Regulated translocation of neutral sphingomyelinase-2 to the plasma membrane drives insulin resistance in steatotic hepatocytes.

Obesity-associated diabetes is linked to the accumulation of ceramide in various organs, including the liver. The exact mechanisms by which ceramide contributes to diabetic pathology are unclear, but one proposed scenario is that ceramide accumulation may inhibit insulin signaling pathways. It is unknown however whether the excess ceramide is generated proximal to the insulin receptor, that is, at the plasma membrane (PM), where it could affect the insulin signaling pathway directly, or the onset of insulin resistance is due to ceramide-induced mitochondrial dysfunction and/or lipotoxicity. Using hepatic cell lines and primary cultures, gain- and loss- of function approach, and state-of-the art lipid imaging, this study shows that PM-associated neutral sphingomyelinase 2 (nSMase2) regulates ceramide homeostasis in fat-loaded hepatocytes and drives the onset of insulin resistance. Our results provide evidence of a regulated translocation of nSMase2 to the PM which leads to local generation of ceramide and insulin resistance in cells treated with palmitic acid (PAL), a type of fat commonly found in diabetogenic diets. Oleic acid, which also causes accumulation of lipid droplets, does not induce nSMase2 translocation and insulin resistance. Experiments using the acyl-biotin exchange method to quantify protein palmitoylation show that cellular PAL abundance regulates the rate of nSMase2 palmitoylation. Furthermore, while inhibition of nSMase2 with GW4869 prevents PAL-induced insulin resistance, the overexpression of wild type nSMase2 but not palmitoylation-defective mutant protein potentiates the suppressive effect of PAL on insulin signaling. Overall, this study identifies nSMase2 as a novel component of the mechanism of insulin resistance onset in fat-loaded hepatocytes, that is, cell-autonomous and driven by PAL.

Incorporation and visualization of azido-functionalized N-oleoyl serinol in Jurkat cells, mouse brain astrocytes, 3T3 fibroblasts and human brain microvascular endothelial cells.

The synthesis and biological evaluation of azido-N-oleoyl serinol is reported. It mimicks biofunctional lipid ceramides and has shown to be capable of click reactions for cell membrane imaging in Jurkat and human brain microvascular endothelial cells.

Prenatal alcohol exposure triggers ceramide-induced apoptosis in neural crest-derived tissues concurrent with defective cranial development.

Fetal alcohol syndrome (FAS) is caused by maternal alcohol consumption during pregnancy. The reason why specific embryonic tissues are sensitive toward ethanol is not understood. We found that in neural crest-derived cell (NCC) cultures from the first branchial arch of E10 mouse embryos, incubation with ethanol increases the number of apoptotic cells by fivefold. Apoptotic cells stain intensely for ceramide, suggesting that ceramide-induced apoptosis mediates ethanol damage to NCCs. Apoptosis is reduced by incubation with CDP-choline (citicoline), a precursor for the conversion of ceramide to sphingomyelin. Consistent with NCC cultures, ethanol intubation of pregnant mice results in ceramide elevation and increased apoptosis of NCCs in vivo. Ethanol also increases the protein level of prostate apoptosis response 4 (PAR-4), a sensitizer to ceramide-induced apoptosis. Prenatal ethanol exposure is concurrent with malformation of parietal bones in 20% of embryos at day E18. Meninges, a tissue complex derived from NCCs, is disrupted and generates reduced levels of TGF-ß1, a growth factor critical for bone and brain development. Ethanol-induced apoptosis of NCCs leading to defects in the meninges may explain the simultaneous presence of cranial bone malformation and cognitive retardation in FAS. In addition, our data suggest that treatment with CDP-choline may alleviate the tissue damage caused by alcohol.

A novel isoform of prostate apoptosis response 4 (PAR-4) that co-distributes with F-actin and prevents apoptosis in neural stem cells.

