Jose L. Daniotti

Enhanced insulin sensitivity in mice lacking ganglioside GM3.

Gangliosides are sialic acid-containing glycosphingolipids that are present on all mammalian plasma membranes where they participate in recognition and signaling activities. We have established mutant mice that lack GM3 synthase (CMP-NeuAc:lactosylceramide α2,3-sialyltransferase; EC 2.4.99.-). These mutant mice were unable to synthesize GM3 ganglioside, a simple and widely distributed glycosphingolipid. The mutant mice were viable and appeared without major abnormalities but showed a heightened sensitivity to insulin. A basis for the increased insulin sensitivity in the mutant mice was found to be enhanced insulin receptor phosphorylation in skeletal muscle. Importantly, the mutant mice were protected from high-fat diet-induced insulin resistance. Our results show that GM3 ganglioside is a negative regulator of insulin signaling, making it a potential therapeutic target in type 2 diabetes.

Acyl-Protein Thioesterase 2 Catalizes the Deacylation of Peripheral Membrane-Associated GAP-43

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.

Understanding the stepwise synthesis of glycolipids.

Glycolipid expression is highly regulated during development and differeniation. The control relies mainly on transcriptional modulation of key glycosyltransferases acting at the branching points of the pathway of biosynthesis. Transferases are Golgi residents that depend on N-glycosylation and oligosaccharide processing for proper folding in the endoplasmic reticulum. The N-terminal domain bears information for their transport to the Golgi, retention in the organelle and differential concentration in sub-Golgi compartments. In the Golgi, some transferases associate forming functional multienzyme complexes. It is envisaged that the machinery for synthesis in the Golgi complex, and its dynamics, constitute a potential target for fine tuning of the control of glycolipid expression according to cell demands.

Chinese hamster ovary cells lacking GM1 and GD1a synthesize gangliosides upon transfection with human GM2 synthase

GM3-positive Chinese hamster ovary cells (CHO-K1 cells) lack the ability to synthesize GM2 and the complex gangliosides GM1 and GD1a from [3H]Gal added to the culture medium. However, they acquire the ability to synthesize GM2 and to synthesize and immunoexpress complex gangliosides upon transient transfection with a cDNA encoding the human GM3:N-acetylgalactosaminyl transferase (GM2 synthase). The activities of endogenous GM1- and GD1a-synthases in the parental cell line and in cells transfected with the plasmid with or without the GM2 synthase cDNA were essentially identical and comparable in terms of specific activity with the endogenous GM3 synthase. Results indicate that glycosyltransferases acting on GM2 to produce GM1 and GD1a are constitutively present in CHO-K1 cells, and that the expression of their activities depend on the supply of the acceptor GM2. In addition, these results lend support to the notion that GM2 synthase is a key regulatory enzyme influencing the balance between simple and complex gangliosides.

GM3 α2,8‐Sialyltransferase (GD3 Synthase)

Abstract: GD3 synthase (Sial‐T2) is a key enzyme of ganglioside synthesis that, in concert with GM2 synthase (GAlNAc‐T), regulates the ratio of a‐ and b‐pathway gangliosides. In this work, we study the sub‐Golgi location of an epitope‐tagged version of chicken Sial‐T2 transfected to CHO‐K1 cells. The expressed protein was enzymatically active both in vitro and in vivo and showed a molecular mass of ∼47 or ∼95 kDa on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the presence or absence of, respectively, β‐mercaptoethanol. The 95‐kDa form of Sial‐T2 was also detected if the protein was retained in the endoplasmic reticulum (ER) due to impaired glycosylation, indicating that it was formed in the ER. Confocal immunofluorescence microscopy showed Sial‐T2 localized to the Golgi complex and, within the organelle, partially co‐localizing with the mannose‐6‐phosphate receptor, a marker of the trans‐Golgi network (TGN). In cells treated with brefeldin A, a major fraction of Sial‐T2 redistributed to the ER, even under controlled expression to control for mislocalization due to protein overloading. In experiments of incorporation of sugars into endogenous acceptors of Golgi membranes in vitro, GD3 molecules formed by incubation with CMP‐NeuAc were converted to GD2 upon incubation with UDP‐GalNAc. These results indicate that Sial‐T2 localizes mainly to the proximal Golgi, although a fraction is located in the TGN functionally coupled to GalNAc‐T. Consistent with this, most of the enzyme was in an endoglycosidase H (Endo‐H)‐sensitive, neuraminidase (NANase)‐insensitive form. A minor secreted form lacking ∼40 amino acids was Endo‐H‐resistant and NANase‐sensitive, indicating that the cells were able to process N‐glycans to Endo‐H‐resistant forms. Taken together, the results of these biochemical and immunocytochemical experiments indicate that in CHO‐K1 cells, most Sial‐T2 localizes in the proximal Golgi and that a functional fraction is also present in the TGN.

