Glycolipid Glycosyltransferases Research Articles

Golgi Phosphoprotein 3 Regulates the Physical Association of Glycolipid Glycosyltransferases.

Glycolipid glycosylation is an intricate process that mainly takes place in the Golgi by the complex interplay between glycosyltransferases. Several features such as the organization, stoichiometry and composition of these complexes may modify their sorting properties, sub-Golgi localization, enzymatic activity and in consequence, the pattern of glycosylation at the plasma membrane. In spite of the advance in our comprehension about physiological and pathological cellular states of glycosylation, the molecular basis underlying the metabolism of glycolipids and the players involved in this process remain not fully understood. In the present work, using biochemical and fluorescence microscopy approaches, we demonstrate the existence of a physical association between two ganglioside glycosyltransferases, namely, ST3Gal-II (GD1a synthase) and β3GalT-IV (GM1 synthase) with Golgi phosphoprotein 3 (GOLPH3) in mammalian cultured cells. After GOLPH3 knockdown, the localization of both enzymes was not affected, but the fomation of ST3Gal-II/β3GalT-IV complex was compromised and glycolipid expression pattern changed. Our results suggest a novel control mechanism of glycolipid expression through the regulation of the physical association between glycolipid glycosyltransferases mediated by GOLPH3.

Spatial Organization and Stoichiometry of N-Terminal Domain-Mediated Glycosyltransferase Complexes in Golgi Membranes Determined by Fret Microscopy

The functional link between glycolipid glycosyltransferases (GT) relies on the ability of these proteins to form organized molecular complexes. The organization, stoichiometry and composition of these complexes may impact their sorting properties, sub-Golgi localization, and may determine relative efficiency of GT in different glycolipid biosynthetic pathways. In this work, by using Förster resonance energy transfer microscopy in live CHO-K1 cells, we investigated homo- and hetero-complex formation by different GT as well as their spatial organization and molecular stoichiometry on Golgi membranes. We find that GalNAcT and GalT2 Ntd are able to form hetero-complexes in a 1:2 molar ratio at the trans-Golgi network and that GalT2 but not GalNAcT forms homo-complexes. Also, GalNAcT/GalT2 complexes exhibit a stable behavior reflected by its clustered lateral organization. These results reveals that particular topological organization of GTs may have functional implications in determining the composition of glycolipids in cellular membranes.

Defective GM3 Synthesis in Cog2 Null Mutant CHO Cells Associates to Mislocalization of Lactosylceramide Sialyltransferase in the Golgi Complex

The conserved oligomeric Golgi (COG) complex is a eight subunit (COG1 to 8) tethering complex involved in the retrograde trafficking of multiple Golgi processing proteins. Here we studied the glycolipid synthesis status in ldlC cells, a Cog2 null mutant CHO cell line. Biochemical studies revealed a block in the coupling between LacCer and GM3 synthesis, resulting in decreased levels of GM3 in these cells. Uncoupling was not attributable to decreased activity of the glycosyltransferase that uses LacCer as acceptor substrate (SialT1). Rather, immunocytochemical experiments evidenced a mislocalization of SialT1 as consequence of the lack of Cog2 in these cells. Co-immunoprecipitation experiments disclose a Cog2 mediated interaction of SialT1 with the COG complex member Cog1. Results indicate that cycling of some Golgi glycolipid glycosyltransferases depends on the participation of the COG complex and that deficiencies in COG complex subunits, by altering their traffic and localization, affect glycolipid composition.

Identification of a Site in Sar1 Involved in the Interaction with the Cytoplasmic Tail of Glycolipid Glycosyltransferases

Glycolipid glycosyltransferases (GGT) are transported from the endoplasmic reticulum (ER) to the Golgi, their site of residence, via COPII vesicles. An interaction of a (R/K)X(R/K) motif at their cytoplasmic tail (CT) with Sar1 is critical for the selective concentration in the transport vesicles. In this work using computational docking, we identify three putative binding pockets in Sar1 (sites A, B, and C) involved in the interaction with the (R/K)X(R/K) motif. Sar1 mutants with alanine replacement of amino acids in site A were tested in vitro and in cells. In vitro, mutant versions showed a reduced ability to bind immobilized peptides with the CT sequence of GalT2. In cells, Sar1 mutants (Sar1(D198A)) specifically affect the exiting of GGT from the ER, resulting in an ER/Golgi concentration ratio favoring the ER. Neither the typical Golgi localization of GM130 nor the exiting and transport of the G protein of the vesicular stomatitis virus were affected. The protein kinase inhibitor H89 produced accumulation of Sec23, Sar1, and GalT2 at the ER exit sites; Sar1(D189A) also accumulated at these sites, but in this case GalT2 remained disperse along ER membranes. The results indicate that amino acids in site A of Sar1 are involved in the interaction with the CT of GGT for concentration at ER exiting sites.

