Immunology and Cell Biology (1996) 74, A1-A7
The structure and function of hyaluronan: An overview
TORVARD C LAURENT.' ULLA BG LAURENT- and J ROBERT E FRASER-'
Departments of' Medical and Physiological Chemistry and-Ophthalmology. University of Uppsala. Sweden, and
•^Departtnent of Biochemistry. Monash University, Clayton. Victoria. Australia
Summary
Hyaluronan is a major component of synovial tissue and fluid as well as other soft connective tissues. It is a
high-Mr polysaccharide which forms entangled networks already at dilute concentrations (< I mg/mL) and
endows its solutions with unique rheological properties. Physiological functions of hyaluronan (lubrication,
water homeostasis. macromolecular filtering, exclusion, etc.) have been ascribed to the properties of these
networks. Recently a number of specific interactions between hyaluronan and a group of proteins named
hyaladhcrins have also pointed towards a role of hyaluronan in recognition and the regulation of cellular
activities. Many more or less well documented hypotheses have been proposed for the function of hyaluronan
in joints, for example, that it should lubricate, protect cartilage surfaces, scavenge free radicals and debris,
keep the joint cavities open, form flow barriers in the synovium and prevent capillary growth.
Key words: hyaladherins. hyaluronan, hyaluronan receptors, molecular networks, molecular exclusion, pericellular layer, recognition, rheological properties, sieve effects, water homeostasis.
Introduction
Hyaluronan'- is a high molecular weight linear polysaccharide containing alternating N-acetyl-D-glucosamine and
D-glucuronic acid residues linked by p( 1-4) and P(l-3) bonds. Its molecular mass in human normal synovial fluid has been
estimated to be 6-7 x 10^ and in rheumatoid fluid 3-5 x 10^ Dalton.-^"* The remarkable rheological properties of synovial
fluid are due to hyaluronan and its interaction with neighbouring macromolecules. It was noted early tbat the viscosity of
synovial fluid may be lowered in joint disorders. Hyaluronan therefore understandably, plays a part in many hypotheses
on pathophysiology of joint disease and it is not surprising that hyaluronan was tried early in the therapy ofjoint disease.^
Unspecific interactions of hyaluronan with solvent and macromolecules
Hyaluronan networks
The physico-chemical properties of hyaluronan were studied in detail from 1950 onwards (for a review sec reference 6).
The molecules behave in solution as highly hydrated randomly kinked coils, which start to entangle at concentrations of
less than 1 mg/mL. The entanglement point can be seen both by sedimentation analysis'* and viscosity.** More recently
Scott et al.*^ have given evidence that the chains when entangling also interact with each other and form stretches of double
helices so that the network becomes mechanically more firm.
Rheological properties
Solutions of hyaluronan are viscoelastic and the viscosity is markedly shear dependent." "*" Above the entanglement
point the viscosity increases rapidly and exponentially with concentration (-c^^^)** and a solution of 10 g/L may have a
viscosity at low shear of -10^ times the viscosity ofthe solvent. At high shear the viscosity may drop as much as - 1 0 '
times." The elasticity ofthe system increases with increasing molecular weight and concentration of hyaluronan as
expected fora molecular tietwork. The rheological properties of hyaluronan have been connected with lubrication of joints
and tissues and hyaluronan is commonly found in the body between surfaces thai move along each other, for example,
cartilage surfaces and muscle bundles.'-^
Water homeostasis
A fixed polysaccharide network offers a high resistance to bulk flow of solvent.^ This was beautifully demonstrated by
Day,'-* who showed that hyaluronidase treatment removes a strong hindrance to water flow through a fascia. Thus
hyaluronan and other polysaccharidcs prevent excessive fluid fluxes through tissue compartments. Furthermore, the
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TC Laurent e{ al
osmotic pressure ofa hyaluronan solution is non-ideal and increases exponentially with the concentration.^ In spite ofthe
high molecular weight ofthe polymer the osmotic pressure ofa 10 g/L hyaluronan solution is of the same order as a lOg/L
albumin solution. The exponential relationship makes hyaluronan and other polysaccharides excellent osmotic buffering
substances — moderate changes in concentration lead to marked changes in osmotic pressure. Flow resistance together
with osmotic buffering makes hyaluronan an ideal regulator ofthe water homeostasis in the body.
