Synthesis of high mannose, hybrid, and complex type glycans and glycoconjugates in order to study the role of glycosylation of interleukin-1 receptor antagonist

Approximately 1% of the human genome is dedicated to the biosynthesis and diversification of oligosaccharides, where over half of mammalian expressed proteins are glycosylated. Protein glycosylation is divided into N-Linked and O-linked glycosylation depending on the linkage to the polypeptide backbone. N-linked glycosylation has been found to be involved in a multitude of biological processes that include, cell-cell adhesion, cell growth, and immunity. N-linked glycosylation has also been found to increase stability, prevent aggregation, and increase the half-life of the glycoprotein. Due to their significance in biological systems, glycosylation has become an attractive target for the development of more effective therapeutics. Therefore, continued studies are needed to better understand the role of glycosylation in biological systems.;Research in the field of glycobiology has been hampered due to insufficient quantities of the materials needed to conduct biochemical studies. The difficulties in the production of N-linked oligosaccharides and glycoconjugates are mainly due to microheterogeneity and the sheer complexity of the biomolecule. Our laboratory has engineered a glycosylation deficient yeast strain that allows for the production of N-linked oligosaccharides and glycoconjugates that are sensitive to mammalian glycosidases and glycotransferases. Production of these glycosidases and glycotransferases allowed for the synthesis of high mannose, hybrid, and complex type oligosaccharides and glycoconjugates in quantities suitable for biochemical studies. This method permits the synthesis of a variety of N-linked oligosaccharides and glycoconjugates without the difficulties associated with other methods.;The role of glycosylation of interleukin-1 receptor antagonist (IL-1ra) is poorly understood and must be studied in order to determine the effects of glycosylation of IL-1ra. IL-1ra functions to competitively inhibit the pro-inflammatory action of interleukin-1beta, where the non-glycosylated form of IL-1ra is a current therapeutic for rheumatoid arthritis. Although IL-1ra is a potent inhibitor of interleukin-1beta binding, difficulties in storage and the low half-life of the protein still remain. Biophysical studies on the glycosylated form of IL-1ra determined that N-linked glycosylation increases the stability and prevents aggregation of the protein when compared to the non-glycosylated form. Production of homogeneous, human-like glycosylations of proteins, such as IL-1ra, will facilitate studies to determine the role of glycosylation of proteins.