Post-translational and transcriptional regulation of glycolipid glycosyltransferase genes in apoptotic breast carcinoma cells: VII. Studied by DNA-microarray after treatment with l-PPMP

Functions of glycosphingolipids on the eukaryotic cell membranes during the onset of oncogenic processes and cell death are not well understood. Inhibitors of glycosphingolipid biosynthesis were recently found to trigger apoptosis in many carcinoma including breast cancer SKBR-3, MCF-7, and MDA-468 cells through either intrinsic or extrinsic apoptotic pathways as we previously reported. These anti-cancer inhibitors could increase ceramide concentration by blocking functions of glycolipid glycosyltransferases (GLTs). In this study, using a novel fluorescent dye (ASK-0) revealed the damage of cell organelle membranes by an inhibitor of glucosylceramide biosynthesis (L-PPMP). A highly drug- and cell-dependent regulation of MAPKs was also found by cis-platin and L-PPMP when inducing apoptosis in SKBR-3, MCF-7, and MDA-468 cells. A dose and time-dependent regulation of GLTs were investigated by enzymatic assay and DNA microarray analyses. These GLTs are involved in biosynthesis of Le(X) and sialosyl-Le(X) (neolactosyl-ceramide series) such as GalT-4 (UDP-Gal: LcOse3cer beta-galactosyltransferase, GalT-5 (UDP-Gal: nLcOse4Cer alpha1, 3galactosyltransferase, FucT-3 (GDP-Fucose: LM1 alpha1, 4fucosyltransferase). A similar effect was observed with the GLTs involved in the biosyntheses of Gg-series gangliosides, such as SAT-4 (CMP-NeuAc: GgOse4Cer alpha2, 3sialyltransferase, and SAT-2 (CMP-NeuAc: GM3 alpha2, 8sialyltransferase). The glycol-related gene DNA-microarrays also suggested the transcriptional regulation of several GLTs involved in the biosynthesis of neolactosylceramide containing cell-surface antigens in these apoptotic breast carcinoma cells. In the early apoptotic stages (2 to 6 h after L-PPMP treatment) in addition to GlcT-1 gene, several genes (betaGalTs and betaGlcNAcTs) in the SA-Le(a) pathway were stimulated.

Glycosylation of glycolipids in the Golgi complex

Gangliosides are a family of glycolipids characterized by containing a variable number of sialic acid residues. Nearly, all animal cells contain at least some class of ganglioside in their membranes, but membranes from the CNS are characterized by their high content of these lipids. The synthesis of the oligosaccharide moiety of glycolipids is carried out in the Golgi complex. In this study, I will discuss the cellular and molecular basis of the organization of the glycosylating machinery in the Golgi complex, with particular attention to the mutual relationships, sub-Golgi localization, and intracellular trafficking of glycolipid glycosyltransferases, and to their relationships with the corresponding glycolipid acceptors and sugar nucleotide donors. I will also discuss how the organization of the glycosylating machinery in the Golgi may adapt to events controlling glycolipid expression.

Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus.

