Neutral Glycosphingolipids Research Articles

Characterization and Modeling of the Collision Induced Dissociation Patterns of Deprotonated Glycosphingolipids: Cleavage of the Glycosidic Bond.

Glycosphingolipid fragmentation behavior was investigated by combining results from analysis of a series of negative ion tandem mass spectra and molecular modeling. Fragmentation patterns extracted from 75 tandem mass spectra of mainly acidic glycosphingolipid species (gangliosides) suggest prominent cleavage of the glycosidic bonds with retention of the glycosidic oxygen atom by the species formed from the reducing end (B and Y ion formation). Dominant product ions arise from dissociation of sialic acids glycosidic bonds whereas product ions resulting from cleavage of other glycosidic bonds are less abundant. Potential energy surfaces and unimolecular reaction rates of several low-energy fragmentation pathways leading to cleavage of glycosidic bonds were estimated in order to explain observed dissociation patterns. Glycosidic bond cleavage in both neutral (unsubstituted glycosyl group) and acidic glycosphingolipids was the outcome of the charge-directed intramolecular nucleophilic substitution (SN2) mechanism. According to the suggested mechanism, the nucleophile in a form of carboxylate or oxyanion attacks the carbon at position one of the sugar ring, simultaneously breaking the glycosidic bond and yielding an epoxide. For gangliosides, unimolecular reaction rates suggest that dominant product ions related to the cleavage of sialic acid glycosidic bonds are formed via direct dissociation channels. On the other hand, low abundant product ions related to the dissociation of other glycosidic bonds are more likely to be the result of sequential dissociation. Although results from this study mainly contribute to the understanding of glycosphingolipid fragmentation chemistry, some mechanistic findings regarding cleavage of the glycosidic bond may be applicable to other glycoconjugates.

Structural analysis of glucosylceramides (GlcCer) from species of the Pseudallescheria/Scedosporium complex

Glucosylceramides (GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this work, glucosylceramides (GlcCer) were extracted from three strains of Scedosporium (Pseudallescheria) boydii, one strain of Pseudallescheria ellipsoidea and one strain of Pseudallescheria angusta and purified by several chromatographic steps. Using high-performance thin layer chromatography (HPTLC), we found a similarity between GlcCer obtained from all of the analysed strains. A detailed structural analysis of the P. ellipsoidea GlcCer was performed via electrospray ionization mass spectrometry (ESI-MS) and confirmed in 1- and 2-D heteronuclear NMR experiments (1H-13 C HSQC). GlcCer species produced by mycelial forms of these strains displayed the same structure previously demonstrated by our group for P. boydii, Cryptococcus neoformans, Pseudallescheria minustipora, Fusarium solani, and Colletotrichum gloesporioides. A monoclonal antibody (mAb) against GlcCer was used for immunofluorescence experiments. Our results revealed that GlcCer is present on the surface of these fungi, and no difference was observed in the GlcCer structure of the present set of strains in terms of geographic or clinical origin, suggesting a conserved GlcCer structure similar to those previously described for Scedosporium apiospermum, Scedosporium aurantiacum, and P. minutispora. The surface distribution of GlcCer in these fungi is suggestive of the involvement of this molecule in fungal growth.

Structural Characterization of Neutral Glycosphingolipids from 3T3-L1 Adipocytes.

In recent years, obesity has been considered a pathological stage of early lifestyle-related diseases, and adipose tissue and adipocyte research has been active. Glycosphingolipids are involved in the pathogenesis of type 2 diabetes induced by insulin resistance, but the details of the glycosphingolipid molecular species composition of adipocytes have yet to be elucidated. We used 3T3-L1 adipocytes and the 1,2-dichloroethane-wash method to remove triacylglycerols, which are abundant in adipocytes, and analyzed the structures of glycosphingolipids, particularly neutral glycosphingolipids, using liquid chromatography–mass spectrometry.Electronic supplementary materialThe online version of this article (doi:10.1007/s11745-015-4035-7) contains supplementary material, which is available to authorized users.

Fabry disease in a male patient: De novo mutation, germline mosaicism or both?

