379

15
Hyaluronan

Prof. Dr. Peter Prehm

Institut f¸r Physiologische Chemie und Pathobiochemie, Waldeyerstr. 15,
D-48129 M¸nster, Germany; Tel.: ‡ 49-251-8355579; Fax: ‡ 49-251-8355596;
E-mail: prehm@uni-muenster.de

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

2 Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

3 Chemical Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383

4 Occurrence of Hyaluronan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383

5 Mechanism of Hyaluronan Synthesis . . . . . . . . . . . . . . . . . . . . . . . . 384

5.1 Chain Elongation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
5.2 Chain Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
5.3 Chain Export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
5.4 Swelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
5.5 Macromolecular Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385

6 Hyaluronan Synthases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385

7 Hyaluronan-binding Proteins and Receptors . . . . . . . . . . . . . . . . . . . . 386

7.1 CD44 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
7.2 RHAMM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
7.3 Other Hyaluronan-binding Proteins . . . . . . . . . . . . . . . . . . . . . . . . 388

8 Mechanisms of Hyaluronan Release from the Cell Surface . . . . . . . . . . . 388

9 Regulation of Hyaluronan Synthesis . . . . . . . . . . . . . . . . . . . . . . . . 389

9.1 Expression of the Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
9.2 Stimulation and Inhibition of the Synthase . . . . . . . . . . . . . . . . . . . . 389
9.2.1 Signal Transduction at Membranes . . . . . . . . . . . . . . . . . . . . . . . . 390
380 15 Hyaluronan

9.2.2 Intracellular Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . 390

9.3 Influence of Chain Length on Further Elongation . . . . . . . . . . . . . . . . 390

10 Turnover and Catabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391

11 Functions of Hyaluronan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391

11.1 Cellular Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
11.2 Physiological Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
11.2.1 Differentiation and Morphogenesis . . . . . . . . . . . . . . . . . . . . . . . . 392
11.2.2 Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
11.2.3 Synovia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
11.3 Pathological Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
11.3.1 Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
11.3.2 Edema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
11.3.3 Streptococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

12 Hyaluronan Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394

12.1 Degradation by Free Radicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
12.2 Degradation by Hyaluronidases . . . . . . . . . . . . . . . . . . . . . . . . . . . 395

13 Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
13.1 Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
13.2 Market . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396

14 Medical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397

14.1 Ophthalmics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
14.2 Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
14.3 Wound Healing and Scarring . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
14.4 Adhesion Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398
14.5 Drug Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398

15 Effects of Hyaluronan Oligosaccharides . . . . . . . . . . . . . . . . . . . . . . 398

16 Outlook and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398

17 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399

CHO Chinese hamster ovary

PMA phorbol-12-myristate-13-acetate
RHAMM receptor for hyaluronan-mediated motility

