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キーワード:
FLUORESCENCE CORRELATION SPECTROSCOPY; HYALURONIC-ACID; MEMBRANE;
DIFFUSION; GLYCOPOLYMERS; ORIENTATION; INSIGHTS; COMPLEX; HEPARIN; ROLES
要旨:
Glycosaminoglycans (GAGs) are important constituents of extracellular matrices (ECMs). As charged polymers, they do most likely influence lipid and protein dynamics in the outer leaflet of plasma membranes. In this study, we investigated their specific effect, depending on concentration, on lipid diffusion in model membranes. In our assay, GAGs are simply attached electrostatically to supported phospholipid (DOPC) bilayers doped with small amounts of cationic lipid (DOTAP) at physiological pH. Lipid dynamics are characterized via the diffusion of fluorescent lipid analogs (DiD/DiO), determined by fluorescence correlation spectroscopy (FCS). We find that diffusion of DiD is significantly affected by the attachment of GAG. Quite surprisingly, short chains (<= 10 disaccharide units) of hyaluronic acid (unsulfated GAG) on the membrane surface affect the DiD diffusion coefficients stronger than medium or long chains (>= 100 disaccharide units). In particular, short chains of hyaluronic acids at micromolar concentrations display a 2-fold decrease of the diffusion coefficients compared to the situation without GAG. At nanomolar concentrations of hyaluronic acid of both short and long chains, DiD diffusion remains unaltered. In contrast, sulfated GAGs, such as heparan sulfate (HS) and heparin, affect the lipid diffusion already at sub-micromolar concentrations, albeit not as strongly, with a less than 1.5 fold reduction of the diffusion coefficient. Chondroitin sulfate, another class of sulfated GAGs, did not impose any effect on DiD diffusion in the supported phospholipid bilayer at the concentrations studied. We also investigated desulfated heparin, to explore the role of sulfation and to compare its effect with HA. It is observed that heparin derivatives with lower degrees of sulfation have little effect on the lipid diffusion. Altogether, our results suggest that the presence of certain carbohydrate polymers in the ECM does have a noticeable effect on lipid dynamics in biological membranes.
