Stress Impairs Skin Barrier Function and Induces α2-3 Linked N-Acetylneuraminic Acid and Core 1 O-Glycans on Skin Mucins in Atlantic Salmon, Salmo salar.

The skin barrier consists of mucus, primarily comprising highly glycosylated mucins, and the epithelium. Host mucin glycosylation governs interactions with pathogens and stress is associated with impaired epithelial barrier function. We characterized Atlantic salmon skin barrier function during chronic stress (high density) and mucin O-glycosylation changes in response to acute and chronic stress. Fish held at low (LD: 14-30 kg/m3) and high densities (HD: 50-80 kg/m3) were subjected to acute stress 24 h before sampling at 17 and 21 weeks after start of the experiment. Blood parameters indicated primary and secondary stress responses at both sampling points. At the second sampling, skin barrier function towards molecules was reduced in the HD compared to the LD group (Papp mannitol; p < 0.01). Liquid chromatography-mass spectrometry revealed 81 O-glycan structures from the skin. Fish subjected to both chronic and acute stress had an increased proportion of large O-glycan structures. Overall, four of the O-glycan changes have potential as indicators of stress, especially for the combined chronic and acute stress. Stress thus impairs skin barrier function and induces glycosylation changes, which have potential to both affect interactions with pathogens and serve as stress indicators.

Sialic acids expression in newborn rat lungs: implications for pulmonary developmental biology.

Mammalian lung development proceeds during the postnatal period and continues throughout life. Intricate tubular systems of airways and vessels lined by epithelial cells are developed during this process. All cells, and particularly epithelial cells, carry an array of glycans on their surfaces. N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic (Neu5Gc) acids, two most frequently-occurring sialic acid residues, are essential determinants during development and in the homeostasis of cells and organisms. However, systematic data about the presence of cell surface sialic acids in the postnatal lung and their content is still scarce. In the present study, we addressed the histochemical localization of Neu5Ac > Neu5Gc in 0-day-old rat lungs. Furthermore, both residues were separated, identified and quantified in lung membranes isolated from 0-day-old rat lungs using high-performance liquid chromatography (HPLC) methodologies. Finally, we compared these results with those previously reported by us for adult rat lungs. The Neu5Ac > Neu5Gc residues were located on the surface of ciliated and non-ciliated cells and the median values for both residues in the purified lung membranes of newborn rats were 5.365 and 1.935 µg/mg prot., respectively. Comparing these results with those reported for the adults, it was possible to observe a significant difference between the levels of Neu5Ac and Neu5Gc (p < 0.001). A more substantial change was found for the case of Neu5Ac. The preponderance of Neu5Ac and its expressive increase during the postnatal development points towards a more prominent role of this residue. Bearing in mind that sialic acids are negatively charged molecules, the high content of Neu5Ac could contribute to the formation of an anion "shield" and have a role in pulmonary development and physiology.

Improved sensitivity by post-column chemical environment modification of CE-ESI-MS using a flow-through microvial interface.

Post-column chemical environment modification can affect detection sensitivity and signal appearance when capillary electrophoresis is coupled through electrospray ionization to mass spectrometry (CE-ESI-MS). In this study, changes in the signal intensity and peak shape of N-Acetylneuraminic acid (Neu5Ac) were examined when the modifier solution used in a flow-through microvial interface for CE-ESI-MS was prepared using an acidic or basic background electrolyte (BGE) composition. The use of a basic modifier resulted in improved detection compared to the results obtained when an acidic modifier was used in negative ion mode. Increased sensitivity and more symmetrical peak shape were obtained. Using an acidic modifier, the LOD of Neu5Ac was 47.7 nM, whereas for a basic modifier, the LOD of Neu5Ac was 5.20 nM. The calculated asymmetry factor at 100 nM of Neu5Ac ranged from 0.71 to 1.5 when an acidic modifier was used, while the factor ranged from 1.0 to 1.1 when a basic modifier was used. Properly chosen post-column chemical modification can have a significant effect on the performance of the CE-MS system.

A novel sugar analog enhances sialic acid production and biotherapeutic sialylation in CHO cells.

