The long term objective of this project is to understand the structure, function and mechanism of the oligosaccharyltransferase (OST). The OST transfers high mannose oligosaccharides onto the asparagine residues of nascent proteins in the lumen of the rough endoplasmic reticulum. During the proposed funding period, particular emphasis will be placed on a biochemical, structural and molecular characterization of the mammalian and fungal OST complexes. The canine OST has been resolved into several forms that differ with respect to subunit composition and enzymatic activity. A high turnover form of the canine OST has been detected that lacks one or more subunits, hence it appears to be the catalytic core of the native enzyme. One objective of this project is to compare the enzymatic activity of the core enzyme and the native enzyme using biochemically homogeneous dolichol-oligosaccharides as donor substrates. Genetic and biochemical studies indicate that the yeast oligosaccharyltransferase is a hetero-ocatamer composed of the following three subcomplexes: Ost1p-Ost5p, Wbp1p-Swp1p-Ost2p and Stt3p- Ost3p-Ost4p. Mutations in the Stt3p-Ost3p-Ost4p subcomplex cause 2-30 fold reductions in the in vitro OST activity. The role of the Stt3p- Ost3p-Ost4p subcomplex will be analyzed in vitro using purified wild type and mutant OST complexes and purified donor substrates. The objective of these experiments is to determine whether the Stt3p-Ost3p- Ost4p subcomplex selects the fully assembled donor substrate. Novel assays will be developed to identify donor and acceptor substrate binding sites in the OST using methods that are not dependent upon glycopeptide formation. A second major objective is to investigate a quality control pathway in the yeast endoplasmic reticulum that mediates the folding of hypoglycosylated proteins. Yeast strains that do not express the non-essential DnaJ homologue Scj1p are hypersensitive to tunicamycin, mutations in the oligosaccharyltransferase, or mutations in the ER glucosidases. The extent of hypoglycosylation and precise structure of the asparagine-linked oligosaccharides contribute to the protein folding stress caused by hypoglycosylation. A combination of yeast molecular biological, biochemical and cell biological methods will be used to investigate how the transient display or permanent retention of glucose residues on N-linked oligosaccharides influences the folding and maturation of hypoglycosylated proteins in the yeast endoplasmic reticulum.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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University of Massachusetts Medical School Worcester
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Lu, Hua; Fermaintt, Charles S; Cherepanova, Natalia A et al. (2018) Mammalian STT3A/B oligosaccharyltransferases segregate N-glycosylation at the translocon from lipid-linked oligosaccharide hydrolysis. Proc Natl Acad Sci U S A 115:9557-9562
Rinis, Natalia; Golden, Jennifer E; Marceau, Caleb D et al. (2018) Editing N-Glycan Site Occupancy with Small-Molecule Oligosaccharyltransferase Inhibitors. Cell Chem Biol 25:1231-1241.e4
Wei, Wei; Misra, Saurav; Cannon, Matthew V et al. (2018) Molecular mechanisms of missense mutations that generate ectopic N-glycosylation sites in coagulation factor VIII. Biochem J 475:873-886
Shrimal, Shiteshu; Cherepanova, Natalia A; Gilmore, Reid (2017) One flexible loop in OST lassos both substrates. Nat Struct Mol Biol 24:1009-1010
Shrimal, Shiteshu; Cherepanova, Natalia A; Gilmore, Reid (2017) DC2 and KCP2 mediate the interaction between the oligosaccharyltransferase and the ER translocon. J Cell Biol 216:3625-3638
Lin, David L; Cherepanova, Natalia A; Bozzacco, Leonia et al. (2017) Dengue Virus Hijacks a Noncanonical Oxidoreductase Function of a Cellular Oligosaccharyltransferase Complex. MBio 8:
Cherepanova, Natalia; Shrimal, Shiteshu; Gilmore, Reid (2016) N-linked glycosylation and homeostasis of the endoplasmic reticulum. Curr Opin Cell Biol 41:57-65
Cherepanova, Natalia A; Gilmore, Reid (2016) Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation. Sci Rep 6:20946
Lopez-Sambrooks, Cecilia; Shrimal, Shiteshu; Khodier, Carol et al. (2016) Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells. Nat Chem Biol 12:1023-1030
Shrimal, Shiteshu; Gilmore, Reid (2015) Reduced expression of the oligosaccharyltransferase exacerbates protein hypoglycosylation in cells lacking the fully assembled oligosaccharide donor. Glycobiology 25:774-83

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