The elevated expression of prostate apoptosis response-4 (PAR-4) induces apoptosis in differentiating mouse embryonic stem (ES) cells. In embryoid body (EB) cells and the E15.5 stage of embryonic mouse brain, PAR-4 is expressed as two isoforms (38 and 33 kDa). Using mouse EB-derived RNA as a template we have cloned and characterized a novel isoform of PAR-4 (PAR-4/p33) that lacks exon 3 and shows a bona fide splice junction of exons 2 and 4. The molecular mass for PAR-4/p33 is estimated to be 33 kDa, corresponding to the short form found in the EB cells and E15.5 mouse brain. The fluorescent fusion protein of PAR-4/p33 is mainly found in the cytosol and is co-distributed with F-actin filaments, while that of the 38 kDa full length PAR-4/p38 is predominantly translocated to the nucleus. In contrast to the full length PAR-4 (PAR-4/p38), ectopic expression of PAR-4/p33 does not result in the activation of caspase 3 and the induction of apoptosis. PAR-4/p33 forms a complex with PAR-4/p38, which inhibits its nuclear translocation and the induction of apoptosis. PAR-4/p33 is suggested to be a dominant negative isoform of PAR-4/p38 and may regulate PAR-4-dependent apoptosis.

Regulation of apoptosis during neuronal differentiation by ceramide and b-series complex gangliosides.

Lipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of b-series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F-11) cells with sialyltransferase-II (ST2) resulted in a 70% reduction of ceramide-induced apoptosis. This was most likely due to an 80% reduced expression of prostate apoptosis response-4 (PAR-4). PAR-4 expression and apoptosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 mitogen-activated protein (MAPK) kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR-4. Apoptosis and PAR-4 expression, however, were elevated in the ventricular zone which only weakly stained for complex gangliosides. Whole cell patch clamping revealed a 2-fold increased calcium influx in ST2-transfected cells, the blocking of which with nifedipine restored apoptosis to the level of untransfected cells. In serum-free culture, supplementation of the medium with IGF-1 was required to maintain MAPK phosphorylation and the anti-apoptotic effect of BCG expression. BCG-enhanced calcium influx and the presence of insulin-like growth factor-1 may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide-induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expression.

Regulation of glycosyltransferases in ganglioside biosynthesis by phosphorylation and dephosphorylation.

The biosynthesis of gangliosides is known to be under strict metabolic control. One level of control is through post-translational modification of the glycosyltransferases responsible for their biosynthesis. Thus, the activities of several sialyltransferases have been demonstrated to be downregulated by the action of protein kinase C (PKC) in cell-free and intact cell systems. This modulatory effect can be reversed at least in part by the action of membrane-bound phosphatases. In contrast, the activity of N-acetylgalactosaminyltransferase can be upregulated by the action of protein kinase A (PKA) in cultured cells. In addition, studies from several laboratories have demonstrated that phosphorylation of certain glycosyltransferases can affect their intracellular processing and translocation. Thus, modulation of glycosyltransferases by phosphorylation and dephosphorylation should represent an important regulatory mechanism for ganglioside biosynthesis.

Metabolic studies of glycosphingolipid accumulation in mucopolysaccharidosis IIID.

