Comprehensive characterization of complex glycosphingolipids in human pancreatic cancer tissues.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related deaths worldwide, accounting for 90% of primary pancreatic tumors with an average 5-year survival rate of less than 10%. PDAC exhibits aggressive biology, which, together with late detection, results in most PDAC patients presenting with unresectable, locally advanced, or metastatic disease. In-depth lipid profiling and screening of potential biomarkers currently appear to be a promising approach for early detection of PDAC or other cancers. Here, we isolated and characterized complex glycosphingolipids (GSL) from normal and tumor pancreatic tissues of patients with PDAC using a combination of TLC, chemical staining, carbohydrate-recognized ligand-binding assay, and LC/ESI-MS2. The major neutral GSL identified were GSL with the terminal blood groups A, B, H, Lea, Leb, Lex, Ley, P1, and PX2 determinants together with globo- (Gb3 and Gb4) and neolacto-series GSL (nLc4 and nLc6). We also revealed that the neutral GSL profiles and their relative amounts differ between normal and tumor tissues. Additionally, the normal and tumor pancreatic tissues differ in type 1/2 core chains. Sulfatides and GM3 gangliosides were the predominant acidic GSL along with the minor sialyl-nLc4/nLc6 and sialyl-Lea/Lex. The comprehensive analysis of GSL in human PDAC tissues extends the GSL coverage and provides an important platform for further studies of GSL alterations; therefore, it could contribute to the development of new biomarkers and therapeutic approaches.

Sphingomonas cannabina sp. nov., isolated from Cannabis sativa L. 'Cheungsam'.

A Gram-negative, aerobic, rod-shaped bacterium, designated DM2-R-LB4T was isolated from Cannabis sativa L. 'Cheungsam' in Andong, Republic of Korea. The strain DM2-R-LB4T grew at temperatures of 15-45 °C (optimum, 30-37 °C), pH of 5.5-9 (optimum, 8.0), and 0-2 % (w/v) NaCl concentration (optimum, 0%). Phylogenetic analyses based on the 16S rRNA gene sequences revealed that strain DM2-R-LB4T is related to species of the genus Sphingomonas, and shared 97.8 and 97.5% similarity to Sphingomonas kyenggiensis KCTC 42244T and Sphingomonas leidyi DSM 4733T, respectively. The DNA G+C content was 67.9 mol% and genome analysis of the strain DM2-R-LB4T revealed that the genome size was 4 386 171 bp and contained 4 009 predicted protein-coding genes. The average nucleotide identity (ANI) values between strain DM2-R-LB4T and S. kyenggiensis KCTC 42244T, and S. leidyi DSM 4733T was 76.8 and 76.7 %, respectively, while the values of digital DNA-DNA hybridization (dDDH) were 20.7 and 20.6 %, respectively. C14 : 0 2-OH, C16 : 0, and summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c) were the major fatty acids (>10 %) in the strain DM2-R-LB4T. The polar lipids comprised diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), sphingoglycolipid (SGL), glycolipid (GL), phospholipid (PL), and two unidentified polar lipids (L1 and L2). Ubiquinone-10 (Q-10) was the only respiratory quinone. The polyamine pattern was found to contain homospermidine, putrescine, and spermidine. The results of phylogenetic anlayses, polyphasic studies, revealed that strain DM2-R-LB4T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas cannabina sp. nov., is proposed. The type strain is DM2-R-LB4T (=KCTC 92075T = GDMCC 1.3018T).

Glycosphingolipid Changes in Plasma in Parkinson's Disease Independent of Glucosylceramide Levels.