Background: Fabry disease (FD) is an innate error of glycosphingolipid catabolism due to a genetic defect in the gene which codifies for alphagalactosidase A (α-Gal A) production (GLA gene). Mutations affecting the GLA gene result in deficiency of this lysosomal enzyme, which in turn leads to progressive accumulation of neutral glycosphingolipids, primarily globotriaosylceramide (GL-3), in different cell types throughout the body, including renal glomerular and tubular epithelial cells, myocardial cells and valvular fibrocytes, and nervous system. FD has an X linked inheritance. Currently, more than 600 disease-causing mutations in the GLA gene have been described. Most of these mutations are private, restricted to an individual family. De novo mutations are rare; population reports establish their frequency between 4 and 7%. We present the case of a pediatric male patient with a previously unreported de novomutation, which during family tree analysis, revealed a positive sibling, but nomutation found in the mother. Case presentation: A 17 year old male patient from Durango, Mexico with a negative family history presented at our ward. His initial symptoms began at 10 years of age and were primarily neurological: recurrent and progressive acroparesthesias several times a week, upper and lower extremity weaknesswithmovement limitations and fainting spells; he also complained of intermittent fever, hypohidrosis, abdominal pain and diarrhea. Clinical evaluation of the patient revealed tortuosity of retinal vessels, sinus bradycardia and hypotension and distal hyperesthesia in arms and legs. Fabry disease was suspected; αGal A enzyme activity, GLA gene analysis and lysoGL-3 measurement confirmed the diagnosis. Family tree evaluation was carried out; one six-year-old brother also had the mutation; however no mutation was found in the mother’s molecular analysis. FD specific evaluation of the index case revealed positive 24-hour proteinuria (6.5 mg/kg/h reference b4 ml/kg/h); CNSMRI was normal. Kidney biopsy was conducted; electron microscopy analysis of specimens revealed dense lamellar bodies corresponding to lipid material deposits in all cell groups, confirming the presence of a pathological mutation. Conclusions: De novo onset mutations in FD are rare; however, even more infrequent is the presence of germline mutations, which have been reported only periodically in case reports. Unfortunately, female germline cells are difficult to obtain to confirm this diagnosis. doi:10.1016/j.ymgme.2014.12.069

Spatial distribution of brain ceramides in an acid ceramidase deficient murine model: Subsequent histological manifestations and functional deficits

Background: Fabry disease (FD) is an innate error of glycosphingolipid catabolism due to a genetic defect in the gene which codifies for alphagalactosidase A (α-Gal A) production (GLA gene). Mutations affecting the GLA gene result in deficiency of this lysosomal enzyme, which in turn leads to progressive accumulation of neutral glycosphingolipids, primarily globotriaosylceramide (GL-3), in different cell types throughout the body, including renal glomerular and tubular epithelial cells, myocardial cells and valvular fibrocytes, and nervous system. FD has an X linked inheritance. Currently, more than 600 disease-causing mutations in the GLA gene have been described. Most of these mutations are private, restricted to an individual family. De novo mutations are rare; population reports establish their frequency between 4 and 7%. We present the case of a pediatric male patient with a previously unreported de novomutation, which during family tree analysis, revealed a positive sibling, but nomutation found in the mother. Case presentation: A 17 year old male patient from Durango, Mexico with a negative family history presented at our ward. His initial symptoms began at 10 years of age and were primarily neurological: recurrent and progressive acroparesthesias several times a week, upper and lower extremity weaknesswithmovement limitations and fainting spells; he also complained of intermittent fever, hypohidrosis, abdominal pain and diarrhea. Clinical evaluation of the patient revealed tortuosity of retinal vessels, sinus bradycardia and hypotension and distal hyperesthesia in arms and legs. Fabry disease was suspected; αGal A enzyme activity, GLA gene analysis and lysoGL-3 measurement confirmed the diagnosis. Family tree evaluation was carried out; one six-year-old brother also had the mutation; however no mutation was found in the mother’s molecular analysis. FD specific evaluation of the index case revealed positive 24-hour proteinuria (6.5 mg/kg/h reference b4 ml/kg/h); CNSMRI was normal. Kidney biopsy was conducted; electron microscopy analysis of specimens revealed dense lamellar bodies corresponding to lipid material deposits in all cell groups, confirming the presence of a pathological mutation. Conclusions: De novo onset mutations in FD are rare; however, even more infrequent is the presence of germline mutations, which have been reported only periodically in case reports. Unfortunately, female germline cells are difficult to obtain to confirm this diagnosis. doi:10.1016/j.ymgme.2014.12.069

Globosides as key players in the pathophysiology of Shiga toxin-associated acute kidney failure and Fabry disease