Severe neurological deficits and mental retardation are frequently associated with disrupted ganglioside metabolism in a variety of gangliosidoses and lysosomal storage disorders. Accumulation of glycosphingolipids (GSLs) in the central nervous system (CNS) of humans and animals affected with several types of mucopolysaccharidoses (MPS) also correlates with the severity of neurological dysfunction. Mucopolysaccharidosis type IIID (MPS IIID) is characterized by deficiency in lysosomal N-acetylglucosamine 6-sulfatase activity and the accumulation and excretion of heparan sulfates and N-acetylglucosamine 6-sulfate. We investigated the metabolism of GSLs in the prenatal, neonatal, and adult MPS IIID caprine brains and an MPS experimental cell culture model. The amounts of total glycolipids in prenatal, neonatal, and adult MPS IIID caprine brains were about 2-fold higher than those in control samples. GM3, GD3, and lactosyl ceramide were the principal GSLs which abnormally accumulated in caprine MPS IIID brains. These changes may be, in part, due to the reduction of sialidase and UDP-N-acetylgalactosamine:GM3 N-acetylgalactosaminyltransferase (GalNAc-T) activities in MPS IIID caprine brain. To further examine the possible mechanism of GSL accumulation in MPS IIID brains, we employed a cell culture model using suramin-treated neuronal cultures of differentiated P19 cells. HPTLC analysis showed elevated GSLs in suramin-treated cells. Metabolic pulse-chase labeling study revealed that the GSL accumulation in suramin-treated cells may be attributed to both disturbed biosynthesis and significantly slower degradation of GSLs. In addition, the consistency of observations in the cell culture and caprine models supports the cell culture system as a means of evaluating GSL metabolic perturbations.

Disialoganglioside GD3 is selectively expressed by developing and mature human mast cells.

BACKGROUND: Disialoganglioside GD3 is expressed on the surface of selected cell types. Anti-GD3 mAb administered to human subjects with malignant melanoma produces signs and symptoms of immediate hypersensitivity reactions. OBJECTIVE: The expression of GD3 by human mast cells was assessed during mast cell development in vitro and in samples of lung and skin. METHODS: GD3 on tissue- and in vitro-derived mast cells was analyzed after double labeling of cells for tryptase (G3 mAb) or Kit (YB5.B8 mAb) and GD3 (R24 mAb). Glycolipids in extracts of fetal liver-derived mast cells were examined by using high-performance thin-layer chromatography. RESULTS: Flow cytometry showed that the percentage of GD3+ cells increased in parallel to Kit+ cells during the recombinant human stem cell factor-dependent development of fetal liver-derived mast cells. Double-labeling experiments showed that GD3+ cells were also surface Kit+ and granule tryptase positive, identifying them as mast cells in preparations of lung-, skin-, fetal liver-, and cord blood-derived cells. The major acidic glycolipid detected was NeuAcalpha2-8NeuAcalpha2-3Galbeta1-4Glcbeta1-1'Cer (GD3). Among peripheral blood leukocytes, only basophils and about 10% of the T cells were labeled with anti-GD3 mAb. Anti-GD3 mAb-conjugated magnetic beads were used to purify mast cells to greater than 90% purity from dispersed skin cells enriched to approximately 12% purity by means of density-dependent sedimentation but were less proficient for dispersed human lung mast cells, most likely because of other cell types that express GD3. CONCLUSION: GD3 is expressed on the surface of developing human mast cells in parallel to tryptase in secretory granules and, like Kit, can serve as a target for their enrichment by immunoaffinity techniques.

Protein-ribosome-mRNA display: affinity isolation of enzyme-ribosome-mRNA complexes and cDNA cloning in a single-tube reaction.

An enzyme-ribosome-mRNA complex was specifically purified by binding to the immobilized enzyme substrate and the cDNA was cloned in a single-tube reaction by one-step reverse transcription-PCR. The ganglioside GM3, used by sialyltransferase II (ST-II) as a substrate, was coated on a 96-well microtiter plate and ST-II was in vitro transcribed and translated from a cDNA library. The isolation of an enzyme-specific protein-ribosome (PRIME) complex was achieved with as little as 0.1 ng ST-II-specific cDNA in 5 microg of a total plasmid preparation or with the cDNA prepared from sublibraries previously inoculated at a density of 2000 clones/culture well. The affinity purification of the PRIME complex was highly specific for GM3 and did not result in cDNA amplification when a different ganglioside (GM1) was used for coating of the microtiter plate. The amplified cDNA was used for cloning or a second round of ribosome display, providing a fast analysis of enzyme affinity to multiple substrates. PRIME display can be used for host-free cDNA cloning from mRNA or cDNA libraries and for binding site mapping of the in vitro translated protein. The use of a single-tube reaction in ligand-coated microtiter plates indicates the versatility of PRIME display for cDNA cloning by automated procedures.

