Novel functions of complex carbohydrates elucidated by the mutant mice of glycosyltransferase genes

Complex carbohydrates consist of carbohydrate moieties and protein or lipid portions, resulting in the formation of glycoproteins, proteoglycans or glycosphingolipids. The polymorphic carbohydrate structures are believed to contain profound biological implications which are important in cell-cell or cell-extracellular matrix interactions. A number of studies to delineate the roles of carbohydrates have been performed, and demonstrated definite changes in their profiles, cellular phenotypic changes or, sometimes, morphological and functional changes in tissues after modification of their structures. Recent successes in the isolation of glycosyltransferase genes and their modification enzyme genes has enabled clearer demonstrations of the roles of complex carbohydrates. In particular, genetic modification of glycosyltransferase genes in mice can elucidate the biological significances of their products in vivo. Here, we summarize recent advances in the understanding of the roles of complex carbohydrates provided from studies of gene knock-out mice of glycosyltransferase and modification enzyme genes focusing on novel functions which had not been expected.     

Mast cell heterogeneity. Differential synthesis and expression of glycosphingolipids by mouse serosal mast cells as compared to IL-3-dependent bone marrow culture-derived mast cells before or after coculture with 3T3 fibroblasts

The synthesis and intracellular expression of glycosphingolipids by mouse serosal mast cells (SMC) have been characterized by radiolabeling and TLC and by immunodetection in situ. Chromatographic analysis of purified glycosphingolipids from SMC intrinsically labeled with [14C]galactose and [14C]glucosamine hydrochloride revealed the predominant synthesis of only the simplest neutral glycosphingolipid and ganglioside, glucosylceramide and ganglioside GM3, respectively. Intracellular indirect immunofluorescence staining of permeabilized SMC demonstrated the absence of the more complex neutral glycosphingolipids lactosylceramide, globotriosylceramide, globotetraosylceramide, and globopentaosylceramide, the absence of ganglioside GM1, and the presence of ganglioside GM3. By contrast, permeabilized mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mast cells recovered after 21 days of coculture of BMMC with mouse 3T3 fibroblasts expressed lactosylceramide, globotriosylceramide, globotetraosylceramide, ganglioside GM1, and ganglioside GM3, but not globopentaosylceramide intracellularly as determined by immunofluorescence. The findings indicate a loss of biosynthetic capacity and epitope maintenance for glycosphingolipids with in vivo differentiation of SMC from IL-3-dependent BMMC progenitors. Thus, although mast cells derived after coculture of these progenitors for 21 days with fibroblasts assume multiple SMC-like properties in terms of their histochemical staining and their secretory granule proteoglycan and neutral protease constituents, they do not lose the ability to express complex glycosphingolipids. The finding that glycosphingolipid composition does not change coordinately with other secretory granule markers defines a new stage of mouse mast cell development between the BMMC and SMC and provides evidence that mast cell development is more complex than previously appreciated.     

Glycosphingolipid specificity of the human sulfatide activator protein

The interaction of the sulfatide activator protein with different glycosphingolipids have been studied in detail. The following findings were made. 1. The sulfatide activator protein forms water-soluble complexes with sulfatides [Fischer, G. and Jatzkewitz, H. (1977) Hoppe-Seyler's Z. Physiol. Chem. 356, 6588-6591] and various other glycospingolipids. 2. In the absence of degrading enzymes the activator protein acts in vitro as a glycosphingolipid transfer protein, transporting glycosphingolipids from donor to acceptor liposomes. Lipids having less than three hexoses, e.g. galactosylceramide, sulfatide and ganglioside GM3 were transferred at very slow rates, whereas complex lipids such as gangliosides GM2, GM1 and GD1a were transferred much faster than the former. The transfer rate increased with increasing length of the carbohydrate chain of the lipid molecules. 3. Both the acyl residue in the ceramide moiety and the nature of the carbohydrate chain are significant for recognition of the glycosphingolipids by the sulfatide activator protein. Apparently, both residues serve as an anchor and the longer they are the better they are recognized by the protein. 4. In the absence of activator protein, degradation rates of sulfatide derivatives by arylsulfatase A, and of ganglioside GM1 derivatives by beta-galactosidase, increase with decreasing length of acyl residues in their hydrophobic ceramide moiety. Addition of activator protein stimulates the degradation of only those GM1 and sulfatide derivatives that have long-chain fatty acids in their hydrophobic ceramide anchor.     

