The analysis in the previous section suggests that branching structures generated by molecular glycosylation may alter protein properties. Such changes may be important for the roles of glycans in infection inhibition (Figure 4C). In the context of polymer brush theory, such changes could modulate molecular packing. Then changes in molecular packing may alter U* associated with molecular exclusion, if that occurs in the formation of the interface (Figure 3F).

Under this hypothesis, we next investigated whether glycosylation affects protein packing and intermolecular interactions on the plasma membrane. To directly address this question, we biochemically reconstituted membrane protein systems in vitro and quantified protein packing. The ectodomain of the membrane proteins used in the viral infection assay was purified and introduced into fluid lipid bilayers formed on silica beads via affinity tag (Figure 5A). In this geometry, the membrane surface area was fixed and unchanged, allowing the molecular density to be measured without the influence of membrane structure (Lee et al., 2021). A non-glycosylated protein ectodomain produced in E. coli was also used for comparison. Molecular binding density was measured for each lipid-coated bead by flow cytometry.

Biochemical reconstitution of protein packing in membrane surface.

(A) Lipid bilayers coated on silica beads for incorporating bacterial (b-) and glycosylated (mammalian expressed, g-) proteins, schematic and fluorescent images. (B) Representative result for flow cytometry analyses of protein binding to lipid bilayer coated silica beads. Bar is the standard deviation in each measurement. Lines are regression curves to receptor binding model Bx/(KD +x), where x is protein concentration, KD is the dissociation constant, and B is the saturated density. (C) Relations of surface area coverage by bound proteins and concentrations of proteins used for membrane binding. Surface area was normalized by assuming all bound proteins were in a hemisphere of radius RF and the ratio of coverage was calculated. Protein concentrations were normalized by KD. The dot line in the plot indicates the coverage when the hemisphere of radius RF aligned in a hexagonal close packing. (D) Schematic for structures and free energies for glycosylated and non-glycosylated proteins with similar RF that are at diluted and more condensed densities on the membrane surface.

The non-glycosylated recombinant CD43 and MUC1(14TR) ectodomain produced in E. coli bound at an estimated average saturation density of more than 10,000 molecules μm–2, while the glycosylated CD43 and MUC1 ectodomain produced in HEK 293T cells bound at much lower saturation densities (Figure 5B). Thus, the saturation binding densities of these proteins reduced significantly by glycosylation. The ectodomain of EPHB1, a low-level glycosylated protein, bound in the intermediate density range. The binding density of non-glycosylated molecules was comparable to the saturation binding density shown for green fluorescent protein (GFP) on planar lipid bilayers (~7000 μm–2; Nye and Groves, 2008). Dissociation constants for membrane-bound glycosylated (g-) proteins (0.61 μM and 0.48 μM for CD43 and MUC1, respectively) were similar to or lower than those for bacterial (b-) proteins (3.84 μM and 12.6 μM for CD43 and MUC1, respectively), indicating that differences in molecular affinity for the lipid bilayer are not responsible for the large differences in saturation binding densities. The binding of all tested molecules was well explained by the standard first-order receptor-ligand binding reaction (Figure 5B), and single molecule tracking experiments confirmed that both glycosylated (g-) and non-glycosylated and bacterial (b-) proteins were equally diffusible as individual molecules (Figure 5—figure supplement 1A). Therefore, it is unlikely that there are protein-protein or protein-layer interactions that would allosterically affect protein packing.

To compare the packing of proteins with different molecular weights and RF, we introduce the projected coverage area on the membrane as a normalized parameter, assuming that these molecules could be approximated by a hemisphere of RF, when they are in the mushroom regime (Halperin, 1999; Milner, 1991; Figure 5C). Under this assumption, the coverage of highly glycosylated proteins at binding saturation was ~20% for g-MUC1 and ~40% for g-CD43, respectively. These were smaller than the coverage of molecules at hexagonal close packing that is ~90.7%. In contrast, the coverage of b-CD43 and b-MUC1 at saturated binding was estimated to be greater than 100% under this normalization standard, indicating that the mean projected sizes of these molecules in surface direction were smaller than those expected from their RF. Thus, it is clear that glycosylation reduces the saturation density of membrane proteins, regardless of molecular size.

