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Abstract

Sphingolipids that differ in their acyl chains have different effects on physiological and pathophysiological processes. Some of the roles can be explained by the biophysical characteristics of sphingolipids with different acyl chain lengths, but direct intracellular targets of especially very long chain fatty acid-containing sphingolipids remain largely unknown. Therefore we previously established a proteomic approach to identify direct interaction partners of very long chain fatty acid-containing sphingolipids in a cellular system. In this PhD thesis, the data obtained from the proteomic approach were bioinformatically analysed and validated. Biochemical assays were performed to further characterise a subset of the identified proteins, including the membrane protein dystroglycan, with respect to structural and functional aspects of their interactions with sphingolipids. Functional studies in oligodendrocytes were performed to investigate a specific role of dystroglycan with very long chain fatty acid-containing sphingolipids in myelin. The obtained bioinformatic analyses revealed a broad spectrum of functions of very long chain fatty acid-containing sphingolipids. The identified interaction appeared to play roles in e.g. membrane/vesicle fusion and diverse adhesion processes. Several of the identified proteins were functionally connected, indicating a preference for the same local lipids within multiprotein complexes. Selected protein-lipid interactions from the proteomic approach were successfully validated. To define the interacting lipid species of these interactions, different assays were performed. The sphingolipid metabolism was modified by two different approaches, siRNA-mediated knockdowns and knockouts of targeting enzymes of the sphingolipid biosynthesis pathway. The results were not unambiguous due to a lack of specificity within the highly connected sphingolipid network. Among the multiple suggested functions of very long chain fatty acid-containing sphingolipids, they fulfil an important function in myelin stability, however the underlying molecular mechanisms are not yet understood. Like very long chain fatty acid-containing sphingolipids, dystroglycan was suggested to be important for myelin stability and the myelination process itself. Therefore dystroglycan-very long chain fatty acid-containing sphingolipids interactions and their putative functions were investigated in oligodendrocytes. An interaction of dystroglycan with sphingolipids was observed and additional data suggested a role of very long chain fatty acid-containing sphingolipids in dystroglycan trafficking to the plasma membrane. The cellular system established in this work will help to elucidate specific interactions of sphingolipids with dystroglycan and to understand their functional implications during myelination in oligodendrocytes. Concluding, these data provide novel insights into the role of very long chain fatty acid-containing sphingolipids in multiple biological processes and hinted at a role of these lipids in trafficking of dystroglycan to the plasma membrane in oligodendrocytes.