Probing quantum coherence in a biological system by means of DNA amplification.

As a result of rapid decoherence, quantum effects in biological systems are usually confined to single electron or hydrogen delocalizations. In principle, molecular interactions at high temperatures can be guided by quantum coherence if embedded in a dynamics preventing decoherence. This was experimentally investigated by analyzing the thermodynamics, kinetics, and quantum mechanics of the primer/template duplex formation during DNA amplification by polymerase chain reaction. The structures of the two oligonucleotide primers used for amplification of a cDNA template were derived either from a repetitive motif or a fractal distribution of nucleotide residues. Contrary to the computer-based calculation of the primer melting temperatures (T(m)) that predicted a higher T(m) for the non-fractal primer due to nearest-neighbor effects, it was found that the T(m) of the non-fractal primer was actually 2 degrees C lower than that of its fractal counterpart. A thermodynamic analysis of the amplification reaction indicated that the primer annealing process followed Bose-Einstein instead of Boltzmann statistics, with an additional binding potential of mu=500 J/mol or 10(-21) J/molecule due to a superposition of binding states within the primer/template duplex. The temporal evolution of the Bose-Einstein state was determined by enzyme kinetic analysis of the association of the primer/template duplex to Taq polymerase. Assuming that collision with the enzyme interrupted the superposition, it was found that the Bose-Einstein state lasted for t(dec)=0.7x10(-12) s, corresponding to the energy dispersion (DeltaE) of quantum coherent states (mu=DeltaE>/=h/t(dec)). A quantum mechanical analysis revealed that the coherent state was stabilized by almost vanishing separation energies between distinct binding states during a temperature-driven shifting of the two DNA strands in the primer/template duplex. The additional binding potential is suggested to arise from a short-lived electron tunneling as the result of overlapping orbitals along the axis of the primer/template duplex. This effect was unique to the fractal primer due to the number of binding states that remained almost constant, irrespective of the size of shifting. It is suggested that fractal structures found in proteins or other macromolecules may facilitate a short-lived quantum coherent superposition of binding states. This may stabilize molecular complexes for rapid sorting of correct-from-false binding, e.g. during folding or association of macromolecules. The experimental model described in this paper provides a low-cost tool for simulating and probing quantum coherence in a biological system.

Effect of N-glycosylation on turnover and subcellular distribution of N-acetylgalactosaminyltransferase I and sialyltransferase II in neuroblastoma cells.

Gangliosides are sialylated glycosphingolipids whose biosynthesis is catalyzed by a series of endoplasmic reticulum (ER)- and Golgi-resident glycosyltransferases. Protein expression, processing, and subcellular localization of the key regulatory enzymes for ganglioside biosynthesis, sialyltransferase II (ST-II) and N-acetylgalactosaminyltransferase I (GalNAcT), were analyzed upon transient expression of the two enzymes in the neuroblastoma cell lines NG108-15 and F-11. The enzymes were endowed with a C-terminal epitope tag peptide (FLAG) for immunostaining and immunoaffinity purification using a FLAG-specific antibody. Mature ST-II-FLAG and GalNAcT-FLAG were expressed as N-glycoproteins with noncomplex oligosaccharides. ST-II-FLAG was distributed to the Golgi apparatus, whereas GalNAcT-FLAG was found in the ER and Golgi. Inhibition of early N-glycoprotein processing with castanospermine resulted in a distribution of ST-II-FLAG to the ER, whereas that of GalNAcT-FLAG remained unaltered. In contrast to GalNAcT, the activity of ST-II and the amount of immunostained enzyme were reduced concomitantly by 75% upon incubation with castanospermine. This was due to a fourfold increased turnover of ST-II-FLAG, which was not found with GalNAcT-FLAG. The ER retention and increased turnover of ST-II-FLAG were most likely due to its inability to bind to calnexin upon inhibition of early N-glycoprotein processing. Calnexin binding was not observed for GalNAcT-FLAG, indicating a differential effect of N-glycosylation on the turnover and subcellular localization of the two glycosyltransferases.

