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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043768-23
Application #
8316232
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Marino, Pamela
Project Start
1990-04-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
23
Fiscal Year
2012
Total Cost
$389,865
Indirect Cost
$152,865
Name
University of Massachusetts Medical School Worcester
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
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
Cherepanova, Natalia; Shrimal, Shiteshu; Gilmore, Reid (2016) N-linked glycosylation and homeostasis of the endoplasmic reticulum. Curr Opin Cell Biol 41:57-65
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
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; Gilmore, Reid (2013) Glycosylation of closely spaced acceptor sites in human glycoproteins. J Cell Sci 126:5513-23
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
Shrimal, Shiteshu; Ng, Bobby G; Losfeld, Marie-Estelle et al. (2013) Mutations in STT3A and STT3B cause two congenital disorders of glycosylation. Hum Mol Genet 22:4638-45
Bas, Tuba; Gao, Grace Y; Lvov, Anatoli et al. (2011) Post-translational N-glycosylation of type I transmembrane KCNE1 peptides: implications for membrane protein biogenesis and disease. J Biol Chem 286:28150-9
Gilmore, Reid (2011) Structural biology: Porthole to catalysis. Nature 474:292-3
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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