| Summary: | The central dogma of molecular biology outlines the process of information transfer from a DNA sequence, to a protein chain. Beyond the step of protein synthesis, there are a variety of post-translational modifications that can take place, one of which is addition of carbohydrate chains to nascent proteins, known as glycosylation. The N-linked glycosylation pathway is responsible for the covalent attachment of multifunctional carbohydrate chains on asparagine residues of nascent proteins at Asn-X-Ser/Thr consensus sequences. These carbohydrate chains are thought to aid in cell signaling, immune recognition, and other processes.
Golgi alpha-mannosidase II (GMII) is the enzyme in the N-glycosylation pathway that is responsible for cleaving two mannose linkages in the oligosaccharide GnMan5Gn2 (where Gn is N-acetylglucosamine and Man is mannose), thereby producing GnMan3Gn2 , which is the committed step in complex N-glycan synthesis. It has been speculated that GMII is an excellent therapeutic target for cancer treatment, as the unusual distribution of carbohydrates on the surface of tumour cells has been characterized in many cancers. In addition, swainsonine-—a strong, yet nonspecific inhibitor of GMII—-has been shown to block metastasis and improve the clinical outcome of patients with certain cancers, including those of the colon, breast and skin.
This thesis examines Golgi alpha-mannosidase II from Drosophila melanogaster (dGMII) as a model for all GMII enzymes. First, a 1.80 Angstrom resolution crystal structure of a weak inhibitor, kifunensine, binding to dGMII provides mechanistic insights into the substrate distortion in the GMII reaction. It is hypothesized that the GMII reaction proceeds via a 1 Sinterintermedi-ate. Second, a 1.40 Angstrom resolution structure of a mutant dGMII bound to its natural substrate, GnMan5Gn, identifies key substrate binding and catalytic residues, as well as expanding the definition of the GMII active site to include two distant sugar−binding subsites. Finally, the results are taken together, with knowledge of other related enzymes to synthesize a plausible itinerary for the GMII reaction.
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