Direct assay of glycosphingolipid glycosyltransferase activities on thin-layer chromatogram

A modified method for the determination of glycosphingolipid glycosyltransferase activity using high-performance thin-layer chromatographic (HPTLC) plates has been developed. An acceptor glycosphingolipid was chromatographed on an HPTLC plate and was incubated with an enzyme mixture and an appropriate radioactive sugar nucleotide. After incubation, the plate was washed with phosphate buffer and 2% Tween 80. The radiolabeled reaction product was scrapped off the plate and the radioactivity determined using a liquid scintillation counter or, alternatively, the plate was exposed to an X-ray film to reveal the radioactive product. We have used this assay method to determine the activities of rat brain cytidine 5'-monophosphate-N-acetylneuraminic acid: LacCer-, GM3-, GM1-, or GD3-sialyltransferases. This method is sensitive, fast, and reliable and is capable of assaying simultaneously the activities of glycosyltransferases with multiple acceptor specificity. It should be useful in monitoring the enzyme activities present in various column fractions during chromatographic fractionation of glycosyltransferases with different substrate specificities.     

Total metabolic flow of glycosphingolipid biosynthesis is regulated by UDP-GlcNAc:lactosylceramide beta 1-->3N-acetylglucosaminyltransferase and CMP-NeuAc:lactosylceramide alpha 2-->3 sialyltransferase in human hematopoietic cell line HL-60 during differentiation.

We have previously reported that ganglioside GM3 was remarkably increased during monocytoid differentiation of human myelogenous leukemia cell line HL-60 cells and that neolacto series gangliosides (NeuAc-nLc) were enriched during granulocytoid differentiation. In addition, HL-60 was differentiated into monocytic lineage by exogenous GM3 and into granulocytoid by NeuAc-nLc. In the present report, the enzymatic bases of glycosphingolipid biosynthesis in HL-60 during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid were investigated. The following results were of particular interest. (i) Lactosylceramide alpha 2-->3 sialyltransferase (GM3 synthase) was remarkably up-regulated during monocyte differentiation, while the GM3 synthase level did not change in granulocytic differentiation. (ii) By contrast, lactosylceramide beta 1-->3N-acetylglucosaminyltransferase (Lc3Cer synthase) was down-regulated during monocytic differentiation, while the activity of Lc3Cer synthase was found to increase in granulocytic differentiation. (iii) The activities of four downstream glycosyltransferases (for synthesis of NeuAc-nLc) were found to increase or to remain unchanged during monocytic and granulocytic differentiation. These results strongly suggested the following. The dramatic GM3 increase and the decrease of NeuAc-nLc during monocytic differentiation are the consequences of the up-regulation of GM3 synthase and the down-regulation of Lc3Cer synthase, although the downstream enzymes are ready to catalyze their enzyme reactions. The notable increase of NeuAc-nLc and the relative decrease of GM3 during granulocytic differentiation are the results of the unchanged level of GM3 synthase and the up-regulation of Lc3Cer synthase together with the activation of the downstream glycosyltransferases. These results suggest that these two key upstream glycosyltransferases, GM3 synthase and Lc3Cer synthase, play critical roles in regulating the glycosphingolipid biosynthesis in HL-60 cells during differentiation. This switching mechanism of these two glycosyltransferases, together with our previous findings, might be one of the most important parts of the determining system of differentiation direction in human myeloid cells into monocytic or granulocytic lineages.     