1 cule is most frequently referred to as
Introduction
Although hyaluronan has a very simple
structure, almost everything else concerning
the molecule is unusual. Sometimes its role
is mechanical and structural (as in the
synovial fluid, the vitreous humor, or the
umbilical cord), whereas sometimes it in-
teracts in tiny concentrations in cells to
trigger important responses. Hyaluronan
has an unusual mechanism of biosynthesis
and exceptional physical properties; conse-
quently, research on this compound was
cumbersome, with progress often impeded
by failures ± often because established
procedures from other fields were not
applicable and new techniques needed to
be developed before any progress could be
made.
During the decades of hyaluronan re-
search, several books and reviews have
marked such progress including Balazs
(1970), Laurent (1989, 1998), Laurent and
Fraser (1992), Goa and Benfield (1994),
Lapcik et al. (1998), and Abatangelo and
Weigel (2000), whilst reviews are published
continually on a new web-site: http://
www.glycoforum.gr.jp/science/hyaluronan.
2 ered during the 1990s.
Historical Outline
In 1934, Karl Meyer described a procedure
for isolating a novel glycosaminoglycan from
the vitreous humor of bovine eyes, and
named it hyaluronic acid (from the Greek,
hyalos ˆ glassy, vitreous) (Meyer and Pal-
mer, 1934). These authors showed that this
substance contained a uronic acid and an
amino sugar, but no sulfoesters. At physio-
logical pH all carboxyl groups are dissoci-
ated, and hence the polysaccharide should be
called hyaluronate. Today, this macromole-
2 Historical Outline 381
hyaluronan, in order to emphasize its poly-
saccharide nature. During the 1930s and
1940s, hyaluronan was isolated from many
sources such as the vitreous body, synovial
fluids, umbilical cord, skin, and rooster
comb (Meyer, 1947) and also from strepto-
cocci (Kendall et al., 1937).
The physico-chemical characterization of
hyaluranon was carried out during the 1950s
and 1960s. The molecular weight is in the
order of several millions, whilst in solution
the chain behaves as an extended random
coil, with a diameter of ~ 500 nm. At
concentrations as low as 0.1%, the chains
are entangled, and this results in extremely
high, shear-dependent viscosity (Laurent,
1970). These properties enable hyaluronan
to regulate water balance, osmotic pressure
and flow resistance, to interact with proteins,
and also to act as a sieve, as a lubricant, and
to stabilize structures by virtue of electro-
static interactions (Comper and Laurent,
1978).
In 1972, Hardingham and Muir discov-
ered that hyaluronan interacts with cartilage
proteoglycans and serves as the central
structural backbone of cartilage. This was
the first example of a specific interaction
between hyaluronan and a protein, and
many more such interactions were discov-
After 1980, the research spread in many
directions, mainly because until that time it
had been assumed that hyaluronan be-
longed to the proteoglycans, and that its
biosynthesis proceeded in a similar manner.
In fact, many studies were conducted to
detect the protein moiety, but this assump-
tion was disproved when a plausible mech-
anism of biosynthesis was proposed (Prehm,
1983a,b). It had also been assumed that the
synthesis of hyaluronan occurred in the
Golgi body ± as was the case for all other
secretory eucaryotic polysaccharides ± until
382 15 Hyaluronan
it was shown that hyaluronan was in fact
synthesized at plasma membranes and the
chains were extruded directly into the extra-
