O-acetylation of G d 3

167

The supplementation of the sialic acid biosynthetic pathway with exogenously supplied N-acetylmannosamine (ManNAc) analogs has many potential biomedical and biotechnological applications. In this work, we explore the structure-activity relationship of ManNAc analogs on cell viability and metabolic flux into the sialic acid biosynthetic pathway to gain a better understanding of the fundamental biology underlying "glycosylation engineering" technology. A panel of ManNAc analogs bearing various modifications on the hydroxyl groups as well as substitutions at the N-acyl position was investigated. Increasing the carbon chain length of ester derivatives attached to the hydroxyl groups increased the metabolic efficiency of sialic acid production, whereas similar modification to the N-acyl group decreased efficiency. In both cases, increases in chain length decreased cell viability; DNA ladder formation, Annexin V-FITC two-dimensional flow cytometry assays, caspase-3 activation, and down-regulation of sialoglycoconjugate-processing enzymes established that the observed growth inhibition and toxicity resulted from apoptosis. Two of the panel of 12 analogs tested, specifically Ac 4 ManNLev and Ac 4 ManNHomoLev, were highly toxic. Interestingly, both of these analogs maintained a ketone functionality in the same position relative to the core monosaccharide structure, and both also inhibited flux through the sialic acid pathway (the remainder of the less toxic analogs either increased or had no measurable impact on flux). These results provide fundamental insights into the role of sialic acid metabolism in apoptosis by demonstrating that ManNAc analogs can modulate apoptosis both indirectly via hydroxylgroup effects and directly through N-acyl-group effects.The term "sialic acid engineering" refers to a technique where non-natural N-acetylmannosamine (ManNAc) 1 analogs intercept the sialic acid biosynthetic pathway and are incorporated into cellular sialoglycoconjugates in the place of sialic acid residues (Fig. 1) (1, 2). The impetus behind this strategy is to mimic nature, which uses Ͼ50 different forms of sialic acid to modulate the structure and function of sialic acid-bearing glycoproteins and lipids (3). By using synthetic N-acyl-modified ManNAc analogs, the surfaces of living cells can be endowed with novel properties not found in nature (4) that, depending on the exact analog used to perform this "submolecular microsurgery" (5), have the potential to elicit a variety of changes in the behavior of the host cell. Theoretically, the ability to modify the cell surface and recombinant sialoglycoconjugates with molecular precision has the potential to regulate any biological process governed by sialic acid, such as cell growth and differentiation, communication among different cells, recognition of soluble factors, and attachment to, or disengagement from, the extracellular matrix (6). In practice, sialic acid engineering methods have already been demonstrated to regulate cellular responses ranging from adhesion to prolif...

Section: Discussionmentioning

confidence: 49%

mentioning

confidence: 99%

Characterization of the Metabolic Flux and Apoptotic Effects of O-Hydroxyl- and N-Acyl-modified N-Acetylmannosamine Analogs in Jurkat Cells

Kim

Sampathkumar

Jones

et al. 2004

167

“…GNE mRNA Levels Modulate Apoptosis and ProliferationBased on reports that GM3 and GD3 have an impact on apoptosis and proliferation (53)(54)(55)(56), combined with the connections between GNE and these gangliosides uncovered in this study, we tested these two parameters in cells overexpressing rGNE and in cells treated with shGNE760 to reduce GNE expression. The overexpression of rGNE resulted in a moderate increase in apoptotic cells (Fig.…”

Section: Verification Of the Flow Cytometry Detection Methods By Analymentioning

confidence: 99%

Roles for UDP-GlcNAc 2-Epimerase/ManNAc 6-Kinase outside of Sialic Acid Biosynthesis