BACKGROUND: Alteration in glycosphingolipids (GSLs) in Parkinson's disease (PD) still needs to be determined. OBJECTIVES: We evaluated if PD subjects show abnormal GSLs levels compared to healthy controls (HC) and if GSLs correlate with clinical features. METHODS: We analyzed GSLs and glucosylceramide (GlcCer) in plasma using two normal-phase high-performance liquid chromatography assays; clinico-demographic data were extracted. RESULTS: Eighty PD subjects and 25 HCs were analyzed. Levels of GlcCer, GD1b, Gb4, GalNAcGA1, and b-series were higher in PD patients than in HCs; total GSLs, GT1b, GM1a, GM3, GM2, and a-series levels were lower in PD patients than in HCs. Changes in GSLs were present in PD subjects, with GlcCer levels similar to those in HCs. The results were similar after excluding certain GBA1 mutation carriers. Movement Disorder Society Unified Parkinson's Disease Rating Scale, Part III, correlated with Gb4 and Montreal Cognitive Assessment with GD1b levels. CONCLUSIONS: Multiple GSL abnormalities in plasma were detected in patients with and without GlcCer changes, indicating a broader shift in lipid homeostasis. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

Enterohemorrhagic Escherichia coli and a Fresh View on Shiga Toxin-Binding Glycosphingolipids of Primary Human Kidney and Colon Epithelial Cells and Their Toxin Susceptibility.

Enterohemorrhagic Escherichia coli (EHEC) are the human pathogenic subset of Shiga toxin (Stx)-producing E. coli (STEC). EHEC are responsible for severe colon infections associated with life-threatening extraintestinal complications such as the hemolytic-uremic syndrome (HUS) and neurological disturbances. Endothelial cells in various human organs are renowned targets of Stx, whereas the role of epithelial cells of colon and kidneys in the infection process has been and is still a matter of debate. This review shortly addresses the clinical impact of EHEC infections, novel aspects of vesicular package of Stx in the intestine and the blood stream as well as Stx-mediated extraintestinal complications and therapeutic options. Here follows a compilation of the Stx-binding glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) and their various lipoforms present in primary human kidney and colon epithelial cells and their distribution in lipid raft-analog membrane preparations. The last issues are the high and extremely low susceptibility of primary renal and colonic epithelial cells, respectively, suggesting a large resilience of the intestinal epithelium against the human-pathogenic Stx1a- and Stx2a-subtypes due to the low content of the high-affinity Stx-receptor Gb3Cer in colon epithelial cells. The review closes with a brief outlook on future challenges of Stx research.

High Diversity of Glycosphingolipid Glycans of Colorectal Cancer Cell Lines Reflects the Cellular Differentiation Phenotype.

Colorectal cancer (CRC)-associated changes of protein glycosylation have been widely studied. In contrast, the expression of glycosphingolipid (GSL) patterns in CRC has, hitherto, remained largely unexplored. Even though GSLs are major carriers of cell surface carbohydrates, they are understudied due to their complexity and analytical challenges. In this study, we provide an in-depth analysis of GSL glycans of 22 CRC cell lines using porous graphitized carbon nano-liquid chromatography coupled with electrospray ionization-mass spectrometry. Our data revealed that the GSL expression varies among different cell line classifications, with undifferentiated cell lines showing high expression of blood group A, B, and H antigens while for colon-like cell lines the most prominent GSL glycans contained (sialyl)-LewisA/X and LewisB/Y antigens. Moreover, the GSL expression correlated with relevant glycosyltransferases that are involved in their biosynthesis as well as with transcription factors (TFs) implicated in colon differentiation. Additionally, correlations between certain glycosyltransferases and TFs at mRNA expression level were found, such as FUT3, which correlated with CDX1, ETS2, HNF1A, HNF4A, MECOM, and MYB. These TFs are upregulated in colon-like cell lines pointing to their potential role in regulating fucosylation during colon differentiation. In conclusion, our study reveals novel layers of potential GSL glycans regulation relevant for future research in colon differentiation and CRC.

Carbohydrate-carbohydrate interaction drives the preferential insertion of dirhamnolipid into glycosphingolipid enriched membranes.

Rhamnolipids (RLs) are among the most important biosurfactants produced by microorganisms, and have been widely investigated because of their multiple biological activities. Their action appears to depend on their structural interference with lipid membranes, therefore several studies have been performed to investigate this aspect. We studied by X-ray scattering, neutron reflectometry and molecular dynamic simulations the insertion of dirhamnolipid (diRL), the most abundant RL, in model cellular membranes made of phospholipids and glycosphingolipids. In our model systems the affinity of diRL to the membrane is highly promoted by the presence of the glycosphingolipids and molecular dynamics simulations unveil that this evidence is related to sugar-sugar attractive interactions at the membrane surface. Our results improve the understanding of the plethora of activities associated with RLs, also opening new perspectives in their selective use for pharmaceutical and cosmetics formulations. Additionally, they shed light on the still debated role of carbohydrate-carbohydrate interactions as driving force for molecular contacts at membrane surface.

