The expanding universe of alkaloid biosynthesis

Characterization of many of the major gene families responsible for the generation of central intermediates and for their decoration, together with the development of large genomics and proteomics databases, has revolutionized our capability to identify exotic and interesting natural-product pathways. Over the next few years, these tools will facilitate dramatic advances in our knowledge of the biosynthesis of alkaloids, which will far surpass that which we have learned in the past 50 years. These tools will also be exploited for the rapid characterization of regulatory genes, which control the development of specialized cell factories for alkaloid biosynthesis.     

Effect of puromycin and cycloheximide on glycosphingolipid biosynthesis by PHA stimulated human lymphocytes

The effect of puromycin and cycloheximide on the biosynthesis of neutral glycosphingolipids and gangliosides by PHA stimulated lymphocytes is studied. Under conditions of permanent inhibition of protein synthesis, and depending on the time of lymphocyte stimulation, inhibition of the biosynthesis of neutral glycosphingolipids was either restricted only to certain species or was very low for all glycosphingolipid species. The degree of inhibition of the various ganglioside species was affected by the time of incubation. After transient inhibition of protein synthesis and at times when protein biosynthesis had recovered, neutral glycosphingolipid and ganglioside biosynthesis inhibition was very prominent and did not recover. The possibility is discussed that glycosphingolipid biosynthesis does not depend directly on concurrent nascent peptide formation and it is proposed that inhibition of glycosphingolipid biosynthesis is related primarily to impairment of the endoplasmic reticulum and to inhibition of galactose transferases, secondary to the binding of the inhibitors.     

Cell density-dependent changes of glycosphingolipid biosynthesis in cultured human skin fibroblasts

In this study, the glycosphingolipid biosynthesis was investigated in the sparse and the confluent cell populations of cultured human skin fibroblasts.The human skin fibroblast cell populations were metabolically pulse labeled with ¹⁴C-galactose (48 h). The amounts of ¹⁴C-radioactivity (cpm) incorporated into extracted and purified total cellular glycosphingolipid fractions were counted by -scintillation and the individual glycosphingolipid species were separated by high performance thin layer chromatography and visualized by autoradiography. The relative labeling (%) of individual newly synthesized glycosphingolipid species was detected by densitometric scanning of autoradiographic glycosphingolipid patterns.The incorporation of ¹⁴C-label into total glycosphingolipids per cell increased significantly as the cell-density increased, referring to five fold higher rate of glycosphingolipid biosynthesis de novo in cells at confluency vs. sparse populations. The total newly synthesized glycosphingolipid pattern (100%) of sparse cell populations showed a significant predominance of the gangliosides (70%) over the neutral glycosphingolipids (30%), with ganglioside GM2 as the major species followed by monohexosyl-ceramide. Oppositely, the newly synthesized neutral glycosphingolipids (67%) predominated over the gangliosides (33%) in cells at confluency (contact inhibition). Cells reaching confluency were characterized by: (a) a dramatic increase of absolute amount of all newly synthesized neutral glycosphingolipid species, particularly the most abundant monohexosyl-ceramide and trihexosyl-ceramide, but also of the ganglioside GM3; (b) a drastic decrease of absolute amount of newly synthesized ganglioside GM2. The specific shift in newly synthesized glycosphingolipid pattern in cells reaching confluency suggests a down-regulation of biosynthetic pathway primarily at the level of N-acetylgalactosaminyl-transferase. A possible involvement of glycosphingolipids in cell density-dependent regulation of cell growth through establishment of the direct intermolecular intermembrane interactions is discussed.     

Biochemical diversity of glycosphingolipid biosynthesis as a driver of Coccolithovirus competitive ecology

Coccolithoviruses (EhVs) are large, double-stranded DNA-containing viruses that infect the single-celled, marine coccolithophore Emiliania huxleyi. Given the cosmopolitan nature and global importance of E. huxleyi as a bloom-forming, calcifying, photoautotroph, E. huxleyi–EhV interactions play a key role in oceanic carbon biogeochemistry. Virally-encoded glycosphingolipids (vGSLs) are virulence factors that are produced by the activity of virus-encoded serine palmitoyltransferase (SPT). Here, we characterize the dynamics, diversity and catalytic production of vGSLs in an array of EhV strains in relation to their SPT sequence composition and explore the hypothesis that they are a determinant of infectivity and host demise. vGSL production and diversity was positively correlated with increased virulence, virus replication rate and lytic infection dynamics in laboratory experiments, but they do not explain the success of less-virulent EhVs in natural EhV communities. The majority of EhV-derived SPT amplicon sequences associated with infected cells in the North Atlantic derived from slower infecting, less virulent EhVs. Our lab-, field- and mathematical model-based data and simulations support ecological scenarios whereby slow-infecting, less-virulent EhVs successfully compete in North Atlantic populations of E. huxleyi, through either the preferential removal of fast-infecting, virulent EhVs during active infection or by having access to a broader host range.     

