Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity.

Innate immune signaling via TLR4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM3 ganglioside in human serum is composed of a variety of fatty acids, including long-chain (LCFA) and very-long-chain (VLCFA). Analysis of circulating levels of human serum GM3 species from patients at different stages of insulin resistance and chronic inflammation reveals that levels of VLCFA-GM3 increase significantly in metabolic disorders, while LCFA-GM3 serum levels decrease. Specific GM3 species also correlates with disease symptoms. VLCFA-GM3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR4-mutant mice. In cultured monocytes, GM3 by itself has no effect on TLR4 activation; however, VLCFA-GM3 synergistically and selectively enhances TLR4 activation by LPS/HMGB1, while LCFA-GM3 and unsaturated VLCFA-GM3 suppresses TLR4 activation. GM3 interacts with the extracellular region of TLR4/MD2 complex to modulate dimerization/oligomerization. Ligand-molecular docking analysis supports that VLCFA-GM3 and LCFA-GM3 act as agonist and antagonist of TLR4 activity, respectively, by differentially binding to the hydrophobic pocket of MD2. Our findings suggest that VLCFA-GM3 is a risk factor for TLR4-mediated disease progression.

Protecting-Group-Free Synthesis of ADP-Ribose and Dinucleoside Di-/Triphosphate Derivatives via P(V)-P(V) Coupling Reaction.

Biomolecules containing adenosine di- or triphosphate (ADP or ATP) are crucial for diverse biological processes. Synthesis of these biomolecules and development of their chemical probes are important to elucidate their functions. Enabling reproducible and high-yielding access to these ADP- and ATP-containing molecules via conventional P(III)-P(V) and P(V)-P(V) coupling reactions is challenging owing to water content in highly polar phosphate-containing substrates. Herein, we report an efficient and reliable method for protecting-group-free P(V)-P(V) coupling reaction through in situ activation of phosphates using hydrolysis-stable 2-[N-(2-methylimidazoyl)]-1,3-dimethylimidazolinium chloride (2-MeImIm-Cl), providing the corresponding electrophilic P(V) intermediates for subsequent nucleophilic attack using their coupling partners. This P(V)-P(V) coupling reaction proceeded even in a wet reaction medium and showed a broad substrate scope, accommodating protecting-group-free synthesis of ADP-ribose and nicotinamide adenine diphosphate analogs, ATP-containing biomolecules, and ADP-ribosyl peptides.

Improved synthesis of CD22-binding sialosides and its application for further development of potent CD22 inhibitors.

CD22, one of the sialic acid-binding immunoglobulin-like lectins (Siglecs), regulates B lymphocyte signaling via its interaction with glycan ligands bearing the sequence Neu5Ac/Gcα(2→6)Gal. We have developed the synthetic sialoside GSC-718 as a ligand mimic for CD22 and identified it as a potent CD22 inhibitor. Although the synthesis of CD22-binding sialosides including GSC-718 has been reported by our group, the synthetic route was unfortunately not suitable for large-scale synthesis. In this study, we developed an improved scalable synthetic procedure for sialosides which utilized 1,5-lactam formation as a key step. The improved procedure yielded sialosides incorporating a series of aglycones at the C2 position. Several derivatives with substituted benzyl residues as aglycones were found to bind to mouse CD22 with affinity comparable to that of GSC-718. The new procedure developed in this study affords sialosides in sufficient quantities for cell-based assays, and will facilitate the search for promising CD22 inhibitors that have therapeutic potential.

Fluorescent GD2 analog for single-molecule imaging.

Ganglioside GD2 is associated with the proliferation and migration of breast cancer cells. However, the precise role of GD2 is unclear because its tendency to form dynamic and transient domains in cell plasma membranes (PMs), called lipid rafts, makes it difficult to observe. Previously, we developed fluorescent analogs of gangliosides (e.g., GM3 and GM1), which enabled the observation of lipid raft formation for the first time using single-molecule imaging. In this report, we describe the first chemical synthesis of a fluorescent ganglioside, GD2. A biophysical analysis of the synthesized analog revealed its raft-philic character, suggesting its potential to aid single-molecule imaging-based investigations into raft-associated interactions.

GM1 structural requirements to mediate neuronal functions.