A desirable feature of many therapeutic glycoprotein production processes is to maximize the final sialic acid content. In this study, the effect of applying a novel chemical analog of the sialic acid precursor N-acetylmannosamine (ManNAc) on the sialic acid content of cellular proteins and a model recombinant glycoprotein, erythropoietin (EPO), was investigated in CHO-K1 cells. By introducing the 1,3,4-O-Bu3 ManNAc analog at 200-300 µM into cell culture media, the intracellular sialic acid content of EPO-expressing cells increased ∼8-fold over untreated controls while the level of cellular sialylated glycoconjugates increased significantly as well. For example, addition of 200-300 µM 1,3,4-O-Bu3 ManNAc resulted in >40% increase in final sialic acid content of recombinant EPO, while natural ManNAc at ∼100 times higher concentration of 20 mM produced a less profound change in EPO sialylation. Collectively, these results indicate that butyrate-derivatization of ManNAc improves the capacity of cells to incorporate exogenous ManNAc into the sialic acid biosynthetic pathway and thereby increase sialylation of recombinant EPO and other glycoproteins. This study establishes 1,3,4-O-Bu3 ManNAc as a novel chemical supplement to improve glycoprotein quality and sialylation levels at concentrations orders of magnitude lower than alternative approaches. Biotechnol. Bioeng. 2017;114: 1899-1902. © 2017 Wiley Periodicals, Inc.

Structure of the LPS O-chain from Fusobacterium nucleatum strain 10953, containing sialic acid.

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. Recently, it has been gaining attention as a potential causative agent for colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about virulence factors of this organism and thus we have initiated studies to examine the bacterial surface glycochemistry. Consistent with a recent paper suggesting that F. nucleatum strain 10593 can synthesize sialic acid, a staining technique identified sialic acid on the bacterial surface. We isolated lipopolysaccharide from this F. nucleatum strain and performed structural analysis on the O-antigen. Our studies identified a trisaccharide repeating unit of the O-antigen with the following structure: -[→4)-α-Neup5Ac-(2 â†’ 4)-ß-d-Galp-(1 â†’ 3)-α-d-FucpNAc4NAc-(1-]- where Ac indicates 4-N-acetylation of ∼30% FucNAc4N residues. The presence of sialic acid as a constituent of the O-antigen is consistent with recent data identifying de novo sialic acid synthesis in this strain.

Isotype-specific glycosylation analysis of mouse IgG by LC-MS.

With mice being the top model organism in immunology and with Fc glycosylation being increasingly recognized as important modulator of antibody function, the time has come to take a look at the glycosylation of mouse IgG isotypes. Tryptic glycopeptides of mouse IgG1, IgG2, and IgG3 differ in mass and so these three isoforms can be easily discriminated by MS. Commercial IgG contained a rare IgG1 variant but no IgG3, which, however, was found in sera of C57BL/6 and BALB/c mice. These strains deviated with regard to IgG2a and IgG2b alleles. The Ig2a B allele was not observed in any of the four samples investigated. All a/c isotypes contain the same glycopeptide sequence, which deviates from that of IgG2b by containing Leu instead of Ile. The Leu/Ile glycopeptide variants were separated by RP chromatography and the order of elution was determined. The major glycoforms on all isotypes were fucosylated with no and one galactose (GnGnF and GnAF) followed by fully galactosylated AAF and smaller amounts of mono- and disialylated N-glycans. In the commercial serum pool, the relative ratios of glycans differed between isotypes. Sialic acid exclusively occurred as N-glycolylneuraminic acid. Fucosylation was essentially complete. No bisected and no α1,3-galactosylated glycans were found.

A sensitive and efficient method for determination of N-acetylhexosamines and N-acetylneuraminic acid in breast milk and milk-based products by high-performance liquid chromatography via UV detection and mass spectrometry identification.