N-acylated serinol is a novel ceramide mimic inducing apoptosis in neuroblastoma cells.

A novel structural analog of ceramide was synthesized by N-acylation of serinol (2-amino-1,3-propanediol) and studied for its effects on glycolipid biosynthesis and cell differentiation of neuroblastoma cells. Incubation with N-palmitoylated serinol (C16-serinol) increased the concentration of endogenous ceramide by 50-80% and caused apoptosis in rapidly dividing low density cells but not in confluent cells. Cell death was not suppressed by simultaneous incubation with phorbol ester, known to antagonize ceramide-induced apoptosis by activation of protein kinase C (PKC). Purification of potential target proteins of C16-serinol was achieved by affinity chromatography of a protein preparation from rat brain on immobilized C16-serinol. A gel activity assay revealed that the eluate from C16-serinol-Sepharose contained three serine/threonine-specific protein kinases with molecular masses of 50, 70, and 95 kDa. The 70-kDa protein was immunostained on a Western blot using a PKCzeta-specific antibody. The purified PKCzeta could be activated directly by C16-serinol in an in vitro phosphorylation assay. Induction of apoptosis in neuroblastoma cells was suppressed by inhibition of PKCzeta with Gö 6983. Our overall results indicate that apoptosis in neuroblastoma cells induced by C16-serinol was at least partially mediated by activation of PKCzeta on condition of ongoing cell division. N-Acylated serinols may thus be useful for induction of apoptosis in mitotic cells and may be of therapeutic potential for treatment of cancer in the nervous system.

Alteration of ganglioside composition by stable transfection with antisense vectors against GD3-synthase gene expression.

Gangliosides are ubiquitous components of mammalian cells. Their expression is frequently altered in many tumor types. We previously showed that alteration of the ganglioside composition often resulted in changes in cellular morphology and differentiation of cultured cells. In this study, we targeted sialyltransferase gene expression by the antisense knockdown experiment, and the results showed that inhibition of the expression of gangliosides GD3 and O-acetylated GD3 (OAc-GD3) in the neuroblastoma F-11 cells greatly reduced the tumor growth in nude mice. The sense and antisense vectors containing either a 5' end fragment or the entire sequence of the cDNA coding for GD3-synthase were prepared and used in separate experiments to transfect the F-11 cells which express high levels of gangliosides GD3 and OAc-GD3. Single clones were isolated and expanded. Both the activity of the GD3-synthase and the concentrations of GD3 and OAc-GD3 in the antisense-transfected cells were dramatically decreased as a result of transfection with the antisense expression vectors. Further characterization of the antisense-transfected cells showed reduced rates of cell growth and neurite formation and changes in cellular morphology. When the cells were inoculated in athymic nude mice, the tumor growth rate was remarkably suppressed although the tumor incidence was not affected by the altered ganglioside composition. These results indicate that the tumor-associated ganglioside(s) is(are) involved in regulation of tumor growth, probably through the stimulation of angiogenesis of the tumor.

Multi-enzyme kinetic analysis of glycolipid biosynthesis.