Glycolipid glycosyltransferases catalyze the stepwise transfer of monosaccharides from sugar nucleotides to proper glycolipid acceptors. They are Golgi resident proteins that colocalize functionally in the organelle, but their intimate relationships are not known. Here, we show that the sequentially acting UDP-GalNAc:lactosylceramide/GM3/GD3 beta-1,4-N-acetyl-galactosaminyltransferase and the UDP-Gal:GA2/GM2/GD2 beta-1,3-galactosyltransferase associate physically in the distal Golgi. Immunoprecipitation of the respective epitope-tagged versions expressed in transfected CHO-K1 cells resulted in their mutual coimmunoprecipitation. The immunocomplexes efficiently catalyze the two transfer steps leading to the synthesis of GM1 from exogenous GM3 in the presence of UDP-GalNAc and UDP-Gal. The N-terminal domains (cytosolic tail, transmembrane domain, and few amino acids of the stem region) of both enzymes are involved in the interaction because (i) they reproduce the coimmunoprecipitation behavior of the full-length enzymes, (ii) they compete with the full-length counterpart in both coimmunoprecipitation and GM1 synthesis experiments, and (iii) fused to the cyan and yellow fluorescent proteins, they localize these proteins to the Golgi membranes in an association close enough as to allow fluorescence resonance energy transfer between them. We suggest that these associations may improve the efficiency of glycolipid synthesis by channeling the intermediates from the position of product to the position of acceptor along the transfer steps.

From the Cover: Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus

Glycolipid glycosyltransferases catalyze the stepwise transfer of monosaccharides from sugar nucleotides to proper glycolipid acceptors. They are Golgi resident proteins that colocalize functionally in the organelle, but their intimate relationships are not known. Here, we show that the sequentially acting UDP-GalNAc:lactosylceramide/GM3/GD3 beta-1,4-N-acetyl-galactosaminyltransferase and the UDP-Gal:GA2/GM2/GD2 beta-1,3-galactosyltransferase associate physically in the distal Golgi. Immunoprecipitation of the respective epitope-tagged versions expressed in transfected CHO-K1 cells resulted in their mutual coimmunoprecipitation. The immunocomplexes efficiently catalyze the two transfer steps leading to the synthesis of GM1 from exogenous GM3 in the presence of UDP-GalNAc and UDP-Gal. The N-terminal domains (cytosolic tail, transmembrane domain, and few amino acids of the stem region) of both enzymes are involved in the interaction because (i) they reproduce the coimmunoprecipitation behavior of the full-length enzymes, (ii) they compete with the full-length counterpart in both coimmunoprecipitation and GM1 synthesis experiments, and (iii) fused to the cyan and yellow fluorescent proteins, they localize these proteins to the Golgi membranes in an association close enough as to allow fluorescence resonance energy transfer between them. We suggest that these associations may improve the efficiency of glycolipid synthesis by channeling the intermediates from the position of product to the position of acceptor along the transfer steps.

Activities of glycolipid glycosyltransferases and sialidases during the early development of Xenopus laevis.

The activities of glycosyltransferases and sialidases, together with the ganglioside content and distribution, have been extensively studied in mammals, while the informations on tissues of other animals, including amphibian, are scarce. In this paper we present data on the activities of SAT-1, SAT-2, SAT-4, SAT-5, GlcNAcT-1, GalNAcT-1, GalT-6, and sialidases studied in Xenopus laevis embryos at different stages of development. The highest activity was found at days 4 and 5 of embryogenesis for glycosyltransferases and sialidases respectively; a tentative correlation between the in vitro activity of these enzymes and the content of neutral and acidic glycolipids is discussed.

Molecular cloning of eukaryotic glycoprotein and glycolipid glycosyltransferases; a survey

Molecular cloning of eukaryotic glycoprotein and glycolipid glycosyltransferases; a survey

Molecular cloning of eukaryotic glycoprotein and glycolipid glycosyltransferases: a survey.

The rapidity with which molecular sequence data are gathered continues to grow. The result is that, for many workers, it is increasingly difficult to keep abreast of the current state of play of molecular cloning, even for those genes that encode proteins of special interest. The clear success of the various worldwide genome projects has made this even more apparent, and by the end of 1996 the complete determination of the nucleotide sequences of the genomes of two eukaryotes, Saccharomyces cerevisiae and Caenorhabditis elegans, will have either been completed or will be nearing completion. This article is an attempt to provide, in an easily accessible format, a compilation of genes and cDNAs that have been sequenced and deposited in GenBank that encode transferase enzymes involved in eukaryotic glycoprotein or glycolipid biosynthesis. The full sequence information can be easily retrieved from a databank, e.g. GenBank, using the relevant accession number(s).