A sensitive and efficient method of high performance liquid chromatography using 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) as pre-column derivatization reagent coupled with UV detection (HPLC-UV) and online mass spectrometry identification was established for determination of the most common N-Acetylhexosamines (N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-galactosamine (GalNAc)) and N-acetylneuraminic acid (Neu5Ac). In order to obtain the highest liberation level of the three monosaccharides without destruction of Neu5Ac or conversion of GlcNAc/GalNAc to GlcN/GalN in the hydrolysis procedure, the pivotal parameters affecting the liberation of N-acetylhexosamines/Neu5Ac from sample were investigated with response surface methodology (RSM). Under the optimized condition, maximum yield was obtained. The effects of key parameters on derivatization, separation and detection were also investigated. At optimized conditions, three monosaccharides were labeled fast and entirely, and all derivatives exhibited a good baseline resolution and high detection sensitivity. The developed method was linear over the calibration range 0.25-12µM, with R(2)>0.9991. The detection limits of the method were between 0.48 and 2.01pmol. Intra- and inter-day precisions for the three monosaccharides (GlcNAc, GalNAc and Neu5Ac) were found to be in the range of 3.07-4.02% and 3.69-4.67%, respectively. Individual monosaccharide recovery from spiked milk was in the range of 81%-97%. The sensitivity of the method, the facility of the derivatization procedure and the reliability of the hydrolysis conditions suggest the proposed method has a high potential for utilization in routine trace N-acetylhexosamines and Neu5Ac analysis in biological samples.

Total and free serum sialic acid concentration in liver diseases.

BACKGROUND: The objective of this study was to compare the levels of total (TSA) and free (FSA) sialic acid in acute and chronic liver diseases. MaTERIALS AND METHODS: The serum TSA and FSA levels were determined in 278 patients suffering from acute and chronic liver diseases of different etiologies. TSA was estimated by enzymatic method and FSA by the thiobarbituric method modified by Skoza and Mohos. RESULTS: There were no significant differences in the serum TSA concentration between liver diseases of different etiologies, although in most of the liver diseases the mean TSA level was significantly lower than that in the control group. In contrast to TSA, the concentration of FSA appears to differ between liver diseases. In toxic hepatitis it was higher than that in nonalcoholic cirrhosis. However, neither of them differs between alcoholic and nonalcoholic cirrhosis or between liver tumors and tumors with cirrhosis. CONCLUSIONS: We conclude that the changes in concentrations of TSA and FSA during the same liver diseases indicate significant disturbances in sialylation of serum glycoproteins.

Desialylation improves the detection of recombinant erythropoietins in urine samples analyzed by SDS-PAGE.

Recombinant erythropoietin (rhEPO) has been misused for over two decades by athletes, mainly but not only in endurance sports. A direct rhEPO detection method in urine by isoelectric focusing (IEF) was introduced in 2000, but the emergence of third-generation erythropoiesis-stimulating agents and so-called biosimilar rhEPOs, together with the sensitivity of human endogenous EPO (huEPO) pattern to enzymatic activities and its modification following short strenuous exercise, prompted the development of a complementary test based on SDS-PAGE analysis. While Mircera and NESP are easily detected with the existing IEF and SDS-PAGE methods, some samples containing both epoetin-α/ß and huEPO present profiles that are still difficult to interpret. As doping practices have moved to micro-dosing, these mixed patterns are more frequently observed. We investigated the impact of enzymatic desialylation on the urinary and serum EPO profiles obtained by SDS-PAGE with the aim of improving the separation of the bands in these mixed EPO populations. We observed that the removal with neuraminidase of the sialic acid moieties from the different EPOs studied reduced their apparent molecular weight (MW) and increased the migration distance between huEPO and rhEPO centroids, therefore eliminating the size overlaps between them and improving the detection of rhEPO.

Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells.

The baculovirus/insect cell system is widely used for recombinant protein production, but it is suboptimal for recombinant glycoprotein production because it does not provide sialylation, which is an essential feature of many glycoprotein biologics. This problem has been addressed by metabolic engineering, which has extended endogenous insect cell N-glycosylation pathways and enabled glycoprotein sialylation by baculovirus/insect cell systems. However, further improvement is needed because even the most extensively engineered baculovirus/insect cell systems require media supplementation with N-acetylmannosamine, an expensive sialic acid precursor, for efficient recombinant glycoprotein sialylation. Our solution to this problem focused on E. coli N-acetylglucosamine-6-phosphate 2'-epimerase (GNPE), which normally functions in bacterial sialic acid degradation. Considering that insect cells have the product, but not the substrate for this enzyme, we hypothesized that GNPE might drive the reverse reaction in these cells, thereby initiating sialic acid biosynthesis in the absence of media supplementation. We tested this hypothesis by isolating transgenic insect cells expressing E. coli GNPE together with a suite of mammalian genes needed for N-glycoprotein sialylation. Various assays showed that these cells efficiently produced sialic acid, CMP-sialic acid, and sialylated recombinant N-glycoproteins even in growth media without N-acetylmannosamine. Thus, this study demonstrated that a eukaryotic recombinant protein production platform can be glycoengineered with a bacterial gene, that a bacterial enzyme which normally functions in sialic acid degradation can be used to initiate sialic acid biosynthesis, and that insect cells expressing this enzyme can produce sialylated N-glycoproteins without N-acetylmannosamine supplementation, which will reduce production costs in glycoengineered baculovirus/insect cell systems.