Globosides and their isomeric counterparts isoglobosides belong to the class of neutral glycosphingolipids with an as yet undefined physiological function. In the pathogenesis of human diseases, globosides play an important role as cellular receptors for Shiga toxins which are produced by certain strains of S. dysenteriae and E. coli. In order to elucidate the pathogenesis of Shiga toxin-associated kidney failure, we studied human kidney biopsies and animal models. Our work showed that in patients suffering from Shiga toxin-elicited kidney failure, no complement activation could be demonstrated by immunohistochemical analysis of kidney biopsies. Therefore, complement activation is unlikely to play a major role in mediating thrombotic microangiopathy on exposure to Shiga toxin. Moreover, analysis of the human biopsies and of a murine model of Shiga toxin-associated disease pinpointed acute tubular damage as an important and previously neglected contributor to acute kidney failure in patients infected with Shiga toxin-producing E. coli. Furthermore, globosides play a decisive role in the pathogenesis of Fabry disease which results from a decreased or absent activity of the lysosomal enzyme α-galactosidase A. The results on transgenic mice showed that in vital organs, such as the heart, kidneys and liver, it was possible to revert the phenotype of Fabry disease by eliminating the synthesis of globosides. This implicates that substrate reduction therapy through inhibition of globosides might represent a new therapeutic option for Fabry disease, all the more so as globosides seem to be dispensable.

Structural diversity and biological significance of glycosphingolipids in pathogenic and opportunistic fungi.

Glycosphingolipids (GSLs) are ubiquitous membrane components and have key roles in biological systems, acting as second messengers or modulators of signal transduction by affecting several events, ranging from cell adhesion, cell growth, cell motility, regulation of apoptosis and cell cycle. Over the last 20 years our laboratory and other research groups determined the glycan and ceramide structures of more than 20 GSLs from several pathogenic/opportunistic fungi, using a combination of gas chromatography, mass spectrometry, nuclear magnetic resonance as well as other immunochemical and biochemical techniques. Fungal GSLs can be divided in two major classes: neutral GSLs, galactosyl- and glucosylceramide (GlcCer), and acidic GSLs, the glycosylinositol-phosphorylceramides (GIPCs). Glycosyl structures in fungal GIPCs exhibited significant structural diversity and distinct composition when compared to mammalian GSLs, e.g., the expression of inositol-mannose and inositol-glucosamine cores and the terminal residue of β-D-galactofuranose which are absent in mammalian cells. Studies performed by our group demonstrated that GIPC (Galfβ 6[Manα3]Manα2InsPCer) elicited in patients with paracoccidioidomycosis an immune response with production of antibodies directed to the terminal residue of β-D-galactofuranose. Further studies also showed that inhibition of GlcCer biosynthetic pathways affects fungal colony formation, spore germination and hyphal growth, indicating that enzymes involved in GlcCer biosynthesis may represent promising targets for the therapy of fungal infections. Recently, it was shown that GlcCer and GIPCs are preferentially localized in membrane microdomains and monoclonal antibodies directed to these GSLs interfere in several fungal biological processes such as growth and morphological transition. This review focuses on glycan structures carried on sphingolipids of pathogenic/opportunistic fungi, and aspects of their biological significance are discussed.

Glycolipid sensing and innate immunity in paracoccidioidomycosis.

Distinct glycolipid profiles are described in microorganisms, which have been shown to modulate the innate immune system. We tested the hypothesis that glycosphingolipids from Paracoccidioides brasiliensis have immunomodulatory properties on monocytes and dendritic cells of two groups of healthy individuals, one cured of paracoccidioidomycosis in the past (CUR-I) and the other nonexposed to P. brasiliensis (HNE-I). Two classes of glycosphingolipids purified from yeast cells were evaluated: a neutral glycosphingolipid, monohexosylceramide (CMH), and acidic glycosylinositolphosphorylceramides (GIPCs). Both glycosphingolipids affected the functioning of innate immunity cells, interfering with the antigen presenting process: P. brasiliensis yeast cells phagocytosis, IL-10 secretion, and costimulatory molecules and recognition receptors expression by monocytes were altered, while dendritic cell antigen presentation to autologous T cells was markedly down-modulated as shown by reduced T-cell proliferative responses. The mechanisms by which CMH and GIPCs exert their effects differ since the target cells did not always respond similarly to the challenge with the glycosphingolipids. Moreover, CUR-I and HNE-I presented different responses to the glycosphingolipids. Differences not only in the glycosphingolipid structure (such as the polar head group or the ceramide moiety), but also in the innate immunity properties of CUR-I and HNE-I, may underlie these differences and contribute to individual's susceptibility or resistance to develop paracoccidioidomycosis.

