Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes.

Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes.

The SPPL3-Defined Glycosphingolipid Repertoire Orchestrates HLA Class I-Mediated Immune Responses.

HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.

Glycosphingolipid synthesis mediates immune evasion in KRAS-driven cancer.

Cancer cells frequently alter their lipids to grow and adapt to their environment1-3. Despite the critical functions of lipid metabolism in membrane physiology, signalling and energy production, how specific lipids contribute to tumorigenesis remains incompletely understood. Here, using functional genomics and lipidomic approaches, we identified de novo sphingolipid synthesis as an essential pathway for cancer immune evasion. Synthesis of sphingolipids is surprisingly dispensable for cancer cell proliferation in culture or in immunodeficient mice but required for tumour growth in multiple syngeneic models. Blocking sphingolipid production in cancer cells enhances the anti-proliferative effects of natural killer and CD8+ T cells partly via interferon-γ (IFNγ) signalling. Mechanistically, depletion of glycosphingolipids increases surface levels of IFNγ receptor subunit 1 (IFNGR1), which mediates IFNγ-induced growth arrest and pro-inflammatory signalling. Finally, pharmacological inhibition of glycosphingolipid synthesis synergizes with checkpoint blockade therapy to enhance anti-tumour immune response. Altogether, our work identifies glycosphingolipids as necessary and limiting metabolites for cancer immune evasion.

Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells.

Coevolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Here, we report that the intestinal microbe Bacteroides fragilis modifies the homeostasis of host invariant natural killer T (iNKT) cells by supplementing the host's endogenous lipid antigen milieu with unique inhibitory sphingolipids. The process occurs early in life and effectively impedes iNKT cell proliferation during neonatal development. Consequently, total colonic iNKT cell numbers are restricted into adulthood, and hosts are protected against experimental iNKT cell-mediated, oxazolone-induced colitis. In studies with neonatal mice lacking access to bacterial sphingolipids, we found that treatment with B. fragilis glycosphingolipids-exemplified by an isolated peak (MW = 717.6) called GSL-Bf717-reduces colonic iNKT cell numbers and confers protection against oxazolone-induced colitis in adulthood. Our results suggest that the distinctive inhibitory capacity of GSL-Bf717 and similar molecules may prove useful in the treatment of autoimmune and allergic disorders in which iNKT cell activation is destructive.

Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions.

The embryonic cell surface is rich in glycosphingolipids (GSLs), which change during differentiation. The reasons for GSL subgroup variation during early embryogenesis remain elusive. By combining genomic approaches, flow cytometry, confocal imaging, and transcriptomic data analysis, we discovered that α1,2-fucosylated GSLs control the differentiation of human pluripotent cells (hPCs) into germ layer tissues. Overexpression of α1,2-fucosylated GSLs disrupts hPC differentiation into mesodermal lineage and reduces differentiation into cardiomyocytes. Conversely, reducing α1,2-fucosylated groups promotes hPC differentiation and mesoderm commitment in response to external signals. We find that bone morphogenetic protein 4 (BMP4), a mesodermal gene inducer, suppresses α1,2-fucosylated GSL expression. Overexpression of α1,2-fucosylated GSLs impairs SMAD activation despite BMP4 presence, suggesting α-fucosyl end groups as BMP pathway regulators. Additionally, the absence of α1,2-fucosylated GSLs in early/late mesoderm and primitive streak stages in mouse embryos aligns with the hPC results. Thus, α1,2-fucosylated GSLs may regulate early cell-fate decisions and embryo development by modulating cell signaling.

(Sialyl)Lewis Antigen Expression on Glycosphingolipids, N-, and O-Glycans in Colorectal Cancer Cell Lines is Linked to a Colon-Like Differentiation Program.