Inhibition of GM3 Synthase Attenuates Neuropathology of Niemann-Pick Disease Type C by Affecting Sphingolipid Metabolism

In several lysosomal storage disorders, including Niemann-Pick disease Type C (NP-C), sphingolipids, including glycosphingolipids, particularly gangliosides, are the predominant storage materials in the brain, raising the possibility that accumulation of these lipids may be involved in the NP-C neurodegenerative process. However, correlation of these accumulations and NP-C neuropathology has not been fully characterized. Here we derived NP-C mice with complete and partial deletion of the Siat9 (encoding GM3 synthase) gene in order to investigate the role of ganglioside in NP-C pathogenesis. According to our results, NPC mice with homozygotic deletion of GM3 synthase exhibited an enhanced neuropathological phenotype and died significantly earlier than NP-C mice. Notably, in contrast to complete depletion, NP-C mice with partial deletion of the GM3 synthase gene showed ameliorated NP-C neuropathology, including motor disability, demyelination, and abnormal accumulation of cholesterol and sphingolipids. These findings indicate the crucial role of GM3 synthesis in the NP-C phenotype and progression of CNS pathologic abnormality, suggesting that well-controlled inhibition of GM3 synthesis could be used as a therapeutic strategy.     

Functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains

At cell surface microdomains, glycosyl epitopes, carried either by glycosphingolipids, N- or O-linked oligosaccharides, are recognized by carbohydrate-binding proteins or complementary carbohydrates. In both cases, the carbohydrate epitopes may be clustered with specific signal transducers, tetraspanins, adhesion receptors or growth factor receptors. Through this framework, carbohydrates can mediate cell signaling leading to changes in cellular phenotype. Microdomains involved in carbohydrate-dependent cell adhesion inducing cell activation, motility, and growth are termed "glycosynapse". In this review a historical synopsis of glycosphingolipids-enriched microdomains study leading to the concept of glycosynapse is presented. Examples of glycosynapse as signaling unit controlling the tumor cell phenotype are discussed in three contexts: (i) Cell-to-cell adhesion mediated by glycosphingolipids-to-glycosphingolipids interaction between interfacing glycosynaptic domains, through head-to-head (trans) carbohydrate-to-carbohydrate interaction. (ii) Functional role of GM3 complexed with tetraspanin CD9, and interaction of such complex with integrins, or with fibroblast growth factor receptor, to control tumor cell phenotype and its reversion to normal cell phenotype. (iii) Inhibition of integrin-dependent Met kinase activity by GM2/tetraspanin CD82 complex in glycosynaptic microdomain. Data present here suggest that the organizational status of glycosynapse strongly affects cellular phenotype influencing tumor cell malignancy.     

Mice expressing only monosialoganglioside GM3 exhibit lethal audiogenic seizures

Gangliosides are a family of glycosphingolipids that contain sialic acid. Although they are abundant on neuronal cell membranes, their precise functions and importance in the central nervous system (CNS) remain largely undefined. We have disrupted the gene encoding GD3 synthase (GD3S), a sialyltransferase expressed in the CNS that is responsible for the synthesis of b-series gangliosides. GD3S-/- mice, even with an absence of b-series gangliosides, appear to undergo normal development and have a normal life span. To further restrict the expression of gangliosides, the GD3S mutant mice were crossbred with mice carrying a disrupted GalNAcT gene encoding beta1,4-N-acetylgalactosaminyltransferase. These double mutant mice expressed GM3 as their major ganglioside. In contrast to the single mutant mice, the double mutants displayed a sudden death phenotype and were extremely susceptible to induction of lethal seizures by sound stimulus. These results demonstrate unequivocally that gangliosides play an essential role in the proper functioning of the CNS.     

