The long-term goal of the proposed research is to provide new information about the mechanisms involved in glycoprotein N-linked and O-linked oligosaccharide synthesis and subsequent processing reactions. Addition of carbohydrates to proteins represents the most diverse and complex co- and post-translational modification found in nature, and failure to properly synthesize, target, and degrade glycoprotein glycans leads to numerous identifiable disease states in humans. Recently, a new constellation of human diseases, termed Carbohydrate Deficient Glycoprotein Syndromes, has become a major focus, because the disease incidence is much higher than originally believed due to a growing understanding of its biochemical basis, better recognition of the symptoms, and proper diagnosis. The syndrome is frequently associated with dysmorphism, hypotonia, and neural development disorders that have been attributed to other diseases. Type I CDGS results from any genetic defect that limits formation of the GIcMan9GIcNAC2-PP-dolichol, which serves as the N-glycan donor in the endoplasmic reticulum of essentially all eukaryotic cells. As a result, the yeast, Saccharomyces cerevisiae, has become an important tool in understanding CDGS, because of the ability to identify and biochemically characterize mutations in the biosynthetic pathway steps common to humans. Currently, there are still unidentified genes involved in oligosaccharide-lipid formation that could be the genetic basis for forms of CDGS. This work will determine the role of a newly discovered gene, ALG12, which appears to specify a late-acting sugar transferase in oligosaccharide-lipid synthesis, in downstream processing events (Aim 1). In addition, a bioinformatic approach has identified a unique ORF in S. cerevisiae, potentially encoding one of the remaining sugar transferases involved in N-glycan or GPI anchor precursor synthesis, which will be characterized (Aim 2). Several additional genes responsible for cell wall mannoprotein synthesis in Schizosaccharomyces pombe will be examined to determine their biosynthetic roles (Aim 3) and the biosynthetic route of a novel cell wall sugar epitope, pyruvylGalbeta1,3Gal will be determined (Aim 4). Finally, a novel S. pombe alpha1,3-galactosyltransferase which may be related to enzymes in non-primate mammals and new world monkeys that synthesize the alpha1,3Gal epitope on tissues leading to xenograft rejection in humans, will be characterized (Aim 5). Methods employed include high-field NMR spectroscopy, mass spectrometry, protein separations techniques, molecular genetics and biochemical analyses.
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