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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:
[email protected] [email protected] 1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 81
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Hist Histo oric rical Outli tline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
38 1
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Chem hemica ical Stru Struccture ture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 83
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Occu Occurr rren ence ce of Hyal Hyalur uron onan an . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 83
5 5.1 5.2 5.3 5.4 5.5
Mech Mechan anis ism m of Hyal Hyalur uron onan an Synt Synthe hesi siss Chain Elongation . . . . . . . . . . . Chain Size . . . . . . . . . . . . . . . Chain Export . . . . . . . . . . . . . . Swelling . . . . . . . . . . . . . . . . Macromolecular Assem sembly . . . . . .
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3 84 3 84 3 85 385 38 5 385
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Hyal Hyalur uron onan an Synt Syntha hase sess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
385
7 7.1 7.2 7.3 7.3
Hyalur Hyalurona onan-b n-bind inding ing Protei Proteins ns and Recept Receptors ors CD44 . . . . . . . . . . . . . . . . . . . . . . RHAMM . . . . . . . . . . . . . . . . . . . . Othe Otherr Hyal Hyalur uron onan an-b -bin ind ding Prot Protei eins ns . . . .
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3 86 387 38 8 388 388
8
Mechan Mechanism ismss of Hyalur Hyalurona onan n Releas Releasee from from the Cell Cell Surfac Surfacee . . . . . . . . . . .
38 8
9 9.1 9.2 9.2 9.2. 9.2.11
Regu Regula lati tion on of Hyal Hyalur uron onan an Synt Synthe hesi siss . . . . Expressi ssion of the Syntha thase . . . . . . . . . Stim Stimul ulat atio ion n and and Inhi Inhibi biti tion on of the the Synt Syntha hase se Signa ignall Trans ransd ducti uction on at Memb embrane raness . . . .
3 89 3 89 389 389 390 390
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15 Hyaluronan
9.2.2 9.3
Intracellular Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . Influence of Chain Length on Further Elongation . . . . . . . . . . . . . . . .
390 390
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Turnover and Catabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
391
11 11.1 11.2 11.2.1 11.2.2 11.2.3 11.3 11.3.1 11.3.2 11.3.3
Functions of Hyaluronan . . . . . . . . Cellular Functions . . . . . . . . . . . Physiological Functions . . . . . . . . Differentiation and Morphogenesis . Wound Healing . . . . . . . . . . . . . Synovia . . . . . . . . . . . . . . . . . . Pathological Functions . . . . . . . . . Metastasis . . . . . . . . . . . . . . . . Edema . . . . . . . . . . . . . . . . . . Streptococci . . . . . . . . . . . . . . .
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391 391 392 392 392 393 393 393 393 393
12 12.1 12.2
Hyaluronan Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Degradation by Free Radicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . Degradation by Hyaluronidases . . . . . . . . . . . . . . . . . . . . . . . . . . .
394 394 395
13 13.1 13.2
Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Market . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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14 14.1 14.2 14.3 14.4 14.5
Medical Applications . . . . . Ophthalmics . . . . . . . . . . Arthritis . . . . . . . . . . . . . Wound Healing and Scarring Adhesion Prevention . . . . . Drug Delivery . . . . . . . . .
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Effects of Hyaluronan Oligosaccharides . . . . . . . . . . . . . . . . . . . . . .
398
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Outlook and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
398
17
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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CHO Chinese hamster ovary PMA phorbol-12-myristate-13-acetate RHAMM receptor for hyaluronan-mediated motility
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2 Historical Outline
1
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 interacts in tiny concentrations in cells to trigger important responses. Hyaluronan has an unusual mechanism of biosynthesis and exceptional physical properties; consequently, 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 research, 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
Historical Outline
In 1934, Karl Meyer described a procedure for isolating a novelglycosaminoglycan from the vitreous humor of bovine eyes, and named it hyaluronic acid (from the Greek, hyalos glassy, vitreous) (Meyer and Palmer, 1934). These authors showed that this substance contained a uronic acid and an amino sugar, but no sulfoesters. At physiological pH all carboxyl groups are dissociated, andhence the polysaccharide shouldbe called hyaluronate. Today, this macromole-
cule is most frequently referred to as hyaluronan, in order to emphasize its polysaccharide 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 streptococci (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 electrostatic interactions (Comper and Laurent, 1978). In 1972, Hardingham and Muir discovered 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 discovered during the 1990s. After 1980, the research spread in many directions, mainly because until that time it had been assumed that hyaluronan belonged 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 assumption was disproved when a plausible mechanism 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 ~
381
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15 Hyaluronan
it was shown that hyaluronan was in fact synthesized at plasma membranes and the chains were extruded directly into the extracellular matrix (Prehm, 1984). The catabolic pathways of hyaluranon were also elucidated at about this time (Fraser et al., 1981). Subsequently, it became possible to measure 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 hyaluronan for medical purposes, and produced highly viscous and noninflammatory preparations on a commercial scale both as an aid for ophthalmic surgery and as viscosupplementationfor 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 (Turley et al., 1987; Aruffo et al., 1990), and studied intensively during the 1990s. Although 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 molecular biology of the receptors which led to a fundamental understanding of these processes. In particular, the receptors CD44 and RHAMM (Receptor for Hyaluronan-Mediated Motility) have attracted much enthusiasm, 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 impairments (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). Subsequently, 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 inactive. 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 biological functions. Oligosaccharides of hyaluronan induce angiogenesis (West et al., 1985) and also activate lymphocytes (McKee et al., 1997; Termeer et al., 2000). Radical degradation generates reactive aldehydes (Uchiyama et al., 1990) which modify proteins into the main antigenic structures of rheumatoid arthritis (Prehm, 2000). This discovery finally terminated a long and oppressive period of ignorance in a medically important
4 Occurrence of Hyaluronan
problem, and may eventually lead to a curative treatment of these diseases that currently are treated only symptomatically.