Since the 1980s, it has been known that the administration of ganglioside GM1 to cultured cells induced or enhanced neuronal differentiation. GM1 mechanism of action relies on its direct interaction and subsequent activation of the membrane tyrosine kinase receptor, TrkA, which naturally serves as NGF receptor. This process is mediated by the sole oligosaccharide portion of GM1, the pentasaccharide ß-Gal-(1-3)-ß-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-ß-Gal-(1-4)-ß-Glc. Here we detailed the minimum structural requirements of the oligosaccharide portion of GM1 for mediating the TrkA dependent neuritogenic processing. By in vitro and in silico biochemical approaches, we demonstrated that the minimal portion of GM1 required for the TrkA activation is the inner core of the ganglioside's oligosaccharide ß-Gal-(1-3)-ß-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-ß-Gal. The addition of a sialic acid residue at position 3 of the outer galactose of the GM1 oligosaccharide, which forms the oligosaccharide of GD1a, prevented the interaction with TrkA and the resulting neuritogenesis. On the contrary, the addition of a fucose residue at position 2 of the outer galactose, forming the Fucosyl-GM1 oligosaccharide, did not prevent the TrkA-mediated neuritogenesis.

The Protein Tyrosine Phosphatase SHP-1 (PTPN6) but Not CD45 (PTPRC) Is Essential for the Ligand-Mediated Regulation of CD22 in BCR-Ligated B Cells.

CD22 is an inhibitory B cell coreceptor that regulates B cell development and activation by downregulating BCR signaling through activation of SH2-containing protein tyrosine phosphatase-1 (SHP-1). CD22 recognizes α2,6 sialic acid as a specific ligand and interacts with α2,6 sialic acid-containing membrane molecules, such as CD45, IgM, and CD22, expressed on the same cell. Functional regulation of CD22 by these endogenous ligands enhances BCR ligation-induced signaling and is essential for normal B cell responses to Ags. In this study, we demonstrate that CD45 plays a crucial role in CD22-mediated inhibition of BCR ligation-induced signaling. However, disruption of ligand binding of CD22 enhances CD22 phosphorylation, a process required for CD22-mediated signal inhibition, upon BCR ligation in CD45-/- as well as wild-type mouse B cells but not in mouse B cells expressing a loss-of-function mutant of SHP-1. This result indicates that SHP-1 but not CD45 is required for ligand-mediated regulation of CD22. We further demonstrate that CD22 is a substrate of SHP-1, suggesting that SHP-1 recruited to CD22 dephosphorylates nearby CD22 as well as other substrates. CD22 dephosphorylation by SHP-1 appears to be augmented by homotypic CD22 clustering mediated by recognition of CD22 as a ligand of CD22 because CD22 clustering increases the number of nearby CD22. Our results suggest that CD22 but not CD45 is an endogenous ligand of CD22 that enhances BCR ligation-induced signaling through SHP-1-mediated dephosphorylation of CD22 in CD22 clusters.

Ligand-Controlled Stereoselective Synthesis and Biological Activity of 2-Exomethylene Pseudo-glycoconjugates: Discovery of Mincle-Selective Ligands.

Glycoconjugate analogues in which the sp3 -hybridized C2 position of the carbohydrate structure (normally bearing a hydroxy group) is converted into a compact sp2 -hybridized exomethylene group are expected to have unique biological activities. We established ligand-controlled Tsuji-Trost-type glycosylation methodology to directly prepare a variety of these 2-exomethylene pseudo-glycoconjugates, including glucosylceramide analogues, in an α- or ß-selective manner. Glucocerebrosidase GBA1 cleaves these synthetic pseudo-ß-glucosylceramides similarly to native glucosylceramides. The pseudo-glucosylceramides exhibit selective ligand activity towards macrophage-inducible C-type lectin (Mincle), but unlike native glucosylceramides, are inactive towards CD1d.

The sialidase NEU1 directly interacts with the juxtamembranous segment of the cytoplasmic domain of mucin-1 to inhibit downstream PI3K-Akt signaling.