Characterization of Human Medullary Thyroid Carcinoma Glycosphingolipids Identifies Potential Cancer Markers.

Medullary thyroid carcinoma (MTC) accounts for only 1-2% of thyroid cancers; however, metastatic MTC is a mortal disease with no cure. In this study, glycosphingolipids were isolated from human MTCs and characterized by mass spectrometry and binding of carbohydrate recognizing ligands. The tissue distribution of selected compounds was investigated by immunohistochemistry. The amount of acid glycosphingolipids in the MTCs was higher than in the normal thyroid glands. The major acid glycosphingolipid was the GD3 ganglioside. Sulfatide and the gangliosides GM3 and GD1a were also present. The majority of the complex non-acid glycosphingolipids had type 2 (Galß4GlcNAc) core chains, i.e., the neolactotetraosylceramide, the Lex, H type 2 and x2 pentaosylceramides, the Ley and A type 2 hexaosylceramides, and the A type 2 heptaosylceramide. There were also compounds with globo (GalαGalß4Glc) core, i.e., globotriaosylceramide, globotetraosylceramide, the Forssman pentaosylceramide, and the Globo H hexaosylceramide. Immunohistochemistry demonstrated an extensive expression av Ley in the MTC cells and also a variable intensity and prevalence of Globo H and Lex. One individual with multiple endocrine neoplasia type 2B expressed the Forssman determinant, which is rarely found in humans. This study of human MTC glycosphingolipids identifies glycans that could serve as potential tumor-specific markers.

Profiling of glycosphingolipids with SCDase digestion and HPLC-FLD-MS.

Lipid components of cells and tissues feature a large diversity of structures that present a challenging problem for molecular analysis. Glycolipids from mammalian cells contain glycosphingolipids (GSLs) as their major glycolipid component, and these structures vary in the identity of the glycan headgroup as well as the structure of the fatty acid and sphingosine (Sph) tails. Analysis of intact GSLs is challenging due to the low abundance of these species. Here, we develop a new strategy for the analysis of lyso-GSL (l-GSL), GSL that retain linkage of the glycan headgroup with the Sph base. The analysis begins with digestion of a GSL sample with sphingolipid ceramide N-deacylase (SCDase), followed by labelling with an amine-reactive fluorophore. The sample was then analyzed by HPLC-FLD-MS and quantitated by addition of an external standard. This method was compared to analysis of GSL glycans after cleavage by an Endoglycoceramidase (EGCase) enzyme and labeling with a fluorophore (2-anthranilic acid, 2AA). The two methods are complementary, with EGCase providing improved signal (due to fewer species) and SCDase providing analysis of lyso-GSL. Importantly the SCDase method provides Sph composition of GSL species. We demonstrate the method on cultured human cells (Jurkat T cells) and tissue homogenate (porcine brain).

Characterization of Glycosphingolipids in the Human Parathyroid and Thyroid Glands.

As part of a systematic investigation of the glycosphingolipids in human tissues, acid and non-acid glycosphingolipids from human thyroid and parathyroid glands were isolated and characterized with mass spectrometry and binding of carbohydrate-recognizing ligands, with a focus on complex compounds. The glycosphingolipid patterns of the human parathyroid and thyroid glands were very similar. The major acid glycosphingolipids were sulfatide and the gangliosides GM3, GD3, GD1a, GD1b, GT1b and Neu5Ac-neolactotetraosylceramide, and the major non-acid glycosphingolipids were globotriaosylceramide and globoside. We also found neolactotetra- and neolactohexaosylceramide, the x2 glycosphingolipid, and complex glycosphingolipids with terminal blood group O and A determinants in both tissues. A glycosphingolipid with blood group Leb determinant was identified in the thyroid gland, and the parathyroid sample had a glycosphingolipid with terminal blood group B determinant. Immunohistochemistry demonstrated the expression of blood group A antigens in both the thyroid and parathyroid glands. A weak cytoplasmatic expression of the GD1a ganglioside was present in the thyroid, while the parathyroid gland had a strong GD1a expression on the cell surface. Thus, the glycosylation of human thyroid and parathyroid glands is more complex than previously appreciated. Our findings provide a platform for further studies of alterations of cell surface glycosphingolipids in thyroid and parathyroid cancers.