Characterization of adrenergic receptors on proximal tubular basolateral membranes

Alpha adrenergic receptors are identified on basolateral plasma membranes derived from proximal tubular epithelial cells. The density of alpha 2 receptors was over two-fold greater than alpha 1 receptors. The basolateral membranes were devoid of beta receptors. These results support previous demonstrations of alpha adrenergic receptors in rat renal cortex and concur with studies which suggest a limited presence of beta receptors on rat proximal tubules.     

Differential effects of three inhibitors of glycosphingolipid biosynthesis on neuronal differentiation of embryonal carcinoma stem cells

Gangliosides have been implicated in having important roles in neural development. It has been shown that disruption of ganglioside biosynthesis inhibits neurite outgrowth. However, many contradictory results have been reported. The inconsistency of these reports may result from the differential use of neuronal cell lines and inhibitors for ganglioside biosynthesis. In order to clarify the inconsistency in these studies, we utilized an in vitro neuronal differentiation model using an embryonic caricinoma (EC) stem cell line to elucidate the relationship between ganglioside expression and neural development. These cells were exposed to three different inhibitors of glucosylceramide synthase, the first enzyme committed for the biosynthesis of most of the brain gangliosides. All three inhibitors, d-threo-1-phenyl-2-decanoylamino-3-morphlino-1-propanol (D-PDMP), d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (D-PPPP), and N-butydeoxynojirimycin (NB-DNJ) can inhibit greater than 90% of ganglioside biosynthesis at certain concentrations, respectively. D-PDMP significantly slowed down cellular proliferation in undifferentiated P19 EC cells, inhibited neurite outgrowth, and eventually caused cell death in differentiated cells. However, no retardation in cell growth, neuronal differentiation, and neurite outgrowth was observed in cultures treated with D-PPPP or NB-DNJ despite the depletion of gangliosides. These results indicate that the effect of D-PDMP on cellular proliferation, neurite outgrowth, and survival of differentiated cells is independent of the inhibition of ganglioside biosynthesis.     

Isolation of renal proximal tubular brush-border membranes

This protocol describes a method for the isolation and purification of renal proximal tubular brush-border membranes in high yield and high purity. Based on a different reactivity of the brush-border membrane compared to other cellular membranes with divalent cations, such as Mg2+, purified membrane vesicles can be obtained after a few differential centrifugation steps (within approximately 3 h) that are suitable for in vitro studies, such as transport experiments or protein and lipid analysis.     

Biochemical properties of isolated transverse tubular membranes

This review addresses the major biochemical and structural characteristics of isolated transverse tubule (T-tubule) membranes, including methods of isolation and morphology of purified membranes, evaluation of attendant membrane activities, including ion pumps and channels, and structural and compositional analyses of functionally relevant components. Particular emphasis is placed on the Mg²⁺-ATPase, its localization in the T-system, its unusual kinetic properties, its possible functions, and its potential regulation by diacylglycerol and other biologically-relevant lipids. Conclusions are drawn with respect to the biochemical markers characteristic of T-tubule membranes and the criteria to be applied in the assessment of isolated T-tubule membrane purity.Abbreviations CMC critical micelle concentration - Con-A concanavalin A - DHP dihydropyridine - E-C excitation-contraction - E-P phosphoenzyme - FITC fluorescein isothiocyanate - IgG immunoglobulin G - immuno-EM immunoelectron microscopic - IP₃ inositol 1,4,5-triphosphate - LPC lysophosphatidylcholine - NBD-C1 7-chloro-4-nitrobenz-2-oxa-1,3-diozole - NBD-F 7-fluoro-NBD - NEM N-ethylmaleimide - PL phospholipid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SL sarcolemma - SR sarcoplasmic reticulum - STX saxitoxin - TTX tetrodotoxin - T-tubule transverse tubule - WGA wheatgerm agglutinin     