The extracellular domain (ED) of the membrane-spanning sialoglycoprotein, mucin-1 (MUC1), is an in vivo substrate for the lysosomal sialidase, neuraminidase-1 (NEU1). Engagement of the MUC1-ED by its cognate ligand, Pseudomonas aeruginosa-expressed flagellin, increases NEU1-MUC1 association and NEU1-mediated MUC1-ED desialylation to unmask cryptic binding sites for its ligand. However, the mechanism(s) through which intracellular NEU1 might physically interact with its surface-expressed MUC1-ED substrate are unclear. Using reciprocal coimmunoprecipitation and in vitro binding assays in a human airway epithelial cell system, we show here that NEU1 associates with the MUC1-cytoplasmic domain (CD) but not with the MUC1-ED. Prior pharmacologic inhibition of the NEU1 catalytic activity using the NEU1-selective sialidase inhibitor, C9-butyl amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid, did not diminish NEU1-MUC1-CD association. In addition, glutathione-S-transferase (GST) pull-down assays using the deletion mutants of the MUC1-CD mapped the NEU1-binding site to the membrane-proximal 36 aa of the MUC1-CD. In a cell-free system, we found that the purified NEU1 interacted with the immobilized GST-MUC1-CD and the purified MUC1-CD associated with the immobilized 6XHis-NEU1, indicating that the NEU1-MUC1-CD interaction was direct and independent of its chaperone protein, protective protein/cathepsin A. However, the NEU1-MUC1-CD interaction was not required for the NEU1-mediated MUC1-ED desialylation. Finally, we demonstrated that overexpression of either WT NEU1 or a catalytically dead NEU1 G68V mutant diminished the association of the established MUC1-CD binding partner, PI3K, to MUC1-CD and reduced downstream Akt kinase phosphorylation. These results indicate that NEU1 associates with the juxtamembranous region of the MUC1-CD to inhibit PI3K-Akt signaling independent of NEU1 catalytic activity.

Protecting-group- and microwave-free synthesis of ß-glycosyl esters and aryl ß-glycosides of N-acetyl-d-glucosamine.

A protecting-group-free method for synthesis of ß-glycosyl esters and aryl ß-glycosides was developed by using latent chemical reactivity of N-acetyl-d-glucosamine (GlcNAc) oxazoline. The GlcNAc oxazoline was spontaneously reacted with carboxylic acids and phenol derivatives via the oxazoline ring opening without the use of a catalyst or heating conditions (i.e., microwave irradiation), affording the desired products in moderate to excellent yields with ß-selectivity. This simple protecting-group-free method exhibits a wide substrate scope and good functional group tolerance, and it allows the efficient production of a novel class of GlcNAc-conjugated biomaterials and prodrug candidates.

Investigation of the Protection of the C4 Hydroxyl Group in Macrobicyclic Kdo Donors.

Chemical synthesis of 3-deoxy-d-manno-2-octulosonic acid (Kdo)-containing glycans, such as bacterial lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs), is in high demand for the development of vaccines against pathogenic bacteria. We have recently achieved the complete α-stereoselective glycosidation of Kdo using a macrobicyclic donor tethered at the C1 and C5 positions. In this study, to expand the scope of Kdo glycosidation, we sought to protect the 4-OH group, thereby shortening the reaction time and ensuring the conversion of the glycosyl acceptor via its selective removal. The protection of the 4-OH group influenced the reactivity of the Kdo donor, and the triisopropylsilyl (TIPS) group acted as a selectively removable booster. The 4-O-TIPS donor allowed the synthesis of the α(2,4)-linked dimeric Kdo sequence, which is widely found in bacterial LPSs.

Structural insight into the recognition of pathogen-derived phosphoglycolipids by C-type lectin receptor DCAR.