Gangliosides are acidic glycosphingolipids synthesized sequentially by a series of glycosyltransferases acting in parallel biosynthetic pathways. While most glycosyltransferases are highly specific, some, however, may catalyze equivalent steps in each pathway using different gangliosides as substrates (e.g. N-acetylgalactosaminyltransferase, sialyltransferase-IV). A multi-enzyme kinetic analysis was developed on the condition that serial enzymatic reactions operate below substrate saturation. A multi-enzyme kinetic analysis enabled a simultaneous calculation of the Vmax/Km value of each enzyme derived from the equilibrium concentration of the respective substrate. Substrate concentrations [S] were determined by radioactive labelling of gangliosides in intact cells with the precursor sugars [14C]galactose and [14C]glucosamine, followed by high-performance thin-layer chromatography and autoradiography of the radiolabelled glycolipids. On the basis of Michaelis-Menten kinetics, Vmax/Km values were derived from [S] by a system of linear equations. The procedure was used to analyze the development of the glycolipid composition during differentiation of rat gliomaxmurine neuroblastoma (NG108-15) cells. The Vmax/Km values calculated by multi-enzyme kinetic analysis were consistent with the kinetic data obtained with solubilized enzymes. Application of multi-enzyme kinetic analysis to published data on the correlation of enzyme activities with ganglioside levels in various cell lines and tissues indicated the validity of this method for analysis of the glycolipid biosynthesis, in particular, of its initial steps. On the basis of the kinetic analysis, it is suggested that the cell lines can be divided into two groups with respect to the substrate pools of GM3 used by sialyltransferase-II and N-acetylgalactosaminyltransferase-I. The first group encompasses the majority of the neuroblastoma cell lines and the embryonic rat brain where the two enzymes share a common pool of GM3. In the second group, the two enzymes do not compete for the same pool of GM3, indicating a different subcellular localization of CMP-NeuAc:GM3 alpha2-8-sialyltransferase and UDP-N-acetylgalactosaminyl:GM3 N-acetylgalactosaminyltransferase. In this study, the theory of a multi-enzyme kinetic analysis is discussed and its application to analysis of the glycolipid biosynthesis in neuroblastoma cells is demonstrated. A multi-enzyme kinetic analysis can be applied to other biosynthetic pathways and provides the advantage of analyzing kinetic data with intact cells or tissue samples.

Differential effects of glycolipid biosynthesis inhibitors on ceramide-induced cell death in neuroblastoma cells.

An in vitro model of Gaucher's disease in murine neuroblastoma x rat glioma NG108-15 cells was used to investigate the physiological effects of two specific inhibitors of glucosylceramide synthase, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d,l-PDMP) and N-butyldeoxynojirimycin (NB-DNJ), which have been suggested as agents for treatment of glycolipid storage disorders. Incubation of NG108-15 cells with conduritol-B-epoxide, a covalent inhibitor of glucosylceramidase, raised the intracellular concentration of glucosylceramide (GC) by more than fourfold, indicating a glycolipid composition equivalent to that of Gaucher's cells. The level of GC was decreased, and the cells were depleted of gangliosides by postincubation with d,l-PDMP or NB-DNJ. Treatment with d,l-PDMP, but not with NB-DNJ, resulted in a dose-dependent reduction of the growth rate and eventually caused cell death in NG108-15 cells on reaching confluency. An in situ detection assay using terminal nucleotidyltransferase indicated that cell degeneration was accompanied by apoptosis. Lipid analysis by high-performance TLC revealed that on incubation with d,l-PDMP, but not with NB-DNJ, the concentration of endogenous ceramide was elevated by threefold. Ceramide elevation and apoptosis were also observed when NG108-15 cells were incubated with daunorubicin, which was previously reported to induce programmed cell death by stimulation of ceramide synthesis. Structural characterization by HPLC and subsequent laser desorption mass spectrometry revealed that the endogenous ceramide contained fatty acids with chain lengths ranging from C14:0 to C24:0. The results indicate that elevation of levels of these ceramide species by incubation with d,l-PDMP or daunorubicin induces programmed cell death in NG108-15 cells. Because ceramide accumulation and cell death were not observed on incubation with NB-DNJ, its use is suggested to be less toxic than that of d,l-PDMP for treatment of Gaucher's disease and other sphingolipid storage disorders.

Combinatorial PCR approach to homology-based cloning: cloning and expression of mouse and human GM3-synthase.