Characterization of Scedosporium apiospermum glucosylceramides and their involvement in fungal development and macrophage functions.

Scedosporium apiospermum is an emerging fungal pathogen that causes both localized and disseminated infections in immunocompromised patients. Glucosylceramides (CMH, GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this study, glucosylceramides (GlcCer) were extracted and purified in several chromatographic steps. Using high-performance thin layer chromatography (HPTLC) and electrospray ionization mass spectrometry (ESI-MS), N-2′-hydroxyhexadecanoyl-1-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine was identified as the main GlcCer in S. apiospermum. A monoclonal antibody (Mab) against this molecule was used for indirect immunofluorescence experiments, which revealed that this CMH is present on the surface of the mycelial and conidial forms of S. apiospermum. Treatment of S. apiospermum conidia with the Mab significantly reduced fungal growth. In addition, the Mab also enhanced the phagocytosis and killing of S. apiospermum by murine cells. In vitro assays were performed to evaluate the CMHs for their cytotoxic activities against the mammalian cell lines L.929 and RAW, and an inhibitory effect on cell proliferation was observed. Synergistic in vitro interactions were observed between the Mab against GlcCer and both amphotericin B (AmB) and itraconazole. Because Scedosporium species develop drug resistance, the number of available antifungal drugs is limited; our data indicate that combining immunotherapy with the available drugs might be a viable treatment option. These results suggest that in S. apiospermum, GlcCer are most likely cell wall components that are targeted by antifungal antibodies, which directly inhibit fungal development and enhance macrophage function; furthermore, these results suggest the combined use of monoclonal antibodies against GlcCer and antifungal drugs for antifungal immunotherapy.

Direct analysis of sialylated or sulfated glycosphingolipids and other polar and neutral lipids using TLC-MS interfaces

Gangliosides and sulfatides (STs) are acidic glycosphingolipids (GSLs) that have one or more sialic acids or sulfate substituents, in addition to neutral sugars, attached to the C-1 hydroxyl group of the ceramide long chain base. TLC is a widely employed and convenient technique for separation and characterization of GSLs. When TLC is directly coupled to MS, it provides both the molecular mass and structural information without further purification. Here, after development of the TLC plates, the structural analyses of acidic GSLs, including gangliosides and STs, were investigated using the liquid extraction surface analysis (LESA™) and CAMAG TLC-MS interfaces coupled to an ESI QSTAR Pulsar i quadrupole orthogonal TOF mass spectrometer. Coupling TLC with ESI-MS allowed the acquisition of high resolution mass spectra of the acidic GSLs with high sensitivity and mass accuracy, without the loss of sialic acid residues that frequently occurs during low-pressure MALDI MS. These systems were then applied to the analysis of total lipid extracts from bovine brain. This allowed profiling of many different lipid classes, not only gangliosides and STs, but also SMs, neutral GSLs, and phospholipids.

Application of direct HPTLC‐MALDI for the qualitative and quantitative profiling of neutral and acidic glycosphingolipids: The case of NEU3 overexpressing C2C12 murine myoblasts

Glycosphingolipids (GSLs) are a class of ubiquitous lipids characterized by a wide structural repertoire and a variety of functional implications. Importantly, altered levels have been correlated with different diseases, suggesting their crucial role in health. Conventional methods for the characterization and quantification are based on high-performance TLC (HPTLC) separation and comparison with the migration distance of standard samples or on MS. We set up and herein report the application of an ImagePrep method for glycosphingolipids qualitative and quantitative profiling through direct HPTLC-MALDI with particular application to wild-type and NEU3 sialidase-overexpressing C2C12 myoblasts. Lipids were analyzed by HPTLC, coupled with MALDI-TOF, and the resulting GSLs profiles were compared to the [³H]sphingolipids HPTLC patterns obtained after metabolic radiolabeling. GSLs detection by HPTLC-MALDI was optimized by testing different methods for matrix delivery and by performing quantitative analyses using serial dilutions of GSLs standards. Through this approach an accurate analysis of each variant of neutral and acidic GSLs, including the detection of different fatty-acid chain variants for each GSL, was provided and these results demonstrated that HPTLC-MALDI is an easy and high-throughput analytical method for GSLs profiling, suggesting its use for an early detection of markers in different diseases, including cancer and heart ischemia.