Inhibition of glycosphingolipid biosynthesis reduces secretion of the beta-amyloid precursor protein and amyloid beta-peptide

Alzheimer disease is associated with extracellular deposits of amyloid beta-peptides in the brain. Amyloid beta-peptides are generated by proteolytic processing of the beta-amyloid precursor protein by beta- and gamma-secretases. The cleavage by secretases occurs predominantly in post-Golgi secretory and endocytic compartments and is influenced by cholesterol, indicating a role of the membrane lipid composition in proteolytic processing of the beta-amyloid precursor protein. To analyze the role of glycosphingolipids in these processes we inhibited glycosyl ceramide synthase, which catalyzes the first step in glycosphingolipid biosynthesis. The depletion of glycosphingolipids markedly reduced the secretion of endogenous beta-amyloid precursor protein in different cell types, including human neuroblastoma SH-SY5Y cells. Importantly, secretion of amyloid beta-peptides was also strongly decreased by inhibition of glycosphingolipid biosynthesis. Conversely, the addition of exogenous brain gangliosides to cultured cells reversed these effects. Biochemical and cell biological experiments demonstrate that the pharmacological reduction of cellular glycosphingolipid levels inhibited maturation and cell surface transport of the beta-amyloid precursor protein. In the glycosphingolipid-deficient cell line GM95, cellular levels and maturation of beta-amyloid precursor protein were also significantly reduced as compared with normal B16 cells. Together, these data demonstrate that glycosphingolipids are implicated in the regulation of the subcellular transport of the beta-amyloid precursor protein in the secretory pathway and its proteolytic processing. Thus, enzymes involved in glycosphingolipid metabolism might represent targets to inhibit the production of amyloid beta-peptides.     

Reconstitution of glucosylceramide flip-flop across endoplasmic reticulum: implications for mechanism of glycosphingolipid biosynthesis

Most glycosphingolipids are synthesized by the sequential addition of monosaccharides to glucosylceramide (GlcCer) in the lumen of the Golgi apparatus. Because GlcCer is synthesized on the cytoplasmic face of Golgi membranes, it must be flipped to the non-cytoplasmic face by a lipid flippase in order to nucleate glycosphingolipid synthesis. Halter et al. (Halter, D., Neumann, S., van Dijk, S. M., Wolthoorn, J., de Mazière, A. M., Vieira, O. V., Mattjus, P., Klumperman, J., van Meer, G., and Sprong, H. (2007) Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. J. Cell Biol. 179, 101–115) proposed that this essential flipping step is accomplished via a complex trafficking itinerary; GlcCer is moved from the cytoplasmic face of the Golgi to the endoplasmic reticulum (ER) by FAPP2, a cytoplasmic lipid transfer protein, flipped across the ER membrane, then delivered to the lumen of the Golgi complex by vesicular transport. We now report biochemical reconstitution studies to analyze GlcCer flipping at the ER. Using proteoliposomes reconstituted from Triton X-100-solubilized rat liver ER membrane proteins, we demonstrate rapid (t_½ < 20 s), ATP-independent flip-flop of N-(6-((7-nitro-2–1,3-benzoxadiazol-4-yl)amino)hexanoyl)-d-glucosyl-β1–1′-sphingosine, a fluorescent GlcCer analog. Further studies involving protein modification, biochemical fractionation, and analyses of flip-flop in proteoliposomes reconstituted with ER membrane proteins from yeast indicate that GlcCer translocation is facilitated by well characterized ER phospholipid flippases that remain to be identified at the molecular level. By reason of their abundance and membrane bending activity, we considered that the ER reticulons and the related Yop1 protein could function as phospholipid-GlcCer flippases. Direct tests showed that these proteins have no flippase activity.     

Role of multiple drug resistance protein 1 in neutral but not acidic glycosphingolipid biosynthesis

Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246-6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis.     

Inhibitors of glycosphingolipid biosynthesis reduce transepithelial electrical resistance in MDCK I and FRT cells

Madin-Darby canine kidney (MDCK)I and Fisher rat thyroid (FRT) cells exhibit transepithelial electricalresistance (TER) values in excess of 5,000  · cm². When these cells wereincubated in the presence of various inhibitors of sphingolipidbiosynthesis, a >5-fold reduction of TER was observed without changesin the gate function for uncharged solutes or the fence function forapically applied fluorescent lipids. The localization of ZO-1 andoccludin was not altered between control and inhibitor-treated cells,indicating that the tight junction was still intact. Furthermore, thecomplexity of tight junction strands, analyzed by freeze-fracturemicroscopy, was not reduced. Once the inhibitor was removed and thecells were allowed to synthesize sphingolipids, a gradual recovery ofthe TER was observed. Interestingly, these inhibitors did not attenuatethe TER of MDCK II cells, a cell line that typically exhibits valuesbelow 800  · cm^2. These resultssuggest that glycosphingolipids play a role in regulating theelectrical properties of epithelial cells.