Engineering of an N-acetylneuraminic acid synthetic pathway in Escherichia coli.

N-acetylneuraminic acid (NeuAc) has recently drawn much attention owing to its wide applications in many aspects. Besides extraction from natural materials, production of NeuAc was recently focused on enzymatic synthesis and whole-cell biocatalysis. In this study, we designed an artificial NeuAc biosynthetic pathway through intermediate N-acetylglucosamine 6-phosphate in Escherichia coli. In this pathway, N-acetylglucosamine 2-epimerase (slr1975) and glucosamine-6-phosphate acetyltransferase (GNA1) were heterologously introduced into E. coli from Synechocystis sp. PCC6803 and Saccharomyces cerevisiae EBY100, respectively. By derepressing the feedback inhibition of glucosamine-6-phosphate synthase, increasing the accumulation of N-acetylglucosamine and pyruvate, and blocking the catabolism of NeuAc, we were able to produce 1.62 g l⁻¹ NeuAc in recombinant E. coli directly from glucose. The NeuAc yield reached 7.85g l⁻¹ in fed-batch fermentation. This process offered an efficient fermentative method to produce NeuAc in microorganisms using glucose as carbon source and can be optimized for further improvement.

Evaluation of sialic acid-analogs for the attenuation of amyloid-beta toxicity.

BACKGROUND: Amyloid-beta peptide (Aß) is the main constituent of senile plaques and is implicated in the pathogenesis of Alzheimer's disease (AD). To that end, agents which either sequester Aß or interfere with Aß interaction/binding to cells have been investigated as a means to reduce the pathological effects of Aß. METHODS: Different structural analogs of sialic acid (N-acetylneuramic acid) were used to decorate a chitosan backbone using EDC chemistry. FTIR and colorimetric assays were used to characterize the complexes. The ability of these complexes to attenuate Aß toxicity was investigated in vitro using a model neuroblastoma cell line SH-SY5Y. RESULTS: Oxygen substitution in ring structure is responsible for the increase in toxicity and increase in protective properties of the complexes. Also, the multi OH tail present in sialic acid is critical to attenuate toxicity. Analogs show no protective properties which reinforces the conclusion that clustering of sugars in cellular membranes play a significant role in Aß binding. CONCLUSIONS: Successfully produced compounds that showed varying degree of efficacy in attenuating Aß toxicity to cells in culture. This work elucidates the impact that certain structures of sialic acid and its analogs can have on Aß binding. It will allow for more specific and detailed improvements in the therapeutic polysaccharide structures that can be developed and modified to overcome other shortcomings of AD therapeutic development, particularly of penetrating the blood-brain barrier. GENERAL SIGNIFICANCE: Oxygen atom plays crucial role on therapeutic effectiveness. This work can help as a general guideline for further therapeutic development.

Specific extraction of sialic-acid-containing glycans and glycopeptides using serotonin-bonded silica.

Serotonin-bonded silica was developed for specific extraction of sialic-acid-containing glycans and glycopeptides. Serotonin, coupled by reductive amination with aldehyde silica particles via its ethylamino group, has strong affinity to sialic acid in glycan chain termini. Sialylated glycans trapped on serotonin silica particles are released by washing with ammonium acetate solution, providing highly efficient specific trap and release glycoconjugates. With >100 micromol/g adsorption capacity, the particles are applicable to purify labeled glycans after derivatization with 2-aminopyridine to remove excess reagents. Serotonin silica efficiently enriches sialic-acid-containing glycopeptides from tryptic digests for LC/MS analysis of glycans' heterogeneity in glycoproteins.