Degradation of dilauroylphosphatidylcholine by phospholipase A2 in monolayers containing glycosphingolipids

The ability of phospholipase A2 from porcine pancreas to degrade all of the available dilauroylphosphatidylcholine in mixed monolayers with galactocerebroside, sulfatide, or ganglioside GM1 was investigated at different constant surface pressures. Under the conditions used the interfacial glycosphingolipid composition was continuously enriched as the enzyme action proceeded. The total percentage of phospholipid degradation depends on the surface pressure and on the type of glycosphingolipid. The presence of sulfatide activates the enzyme while galactocerebroside and ganglioside GM1 are inhibitory. The extent of phospholipid hydrolysis is independent of the effect of glycosphingolipids on the enzyme velocity. This is so when the latter is measured either in conditions of constant glycosphingolipid composition and zero-order kinetics [Bianco, I.D., Fidelio, G.D., & Maggio, B. (1989) Biochem. J. 258, 95-99] or under variable surface composition as in the present work. The modulation of phospholipase A2 activity by glycosphingolipids operates at two independent levels. One controls the rate of enzyme activity, and the other modulates the total extent of substrate degradation. This depends on the initial interaction of the enzyme with the interface. The glycosphingolipid effect on the activity is different depending on whether the enzyme has access to the substrate from the subphase or is already adsorbed to the lipid interface.     

Cellular and molecular biology of glycosphingolipid glycosylation

Brain tissue is characterized by its high glycosphingolipid content, particularly those containing sialic acid (gangliosides). As a result of this observation, brain tissue was a focus for studies leading to the characterization of the enzymes participating in ganglioside biosynthesis, and their participation in driving the compositional changes that occur in glycolipid expression during brain development. Later on, this focus shifted to the study of cellular aspects of the synthesis, which lead to the identification of the site of synthesis in the neuronal soma and their axonal transport toward the periphery. In this review article, we will focus in subcellular aspects of the biosynthesis of glycosphingolipid oligosaccharides, particularly the mechanisms underlying the trafficking of glycosphingolipid glycosyltransferases from the endoplasmic reticulum to the Golgi, those that promote their retention in the Golgi and those that participate in their topological organization as part of the complex membrane bound machinery for the synthesis of glycosphingolipids.     

Uncoupling of ganglioside biosynthesis by Brefeldin A

We have studied the effect of Brefeldin A (BFA), an antiviral antibiotic, on glycosphingolipid metabolism in primary cultured cerebellar cells. Cells were labeled metabolically with [14C]galactose, or pulse-labeled with precursors of glycosphingolipid biosynthesis; i.e., [14]serine, [3H]palmitic acid or [3H]sphingosine. In all cases BFA (1 microgram/ml) strongly inhibited (75-95%) ganglioside biosynthesis beyond the stage of GM3 and GD3, that is the formation of GM1, GD1a, GT1b and GQ1b. Simultaneously an accumulation of GlcCer, LacCer, GM3 and GD3 was observed (up to 2000%). These effects could be reversed fully by removal of the BFA from the culture medium. These results indicate that the LacCer-, GM3- and GD3-synthases of murine cerebellar cells are localized together on the proximal site of the Golgi apparatus, probably in the cis-Golgi compartment. It is probable that sphingomyelin synthase and some of the other glycosyltransferases involved in ganglioside biosynthesis are localized in distinct compartments beyond the cis Golgi.     

Effect of a fatty acid moiety of phospholipid and ceramide on purified GalT-3 (UDP-Gal:GM2 beta 1-3 galactosyltransferase) activity from embryonic chicken brain

Galactosyltransferase, GalT-3 (UDP-Gal:GM2 beta 1-3 galactosyltransferase) has been characterized and solubilized from 19-day-old embryonic chicken brain, and purified to over 2000-fold using mixed-modal chromatography on a omega-aminohexyl Sepharose column and affinity chromatography on a UDP-hexanolamine Sepharose column. The activity of purified GalT-3 was modulated by phospholipids in vitro with stimulation observed specifically with dipalmitoyl phosphatidylethanolamine (PE). All natural phospholipids tested (PE, PC and PI) inhibited GalT-3 activity. Enzyme activity was affected by the structure of the phospholipid vesicle. It was stabilized by the hexagonal (dipalmitoyl PE) structure and inhibited by the bilayer (dielaidoyl PE) structure. The long-chain fatty acid moiety of the glycosphingolipid substrate, GM2, was found to be necessary for optimum enzyme activity. In the absence of fatty acid, the modified substrates, lyso-GM2 and acetyl-GM2, had a 10-fold increased Km and a 4-8 fold decreased Vmax compared to the normal substrate. We postulate that GalT-3 belongs to a group of glycosyltransferases having recognition for both the carbohydrate as well as the hydrophobic domains (HY-CARS) of their substrates and that the fatty acid moiety of either the substrate (GM2) or a heterotropic effector (phospholipid) plays an important role in regulating the activity of this enzyme.     

