Characterization of methylprednisolone esters of hyaluronan in aqueous solution: conformation and aggregation behavior

Methylprednisolone steroid esters of hyaluronan differing in degree of functionalization and molecular weight were investigated in aqueous solution. Conformation and aggregation phenomena were elucidated by means of circular dichroism, viscometry, rheology, and nuclear magnetic resonance, mainly by (1)H pulsed field gradient (PFG) NMR, which allows the determination of the diffusion coefficient of the species under investigation. The functionalization of hyaluronan with the steroid induces a reduction of the molecular volume, as a consequence of intramolecular hydrophobic interactions. For concentrated samples we have observed the coexistence of unimolecular collapsed chains and of aggregates, the latter disappearing upon dilution. The methylprednisolone ester of lower molecular weight hyaluronan has a larger molecular volume than its higher molecular weight analogue, even though still smaller than the underivatized polymer. This effect can be explained with the reduced flexibility of the polymer backbone probably impairing intramolecular interactions.     

Glycosaminoglycan conformation: do aqueous molecular dynamics simulations agree with x-ray fiber diffraction?

Glycosaminoglycan-protein interactions are biologically important and require an appreciation of glycan molecular shape in solution, which is presently unavailable. In previous studies we found strong similarity between aqueous molecular dynamics (MD) simulations and published x-ray diffraction refinements of hyaluronan. We have applied a similar approach here to chondroitin and dermatan, attempting to clarify some of the issues raised by the x-ray diffraction literature relating to chondroitin and dermatan sulfate. We predict that chondroitin has the same beta(1-->4) linkage conformation as hyaluronan, and that their average beta(1-->3) conformations differ. This is explained by changes in hydrogen-bonding across this linkage, resulting from its axial hydroxyl, causing a different sampling of left-handed helices in chondroitin (2.5- to 3.5-fold) as compared with hyaluronan (3.0- to 4.0-fold). Few right-handed helices, which lack intramolecular hydrogen-bonds, were sampled during our MD simulations. Thus, we propose that the 8-fold helix observed in chondroitin-6-sulfate, represented in the literature as an 8(3) helix (right-handed), though it has never been refined, is more likely to be 8(5) (left-handed) helix. Molecular dynamics simulations implied that (4)C(1) and (2)S(O), but not (1)C(4), forms of iduronate could be used in refinements of dermatan x-ray fiber diffraction patterns. Current models of 8-fold dermatan sulfate chains containing (4)C(1) iduronate refine to right-handed helices, which possess no intramolecular hydrogen-bonds. However, MD simulations predict that models containing (2)S(O) iduronate could provide better (8(5) helix) starting structures for refinement. Thus, the 8-fold dermatan sulfate refinement (8(3) helix) could be in error.     

Molecular interaction studies of the hyaluronan derivative, hylan A using atomic force microscopy

Intermolecular self-association of hylan chains can be observed in hylan of molecular weight ca. 1×10⁷, with an indication of specific cross-linking protein points and inter-chain cross-links of molecular weight of between 10,000 and 80,000. When this high molecular weight hylan is autoclaved to M_w 1.8×10⁶, to yield a molecular size of the same order as a conventional hyaluronan, the structural features of hylan are retained, with regions of network disintegration having single chains to which one or two chains are joined. After degradation by ^√OH radicals, extended linear chains are found with some of the straight chains having branch points. These can be attributed to the unwinding of the hylan coils by the movement of a droplet of water across the mica surface. The effect of filtration by 1 μm filter does not reduce the measured M_w (corresponding to an intrinsic viscosity of 8188 at low shear rate). However, when stressed through a 0.45 μm filter the M_w falls to a quarter of its previous value. The cross-linked structure of the original hylan is shown to be equivalent to a hyaluronan of ca. 10×10⁶, based on rheological measurements. The cross-linked structure confers stability to degradation by ^√OH radicals not observed for hyaluronan. This distinctive behaviour of hylan is maintained for the entire range of molecular weights studied. The results confirm the tendency of hylan chains to readily undergo chain–chain association.     