Determination of sialic acid and gangliosides in biological samples and dairy products: a review.

Gangliosides are sphingolipids containing one or more moieties of sialic acid in their structure. Both gangliosides and sialic acid are bioactive compounds related to animal physiology. Due to their biological relevance, analytical methods adapted to each type of matrix have been developed over time. The present study reviews the main methods applied to the analysis of sialic acid and gangliosides in biological samples and dairy products.

Sialic acid content of tissue-specific gp96 and its potential role in modulating gp96-macrophage interactions.

Cancer-derived heat shock protein gp96 induces a tumor-specific protective immune response primarily mediated by cytotoxic T lymphocytes (CTL) directed toward cancer-associated peptides associated with gp96. Both innate and adaptive immune responses have been demonstrated using a cell culture-based signaling mechanism. When used as an extraneous vaccine, one critical interaction which must occur for an immune response to be generated is the interaction between gp96 and the antigen presenting cell (APC) surface receptors (CD91, SR-A, TLR-2, and TLR-4). Our previous study concluded that gp96 purified from various rat and human prostate cancers is differentially glycosylated based on the amino and neutral monosaccharide content, and it was postulated that the monosaccharides may play a role in its biological activity. In this report, we report differences in the cancer-specific sialic acid content of gp96 purified from normal rat prostate compared to two rat prostate cancers, MAT-LyLu and Dunning G, as well as between two human prostate cancer cells, LnCaP and DU145. We also examined the modulatory effect of sialic acid residues on the binding of gp96 to APCs and its subsequent activation. Our results supported the contention that significant differences in the sialic acid content exist between Dunning G, MAT-LyLu, and normal rat prostate gp96, which affected its binding and biochemical activity to APCs. We therefore postulate that varied glycans of HPS96, a hitherto neglected structural component, may play a pivotal role in its anticancer activity. We suggest that construction of the glycan tree is a key to identification of the necessary and sufficient elements in the structure-function activity of HSP96.

Purification and structural characterization of de-N-acetylated form of GD3 ganglioside present in human melanoma tumors.

The presence of gangliosides containing de-N-acetylated sialic acids in human tissues has been so far shown by using mouse monoclonal antibodies specific for the de-N-acetylated forms, but the isolation and chemical characterization of such compounds have not yet been performed. Since indirect evidence suggested that de-N-acetylGD3 ganglioside could be present in human melanoma tumors, we analyzed the gangliosides purified from a 500-g pool of those tumors. The de-N-acetylGD3 that was found to migrate just below GD2 in thin-layer chromatography was isolated from the disialogangliosides by high-pressure liquid chromatography using the specific antibody SGR37 to monitor the elution. The amount of antigen was found to be 320 ng per gram of fresh tumor or 0.1% of total gangliosides. Gas chromatography-mass spectrometry analysis of the antibody-positive ganglioside showed that sialic acids were formed of one molecule of N-acetylneuraminic acid and one molecule of neuraminic acid. Radioactive re-N-acetylation of the antigen yielded a GD3-like ganglioside with the radioactive label on the external sialic acid. The constitutive fatty acids were found to differ markedly from those of GD3 and 9-O-acetylGD3 isolated from the same pool of tumors. The major fatty acids were C16:0 and C18:0 in de-N-acetylGD3, whereas GD3 and its 9-O-acetylated derivative contained a large amount of C24:1. These data show that de-N-acetylGD3 ganglioside is indeed present in human melanoma tumors, and the fatty acid content suggests the existence of a de-N-acetylase mostly active on the molecular species of gangliosides with short-chain fatty acids.

RNA interference of sialidase improves glycoprotein sialic acid content consistency.

