Protein glycosylation in eukaryotic cells is important for numerous cellular processes, including protein folding, lysosomal targeting, receptor signaling and cell-cell adhesion. Glycoproteins, therefore, are involved in most physiological processes, and aberrant protein glycosylation is observed in almost all major human diseases. Despite the pathophysiological significance of glycoproteins, the vast majority of them have not been characterized at the molecular level, and the functions of their glycans are poorly understood. Cell surface glycosylation is dynamic and changes in glycan structure accompany cell transformation in cancer progression. However, because of current limitations in molecular tools it is not yet possible to follow the dynamic changes of cell-surface glycans with high spatia and temporal resolution. The long-term objective of this project is to develop new imaging and proteomic tools to analyze changes in naturally- occurring glycans in disease-related processes. Our central hypothesis is that bioorthogonal click reactions with fast kinetics, high specificity ad biocompatibility can serve as the foundation upon which tools can be developed and applied to decipher the functional roles of glycans in human disease.
In Aim 1, we will develop labeling methods based on copper-catalyzed azide-alkyne cycloaddition (CuAAC) to incorporate small-molecule fluorescent probes on to cell-surface glycoconjugates. These methods will be combined with modified, stroboscopic, time-lapse imaging to achieve a fast form of localization microscopy for sub-diffraction-limit imaging of glycoconjugates in living systems. Methods will be developed to enable single molecule tracking of glycans on a specific membrane protein. Studies have shown that abnormal glycosylation in tumor cells is associated with cancer progression and malignancy by regulating adhesion, receptor signaling and protein expression. Terminal sialylation and fucosylation are key contributors to these processes.
In Aim 2, we will develop a general glycoproteomic approach to identify sialylated glycoproteins in cancer cells. We will apply this approach for comparative analysis of the sialylated proteomes of cancer cells with distinct metastatic potentials. We hypothesize that abnormal glycosylation in tumor cells will affect the dynamic behaviors of membrane proteins, and thus adhesion and cancer metastasis. Glycoproteins identified from Aim 2 with unique expression patterns will be selected for further biological studies. We will alter glycosylation status of these proteins directly on th cell surface by in situ glycosylation reactions and by glycosidase treatment. The impact of the altered dynamics of the selected proteins on the membrane will be quantified, and the resulting influence, if any, upon cell adhesion and migration will be evaluated (Aim 3).

Public Health Relevance

Protein glycosylation is the most complex and prevalent posttranslational modification. Most cellular processes are mediated by or interact with glycoproteins on some level. It is therefore not surprising that changes in cell surface glycosylation have been observed in almost every major human disease, including cancer and inflammation. The goal of this project is to develop chemical tools for imaging glycans in live cells, and for proteomic analysis of glycoproteins in cancer cells with distinct metastatic potentials to elucidate their functional roles. The glycan-based diagnostic tools developed in this project will be applied to improve our fundamental understanding of how changes to glycosylation status contribute to the process of cancer metastasis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM111938-05
Application #
9308990
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Marino, Pamela
Project Start
2015-04-15
Project End
2019-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
5
Fiscal Year
2017
Total Cost
$310,900
Indirect Cost
$109,300
Name
Scripps Research Institute
Department
Type
Research Institutes
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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