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

Abstract

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...

UDP-GlcNAc 2-Epimerase/ManNAc Kinase (GNE): A Master Regulator of Sialic Acid Synthesis

“…However, sialuria cell lines have been employed for a variety of in vitro studies, including Jurkat cells [26], Chinese Hamster Ovary (CHO) cells [85, 86], Spodoptera frugiperda (Sf9) insect cells [42], and human epithelial kidney (HEK) cells [87]. These mutant cell lines either arose through mutagenesis screens, or were created by transfection of a GNE plasmid containing a sialuria mutation into normal cells.…”

Activation of the Unfolded Protein Response in Sporadic Inclusion-Body Myositis but Not in HereditaryGNEInclusion-Body Myopathy

“…It is known that different GNE mutations cause different clinical phenotypes (21, 47). Accordingly, it is conceivable that different mutations in the GNE gene may cause different intracellular consequences, considering that additional functions beyond its enzymatic involvement in sialic acid synthesis have been proposed (48). …”

Hereditary Inclusion Body Myopathy (HIBM2)

“…The reactivities to lectins are also variable in some myofibers, suggesting that hyposialylation in muscles may contribute to the focal accumulations of autophagic vacuoles and/or amyloid deposits in affected muscle tissue. Although sialic acid dysregulation is likely primary to disease pathogenesis, recent assessments of myoblast cellular sialylation patterns,14,15 suggest the possible role of other GNE-related contributing mechanisms 54,55. Eisenberg et al looked at the role of GNE gene and other neighboring genes, such as, clathrin light chainA (CLTA)56 which is a regulatory element in clathrin gene function, known to be involved in several pathways of lysosomal proteolysis, and, reversion-inducing cysteine-rich protein with Kazal motifs (RECK)57 which is a membrane-anchored glycoprotein with transformation suppressor activity both located close to the GNE gene 58.…”