Wang

Sun

et al. 2006

Roles for UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE) beyond controlling flux into the sialic acid biosynthetic pathway by converting UDP-GlcNAc to N-acetylmannosamine are described in this report. Overexpression of recombinant GNE in human embryonic kidney (HEK AD293) cells led to an increase in mRNA levels for ST3Gal5 (GM3 synthase) and ST8Sia1 (GD3 synthase) as well as the biosynthetic products of these sialyltransferases, the GM3 and GD3 gangliosides. Conversely, down-regulation of GNE by RNA interference methods had the opposite, but consistent, effect of lowering ST3Gal5 and ST8Sia1 mRNAs and reducing GM3 and GD3 levels. Control experiments ensured that GNE-mediated changes in sialyltransferase expression and ganglioside biosynthesis were not the result of altered flux through the sialic acid pathway. Interestingly, exogenous GM3 and GD3 also changed the expression of GNE and led to reduced ST3Gal5 and ST8Sia1 mRNA levels, demonstrating a reciprocating feedback mechanism where gangliosides regulate upstream biosynthetic enzymes. Cellular responses to the GNE-mediated changes in ST3Gal5 and ST8Sia1 expression and GM3 and GD3 levels were investigated next. Conditions that led to reduced ganglioside production (e.g. short hairpin RNA exposure) stimulated proliferation, whereas conditions that resulted in increased ganglioside levels (e.g. recombinant GNE and exogenous gangliosides) led to reduced proliferation with a concomitant increase in apoptosis. Finally, changes to BiP expression and ERK1/2 phosphorylation consistent with apoptosis and proliferation, respectively, were observed. These results provide examples of specific biochemical pathways, other than sialic acid metabolism, that are influenced by GNE.Sialic acids, 9-carbon amino-monosaccharides, are ubiquitously displayed on mammalian cell surfaces. These sugars modulate biological and pathological events ranging from development and immune function to tumor biology and the viral and bacterial infection of cells (1-3). Not surprisingly, factors that influence the biosynthesis of sialic acid-containing glycoconjugates are of great interest due to the ever growing list of cellular functions influenced by this sugar. The biochemical steps that control sialic acid production, outlined in Fig. 1, have been intensely investigated in recent years (4) with a particular focus given to the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE).2 Whereas GNE is known to control flux into the sialic acid biosynthetic pathway through conversion of UDP-GlcNAc to ManNAc (5) and has been described as the key determinant in the surface display of sialoglycans (6), the biological activity of this protein and its impact in health and disease remain far from understood. In particular, accumulating evidence that healthy cells maintain higher levels of GNE than required for sialic acid production raises the possibility that GNE may be a "moonlighting" protein that serves multiple functions within a cell (7,8).The hypothesis that cells have "excess" GNE is based...

“…The CD60 subset of human T-lymphocyte markers includes GD3 and 9AcGD3 (25). Also, although certain cells are susceptible to GD3-mediated apoptosis, administration of 9AcGD3 can apparently prevent this process (26,27).It remains unclear why most cells that express GD3 also 9-O-acetylate GD3. Unfortunately, the enzymatic machinery involved has proven very labile and difficult to directly assay, purify, or clone.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

9-O-Acetylation of Exogenously Added Ganglioside GD3

Chen

Challa

Varki

2006

Self Cite

Sialic acids are sometimes 9-O-acetylated in a developmentally regulated and cell-type-specific manner. Cells naturally expressing the disialoganglioside GD3 often O-acetylate the terminal sialic acid residue, giving 9-O-acetyl-GD3 (9AcGD3), a marker of neural differentiation and malignant transformation. We also reported that Chinese hamster ovary cells transfected with GD3 synthase can spontaneously O-acetylate some of the newly synthesized GD3. It is unclear whether such phenomena result from induction of the 9-Oacetylation machinery and whether induction is caused by the GD3 synthase protein or by the GD3 molecule itself. We now show that exogenously added GD3 rapidly incorporates into the plasma membrane of Chinese hamster ovary cells, and 9AcGD3 is detected after ϳ6 h. The incorporated GD3 and newly synthesized 9AcGD3 have a half-life of ϳ24 h. This phenomenon is also seen in other cell types, such as human diploid fibroblasts. Inhibitors of gene transcription, protein translation, or endoplasmic reticulum-to-Golgi transport each prevent induction of 9-O-acetylation, without affecting GD3 incorporation. Inhibition of the initial clathrin-independent internalization of incorporated GD3 also blocks induction of 9-O-acetylation. Thus, new synthesis of one or more components of the 9-O-acetylation machinery is induced by incorporation and internalization of GD3. Prepriming with structurally related gangliosides fails to accelerate the onset of 9-O-acetylation of subsequently added GD3, indicating a requirement for specific recognition of GD3. To our knowledge, this is the first example wherein a newly expressed or exogenously introduced ganglioside induces de novo synthesis of an enzymatic machinery to modify itself, and the first evidence for a mechanism of induction of sialic acid O-acetylation.Gangliosides are glycosphingolipids containing one or more sialic acids and are commonly found on the outer leaflet of the plasma membrane (1, 2). With ceramide tails embedded in the membrane bilayer and glycans protruding from the surface, gangliosides are often clustered in microdomains and lipid rafts. Besides being major structural components in neural cell membranes, gangliosides have been found to play important roles in cell adhesion, cell recognition, signal transduction, and neural development (3-6). Early studies showed that when gangliosides in micellar form are exogenously added to cell culture media, they are capable of first becoming associated with cell surface proteins (a trypsin-sensitive component) and subsequently becoming inserted into the plasma membrane (becoming trypsin-resistant) (7).The latter molecules are then indistinguishable from their endogenous counterparts that are synthesized in the same cell (8,9). This technique has since been widely employed to study the metabolic fate and functions of gangliosides. Radioactive and fluorescently labeled gangliosides have been used to elegantly trace the internalization route of exogenous gangliosides, demonstrating that such gangliosides are intern...