GD3 Prevalence in Adult Rat Retina Correlates with the Maintenance of a High GD3‐/GM2‐Synthase Activity Ratio Throughout Development

Unlike neurons from avian retina and other regions of avian and mammalian brain, neurons from mammalian retina not only contain gangliosides of the gangliotetraosyl ceramide series but also maintain a prevalence of GD3, a gangliosidc of the lactosylceramidc series characteristic of proliferative neural cells, when they are fully differentiated. We show here that GD3 is prevalent at all developmental periods of the rat retina from birth [50% of total gangliosidic N‐acetylneuraminic acid (Neu NAc)] to adult (30% of total gangliosidic Neu NAc). GD3‐synthase specific activity increased about 1.5‐fold from birth to day 7 and essentially plateaued thereafter. The GD3‐/GM2‐synthase specific activity ratio was compared in rat and chicken retina at early and late developmental stages. In chicken retina the ratio was about 0.7 at early (when GD3 is prevalent) and decreased to 0.07 at late (when GDI a is prevalent) developmental stages. In rat retina the ratio was about 13 and 6 at, respectively, early and late developmental stages. These findings suggest that the prevalence of GD3 and of other “b” pathway gangliosides in adult rat retina neurons could be due in part to the maintenance of a high GD3‐/GM2‐synthase activity ratio throughout development of the tissue.

Regulation of Ganglioside Composition and Synthesis Is Different in Developing Chick Retinal Pigment Epithelium and Neural Retina

Abstract: We examined the immunocytochemical expression of GM3 and QD3 in 3‐day‐old chick embryo retinal pigment epithelium (RPE) and neural retina (NR). We also compared the composition of gangliosides and the activities of key ganglioside glycosyltransferases of the RPE and NR of 8‐, 12‐, and 15‐day old embryos. The immunocytochemical studies in 3‐day‐old embryos showed heavy expression of GM3 and GD3 at the inner and outer layers of the optic vesicle that are the precursors of the RPE and NR, respectively. The compositional and enzymatic studies showed pronounced differences between RPE and NR of 8‐day and older embryos. HPTLC showed that at 8 days the major species were GM3 and GD3 in RPE and GD3 and GT3 in NR. As development proceeded, GD3 decreased in both tissues, GM3 became the major ganglioside in RPE, and ganglio‐series gangliosides (mainly GD1a) became the major species in NR. At 15 days the major species were GD1 a in NR and GM3 in RPE. Enzyme determinations showed that whereas in RPE from 12‐day‐old embryos GM2 synthase was under the limit of detection and GD3 synthase activity was about sixfold lower than GM3 synthase, in NR the activities of GM3 and GD3 synthases were similar and both six‐to ninefold lower than GM2 synthase. These results evidence a markedly different modulation of the ganglioside glycosylating system in cells of a common origin that through distinct differentiation pathways originate two closely related tissues of the optic system. In addition, they reinforce the relevance of the relative activities of key transferases in determining the pattern of gangliosides in different cell types.