Sequential changes in glycolipid expression during human B cell differentiation: enzymatic bases

We have previously reported that human B cell differentiation is accompanied by sequential changes in glycosphingolipid expression. Pre-B cells contain lacto-series type II chain-based glycolipids and GM3 ganglioside; mature/activated B cells do not synthesize lacto-series compounds but express GM3 and globo-series glycolipids (Gb3 and Gb4); terminally differentiated B cells, in addition to these compounds, also contain GM2 ganglioside. At the cell surface, Gb3, Gb4 and GM2 constitute stage-specific antigens. To elucidate the biosynthetic mechanism leading to these modifications we have compared activities of the glycosyltransferases involved in the core structure assembly and the first elongation steps of neo-lacto, ganglio- and globo-series glycolipids. These glycosyltransferase activities have been measured in B cell lines and normal B lymphocytes at various stages of differentiation. We first determined the optimal requirements of the four glycosyltransferases which wynthesize Lc3, GM3, Gb4 and GM2 glycolipids in B lymphocytes and then tested these enzymes and the Gb3 synthetase in the selected B cells. The following results were obtained: ß1 → 3N-Acetylglucosaminyltransferase (Lc3 synthetase) has a high activity in pro- and pre-B cells whereas it is undetectable in more differentiated cells; α2 → 3 sialyltransferase (GM3 synthetase) is activated from the pre-B cell stage to the terminally differentiated myeloma cells; α → 4 galactosyltransferase (Gb3 synthetase) is only detected in cells representing the late stages of B cell differentiation; ß1 → 3N-Acetylgalactosaminyltransferase (Gb4 synthetase) is only found in some lymphoblastoid cell lines, representative of activated B cells whereas the ß1 → 4 N-Acetylgalactosaminyltransferase (GM2 synthetase) has a high activity in these lymphoblastoid cell lines and in terminally differentiated myeloma cells. These results suggest that the sequential shifts in the three major glycosphingolipid series observed during B cell differentiation are mostly due to sequential activations of the corresponding glycosyltransferases.     

Egghead and brainiac are essential for glycosphingolipid biosynthesis in vivo

The Drosophila genes, brainiac and egghead, encode glycosyltransferases predicted to act sequentially in early steps of glycosphingolipid biosynthesis, and both genes are required for development in Drosophila. egghead encodes a beta4-mannosyltransferase, and brainiac encodes a beta3-N-acetylglucosaminyltransferase predicted by in vitro analysis to control synthesis of the glycosphingolipid core structure, GlcNAcbeta1-3Manbeta1-4Glcbeta1-Cer, found widely in invertebrates but not vertebrates. In this report we present direct in vivo evidence for this hypothesis. egghead and brainiac mutants lack elongated glycosphingolipids and exhibit accumulation of the truncated precursor glycosphingolipids. Furthermore, we demonstrate that despite fundamental differences in the core structure of mammalian and Drosophila glycosphingolipids, the Drosophila egghead mutant can be rescued by introduction of the mammalian lactosylceramide glycosphingolipid biosynthetic pathway (Galbeta1-4Glcbeta1-Cer) using a human beta4-galactosyltransferase (beta4Gal-T6) transgene. Conversely, introduction of egghead in vertebrate cells (Chinese hamster ovary) resulted in near complete blockage of biosynthesis of glycosphingolipids and accumulation of Manbeta1-4Glcbeta1-Cer. The study demonstrates that glycosphingolipids are essential for development of complex organisms and suggests that the function of the Drosophila glycosphingolipids in development does not depend on the core structure.     

