Asparagine (N-linked) glycosylation is a ubiquitous modification of eukaryotic secretory and membrane proteins. N-glycosylation is the most common type of glycosylation, with 90% of glycoproteins being N- glycosylated. The N-glycans are initially added to a triplet sequence N-X-S/T by a multi-protein transmembrane complex called oligosaccharyl transferase (OST). Most of the protein N-glycosylation occurs while a protein is being synthesized by the ribosome and being transported through the translocon. Hence, OST physically interacts and forms a super-complex with the ribosome and translocon. The mechanisms of protein synthesis and nascent peptide translocation are better understood thanks to available structures of ribosomes and translocons. However, our understanding of eukaryotic protein N-glycosylation is very limited due to the lack of high-resolution OST structures. Our goal is to bridge this major knowledge gap by characterizing the structure and function of the eight-protein OST complex of yeast. A recent study demonstrated that inhibiting human OST induces senescence in receptor tyrosine kinase?driven tumor cells, suggesting that OST may be a target for the development of anti-tumor agents. Furthermore, because transformation of a normal cell to a cancer cell is usually accompanied by N-glycan branching and extension, several N-glycans have been widely used as tumor markers: for example, carbohydrate antigen (CA) CA19-9 for detecting pancreatic cancer, and CA125, which is considered the gold standard marker for diagnosing ovarian cancer. We propose a comprehensive structure and function study of the yeast OST complex, with a combined approach of cryo-EM, X-ray crystallography, structure-based mutagenesis and cell biology, and in vitro activity assays. Our work has important implications in tumorigenesis and cancer diagnosis and treatment.

Public Health Relevance

Protein glycosylation is a post- or co-translational protein modification that plays many essential roles in the vast majority of secreted and transmembrane proteins and enzymes. Aberrant glycosylation is a hallmark of tumorigenesis, and in fact several N-glycans are well-established tumor diagnosis and progression markers. This proposal is to investigate the structure and function of a multi-protein complex called oligosaccharyltransferase (OST), which is solely responsible for en bloc transfer of 14-sugar oligosaccharides to nascent polypeptides before the proteins are fully folded.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA231466-03
Application #
9938510
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Amin, Anowarul
Project Start
2018-06-18
Project End
2023-05-31
Budget Start
2020-06-01
Budget End
2021-05-31
Support Year
3
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Van Andel Research Institute
Department
Type
DUNS #
129273160
City
Grand Rapids
State
MI
Country
United States
Zip Code
49503