Comprehensive analysis of glycosphingolipid glycans by lectin microarrays and MALDI-TOF mass spectrometry.

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates present on the cell membrane; they play significant roles in many bioprocesses such as cell adhesion, embryonic development, signal transduction and carcinogenesis. Analyzing such amphiphilic molecules is a major challenge in the field of glycosphingolipidomics. We provide a step-by-step protocol that uses a lectin microarray to analyze GSL glycans from cultured cells. The procedure describes (i) extraction of GSLs from cell pellets, (ii) N-monodeacylation using sphingolipid ceramide N-deacylase digestion to form lyso-GSLs, (iii) fluorescence labeling at the newly exposed amine group, (iv) preparation of a lectin microarray, (v) GSL-glycan analysis by a lectin microarray, (vi) complementary mass spectrometry analysis and (vii) data acquisition and analysis. This method is high-throughput, low cost and easy to conduct, and it provides detailed information about glycan linkages. This protocol takes ~10 d.

Protocol for measuring sphingolipid metabolism in budding yeast.

Sphingolipid biosynthesis occurs in both the endoplasmic reticulum (ER) and the Golgi apparatus. Ceramide synthesized in the ER is transported to the Golgi and incorporated into complex sphingolipids. Here, we present a step-by-step protocol to analyze sphingolipid metabolism in budding yeast. Ceramide and inositolphosphorylceramide (IPC) are classes of sphingolipids present in yeast and are metabolically labeled with radioactive precursors. This protocol for metabolic labeling can be used to investigate ceramide transport in an in vivo environment. For complete details on the use and execution of this protocol, please refer to Ikeda et al. (2020).

Qualitative and Quantitative Study of Glycosphingolipids in Human Milk and Bovine Milk Using High Performance Liquid Chromatography-Data-Dependent Acquisition-Mass Spectrometry.

Cerebrosides (Crb; including glucosylceramide and galactosylceramide) and lactosylceramide (LacCer) are structurally complex lipids found in many eukaryotic cell membranes, where they play important roles in cell growth, apoptosis, cell recognition and signaling. They are also found in mammalian milk as part of the milk fat globule membrane (MFGM), making milk an important dietary component for the rapidly growing infant. This study reports the development of a robust analytical method for the identification and characterization of 44 Crb and 23 LacCer molecular species in milk, using high performance liquid chromatography-tandem mass spectrometry in data-dependent acquisition mode. For the first time, it also compares the distributions of these species in human and bovine milks, a commercial MFGM-enriched dairy ingredient (MFGM Lipid 100) and commercial standards purified from bovine milk. A method for quantifying Crb and LacCer in milk using mass spectrometry in neutral loss scan mode was developed and validated for human milk, bovine milk and MFGM Lipid 100. Human milk was found to contain approximately 9.9-17.4 µg Crb/mL and 1.3-3.0 µg LacCer/mL, whereas bovine milk (pooled milk from a Friesian herd) contained 9.8-12.0 and 14.3-16.2 µg/mL of these lipids, respectively. The process used to produce MFGM Lipid 100 was shown to have enriched these components to 448 and 1036 µg/g, respectively. No significant changes in the concentrations of both Crb and LacCer were observed during lactation.

Differentiation and Quantification of Diastereomeric Pairs of Glycosphingolipids Using Gas-Phase Ion Chemistry.