Alterations in the glycomic profile are a hallmark of cancer, including colorectal cancer (CRC). While, the glycosylation of glycoproteins and glycolipids has been widely studied for CRC cell lines and tissues, a comprehensive overview of CRC glycomics is still lacking due to the usage of different samples and analytical methods. In this study, we compared glycosylation features of N-, O-glycans, and glycosphingolipid glycans for a set of 22 CRC cell lines, all measured by porous graphitized carbon nano-liquid chromatography-tandem mass spectrometry. An overall, high abundance of (sialyl)Lewis antigens for colon-like cell lines was found, while undifferentiated cell lines showed high expression of H blood group antigens and α2-3/6 sialylation. Moreover, significant associations of glycosylation features were found between the three classes of glycans, such as (sialyl)Lewis and H blood group antigens. Integration of the datasets with transcriptomics data revealed positive correlations between (sialyl)Lewis antigens, the corresponding glycosyltransferase FUT3 and transcription factors CDX1, ETS, HNF1/4A, MECOM, and MYB. This indicates a possible role of these transcription factors in the upregulation of (sialyl)Lewis antigens, particularly on glycosphingolipid glycans, via FUT3/4 expression in colon-like cell lines. In conclusion, our study provides insights into the possible regulation of glycans in CRC and can serve as a guide for the development of diagnostic and therapeutic biomarkers.

Altered plasma membrane abundance of the sulfatide-binding protein NF155 links glycosphingolipid imbalances to demyelination.

Myelin is a multilayered membrane that tightly wraps neuronal axons, enabling efficient, high-speed signal propagation. The axon and myelin sheath form tight contacts, mediated by specific plasma membrane proteins and lipids, and disruption of these contacts causes devastating demyelinating diseases. Using two cell-based models of demyelinating sphingolipidoses, we demonstrate that altered lipid metabolism changes the abundance of specific plasma membrane proteins. These altered membrane proteins have known roles in cell adhesion and signaling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule neurofascin (NFASC), a protein critical for the maintenance of myelin-axon contacts, changes following disruption to sphingolipid metabolism. This provides a direct molecular link between altered lipid abundance and myelin stability. We show that the NFASC isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferentially binds sulfatide-containing membranes in cis, with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of galactosphingolipidoses.

Neural-specific alterations in glycosphingolipid biosynthesis and cell signaling associated with two human ganglioside GM3 synthase deficiency variants.

GM3 Synthase Deficiency (GM3SD) is a neurodevelopmental disorder resulting from pathogenic variants in the ST3GAL5 gene, which encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. This enzyme adds a sialic acid to the terminal galactose of lactosylceramide (LacCer) to produce the monosialylated ganglioside GM3. In turn, GM3 is extended by other glycosyltransferases to generate nearly all the complex gangliosides enriched in neural tissue. Pathogenic mechanisms underlying the neural phenotypes associated with GM3SD are unknown. To explore how loss of GM3 impacts neural-specific glycolipid glycosylation and cell signaling, GM3SD patient fibroblasts bearing one of two different ST3GAL5 variants were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to neural crest cells (NCCs). GM3 and GM3-derived gangliosides were undetectable in cells carrying either variant, while LacCer precursor levels were elevated compared to wildtype (WT). NCCs of both variants synthesized elevated levels of neutral lacto- and globo-series, as well as minor alternatively sialylated GSLs compared to WT. Ceramide profiles were also shifted in GM3SD variant cells. Altered GSL profiles in GM3SD cells were accompanied by dynamic changes in the cell surface proteome, protein O-GlcNAcylation, and receptor tyrosine kinase abundance. GM3SD cells also exhibited increased apoptosis and sensitivity to erlotinib-induced inhibition of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase rescued baseline and erlotinib-induced apoptosis. Collectively, these findings indicate aberrant cell signaling during differentiation of GM3SD iPSCs and also underscore the challenge of distinguishing between variant effect and genetic background effect on specific phenotypic consequences.

GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis.

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.

Plant cell-surface GIPC sphingolipids sense salt to trigger Ca2+ influx.

