The Hypothesis on Function of Glycosphingolipids and ABO Blood Groups Revisited

Twenty-five years ago the author proposed new ideas of glycoprotein (GPs) and glycosphingolipid (GSLs) functions at the cell membrane. The GPs, apart from their glycan carrying capacity, were assumed to have specific, protein associated, functions. In contrast, GSLs such as those of globo and neolacto/lacto series, were considered to be energetically cheap membrane packing substances, filling in membrane spaces not covered with functional GPs. The terminal carbohydrate structures of the neolacto/lacto GSLs, i.e., sialic acid residues and ABH glycotopes, were postulated to have either regulatory or protective functions, respectively. A special active role was ascribed to terminal β-galactosyl residues of GSLs and GPs. Gangliosides were considered to be functional GSLs. In the present review the author discusses these old ideas in context of the contemporary knowledge and comes to the conclusion that they have not aged.     

Thermotropic behavior of glycosphingolipids in aqueous dispersions

The thermotropic behavior of 20 chemically related glycosphingolipids (GSLs) of high purity, containing neutral and anionic carbohydrate residues in their oligosaccharide chains, was studied by high-sensitivity differential scanning calorimetry. In general, the polar head group of GSLs appears to be one of the major determinants of their phase behavior. Compared to phospholipids, the presence of the carbohydrate rather than the phosphorylcholine moiety in the polar head group and a sphingosine base in the hydrocarbon portion of GSLs reduces the effect on the transition temperature (Tm) brought about by increasing the number of methylene groups in the amide-linked fatty acyl chains. For simple neutral GSLs, the Tm's were 20-40 degrees C higher than those of phospholipids with comparable hydrocarbon chains. As the oligosaccharide chain of GSLs becomes more complex, the excess heat capacity, Tm, enthalpy (delta Hcal), and entropy of the transition decrease proportionally to the number of carbohydrate residues present in the polar head group. The Tm and delta Hcal for anionic GSLs were 16-25 degrees C and 1-3 kcal mol-1 lower than those of neutral GSLs with comparable oligosaccharide chains. A linear dependence of delta Hcal with Tm was found. However, the slopes of these plots were different for neutral and for anionic GSLs, suggesting different types of intermolecular organizations for the two. The Tm and delta Hcal were linearly dependent on the molecular area of both neutral and anionic GSLs; this indicated that the influence of the complexity of the polar head group in GSLs for establishing the thermodynamic behavior may be mediated by the intermolecular spacings.     

Linking glycosphingolipids to Alzheimer’s amyloid-ß: extracellular vesicles and functional plant materials

Glycoconjugate Journal - Glycosphingolipids (GSLs) are a specialized class of membrane lipids composed of a ceramide and a carbohydrate head group. GSLs are localized in cell membranes and were...     

Stemming the tide: glycosphingolipid synthesis inhibitors as therapy for storage diseases

Glycosphingolipids (GSLs) are plasma membrane components of every eukaryotic cell. They are composed of a hydrophobic ceramide moiety linked to a glycan chain of variable length and structure. Once thought to be relatively inert, GSLs have now been implicated in a variety of biological processes. Recent studies of animals rendered genetically deficient in various classes of GSLs have demonstrated that these molecules are important for embryonic differentiation and development as well as central nervous system function. A family of extremely severe diseases is caused by inherited defects in the lysosomal degradation pathway of GSLs. In many of these disorders GSLs accumulate in cells, particularly neurons, causing neurodegeneration and a shortened life span. No effective treatment exists for most of these diseases and little is understood about the mechanisms of pathogenesis. This review will discuss the development of a new approach to the treatment of GSL storage disorders that targets the major synthesis pathway of GSLs to stem their cellular accumulation.     