The glycosphingolipid composition and glycosyltransferase activities of the small intestinal mucosa of testosterone-treated rats

Prior studies have demonstrated that sex hormones can influence the glycosphingolipid composition of different organs, including small intestine. However, to date, the effects of testosterone on glycosphingolipids of rat small intestinal mucosa have not been examined. Experiments were conducted to examine the effect of subcutaneous administration of synthetic testosterone (500 micrograms/100 g body wt.) on the gangliosides and neutral glycosphingolipids of rat small intestinal mucosa. Their results demonstrated that testosterone administrations: (i) increased the ganglioside content including hematoside (GM3); (ii) increased the total content of neutral glycosphingolipids, which was due to the increases in glucosylceramide and globotriaosylceramide; (iii) increased the activities of cytidine 5'-monophosphate-N-acetylneuraminic acid: lactosylceramide sialyltransferase, and UDPgalactose: lactosylceramide galactosyltransferase; (iv) increased the percentage of the long chain base phytosphingosine in hematoside, glucosyl-, and globotriaosylceramide; and (v) significantly altered the fatty acid composition of each of these glycosphingolipids. These results demonstrate that administration of testosterone induces alterations in glycosphingolipid composition and glycosyltransferases activities in rat small intestinal mucosa.     

Carbohydrate and hydrophobic-carbohydrate recognition sites (CARS and HY-CARS) in solubilized glycosyltransferases

Six different glycosyltransferases that are active with glycosphingolipid substrates have been purified from Golgi-membranes after solubilization with detergents. It appears that GalT-4(UDP-Gal:GlcNAc-R1 beta 1-4GalT), GalNAcT-2(UDP-Gal:Gal alpha-R2 beta 1-3GalNAcT) and FucT-2(GDP-Fuc:Gal beta GlcNAc-R3 alpha 1-2FucT) are specific for oligosaccharides bound to ceramide or to a protein moiety. These are called CARS (carbohydrate recognition sites) glycosyltransferases (GLTs). On the other hand, GalT-3(UDP-Gal:GM2 beta 1-3GalT), GalNAcT-1(UDP-GalNAc:GM3 beta 1-4GalNAcT) and FucT-3 (GDP-Fuc:LM1 alpha 1-3FucT) recognize both hydrophobic moieties (fatty acid of ceramide) as well as the oligosaccharide chains of the substrates. These GLTs are called HY-CARS (hydrophobic and carbohydrate recognition sites). D-Erythro-sphingosine (100-500 microM) modulates the in vitro activities of these GLTs. Modulation depends on the binding of D-sphingosine to a protein backbone, perhaps on more than one site and beyond transmembrane hydrophobic domains. Control of GLTs by free D-sphingosine was suggested with the concomitant discovery of ceramide glycanase in rabbit mammary tissues. The role of free sphingosine as an in vivo homotropic modulator of glycosyltransferases is becoming apparent.     

Accumulation of glycosphingolipids in human atherosclerotic plaque and unaffected aorta tissues

We have measured the levels of glycosphingolipids and the activityof glycosphingolipid glycosyltransferases in human aortic intimaand media from patients who died of atherosclerosis. The effectsof lactosylceramide (LacCer) and glucosylceramide (GlcCer) fromplaque intima on smooth muscle cell proliferation were assessed.When the GIcCer data was expressed as (pg GlcCer/mg cholesteroland/mg total phospholipid, a 28-fold and 7-told increase inplaque intima compared to normal intima was observed. Similarly,the level of LacCer was elevated 5-fold and 4-fold, respectively,compared to unaffected intima. The activity of UDPGicCer: ceramideß1     