GM3-synthase, also known as sialyltransferase I (ST-I), catalyzes the transfer of a sialic acid residue from CMP-sialic acid onto lactosylceramide to form ganglioside GM3. In order to clone this enzyme, as well as other sialyltransferases, we developed an approach that we termed combinatorial PCR. In this approach, degenerate primers were designed on the basis of conserved sequence motifs of the ST3 family of sialyltransferases (STs). The nucleotide sequence of the primers was varied to cover all amino acid variations occurring in each motif. In addition, in some primers the sequence was varied to cover possible homologous substitutions that are absent in the available motifs. A panel of cDNA from 12 mouse and 8 human tissues was used to enable cloning of tissue- and stage-specific sialyltransferases. Using this approach, the fragments of 11 new putative sialyltransferases were isolated and sequenced so far. Analysis of the expression pattern of a particular sialyltransferase across the panel of cDNA from the different tissues provided information about the tissue specificity of ST expression. We chose two new ubiquitously expressed human and mouse STs to clone full-length copies and to assay for GM3-synthase activity. One of the STs, which exhibited the highest homology to ST3 Gal III, showed activity toward lactosylceramide (LacCer) and was termed ST3 Gal V according to the suggested nomenclature [1]. The other ubiquitously expressed sialyltransferase was termed ST3Gal VI. All isolated sialyltransferases were screened for alternatively spliced forms (ASF). Such forms were found for both human ST3Gal V and ST3Gal VI in human fetal brain cDNA library. The detailed cloning strategy, functional assay, and full length cDNA and protein sequences of GM3 synthase (ST3Gal V, or ST-I) are presented.

Regulation of ganglioside metabolism by phosphorylation and dephosphorylation.

Cell differentiation is frequently accompanied by alterations in the composition of gangliosides in the plasma membrane resulting from a regulation of the enzyme activities involved. The regulation of CMP-NeuAc:GM1 alpha2-3-sialyltransferase (ST-IV) and UDP-GalNAc:GM3 N-acetylgalactosaminyltransferase (Gal-NAc-T) by the degree of enzyme phosphorylation was analyzed by determination of the enzyme activity on incubation of NG108-15 cells with various protein phosphatase inhibitors (okadaic acid and orthovanadate) or protein kinase activators (phorbol ester and forskolin). Incubation with okadaic acid, but not with orthovanadate, inhibited the ST-IV activity to 45% of that of control cells with t(1/2) = 60 min for the inactivation reaction. This indicates a rapid hyperphosphorylation of ST-IV due to the inhibition of a serine/threonine-specific phosphatase. A similar rate of inactivation was found on stimulation of protein kinase C with phorbol ester. In contrast to ST-IV, the activity of GalNAc-T was increased on stimulation of intracellular phosphorylation systems. The fastest activation of GalNAc-T was achieved with forskolin, yielding up to 160% of the initial activity within 30 min of effector incubation. Up-regulation of GalNAc-T in conjunction with down-regulation of ST-IV by stimulation of phosphorylation is suggested to serve as a physiological mechanism to increase the concentration of GM1, which was found to be elevated in correlation with the cell density. This assumption was corroborated by metabolic labeling studies with radioactive ganglioside precursors indicating an enhancement of the relative amount of a-series gangliosides subsequent to GM3 on phosphorylation stimulation. In particular, the biosynthesis of GM1 was specifically elevated within 2 h of incubation with forskolin. We conclude from the overall data that the ganglioside composition during the cell differentiation of NG108-15 cells can be specifically regulated by both protein kinase A- and protein kinase C-related phosphorylation systems.

Man9-mannosidase from pig liver is a type-II membrane protein that resides in the endoplasmic reticulum. cDNA cloning and expression of the enzyme in COS 1 cells.