An important challenge facing therapeutic protein production in mammalian cell culture is the cleavage of terminal sialic acids on recombinant protein glycans by the glycosidase enzymes released by lysed cells into the supernatant. This undesired phenomenon results in a protein product which is rapidly cleared from the plasma by asialoglycoprotein receptors in the liver. In this study, RNA interference was utilized as a genetic approach to silence the activity of sialidase, a glycosidase responsible for cleaving terminal sialic acids on IFN-gamma produced by Chinese Hamster Ovary (CHO) cells. We first identified a 21-nt double stranded siRNA that reduced endogenous sialidase mRNA and protein activity levels. Potency of each siRNA sequences was compared using real time RT-PCR and a sialidase activity assay. We next integrated the siRNA sequence into CHO cells, allowing production and selection of stable cell lines. We isolated stable clones with sialidase activity reduced by over 60% as compared to the control cell line. Micellar electrokinetic chromatography (MEKC), thiobarbituric acid assay (TAA), and high performance anion exchange chromatography (HPAEC) coupled to amperometric detection were performed to analyze glycan site occupancy, sialic acid content, and distribution of asialo-/sialylated-glycan structures, respectively. Two of the stable clones successfully retained the full sialic acid content of the recombinant IFN-gamma, even upon cells' death. This was comparable to the case where a chemically synthesized sialidase inhibitor was used. These results demonstrated that RNA interference of sialidase can prevent the desialylation problem in glycoprotein production, resulting improved protein quality during the entire cell culture process.

Sialic acid concentrations in plants are in the range of inadvertent contamination.

The long held but challenged view that plants do not synthesize sialic acids was re-evaluated using two different procedures to isolate putative sialic acid containing material from plant tissues and cells. The extracts were reacted with 1,2-diamino-4,5-methylene dioxybenzene and the fluorescently labelled 2-keto sugar acids analysed by reversed phase and normal phase HPLC and by HPLC-electrospray tandem mass spectrometry. No N-glycolylneuraminic acid was found in the protein fraction from Arabidopsis thaliana MM2d cells. However, we did detect 3-deoxy-D: -manno-octulosonic acid and trace amounts (3-18 pmol/g fresh weight) of a compound indistinguishable from N-acetylneuraminic acid by its retention time and its mass spectral fragmentation pattern. Thus, plant cells and tissues contain five orders of magnitude less sialic acid than mammalian tissues such as porcine liver. Similar or lower amounts of N-acetylneuraminic acid were detected in tobacco cells, mung bean sprouts, apple and banana. Yet even yeast and buffer blanks, when subjected to the same isolation procedures, apparently contained the equivalent of 5 pmol of sialic acid per gram of material. Thus, we conclude that it is not possible to demonstrate unequivocally that plants synthesize sialic acids because the amounts of these sugars detected in plant cells and tissues are so small that they may originate from extraneous contaminants.

Differential distribution of the JC virus receptor-type sialic acid in normal human tissues.

JC virus (JCV), a member of the polyomavirus family, causes a demyelinating disease of the central nervous system (CNS) in humans known as progressive multifocal leukoencephalopathy. Although glial cells are the principal target of JCV productive infection in progressive multifocal leukoencephalopathy patients, little is known regarding the site of JCV persistence and the mechanisms by which the virus spreads to the CNS to cause disease. Previous work has demonstrated the presence of replicating JCV DNA in B lymphocytes from peripheral blood, tonsil, and spleen and it has been hypothesized that lymphocytes may be one site of JCV persistence. Detection of viral gene products in renal tubules and excretion of JC virions in the urine suggests JCV persistence in the kidney. A respiratory route of viral transmission has also been hypothesized implicating the lung as another possible site of persistent JCV infection. Earlier studies from our laboratory have shown that terminal alpha 2,6-linked sialic acid is a critical component of the JCV receptor. In this report we examined the tissue distribution of this JCV receptor-type sialic acid in a panel of normal human tissues. Our results demonstrate that in normal brain JCV receptor-type sialic acids are expressed on oligodendrocytes and astrocytes, but not on cortical neurons. The receptor-type sialic acid is also more highly expressed on B lymphocytes than on T lymphocytes in normal human spleen and tonsil. In addition, both the kidney and lung express abundant levels of alpha 2-6-linked sialic acids. Our data show a striking correlation between the expression of the JCV receptor-type sialic acid on cells and their susceptibility to infection by the virus. These findings also support the hypothesis of JCV persistence in lymphoid tissue and B-cell-facilitated viral dissemination to the CNS.

Presence of an unusual GM2 derivative, taurine-conjugated GM2, in Tay-Sachs brain.

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.