Engineering of spectator glycocalyx structures to evaluate molecular interactions at crowded cellular boundaries

Abstract

In the mucosal epithelium, the cellular glycocalyx can project tens to hundreds of nanometers into the extracellular space, erecting a physical barrier that provides protective functions, mediates the exchange of nutrients and regulates cellular interactions. Little is understood about how the physical properties of the mucosal glycocalyx influence molecular recognition at the cellular boundary. Here, we report the synthesis of PEG-based glycopolymers with tunable glycan composition, which approximate the extended architecture of mucin glycoproteins, and tether them to the plasma membranes of red blood cells (RBC) to construct an artificial mucin brush-like glycocalyx. We evaluated the association of two lectins, ConA and SNA, with their endogenous glycan ligands on the surface of the remodelled cells. The extended glycocalyx provided protection against agglutination of RBCs by both lectins; however, the rate and magnitude of ConA binding were attenuated to a greater degree in the presence of the glycopolymer spectators compared to those measured for SNA. The different sensitivity of ConA and SNA to glycocalyx crowding likely arises from the distinct presentation of their mannoside and sialoside receptors, respectively, within the native RBC glycocalyx.

Figure 1.

Recognition of glycans by protein receptors occurs in the dynamic and compositionally heterogeneous environment of the cellular glycocalyx. These interactions can be influenced by the presence and physical properties of non-binding, spectator, glycoconjugates. Synthetic mimetics of membrane associated glycoproteins, such as mucins, can be generated and installed into the native glycocalyx to evaluate how changes in glycocalyx crowding and physical properties impact ligand-receptor interactions.

Figure 2.

Synthesis and characterization of mucin mimetics 5. (A) Glycopolymers 5 were generated from poly(epichlorohydrin) 1 via chloride-to-azide sidechain substitution followed by the CuAAC conjugation of propargyl β-O-glycosides (Glc, Gal, Fuc, and GlcA). The polymers were furnished with a hydrophobic cholestanone moiety for anchoring into cell membranes and a Cy5 fluorescent tag for imaging and quantification. (B) SEC analysis indicated narrow molecular weight and chain-length distributions before and after the chloride-to-azide exchange. (C) Glycopolymers 5 exhibited increased retention in aqueous SEC compared to a PEO standard of similar molecular weight (122 kDa), a characteristic behavior of glycopolymers with extended molecular conformations. (D) AFM imaging of lactose-modified glycopolymers confirmed elongated, mucin-like morphology of the PEG-based glycopolymers.

Figure 3.

RBC glycocalyx remodeling with glycopolymers 5. (A) Polymers 5 incorporate dose-dependently into RBC membranes according to their pendant glycans. Increasing glycopolymer density in RBC membranes induces cell rounding, swelling, and lysis at polymer concentrations above 2.5 uM via brightfield microscopy (B) or SEM (C).

Figure 4.

Association of ConA and SNA lectins with glycocalyx-remodeled RBCs. (A) Binding of fluorescein (FL)-labeled lectins to remodeled cells were assessed via flow cytometry. The presence of spectator glycopolymers 5 at the surface of RBCs attenuates both the initial rate as well as saturation binding of ConA to cell surface glycans. (B) Glycopolymers 5 have no effect on the initial rates of SNA binding but inhibit lectin association near saturation. Relative initial rates were calculated from the linear regions of lectin binding curves and normalized to control cells without polymer treatment. Δ MFI_max corresponds to the change in median fluorescence intensity of cells at saturation lectin binding. (ANOVA, Tukey’s multiple comparisons test; p*** < 0.001).

Figure 5.

Glycocalyx crowding with spectator glycoconjugates differentially affects the association of ConA and SNA with endogenous glycans depending on their distribution throughout the glycocalyx. The glycopolymer spectators inhibit both the initial rates and saturation binding of ConA to the less accessible mannose structures. Glycocalyx crowding influences the association of SNA with sialic acid residues only at later time points near saturation binding, once the more available peripheral glycans have become occupied.