Glycosphingolipids (GSLs), including lyso-glycosphingolipids (lyso-GSLs) and cerebrosides (HexCer), constitute a sphingolipid subclass. The diastereomerism between their monosaccharide head groups, glucose and galactose in mammalian cells, gives rise to an analytical challenge in the differentiation of their biological roles in healthy and disease states. Shotgun tandem mass spectrometry has been demonstrated to be a powerful tool in lipidomics analysis in which the differentiation of the diastereomeric pairs of GSLs could be achieved with offline chemical modifications. However, the limited number of standards, as well as the lack of the comprehensive coverage of the GSLs, complicates the qualitative and quantitative analysis of GSLs. In this work, we describe a novel strategy that couples shotgun tandem mass spectrometry with gas-phase ion chemistry to achieve both differentiation and quantification of the diastereomeric pairs of GSLs. In brief, deprotonated GSL anions, [GSL-H]-, and terpyridine-magnesium complex dications, [Mg(Terpy)2]2+, are sequentially injected and mutually stored in a linear ion trap to form charge-inverted complex cations, [GSL-H + MgTerpy]+. The collision-induced dissociation of the charge-inverted complex cations leads to significant spectral differences between the diastereomeric pairs of GSLs, which permits their distinction. Moreover, we describe a relative quantification strategy with the normalized %Area extracted from selected diagnostic ions in binary mixtures. Analytical performance with the selected pure-component pairs, lyso-GSLs and HexCer(d18:1/18:0), was also evaluated in terms of accuracy, repeatability, and interday precision. The pure components could be extended to different fatty acyl chains on cerebrosides with a limited error, which allows for the relative quantitation of the diastereomeric pairs without all standards. We successfully applied the presented method to identify and quantify, on a relative basis, the GSLs in commercially available total cerebroside extracts from the porcine brain.

The binding mechanism of the virulence factor Streptococcus suis adhesin P subtype to globotetraosylceramide is associated with systemic disease.

Streptococcus suis is part of the pig commensal microbiome but strains can also be pathogenic, causing pneumonia and meningitis in pigs as well as zoonotic meningitis. According to genomic analysis, S. suis is divided into asymptomatic carriage, respiratory and systemic strains with distinct genomic signatures. Because the strategies to target pathogenic S. suis are limited, new therapeutic approaches are needed. The virulence factor S. suis adhesin P (SadP) recognizes the galabiose Galα1-4Gal-oligosaccharide. Based on its oligosaccharide fine specificity, SadP can be divided into subtypes PN and PO We show here that subtype PN is distributed in the systemic strains causing meningitis, whereas type PO is found in asymptomatic carriage and respiratory strains. Both types of SadP are shown to predominantly bind to pig lung globotriaosylceramide (Gb3). However, SadP adhesin from systemic subtype PN strains also binds to globotetraosylceramide (Gb4). Mutagenesis studies of the galabiose-binding domain of type PN SadP adhesin showed that the amino acid asparagine 285, which is replaced by an aspartate residue in type PO SadP, was required for binding to Gb4 and, strikingly, was also required for interaction with the glycomimetic inhibitor phenylurea-galabiose. Molecular dynamics simulations provided insight into the role of Asn-285 for Gb4 and phenylurea-galabiose binding, suggesting additional hydrogen bonding to terminal GalNAc of Gb4 and the urea group. Thus, the Asn-285-mediated molecular mechanism of type PN SadP binding to Gb4 could be used to selectively target S. suis in systemic disease without interfering with commensal strains, opening up new avenues for interventional strategies against this pathogen.

Isolation of glycosylinositol phosphoceramide and phytoceramide 1-phosphate in plants and their chemical stabilities.