cellular matrix (Prehm, 1984). The catabolic
pathways of hyaluranon were also elucidated
at about this time (Fraser et al., 1981).
Subsequently, it became possible to mea-
sure hyaluronan specifically in body fluids
with high sensitivity (Tengblad et al., 1980),
and also to visualize it histochemically.
These advances opened the way to assess
the role of hyaluronan in many pathological
disturbances.
Balazs pioneered the application of hya-
luronan for medical purposes, and produced
highly viscous and noninflammatory prep-
arations on a commercial scale both as an aid
for ophthalmic surgery and as viscosupple-
mentation for synovial fluids in patients with
osteoarthritis (Balazs, 1982; Balazs and
Denlinger, 1989).
Although the importance of hyaluronan in
cellular behavior had been recognized for
decades, it was not until 1986 that its
requirement for detachment in mitotic cell
division was proven (Brecht et al., 1986).
Underhill and Toole (1979, 1982) reported
that hyaluronan was an adhesive cell surface
component that formed large coats around
untransformed fibroblasts, and smaller
coats around transformed cells.
Cell surface hyaluronan-binding proteins
were discovered during the late 1980s (Tur-
ley et al., 1987; Aruffo et al., 1990), and
studied intensively during the 1990s. Al-
though hyaluranon was already known to be
involved in both metastasis (Toole et al.,
1979) and cell differentiation (Toole et al.,
1977), it was investigations into the molec-
ular biology of the receptors which led to a
fundamental understanding of these proc-
esses. In particular, the receptors CD44 and
RHAMM (Receptor for Hyaluronan-Medi-
ated Motility) have attracted much enthusi-
asm, mainly because they are believed to be
involved in cancer metastasis (Arch et al.,
1992; Hall et al., 1995). However, a sobering
shock reached the scientific community,
when CD44-deficient mice were bred that
had only marginal physiological impair-
ments (Schmits et al., 1997). In addition,
the receptor RHAMM became a matter of
bitter scientific debate when it was found to
be located mainly intracellularly (Hofmann
et al., 1998a; Turley et al., 1998). Subse-
quently, a number of other intracellular
hyaluronan-binding proteins have been
found (Huang et al., 2000), though their
function remains somewhat of a mystery.
During the 1990s, hyaluronan synthases
were cloned from different sources ( Weigel
et al., 1997), each capable of producing
hyaluronan of different chain lengths and
quantities (Itano et al., 1999).
The actions of hyaluronan as an adhesive
component and also as a detachment factor
appeared paradoxical. This paradox has
recently been solved however, when it was
realized that the cellular functions are
mediated through cell-surface receptors that
are susceptible to proteases (Dube et al.,
2001). It appears that hyaluronan acts as an
amplifier for active proteases, but as an
attachment factor when proteases are inac-
tive.
It has long been known that hyaluronan is
very sensitive to breakdown by oxygen
radicals ( Wong et al., 1981), and it has
become clear that it is the breakdown
products which mediate important biolog-
ical functions. Oligosaccharides of hyalur-
onan induce angiogenesis ( West et al., 1985)
and also activate lymphocytes (McKee et al.,
1997; Termeer et al., 2000). Radical degra-
dation generates reactive aldehydes (Uchiya-
ma et al., 1990) which modify proteins into
the main antigenic structures of rheumatoid
arthritis (Prehm, 2000). This discovery fi-
nally terminated a long and oppressive
period of ignorance in a medically important

