Research
The next challenge. Unlike the relationships between DNA, RNA and protein, which are relatively well understood (green tick) the rules governing both GAG biosynthesis and the structural control of GAG-related function remain poorly understood (red question mark). This has limited our fundamental understanding of cellular physiology and our ability to explore clinical/industrial applications.
Glycosaminoglycans (GAGs) are polysaccharides present on the surface of most cells and in the extracellular matrix (ECM) of all tissues that influence how cells interact with their pericellular environment. GAG activities are mediated via the binding of protein ligands (growth factors, cytokines, matrix components etc.) to motifs encoded within the GAG chains. The chains are constructed through the action of multiple enzymes which themselves are under transcriptional, metabolic, and epigenetic control.
Currently, we lack a thorough understanding of how the functional activity of a GAG links to its structure and, critically, how GAG structure is controlled by cells. This prevents the harnessing of GAGs for multiple bioscience applications: it is currently impossible to predict changes in GAG motifs/activity from transcriptomic datasets, or to select which enzymes should be combined to synthesise a GAG with a specific function.
We have assembled an interdisciplinary team with complementary approaches, including the use of 3D in vitro models of development (gastruloids) to provide material for coordinated analyses, creating an interconnected web of information that we will then use to generate and test hypotheses. Transcriptomic and proteomic analyses linked to detailed structural and spatial analysis of GAGs will allow us to build and then refine models of how the biosynthetic machinery creates particular GAG structures, allowing us to generate and apply a novel toolbox of GAG-binding probes. Coordinating this information with defined developmental stages and responses to known GAG-dependent ligands (e.g., BMP, FGF family members etc.) will directly link structure to function. To test emergent hypotheses, we will use gene editing to target key regulatory hotspots in the 3D gastruloid models, allowing the refinement of our understanding of the critical factors controlling GAG structure and function and how this maps onto molecular and biological function.
Objectives
01
Discovery
Our aim is to utilise the 3-D gastruloid model of early development to link GAG structure to function. By targeting known biological pathways linked to GAG biosynthesis and combining this with multiple layers of analysis including the use of established GAG detection methods alongside novel techniques such as time-of-flight secondary ion MS (ToF-SIMS) we will transform our understanding of GAG structure/function relationships.
In addition, by generating novel proteomic and transcriptomic datasets of GAG biosynthetic components linked to developmental stage using the gastruloid model, we will integrate transcript, protein and GAG datasets to identify potential key regulators of GAG biosynthesis and identify potential targets for exploitation as outlined in objective 3.
The lead is: Cathy Merry
Contact: Cathy.Merry@nottingham.ac.uk
02
Tool development
Using the latest developments in the fields of phage and yeast display, such as utilising next-generation sequencing, our aim is to create probes capable of discriminating between subtle differences in GAGs of the same family. These can, in turn, provide crucial information on GAG involvement in developmental processes and beyond.
The lead is: Tony Day
Contact: Anthony.Day@manchester.ac.uk
03
Challenging the system
We will identify targets in the family of GAG biosynthetic genes, core proteins and key transcriptional regulators to be disrupted in both the human and mouse gastruloid system. To assess the impact of each targeted gene on patterning, we will monitor gastruloid morphology, reporter expression and localisation of stage-specific markers. This will demonstrate how changes in GAG structure alter the key signalling pathways driving early development and, in turn, inform us of the regulatory mechanisms of GAG structure and biosynthesis.
The lead is: David Turner
Contact: David.Turner@liverpool.ac.uk