Molecular dynamics simulations of the two disaccharides of hyaluronan in aqueous solution

Hyaluronan is an unusually stiff polymer when in aqueous solution,which has important consequences for its biological function.Molecular dynamics simulations of hyaluronan disaccharides havebeen performed, with explicit inclusion of water, to determinethe molecular basis of this stiffness, and to investigate thedynamics of the glycosidic linkages. Our simulations revealthat stable sets of hydrogen bonds frequently connect the neighboringresidues of hyaluronan. Water caging around the glycosidic linkagewas observed to increase the connectivity between sugars, andfurther constrain them. This, we propose, explains the unusualstiffness of polymeric hyaluronan. It would allow the polysaccharideto maintain local secondary structure, and occupy large solutiondomains consistent with the visco-elastic nature of hyaluronan.Simulations in water showed no significant changes on inclusionof the exo-anomeric effect. This, we deduced, was due to hyaluronandisaccharides ordering first shell water molecules. In somecases these waters were observed to transiently induce con-formationalchange, by breaking intramolecular hydrogen bonds. conformation hyaluronan hydrogen bonds molecular dynamics water     

Mammalian vitreous humor contains networks of hyaluronan molecules: electron microscopic analysis using the hyaluronan-binding region (G1) of aggrecan and link protein.

Vitreous humor from human, bovine, and chicken eyes was analyzed by rotary shadowing to characterize further the supramolecular organization of the gel-like matrix which forms this tissue. Extensive filamentous networks, distinct from collagen fibrils, were found in both human and bovine vitreous but not in chicken vitreous. The networks consisted of branching structures of various diameters, due to variable numbers of hyaluronan molecules being laterally associated with each other and apparently giving rise to a three-dimensional lattice. These networks could be decorated in a specific and regular manner by the hyaluronan-binding region called G1 purified from bovine nasal septum cartilage. The extent of decoration of hyaluronan was dependent on the relative concentration of G1. In the presence of an excess of G1 the networks were destabilized giving rise to individual unbranched hyaluronan chains of varying length that were saturated with G1. One or more globular proteins, as yet uncharacterized, were seen interacting with the hyaluronan networks, often at branch points. These proteins may serve to stabilize the three-dimensional structure of the matrix although highly ordered networks were also observed without globular proteins. Link protein, which also binds to hyaluronan, bound to the networks in a fashion clearly distinct from G1. Neither G1 nor link protein bound directly to human or bovine vitreous collagen fibrils. However, link protein did bind extensively to the glycosaminoglycan coat of chicken vitreous collagen fibrils described previously (D. W. Wright, and R. Mayne J. Ultrastruct. Mol. Struct. Res. 100, 224-234, 1988), while G1 did not. Digestion of the chicken vitreous collagen fibrils with Streptomyces hyaluronidase did not result in the removal of the glycosaminoglycan coat of the collagen fibrils nor did it affect the binding of G1 or link protein to the fibrils, indicating that hyaluronan is not a component of this structure. These studies demonstrate that proteins with specific binding properties can be used as probes to investigate the structure of the native vitreous humor gel from several species and suggest that this method potentially can be used for structural studies of other connective tissue matrices.     

Inter-alpha-inhibitor, hyaluronan and inflammation

Inter-alpha-inhibitor is an abundant plasma protein whose physiological function is only now beginning to be revealed. It consists of three polypeptides: two heavy chains and one light chain called bikunin. Bikunin, which has antiproteolytic activity, carries a chondroitin sulphate chain to which the heavy chains are covalently linked. The heavy chains can be transferred from inter-alpha-inhibitor to hyaluronan molecules and become covalently linked. This reaction seems to be mediated by TSG-6, a protein secreted by various cells upon stimulation by inflammatory cytokines. Inter-alpha-inhibitor has been shown to be required for the stabilization of the cumulus cell-oocyte complex during the expansion that occurs prior to ovulation. Hyaluronan-linked heavy chains in the extracellular matrix of this cellular complex have recently been shown to be tightly bound to TSG-6. Since TSG-6 binds to hyaluronan, its complex with heavy chains could stabilize the extracellular matrix by cross-linking hyaluronan molecules. Heavy chains linked to hyaluronan molecules have also been found in inflamed tissues. The physiological role of these complexes is not known but there are indications that they might protect hyaluronan against fragmentation by reactive oxygen species. TSG-6 also binds to bikunin thereby enhancing its antiplasmin activity. Taken together, these results suggest that inter-alpha-inhibitor is an anti-inflammatory agent which is activated by TSG-6.     