The C-type lectin receptors (CLRs) form a family of pattern recognition receptors that recognize numerous pathogens, such as bacteria and fungi, and trigger innate immune responses. The extracellular carbohydrate-recognition domain (CRD) of CLRs forms a globular structure that can coordinate a Ca2+ ion, allowing receptor interactions with sugar-containing ligands. Although well-conserved, the CRD fold can also display differences that directly affect the specificity of the receptors for their ligands. Here, we report crystal structures at 1.8-2.3 Å resolutions of the CRD of murine dendritic cell-immunoactivating receptor (DCAR, or Clec4b1), the CLR that binds phosphoglycolipids such as acylated phosphatidyl-myo-inositol mannosides (AcPIMs) of mycobacteria. Using mutagenesis analysis, we identified critical residues, Ala136 and Gln198, on the surface surrounding the ligand-binding site of DCAR, as well as an atypical Ca2+-binding motif (Glu-Pro-Ser/EPS168-170). By chemically synthesizing a water-soluble ligand analog, inositol-monophosphate dimannose (IPM2), we confirmed the direct interaction of DCAR with the polar moiety of AcPIMs by biolayer interferometry and co-crystallization approaches. We also observed a hydrophobic groove extending from the ligand-binding site that is in a suitable position to interact with the lipid portion of whole AcPIMs. These results suggest that the hydroxyl group-binding ability and hydrophobic groove of DCAR mediate its specific binding to pathogen-derived phosphoglycolipids such as mycobacterial AcPIMs.

Therapeutic Effect of Neuraminidase-1-Selective Inhibition in Mouse Models of Bleomycin-Induced Pulmonary Inflammation and Fibrosis.

Pulmonary fibrosis remains a serious biomedical problem with no cure and an urgent need for better therapies. Neuraminidases (NEUs), including NEU1, have been recently implicated in the mechanism of pulmonary fibrosis by us and others. We now have tested the ability of a broad-spectrum neuraminidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), to modulate the in vivo response to acute intratracheal bleomycin challenge as an experimental model of pulmonary fibrosis. A marked alleviation of bleomycin-induced body weight loss and notable declines in accumulation of pulmonary lymphocytes and collagen deposition were observed. Real-time polymerase chain reaction analyses of human and mouse lung tissues and primary human lung fibroblast cultures were also performed. A predominant expression and pronounced elevation in the levels of NEU1 mRNA were observed in patients with idiopathic pulmonary fibrosis and bleomycin-challenged mice compared with their corresponding controls, whereas NEU2, NEU3, and NEU4 were expressed at far lower levels. The levels of mRNA for the NEU1 chaperone, protective protein/cathepsin A (PPCA), were also elevated by bleomycin. Western blotting analyses demonstrated bleomycin-induced elevations in protein expression of both NEU1 and PPCA in mouse lungs. Two known selective NEU1 inhibitors, C9-pentyl-amide-DANA (C9-BA-DANA) and C5-hexanamido-C9-acetamido-DANA, dramatically reduced bleomycin-induced loss of body weight, accumulation of pulmonary lymphocytes, and deposition of collagen. Importantly, C9-BA-DANA was therapeutic in the chronic bleomycin exposure model with no toxic effects observed within the experimental timeframe. Moreover, in the acute bleomycin model, C9-BA-DANA attenuated NEU1-mediated desialylation and shedding of the mucin-1 ectodomain. These data indicate that NEU1-selective inhibition offers a potential therapeutic intervention for pulmonary fibrotic diseases. SIGNIFICANCE STATEMENT: Neuraminidase-1-selective therapeutic targeting in the acute and chronic bleomycin models of pulmonary fibrosis reverses pulmonary collagen deposition, accumulation of lymphocytes in the lungs, and the disease-associated loss of body weight-all without observable toxic effects. Such therapy is as efficacious as nonspecific inhibition of all neuraminidases in these models, thus indicating the central role of neuraminidase-1 as well as offering a potential innovative, specifically targeted, and safe approach to treating human patients with a severe malady: pulmonary fibrosis.

Lipoteichoic acid anchor triggers Mincle to drive protective immunity against invasive group A Streptococcus infection.

Group A Streptococcus (GAS) is a Gram-positive bacterial pathogen that causes a range of diseases, including fatal invasive infections. However, the mechanisms by which the innate immune system recognizes GAS are not well understood. We herein report that the C-type lectin receptor macrophage inducible C-type lectin (Mincle) recognizes GAS and initiates antibacterial immunity. Gene expression analysis of myeloid cells upon GAS stimulation revealed the contribution of the caspase recruitment domain-containing protein 9 (CARD9) pathway to the antibacterial responses. Among receptors signaling through CARD9, Mincle induced the production of inflammatory cytokines, inducible nitric oxide synthase, and reactive oxygen species upon recognition of the anchor of lipoteichoic acid, monoglucosyldiacylglycerol (MGDG), produced by GAS. Upon GAS infection, Mincle-deficient mice exhibited impaired production of proinflammatory cytokines, severe bacteremia, and rapid lethality. GAS also possesses another Mincle ligand, diglucosyldiacylglycerol; however, this glycolipid interfered with MGDG-induced activation. These results indicate that Mincle plays a central role in protective immunity against acute GAS infection.