problem, and may eventually lead to a
curative treatment of these diseases that
currently are treated only symptomatically.
3 at a hyaluronan concentration of 1 mg mL 1,
Chemical Structure
The complete structure of hyaluranon was
elucidated by the group of Karl Meyer, who
characterized the oligosaccharides obtained
by the action of testicular hyaluronidases
( Weissman and Meyer, 1954). Hyaluronan
consists of basic disaccharide units of D-
glucuronic acid and D-N-acetylglucosamine,
these being linked together through alter-
nating b-1,4 and b-1,3 glycosidic bonds
(Figure 1).
The number of repeat disaccharides in a
completed hyaluronan molecule can reach
10,000 or more, with a molecular mass of
~ 4 î 106 daltons (each disaccharide is
~ 400 daltons). In a physiological solution,
the backbone of a hyaluronan molecule is
stiffened by a combination of the chemical
structure of the disaccharide, internal hydro-
gen bonds, and interactions with solvent. In
addition, the preferred shape in water
features hydrophobic patches on alternating
sides of the flat, tape-like secondary struc-
ture. The two sides are identical, so that
hyaluronan molecules are ambidextrous,
enabling them to aggregate via specific
interactions in water to form meshworks,
even at low concentrations (Scott et al.,
Fig. 1 Repeating unit of hyaluranon.
4 Occurrence of Hyaluronan 383
1991). In physiological solutions a hyalur-
onan molecule assumes an expanded ran-
dom coil structure which occupies a very
large domain. The actual mass of hyalur-
onan within this domain is very low, and
~ 0.1% molecules would overlap each other
or higher. This domain structure of hyalur-
onan has interesting and important conse-
quences. Small molecules such as water,
electrolytes and nutrients can diffuse freely
through the solvent, within the domain;
however, large molecules such as proteins
will be partially excluded from the domain
because of their hydrodynamic sizes in
solution. This leads both to slower diffusion
of macromolecules through the network and
to their lower concentration in the network
compared with the surrounding hyaluronan-
free compartments. At pH 7, the carboxyl
groups are predominantly ionized, and the
hyaluronan molecule is a polyanion that has
associated exchangeable cation counterions
to maintain charge neutrality.
4
Occurrence of Hyaluronan
Hyaluronan is present in all vertebrates, and
also in the capsule of some pathogenic
bacteria such as Streptococcus sp. and Pas-
teurella. It is a component of extracellular
matrices in most tissues, and in some tissues
it is a major constituent. The concentration
of hyaluronan is particularly high in rooster
comb (7.5 mg mL 1), in the synovial fluid
(3 ± 4 mg mL 1), in umbilical cord (3 mg
mL 1), in the vitreous humor of the eye
(0.2 mg mL 1), and in skin (0.5 mg mL 1).
In other tissues that contain less hyaluronan,
it forms an essential structural component
of the matrix. In cartilage it forms the
aggregation center for aggrecan, the large
chondroitin sulfate proteoglycan, and re-
384 15 Hyaluronan
tains this macromolecular assembly in the
matrix by specific hyaluronan±protein inter-
actions. It also forms a scaffold for binding of
other matrix components around smooth
muscle cells on the aorta, and on fibroblasts
in the dermis of skin. The largest deposit of
hyaluronan resides in the skin; in an adult
human this totals ~ 8 g. Hyaluronan has
also been detected intracellularly in prolifer-
ating cells (Evanko and Wight, 1999)
5 protein backbone. Hyaluronan is synthe-
Mechanism of Hyaluronan Synthesis
The unusual mechanism of hyaluranon
synthesis has impeded progress for a long
time ± a situation which has also occurred
with other important polysaccharides such
as cellulose and chitin. However, it now
appears that two mechanisms have evolved
independently ± for mammalian cells and
for streptococci on the one hand, and for
Pasteurella on the other hand.
Fig. 2 Mechanism of hyaluranon synthesis.
5.1
Chain Elongation
Hyaluronan synthesis in mammalian cells
differs from that of other polysaccharides in
many aspects. The molecule is elongated at
the reducing end by alternate transfer of
UDP-hyaluronan to the substrates UDP-
GlcNac and UDP-GlcA, thereby liberating the
UDP-moiety (Figure 2) (Prehm, 1983a,b).
Other glucosaminoglycans grow at the
non-reducing end and hence require a
sized at plasma membranes, the nascent
chains being extruded directly into the
extracellular matrix (Prehm, 1984). In con-
trast, other glucosaminoglycans are made in
the Golgi body. Chain initiation does not
require either a protein backbone (as for
proteoglycans) or preformed oligosacchari-
des as starters; only the presence of the
nucleotide sugar precursors is needed to
initiate new chains. During elongation the
chain is retained on the membrane-integrat-
ed synthase, this mechanism of synthesis
being in operation for hyaluranon synthesis
in both vertebrates and in Gram-positive

