Influence of glycosaminoglycans on lipid dynamics in supported phospholipid bilayers

Harekrushna Sahoo,Petra Schwille

doi:10.1039/c3sm26906j

Abstract

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.

Highlights

IntroductionGAGs, together with other components of the extracellular matrix, are supposed to be involved in many biological functions at and across the cell membrane
Glycosaminoglycans (GAGs), an evolutionary well-conserved class of carbohydrates, are important components of the extracellular matrix (ECM), attached to the outer surface of the cell membrane.[1,2] Structurally, GAG, a polymer chain of repeated disaccharide units, is covalently linked to a protein to form proteoglycans or glycoproteins, depending on the ratio between GAG and the protein.[3]
The effect of different GAGs on lipid diffusion was studied by fluorescence correlation spectroscopy (FCS) in terms of diffusion coefficient ratio (Dratio), providing qualitative information about the interaction between the GAG polymer and the membrane, in

Summary

Introduction

GAGs, together with other components of the extracellular matrix, are supposed to be involved in many biological functions at and across the cell membrane. In 2007, Zhang and coworkers reported that the presence of a polymer, quaternized poly(4-vinylpyridine), on the membrane surface creates heterogeneity in the lipid diffusion of the bilayer. They observed a signi cant difference between the diffusion coefficients of the lipids which are in contact with the polymer (0.50 Æ 0.12 mm[2] sÀ1) and those which are not (2.62 Æ 0.18 mm[2] sÀ1).[14] Since little more has been reported on carbohydrate attachment to membranes and their effects on the lipid dynamics. FCS has several advantages over FRAP, which is used to determine diffusion parameters, such as the requirement of lower uorescent probe concentration (0.005 to 0.01%), higher spatial resolution, and a better precision in determining diffusion heterogeneity.[20,21,22,23]

Material and methods

Results and discussions

Conclusions