Recombinant glycosidases as tools in research

The importance of glycosylation events and resulting glycoconjugates in health and disease is well-known. The variability of glycoconjugates in nature, including also the more complex than previously believed glycomes of invertebrates, presents a considerable challenge as regards their structural analysis. Even the release of glycans from proteins or peptides requires careful consideration and various enzymatic or chemical approaches can be employed, which may result in a different set of glycans being isolated. Often the N-glycosidase from almonds is used to release N-glycans from glycopeptides. Furthermore, another glycosidase, Drosophila Fdl, removes specifically the N-acetylglucosamine attached to the alpha1,3-arm of N-glycans. In order to optimise the use of these enzymes, I propose to prepare recombinant forms of them and examine further their substrate requirements and optimal conditions. The end result is intended to be highly pure, well characterized and, if possible, immobilized forms of these enzymes, which should facilitate development of new, faster and robust methods for N- glycan analysis.

Various analytical tools are necessary for studying complex macromolecules found in living organisms. Although different modern analytical instruments facilitate detailed examination of a broad range of samples, very often a confirmation of the performed analyses is desired or even necessary. For this purpose, researchers also make use of well-characterised enzymes which are able to remove specific building stones of the studied macromolecules. In this project, for enzymes involved in removal of single monosaccharides or large saccharide chains from proteins carrying such structures (for example, antibody IgG), a cost-effective scheme for their production in yeast and insect cells has been established. The relevant properties of the produced enzymes have been studied thoroughly including a range of custom-made artificial and natural substrates. The properties of these enzymes were compared to either already known related enzymes or native enzymes purified directly from natural sources, gaining insight into their specificity and sensitivity towards various relevant compounds and conditions used defining the limitations of their use. As the result of this project, the repertoire of well- defined enzymatic tools available to researchers has therefore been enriched. Moreover, the results on a specific group of enzymes studied in the course of this project also serves to further efforts towards alleviating issues related to lactose intolerance in both food technology and medicine.