Ganglioside GM1 Contributes to the State of Insulin Resistance in Senescent Human Arterial Endothelial Cells

Vascular endothelial cells (ECs) play central roles in physiologically important functions of blood vessels and contribute to the maintenance of vascular integrity. Therefore, it is considered that the impairment of EC functions leads to the development of vascular diseases. However, the molecular mechanisms of the EC dysfunctions that accompany senescence and aging have not yet been clarified. The carbohydrate antigens carried by glycoconjugates (e.g. glycoproteins, glycosphingolipids, and proteoglycans) mainly present on the cell surface serve not only as marker molecules but also as functional molecules. In this study, we have investigated the abundance and functional roles of glycosphingolipids in human ECs during senescence and aging. Among glycosphingolipids, ganglioside GM1 was highly expressed in abundance on the surface of replicatively and prematurely senescent ECs and also of ECs derived from an elderly subject. Insulin signaling, which regulates important functions of ECs, is impaired in senescent and aged ECs. Actually, by down-regulating GM1 on senescent ECs and overloading exogenous GM1 onto non-senescent ECs, we showed that an increased abundance of GM1 functionally contributes to the impairment of insulin signaling in ECs. Taken together, these findings provide the first evidence that GM1 increases in abundance on the cell surface of ECs under the conditions of cellular senescence and aging and causes insulin resistance in ECs. GM1 may be an attractive target for the detection, prevention, and therapy of insulin resistance and related vascular diseases, particularly in older people.     

A possible biological function of carbohydrate structures which are typical of erythrocytes

Carbohydrates of erythrocyte glycoconjugates seem to be specifically designed so that they do not appreciably interact with other types of cells or ligands. This applies to glycosphingolipids (GSLs) and glycoproteins (GPs). An important distinction between the two types of glycoconjugates seems to be that GPs, apart from carrying carbohydrates, have some biological function which is most likely associated with their protein moieties. GSLs of erythrocytes, including the ABH, PP1 and Pk blood group substances, are viewed as energetically cheap membrane-packing substances filling in the membrane areas not covered by functional GPs. Their sole function should be the formation of inert carbohydrate protective layer at the membrane. The role of the inert carbohydrate structures in the development, tumorigenesis and evolution of blood group polymorphism is discussed.     

Novel class of glycosphingolipids involved in male fertility

Mice require testicular glycosphingolipids (GSLs) for proper spermatogenesis. Mutant mice strains deficient in specific genes encoding biosynthetic enzymes of the GSL pathway including Galgt1 (encoding GM2 synthase) and Siat9 (encoding GM3 synthase) have been established lacking various overlapping subsets of GSLs. Although male Galgt1-/- mice are infertile, male Siat9-/- mice are fertile. Interestingly, GSLs thought to be essential for male spermatogenesis are not synthesized in either of these mice strains. Hence, these GSLs cannot account for the different phenotypes. A novel class of GSLs was observed composed of eight fucosylated molecules present in fertile but not in infertile mutant mice. These GSLs contain polyunsaturated very long chain fatty acid residues in their ceramide moieties. GSLs of this class are expressed differentially in testicular germ cells. More importantly, the neutral subset of this new GSL class strictly correlates with male fertility. These data implicate polyunsaturated, fucosylated GSLs as essential for spermatogenesis and male mouse fertility.     

Glycosphingolipids of human plasma

A number of glycosphingolipids, including 10 gangliosides, not previously identified in human plasma have been characterized. The plasma contains 2 micrograms of lipid-bound sialic acid/ml plasma and 54% of the gangliosides are monosialo, 30% disialo, 10% trisialo, and 6% tetrasialo. Individual glycosphingolipids were purified by high-performance liquid chromatography and thin-layer chromatography, and were characterized on the basis of their chromatographic mobility, carbohydrate composition, hydrolysis by glycosidases, methylation analysis, and immunostaining with anti-glycosphingolipid antibodies. The monosialogangliosides were identified as GM3, GM2, sialosyl(2-3)- and sialosyl(2-6)lactoneotetraosylceramides, sialosyllacto-N-nor-hexaosylceramide, and sialosyllacto-N-isooctaosylceramide. The major gangliosides in the polysialo fractions contained a ganglio-N-tetraose backbone and were identified as GD3, GD1a, GD1b, and GQ1b. The most abundant neutral glycosphingolipids were glucosyl, lactosyl, globotriaosyl, globotetraosyl and lactoneotetraosylceramides. The other neutral glycosphingolipids, tentatively identified by immunostaining with monoclonal antibodies, contained H1, Lea, Leb, and lacto-N-fucopentose III (X hapten) structures.     