The structural and compositional changes of glycosaminoglycans are closely associated with tissue type in human laryngeal cancer

Hyaluronan and sulfated glycosaminoglycans, as intrinsic components of proteoglycans, are playing important roles in cancer biology. In the present study, we investigated in detail the glycosaminoglycans on both fine chemical and structural levels in laryngeal cartilaginous and non-cartilaginous tissues at different stages of laryngeal cancer. The results indicated that in cartilaginous tissues the amounts of chondroitin sulfate, keratan sulfate, dermatan sulfate and hyaluronan presented a dramatic decrease in contrast to the non-cartilaginous tissues, which showed a significant increase of these glycosaminoglycans compared to their normal counterparts. On fine chemical structure, the molar ratios of 4-sulfated to 6-sulfated and non-sulfated to sulfated disaccharides from both cartilaginous and non-cartilaginous cancerous tissues showed a significant increase. On molecular-size level, in laryngeal cancer, the chromatographic behaviour of the sulfated glycosaminoglycan chains from both tissue-types revealed their lower M(r) with a more polydisperse and heterogeneous distribution compared to the normal ones. In addition, in both tissues, a significant decrease of high molecular-size hyaluronan was observed. Of particular interest was the great increase of hyaluronan of low molecular mass in the laryngeal non-cartilaginous tissues, which ranged from 330 to 890 kDa. The kind and the extent of these alterations, which presented an intense stage-related behaviour, depended on the tissue origin and could be associated with the malignant phenotype of human laryngeal cancer.     

Architecture of Peptide Assembly in Liposome

Abstract

Two chains of amphiphilic α-helical dodecapeptides were connected to a cyclic octapeptide in a parallel or antiparallel arrangement. Conformation, orientation, and association of the dodeca-peptide chains in the presence of dimyristoylphosphatidylcholine (DMPC) liposome were studied. The peptides were easily incorporated into the bilayer membrane. CD measurements revealed that the dodecapeptides took a distorted a-helix conformation due to intramolecular association. Fluorescence quenching measurement of a probe connected to each terminal region of the dodecapeptides revealed that the dodecapeptide chain was located on the membrane surface with a parallel orientation to the surface. Thermal denaturation of the helical structure was suppressed by connecting the two dodecapeptide chains to the cyclic peptide. These experimental results indicate that a supersecondary structure composed of associated α-helices was constructed on the surface of DMPC liposome by using a cyclic peptide as a template.     

Circulating hyaluronan, chondroitin sulphate and dextran sulphate bind to a liver receptor that does not recognize heparin

Chondroitin sulphate, injected intravenously into rats and given prior to intravenous ¹²⁵I-labelled hyaluronan with a mean Mw of about 400 kDa, was shown to inhibit the rapid receptor-mediated uptake of hyaluronan by the liver. The labelled hyaluronan that remained in the circulation was shown, by size exclusion chromatography of serum and urine, to be rapidly degraded down to fragments of lower Mw and filtered out into the urine and tissues. When the uptake of ¹²⁵I-hyaluronan was inhibited by unlabelled hyaluronan, only very low degradation and urinary excretion were found. Liver uptake could also be inhibited by dextran sulphate but not by heparin. Unlabelled hyaluronan could inhibit the liver uptake of labelled chondroitin sulphate but not labelled heparin. Unlabelled chondroitin sulphate and dextran sulphate inhibited cell association of labelled hyaluronan to liver endothelial cells in culture more effectively than unlabelled hyaluronan. Our data show that the liver hyaluronan receptors also recognize and effectively bind chondroitin sulphate and dextran sulphate but not heparin and that a hyaluronan-specific saturable degradative mechanism exists in the circulation. Such a mechanism could explain why hyaluronan in the general circulation has a much lower Mw than the hyaluronan in lymph. The results also indicate that increased hyaluronan levels in serum, and increased urinary excretion of hyaluronan, may be secondary to increased outflow of chondroitin sulphate from the tissues during some pathological conditions. This revised version was published online in November 2006 with corrections to the Cover Date.     

Expression of Recombinant Hyaluronan Synthase (HAS) Isoforms in CHO Cells Reduces Cell Migration and Cell Surface CD44

