The analysis of gangliosides and glycosphingolipids is crucial for understanding cellular membrane structure and function as well as to accurately diagnose certain inborn errors of metabolism. GM2-gangliosidosis represents a rare and fatal group of lysosomal storage disorders characterized by accumulation of GM2 gangliosides in various tissues and organs. These disorders arise due to deficiency or functional impairment of the β-hexosaminidase A or B enzymes, which are responsible for degradation of GM2 ganglioside. Deficient enzyme activity primarily leads to the accumulation of GM2 gangliosides within the lysosomes of cells. Accurate and rapid diagnostic methods that detect increased levels of GM2 gangliosides in patients with GM2-gangliosidosis can play a significant role in early diagnosis and appropriate treatment of this condition. To address this need, we developed a multiplexed liquid chromatography-tandem mass spectrometry method targeting 84 species of gangliosides and other glycosphingolipids involved in ganglioside metabolism. Reproducibility, linearity, extraction efficiency, and sample stability were evaluated and proof-of-concept data obtained from analysis of serum samples from confirmed cases of GM2-gangliosidosis. This method has the potential to simultaneously monitor the biosynthesis of gangliosides and the lysosomal catabolic pathway serving as a valuable tool for screening and diagnosing an important group of lysosomal storage disorders.

Glycosphingolipids (GSL) are a highly heterogeneous class of lipids representing the majority of the sphingolipid category. GSL are fundamental constituents of cellular membranes that have key roles in various biological processes, such as cellular signaling, recognition, and adhesion. Understanding the structural complexity of GSL is pivotal for unraveling their functional significance in a biological context, specifically their crucial role in the pathophysiology of various diseases. Mass spectrometry (MS) has emerged as a versatile and indispensable tool for the structural elucidation of GSL enabling a deeper understanding of their complex molecular structures and their key roles in cellular dynamics and patholophysiology. Here, we provide a thorough overview of MS techniques tailored for the analysis of GSL, emphasizing their utility in probing GSL intricate structures to advance our understanding of the functional relevance of GSL in health and disease. The application of tandem MS using diverse fragmentation techniques, including novel ion activation methodologies, in studying glycan sequences, linkage positions, and fatty acid composition is extensively discussed. Finally, we address current challenges, such as the detection of low-abundance species and the interpretation of complex spectra, and offer insights into potential solutions and future directions by improving MS instrumentation for enhanced sensitivity and resolution, developing novel ionization techniques, or integrating MS with other analytical approaches for comprehensive GSL characterization.

Glycosphingolipids (GSLs) are abundantly expressed in cancer cells. The effects of GSL-targeted immunotherapies are not fully understood. Here, we show that the inhibition of GSL synthesis with the UDP-glucose ceramide glucosyltransferase inhibitor eliglustat can increase the exposure of the major histocompatibility complex (MHC) and tumour antigen peptides, enhancing the antitumour response of CD8+ T cells in a range of tumour models. We therefore conducted a proof-of-concept phase I trial on the combination of eliglustat and an anti-PD-1 antibody for the treatment of advanced cancers (NCT04944888). The primary endpoints were safety and feasibility, and the secondary endpoint was antitumor activity. All prespecified endpoints were met. Among the 31 enrolled patients, only 1 patient experienced a grade 3 adverse event (AE), and no grade 4 AEs were observed. The objective response rate was 22.6% and the disease control rate reached 71%. Of the 8 patients with proficient mismatch repair/microsatellite stable (pMMR/MSS) colorectal cancer, one achieved complete response and two each had partial response and stable disease. In summary, inhibiting the synthesis of GSLs might represent an effective immunotherapy approach.

GSLs are the major glycolipids in vertebrates and mediate many key biological processes from intercellular recognition to cis regulation of signal transduction. The fast-expanding field of glycobiology has led to a growing demand for diverse and structurally defined GSLs, and enzymatic GSL synthesis is developing rapidly in accordance. This article provides an overview of natural GSL biosynthetic pathways and surveys the bacterial enzymes applied to GSL synthesis and recent progress in synthesis strategies. By correlating these three areas, this article aims to define the gaps between GSL biosynthesis and chemoenzymatic synthesis and evaluate the opportunities for harnessing natural forces to access GSLs efficiently.

