the regulation of dendritogenesis in cortical pyramidal neurons.