Structural and biochemical insights into biosynthesis and degradation of N-glycans
Sammanfattning: Carbohydrates are a primary energy source for all living organisms, but importantly, they also participate in a number of life-sustaining biological processes, e.g. cell signaling and cell-wall synthesis. The first part of the thesis examines glycosyltransferases that play a crucial role in the biosynthesis of N-glycans. Precursors to eukaryotic N-glycans are synthesized in the endoplasmic reticulum (ER) in the form of a lipid-bound oligosaccharide, which is then transferred to a nascent protein. The first seven sugar units are assembled on the cytoplasmic side of the ER, which is performed by glycosyltransferases that use nucleotide sugars as donors. The mannosyl transferase PcManGT is produced by the archaeon Pyrobaculum calidifontis, and the biochemical and structural results presented in the thesis suggest that the enzyme may be a counterpart to the glycosyltransferase Alg1 that participates in the biosynthesis of N-glycans in eukaryotes. Within the ER (in the lumen), activated dolichol-bound sugars are used as donor substrates instead of nucleotide sugars for glycosyltransferases that synthesize N-glycans. The glycosyltransferase dolichylphosphate mannose synthase (DPMS) catalyzes the formation of dolichylphosphate mannose, which is one of these dolichyl-bound sugars. The structure and function were studied for DPMS from Pyrococcus furiosus using protein X-ray crystallographic and biochemical methods and a new assay based on proteoliposomes was designed. The second part of the thesis focuses on glycoside hydrolases from bacteria that break down oligo- and polysaccharides. In one of the studies, a bacterial glycoside hydrolase from the acne bacterium Cutibacterium acnes was characterized. The enzyme was shown to be able to break down the host's N-glycans, which can be used as nutrients or perhaps even evade detection of the immune system. This study also suggests a cytoplasmic biosynthetic pathway for the formation of N-glycans in the acne bacterium. In another study, a glycoside hydrolase from a bacterium living in the moose rumen was characterized. The enzyme was shown to be able to break down β-1,3-glucans, which is a property that can be used industrially for biomass treatment.
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