This is a competing renewal application for R01 GM066047 entitled Chemical Glycoproteomics. The broad objective of this project, now it its 12th year, is to develop chemical tools for studying mucin-type O- glycosylation and the polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) that initiate this widespread posttranslational modification. Mucin-type O-glycosylation has proven far more difficult to study than other prevalent forms, such as N-glycosylation and O-GlcNAcylation. Our parts list of proteins and sites modified with O-glycans is far from complete, and we have little information regarding the biological substrates of the 20 human ppGalNAcTs. Yet, genetic studies have linked dysregulated expression of ppGalNAcTs to many human diseases, and mutations in O-glycosites can lead to profound pathologies. Thus there is a need for new tools that accelerate research into the molecular basis and biological significance of mucin-type O- glycosylation. This project builds on the work from previous granting periods with three Specific Aims.
In Aim 1, we will develop a glycoproteomics platform for identifying O-glycosylated proteins and mapping O-glycosites. We will employ metabolic labeling with an azide-functionalized GalNAc derivative (GalNAz) followed by click chemistry with biotin probes for selective enrichment of mucin-type O-glycoproteins. After trypsinolysis, glycosylated peptides will be identified and sequenced using our mass-independent IsoStamp technology, developed in the previous funding period. The glycoproteomics platform will be used to identify O-glycoproteins and map O- glycosites from cancerous and normal human prostate tissues, with the long-term goal of biomarker discovery.
In Aim 2, we will develop a chemical genetic approach for identifying biological substrates of ppGalNAcT family members. We will employ the bump-hole strategy to orthogonalize individual ppGalNAcTs so they can uniquely mark their cellular substrates with azidosugars. The azide-modified glycoproteins and glycosites will be identified using the glycoproteomics platform from Aim 1. Finally, in Aim 3 we will identify biological substrates of ppGalNAcT-7, the expression of which is correlated with tumor progression. Using the tools of Aims 1 and 2, we will map O-glycosites initiated by ppGalNAcT-7 in cervical cancer cells. This dataset will fuel hypotheses regarding the enzyme's role in disease progression.

Public Health Relevance

Most human proteins are decorated with sugar molecules, which is why we call them 'glycoproteins'. The shapes, sizes and attachment sites of the sugars change when the cell making the glycoprotein transforms from healthy to diseased. The goal of this project is to develop chemistry-based technologies for detecting changes in glycoproteins associated with cancer, which will aid in cancer research and diagnosis down the line.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA200423-14
Application #
9096777
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Krueger, Karl E
Project Start
2002-08-01
Project End
2020-05-31
Budget Start
2016-06-01
Budget End
2017-05-31
Support Year
14
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
Spiciarich, David R; Oh, Stephen T; Foley, Amy et al. (2018) A Novel Germline Variant in CSF3R Reduces N-Glycosylation and Exerts Potent Oncogenic Effects in Leukemia. Cancer Res 78:6762-6770
Woo, Christina M; Lund, Peder J; Huang, Andrew C et al. (2018) Mapping and Quantification of Over 2000 O-linked Glycopeptides in Activated Human T Cells with Isotope-Targeted Glycoproteomics (Isotag). Mol Cell Proteomics 17:764-775
Spiciarich, David R; Nolley, Rosalie; Maund, Sophia L et al. (2017) Bioorthogonal Labeling of Human Prostate Cancer Tissue Slice Cultures for Glycoproteomics. Angew Chem Int Ed Engl 56:8992-8997
Tomlin, Frederick M; Gerling-Driessen, Ulla I M; Liu, Yi-Chang et al. (2017) Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity. ACS Cent Sci 3:1143-1155
Woo, Christina M; Felix, Alejandra; Byrd, William E et al. (2017) Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes. J Proteome Res 16:1706-1718
Jolly, Amber L; Agarwal, Paresh; Metruccio, Matteo M E et al. (2017) Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding. FASEB J 31:2393-2404
Andres, Lissette M; Blong, Ian W; Evans, Angela C et al. (2017) Chemical Modulation of Protein O-GlcNAcylation via OGT Inhibition Promotes Human Neural Cell Differentiation. ACS Chem Biol 12:2030-2039
Woo, Christina M; Felix, Alejandra; Zhang, Lichao et al. (2017) Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars. Anal Bioanal Chem 409:579-588
Palaniappan, Krishnan K; Bertozzi, Carolyn R (2016) Chemical Glycoproteomics. Chem Rev 116:14277-14306
Sheta, Razan; Roux-Dalvai, Florence; Woo, Christina M et al. (2016) Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells. Data Brief 8:342-9

Showing the most recent 10 out of 13 publications