The research described in this proposal is directed towards understanding the mechanism of asparagine- linked glycosylation of proteins in the endoplasmic reticulum (ER) by the oligosaccharyltransferase (OST). Particular emphasis will be placed upon (i) determining whether the STT3B isoform of the OST performs a general role in posttranslational glycosylation of sites that are skipped by the STT3A isoform of the OST, (ii) elucidating how the two OST isoforms contribute to hypoglycosylation of proteins in cells with defects in the assembly of the dolichol-linked oligosaccharide donor for N-linked glycosylation, and (iii) obtaining a cryo-electron microscopy structure of the OST in a complex with a translating ribosome and a protein translocation channel. Current evidence indicates that the STT3A isoform of the OST is primarily responsible for cotranslational N-glycosylation of nascent polypeptides as they pass through the protein translocation channel. The STT3B isoform can mediate posttranslational glycosylation of unfolded proteins. Novel insight into the in vivo role of the STT3B isoform of the OST will be obtained by identifying additional classes of STT3B substrates. Integral membrane proteins with glycosylation sites near the membrane bilayer, proteins with carboxyl-terminal glycosylation sites or multiple closely spaced glycosylation sites will be tested as potential STT3B substrates. Co-immunoprecipitation experiments will be performed to determine whether hypoglycosylated proteins are delivered to the STT3B isoform of the OST by lumenal ER chaperones or ER-localized lectins. Inherited defects in the assembly pathway for the dolichol-linked oligosaccharide donor (Dol-PP-OS) for N-linked glycosylation cause congenital disorders of glycosylation (CDG-I). Assembly of Dol-PP-OS will be perturbed in HeLa cells by si-RNA mediated depletion of the ALG6 glucosyltransferase. Simultaneous depletion of ALG6 and STT3B will be used to test whether the STT3B isoform of the OST modifies glycosylation sites that are skipped by the STT3A isoform of the OST in ALG6-deficient cells. Recent reports indicate that mutations in the TUSC3 gene can cause autosomal recessive mental retardation. Si-RNA mediated depletion of TUSC3 will be used to determine whether this OST accessory subunit is required for efficient glycosylation of certain proteins. The yeast and mammalian OST are large (240-270 kD) hetero-oligomeric membrane proteins that may interact directly with the protein translocation channel in the endoplasmic reticulum. Complexes of the OST, the Sec61 protein translocation channel and a translating ribosome will be prepared for cryoelectron microscopy. Structural analysis of these complexes should provide insight into the location of the STT3 active site in the OST complex and the path taken by the nascent polypeptide between the lumenal face of the protein translocation channel and the OST active site.

Public Health Relevance

Glycosylation of secretory proteins, lysosomal proteins and integral membrane proteins is of fundamental importance to human health. Hypoglycosylation of glycoproteins in the endoplasmic reticulum is responsible for the family of diseases known as congenital disorders of glycosylation (CDG-I). Mutations in the TUSC3 gene, which encodes a non-catalytic subunit of the oligosaccharyltransferase, can cause autosomal recessive mental retardation. Our studies address the role of oligosaccharyltransferase isoforms in achieving efficient glycosylation of proteins.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Marino, Pamela
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University of Massachusetts Medical School Worcester
Schools of Medicine
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Shrimal, Shiteshu; Cherepanova, Natalia A; Gilmore, Reid (2015) Cotranslational and posttranslocational N-glycosylation of proteins in the endoplasmic reticulum. Semin Cell Dev Biol 41:71-8
Shrimal, Shiteshu; Trueman, Steven F; Gilmore, Reid (2013) Extreme C-terminal sites are posttranslocationally glycosylated by the STT3B isoform of the OST. J Cell Biol 201:81-95
Ruiz-Canada, Catalina; Kelleher, Daniel J; Gilmore, Reid (2009) Cotranslational and posttranslational N-glycosylation of polypeptides by distinct mammalian OST isoforms. Cell 136:272-83
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Karamyshev, Andrey L; Kelleher, Daniel J; Gilmore, Reid et al. (2005) Mapping the interaction of the STT3 subunit of the oligosaccharyl transferase complex with nascent polypeptide chains. J Biol Chem 280:40489-93
Nilsson, IngMarie; Kelleher, Daniel J; Miao, Yiwei et al. (2003) Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex. J Cell Biol 161:715-25
Kelleher, Daniel J; Karaoglu, Denise; Mandon, Elisabet C et al. (2003) Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties. Mol Cell 12:101-11
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Karaoglu, D; Kelleher, D J; Gilmore, R (1997) The highly conserved Stt3 protein is a subunit of the yeast oligosaccharyltransferase and forms a subcomplex with Ost3p and Ost4p. J Biol Chem 272:32513-20
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