Addition of the saccharide components to classical N- or O-linked glycoproteins is known to occur in the lumen of the endoplasmic reticulum and Golgi apparatus, and as a result, the vast majority of glycoproteins are found in these lumenal compartments, on the cell surface, or in the extracellular spaces. However, several laboratories have recently presented compelling evidence for the presence of glycoproteins in the nuclear and cytoplasmic compartments of the cell. Very little is known about the structure(s) of the oligosaccharide chains on these glycoproteins, their mode of biosynthesis, or how they affect the function(s) of the proteins to which they are attached. The goal of this proposal is to study the glycosylation of three biologically important proteins which are known to be in the nucleus and have recently been shown to be glycosylated: SV 40 large T antigen, the high mobility group proteins, and DNA polymerase alpha. Initial studies will involve structural analysis of the carbohydrates, determination of the chemical nature of the carbohydrate-peptide linkage, and identification of the sites of glycosylation along the polypeptide backbone. Knowledge of the structures present and of the sites of glycosylation will provide essential data for determination of how and where the saccharides are added to the proteins. Previously described forms of nuclear and cytoplasmic glycosylation, such as O-linked N- acetylglucosamine, have been demonstrated to have novel carbohydrate structures and linkages distinct from classic N- and O-linked oligosaccharides. Thus, novel structures will likely be found on the three proteins examined in this proposal. Novel structures imply the existence of novel enzymes for their synthesis. Experiments are proposed for the identification and characterization of any such previously undescribed enzymes. Alternatively, if classic N- and/or O-linked oligosaccharides are found to be present on these proteins, then the topological problem of how proteins located on the cytoplasmic side of the endoplasmic reticulum and Golgi apparatus are modified by enzymes present in the lumen of these same vesicles will be addressed. Post-translational modifications provide an important means of regulating function, stability, and localization of proteins. The three proteins to be studied in this proposal are involved in control of the cell cycle, transcriptional regulation, and DNA metabolism. The regulation of these proteins by post-translational modifications is especially relevant to disease states such as cancer, which may be caused by defects in these processes. The studies outlined in this proposal will form the basis for future work on the role glycosylation plays in the function of these and other nuclear and cytoplasmic proteins.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Physiological Chemistry Study Section (PC)
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State University New York Stony Brook
Schools of Arts and Sciences
Stony Brook
United States
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Haltiwanger, Robert S; Lowe, John B (2004) Role of glycosylation in development. Annu Rev Biochem 73:491-537
Moloney, D J; Haltiwanger, R S (1999) The O-linked fucose glycosylation pathway: identification and characterization of a uridine diphosphoglucose: fucose-beta1,3-glucosyltransferase activity from Chinese hamster ovary cells. Glycobiology 9:679-87
Medina, L; Haltiwanger, R S (1998) Calf thymus high mobility group proteins are nonenzymatically glycated but not significantly glycosylated. Glycobiology 8:191-8
Medina, L; Grove, K; Haltiwanger, R S (1998) SV40 large T antigen is modified with O-linked N-acetylglucosamine but not with other forms of glycosylation. Glycobiology 8:383-91
Haltiwanger, R S; Grove, K; Philipsberg, G A (1998) Modulation of O-linked N-acetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-N-acetylglucosaminidase inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate. J Biol Chem 273:3611-7
Moloney, D J; Lin, A I; Haltiwanger, R S (1997) The O-linked fucose glycosylation pathway. Evidence for protein-specific elongation of o-linked fucose in Chinese hamster ovary cells. J Biol Chem 272:19046-50
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