Mucin-like glycocalyx modules for creating complex artificial glycocalyxes

The glycocalyx on the outer surface of cells is made of glycoproteins, glycolipids, and proteoglycans (along with hyaluronan), and accomplishes many crucial functions in the communication of the cell with its environment. Mucins are one major class of glycoproteins in the glycocalyx; they are highly glycosylated (with sugars typically representing around 80% of the molecular mass) and accomplish important functions in the defence against pathogens. Other important glycocalyx elements are Lewis carbohydrate antigens, a group of oligosaccharides which contain Fucose. Two of them, Lewisy and Lewisx, are also targets for cholera toxin (CT), a lectin secreted by Vibrio cholerae that causes life-threating diarrhoea. Finally, on the cell membrane there is also the presence of the Gb3 glycosphingolipid, which its overexpression leads to Fabry’s disease. This glycolipid is also the target for shiga toxin (STx), a lectin secreted by Shigella dysenteriae type 1 (S. dysenteriae) and some strains of Escherichia coli, and it causes abdominal pain, watery diarrhoea, haemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Even though the general mechanism of infection for both cholera toxin and shiga toxin are well known, how these proteins interact with the glycocalyx toward infecting its host cell remains poorly understood. Recent discoveries have shown the need of fucosylated structures to perform a first binding between the cell surface and cholera toxin, while for shiga toxin is well known the need of high concentration of Gb3 on the cell membrane but it is unknown the interactions between the protein and the glycocalyx. The complexity of the glycocalyx, and the scarcity of tools to control and analyse glycocalyx composition and organisation, make mechanistic studies in vivo and in vitro challenging. Our work aims to generate well-defined glycocalyx models to understand how cholera and shiga toxins interact with the cell surface. To synthesize a well-defined structure suitable to perform binding studies with these lectins, we are building mucin-like structures that have hyaluronic acid as a backbone and present a single type of pendant oligosaccharide, Lewisx for cholera toxin and Gb3 for shiga toxin, at defined densities along the hyaluronic acid contour. To this end, hyaluronic acid and oligosaccharides were prepared with appropriate bio-orthogonal reactive groups to allow their conjugation. This was followed by the incorporation of a biotin (or His-tag) on the reducing end of hyaluronic acid to build a well-defined molecular structure suitable for anchorage to cell membrane models, and to perform quantitative binding studies using quartz crystal microbalance (QCM-D) or spectroscopic ellipsometry (SE). The data obtained with the mucin-like structures offered a better understanding about how the glycocalyx interacts with cholera toxin and shiga toxin.