Metabolic pathways and intracellular trafficking of gangliosides

Gangliosides constitute a large and heterogeneous family of acidic glycosphingolipids that contain one or more sialic acid residues and are expressed in nearly all vertebrate cells. Their de novo synthesis starts at the endoplasmic reticulum and is continued by a combination of glycosyltransferase activities at the Golgi complex, followed by vesicular delivery to the plasma membrane. At the cell surface, gangliosides participate in a variety of physiological as well as pathological processes. The cloning of genes for most of the glycosyltransferases responsible for ganglioside biosynthesis has produced a better understanding of the cellular and molecular basis of the ganglioside metabolism. In addition, the ability to delete groups of glycosphingolipid structures in mice has been enormously important in determining their physiological roles. Recently, a number of enzymes for ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane, which might contribute to modulate local glycolipid composition, and consequently, the cell function.     

Glycosphingolipid metabolic reprogramming drives neural differentiation

Neural development is accomplished by differentiation events leading to metabolic reprogramming. Glycosphingolipid metabolism is reprogrammed during neural development with a switch from globo‐ to ganglio‐series glycosphingolipid production. Failure to execute this glycosphingolipid switch leads to neurodevelopmental disorders in humans, indicating that glycosphingolipids are key players in this process. Nevertheless, both the molecular mechanisms that control the glycosphingolipid switch and its function in neurodevelopment are poorly understood. Here, we describe a self‐contained circuit that controls glycosphingolipid reprogramming and neural differentiation. We find that globo‐series glycosphingolipids repress the epigenetic regulator of neuronal gene expression AUTS2. AUTS2 in turn binds and activates the promoter of the first and rate‐limiting ganglioside‐producing enzyme GM3 synthase, thus fostering the synthesis of gangliosides. By this mechanism, the globo–AUTS2 axis controls glycosphingolipid reprogramming and neural gene expression during neural differentiation, which involves this circuit in neurodevelopment and its defects in neuropathology.     

Use of the enzyme-linked immunoadsorbent assay to monitor the purification of glycosphingolipid antigens by high-performance liquid chromatography

An enzyme-linked immunoadsorbent assay (ELISA) technique has been applied to the analysis of glycosphingolipid fractions separated by high-performance liquid chromatography. Nanogram amounts of selected fractions were placed in microtiter wells and analyzed for glycosphingolipids carrying carbohydrate epitopes recognized by monoclonal antibodies using an avidin-biotin enzyme system (ABC reagents). A large number of fractions (more than 100) can be conveniently evaluated for the presence of glycosphingolipids recognized by one or more monoclonal antibodies in a single analysis. This method is a rapid and sensitive procedure for monitoring the purification of glycosphingolipid antigens and can be used in conjunction with immunostaining of glycosphingolipids separated by thin-layer chromatography.     

Engineered Biosynthesis of Disaccharide-Modified Polyene Macrolides

Recent work has uncovered genes for two glycosyltransferases that are thought to catalyze mannosylation of mycosaminyl sugars of polyene macrolides. These two genes are nypY from Pseudonocardia sp. strain P1 and pegA from Actinoplanes caeruleus. Here we analyze these genes by heterologous expression in various strains of Streptomyces nodosus, producer of amphotericins, and in Streptomyces albidoflavus, which produces candicidins. The NypY glycosyltransferase converted amphotericins A and B and 7-oxo-amphotericin B to disaccharide-modified forms in vivo. The enzyme did not act on amphotericin analogs lacking exocyclic carboxyl or mycosamine amino groups. Both NypY and PegA acted on candicidins. This work confirms the functions of these glycosyltransferases and provides insights into their acceptor substrate tolerance. Disaccharide-modified polyenes may have potential as less toxic antibiotics.     

Photoaffinity labelling of glycosyltransferases.

The photoaffinity analogues 5-azido-UDP-glucose and 5-azido-UDP-glucuronic acid have proven to be valuable biochemical tools in the studies of nucleoside diphosphate sugar-utilizing enzymes, especially membrane-associated glycosyltransferases. A summary of the past and current uses of these analogues is presented, as well as photoaffinity data for the enzyme UDP-glucose: dolichylphosphate glucosyltransferase (Glc-P-Dol synthase). This enzyme has served as a model membrane-associated glycosyltransferase for demonstrating the uses of 5-azido-UDP-glucose. The advantages of using photoaffinity analogues for the purification and characterization of glycosyltransferases are presented, as well as an outline of the general procedures which can be used in conjunction with these analogues.     

