Determinants of glycosaminoglycan attachment and elongation

Protein molecules are often modified with chemical groups that help them carry out their many important functions in our body. Proteins that are exposed on the cell surface or are released into the extracellular space are modified with sugars, which are added by an assembly line of enzymes (proteins that speed up chemical reactions) inside the cell. These sugar modifications, also known as glycans, have many functions: they contribute to the protective barrier function of the cell envelope, help cells stick to each other, guide immune cells to their targets, and so on. We are interested in one particular group of glycans called glycosaminoglycans, or GAGs. GAGs are long sugar chains that are decorated with chemical groups that carry negative charges. GAGs are very important in several types of connective tissue. The shock-absorbing properties of cartilage, for instance, derive in large part from the abundant GAG chains present in this tissue. GAGs are also very important in cell-to-cell signalling because many signal molecules bind strongly to the sulfate groups of GAGs.

The DNA in our cells contains the instructions for making proteins, but not for making glycans. This means that the glycan-making enzymes must be very specific to ensure that only the right glycans are made. In the case of GAGs, the enzymes must select the right attachment site in the protein and then add one of two types of GAG chain. Our research is seeking to understand how these processes work at the molecular level.

We have previously worked out the atomic structure of the enzyme that starts the assembly of a GAG chain. Making use of this insight, we will engineer an extra cavity into the enzyme. We will then synthesise a sugar with an extra chemical group that fits into the engineered cavity. In this way, we will be able to label all the proteins that are modified by this enzyme. Using the chemical label as a handle, we will purify the products and analyse them by using a very sensitive technique called mass spectrometry. We expect to find new GAG-modified proteins and to learn what exactly defines a GAG attachment site.

In another strand of the programme, we will add a GAG chain to a protein molecule in a test tube, by using only purified enzymes and sugars. This system is much simpler than what happens inside a cell, and it therefore allows us to pinpoint exactly how the enzymes avoid making errors. We will use a powerful technique called X ray crystallography that allows us to visualise all the atoms in the enzymes. Knowing the three-dimensional structures of enzymes helps us understand how they function, in the same way that detailed drawings help engineers to understand man-made machines.

The results from these experiments will tell us how the GAG-making process works. This will not only answer an important biological question, but also generate new tools for making materials that may one day be used to replace defective tissues in human patients.

Proteoglycans (PGs) consisting of a core protein bearing one or several glycosaminoglycan (GAG) chains are essential for multicellular life: they regulate the activities of many morphogens, growth factors, cytokines and proteases, and they are a key constituent of all types of extracellular matrix. The GAG component, which is responsible for many of the biological activities, is synthesised by enzymes residing in the endoplasmic reticulum and Golgi apparatus. How core proteins are selected for GAG attachment and how the GAG type is determined are important questions for which we currently lack satisfactory answers. In this collaborative project, we aim to answer these questions by combining our expertise in structural enzymology and chemical biology. We will engineer the xylosyltransferase that initiates GAG biosynthesis for bioorthogonal labelling of substrates in living cells and analyse the labelled products by mass spectrometry. This proteomic approach is expected to uncover novel PGs. Following elaboration of the O-linked xylose to a tetrasaccharide, addition of the fifth sugar determines whether the GAG will become heparan sulfate (HS) or chondroitin sulfate (CS). In order to define how the GAG type is specified by the PG core sequence, we will assemble tetrasaccharide linkers on PG-derived (poly)peptides in vitro and react them with the glycosyltransferases that initiate HS synthesis (EXTL3) or CS synthesis (CSGALNACTs). Crystal structures of enzyme-substrate complexes will reveal the specificity-determining interactions. We further aim to determine the structure of the EXT1/EXT2 complex that polymerises the HS chain following initiation by EXTL3 . Collectively, these experiments will define the molecular mechanisms that determine GAG attachment and elongation, which in turn will facilitate the rational design of novel reagents and biomaterials.

The direct beneficiaries during the lifetime of the project are mainly in the academic research community, as the proposed research will provide new knowledge, reagents and novel methodologies to study proteoglycans. Our results will be of direct interest to researchers in the areas of glycobiology, enzymology, structural biology, and chemical biology. We expect to discover novel proteoglycans, and such disoveries would have a significant impact on areas in which proteoglycans are known to play essential roles: matrix biology, cell biology, developmental biology, and neurobiology. We will disseminate the results of our research through presentations at academic conferences and publications in peer-reviewed open access journals. We are committed to communicating our results to a wider audience. In order to do this, we will use our professional websites and engage with the press offices at Imperial College and the Francis Crick Institute. We will showcase our research at public science events, such as the annual Somers Town and Imperial Festivals. We will also provide work experience for school kids from disadvantaged backgrounds.

The pharmaceutical industry will benefit from our progress in characterising posttranslational modifications on the cell surface, which is the site of action of many biopharmaceuticals. Proteoglycans are also essential for cell entry of many viruses, bacteria and other pathogens. Novel glycomimetics may prove useful for vaccine development. The biomaterials sector will benefit from new tools for the synthesis of glycoconjugates, with potential applications in regenerative medicine. We intend to hold formal review meetings in years 2 and 3, in order to identify potentially valuable intellectual property, and will realise any opportunities in consultation with the technology transfer offices at Imperial and the Crick. Both Hohenester and Schumann have experience with patent filings.

This interdisciplinary project will contribute to the UK science base through training of postdoctoral researchers in chemical biology and structural biology. Skills in these areas are highly valued by industrial employers in the UK. In addition to lab skills, the outstanding environment at Imperial and the Crick will provide ample opportunities for the postdoctoral reserachers to develop transferable skills, in particular in project and people management and in communicating with experts and the lay public.