Glycolipid-mediated cell-cell recognition in inflammation and nerve regeneration

Cell surface complex carbohydrates have emerged as key recognition molecules, mediating physiological interactions between cells. Typically, glycans on one cell surface are engaged by complementary carbohydrate binding proteins (lectins) on an apposing cell, initiating appropriate cellular responses. Although many cell surface lectins have been identified in vertebrates, only a few of their endogenous carbohydrate ligands have been established. Each major class of cell surface glycans-glycoproteins, glycolipids, and proteoglycans-has been implicated as physiologically relevant lectin ligands. The current minireview focuses on findings that implicate glycosphingolipids as especially important molecules in cell-cell recognition in two different systems: the recognition of human leukocytes by E-selectin on the vascular endothelium during inflammation and the recognition of nerve cell axons by myelin-associated glycoprotein in myelin-axon stabilization and the regulation of axon regeneration.     

The fate and function of glycosphingolipid glucosylceramide

In higher eukaryotes, glucosylceramide is the simplest member and precursor of a fascinating class of membrane lipids, the glycosphingolipids. These lipids display an astounding variation in their carbohydrate head groups, suggesting that glycosphingolipids serve specialized functions in recognition processes. It is now realized that they are organized in signalling domains on the cell surface. They are of vital importance as, in their absence, embryonal development is inhibited at an early stage. Remarkably, individual cells can live without glycolipids, perhaps because their survival does not depend on glycosphingolipid-mediated signalling mechanisms. Still, these cells suffer from defects in intracellular membrane transport. Various membrane proteins do not reach their intracellular destination, and, indeed, some intracellular organelles do not properly differentiate to their mature stage. The fact that glycosphingolipids are required for cellular differentiation suggests that there are human diseases resulting from defects in glycosphingolipid synthesis. In addition, the same cellular differentiation processes may be affected by defects in the degradation of glycosphingolipids. At the cellular level, the pathology of glycosphingolipid storage diseases is not completely understood. Cell biological studies on the intracellular fate and function of glycosphingolipids may open new ways to understand and defeat not only lipid storage diseases, but perhaps other diseases that have not been connected to glycosphingolipids so far.     

Enzyme-catalyzed synthesis of carbohydrates

Several new enzymes of utility in the synthesis of carbohydrates have been reported during the past year. Additionally, the utility of several well studied enzymes has been expanded. Pyruvate aldolases, aldolase abzymes and both wild-type and mutated glycosidases have found increasing acceptance in the community. Preliminary reports suggest that thermophilic enzymes may possess significant advantages compared to their mesophilic counterparts for carbohydrate synthesis.     

Cloning and expression of genes related to the sucrose-metabolizing enzymes and carbohydrate changes in peach

To shed light on the relationship between sucrose metabolism and expression of genes related to sucrose-metabolizing enzymes, six genes encoding sucrose-metabolizing enzymes were isolated, and the levels of four main carbohydrates and related enzyme activities as well as the expression of these six genes were determined in fruits, leaves and phloem-enriched fraction throughout peach fruit development. Sucrose content in mature fruit ranked first followed by glucose, fructose and sorbitol in that order, while sorbitol was the highest and sucrose lowest in phloem-enriched fraction and leaves. Glucose and fructose had similar change patterns throughout fruit development. Cloning results reveal that the nucleotide sequences of the six genes have high similarity to corresponding genes isolated from other plants. In addition, the expression of these genes and the levels of related enzyme activities varied with tissue and stage of fruit development, suggesting a complexity in relationships between carbohydrates, enzymes activities and related gene expression. Sucrose phosphate synthase maybe a key enzyme involved in sucrose synthesis while sucrose synthase may mainly be responsible for sucrose synthesis in peach fruits at later stages of development. Further studies are needed to genetically and physiologically characterize these genes and enzymes in peach and to gain a better understanding of their functions and relationship with carbohydrate metabolism.     

