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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM023900-23
Application #
6128899
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Marino, Pamela
Project Start
1977-09-09
Project End
2004-04-30
Budget Start
2000-05-01
Budget End
2001-04-30
Support Year
23
Fiscal Year
2000
Total Cost
$211,899
Indirect Cost
Name
Wadsworth Center
Department
Type
DUNS #
110521739
City
Menands
State
NY
Country
United States
Zip Code
12204
Andreishcheva, Ekaterina N; Kunkel, Jeremy P; Gemmill, Trent R et al. (2004) Five genes involved in biosynthesis of the pyruvylated Galbeta1,3-epitope in Schizosaccharomyces pombe N-linked glycans. J Biol Chem 279:35644-55
Trimble, Robert B; Lubowski, Catherine; Hauer 3rd, Charles R et al. (2004) Characterization of N- and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris. Glycobiology 14:265-74
Cipollo, John F; Trimble, Robert B (2002) Hypoglycosylation in the alg12delta yeast mutant destabilizes protease A and causes proteolytic loss of external invertase. Glycobiology 12:30G-3G
Cipollo, John F; Trimble, Robert B (2002) The Saccharomyces cerevisiae alg12delta mutant reveals a role for the middle-arm alpha1,2Man- and upper-arm alpha1,2Manalpha1,6Man- residues of Glc3Man9GlcNAc2-PP-Dol in regulating glycoprotein glycan processing in the endoplasmic reticulum and Golgi appa Glycobiology 12:749-62
Cipollo, J F; Trimble, R B; Chi, J H et al. (2001) The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum. J Biol Chem 276:21828-40
Cipollo, J F; Trimble, R B (2000) The accumulation of Man(6)GlcNAc(2)-PP-dolichol in the Saccharomyces cerevisiae Deltaalg9 mutant reveals a regulatory role for the Alg3p alpha1,3-Man middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing. J Biol Chem 275:4267-77
Cipollo, J F; Trimble, R B; Rance, M et al. (2000) Two-dimensional relayed-rotating-frame overhauser spectroscopy (1)H NMR experiments for the selective identification of 1,2-glycosidic linkages in polysaccharides. Anal Biochem 278:52-8
Verostek, M F; Lubowski, C; Trimble, R B (2000) Selective organic precipitation/extraction of released N-glycans following large-scale enzymatic deglycosylation of glycoproteins. Anal Biochem 278:111-22
Gemmill, T R; Trimble, R B (1999) Schizosaccharomyces pombe produces novel Gal0-2Man1-3 O-linked oligosaccharides. Glycobiology 9:507-15
Gemmill, T R; Trimble, R B (1999) Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim Biophys Acta 1426:227-37

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