In the present study we investigated the functional properties of the three recombinant hyaluronan synthases (HAS proteins) HAS1, HAS2, and HAS3. HAS3-transfected CHO clones exhibited the highest hyaluronan polymerization rate followed by HAS2 transfectants which were more catalytically active than HAS1 transfectants. In living cells all three HAS proteins synthesized hyaluronan chains of high molecular weight (larger than 3.9 x 10(6)). In vitro, the HAS2 isoform produced hyaluronan chains of a molecular weight larger than 3.9 x 10(6), whereas HAS3 produced polydisperse hyaluronan (molecular weight 0.12-1 x 10(6)), and HAS1 synthesized much shorter chains of an average molecular weight of 0.12 x 10(6). Thus, each HAS protein may interact with different cytoplasmic proteins which may influence their catalytic activity. CHO transfectants with the ability to synthesize about 1 microgram hyaluronan/1 x 10 (5) cells/24 h were surrounded by hyaluronan-containing coats, whereas transfectants generating about 4-fold lower amounts of hyaluronan formed coats only in the presence of chondroitin sulfate proteoglycan. An inverse correlation between hyaluronan production on the one hand and cell migration and cell surface CD44 expression on the other was found; a 4-fold lower migration and a 2-fold decrease of cell surface CD44 receptors was seen when hyaluronan production increased 1000-fold over the level in the untransfected cells. The inverse relationships between hyaluronan production and migration and CD44 expression of cells are of importance for the regulation of cell-extracellular matrix interactions.     

Extracellular Processing of the Cartilage Proteoglycan Aggregate and Its Effect on CD44-mediated Internalization of Hyaluronan

In many cells hyaluronan receptor CD44 mediates the endocytosis of hyaluronan and its delivery to endosomes/lysosomes. The regulation of this process remains largely unknown. In most extracellular matrices hyaluronan is not present as a free polysaccharide but often is found in complex with other small proteins and macromolecules such as proteoglycans. This is especially true in cartilage, where hyaluronan assembles into an aggregate structure with the large proteoglycan termed aggrecan. In this study when purified aggrecan was added to FITC-conjugated hyaluronan, no internalization of hyaluronan was detected. This suggested that the overall size of the aggregate prevented hyaluronan endocytosis and furthermore that proteolysis of the aggrecan was a required prerequisite for local, cell-based turnover of hyaluronan. To test this hypothesis, limited C-terminal digestion of aggrecan was performed to determine whether a size range of aggrecan exists that permits hyaluronan endocytosis. Our data demonstrate that only limited degradation of the aggrecan monomer was required to allow for hyaluronan internalization. When hyaluronan was combined with partially degraded, dansyl chloride-labeled aggrecan, blue fluorescent aggrecan was also visualized within intracellular vesicles. It was also determined that sonicated hyaluronan of smaller molecular size was internalized more readily than high molecular mass hyaluronan. However, the addition of intact aggrecan to hyaluronan chains sonicated for 5 and 10 s reblocked their endocytosis, whereas aggregates containing 15-s sonicated hyaluronan were internalized. These data suggest that hyaluronan endocytosis is regulated in large part by the extracellular proteolytic processing of hyaluronan-bound proteoglycan.     

New amphiphilic lactic acid oligomer-hyaluronan conjugates: synthesis and physicochemical characterization

The "grafting onto" strategy was used to conjugate DL-lactic acid oligomers (OLA) to hyaluronan (HA) for the sake of developing novel degradable HA-based self-assembling polymeric systems. Grafting was achieved by reacting COCl-terminated OLA with cetyltrimethylammonium hyaluronate (CTA-HA) in dimethyl sulfoxide (DMSO). The resulting CTA-HAOLA conjugates were purified and turned to sodium form (Na-HAOLA) by dissolution in a phosphate buffer-DMSO mixture and successive dialyses against DMSO, ethanol, and water. In contrast, when the same protocol was applied to CTA-HAOLA, phase separation with gel formation was observed. The solution phase was composed of Na-HAOLA whereas the gel phase was made of mixed CTA-Na-HAOLA salt with ca. 25% of the carboxyl groups neutralized by CTA. Gelation was assigned to intramolecular hydrophobic associations between OLA and cetyl alkyl chains that complemented electrostatic interactions between CTA and HA COO- groups synergistically. Therefore, the corresponding stabilized CTA ions required more drastic conditions to be released. Under the selected dialysis conditions, the CTA-Na-HAOLA gels formed tiny tubes. Na-HAOLA and CTA-Na-HAOLA were characterized by FTIR, one-dimensional 1H and two-dimensional 1H NMR. The extent of grafting was ca. 5% per disaccharidic repeating unit, regardless of the molecular weight, as determined by NMR and capillary zone electrophoresis. Amphiphilic Na-HAOLA molecules were aggregated and formed spherical species in water according to size exclusion chromatography combined with multiangle laser light scattering detection. The critical aggregation concentration ranged between 0.2 and 0.35% (w/v), depending of the molecular weight of the parent hyaluronan.     