Man9-mannosidase, one of three different alpha 1,2-exo-mannosidases known to be involved in N-linked oligosaccharide processing, has been cloned in lambda gt10, using a mixed-primed pig liver cDNA library. Three clones were isolated which allowed the reconstruction of a 2731-bp full-length cDNA. The cDNA construct contained a single open reading frame of 1977 bp, encoding a 659-residue polypeptide with a molecular mass of approximately 73 kDa. The Man9-mannosidase specificity of the cDNA construct was verified by the observation that all peptide sequences derived from a previously purified, catalytically active 49-kDa fragment were found within the coding region. The N-terminus of the 49-kDa fragment aligns with amino acid 175 of the translated cDNA, indicating that the catalytic activity is associated with the C-terminus. Transfection of COS 1 cells with the Man9-mannosidase cDNA gave rise to a > 30-fold over-expression of a 73-kDa protein whose catalytic properties, including substrate specificity, susceptibility towards alpha-mannosidase inhibitors and metal ion requirements, were similar to those of the 49-kDa enzyme fragment. Thus deletion of 174 N-terminal amino acids in the 73-kDa protein appears to have only marginal influence on the catalytic properties. Structural and hydrophobicity analysis of the coding region, as well as the results from tryptic degradation studies, point to pig liver Man9-mannosidase being a non-glycosylated type-II transmembrane protein. This protein contains a 48-residue cytosolic tail followed by a 22-residue membrane anchor (which probably functions as internal and non-cleavable signal sequence), a lumenal approximately 100-residue-stem region and a large 49-kDa C-terminal catalytic domain. As shown by immuno-fluorescence microscopy, the pig liver enzyme expressed in COS 1 cells, is resident in the endoplasmic reticulum, in contrast to COS 1 Man9-mannosidase from human kidney which is Golgi-located [Bieberich, E. & Bause, E. (1995) Eur. J. Biochem. 233, 644-649]. Localization of the porcine enzyme in the endoplasmic reticulum is consistent with immuno-electron-microscopic studies using pig hepatocytes. The different intracellular distribution of pig liver and human kidney Man9-mannosidase is, therefore, enzyme-specific rather than a COS-1-cell-typical phenomenon. Since we observe approximately 81% sequence similarity between the two alpha-mannosidases, we deduce that the localization in either endoplasmic reticulum or Golgi is likely to be sequence-dependent.

Man9-mannosidase from human kidney is expressed in COS cells as a Golgi-resident type II transmembrane N-glycoprotein.

Man9-mannosidase, an alpha 1,2-specific exo-enzyme involved in N-linked oligosaccharide processing, has been cloned recently from a human kidney cDNA library [Bause, E., Bieberich, E., Rolfs, A., Völker, C. & Schmidt, B. (1993) Eur. J. Biochem. 217, 533-540]. Transient expression in COS 1 cells of the enzyme resulted in a more than 20-fold increase of a catalytic activity cleaving specifically alpha 1,2-mannosidic linkages in [14C]Man9-GlcNAc2 or [14C]Man5-GlcNAc2. Man9-mannosidase is expressed as a N-glycoprotein with a molecular mass of 73 kDa. Its enzymic activity is metal ion dependent and inhibited strongly by 1-deoxymannojirimycin (50% at 100 microM). Proteolytic studies with the membrane-associated form of Man9-mannosidase support the view that the enzyme is a type II transmembrane protein as predicted from its cDNA sequence. Several lines of evidence suggest that Man9-mannosidase, as expressed, is N-glycosylated at one of three potential Asn-Xaa-Thr/Ser/Cys acceptor sites. Approximately 50% of the N-linked oligosaccharide chains are removed by endoglycosidase H treatment, whereas complete deglycosylation of the enzyme is observed, when transfected cells were cultured in the presence of the Golgi mannosidase II inhibitor swainsonine, indicating that the sugar moiety of Man9-mannosidase is processed partially by Golgi-resident enzymes. This observation is consistent with the results of indirect immunofluorescence studies, pointing to a localization of the Man9-mannosidase predominantly in the juxtanuclear Golgi region. This localization clearly differs from that of pig liver Man9-mannosidase which appears to be located in the endoplasmic reticulum and transient vesicles.