Our objective is to elucidate the genetic control of early stages of protein glycosylation in eukaryotes. We are particularly interested in processing reactions which involve alpha-mannosidases and mannosyltransferases. We have purified, cloned, sequenced and disrupted the gene from Saccharomyces cerevisiae which encodes a processing alpha- mannosidase of unique specificity since it removes one mannose residue from MangGlcNAc with the formation of a single Man8GlcNAc isomer. We propose to study its role in yeast protein glycosylation in vivo. An unsuspected homology between this yeast gene and a partial mammalian cDNA encoding a processing alpha-mannosidase of different specificity was observed. Highly conserved regions between these alpha-mannosidases from widely divergent species were exploited for PCR reactions. We demonstrated the existence of a novel mouse alpha-mannosidase gene. Evidence was obtained for tissue- specific and developmental regulation of expression of the two different mouse alpha-mannosidases. We propose to isolate the cDNA corresponding to this novel mammalian gene for comparative expression and localization studies, and to use PCR to isolate related alpha-mannosidase genes from different species. In parallel experiments, we have partially purified the alpha-1,6-mannosyltransferase which initiates the formation of yeast- specific oligosaccharides, and plan to isolate the corresponding gene using either amino acid sequence information obtained from the purified protein to design degenerate oligonucleotides for PCR reactions on yeast genomic DNA. Alternatively, a yeast genomic bank will be screened for expression with a novel colony filter assay for the transferase. Disruption of this gene is expected to block yeast-specific glycosylation, and lead to yeast strains for heterologous expression of biologically important glycoproteins lacking immunogenic yeast-specific carbohydrate structures.
| 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 |