Predicting the molecular shape of polysaccharides from dynamic interactions with water

How simple monosaccharides, once polymerized, become the basis for structural materials remains a mystery. A framework is developed to investigate the role of water in the emergence of dynamic structure in polysaccharides, using the important beta(1-->4) linkage as an example. This linkage is studied within decasaccharide fragments of cellulose, chitin, mannan, xylan, and hyaluronan, using molecular simulations in the presence of explicit water solvent. Although cellulose, mannan, chitin, and xylan are chemically similar, their intramolecular hydrogen-bond dynamics and interaction with water are predicted to differ. Cellulose, mannan, and chitin favor relatively static intramolecular hydrogen bonds, xylan prefers dynamic water bridges, and multiple water configurations are predicted at the beta(1-->4) linkages of hyaluronan. With such a variety of predicted dynamics, the hypothesis that the beta(1-->4) linkage is stabilized by intramolecular hydrogen bonds was rejected. Instead, it is proposed that favored molecular configurations are consistent with maximum rotamer and water degrees of freedom, explaining observations made previously by X-ray diffraction. Furthermore, polysaccharides predicted to be conformationally restricted in simulations (cellulose, chitin, and mannan) prefer the solid state in reality, even as oligosaccharides. Those predicted to be more flexible (xylan and hyaluronan) are known to be soluble, even as high polymers. Therefore an intriguing correlation between chemical composition, water organization, polymer properties, and biological function is proposed.     

Spontaneous and promoted association of linear oligoglycines

Linear oligoglycines of various lengths bearing a carboxyl or an amide group at their C-termini and also their poly(acrylamide) conjugates were synthesized. No self-assembly into supramolecular structures was observed for free oligoglycines H-(Gly)m-OH(m = 3-5). At the same time, oligoglycylamides H-(Gly)m-NH2 (m = 3-5) demonstrated ability for both self-assembly in aqueous solution and assembly promoted by an additional interaction with surface. In the case of polymer-bound oligoglycines (and their amides), no intramolecular clustering of peptide chains, as expected, was observed. This means that the presence of several oligoglycine chains bound to each other in one center is not a necessary prerequisite for polyglycine II-type association.     

Effects of Ascorbic Acid and Analogs on the Activity of Testicular Hyaluronidase and Hyaluronan Lyase on Hyaluronan

We have evaluated the inhibition of testicular hyaluronidase and hyaluronan lyase by L-ascorbic acid and chemical analogs. We observed that L-ascorbic acid, D-isoascorbic acid and dehydroascorbic acid inhibited both types of enzymes, but showed stronger effects towards hyaluronan lyase. But these compounds were observed to degrade the substrate, hyaluronan, by themselves. Of the other ascorbic acid analogs tested, saccharic acid inhibited hyaluronan lyase, while not affecting the enzymatic activity of testicular hyaluronidase, nor affecting the physic-chemical stability of hyaluronan. This is the first compound, to our knowledge, to be shown to possess such selective inhibition. Therefore, we propose that saccharic acid could serve as a lead compound for the development of potent and selective inhibitors of bacterial hyaluronan lyase or of polysaccharide lyase enzymes in general as we observed this compound to be capable of inhibiting chondroitinase ABC in addition to hyaluronan lyase.     

Effects of ascorbic acid and analogs on the activity of testicular hyaluronidase and hyaluronan lyase on hyaluronan

We have evaluated the inhibition of testicular hyaluronidase and hyaluronan lyase by L-ascorbic acid and chemical analogs. We observed that L-ascorbic acid, D-isoascorbic acid and dehydroascorbic acid inhibited both types of enzymes, but showed stronger effects towards hyaluronan lyase. But these compounds were observed to degrade the substrate, hyaluronan, by themselves. Of the other ascorbic acid analogs tested, saccharic acid inhibited hyaluronan lyase, while not affecting the enzymatic activity of testicular hyaluronidase, nor affecting the physic-chemical stability of hyaluronan. This is the first compound, to our knowledge, to be shown to possess such selective inhibition. Therefore, we propose that saccharic acid could serve as a lead compound for the development of potent and selective inhibitors of bacterial hyaluronan lyase or of polysaccharide lyase enzymes in general as we observed this compound to be capable of inhibiting chondroitinase ABC in addition to hyaluronan lyase.     