     

Regulation of glycosphingolipid glycosyltransferase by low density lipoprotein receptors in cultured human proximal tubular cells

We have shown previously that low density lipoproteins (LDL) suppressed the synthesis of lactosylceramide in normal human proximal tubular cells, but stimulated such synthesis in proximal tubular cells from LDL receptor negative subjects (Chatterjee, S., Clarke, K., and Kwiterovich, P.O., Jr. (1986) J. Biol. Chem. 261, 13474-13479). To understand the mechanism(s) of this effect of LDL, we have studied here the effects of LDL on the activity of UDP-GalCer:beta-galactosyltransferase (GalT-2). Maximum suppression (70-80%) of the activity of GalT-2 in normal proximal tubular cells at 37 degrees C occurred at a LDL concentration of 25 micrograms/ml medium. Such suppression was not observed either when the cells were incubated with LDL at 4 degrees C, or when the cells were preincubated with leupeptin, followed by incubation with LDL at 37 degrees C. High density lipoproteins and fetuin did not suppress the activity of GalT-2 in normal proximal tubular cells. In contrast LDL modified by reductive methylation (M-LDL, 100 micrograms/ml) stimulated the activity of GalT-2, approximately 3-fold. The effects of LDL and M-LDL were not related to their glycosphingolipid content. Much less suppression and stimulation of the activity of GalT-2 in proximal tubular cells by LDL and M-LDL, respectively, was found in normal human skin fibroblasts, Chinese hamster ovary cells, and bovine smooth muscle cells, suggesting that the LDL-mediated effect may be tissue-specific. In cells grown to very high density, the activity of the LDL receptor is decreased, and there was less suppression of GalT-2 activity by LDL. In normal proximal tubular cells, LDL stimulated the activity of UDP-Gal:LacCer, alpha-galactosyltransferase activity, UDP-Gal:LcOse3Cer, beta-galactosyltransferase, and CMP-NeuAc:LacCer,alpha-sialyltransferase activity but did not alter the activity of sulfotransferase. In conclusion, LDL that entered the normal proximal tubular cells via the LDL receptor-mediated pathway decreased GalT-2 activity, an effect that was dependent upon the binding, internalization, and degradation of receptor-bound LDL. In contrast LDL that entered normal or LDL receptor-negative proximal tubular cells via an LDL receptor-independent pathway failed to suppress GalT-2 activity, and led to a stimulation of LacCer synthesis.     

Lateral diffusion on tubular membranes: quantification of measurements bias

Single Particle Tracking (SPT) is a powerful technique for the analysis of the lateral diffusion of the lipid and protein components of biological membranes. In neurons, SPT allows the study of the real-time dynamics of receptors for neurotransmitters that diffuse continuously in and out synapses. In the simplest case where the membrane is flat and is parallel to the focal plane of the microscope the analysis of diffusion from SPT data is relatively straightforward. However, in most biological samples the membranes are curved, which complicates analysis and may lead to erroneous conclusions as for the mode of lateral diffusion. Here we considered the case of lateral diffusion in tubular membranes, such as axons, dendrites or the neck of dendritic spines. Monte Carlo simulations allowed us to evaluate the error in diffusion coefficient (D) calculation if the curvature is not taken into account. The underestimation is determined by the diameter of the tubular surface, the frequency of image acquisition and the degree of mobility itself. We found that projected trajectories give estimates that are 25 to 50% lower than the real D in case of 2D-SPT over the tubular surface. The use of 3D-SPT improved the measurements if the frequency of image acquisition was fast enough in relation to the mobility of the molecules and the diameter of the tube. Nevertheless, the calculation of D from the components of displacements in the axis of the tubular structure gave accurate estimate of D, free of geometrical artefacts. We show the application of this approach to analyze the diffusion of a lipid on model tubular membranes and of a membrane-bound GFP on neurites from cultured rat hippocampal neurons.