Glycosylinositol phosphoceramide (GIPC) is a sphingophospholipid in plants. Recently, we identified that GIPC is hydrolyzed to phytoceramide 1-phosphate (PC1P) by an uncharacterized phospholipase D activity following homogenization of certain plant tissues. We now developed methods for isolation of GIPC and PC1P from plant tissues and characterized their chemical stabilities. Hydrophilic solvents, namely a lower layer of a mixed solvent system consisting of isopropanol/hexane/water (55:20:25, v/v/v) was efficient solvent for extraction and eluent in column chromatography. GIPC was isolated by Sephadex column chromatography followed by TLC. A conventional method, such as the Bligh and Dyer method, was applicable for PC1P extraction. Specifically, PC1P was isolated by TLC following mild alkali treatment of lipid extracts of plants. The yields of GIPC and PC1P in our methods were both around 50-70%. We found that PC1P is tolerant against heat (up to 125 °C), strong acid (up to 10 M HCl), and mild alkali (0.1 M KOH). In contrast, significant degradation of GIPC occurred at 100 °C and 1.0 M HCl treatment, suggesting the instability of the inositol glycan moiety in these conditions. These data will be useful for further biochemical and nutritional studies on these sphingolipids.

MALDI-2 Mass Spectrometry and Immunohistochemistry Imaging of Gb3Cer, Gb4Cer, and Further Glycosphingolipids in Human Colorectal Cancer Tissue.

The main cellular receptors of Shiga toxins (Stxs), the neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer/CD77) and globotetraosylceramide (Gb4Cer), are significantly upregulated in about half of the human colorectal carcinomas (CRC) and in other cancers. Therefore, conjugates exploiting the Gb3Cer/Gb4Cer-binding B subunit of Stx (StxB) have attracted great interest for both diagnostic and adjuvant therapeutic interventions. Moreover, fucosylated GSLs were recognized as potential tumor-associated targets. One obstacle to a broader use of these receptor/ligand systems is that the contribution of specific GSLs to tumorigenesis, in particular, in the context of an altered lipid metabolism, is only poorly understood. A second is that also nondiseased organs (e.g., kidney) and blood vessels can express high levels of certain GSLs, not least Gb3Cer/Gb4Cer. Here, we used, in a proof-of-concept study, matrix-assisted laser desorption/ionization mass spectrometry imaging combined with laser-induced postionization (MALDI-2-MSI) to simultaneously visualize the distribution of several Gb3Cer/Gb4Cer lipoforms and those of related GSLs (e.g., Gb3Cer/Gb4Cer precursors and fucosylated GSLs) in tissue biopsies from three CRC patients. Using MALDI-2 and StxB-based immunofluorescence microscopy, Gb3Cer and Gb4Cer were mainly found in dedifferentiated tumor cell areas, tumor stroma, and tumor-infiltrating blood vessels. Notably, fucosylated GSL such as Fuc-(n)Lc4Cer generally showed a highly localized expression in dysplastic glands and indian file-like cells infiltrating adipose tissue. Our "molecular histology" approach could support stratifying patients for intratumoral GSL expression to identify an optimal therapeutic strategy. The improved chemical coverage by MALDI-2 can also help to improve our understanding of the molecular basis of tumor development and GSL metabolism.

The occurrence of inositolphosphoceramides in spirulina microalgae.

Spirulina microalga (Arthrospira platensis) is an interesting phototrophic organism because of its high content of nutrients including proteins, lipids, essential amino acids, antioxidants, vitamins, polysaccharides, and minerals. Hydrophilic interaction liquid chromatography (HILIC) coupled to linear ion trap (LIT) and Orbitrap Fourier transform mass spectrometry (FTMS) via ESI was employed for the separation and characterization of lipid species in A. platensis. Inositolphosphoceramides (IPC) are minor but important constituents of spirulina; their investigation was accomplished by HILIC-ESI-MS including collision-induced dissociation (MS2 , MS3 ) of deprotonated molecules in the LIT analyzer and a schematic fragmentation pattern is described. All four commercial spirulina samples revealed the occurrence of the same IPC species at m/z 796.6 (d18:0/16:0;1), 810.6 (d18:0/17:0;1), 824.6 (d18:0/18:0;1), and 826.6 (d18:0/17:0;2) but in diverse relative abundance. This study sets the stage for future investigations on IPC in other algae and microalgae.

Characterization of sheep erythrocyte glycosphingolipids recognized by human anti-Forssman antibodies.