1991). In physiological solutions a hyaluronan molecule assumes an expanded random coil structure which occupies a very large domain. The actual mass of hyaluronan within this domain is very low, and 0.1% molecules would overlap each other 3 at a hyaluronan concentration of 1 mg mLÀ1, Chemical Structure or higher. This domain structure of hyaluronan has interesting and important conseThe complete structure of hyaluranon was quences. Small molecules such as water, elucidated by the group of Karl Meyer, who electrolytes and nutrients can diffuse freely characterized the oligosaccharides obtained through the solvent, within the domain; by the action of testicular hyaluronidases however, large molecules such as proteins (Weissman and Meyer, 1954). Hyaluronan will be partially excluded from the domain consists of basic disaccharide units of D- because of their hydrodynamic sizes in glucuronic acid and D-N -acetylglucosamine, solution. This leads both to slower diffusion these being linked together through alter- of macromolecules through the network and nating b-1,4 and b-1,3 glycosidic bonds to their lower concentration in the network (Figure 1). compared with the surrounding hyaluronanThe number of repeat disaccharides in a free compartments. At pH 7, the carboxyl completed hyaluronan molecule can reach groups are predominantly ionized, and the 10,000 or more, with a molecular mass of hyaluronan molecule is a polyanion that has 4î106 daltons (each disaccharide is associated exchangeable cation counterions 400 daltons). In a physiological solution, to maintain charge neutrality. the backbone of a hyaluronan molecule is stiffened by a combination of the chemical structure of the disaccharide, internal hydro- 4 gen bonds, and interactions with solvent. In Occurrence of Hyaluronan addition, the preferred shape in water features hydrophobic patches on alternating Hyaluronan is present in all vertebrates, and sides of the flat, tape-like secondary struc- also in the capsule of some pathogenic ture. The two sides are identical, so that bacteria such as Streptococcus sp. and Pas- hyaluronan molecules are ambidextrous, teurella. It is a component of extracellular enabling them to aggregate via specific matrices in most tissues, andin some tissues interactions in water to form meshworks, it is a major constituent. The concentration even at low concentrations (Scott et al., of hyaluronan is particularly high in rooster comb (7.5 mg mLÀ1), in the synovial fluid (3± 4 mg mLÀ1), in umbilical cord (3 mg mLÀ1), in the vitreous humor of the eye (0.2 mg mLÀ1), and in skin (0.5 mg mLÀ1). 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 Fig. 1 Repeating unit of hyaluranon. chondroitin sulfate proteoglycan, and re~
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~
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15 Hyaluronan
Fig. 2
tains this macromolecular assembly in the matrix by specific hyaluronan±protein interactions. 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 proliferating cells (Evanko and Wight, 1999) ~
5
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.
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 UDPGlcNac andUDP-GlcA,therebyliberatingthe UDP-moiety (Figure 2) (Prehm, 1983a,b). Other glucosaminoglycans grow at the non-reducing end and hence require a protein backbone. Hyaluronan is synthesized at plasma membranes, the nascent chains being extruded directly into the extracellular matrix (Prehm, 1984). In contrast, other glucosaminoglycans are made in the Golgi body. Chain initiation does not require either a protein backbone (as for proteoglycans) or preformed oligosaccharides 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-integrated synthase, this mechanism of synthesis being in operation for hyaluranon synthesis in both vertebrates and in Gram-positive
6 Hyaluronan Synthases
streptococci. However, a different mechanism seems to exist for hyaluranon synthesis in Gram-negative Pasteurella in which the chains are elongated at the non-reducing end (DeAngelis, 1999).
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.
5.2
Chain Size
5.4
Swelling
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 demonstrated in isolated streptococcal membranes (Nickel et al., 1998) and also in intact fibroblasts (Philipson et al., 1985). It thus appeared that high molecular-weight hyaluronan inhibited its own chain elongation, when it was retained on the synthase. This phenomenon may occur for solely thermodynamic reasons, because the decrease in entropy during the synthesis of a macromolecule 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).
Hydration and swelling of nascent hyaluronan 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 membranes thus ensures that, during synthesis, very large aggregates are formed at the site of final deposition, and not intracellularly.
6
Hyaluronan Synthases 5.3
Chain Export
The growing chain must be exported through a membrane pore, and consequently the proposal was made by Weigel that this pore is formedby the synthase itself, because the inactivation rate of the synthase by irradiation did not permit the participation
Hyaluronan is synthesized at the protoplast membrane of group A and group C streptococci (Markovitz and Dorfman, 1962), the enzymatic activity being solubilized by very mild detergents such as digitonin (Triscott and van de Rijn, 1986). Conventional purification procedures such as ion-exchange chromatography of detergent-solubilized
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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 X114 extract. Phase separation can be induced in 1% Triton X-114 solutions by a temperature shift from 0 C to 37 C, 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 C, and that the synthase will remain in the aqueous phase, where it is associated with hyaluronan. Final purification of the hyaluronan synthase was achieved by ion-exchange chromatography and yielded an electrophoretically homogeneous 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 membrane 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 proteins. Molecular cloning of the streptococcal hyaluronan synthase was reported independently 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 required 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-1phosphate 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 designated 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 constituents of the extracellular matrix, and stabilize its integrity. Hyaluronan receptors are involved in cellular signal transduction; one receptor family includes the binding proteins 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 hyaluronan-binding protein, and the hyaluronanbinding 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).