Carbohydrate antigens as targets for active specific immunotherapy

 Carbohydrate antigens such as GM2, GD2 and GD3 (gangliosides), Lewis^y and globo-H (neutral glycolipids and glycoproteins), and Tn, TF and sTn (glycoproteins) are overexpressed in a variety of cancers. Antibodies against several of these carbohydrate antigens have been detected in sera from patients treated with cancer vaccines, and have been associated with a more favorable prognosis. Clinical responses have been reported after treatment with monoclonal antibodies against some of these antigens. Hence cell-surface carbohydrate antigens have been identified as suitable targets for immune attack by both active and passive immunotherapies. Different approaches have been adopted to induce immune responses against these carbohydrate antigens. These includes vaccination with whole or lysed tumor cells, purified or synthetic carbohydrates, immunogenic carbohydrate derivatives, or carbohydrates conjugated with immunogenic carriers and administered with immunological adjuvants. In the case of gangliosides, immunization with either whole tumor cells or cell lysates has only occasionally induced responses against carbohydrate antigens, and the antibodies were generally IgM antibodies of low titer. Compared with other methods of vaccination, conjugate vaccines have consistently induced the highest titer of IgM and IgG antibodies against gangliosides and other carbohydrate antigens. Preclinical and clinical studies with conjugate carbohydrate vaccines have induced IgM and IgG antibody responses capable of inducing complement-mediated cytotoxicity of tumor cells in vitro and associated with prolonged disease-free and overall survival in patients. Received: 6 August 1996 / Accepted: 20 September 1996     

Demonstration of diet-induced decoupling of fatty acid and cholesterol synthesis by combining gene expression array and 2H2O quantification

The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate-free diet. At the end of the dietary intervention, the two groups received (2)H(2)O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a custom-designed pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydrate-free diet. In support of these findings, (2)H(2)O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered.     

Localization of neutral and acidic glycosphingolipids in rat lens

Rat lens was found to contain several neutral and acidic glycosphingolipidsin lens epithelia, cortex and nucleus, and showed developmentalchanges in their content and localization. TLC-immunostainingof gangliosides revealed the enrichment of some ganglio-seriesgangliosides (GM3, GM1, GD3 and GD1b) in lens epithelia andthe presence of GM3 and GD3 in the lens nucleus. Immunohistochemicalstudies confirmed the distribution of GM3 and GM1 in anteriorlens epithelial cells and the cortex, with expression decreasingtoward the lens nucleus. Immunoreaction to GD3 was more intensein the lens nucleus than in epithelial cells. In contrast, theexpression of neolacto-series glycosphingolipids was restrictedto the lens nucleus. In order to investigate the pathologicalchanges of glycosphingolipids in cataract, galactose-inducedcataractous lenses were examined. However, no significant changeswere observed in the content and composition of glycosphingolipids.In addition, Lewis^x epitopes found in human cataractous lenseswere not detected in the cataractous lenses of galactosaemicrats and hereditary cataractous Emory mice. cataract gangliosides glcosphingolipids Lewis^x rat lens     

Facile and rapid direct gold surface immobilization with controlled orientation for carbohydrates

Effective surface immobilization is a prerequisite for numerous carbohydrate-related studies including carbohydrate-biomolecule interactions. In the present work, we report a simple and rapid modification technique for diverse carbohydrate types in which direct oriented immobilization onto a gold surface is accomplished by coupling the amine group of a thiol group-bearing aminophenyl disulfide as a new coupling reagent with an aldehyde group of the terminal reducing sugar in the carbohydrate. To demonstrate the generality of this proposed reductive amination method, we examined its use for three types of carbohydrates: glucose (monosaccharide), lactose (disaccharide), and GM1 pentasaccharide. Through successful mass identifications of the modified carbohydrates, direct binding assays on gold surface using surface plasmon resonance and electrochemical methods, and a terminal galactose-binding lectin assay using atomic force microscopy, we confirmed several advantages including direct and rapid one-step immobilization onto a gold surface and exposure of functional carbohydrate moieties through oriented modification of the terminal reducing sugar. Therefore, this facile modification and immobilization method can be successfully used for diverse biomimetic studies of carbohydrates, including carbohydrate-biomolecule interactions and carbohydrate sensor or array development for diagnosis and screening.     

Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex.

Although glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.     

Iminosugar-based inhibitors of glucosylceramide synthase increase brain glycosphingolipids and survival in a mouse model of Sandhoff disease

The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects.