Changes in cell, tissue and organism metabolism commonly accompany development, and also major disorders like cancer and cardiovascular diseases. Such metabolic changes result in prominent alterations in cell surface carbohydrate structures. Thus, the measurement of cellular glycan changes can report on the underlying metabolic processes. These changing glycan profiles also regulate cell function, and thus their quantitative measurement can provide insight into human pathophysiology. While the measurement of glycans on naturally occurring proteins is beneficial, the expression of such glycoproteins can be heterogeneous even for the same cell type from different donors, or blood biomarkers from a similar patient population. Further, glycan profiling typically involves sample destruction since glycans are extracted from cells or tissue prior to analyses using mass spectrometry, lectin microarrays or other methods. In this regard, it would be attractive to develop a tool/technology that will enable the non-destructive longitudinal study of tissue specific, glycan structure changes both for ex vivo and in vivo applications. To address this unmet need, this proposal develops a new reagent toolkit called ?synthetic glycan biomarkers? (?SynGlycans?) for the measurement of dynamic changes in cellular carbohydrate profiles. In this regard, SynGlycan are non-natural, designer probes that are optimized for: 1. Secretion from cells, with SynGlycan products being found in either blood or urine in animal models and 2. Targeted mass spectrometry based quantitative analysis. Such reagents may be used for longitudinal studies in a variety of disease contexts, since they can be designed to be expressed at a high level, in a spatially and temporally controlled manner. In order to test this concept, proof-of-principle studies are proposed here to overcome key technical challenges.
The specific aims are:
Aim 1 : To design, develop and iteratively refine synthetic glycan biomarkers using cell based assays.
This aim will refine a family of SynGlycans to identify a few that are expressed at high levels and that describe the major features of cellular metabolic pathways.
Aim 2 : To assay the temporal evolution of carbohydrate structures in murine disease models by applying synthetic glycan biomarkers in longitudinal studies.
This aim uses a murine colorectal cancer model to measure SynGlycan products secreted into blood and urine. If successful, the core technology developed here can be multiplexed with other existing technologies like inducible-promoters, tissue-specific promoters, imaging-constructs, CRISPR and additional synthetic probes to obtain a rich, ?systems-level view? of whole cell/animal physiology. Once the technology is fully developed, we envision that these probes will be broadly useful as they can be introduced in any system or organism of interest. In the long term, safe expression of such molecules in host may also provide novel molecular diagnostic tools for early disease detection that complements the current focus on natural biomarkers. Overall, this application will result in a new molecular technology and broadly useful tool for biomedical research.
This project will develop and characterize a novel technology called ?synthetic glycan biomarkers? that can be broadly useful to study glycosylation pathways in most mammalian cells and also animal models. These non- natural glycopeptides will report on the metabolic status and glycosylation machinery active in any arbitrary cell/tissue. In mouse tumor models, these synthetic markers may inform us on how carbohydrate structures and metabolic pathways evolve in tumors as the cells grow in the in vivo microenvironment and cancer the progresses.
