Accessible And Robust Tools for Glycosaminoglycan Profiling

Glycosaminoglycans (GAGs) are polysaccharides composed of a repeated disaccharide subunit and known to
be important for the maintenance of structure and mechanical properties of the extracellular matrix. More
recently, changes in the content, size, and composition of GAGs have been found to play biological roles in
disease processes as well. For example, chondroitin sulfate (CS) is the most abundant GAG; however, only the
special form of CS, chondroitin 4-sulfate, is produced by cancer cells and is required for cellular attachment,
migration, and invasion of tumor cells. The most studied GAG, hyaluronan (HA), has been found elevated in
many inflammatory diseases. Recent works suggest not only the amount, but the size of the HA polymers is also
altered in pathological states. The molecular weight and disaccharide subunits of GAGs are highly heterogenous
and as such, there is increasing interest in assigning different biological functions to different molecular weight
GAGs. For example, it was found that the low molecular weight component of serum HA can be used to
differentiate metastatic from non-metastatic breast cancer. Even with these exciting new discoveries of GAG
functions, GAGs are still less studied and understood than their proteoglycan partners due to a lack of convenient
research tools. Sulfated GAGs are usually covalently linked to a core protein and form a complex called
proteoglycan, and an even larger complex can also be formed by multiple proteoglycans. This multi-dimensional
complex makes isolating GAGs from their core protein critical in accurate GAG detection. GAG isolation is a
laborious and time-consuming process involving days of protease digestion, chloroform extractions, dialysis, and
ethanol precipitations which do not fully remove impurities. In addition, many established GAG detection methods
require enzymatic digestion in addition to GAGs isolation making such methods unable to detect intact GAGs
(size determination). The primary goal of this project is to develop robust and accessible research tools enabling
widespread investigation of GAGs’ roles in pathological process.
This Phase I project will create simple-to-use tools for analyzing GAGs from complex biological samples; and
make these tools available to any lab with common biology equipment resulting in a significant savings of time
and labor. Aim 1 will develop an easy-to-use GAG prep kit via selective high-throughput mini-chromatography,
aim 2 will focus on development on straight-forward homogenous intact GAGs assays. The developed tools will
then be validated in aim 3, using breast cancer cell lines and patients’ serum. These tools will enable new
explorations in GAG-based cell signaling, drug development, and biomarker discovery.Changes in glycosaminoglycans content, size and composition have been found to play a biological role in
many pathological processes. Many glycosaminoglycans are also found elevated in inflammatory related
diseases. This project will develop novel tools for studying glycosaminoglycans that will enable new diagnostic
and/or therapeutic development and further elucidation of their role in biological processes.