Neuraminidase 1-mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection.

Pseudomonas aeruginosa (Pa) expresses an adhesin, flagellin, that engages the mucin 1 (MUC1) ectodomain (ED) expressed on airway epithelia, increasing association of MUC1-ED with neuraminidase 1 (NEU1) and MUC1-ED desialylation. The MUC1-ED desialylation unmasks both cryptic binding sites for Pa and a protease recognition site, permitting its proteolytic release as a hyperadhesive decoy receptor for Pa. We found here that intranasal administration of Pa strain K (PAK) to BALB/c mice increases MUC1-ED shedding into the bronchoalveolar compartment. MUC1-ED levels increased as early as 12 h, peaked at 24-48 h with a 7.8-fold increase, and decreased by 72 h. The a-type flagellin-expressing PAK strain and the b-type flagellin-expressing PAO1 strain stimulated comparable levels of MUC1-ED shedding. A flagellin-deficient PAK mutant provoked dramatically reduced MUC1-ED shedding compared with the WT strain, and purified flagellin recapitulated the WT effect. In lung tissues, Pa increased association of NEU1 and protective protein/cathepsin A with MUC1-ED in reciprocal co-immunoprecipitation assays and stimulated MUC1-ED desialylation. NEU1-selective sialidase inhibition protected against Pa-induced MUC1-ED desialylation and shedding. In Pa-challenged mice, MUC1-ED-enriched bronchoalveolar lavage fluid (BALF) inhibited flagellin binding and Pa adhesion to human airway epithelia by up to 44% and flagellin-driven motility by >30%. Finally, Pa co-administration with recombinant human MUC1-ED dramatically diminished lung and BALF bacterial burden, proinflammatory cytokine levels, and pulmonary leukostasis and increased 5-day survival from 0% to 75%. We conclude that Pa flagellin provokes NEU1-mediated airway shedding of MUC1-ED, which functions as a decoy receptor protecting against lethal Pa lung infection.

Stereoselective Synthesis of Diglycosyl Diacylglycerols with Glycosyl Donors Bearing a ß-Stereodirecting 2,3-Naphthalenedimethyl Protecting Group.

Diglycosyl diacylglycerols (DGDGs) are major components of Gram-positive bacterial plasma membranes and are involved in the immune response systems. The chemical synthesis of DGDGs has been highly demanded, as it will allow the elucidation of their biological functions at the molecular level. In this study, we have developed a novel ß-stereodirecting 2,3-naphthalenedimethyl (NapDM) protecting group that is orthogonal to protecting groups commonly used in oligosaccharide synthesis. The NapDM group can be easily cleaved under TFA-mediated acidic conditions. Futhermore, we demonstrated the application of this protecting group to an acyl protecting-group-free strategy by utilizing the NapDM group for the synthesis of DGDGs. This strategy features the use of the ß-stereodirecting NapDM group as an acid-cleavable permanent protecting group and late-stage glycosylation of monoglycosyl diacylglycerol acceptors, enabling the stereoselective synthesis of three different bacterial DGDGs with unsaturated fatty acid chain(s).

Development of Fluorescent Ganglioside GD3 and GQ1b Analogs for Elucidation of Raft-Associated Interactions.

b-Series gangliosides are abundant in central nervous tissues and are involved in important nerve processes. However, their functions are complicated because of their properties of forming dynamic domains in cell plasma membranes (PMs), called lipid rafts. In this study, we aim to develop fluorescently labeled b-series gangliosides that are useful for single-molecule imaging. The chemical synthesis of fluorescent GD3 and GQ1b was achieved using sialylation and ganglioside synthetic methods previously developed by our group. Furthermore, biophysical evaluations demonstrated that synthesized fluorescent GD3 and GQ1b behaved as raft molecules on cell PMs, suggesting their applicability to the study of raft-associated interactions.

Indirect synthetic route to α-l-fucosides via highly stereoselective construction of α-l-galactosides followed by C6-deoxygenation.