Infections pose a challenge for the fast growing aquaculture sector. Glycosphingolipids are cell membrane components that pathogens utilize for attachment to the host to initiate infection. Here, we characterized rainbow trout glycosphingolipids from five mucosal tissues using mass spectrometry and nuclear magnetic resonance and investigated binding of radiolabeled Aeromonas salmonicida to the glycosphingolipids on thin-layer chromatograms. 12 neutral and 14 acidic glycosphingolipids were identified. The glycosphingolipids isolated from the stomach and intestine were mainly neutral, whereas glycosphingolipids isolated from the skin, gills and pyloric caeca were largely acidic. Many of the acidic structures were poly-sialylated with shorter glycan structures in the skin compared to the other tissues. The sialic acids found were Neu5Ac and Neu5Gc. Most of the glycosphingolipids had isoglobo and ganglio core chains, or a combination of these. The epitopes on the rainbow trout glycosphingolipid glycans differed between epithelial sites leading to differences in pathogen binding. A major terminal epitope was fucose, that occurred attached to GalNAc in a α1-3 linkage but also in the form of HexNAc-(Fuc-)HexNAc-R. A. salmonicida were shown to bind to neutral glycosphingolipids from the gill and intestine. This study is the first to do a comprehensive investigation of the rainbow trout glycosphingolipids and analyze binding of A. salmonicida to glycosphingolipids. The structural information paves the way for identification of ways of interfering in pathogen colonization processes to protect against infections in aquaculture and contributes towards understanding A. salmonicida infection mechanisms.

Glycosylated sphingolipids (GSLs) are a diverse group of cellular lipids typically reported as being rare in normal mammary tissue. In breast cancer (BCa), GSLs have emerged as noteworthy markers associated with breast cancer stem cells, mediators of phenotypic plasticity, and contributors to cancer cell chemoresistance. GSLs are potential surface markers that can uniquely characterize the heterogeneity of the tumor microenvironment, including cancer cell subpopulations and epithelial-mesenchymal plasticity (EMP). In this study, mass spectrometry analyses of the total sphingolipidome in breast epithelial cells and their mesenchymal counterparts revealed increased levels of Gb3 in epithelial cells and significantly elevated GD2 levels in the mesenchymal phenotype. To elucidate if GSL-related epitopes on BCa cell surfaces reflect EMP and cancer status, we developed and rigorously validated a 12-color spectral flow cytometry panel. This panel enables the simultaneous detection of native GSL epitopes (Gb3, SSEA1, SSEA3, SSEA4, and GD2), epithelial-mesenchymal transition markers (EpCAM, TROP2, and CD9), and lineage markers (CD45, CD31, and CD90) at the single-cell level. Next, the established panel was used for the analysis of BCa primary tumors and revealed surface heterogeneity in SSEA1, SSEA3, SSEA4, GD2, and Gb3, indicative of native epitope presence also on non-tumor cells. These findings further highlighted the phenotype-dependent alterations in GSL surface profiles, with differences between epithelial and stromal cells in the tumor. This study provides novel insights into BCa heterogeneity, shedding light on the potential of native GSL-related epitopes as markers for EMP and cancer status in fresh clinical samples. The developed single-cell approach offers promising avenues for further exploration.

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) present in cell membranes are implicated in a wide range of biological processes. However, studying GSL binding is hindered by the paucity of purified GSLs and the weak affinities typical of monovalent GBP-GSL interactions. Native mass spectrometry (nMS) performed using soluble model membranes is a promising approach for the discovery of GBP ligands, but the detection of weak interactions remains challenging. The present work introduces MEmbrane ANchor-assisted nMS (MEAN-nMS) for the detection of low-affinity GBP-GSL complexes. The assay utilizes a membrane anchor, produced by covalent cross-linking of the GBP and a lipid in the membrane, to localize the GBP on the surface and promote GSL binding. Ligands are identified by nMS detection of intact GBP-GSL complexes (MEAN-nMS) or using a catch-and-release (CaR) strategy, wherein GSLs are released from GBP-GSL complexes upon collisional activation and detected (MEAN-CaR-nMS). To establish reliability, a library of purified gangliosides incorporated into nanodiscs was screened against human immune lectins, and the results compared with affinities of the corresponding ganglioside oligosaccharides. Without a membrane anchor, nMS analysis yielded predominantly false negatives. In contrast, all ligands were identified by MEAN-(CaR)-nMS, with no false positives. To highlight the potential of MEAN-CaR-nMS for ligand discovery, a natural library of GSLs was incorporated into nanodiscs and screened against human and viral proteins to uncover elusive ligands. Finally, nMS-based detection of GSL ligands directly from cells is demonstrated. This breakthrough paves the way for shotgun glycomics screening using intact cells.