Design of a covalently bonded glycosphingolipid microarray

Glycosphingolipids (GSLs) are well known ubiquitous constituents of all eukaryotic cell membranes, yet their normal biological functions are not fully understood. As with other glycoconjugates and saccharides, solid phase display on microarrays potentially provides an effective platform for in vitro study of their functional interactions. However, with few exceptions, the most widely used microarray platforms display only the glycan moiety of GSLs, which not only ignores potential modulating effects of the lipid aglycone, but inherently limits the scope of application, excluding, for example, the major classes of plant and fungal GSLs. In this work, a prototype “universal” GSL-based covalent microarray has been designed, and preliminary evaluation of its potential utility in assaying protein-GSL binding interactions investigated. An essential step in development involved the enzymatic release of the fatty acyl moiety of the ceramide aglycone of selected mammalian GSLs with sphingolipid N-deacylase (SCDase). Derivatization of the free amino group of a typical lyso-GSL, lyso-G_M1, with a prototype linker assembled from succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester and 2-mercaptoethylamine, was also tested. Underivatized or linker-derivatized lyso-GSL were then immobilized on N-hydroxysuccinimide- or epoxide-activated glass microarray slides and probed with carbohydrate binding proteins of known or partially known specificities (i.e., cholera toxin B-chain; peanut agglutinin, a monoclonal antibody to sulfatide, Sulph 1; and a polyclonal antiserum reactive to asialo-G_M2). Preliminary evaluation of the method indicated successful immobilization of the GSLs, and selective binding of test probes. The potential utility of this methodology for designing covalent microarrays that incorporate GSLs for serodiagnosis is discussed.     

Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method

Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma membrane lipid raft, which is clustered with sphingolipids, sterols, and specific proteins, and thereby contributes to membrane physical properties and specific recognition sites for various biological events. These bioactive GSL molecules consequently affect the pathophysiology and pathogenesis of various diseases. Thus, altered expression of GSLs in various diseases may be of importance for disease-related biomarker discovery. However, analysis of GSLs in blood is particularly challenging because GSLs are present at extremely low concentrations in serum/plasma. In this study, we established absolute GSL-glycan analysis of human serum based on endoglycoceramidase digestion and glycoblotting purification. We established two sample preparation protocols, one with and the other without GSL extraction using chloroform/methanol. Similar amounts of GSL-glycans were recovered with the two protocols. Both protocols permitted absolute quantitation of GSL-glycans using as little as 20 μl of serum. Using 10 healthy human serum samples, up to 42 signals corresponding to GSL-glycan compositions could be quantitatively detected, and the total serum GSL-glycan concentration was calculated to be 12.1–21.4 μM. We further applied this method to TLC-prefractionated serum samples. These findings will assist the discovery of disease-related biomarkers by serum GSL-glycomics.     

Stimulation of phagocytosis and phagosome—lysosome (P-L) fusion of human polymorphonuclear leukocytes by sulfatide (galactosylceramide-3-sulfate)

Abstract Recently, extensive attention has been paid to the physiological function of glycosphingolipids (GSLs) of mammalian cell membranes. Among a variety of GSLs, sulfatide (galactosylceramide-3-sulfate) has been proposed to be a specific receptor or binding molecule to microorganisms. However, no report has appeared on the direct stimulation by sulfatide for cellular function differentiation in phagocytic cells. We found that sulfatide showed a marked stimulation for phagocytic processes of human peripheral polymorphonuclear leukocytes (PMN) using heat-killed cells of Staphylococcus aureus coated with isolated lipid. Among mammalian acidic GSLs, sulfatide showed the highest stimulative activity for adhesion, phagocytosis and phagosome—lysosome (P-L) fusion by PMN. On the other hand, neutral GSLs did not stimulate essentially. Relative phagocytic rate of sulfatide-coated staphylococci was six times higher than that of the non-coated control and P-L fusion rate was ten times at maximum, respectively. Although the promotion mechanism of sulfatide for such phagocytosis or P-L fusion is not clear, it was strongly suggested that the existence of negative charges on carbohydrate moiety may be essential for the induction of differentiation of phagocytic cell function via signal transduction systems.     