Hyaluronan chain conformation and dynamics

An overview of the present state of research in the field of hyaluronan chain conformational aspects is presented. The relationship between structure and dynamics are illustrated for a series of hyaluronan oligomers. Conformational characteristics of hyaluronan chains are discussed, together with the dynamic chain patterns, evaluated by using a theoretical approach to diffusive polymer dynamics. The dependence of correlation times and NMR relaxation parameters from the chain dimension are investigated. Topological features and dimensional properties are related to the structural determinants by using classical computational methods of molecular mechanics and Monte Carlo simulation.     

TSG-6 modulates the interaction between hyaluronan and cell surface CD44

Interactions between CD44 and hyaluronan are implicated in the primary adhesion of lymphocytes to endothelium at inflammatory locations. Here we show that preincubation of hyaluronan with full-length recombinant TSG-6 or its Link module domain (Link_TSG6) enhances or induces the binding of hyaluronan to cell surface CD44 on constitutive and inducible cell backgrounds, respectively. These effects are blocked by CD44-specific antibodies and are absent in CD44-negative cells. Enhancement of CD44-mediated interactions of lymphoid cells with hyaluronan by TSG-6 proteins was seen under conditions of flow at shear forces that occur in post-capillary venules. Increases in the number of rolling cells were observed on substrates comprising TSG-6-hyaluronan complexes as compared with a substrate containing hyaluronan alone. In ligand competition experiments, cell surface-bound TSG-6-hyaluronan complexes were more potent than hyaluronan alone in inhibiting cell adhesion to immobilized hyaluronan. Link_TSG6 mutants with impaired hyaluronan binding function had a reduced ability to modulate ligand binding by cell surface CD44. However, some mutants that exhibited close to wild-type hyaluronan binding were found to have either reduced or increased activity, suggesting that some amino acid residues outside of the hyaluronan binding site might be involved in protein self-association, potentially leading to the formation of cross-linked hyaluronan fibers. In turn, cross-linked hyaluronan could increase the binding avidity of CD44 by inducing receptor clustering. The ability of TSG-6 to modulate the interaction of hyaluronan with CD44 has important implications for CD44-mediated cell activity at sites of inflammation, where TSG-6 is expressed.     

Hyaluronan synthesis induces microvillus-like cell surface protrusions

Hyaluronan synthases (HASs) are plasma membrane enzymes that simultaneously elongate, bind, and extrude the growing hyaluronan chain directly into extracellular space. In cells transfected with green fluorescent protein (GFP)-tagged Has3, the dorsal surface was decorated by up to 150 slender, 3-20-microm-long microvillus-type plasma membrane protrusions, which also contained filamentous actin, the hyaluronan receptor CD44, and lipid raft microdomains. Enzymatic activity of HAS was required for the growth of the microvilli, which were not present in cells transfected with other GFP proteins or inactive GFP-Has3 mutants or in cells incubated with exogenous soluble hyaluronan. The microvilli induced by HAS3 were gradually withered by introduction of an inhibitor of hyaluronan synthesis and rapidly retracted by hyaluronidase digestion, whereas they were not affected by competition with hyaluronan oligosaccharides and disruption of the CD44 gene, suggesting independence of hyaluronan receptors. The data bring out the novel concept that the glycocalyx created by dense arrays of hyaluronan chains, tethered to HAS during biosynthesis, can induce and maintain prominent microvilli.     

Depolarization of the membrane potential by hyaluronan

The membrane potential is mainly maintained by the K⁺ concentration gradient across the cell membrane between the cytosol and the extracellular matrix. Here, we show that extracellular addition of high‐molecular weight hyaluronan depolarized the membrane potential of human fibroblasts, human embryonic kidney cells (HEK), and central nervous system neurons in a concentration‐dependent manner, whereas digestion of cell surface hyaluronan by hyaluronidase caused hyperpolarization. This effect could not be achieved by other glycosaminoglycans or hyaluronan oligosaccharides, chondroitin sulfate, and heparin which did not affect the membrane potential. Mixtures of high‐molecular weight hyaluronan and bovine serum albumin had a larger depolarization effect than expected as the sum of both individual components. The different behavior of high‐molecular weight hyaluronan versus hyaluronan oligosaccharides and other glycosaminoglycans can be explained by a Donnan effect combined with a steric exclusion of other molecules from the water solvated chains of high‐molecular weight hyaluronan. Depolarization of the plasma membrane by hyaluronan represents an additional pathway of signal transduction to the classical CD44 signal transduction pathway, which links the extracellular matrix to intracellular metabolism. J. Cell. Biochem. 111: 858–864, 2010. © 2010 Wiley‐Liss, Inc.