Evidence for the presence of an endogenous cytosolic protein inhibitor of intestinal fucosyltransferase activities

Soluble endogenous inhibitory activities for glycoprotein: α(1–2) and α (1–3) fucosyltransferases are demonstrated in rat small intestinal cytosol. These inhibitors are retained on DEAE-cellulose and are eluted as two fractions A and B. Fraction B is non dialyzable, heat stable and pronase-resistant and consists probably of polynucleotides. Fraction A is also non-dialyzable, but is thermolabile and pronase-sensitive, suggesting that it contains proteins. The inhibition of fucosyltransferase activity by fraction A is competitive for GDP-fucose and non-competitive for the glycoprotein substrate. Inhibition is not due to interfering enzymatic activities (glycosyl-nucleotide pyrophosphatases, glycosidases or proteases) and is reversible. This protein inhibitor, with a molecular weight of 60,000, is found only in the intestine and the pancreas and appears to be different from the previously reported inhibitors of brain glycolipid glycosyltransferases.

Glycolipid glycosyltransferases in human embryonal carcinoma cells during retinoic acid induced differentiation

Retinoic acid induced differentiation of TERA-2-derived human embryonal carcinoma cells is accompanied by a dramatic reduction of extended globo-series glycolipids, including galactosyl globoside, sialylgalactosyl globoside, and globo-A antigen (each recognized by specific MoAbs). Associated with these glycolipid changes, the activities of two key enzymes, alpha 1----4 galactosyltransferase (for synthesis of globotriaosyl core structure) and beta 1----3 galactosyltransferase (for synthesis of galactosyl globoside), were found to be reduced 3- to 4-fold. The latter enzyme plays a key role in the synthesis of extended globo-series structures, and its characterization has not been reported previously. Therefore, its catalytic activity was studied in detail, including substrate specificity, detergent and phospholipid effects, pH and cation requirements, and apparent Km. During retinoic acid induced differentiation, a series of Lex glycolipid antigens (recognized by anti-SSEA-1 antibody) and their core structures (lacto-series type 2 chains) increase dramatically. In parallel with these changes in glycolipid expression, the activities of two key enzymes, beta 1----3 N-acetylglucosaminyltransferase (for extension of lacto-series type 2 chain) and alpha 1----3 fucosyltransferase (for synthesis of Lex structure), were found to increase by 4- and 2-fold, respectively. Similarly, an increase in the expression of several gangliosides (e.g., GD3 and GT3) during retinoic acid induced differentiation was mirrored by a 4-fold increase in the activity of alpha 2----3 sialyltransferase (for synthesis of ganglio core structure, GM3). The results suggest a coordinate regulation of key glycosyltransferases involved in core structure assembly and terminal chain modification.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat brain glycosyltransferase activities during postnatal development

Enzymatic activity of seven glycoprotein and glycolipid glycosyltransferases was studied in microsomal fractions during postnatal development of rat brain from 3 to 42 days after birth. Specific enzymatic variations were detected for some glycosyltransferases, i.d. a maxima was shown at 7 days post partum for the glycoprotein galactosyltransferase and another one at 21 days for galactosylceramide biosynthesis, this latter correlated to myelin synthesis. However, a regular and important activity maxima was always detected for six of the enzymes studied (asialofetuin fucosyltransferase, ovomucoid galactosyltransferase, dolichyl phosphate mannose synthase, ceramide, galactosylceramide and glucosylceramide galactosyltransferases) at the period from 35 to 40 days post partum. As this period corresponds to immediate post-puberty, an endocrinoneuronal control of the glycosyltransferases by sexual hormones is suggested.

Differential activities of glycolipid glycosyltransferases in Tay-Sachs disease: studies in cultured cells from cerebrum.

