| Protein glycosylation is an event that occurs in >50% of all proteins and has a decisive influence on protein structure, function and stability. Nevertheless, in the past era of protein research relatively little attention has been paid to the carbohydrate portion of known glycoproteins for several reasons. First of all, protein glycosylation is not under direct genetic control and therefore cannot be predicted from the genome. Secondly, glycosylation is often a dynamic process and therefore it is extremely difficult to isolate a particular glycoform of a protein in substantial and pure amounts. Thirdly, the influence of carbohydrates on proteins has long been strongly underestimated. And finally, research on carbohydrates is hampered by a strong shortage of tools in comparison with the DNA and protein fields. As a consequence, glycoprotein research has developed poorly to date, which hampers our insight in the function of protein glycosylation, further thwarted by the inherent instability of many glycosidic linkages of the glycoprotein of interest. The proposal described here intends to tackle glycoprotein research in several directions, mainly centered around unique, bioorthogonal chemical reactions. In other words, chemistry will be applied or newly developed that is so mild and selective in nature that it is perfectly compatible with sensitive systems like proteins. Nevertheless, despite its mildness, the chemical reaction should proceed with high selectivity and efficiency. One such reaction is the copper(I)-catalyzed azide-acetylene cycloaddition (CuAAC) that has been developed in 2002. We will apply the CuAAC technology for the preparation of homogeneously pure analogs of glycoproteins, so-called neoglycoproteins, in order to shed light on the positioning, nature and length of specific saccharides on protein function. In particular, we will study not only how glycosylation of an esterase, Candida antartica B influences the catalytic properties of the enzyme, but also how trafficking of proteins to specific cell organels can be regulated by glycosylation (using fluorescence microscopy). The latter study will provide important insights for targeted delivery of (extremely expensive) recombinant glycoproteins for enzyme replacement therapy. On a fundamental basis, novel chemistry will be explored for application in bioorthogonal ligation strategies. The current ?chemical biology? toolbox is only poorly filled and relies on only a handful of chemical reactions, all centered around azide functionality. We have discovered that nitrones provide a unique reactivity with readily accessible cyclooctyne probes, which can be perfectly applied for the mild and efficient preparation of glycoproteins under copper-free conditions. The nitrone-acetylene cycloaddition will be applied in unprecedented strategies for N-terminal protein conjugation. Finally, we will apply the tools and strategies described above to shed light on the role of deficient glycosylation in a range of genetic glycosylation disorders, mainly Congenital Disorders of Glycosylation, and Walker-Warburg Syndrome. Thus, a range of unique azide- or nitrone-labeled sugars will be metabolically incorporated into glycoproteins in an in vitro cell system to investigate the (lack of) glycosylation in CDG-related diseases. Such studies may provide unique insights in the molecular mechanisms that underly a range of glycosylation-related disorders and will help to identify new genetic defects. |
