This is a competing renewal application of RO1 GM66047 (""""""""Studies of Protein Glycosylation""""""""). The broad objective of this project is to develop chemical tools for studies of protein glycosylation, the most prevalent and structurally diverse form of posttranslational modification. The next granting period is focused on two forms of Ser/Thr O-glycosylation for which chemical tools are needed: mucin-type O-glycosylation characterized by a conserved core N-acetylgalactosamine (GalNAc) residue, and O-GlcNAcylation in which Ser/Thr residues are modified with a single 2-N-acetylglucosamine (GlcNAc) residue. Changes in mucin-type O-glycoprotein expression have been associated with cancer. We hypothesize that prostate cancer biomarkers exist among this class of glycoproteins. Protein O-GlcNAcylation is a reversible signaling modification that is known to be critical for viability of embryos. We hypothesize that O-GlcNAcylation regulates proteins involved in human embryonic stem cell (hESC) differentiation and that differences in O-GlcNAcylation states contribute to the varied behavior of hESCs and induced pluripotent stem cells (iPSCs). We propose to develop new technologies for """"""""chemically-directed"""""""" glycoproteomics (Aim 1) and to apply these tools to profile changes in mucin-type O-glycosylation associated with prostate cancer (Aim 2) and to profile O-GlcNAcylated proteins in differentiating hESC and iPSCs (Aim 3). The proposed glycoproteomics platform in Aim 1 combines two technologies developed in the last granting period: metabolic labeling of mucin-type O-glycans and O-GlcNAcylated proteins with azidosugars, and isotopic signature transfer and mass pattern prediction (IsoStamp) for chemically-directed mass spectrometry. IsoStamp exploits the perturbing effects of a dibrominated chemical tag on a peptide's mass envelope, which can be extracted from full-scan MS data sets using a computational algorithm we developed in-house. The method enables high- sensitivity detection of chemically tagged peptides from complex mixtures such as cell and tissue lysates. In addition to the use of metabolic labeling and IsoStamp, our glycoproteomics workflow includes glycosylation site-identification via ETD fragmentation, and quantitative comparative MS analysis with light and heavy IsoStamp tags.
In Aim 2, these glycoproteomics tools will be used to identify mucin-type O-glycoproteins from human prostate cancer biopsy tissue as a function of Gleason grade, and for comparative studies with normal human prostate tissue.
In Aim 3, we will probe changes in the O-GlcNAcylation states of cytosolic and nuclear proteins in human embryonic stem cells (hESCs) during neuronal differentiation. We will also perform a comparative analysis of O-GlcNAcylated proteins in hESCs and iPSCs.

Public Health Relevance

Over half of the proteins in human cells are thought to be decorated with sugar molecules, in which case they are called glycoproteins. The structures of the sugars likely undergo changes when the cell changes from healthy to cancerous, and when embryonic stem cells mature into cells of the adult human body. This proposal aims to develop chemical tools for detecting changes in sugars on proteins during cancer progression and maturation of embryonic cells. The results may offer new tools for cancer detection and diagnosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066047-12
Application #
8655161
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Marino, Pamela
Project Start
2002-08-01
Project End
2015-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
12
Fiscal Year
2014
Total Cost
$338,782
Indirect Cost
$93,518
Name
University of California Berkeley
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Palaniappan, Krishnan K; Bertozzi, Carolyn R (2016) Chemical Glycoproteomics. Chem Rev 116:14277-14306
Northcott, Josette M; Northey, Jason J; Barnes, J Matthew et al. (2015) Fighting the force: Potential of homeobox genes for tumor microenvironment regulation. Biochim Biophys Acta 1855:248-53
Pickup, Michael W; Mouw, Janna K; Weaver, Valerie M (2014) The extracellular matrix modulates the hallmarks of cancer. EMBO Rep 15:1243-53
Belardi, Brian; de la Zerda, Adam; Spiciarich, David R et al. (2013) Imaging the glycosylation state of cell surface glycoproteins by two-photon fluorescence lifetime imaging microscopy. Angew Chem Int Ed Engl 52:14045-9
Palaniappan, Krishnan K; Hangauer, Matthew J; Smith, Timothy J et al. (2013) A chemical glycoproteomics platform reveals O-GlcNAcylation of mitochondrial voltage-dependent anion channel 2. Cell Rep 5:546-52
Breidenbach, Mark A; Palaniappan, Krishnan K; Pitcher, Austin A et al. (2012) Mapping yeast N-glycosites with isotopically recoded glycans. Mol Cell Proteomics 11:M111.015339
Yu, Seok-Ho; Boyce, Michael; Wands, Amberlyn M et al. (2012) Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners. Proc Natl Acad Sci U S A 109:4834-9
An, Hyun Joo; Gip, Phung; Kim, Jaehan et al. (2012) Extensive determination of glycan heterogeneity reveals an unusual abundance of high mannose glycans in enriched plasma membranes of human embryonic stem cells. Mol Cell Proteomics 11:M111.010660
Hubbard, Sarah C; Boyce, Michael; McVaugh, Cheryl T et al. (2011) Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg Med Chem Lett 21:4945-50
Boyce, Michael; Bertozzi, Carolyn R (2011) Bringing chemistry to life. Nat Methods 8:638-42

Showing the most recent 10 out of 31 publications