Four different glycolipid:glycosyltransferase activities involved in the biosynthesis in vitro of gangliosides and blood group-related glycosphingolipids have been tested in a simian virus 40-transformed glial cell culture derived from the cerebrum of a fetus with Tay-Sachs disease (TSD). The TSD cultured brain cells contained little activity of either UDP-Gal:GM2(beta 1-3)galactosyltransferase (GalT-3; EC 2.4.1.62), which catalyzes the formation of GM1a from GM2 (tay-Sachs) ganglioside, or GDP-Fuc:nLcOse4Cer (alpha 1-2)fucosyltransferase (FucT-2; EC 2.4.1.89), which catalyzes the formation of H1 glycolipid from nLcOse4Cer. These cells contained a potent inhibitor of the second reaction (catalyzed by a Golgi-rich membrane fraction from bovine spleen), whereas no inhibition of the first reaction (catalyzed by a membrane fraction from 14-day-old embryonic chicken brain) was observed. The activity of UDP-Gal:LcOse3Cer(beta 1-4)galactosyltransferase (GalT-4; EC 2.4.1.86) was 30- to 80-fold higher than the activity of GalT-3. The presence of CMP-AcNeu:nLcOse4Cer sialyltransferase activity and the absence of either GalT-3 or FucT-2 suggested a probable pathway for the synthesis of sialylneolactotetraosylceramide [GM1b(GlcNAc)] in addition to a specific blockage of GM1a ganglioside synthesis from GM2 in these TSD transformed cells.

Glycolipid glycosyl transferases of a hamster cell line in culture I. Kinetic constants substrate and donor nucleotide sugar specificities

The properties of enzymes catalysing the transfer of a galactose from UDP-galactose to exogenous ceramide monohexoside and ceramide di-hexoside derived from the Syrian hamster cell line NIL 2 were studied. The products of these enzymes were characterized by chemical and enzymatic methods. Kinetic analyses showed that the enzymes are susceptible to inhibition and activation by a number of substrate analogues. The kinetic and inhibition constants, glycolipid substrate specificity and nucleotide sugar donor specificity have been studied.

Glycolipid glycosyl transferases of a hamster cell line in culture II. Subcellular distribution and the effect of culture age and density

The activities of two galactosyl transferases catalysing the formation of di- and tri-glycosyl ceramides in NIL-2 hamster cells have been studied with respect to culture age and density, subcellular distribution, and transformation of cells by virus. The activity of the transferases was found to increase considerably as culture density increased, although maximal activities were found before appreciable cell contact occurred. The highest transferase activities were found in the endoplasmic reticulum. Virus transformation reduces the activity of the transferase catalysing triglycosyl ceramide synthesis, while the transferase catalysing diglycosyl ceramide synthesis is slightly increased. There is no evidence that the transformed cells produce a dialysable soluble inhibitor of transferase activities.

Alterations of glycolipid glycosyltrasnferase levels in lymphoma and lymphoid tissues of lymphoma-bearing hamsters.

Thymus from lymphoma-bearing hamsters in advanced stages of tumor growth showed enhanced activities of four glycolipid-glycosyltransferases which lead to the step-wise synthesis of lactosylceramide to Forssman hapten. The alpha-galactosyltransferase activity of the thymus was decreased until day 6 of inoculation but increased thereafter. The activities of all the transferases tested were significantly lowered in lymphoma tissue. The level of UDP-galactose pyrophosphatase did not correlate with the transferase activities.

Changes in glycolipid glycosyltransferases and glutamate decarboxylase and their relationship to differentiation in neuroblastoma cells

Glycolipid glycosyltransferase activities involved in the biosynthesis in vitro of neutral and acidic glycosphingolipids were measured in C-1300 tumors and cloned cells derived therefrom. An adrenergic clone (NIE-115) was grown in tissue culture in the presence of dibutyryl cyclic AMP and the levels of glycosyltransferases were measured before and after differentiation. Increased activities of galactosyltransferases and sialyl-transferases with a concomitant increase in glutamate decarboxylase activity (the enzyme that catalyzes the synthesis of an inhibitory neurotransmitter, γ-aminobutyric acid) were observed.

Biosynthesis of globoside and Forssman-related glycosphingolipid in mouse adrenal Y-1 tumor cells

Summary The activities of four glycolipid glycosyltransferases involved in the biosynthesis in vitro of globoside and Forssman hapten were measured in normal mouse adrenal tissues, Y-1 mouse adrenal tumor cells, and tumors derived therefrom. These enzyme activities, found in Golgi-rich membranes isolated on a sucrose density gradient, were higher in the tumor cell cultures than in normal mouse adrenal tissue. The ratio between long chain oligoglycosylceramides and short chain glycosphingolipids was higher in the case of Y-l-K cells treated with dibutyryl cAMP than in concanavalin A, colchicine treated, or untreated control cells in culture.