We developed an indirect synthetic method for α-l-fucosides. Based on the fact that l-fucose is 6-deoxy-l-galactose, our strategy consists of the stereoselective construction of α-l-galactoside and its conversion to α-l-fucoside via C6-deoxygenation. The formation of α-l-galactoside is strongly directed using 4,6-O-di-tert-butylsilylene(DTBS)-protected l-galactosyl donors. The DTBS-directed α-l-galactosylation showed broad substrate applicability along with excellent coupling yield and α-selectivity. In the C6-deoxygenation of α-l-galactosides, the Barton-McCombie reaction facilitated the conversion to l-fucosides with good yield. To demonstrate the applicability of our method, we synthesized naturally occurring α-l-fucosides.

Efficient diversification of GM3 gangliosides via late-stage sialylation and dynamic glycan structural studies with 19F solid-state NMR.

Sialic acid-containing glycoconjugates are involved in important biological processes such as immune response, cancer metastasis, and viral infection. However, their chemical syntheses have been challenging, mainly due to the difficulties in the α-sialylation of oligosaccharides. Very recently, we established a completely stereoselective sialidation method using a macrobicyclic sialyl donor. Herein, we describe a rational and efficient synthesis of sialoglycolipids via direct sialylation of a glycolipid at a late-stage, based on our novel sialidation method. The synthetic method enabled the development of GM3 ganglioside analogs with various C5-modifications of the sialosyl moiety. Furthermore, the synthesized analog was subjected to solid-state 19F NMR analysis on the model membranes and it revealed the influence of cholesterol on glycan dynamics.

Synthesis of the Core Oligosaccharides of Lipooligosaccharides from Campylobacter jejuni: A Putative Cause of Guillain-Barré Syndrome.

The chemical synthesis of the highly branched core oligosaccharides of lipooligosaccharides (LOSs) found in Campylobacter jejuni, which causes Guillain-Barré syndrome by a preceding infection, is described. The target LOS mimics, consisting of eight or nine monosaccharides, were classified into three groups as key building blocks: ganglioside-core tetra-/pentasaccharides (GM1-/GD1a-like), l-glycero-d-manno-heptose-containing trisaccharides, and 3-deoxy-d-manno-2-octulosonic acid (KDO) residues. These synthetic fragments were obtained from commercially available monosaccharides. Less obtainable l-glycero-d-manno-heptose and KDO residues, as key components of the LOSs, were synthesized from p-methoxyphenyl d-mannoside and di-O-isopropylidene-protected d-mannose, respectively. The synthesis of α-KDO glycoside, as one of the most difficult stereocontrolled glycosidic constructions, was achieved by treating a 2,3-ene derivative of KDO with phenylselenyl trifluoromethanesulfonate as a suitable α-directing reagent. All synthetic blocks were constructed through a convergent synthetic route, which resulted in the first synthesis of structurally challenging LOS core glycans containing ganglioside GM1 and GD1a-core sequences.

Development of Fluorescently Labeled SSEA-3, SSEA-4, and Globo-H Glycosphingolipids for Elucidating Molecular Interactions in the Cell Membrane.

Glycosphingolipids (GSLs), such as the globo-series GSLs stage-specific embryonic antigen 3 (SSEA-3), SSEA-4, and Globo-H, are specifically expressed on pluripotent stem cells and cancer cells, and are known to be associated with various biological processes such as cell recognition, cell adhesion, and signal transduction. However, the behavior and biological roles of these GSLs are still unclear. In our previous study, we observed the interactions between the lipid raft and GSLs in real-time using single-molecule imaging, where we successfully synthesized various fluorescent analogs of GSLs (e.g., GM1 and GM3). Here, we have developed fluorescent analogs of SSEA-3, SSEA-4, and Globo-H using chemical synthesis. The biophysical properties of these analogs as raft markers were examined by partitioning giant plasma membrane vesicles from RBL-2H3 cells into detergent-resistant membrane fractions and liquid-ordered/liquid-disordered phases. The results indicated that the analogs were equivalent to native-type GSLs. The analogs could be used to observe the behavior of globo-series GSLs for detailing the structure and biological roles of lipid rafts and GSL-enriched nanodomains during cell differentiation and cell malignancy.