The lack of effective vaccines and the development of resistance to the current treatments highlight the urgent need for new anti-leishmanials. Sphingolipid metabolism has been proposed as a promising source of Leishmania-specific targets as these lipids are key structural components of the eukaryotic plasma membrane and are involved in distinct cellular events. Inositol phosphorylceramide (IPC) is the primary sphingolipid in the Leishmania species and is the product of a reaction mediated by IPC synthase (IPCS). The antihistamine clemastine fumarate has been identified as an inhibitor of IPCS in L. major and a potent anti-leishmanial in vivo. Here we sought to further examine the target of this compound in the more tractable species L. mexicana, using an approach combining genomic, proteomic, metabolomic and lipidomic technologies, with molecular and biochemical studies. While the data demonstrated that the response to clemastine fumarate was largely conserved, unexpected disturbances beyond sphingolipid metabolism were identified. Furthermore, while deletion of the gene encoding LmxIPCS had little impact in vitro, it did influence clemastine fumarate efficacy and, importantly, in vivo pathogenicity. Together, these data demonstrate that clemastine does inhibit LmxIPCS and cause associated metabolic disturbances, but its primary target may lie elsewhere.

Ovarian cancer is among the most prevalent causes of mortality among women. Despite improvements in diagnostic methods, non-specific symptoms and delayed gynecological exams can lead to late-stage ovarian tumor discovery. In this study, the effect of an anti-cancer compound, 3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide (Compound 1), was examined. The impacts of cytotoxicity, apoptosis, and metabolomic changes in ovarian cancer cell lines SK-OV-3 and OVCAR-3, as well as glycosphingolipid (GSL) expression, on cancer stem cells (CSCs), marked as CD49f+, and non-CSCs (CD49f-) were explored. Treatment with Compound 1 reduced the percentage of CSCs compared to non-treated cells (p < 0.001). The functional impact of eight GSLs on CSCs and non-CSCs was examined using flow cytometry. The glycophenotype changed in both cell lines, with increases or decreases in its expression, after the treatment. These findings raise the possibility of specifically targeting CSCs in ovarian cancer therapy. Additionally, treatment with Compound 1 resulted in statistically meaningful increased apoptosis, including both early and late apoptosis (p < 0.001), suggesting a pivotal role in initiating programmed cell death by the apoptotic pathway. The analysis revealed that the metabolic activity of treated cancer cells was lower compared to those of the control group (p < 0.001).

Glycosphingolipids (GSLs) are essential components of cell membranes, particularly enriched in the nervous system. Altered molecular distributions of GSLs are increasingly associated with human diseases, emphasizing the significance of lipidomic profiling. Traditional GSL analysis methods are hampered by matrix effect from phospholipids and the difficulty in distinguishing structural isomers. Herein, we introduce a highly sensitive workflow that harnesses magnetic TiO2 nanoparticle-based selective enrichment, charge-tagging Paternò-Büchi reaction, and liquid chromatography-tandem mass spectrometry. This approach enables mapping over 300 distinct GSLs in brain tissues by defining sugar types, long chain bases, N-acyl chains, and the locations of desaturation and hydroxylation. Relative quantitation of GSLs across multiple structural levels provides evidence of dysregulated gene and protein expressions of FA2H and CerS2 in human glioma tissue. Based on the structural features of GSLs, our method accurately differentiates human glioma with/without isocitrate dehydrogenase genetic mutation, and normal brain tissue.