Our ultimate objective is to understand the role of protein glycosylation in eukaryotes, and to elucidate the factors which determine the specific carbohydrate structures on glycoprotein. Biosynthesis of N-linked oligosaccharides begins with the transfer of a preformed oligosaccharide containing glucose, mannose and N-acetylglucosamine from dolichyl pyrophosphate to asparagine residues in protein. Following this transfer, the oligosaccharide undergoes processing which requires the concern action of glycosidases at glycosyltransferases. We propose to continue our studies on the early stages of carbohydrate processing in Saccharomyces cerevisise. We have shown the existence of a highly specific alpha-mannosidase which removes one mannose residue from Man9GlcNAc with the formation of a single man8GlcNAc isomer, and have developed a scheme for its purification. We plan to isolate sufficient purified alpha- mannosidase for the preparation of manospecific polyclonal antibodies and for partial amino acid sequencing in order to clone the gene encoding this processing enzyme by screening a lambda gt11 genomic library with the antibodies and with synthetic oligonucleotides. The importance of this processing alpha-mannosidase to mannoprotein synthesis and to yeast physiology will be determined by gene disruption experiments. The yeast DNA probes will also be to identify the putative homologous gene in animal cells. In parallel experiments, we plan to characterize and partially purify the alpha-1,6- mannosyltransferase which initiates the formation of yeast- specific oligosaccharides. These studies will not only increase our understanding of glycosylation, but may also be useful in the development of new antifungal agents, and in the utilization of Saccharomyces cerevisiae for the expression of biologically important heterologous glycoproteins.
| Lobsanov, Yuri D; Yoshida, Takashi; Desmet, Tom et al. (2008) Modulation of activity by Arg407: structure of a fungal alpha-1,2-mannosidase in complex with a substrate analogue. Acta Crystallogr D Biol Crystallogr 64:227-36 |