Experimental evolution and genome sequencing reveal variation in levels of clonal interference in large populations of bacteriophage φX174

Background

The first step of GPI anchor biosynthesis is catalyzed by PIG-A, an enzyme that transfers N -acetylglucosamine from UDP- N -acetylglucosamine to phosphatidylinositol. This protein is present in all eukaryotic organisms ranging from protozoa to higher mammals, as part of a larger complex of five to six 'accessory' proteins whose individual roles in the glycosyltransferase reaction are as yet unclear. The PIG-A gene has been shown to be an essential gene in various eukaryotes. In humans, mutations in the protein have been associated with paroxysomal noctural hemoglobuinuria. The corresponding PIG-A gene has also been recently identified in the genome of many archaeabacteria although genes of the accessory proteins have not been discovered in them. The present study explores the evolution of PIG-A and the phylogenetic relationship between this protein and other glycosyltransferases.

Results

In this paper we show that out of the twelve conserved motifs identified by us eleven are exclusively present in PIG-A and, therefore, can be used as markers to identify PIG-A from newly sequenced genomes. Three of these motifs are absent in the primitive eukaryote, G. lamblia. Sequence analyses show that seven of these conserved motifs are present in prokaryote and archaeal counterparts in rudimentary forms and can be used to differentiate PIG-A proteins from glycosyltransferases. Using partial least square regression analysis and data involving presence or absence of motifs in a range of PIG-A and glycosyltransferases we show that (i) PIG-A may have evolved from prokaryotic glycosyltransferases and lipopolysaccharide synthases, members of the GT4 family of glycosyltransferases and (ii) it is possible to uniquely classify PIG-A proteins versus glycosyltransferases.

Conclusion

Besides identifying unique motifs and showing that PIG-A protein from G. lamblia and some putative PIG-A proteins from archaebacteria are evolutionarily closer to glycosyltransferases, these studies provide a new method for identification and classification of PIG-A proteins.     

Neobiosynthesis of Glycosphingolipids by Plasma Membrane-associated Glycosyltransferases

Gangliosides, complex glycosphingolipids containing sialic acids, are synthesized in the endoplasmic reticulum and in the Golgi complex. These neobiosynthesized gangliosides move via vesicular transport to the plasma membrane, becoming components of the external leaflet. Gangliosides can undergo endocytosis followed by recycling to the cell surface or sorting to the Golgi complex or lysosomes for remodeling and catabolism. Recently, glycosphingolipid catabolic enzymes (glycohydrolases) have been found to be associated with the plasma membrane, where they display activity on the membrane components. In this work, we demonstrated that ecto-ganglioside glycosyltransferases may catalyze ganglioside synthesis outside the Golgi compartment, particularly at the cell surface. Specifically, we report the first direct evidence of expression and activity of CMP-NeuAc:GM3 sialyltransferase (Sial-T2) at the cell surface of epithelial and melanoma cells, with membrane-integrated ecto-Sial-T2 being able to sialylate endogenously synthesized GM3 ganglioside as well as exogenously incorporated substrate. Interestingly, we also showed that ecto-Sial-T2 was able to synthesize GD3 ganglioside at the cell surface using the endogenously synthesized cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) available at the extracellular milieu. In addition, the expression of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase (GalNAc-T) was also detected at the cell surface of epithelial cells, whose catalytic activity was only observed after feeding the cells with exogenous GM3 substrate. Thus, the relative interplay between the plasma membrane-associated glycosyltransferase and glycohydrolase activities, even when acting on a common substrate, emerges as a potential level of regulation of the local glycosphingolipid composition in response to different external and internal stimuli.