Glycosphingolipids of human umbilical vein endothelial cells and smooth muscle cells

Glycosphingolipids (GSLs) represent an important class of immunogens and receptors. Although cell surface antigens and receptors of endothelial cells (ECs) have been the subject of extensive biochemical investigation, no information is available about their GSLs. We report here the characterization by chromatographic and immunological techniques of GSLs of cultured human umbilical vein ECs and, for comparison, umbilical vein smooth muscle cells (SMCs). The most abundant neutral GSLs of both cell types were lactosylceramide, Gb3, and Gb4, and both cells contained complex lacto and globo series compounds. Immunostaining revealed that ECs, but not SMCs, contained long chain GSLs bearing a type 2 blood group H determinant. ECs also contained more long chain GSLs bearing an unsubstituted terminal lactosamine structure than SMCs. Labeling with galactose oxidase/NaB3H4 demonstrated that neutral glycolipids that contained three or more sugars were accessible on the cell surface. The major gangliosides of both cell types were GM3 and IV3NeuAcnLc4. Immunostaining following neuraminidase treatment revealed that most of the long chain gangliosides in both types of cells contained a lacto core structure, and that ganglio series compounds were more abundant in SMCs than ECs. Gangliosides that contain a polyfucosyllactosamine core and a globo core were also present in both cell types. These results demonstrate that endothelial and smooth muscle cells contain a large diversity of GSL structures, and provide the basis for investigation of the role of these GSLs as cell surface antigens and receptors for blood components.     

Distribution of glycoconjugates on the plasma membrane and on membranes of the open-canalicular system in human platelets

Summary Distribution of carbohydrate moieties in the membrane system of the human blood platelet was studied by electron microscopy employing lectins as a probe. Glutaraldehyde-fixed platelets were treated with biotinylatedlectins (ConA, RCA, WGA, PNA, SBA, DBA and UEA-1) and labeled with horseradish peroxidase-conjugated avidin. Among the lectins used, ConA bound uniformly to the plasma membrane as well as to the membrane of the opencanalicular system (OCS). Other lectins showed more or less reduced binding on the OCS membrane compared with that on the plasma membrane, indicating that there exist regional differences in the distribution pattern of glycoconjugates in the membrane system of the platelet. The relationship of the distribution pattern of the glycoconjugates with the distribution of the major platelet glycoproteins GPIb and GPIIbIIIa is discussed.     

Structural characteristics of the blood group N determinant, tested with anti-N lectin from Vicia graminea]

Crude extracts of Vicia graminea seeds agglutinate human N erythrocytes as anti-N immunsera. The anti-N lectin is purified after precipitations with ammonium sulphate of crude extracts, DE52 Whatman chromatography and sephadex G150 gel filtration. Its homogeneity is demonstrated by physical and immunological methods. The structure determinant for the Vicia graminea anti-N activity was investigated: --with the major glycoprotein of N erythrocytes. --with glycoconjugates isolated from urine of normal human N-blood group as urinary glycoconjugates are probably related to the membrane glycoprotein catabolism. Purification and characterization of glycoconjugates are undertaken by gel filtration and non-exchange chromatography. This purification is checked by hemagglutination-inhibition test with V. graminea lectin. Biochemical characterization of active glycoconjugates gives way to the carbohydrate determinant recognized by anti-N antisera and Vicia graminea lectin.     

Two strains of the Madin-Darby canine kidney (MDCK) cell line have distinct glycosphingolipid compositions.

The glycosphingolipids (GSLs) of two sublines of Madin-Darby canine kidney (MDCK) cells, an epithelial cell line, were characterized by t.l.c., antibody overlay and mass spectrometry. The major characteristic which distinguishes the two MDCK cell strains is their trans-epithelial electrical resistance which is typically of the order of 3000 ohm.cm2 for strain I and 100 ohm.cm2 for strain II cells. Strain I and II cells were equally rich in glycolipids, the cellular GSL/phospholipid ratio being 0.04. However, while the phospholipid patterns were identical, the GSLs showed striking differences, and each cell strain expressed appreciable amounts of GSLs that were not found in the other strain. Both cell types possessed neutral GSLs with one, two or three carbohydrate moieties. The monoglycosylceramide accounted for 50% of the total GSLs in each strain. However, while in strain I cells over 90% of this monoglycosylceramide was monoglucosylceramide, in strain II cells over 90% consisted of monogalactosylceramide. In addition, MDCK strain II cells selectively expressed GSLs belonging to the globo series (26% of its neutral GSLs), including globoside and Forssman antigen, a globoside derivative. MDCK strain I cells, on the other hand, expressed another series of GSLs with 4-7 carbohydrate moieties characterized by the common sequence Hex-HexNAc-Hex-Hex-Cer. The presence of two fucosylated GSLs in these series was established. Both MDCK strain I and II cells contained negatively charged GSLs, the major component of which was the ganglioside GM3. MDCK strain II cells in addition expressed sulfatide, the sulfated derivative of galactosylceramide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of glycoconjugates on the plasma membrane and on membranes of the open-canalicular system in human platelets. A cytochemical study

Distribution of carbohydrate moieties in the membrane system of the human blood platelet was studied by electron microscopy employing lectins as a probe. Glutaraldehyde-fixed platelets were treated with biotinylated-lectins (ConA, RCA, WGA, PNA, SBA, DBA and UEA-1) and labeled with horseradish peroxidase-conjugated avidin. Among the lectins used, ConA bound uniformly to the plasma membrane as well as to the membrane of the open-canalicular system (OCS). Other lectins showed more or less reduced binding on the OCS membrane compared with that on the plasma membrane, indicating that there exist regional differences in the distribution pattern of glycoconjugates in the membrane system of the platelet. The relationship of the distribution pattern of the glycoconjugates with the distribution of the major platelet glycoproteins GPIb and GPIIbIIIa is discussed.     

Detection of binding sites for biotinylated neoglycoproteins and heparin (endogenous lectins) during cerebellar ontogenesis in the rat

Endogenous carbohydrate-binding sites were studied during rat cerebellar development on sections of fixed tissue using synthetic tools, biotinylated neoglycoproteins, in conjunction with subsequent avidinperoxidase staining. Neoglycoproteins were constructed by chemically coupling the histochemically pivotal carbohydrate moieties to an inert carrier protein. The sugar part of the neoglycoproteins included common constituents of the carbohydrate part of cellular glycoconjugates, namely mannose, galactose, fucose, N-acetyl-glucosamine, N-acetylgalactosamine and N-acetyl-neuraminic acid to probe for the presence of respective endogenous receptors. Heparin was biotinylated after mild cyanogen bromide activation and aminoalkylation. Specific positive reactions were obtained for all neoglycoproteins and heparin. The staining pattern with the individual probes disclosed variable developmental regulation. Consequently, these results suggest that recognition processes during cerebellar development may include several types of carbohydrate determinants. In two instances, the binding of neoglycoproteins could be compared to endogenous lectin-specific antibodies. Despite a significant extent of accordance the comparison revealed notable differences. These differences were attributed primarily to fixation and the presence of physiological ligands that can mask the active endogenous carbohydrate-binding proteins. In any case, histochemical application of labeled neoglycoproteins is valuable to discern the presence, localization and developmental pattern of binding sites for the carbohydrate part of glycoconjugates, on which further biochemical and cell biological studies can consequently be based.     

A glycosynapse in myelin?

Myelin, the multilayered membrane which surrounds nerve axons, is the only example of a membranous structure where contact between extracellular surfaces of membrane from the same cell occurs. The two major glycosphingolipids (GSLs) of myelin, galactosylceramide (GalC) and its sulfated form, galactosylceramide I(3)-sulfate (SGC), can interact with each other by trans carbohydrate-carbohydrate interactions across apposed membranes. They occur in detergent-insoluble lipid rafts containing kinases and thus may be located in membrane signaling domains. These signaling domains may contact each other across apposed extracellular membranes, thus forming glycosynapses in myelin. Multivalent forms of these carbohydrates, GalC/SGC-containing liposomes, or galactose conjugated to albumin, have been added to cultured oligodendrocytes (OLs) to mimic interactions which might occur between these signaling domains when OL membranes or the extracellular surfaces of myelin come into contact. These interactions between multivalent carbohydrate and the OL membrane cause co-clustering or redistribution of myelin GSLs, GPI-linked proteins, several transmembrane proteins, and signaling proteins to the same membrane domains. They also cause depolymerization of the cytoskeleton, indicating that they cause transmission of a signal across the membrane. Their effects have similarities to those of anti-GSL antibodies on OLs, shown by others, suggesting that the multivalent carbohydrate interacts with GalC/SGC in the OL membrane. Communication between the myelin sheath and the axon regulates both axonal and myelin function and is necessary to prevent neurodegeneration. Participation of transient GalC and SGC interactions in glycosynapses between the apposed extracellular surfaces of mature compact internodal myelin might allow transmission of signals throughout the myelin sheath and thus facilitate myelin-axonal communication.     

Pseudomonas aeruginosa binds to neoglycoconjugates bearing mucin carbohydrate determinants and predominantly to sialyl-Lewis x conjugates.

Pseudomonas aeruginosa plays an important role in the colonization of the airways of patients suffering from cystic fibrosis. It binds to the carbohydrate part of respiratory and salivary mucins and its binding to cystic fibrosis mucins is even higher, suggesting that qualitative or/and quantitative modifications of the carbohydrate chains may be involved in this process. In order to find out the best carbohydrate receptors for P.aeruginosa, a flow cytometry technique using a panel of polyacrylamide based glycoconjugates labeled with fluorescein was developed. The neoglycoconjugates contained neutral, sialylated or sulfated chains analogous to carbohydrate determinants found at the periphery of respiratory mucins (Le(a), Le(y), Le(x), sialyl- and 3'-sulfo-Le(x), and blood group A determinants). We used also neoglycoconjugates containing Gal(alpha1-2)Galbeta and sialyl- N -acetyllactosamine determinants. The interaction of these glycoconjugates with the nonpiliated strain of P.aeruginosa, 1244-NP, was saturable except for the glycoconjugates containing blood group A or sialyl- N -acetyllactosamine epitopes. The measure of Kd indicated that strain 1244-NP had a higher affinity for the glycoconjugate bearing the sialyl-Le(x)determinant than for all the other glycoconjugates studied. The role of sialic acid was confirmed by competition assay using mainly sialylated mucin glycopeptides. In order to find out if this behavior was the same for pathological strains as for the 1244-NP mutant, four mucoid strains of P.aeruginosa isolated from cystic fibrosis patients were analyzed with the Le(x)neoglycoconjugate, its sialylated and its sulfated derivatives. Individual variations in the binding of these strains to the three glycoconjugates were observed. However, three strains out of four had a higher affinity for the sialyl-Le(x)than for the 3'-sulfo-Le(x)derivative.     

Aspects of glycoconjugate production and lysozyme- and defensins-expression of the ceruminous glands of the horse (Equus przewalskii f. dom.)

The distribution of complex glycoconjugates and antimicrobial substances in the ceruminous glands of the horse (Equus przewalskii f. dom., type: pony) was studied using carbohydrate histochemical and immunohistochemical methods. The epithelial cells and luminal secretion of these glands exhibited considerable amounts of glycoconjugates with various saccharide residues, such as alpha-D-mannose, alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine and sialic acid, including O-acetylated sialic acid. Several sugars (alpha-D-mannose, alpha-L-fucose, and beta-D-galactose) were also detectable in the secretion of sebaceous glands present. Additionally, lysozyme and the peptide group of beta-defensins are demonstrated as products of the apocrine ceruminous glands and sebaceous glands. The results obtained are discussed with regard to the functional significance of the glandular secretions. It is suggested that the complex carbohydrates, lysozyme and beta-defensins found in the ceruminous gland secretions are involved in the function of cerumen as a general antimicrobial protective agent in the external auditory canal.     

Shotgun glycomics: a microarray strategy for functional glycomics

Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells and is a strategy for focusing structural analyses on functionally important glycans.     

Drosophila beta 1,4-N-acetylgalactosaminyltransferase-A synthesizes the LacdiNAc structures on several glycoproteins and glycosphingolipids

The GalNAcbeta1,4GlcNAc (LacdiNAc or LDN) structure is a more common structural feature in invertebrate glycoconjugates when compared with the Galbeta1,4GlcNAc structure. Recently, beta1,4-N-acetylgalactosaminyltransferase (beta4GalNAcT) was identified in some invertebrates including Drosophila. However, the LDN structure has not been reported in Drosophila, and the biological function of LDN remains to be determined. In this study, we examined acceptor substrate specificity of Drosophila beta4GalNAcTA by using some N- and O-glycans on glycoproteins and neutral glycosphingolipids (GSLs). GalNAc was efficiently transferred toward N-glycans, O-glycans, and the arthro-series GSLs. Moreover, we showed that dbeta4GalNAcTA contributed to the synthesis of the LDN structure in vivo. The dbeta4GalNAcTA mRNA was highly expressed in the developmental and adult neuronal tissues. Thus, these results suggest that dbeta4GalNAcTA acts on the terminal GlcNAc residue of some glycans for the synthesis of LDN, and the LDN structure may play a role in the physiological or neuronal development of Drosophila.     

Immobilized glycoconjugates for cell recognition studies

Specific cell-cell recognition and adhesion may involve cell surface glycoconjugates on one cell binding the complementary carbohydrate receptors on an apposing cell surface. Such interactions have been modeled by immobilizing simple synthetic glycosides, glycoproteins, glycosaminoglycans, and glycolipids on otherwise inert plastic surfaces and incubating them with intact cells. Using this approach, the ability of several cell types to recognize specific carbohydrates has been demonstrated. This carbohydrate-directed cell adhesion may depend on cell surface carbohydrate receptors which mediate both the initial specific adhesion and complex postrecognition cellular responses. While the relationship of the cell adhesion demonstrated here to cell-cell recognition in vivo has yet to be determined, this well-controlled biochemical approach may reveal new information on the way in which cells analyze and respond to their immediate external environment.     

A hypothesis on the biological role of ABH,lewis and P blood group determinant structures in glycosphingolipids and glycoproteins

A hypothesis is presented that glycosphingolipids of circulating erythrocytes are membrane-packing substances providing for an energetically cheap carbohydrate protective coat at the cell surface. The glycosphingolipids should cover the membrane surface not occupied by functional glycoproteins. This role is envisaged for the globo series of glycosphingolipids which are P^k and P antigens of human blood. Glycosphingolipids of the neolacto series terminated with non-informative A, B, H. Lewis, P₁ antigenic structures as well as with sialic acid residues should serve the same purpose. These carbohydrate structures may be also used for conferring biological inertness on otherwise functionally active carbohydrate structures and provide protection for circulatory and membrane glycoproteins from proteolysis, denaturation and recognition of potentially antigenic sites of protein moieties by the immunosurveillance system of the body. At the external body surface the same carbohydrate structures may protect cells from the action of pathogenic microorganisms and other environmental factors. The roles of the above mentioned carbohydrate sequences on glycosphingolipids and glycoproteins in the development, tumorigenesis and evolution of blood group polymorphism are discussed.Abbreviations GP glycoprotein - GSL glycosphingolipid - GC glycoconjugate