Progress in Detection and Structural Characterization of Glycosphingolipids in Crude Lipid Extracts by Enzymatic Phospholipid Disintegration Combined with Thin-Layer Chromatography Immunodetection and IR-MALDI Mass Spectrometry

In order to proceed in detection and structural analysis of glycosphingolipids (GSLs) in crude lipid extracts, which still remains a challenge in glycosphingolipidomics, we developed a strategy to structurally characterize neutral GSLs in total lipid extracts prepared from in vitro propagated human monocytic THP-1 cells, which were used as a model cell line. The procedure divides into (1) extraction of total lipids from cellular material, (2) enzymatical disintegration of phospholipids by treatment of the crude lipid extract with phospholipase C, (3) subsequent multiple thin-layer chromatography (TLC) overlay detection of individual GSLs with a mixture of various anti-GSL antibodies, and (4) structural analysis of immunostained GSLs directly on the TLC plate using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF MS) in combination with collision-induced dissociation (CID). Whereas GSLs were mostly undetectable in untreated crude lipid extracts, pretreatment with phospholipase C resulted in clear-cut mass spectra. MS(1) and MS(2) analysis gave similar results when compared to those obtained with a highly purified neutral GSL preparation of THP-1 cells, which served as a control. We could demonstrate in this study the feasibility of simultaneous multiple immunodetection of individual neutral GSLs in one and the same TLC run and their structural characterization in crude lipid extracts after phospholipase C treatment, thereby avoiding laborious and long-lasting sample purification. This powerful combinatorial technique allows for efficient structural characterization of GSLs in small tissue samples and takes a step forward in the emerging field of glycosphingolipidomics.

Pierisins and CARP-1: ADP-ribosylation of DNA by ARTCs in butterflies and shellfish.

The cabbage butterfly, Pieris rapae, and related species possess a previously unknown ADP-ribosylating toxin, guanine specific ADP-ribosyltransferase. This enzyme toxin, known as pierisin, consists of enzymatic N-terminal domain and receptor-binding C-terminal domain, or typical AB-toxin structure. Pierisin efficiently transfers an ADP-ribosyl moiety to the N(2) position of the guanine base of dsDNA. Receptors for pierisin are suggested to be the neutral glycosphingolipids, globotriaosylceramide (Gb3), and globotetraosylceramide (Gb4). This DNA-modifying toxin exhibits strong cytotoxicity and induces apoptosis in various human cell lines, which can be blocked by Bcl-2. Pierisin also produces detrimental effects on the eggs and larvae of the non-habitual parasitoids. In contrast, a natural parasitoid of the cabbage butterfly, Cotesia glomerata, was resistant to this toxin. The physiological role of pierisin in the butterfly is suggested to be a defense factor against parasitization by wasps. Other type of DNA ADP-ribosyltransferase is present in certain kinds of edible clams. For example, the CARP-1 protein found in Meretrix lamarckii consists of an enzymatic domain without a possible receptor-binding domain. Pierisin and CARP-1 are almost fully non-homologous at the amino acid sequence level, but other ADP-ribosyltransferases homologous to pierisin are present in different biological species such as eubacterium Streptomyces. Possible diverse physiological roles of the DNA ADP-ribosyltransferases are discussed.

Structural Elucidation of the Neutral Glycosphingolipids, Mono-, Di-, Tri- and Tetraglycosylceramides from the Marine Crab Erimacrus isenbeckii