Network interactions with other macromolecules
The hyaluronan network retards the diffusion of other molecules.* It can be shown that it is the steric hindrance which
restricts the movements and not the viscosity ofthe solution. The larger the molecule the more it will be hindered. /// vivo
hyaluronan will therefore act as a diffusion barrier and regulate the transport of other substances through the intercellular
spaces. Furthermore, the network will exclude a certain volume of solvent for other molecules; the larger the molecule the
less space will be available to it.^ A solution of 10 g/L of hyaluronan will exclude about half of the solvent to serum
albumin. Hyaluronan and other polysaccharides therefore take part in the partition of plasma proteins between the
vascular and extravascular spaces. The excluded volume phenomenon will also affect the solubility of other macromolecules in the interstitium, change chemical equilibria and stabilize the structure of, for example, collagen fibres.
Hyaluronan binding proteins (Hyaladherins)
Matrix interactions
The first specific interaction between hyaluronan and a protein was discovered by Hardingham and Muir'"* when they
showed that cartilage proteoglycans (aggrecans) and hyaluronan bind to each other. Since then detailed information has
been accumulated on the aggregates deposited in cartilage formed by hyaluronan. aggrccan and linkprotein.'"" We now also
know that there are several similar proteoglycans in other tissues (versican, neurocan, brevican. etc.) with potential to form
aggregates with hyaluronan.'^ Lately a number of other extracellular proteins with affinity for hyaluronan have been
discovered. Several have been found in the brain, which seems to be an organ rich in hyaladherins (hyaluronectin. glial
hyaluronan binding protein, brain enriched hyaluronan binding protein, etc.). Others have been found in other tissues.
Some of them may be proteolytic degradation products of proteoglycans.
Collagen type VI seems to have a strong affinity for hyaluronan via an N-terminal globule in the a3 chain.'^ This
interaction is presumably of structural importance in the matrix. When fibroblasts or maerophages are stimulated by TNF
or IL-I they secrete a component named TSG 6 (TSG = TNF Stimulated Gene) with great homology toother hyaluronan
binding proteins.'^ This protein is. for example, synthesized by the synoviocytes in rheumatoid arthritis and can be
detected in notable amounts in synovial fluid of these patients.'^ Its function is unknown. A circulating protein
synthesized by the liver, inter-a-trypsin inhibitor, consists ofa light chain, bikunin, substituted by a chondroitin sulphate
chain, and two heavy chains covalently bound to the polysaccharide. Bikunin carries the protease inhibitor activity while
the heavy chains can be transferred to hyaluronan apparently via a covalcnt linkage-*^. This interaction seems to be
important in stabilizing the cumulus-oocyte extracellular matrix-' and the pericellular hyaluronan layer around cells.-^ It
is also notable that the inter-a-trypsin inhibitor was found very early in excessive amounts in pathological synovial fluid.-•'
Cell surface interactions
It has been known since 1972 that hyaluronan has the ability to aggregate certain types of cells.-•* The first purification of
a cell surface bound hyaluronan binding protein ('hyaluronan receptor') was described thirteen years later by Underhill et
al}^ They isolated an 85 kDa glycoprotein which later turned out to be identical to the lymphocyte homing receptor
CD44.-*'•-'' Sequence analyses on CD44. which showed great homology with hyaluronan binding proteins in cartilage,
were instrumental in this discovery. A large amount of literature exists today on the structure and distribution of CD44.
There are several variants ofthe protein (splicing, glycosylation) and only some of them bind hyaluronan. Many roles of
the CD44-hyaluronan interaction have been proposed, for example, in development, tumour progression and immune
response.^^"^
Another cell surface protein, which has received considerable attention, is RHAMM (receptor for hyaluronan which
mediates motility). It was discovered by Turley and recently cloned by her group.-** RHAMM is a 58 kDa protein which
lacks homology with the other hyaluronan binding proteins. It is present on the leading lamellae of moving rastransformed cells and is responsible for the rapid locomotion in the presence of hyaluronan.-" RHAMM has been
implicated in developmental processes, metastasis of tumours and other pathological and reparative processes.
When investigating the turnover of hyaluronan in blood Fraser et al.^'^ showed the site of uptake to be the liver, and
subsequently Smedsrod et al.^^ described a specific receptor on liver endothelial cells which mediates endocytosis of
hyaluronan. McCourt et al. isolated by affinity chromatography on hyaluronan columns a protein from the liver with all
the prerequisites for being the receptor and identified it as intercellular adhesion molecule-1 (ICAM-l).-^- This protein has
Hyaluronan: An overview^
also been detected in other tissues which bind hyaluronan, but there are now some doubts about it being the true liver
hyaluronan endocytosing receptor.^^
Pericellular Hyaluronan
Various types of cells (fibroblasts, mesothelial cells, etc.) surround themselves in tissue culture by a pericellular layer which
excludes particles such as red blood cells and bacteria.'" This layer is ver\' sensitive to hyaluronidase and therefore
contains hyaluronan. In principle, hyaluronan could be bound to an hyaladherin on the cell surface and it has been shown
in vitro that pericellular layers can be formed from exogenously added components.^^ However, the layer can also be built
from newly synthesized hyaluronan which is formed on the inside of the plasma membrane and translocated to the
pericellular space.•'^ The pericellular layer may have structural functions in the tissues but could also be a means by which
cells isolate themselves from contact with other cells and matrix compounds.
