Background and previous work
Glycosylation is the most abundant enzymatic posttranslational modification of proteins. Sialic acid represents the terminal monosaccharide of most glycoconjugates and is involved in a variety of cellular functions. It is synthesised in the cytosol from UDP-Nacetylglucosamine (UDP-GlcNAc) by UDP-GlcNAc-2-epimerase/ManNAc-kinase.
Mutations of these enzymes cause hereditary inclusion body myopathy, a very serious
age-related and late-onset disease of distal and proximal skeletal muscles. Recent studies have demonstrated that many intracellular proteins, which are normally non-glycosylated, are modified by a single O-linked GlcNAc on serine/threonine residues via O-GlcNActransferase. O-GlcNAcylation influences localisation, activity or interaction of proteins. This is of special importance since in age-dependent degenerating diseases, such as Alzheimer’s disease, false-regulated O-GlcNAcylation occurs. Several authors have suggested that both sialylation and O-GlcNAc modification of proteins correlate with ageing (especially in neuronal, muscle and endothelial cells). Since both UDP-GlcNAc-2-epimerase/ManNAc-kinase and O-GlcNAc-transferase use UDP-GlcNAc as substrate, we assume that it represents a novel age-dependent “metabolic sensor”. UDP-GlcNAc is synthesised from glucose and thus, blood glucose concentration is heavily involved in its formation.
How are sialylation and/or O-GlcNAcylation involved in ageing and/or senescence? Is it
possible to interfere with ageing and/or senescence by modulating sialylation and/or OGlcNAcylation by metabolic sialic acid engineering using natural or unnatural sialic acid
precursors that bypass UDP-GlcNAc?
We have generated two transgenic mice strains (one is overexpressing the mutated key enzyme of the sialic acid biosynthesis, which leads to high sialic acid levels and one has a defect in the sialic acid biosynthesis). Both strains will be compared with wild-type animals. We plan to analyse O-GlcNAc, sialic acid, sialic acid binding proteins and sialic acid-dependent differentiation markers in all organs over the whole lifespan and quantify age-dependent muscle performance in vivo. The outcome of metabolic sialic acid engineering will be analysed in embryonic stem cells (differentiation) and neuronal cells (neurite outgrowth e.g. regeneration). We also plan to analyse the involvement of sialylation and O-GlcNAcylation on the function of endothelial cells. After metabolic engineering we will analyse their barrier capacity and age-related impact on neuronal cells by real-time cell analysis. Since high levels of glucose induce glycation of proteins, we will analyse the function of an artificial blood-brain barrier after glycation in a neuron-dependent system. O-GlcNac and sialic acid analysis as well as application of sialic acid precursors will also be performed in collaboration with other subprojects (SP1: PTMs of tight junction proteins, SP3: PTMs and protein degradation, SP4: stem cell differentiation, SP7: PTMs in diabetic embryos, SP8: AGEs, SP9: PTMs of Klotho, SP10: PTMs of tight junction proteins, SP12: ion channels, SP13: hexosamine pathway and endothelium, SP14: modulation of Wnt signalling).