Salinity is detrimental to plant growth, crop production and food security worldwide. Excess salt triggers increases in cytosolic Ca2+ concentration, which activate Ca2+-binding proteins and upregulate the Na+/H+ antiporter in order to remove Na+. Salt-induced increases in Ca2+ have long been thought to be involved in the detection of salt stress, but the molecular components of the sensing machinery remain unknown. Here, using Ca2+-imaging-based forward genetic screens, we isolated the Arabidopsis thaliana mutant monocation-induced [Ca2+]i increases 1 (moca1), and identified MOCA1 as a glucuronosyltransferase for glycosyl inositol phosphorylceramide (GIPC) sphingolipids in the plasma membrane. MOCA1 is required for salt-induced depolarization of the cell-surface potential, Ca2+ spikes and waves, Na+/H+ antiporter activation, and regulation of growth. Na+ binds to GIPCs to gate Ca2+ influx channels. This salt-sensing mechanism might imply that plasma-membrane lipids are involved in adaption to various environmental salt levels, and could be used to improve salt resistance in crops.

Single-cell profiling of surface glycosphingolipids opens a new dimension for deconvolution of breast cancer intratumoral heterogeneity and phenotypic plasticity.

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.

Bifunctional glycosphingolipid (GSL) probes to investigate GSL-interacting proteins in cell membranes.

Glycosphingolipids (GSLs) are abundant glycolipids on cells and essential for cell recognition, adhesion, signal transduction, and so on. However, their lipid anchors are not long enough to cross the membrane bilayer. To transduce transmembrane signals, GSLs must interact with other membrane components, whereas such interactions are difficult to investigate. To overcome this difficulty, bifunctional derivatives of II3-ß-N-acetyl-D-galactosamine-GA2 (GalNAc-GA2) and ß-N-acetyl-D-glucosamine-ceramide (GlcNAc-Cer) were synthesized as probes to explore GSL-interacting membrane proteins in live cells. Both probes contain photoreactive diazirine in the lipid moiety, which can crosslink with proximal membrane proteins upon photoactivation, and clickable alkyne in the glycan to facilitate affinity tag addition for crosslinked protein pull-down and characterization. The synthesis is highlighted by the efficient assembly of simple glycolipid precursors followed by on-site lipid remodeling. These probes were employed to profile GSL-interacting membrane proteins in HEK293 cells. The GalNAc-GA2 probe revealed 312 distinct proteins, with GlcNAc-Cer probe-crosslinked proteins as controls, suggesting the potential influence of the glycan on GSL functions. Many of the proteins identified with the GalNAc-GA2 probe are associated with GSLs, and some have been validated as being specific to this probe. The versatile probe design and experimental protocols are anticipated to be widely applicable to GSL research.

Characterization of the rainbow trout (Oncorhynchus mykiss) mucosal glycosphingolipid repertoire and Aeromonas salmonicida binding to neutral glycosphingolipids.

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.

Elusive Protein-Glycosphingolipid Interactions Revealed by Membrane Anchor-Assisted Native Mass Spectrometry.

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.

Hepatic Topology of Glycosphingolipids in Schistosoma mansoni-Infected Hamsters.

Schistosomiasis is a neglected tropical disease caused by worm parasites of the genus Schistosoma. Upon infection, parasite eggs can lodge inside of host organs like the liver. This leads to granuloma formation, which is the main cause of the pathology of schistosomiasis. To better understand the different levels of host-pathogen interaction and pathology, our study focused on the characterization of glycosphingolipids (GSLs). For this purpose, GSLs in livers of infected and noninfected hamsters were studied by combining high-spatial-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) with nanoscale hydrophilic interaction liquid chromatography tandem mass spectrometry (nano-HILIC MS/MS). Nano-HILIC MS/MS revealed 60 GSL species with a distinct saccharide and ceramide composition. AP-SMALDI MSI measurements were conducted in positive- and negative-ion mode for the visualization of neutral and acidic GSLs. Based on nano-HILIC MS/MS results, we discovered no downregulated but 50 significantly upregulated GSLs in liver samples of infected hamsters. AP-SMALDI MSI showed that 44 of these GSL species were associated with the granulomas in the liver tissue. Our findings suggest an important role of GSLs during granuloma formation.

Dysregulated lipid metabolism networks modulate T-cell function in people with relapsing-remitting multiple sclerosis.