The neutral glycosphingolipids, mono-, di-, tri- and tetraglycosylceramides (GL-1, GL-2, GL-3, GL-4a and GL-4b), were identified from whole tissues of the marine crab Erimacrus isenbeckii by successive column chromatography with ion exchange Sephadex (QAE-Sephadex), magnesium silicate (Florisil) and silicic acid (Iatrobeads) resins. Through component analysis, sugar analysis, methylation studies, exoglycosidase cleavage, and various chromatographic and spectrometric techniques, their structures were proposed to be as follows: GL-1, Glcβ1-1Cer; GL-2, Manβ1-4Glcβ1-1Cer; GL-3, Galβ1-3Manβ1-4Glcβ1-1Cer; and GL-4a and GL-4b, Gal3Meα1-4Galβ1-3Manβ1-4Glcβ1-1Cer. The main molecular species of the aliphatic moiety in each purified glycolipid were 18:0, 22:0, 22:1-d14:1 (fatty acid-sphingoid) and 18:0-d16:1 for GL-1; 18:0-d16:1 and 22:1-d14:1, d16:1 for GL-2; 22:1, 24:1-d16:1 for GL-3; 22:1, 24:1-d16:1 for GL-4a; and h22:1, h24:1-d16:1 for GL-4b, respectively. By immunological detection, an arthro-series glycosphingolipid (At3Cer; GlcNAcβ1-3Manβ1-4Glcβ1-1Cer) was also detected as a minor component. The characteristic arthro-series glycosphingolipid has been observed in most animals belonging to the phylum Arthropoda.

Individual profiles of free ceramide species and the constituent ceramide species of sphingomyelin and neutral glycosphingolipid and their alteration according to the sequential changes of environmental oxygen content in human colorectal cancer Caco-2 cells

We previously performed a systematic analysis of free ceramide (Cers) species, the constituent ceramide species of sphingomyelins and neutral glycosphingolipids (NGSLs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with high-energy collision-induced dissociation. As a result, distinct species differences were found among Cers, sphingomyelins and NGSLs in the kidneys. Using this method, we investigated various sphingolipid species from human colon cancer Caco-2 cells as well as the influence of environmental oxygen on these species in detail. Unexpectedly, even in normoxia, all Cers species were composed of dihydrosphingosine (d18:0) and non-hydroxy fatty acid (NFA), and 34% of sphingomyelins were composed of dihydrosphingomyelins with NFA. In contrast, major constituent ceramide species of NGSLs were composed of the usual long-chain base of sphingosine (d18:1) and hydroxy fatty acid (HFA). When the cells were cultured under hypoxic condition for 3 days, all the Cers and nearly 80% of the sphingomyelins were dihydrosphingolipids composed of d18:0-NFAs, but a significant proportion of d18:1-HFAs still remained in the NGSLs. When the cells were transferred from conditions of hypoxia to normoxia again (reoxygenation), Cer species composed of d18:1-NFAs, which were not found in Cers under the original normoxic conditions, appeared. Such Cers were probably synthesized as precursors for the constituent ceramides of sphingomyelins and NGSLs.

A symptomatic Fabry disease mouse model generated by inducing globotriaosylceramide synthesis

Fabry disease is a lysosomal storage disorder in which neutral glycosphingolipids, predominantly Gb3 (globotriaosylceramide), accumulate due to deficient α-Gal A (α-galactosidase A) activity. The GLAko (α-Gal A-knockout) mouse has been used as a model for Fabry disease, but it does not have any symptomatic abnormalities. In the present study, we generated a symptomatic mouse model (G3Stg/GLAko) by cross-breeding GLAko mice with transgenic mice expressing human Gb3 synthase. G3Stg/GLAko mice had high Gb3 levels in major organs, and their serum Gb3 level at 5-25 weeks of age was 6-10-fold higher than that in GLAko mice of the same age. G3Stg/GLAko mice showed progressive renal impairment, with albuminuria at 3 weeks of age, decreased urine osmolality at 5 weeks, polyuria at 10 weeks and increased blood urea nitrogen at 15 weeks. The urine volume and urinary albumin concentration were significantly reduced in the G3Stg/GLAko mice when human recombinant α-Gal A was administered intravenously. These data suggest that Gb3 accumulation is a primary pathogenic factor in the symptomatic phenotype of G3Stg/GLAko mice, and that this mouse line is suitable for studying the pathogenesis of Fabry disease and for preclinical studies of candidate therapies.

Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin–Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed.