The FORS histo-blood group system is the most recently discovered carbohydrate-based human blood group system. FORS is a rare blood group system, and most individuals have naturally occurring anti-FORS1 antibodies in plasma. Screening for anti-FORS1 antibodies is often done by hemagglutination assays using FORS1-expressing sheep erythrocytes, since FORS1-positive human erythrocytes are most often not available. Here, we have characterized the non-acid glycosphingolipids from sheep erythrocytes and isolated subfractions, with mass spectrometry, binding of antibodies and lectins, and by enzymatic hydrolysis. This demonstrated the presence of Forssman and Galili pentaosylceramides, and a Galili heptaosylceramide. Two complex glycosphingolipids recognized by human anti-FORS1 antibodies were characterized as a Forssman neolacto hybrid hexaosylceramide (GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer) and a Forssman Galili hybrid heptaosylceramide (GalNAcα3GalNAcß3Galα3Galß4GlcNAcß3Galß4Glcß1Cer). These are novel glycosphingolipid structures, and to our knowledge, the first case of an elongated Galili antigen. Thus, the anti-Forssman antibodies in human serum bind not only to the classical Forssman pentaosylceramide (GalNAcα3GalNAcß3Galα4Galß4Glcß1Cer), but also when the GalNAcα3GalNAcß3 sequence is presented on a neolacto core chain and even on a Galili carbohydrate sequence.

Clinical course and pathological findings of two late-onset Fabry hemizygous patients including mulberry cell counts after enzyme replacement therapy.

Fabry disease is an X-linked inherited lysosomal storage disorder caused by a deficiency of α-galactosidase A activity, resulting in the intracellular accumulation of globotriaosylceramide and related glycosphingolipids. The phenotypes of Fabry disease in both males and females are grouped into two categories: the classical type and the late-onset type. The classical type shows general symptoms including angiokeratoma(s), acroparesthesia, hypohidrosis, corneal opacity, and gastrointestinal symptoms from an early age. The late-onset type shows cardiac or renal (or both) symptoms from a late age. We present herein the clinical course and pathological findings of two late-onset hemizygous Fabry patients after the initiation of enzyme replacement therapy (ERT), along with their mulberry cell counts during treatment. One patient's case was a renal-variant type without general symptoms; he showed stable renal function and mild proteinuria but little histological improvement with no change in the mulberry cell count during ERT. The other patient had a cardiac-variant type with renal pathological abnormality. He achieved a mild improvement of renal pathological findings, and his mulberry cell count gradually decreased during the treatment. These findings indicate that monitoring the mulberry cell count might help assess the efficacy of ERT, as a renal pathology tool.

Mass spectrometry in combination with a chiral column and multichannel-MRM allows comprehensive analysis of glycosphingolipid molecular species from mouse brain.

Glycosphingolipids (GSLs) exist exclusively in the outer leaflet of plasma membrane in mammalian cells and have diverse structures including different classes of sugars and various molecular species of ceramide moieties. Establishing methods that measure each molecular species in GSL classes should aid functional characterization of GSLs and reveal details about the mechanism of pathogenesis in glycosphingolipidoses. Using an IF-3 chiral column that has never been used for lipid analyses, we developed a liquid chromatography-mass spectrometry (LC-MS) method to separate various GSLs based on sugar and ceramide moieties. To examine GSLs in detail a multichannel-multiple reaction monitoring (multichannel-MRM) mode was used and covered a range of 500-2000 Da. Common fragment ions detected with higher collision energy in the positive ion mode were m/z 264 and 292, and are derived from d18:1 and d20:1 ions, respectively. Both species were used as product ions in the multichannel-MRM for the simultaneous measurement of neutral GSLs, gangliosides and sulfatides. Comprehensive analysis of GSLs in mouse brain using this method revealed that for gangliosides and LacCer, d18:1-C18:0 and d20:1-C18:0 were the major molecular species, whereas d18:1-C24:0 and d18:1-C24:1 were the major molecular species of sulfatides. The results revealed a diverse GSL fatty acid profile. In conclusion, by combining IF-3 chiral column and the multichannel-MRM method various molecular species of GSLs were detected successfully, and a metabolomics approach based on this LC-MS method should facilitate functional analysis of GSLs and the discovery of early biomarkers of glycosphingolipidoses at the molecular level.