Altered cholesterol, oxysterol, sphingolipid, and fatty acid concentrations are reported in blood, cerebrospinal fluid, and brain tissue of people with relapsing-remitting multiple sclerosis (RRMS) and are linked to disease progression and treatment responses. CD4 + T cells are pathogenic in RRMS, and defective T-cell function could be mediated in part by liver X receptors (LXRs)-nuclear receptors that regulate lipid homeostasis and immunity. RNA-sequencing and pathway analysis identified that genes within the 'lipid metabolism' and 'signalling of nuclear receptors' pathways were dysregulated in CD4 + T cells isolated from RRMS patients compared with healthy donors. While LXRB and genes associated with cholesterol metabolism were upregulated, other T-cell LXR-target genes, including genes involved in cellular lipid uptake (inducible degrader of the LDL receptor, IDOL), and the rate-limiting enzyme for glycosphingolipid biosynthesis (UDP-glucosylceramide synthase, UGCG) were downregulated in T cells from patients with RRMS compared to healthy donors. Correspondingly, plasma membrane glycosphingolipids were reduced, and cholesterol levels increased in RRMS CD4 + T cells, an effect partially recapitulated in healthy T cells by in vitro culture with T-cell receptor stimulation in the presence of serum from RRMS patients. Notably, stimulation with LXR-agonist GW3965 normalized membrane cholesterol levels, and reduced proliferation and IL17 cytokine production in RRMS CD4 + T-cells. Thus, LXR-mediated lipid metabolism pathways were dysregulated in T cells from patients with RRMS and could contribute to RRMS pathogenesis. Therapies that modify lipid metabolism could help restore immune cell function.

Glycosphingolipids in congenital disorders of glycosylation (CDG).

Congenital disorders of glycosylation (CDG) are a large family of rare disorders affecting the different glycosylation pathways. Defective glycosylation can affect any organ, with varying symptoms among the different CDG. Even between individuals with the same CDG there is quite variable severity. Associating specific symptoms to deficiencies of certain glycoproteins or glycolipids is thus a challenging task. In this review, we focus on the glycosphingolipid (GSL) synthesis pathway, which is still rather unexplored in the context of CDG, and outline the functions of the main GSLs, including gangliosides, and their role in the central nervous system. We provide an overview of GSL studies that have been performed in CDG and show that abnormal GSL levels are not only observed in CDG directly affecting GSL synthesis, but also in better known CDG, such as PMM2-CDG. We highlight the importance of studying GSLs in CDG in order to better understand the pathophysiology of these disorders.

Emerging role of MAPK signaling in glycosphingolipid-associated tumorigenesis.

Glycosphingolipids (GSLs) are a type of amphipathic lipid molecules consisting of hydrophobic ceramide backbone bound to carbohydrate moiety clustered in the cell surface microdomains named 'lipid rafts' and are known to participate in cell-cell communication as well as intra-cellular signaling, thereby facilitating critical normal cellular processes and functions. Over the past several decades, various GSLs have been reported to be aberrantly expressed in different cancers, many of which have been associated with their prognosis. The wide implication of MAPK signaling in controlling tumor growth, progression, and metastasis through activation of an upstream signaling cascade, often originating in the cell membrane, justifies the rationale for its plausible influence on MAPK signaling. This review highlights the role of GSLs and their metabolites in regulating different signaling pathways towards modulation of tumor cell growth, migration, and adhesion by interacting with various receptors [epidermal growth factor receptor (EGFR), and platelet derived growth factor receptor (PDGFR), and other receptor tyrosine kinases (RTKs)] leading to activation of the MAPK pathway. Furthermore, GSLs can influence the activity and localization of downstream signaling components in the MAPK pathway by regulating the activation state of kinases, which in turn, regulate the activity of MAPKs. Additionally, this review further consolidates the GSL-mediated modulation of MAPK pathway components through the regulation of gene expression. Finally, recent findings on GSL-MAPK crosstalk will be explored in this article for the identification of potential anti-cancer therapeutic targets.

Glycosphingolipids: from metabolism to chemoenzymatic total synthesis.

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.

A multiplexed targeted method for profiling of serum gangliosides and glycosphingolipids: application to GM2-gangliosidosis.

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 ß-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.