New methods for the study of O-linked protein glycosylation

The eukaryotic cell surface is littered with receptors, binding epitopes, and recognition motifs that mediate the cell's interactions with its environment. The carbohydrate chains that adorn membrane proteins represent key players in this information transfer. Despite compelling examples of their physiological importance, protein-bound glycans remain elusive molecules. The heterogeneity that characterizes glycoprotein biosynthesis compromises our ability to formulate strict structure-function correlations for glycans. This dissertation describes two projects designed to curtail glycoprotein heterogeneity to facilitate glycan studies.; The complete chemical synthesis of the insect antibacterial glycoprotein, diptericin, provides the homogeneous sample necessary to analyze glycosylation's functional impact and to investigate the glycoprotein's antibacterial mechanism. Diptericin, an 82 amino acid glycopeptide, contains regions similar to two different types of antibacterial peptides. A revised, highly practical synthesis of the precursor Nα-Fmoc-Thr(Ac₃-α-D-GalNAc) allowed us to produce sufficient quantities of the glycoprotein for mechanistic assays. The synthetic, full-length polypeptide proved active in growth inhibition assays at concentrations similar to those found in insect hemolymph. Biological analysis of diptericin fragments indicated that the main determinant of antibacterial activity lay in the C-terminal, “attacin-like” region. Activity appeared glycosylation independent. Preliminary structural and mechanistic studies were performed, suggesting possible mechanisms of action.; Secondly, we developed a strategy to inhibit O-linked glycosylation in cultured cells. A uridine-based library, the cornerstone of our approach, targeted glycosylating enzymes such as the UDP-GlcNAc 4-epimerase. This key enzyme catalyzes the conversion of the common carbohydrate donor, UDP-GlcNAc, to the O-linked precursor, UDP-GalN Ac; its inhibition should arrest O-linked glycosylation in cultured cells. Our synthetic strategy exploited a crucial chemoselective coupling between uridine derivatives functionalized at their 5′ positions by an aminooxy or hydrazide group and a diverse array of aldehydes, yielding the corresponding oxime- or hydrazone-based libraries. The 1338-member library we generated produced a substrate-competitive 4-epimerase inhibitor with a K_i of 11 μM. The compound failed to inhibit enzymes operating on similar substrates—UDP-Glc dehydrogenase, the bacterial UDP-Glc 4-epimerase, and the UDP-GlcNAc/GalNAc pyrophosphorylase—suggesting suitably specific inhibition for use in cultured cells. Further application of the uridine library to the inhibition of the UDP-galactopyranose mutase of Mycobacterium tuberculosis is discussed briefly.