Highly glycosylated proteins resisted densification, indicating that some intermolecular repulsion is occurring. In the framework of polymer brush theory, the intermolecular repulsion of densely packed highly glycosylated proteins is due to an increase in either fel, fint(dF), or both (Hansen et al., 2003; Wu et al., 2002). The term of intermolecular interaction, fint, is regulated by intermolecular steric repulsion, which occurs when neighboring molecules cannot approach the excluded volume created by the stochastic configuration of the polymer chain (Attili et al., 2012; Faivre et al., 2018; Kreussling and Ullman, 1954; Kuo et al., 2018; Paturej et al., 2016). The magnitude of this steric repulsion depends largely on RF in dilute solutions, but the molecular structure may also affect it when molecules are densified on a surface. In other words, the glycans protruding between molecules can cause steric inhibition between neighboring proteins (Figure 5D). Such intermolecular repulsion due to branched side chains occurs only when the molecules are in close proximity and sterically interact on a two-dimensional surface, but not in dilute solution, and does not occur in unbranched polymers such as underglycosylated proteins (Figure 5D). Based on the above, we propose the following model for membrane proteins: Only when the membrane proteins are glycosylated does strong steric repulsion occur between neighboring molecules during the densification process, suppressing densification.

The molecular structural state of these proteins needs to be further discussed to estimate the contribution of fel, which represents resistance to molecular elongation. Our results suggest that these densely packed nonglycosylated molecules are no longer in a free mushroom state. However, their saturation density was several times lower than previously reported brush transition densities, such as 65,000 µm–2 for 17 kDa polyacrylamide (RF ~15 nm) on a solid surface (Wu et al., 2002). To compare our data on fluid bilayers with previously reported data on solid surfaces, we performed additional experiments with lipid bilayers that lost fluidity. No significant changes in protein binding between fluid and nonfluid bilayers were observed for both b-MUC1 and g-MUC1 molecules (Figure 5—figure supplement 1B). This result suggests that membrane fluidity does not affect the average intermolecular distance or other relevant parameters that control molecular binding in the reconstituted system. Based on these, we speculate that the saturated protein density observed in our experiments is lower than or at most comparable to the actual brush transition density. Thus, although these crowded proteins may be restricted from free random motion, they are not significantly extended as in the condensed brush state, in which the contribution of resistance to molecular extension fel is expected to be small relative to the overall free energy of the system.

Note that this does not mean that glycoproteins cannot form condensed brush structures: in fact, highly glycosylated molecules (e.g. MUC1) can form brush structures in cells when such proteins are expressed at very high densities (Shurer et al., 2019). In these cells, plasma membranes were forced to accommodate these large amounts of membrane proteins with hydrophobic transmembrane domains. In contrast, in our experiment, proteins were soluble in the aqueous phase, and we can analyze intermolecular repulsion in ectodomain independently from the effect of protein compartmentalization. In addition, cells containing brushed glycoproteins also transformed the cell membrane into a shape with an enormous amount of tubular structure. Such membrane deformation results in the increase of total surface area to reduce the density of glycoproteins, indicating that there is strong intermolecular repulsion between glycoproteins. In any case, the free energy of the system is determined by the balance between protein binding and insertion into the membrane, protein deformation, and repulsive forces between proteins, which determine the density of proteins depending on the configuration of the system. Thus, although strong intermolecular repulsions were prominently observed in our simplified system, this may not be the case in other systems. We also note that it has been previously shown that membrane tubules generated upon the insertion of polymers in membranes were neither inhibitory nor activating on viral infection (Kaizuka and Machida, 2023). This is simply because these tubular structures are on the ~μm scale, and much larger than viruses, and we assumed that this logic can also be applied to membrane tubules generated by overcrowded glycocalyx (Shurer et al., 2019).