Glycosylation of Glycolipids in Cancer: Basis for Development of Novel Therapeutic Approaches

Altered networks of gene regulation underlie many pathologies, including cancer. There are several proteins in cancer cells that are turned either on or off, which dramatically alters the metabolism and the overall activity of the cell, with the complex machinery of enzymes involved in the metabolism of glycolipids not being an exception. The aberrant glycosylation of glycolipids on the surface of the majority of cancer cells, associated with increasing evidence about the functional role of these molecules in a number of cellular physiological pathways, has received considerable attention as a convenient immunotherapeutic target for cancer treatment. This has resulted in the development of a substantial number of passive and active immunotherapies, which have shown promising results in clinical trials. More recently, antibodies to glycolipids have also emerged as an attractive tool for the targeted delivery of cytotoxic agents, thereby providing a rationale for future therapeutic interventions in cancer. This review first summarizes the cellular and molecular bases involved in the metabolic pathway and expression of glycolipids, both in normal and tumor cells, paying particular attention to sialosylated glycolipids (gangliosides). The current strategies in the battle against cancer in which glycolipids are key players are then described.

Modulation of GalT1 and SialT1 Sub-Golgi Localization by SialT2 Expression Reveals an Organellar Level of Glycolipid Synthesis Control

Ganglioside glycosyltransferases organize as multienzyme complexes that localize in different sub-Golgi compartments. Here we studied whether in CHO-K1 cells lacking CMP-NeuAc: GM3 sialyltransferase (SialT2), the sub-Golgi localization of UDP-Gal:glucosylceramide β-1,4-galactosyltransferase (GalT1) and CMP-NeuAc:lactosylceramide sialyltransferase (SialT1) complex is affected when SialT2, another member of this complex, is coexpressed. GalT1 and SialT1 sub-Golgi localization was determined by studying the effect of brefeldin A (BFA) and monensin on the synthesis of glycolipids and on the sub-Golgi localization of GalT11-52-CFP (cyan fluorescent protein) and SialT11-54-YFP (yellow fluorescent protein) chimeras by single cell fluorescence microscopy and by isopycnic subfractionation. We found that BFA, and also monensin, impair the synthesis of glycolipids beyond GM3 ganglioside in wild type (WT) cells but beyond GlcCer in SialT2+ cells. Although BFA redistributed GalT1-CFP and SialT1-YFP to the endoplasmic reticulum in WT cells, a fraction of these chimeras remained associated with a distal Golgi compartment, enriched in trans Golgi network, and recycling endosome markers in SialT2+ cells. In BFA-treated cells, the percentage of GalT1-CFP and SialT1-YFP associated with Golgi-like membrane fractions separated by isopycnic subfractionation was higher in SialT2+ cells than in WT cells. These effects were reverted by knocking down the expression of SialT2 with specific siRNA. Results indicate that sub-Golgi localization of glycosyltransferase complexes may change according to the relative levels of the expression of participating enzymes and reveal a capacity of the organelle to adapt the topology of the glycolipid synthesis machinery to functional states of the cell.

GM1 synthase depends on N-glycosylation for enzyme activity and trafficking to the Golgi complex.

Glycosyltransferase cDNAs contain a variable number of potential N-glycosylation sites. Here we examined the occupancy and relevance for the activity and intracellular trafficking of the only potential N-glycosylation site of the mouse β1,3galactosyltransferase (Gal-T2 or GA1/GM1/GD1b synthase) in Gal-T2 cDNA transfected CHO-K1 cells. Transfected cells synthesize a Golgi located active enzyme of 43 kDa whose N-glycan was metabolically labeled from [3H]mannose and was Endo-H sensitive. Inhibition of N-glycosylation by Tunicamycin or by point mutation of the N-glycosylation site resulted in the synthesis of a polypeptide of 40 kDa which lacked enzyme activity and was concentrated in the endoplasmic reticulum (ER). Inhibition of ER glucosidases by Castanospermine impaired the exit of a form of Gal-T2 having reduced enzyme activity from the ER. The N-terminal Gal-T2 domain (aa 1–52) was able to direct and to retain the green fluorescence protein in the Golgi complex. Taken together, these results indicate that Gal-T2 depends on N-glycosylation for its activity and for proper trafficking to, but not its retention in, the Golgi complex.