Engineering of lipid membranes asymmetrically functionalized with chondroitin sulfate†

One of the defining properties of the eukaryotic plasma membrane is the glycocalyx, which is formed by glycosylated lipids and proteins. The glycocalyx is arranged asymmetrically, as it is exclusive to the extracellular side of the membrane. Membrane asymmetry therefore includes both lipid and carbohydrate asymmetry, whereby the latter has the most skewed trans-leaflet imbalance. The glycocalyx plays a structural role that protects cell integrity and it also participates in mechanosensing and other cellular processes. However, our understanding of glycocalyx function is hampered by the lack of suitable model systems to perform biophysical investigation. Here, we describe the engineering of lipid bilayers that are chemically conjugated at the outer surface with one of the most abundant glycocalyx components, chondroitin sulfate (CS). Membranes were doped with a reactive phospholipid, which allowed thiol–maleimide conjugation of thiol-modified CS at the lipid headgroup. Our data show that we achieved CS conjugation of large unilamellar vesicles, supported lipid bilayers, and giant unilamellar vesicles. CS conjugation of vesicles allowed electrostatic recruitment of poly-L-lysine, which could recruit other CS-coated vesicles or CS in solution. Overall, we describe a simple and robust method for polysaccharide functionalization of vesicles which can be applied to gain new mechanistic understanding of the pathophysiological role of the glycocalyx.