Structural characterization of neutral glycosphingolipids using high-performance liquid chromatography-electrospray ionization mass spectrometry with a repeated high-speed polarity and MSn switching system

Four types of neutral glycosphingolipids (LacCer, Gb3Cer, Gb4Cer, and IV3αGalNAc-Gb4Cer; 10 pmol each) were analyzed using high-performance liquid chromatography (HPLC)-electrospray ionization quadrupole ion trap time-of-flight (ESI-QIT-TOF) mass spectrometry (MS) with a repeated high-speed polarity and MSn switching system. This system can provide six types of mass spectra, including positive and negative ion MS, MS2, and MS3 spectra, within 1 s per cycle. Using HPLC with a normal-phase column, information on the molecular weights of major molecular species of four neutral glycosphingolipids was obtained by detecting [M+Na]+ in the positive ion mode mass spectra and [M−H]− in the negative ion mode mass spectra. Sequences of glycosphingolipid oligosaccharide were obtained in the negative ion MS2 spectra. In addition, information on the ceramide structures was clearly obtained in the negative ion MS3 mass spectra. GlcCer molecular species were analyzed by HPLC-ESI-QIT-TOF MS with a reversed-phase column using 1 pmole of GlcCer. The structures of the seven molecular species of GlcCer, namely, d18:1-C16:0, d18:1-C18:0, d18:1-C20:0, d18:1-C22:0, d18:1-C23:0, d18:1-C24:1, and d18:1-C24:0, were characterized using positive ion MS and negative ion MS, MS2, and MS3. The established HPLC-ESI-QIT-TOF MS with MSn switching and a normal phase column has been successfully applied to the structural characterization of LacCer and Gb4Cer in a crude mixture prepared from human erythrocytes.

Structural Characterization of Pyridylaminated Oligosaccharides Derived from Neutral Glycosphingolipids by High-Sensitivity Capillary Electrophoresis–Mass Spectrometry

High-sensitivity capillary electrophoresis-electrospray ionization quadrupole ion trap time-of-flight mass spectrometry (CE-ESI-QIT-TOF MS) was developed to structurally characterize four kinds of pyridylaminated (PA) oligosaccharides, i.e., lactose (Lac)-PA, globotriose (Gb3)-PA, globotetraose (Gb4)-PA, and IV(3) αGalNAc-Gb4 (Forssman antigen)-PA, derived from neutral glycosphingolipids. The CE-MS system included the head-column field-amplified sample stacking (HC-FASS) method for effective sample injection into a capillary column in CE, a sheathless interface between CE and a mass spectrometer, and MS and tandem MS (MS(2)) measurements with narrow mass range repeated high-speed switching. The total sensitivity of the developed CE-MS system was about 20,000 times higher than that of the conventional CE-MS system consisting of pressure injection, a sheath-flow interface, and a wide mass range measurement. The MS and MS(2) spectra of the four PA-oligosaccharides at a concentration of 25 amol/μL in mixtures (each 250 amol/10 μL in a tube) clearly showed protonated molecular ions ([M + H](+)) and the fragment ions responsible for the sequential elimination of saccharides. The developed CE-MS system is a powerful method for the structural characterization of glycosphingolipids extracted from very small amounts of biological materials and could be extended to the structural characterization of oligosaccharides derived from glycoproteins.

Fabry Disease and Cardiovascular Involvement

Fabry disease (FD, OMIM 301500) is a rare X-linked lysosomal storage disorder of the glycosphigolipid metabolism caused by total or partial deficiency of the lysosomal enzyme alpha-galactosidase A (α-gal A). Progressive intralysosomal accumulation of neutral glycosphingolipids in a variety of cell types triggers a cascade of pathophysiological events including cellular death, compromised energy metabolism, small vessel injury, K(Ca)3.1 channel dysfunction in endothelial cells, oxidative stress, impaired autophagosome maturation, tissue ischemia and, importantly, development of irreversible cardiac and renal tissue fibrosis, leading to major multisystemic manifestations. Cardiovascular complications of the disease are very frequent and contribute substantially to disease-related morbidity and mortality in men. Cardiovascular involvement is the leading cause of premature death in heterozygous female patients with FD. Left ventricular hypertrophy is the most prominent cardiac manifestation followed by conduction system disease, valve dysfunction, arrhythmias, vessel disease and coronary microvascular dysfunction. The diagnosis of subclinical forms of the disease, before the development of cardiac hypertrophy, using newer techniques (tissue doppler imaging, strain rate and cardiac magnetic resonance) is crucial to the early initation of the treatment. Greatest benefit of the enzyme replacement treatment is achieved when started at an early stage of the disease before extensive fibrosis or other irreversible tissue damage takes place. Fabry disease should be included in the differential diagnosis algorithm of idiopathic hypertrophy. Determination of Alpha-Gal A activity on plasma and peripheral leukocytes in males and genetic testing in females are the diagnostic gold-standards.