streptococci. However, a different mecha-
nism seems to exist for hyaluranon synthesis
in Gram-negative Pasteurella in which the
chains are elongated at the non-reducing
end (DeAngelis, 1999).
5.2
Chain Size
One point for discussion is what determines
the size of the synthesized hyaluronan, and
this aspect of polymerization also applies to
other macromolecular syntheses such as for
proteins, DNA, or RNA. An answer was
provided from experiments on isolated
membranes from fibroblasts or streptococci,
whereby the removal of nascent hyaluronan
from the hyaluronan synthase enzyme
stimulated its production. This was demon-
strated in isolated streptococcal membranes
(Nickel et al., 1998) and also in intact
fibroblasts (Philipson et al., 1985). It thus
appeared that high molecular-weight hyalur-
onan inhibited its own chain elongation,
when it was retained on the synthase. This
phenomenon may occur for solely thermo-
dynamic reasons, because the decrease in
entropy during the synthesis of a macro-
molecule must be compensated by free
energy from cleavage of the nucleotide
sugars and the subsequent formation of
ordered structures. In fact, this explains why
macromolecules such as proteins, RNA or
DNA do not exceed a certain chain length
(Peller, 1980).
5.3
Chain Export
The growing chain must be exported
through a membrane pore, and consequent-
ly the proposal was made by Weigel that this
pore is formed by the synthase itself, because
the inactivation rate of the synthase by
irradiation did not permit the participation
6 Hyaluronan Synthases 385
of other proteins (Tlapak-Simmons et al.,
1998). However, these authors did not show
that transport of hyaluronan through the
vesicle membrane was inactivated, and
methods should be developed to confirm
this finding.
5.4
Swelling
Hydration and swelling of nascent hyalur-
onan occurs at the site of synthesis on the
cell surface. While swelling to enormous
volumes (diameters up to 500 nm), one
molecule of hyaluronan will displace many
other cell-surface components by virtue of
exclusion. Hence, it is conceivable that this
swelling provides a mechanism whereby
adhesive components are disrupted from the
anchored cell.
5.5
Macromolecular Assembly
Macromolecular assembly with other matrix
molecules such as proteoglycans also occurs
at the cell surface. The compartmentation of
hyaluronan and proteoglycan syntheses to
the Golgi complex and the plasma mem-
branes thus ensures that, during synthesis,
very large aggregates are formed at the site of
final deposition, and not intracellularly.
6
Hyaluronan Synthases
Hyaluronan is synthesized at the protoplast
membrane of group A and group C strepto-
cocci (Markovitz and Dorfman, 1962), the
enzymatic activity being solubilized by very
mild detergents such as digitonin (Triscott
and van de Rijn, 1986). Conventional puri-
fication procedures such as ion-exchange
chromatography of detergent-solubilized
386 15 Hyaluronan
membrane proteins yielded inhomogeneous
protein mixtures that could not be separated
into individual constituents without loss of
enzymatic activity. Therefore, a new method,
based on the phase separation of a detergent
solution, was developed to allow purification
of the synthase in its active form (Prehm
et al., 1996). It was known that membrane
proteins can be separated from soluble
proteins by phase separation of a Triton X-
114 extract. Phase separation can be induced
in 1% Triton X-114 solutions by a temper-
ature shift from 0 8C to 37 8C, with soluble
proteins remaining in the aqueous phase
and membrane proteins in the detergent
phase. However, Triton X-114 was shown to
inactivate the hyaluronan synthase. It was
found that digitonin can undergo phase
separation by the addition of polyethylene
glycol 6000 at 0 8C, and that the synthase will
remain in the aqueous phase, where it is
associated with hyaluronan. Final purifica-
tion of the hyaluronan synthase was
achieved by ion-exchange chromatography
and yielded an electrophoretically homoge-
neous protein of 42 kDa. This study proved
that a single protein was sufficient to direct
hyaluronan synthesis, and that the method
may be generally applicable to other mem-
brane proteins that are associated with
polysaccharides, because it combines the
advantages of the mild detergent digitonin
with phase separation of all membrane
proteins from polysaccharide-binding pro-
teins.
Molecular cloning of the streptococcal
hyaluronan synthase was reported inde-
pendently by DeAngelis and van de Rijn
(DeAngelis et al., 1993; Dougherty and van
de Rijn, 1994). The gene was designated
HasA. The Streptococcus pyogenes operon
encodes two other proteins: HasB is a
UDP-glucose-dehydrogenase, which is re-
quired to convert UDP-glucose to UDP-GlcA
(Dougherty and van de Rijn, 1993), while
HasC is a UDP-glucose-pyrophosphorylase,
which is required to convert glucose-1-
phosphate and UTP to UDP-glucose (Crater
et al., 1995). Mammalian synthases were
cloned simultaneously from a mutant
mouse mammary carcinoma (Itano and
Kimata, 1996) and Xenopus laevis (Meyer
and Kreil, 1996). Now, three mammalian
synthases are known and have been de-
signated Has1, Has2, and Has3 (reviewed by
Weigel et al., 1997). Because these proteins
have 30% identity in terms of amino acid
sequence with the streptococcal synthase,
the genes may have a common ancestor. The
synthase from Pasteurella has been cloned by
DeAngelis et al. (1998), and is structurally
unrelated to the other synthases.
7
Hyaluronan-binding Proteins and Receptors
Hyaluronan-binding proteins are constitu-
ents of the extracellular matrix, and stabilize
its integrity. Hyaluronan receptors are in-
volved in cellular signal transduction; one
receptor family includes the binding pro-
teins aggrecan, link protein, versican and
neurocan and the receptors CD44, TSG6
(Lee et al., 1992), hyaluronectin (Delpech
and Halavent, 1981), GHAP (Perides et al.,
1990), and Lyve-1 (Banerji et al., 1999). The
RHAMM receptor is an unrelated hyalur-
onan-binding protein, and the hyaluronan-
binding sites contain a motif of a minimal
site of interaction with hyaluronan. This is
represented by B(X7)B, where B is any basic
amino acid except histidine, and X is at least
one basic amino acid and any other moiety
except acidic residues. CD44 and RHAMM
have attracted much attention, because they
were believed to be involved in metastasis
(Arch et al., 1992; Hall et al., 1995).