The functions of hyaluronan in the joint
Space filler
The specific functions of hyaluronan in joints are still essentially unknown. The simplest explanation for its presence
would be that a flow of hyaluronan through the joint is needed to keep the joint cavity open and thereby allow extended
movements of the joint. Hyaluronan is constantly secreted into the joint and removed by the synovium. The total amount
of hyaluronan in the joint cavity is determined by these two processes. The half-life of the polysaccharidc at steady-state is
in the order of 0.5-1 days in rabbit and sheep.-^^'^ The volume of the cavity is determined by the pressure conditions
(hydrostatic and osmotic) in the cavity and its surroundings. Hyaluronan could, by its osmotic contributions and its
formation of flow barriers in the limiting layers, be a regulator of the pressure and flow rate.-^** It is interesting that in fetal
development the formation of joint cavities is parallel with a local increase in hyaluronan.**"
Lubrication
Hyaluronan has been regarded as an ideal lubricant in the joints due to its shear-dependent viscosity" but its role in
lubrication has been refuted by others."*- However, there are now reasons to believe that the function of hyaluronan is to
form a film between the cartilage surfaces. The load on the joints may press out water and low-molecular solutes from the
hyaluronan layer into the cartilage matrix. As a result the concentration of hyaluronan increases and a gel structure of
micrometre thickness is formed which protects the cartilage surfaces from frictional damage (e.g. see reference 43). This
mechanism to form a protective layer is much less elTective in arthritis when the synovial hyaluronan has both a lower
concentration and a lower molecular weight than normal. Another change in the arthritic joint is the protein composition
of the synovial fluid. Fraser et al.'*'^ showed 25 years ago that addition of various serum proteins to hyaluronan
substantially increased the viscosity and this has received a renewed interest in view of recently discovered hyaladherins
(see above). TSG-6 and intcr-a-trypsin inhibitor and other acute phase reactants such as haptoglobin arc concentrated to
arthritic synovial fluid.''^ It is not known to what extent these arc affecting the rheology and lubricating properties.
Sea vengerfiinct ions
Hyaluronan has also been assigned scavenger functions in the joints. It has been known since the 1940s that hyaluronan is
degraded by various oxidizing systems and ionizing Irradiation and we know today that the common denominator is a
chain cleavage induced by free radicals, essentially hydroxy radicals."^ Through this reaction hyaluronan acts as a very
efficient scavenger of free radicals. Whether this has any biological importance in protecting the joint against free radicals
is unknown. The rapid turnover of hyaluronan in the joints has led to the suggestion that it also acts as a scavenger for
cellular debris.''^ Cellular material could be caught in the hyaluronan network and removed at the same rate as the
polysaccharide.
Regulation of cellular activities
As discussed above, more recently proposed functions of hyaluronan are based on its specific interactions with hyaladherins. One interesting aspect is the fact that hyaluronan influences angiogenesis but the effect is different depending on its
concentration and molecular weight.''^ High molecular weight and high concentrations of the polymer inhibit the
formation of capillaries, while oligosaccharides can induce angiogenesis. There are also reports of hyaluronan receptors on
vascular endothelial cells by which hyaluronan could act on the cells."* The avascularity of the joint cavity could be a result
of hyaluronan inhibition of angiogenesis.
Another interaction of some interest in the joint is the binding of hyaluronan to cell surface proteins. Lynnphocytes and
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TC Laurent et al.
other cells may find their way to joints through this interaction. Injection of high doses of hyaluronan intra-articularly
could attract cells expressing these proteins. Cells can also change their expression of hyaluronan-binding proteins in states
of disease whereby hyaluronan may influence immunological reactions and cellular traffic m the pathophysiological
processes.'*^ The observation often reported that intra-articular injections of hyaluronan alleviates pain in joint disease^^
may indicate a direct or indirect interaction with pain receptors.
Acknowledgements
This project has been supported by the Swedish Medical Research Council (project 3X-4).
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