Hyaluronan fragments: An information-rich system

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Abstract

Hyaluronan is a straight chain, glycosaminoglycan polymer of the extracellular matrix composed of repeating units of the disaccharide [-d-glucuronic acid-β1,3-N-acetyl-d-glucosamine-β1,4-]n. Hyaluronan is synthesized in mammals by at least three synthases with products of varying chain lengths. It has an extraordinary high rate of turnover with polymers being funneled through three catabolic pathways. At the cellular level, it is degraded progressively by a series of enzymatic reactions that generate polymers of decreasing sizes. Despite their exceedingly simple primary structure, hyaluronan fragments have extraordinarily wide-ranging and often opposing biological functions. There are large hyaluronan polymers that are space-filling, anti-angiogenic, immunosuppressive, and that impede differentiation, possibly by suppressing cell–cell interactions, or ligand access to cell surface receptors. Hyaluronan chains, which can reach 2×104 kDa in size, are involved in ovulation, embryogenesis, protection of epithelial layer integrity, wound repair, and regeneration. Smaller polysaccharide fragments are inflammatory, immuno-stimulatory and angiogenic. They can also compete with larger hyaluronan polymers for receptors. Low-molecular-size polymers appear to function as endogenous “danger signals”, while even smaller fragments can ameliorate these effects. Tetrasaccharides, for example, are anti-apoptotic and inducers of heat shock proteins. Various fragments trigger different signal transduction pathways. Particular hyaluronan polysaccharides are also generated by malignant cells in order to co-opt normal cellular functions. How the small hyaluronan fragments are generated is unknown, nor is it established whether the enzymes of hyaluronan synthesis and degradation are involved in maintaining proper polymer sizes and concentration. The vast range of activities of hyaluronan polymers is reviewed here, in order to determine if patterns can be detected that would provide insight into their production and regulation.

Introduction

Hyaluronan (HA) occurs typically as a high-molecular-size polymer of the extracellular matrix (ECM) of up to 2×104 kDa, composed of repeating disaccharides of N-acetyl-glucosamine and glucuronic acid, connected exclusively by β-linkages (Meyer and Palmer, 1934; Fraser et al., 1997; Lee and Spicer, 2000). In marked contrast with all other glycosaminoglycans (GAGs), HA is synthesized, not in the Golgi apparatus, but on the cytoplasmic surface of the plasma membrane (Prehm, 1984). A number of HA synthases (HAS) are involved in HA synthesis (Weigel et al., 1997; Sugiyama et al., 1998). The polymer is transported out of the cell, possibly by way of a multi-drug resistance (MDR) transporter system (Prehm and Schumacher, 2004; Misra et al., 2005). Because of its enormous size, HA is transported out of the cell as it is being synthesized. This simple polymer does not undergo sulfation or epimerization, as do all other GAGs. These post-synthetic modifications endow other GAGs with complex patterns that are involved in a vast array of functions. However, HA is also involved in widely varying biological activities without benefit of such distinguishing modifications.

Hyaluronan is degraded through three different pathways, though the hyaluronidase (Hyal) enzyme family appear to be prominent in HA catabolism. It is not known whether the enzymes of synthesis or degradation are involved in generating and maintaining specific HA polymer sizes. Polymer size alone appears to confer specific functions on HA fragments. Location and polymer concentration, as well as HA size-specific binding proteins are other variables (Day and Prestwich, 2002). However, a recent plethora of references suggest that size alone is an essential element.

HA fragments of defined sizes are being used increasingly to study specific biological activities, by using them in vitro. The number of such studies is increasing rapidly. The time is right, perhaps, to review these studies, in an attempt to understand how these HA fragments are generated and regulated.

Section snippets

Caveats

There are several serious problems intrinsic to the study of shortened HA polymers that are tabulated here.

  • 1.

    In many studies, sizes of oligosaccharides are not measured accurately. Also, an effect may be attributed not to the major population, but to minor oligosaccharide components. Techniques for the precise measure and evaluation of HA oligomer preparations are now available (Lee and Cowman, 1994; Mahoney et al., 2001; Jing and DeAngelis, 2004). A list of current suppliers for HA, some of

HA synthesis

HAS are glycosyl transferases that occur in vertebrates, bacteria, and algal viruses (DeAngelis, 1999a). There are four HAS genes in most vertebrate genomes, with only three in mammals, expressing enzymes with different properties. They have distinct expression patterns controlled in part by various growth factors and cytokines (Sugiyama et al., 1998; Kennedy et al., 2000; Recklies et al., 2001; Itano et al., 1999, Itano et al., 2004). Expression of the HAS genes also appear to be tissue- and

Catabolism of HA

The degradation of HA occurs in a step-wise process (Roden et al., 1989; Lepperdinger et al., 2004), but details have defied explication. HA turnover in the body occurs through three separate pathways. The relative contribution of each of these pathways to total turnover is unknown.

  • 1.

    There is local cellular turnover that includes binding, internalization, and degradation within cells. Binding is by the predominant HA receptors, CD44 (Lesley et al., 2000; Ponta et al., 2003) and receptor for

The cellular pathway for HA catabolism

The degradation of HA occurs in cells by a series of coordinated enzymatic reactions. A recently formulated catabolic scheme (Stern, 2003, Stern, 2004), proposes that the high-molecular-mass polymer is cleaved progressively by a series of enzymes in which the product of one reaction becomes the substrate for the subsequent one. These successive enzymatic events generate HA fragments of ever-decreasing size. It is reasonable to assume that these same enzymes can conduct some of the business of

HA polymers of varying sizes

HA polymers occur in a variety of sizes that have a vast array of properties, some of which appear to be contradictory. The very large HA polymers are extracellular, are space-occupying and have an array of regulatory and structural functions. The small polymer fragments are angiogenic, inflammatory, and immuno-stimulatory. In general these short oligosaccharides tend to be involved in the body's alarm system, transmitting various modes of “danger signals” (Powell and Horton, 2005). However,

High-molecular-size HA (4×102−2×104 kDa; 2×103−105 sugars)

The very-high-molecular-size HA polymers are among the largest of matrix molecules. Their apparent size is even greater when the solvent volume of surrounding water is considered. It is also one of the most highly charged of molecules, accounting in part for its unusual properties (Lee and Spicer, 2000; Toole, 2000). High-molecular-size HA can function as a lubricant, as a shock absorber, as in the fluid within the joint capsule, and a space-occupying material, as in the vitreous of the

Inside-out matters

A major difference may exist between high-molecular-size HA in vivo and such HA chains after they have been extricated. Very little is known of the actual properties of the high-molecular-size HA within the narrow confines of the ECM or the restricted volume of the intercellular space. When HA undergoes aqueous extraction from major sources, such as from rooster comb, joint fluid, Wharton's jelly or from bacterial capsules, the extraction fluid has very high elastovasticity. This refers to the

Forms and functions

High-molecular-size HA can attain a length of 105 saccharides (2×104 kDa), depending on tissue source and on physiological conditions (Laurent and Fraser, 1992). The polymer is able to incorporate into its solvent domain a large volume of water that is more than 1000 times greater than the volume of the original material (Granger et al., 1984). These are space-filling molecules that hydrate tissues, are able to exclude other molecules and cells, and that are anti-angiogenic (Feinberg and Beebe,

HA fragments

Smaller fragments of HA polymers are involved in a variety of normal and pathological processes. HA fragments have sizes that overlap in the functions they perform. It would seem to be more expedient henceforth, to review their physiological roles and their participation in various physiological and pathological processes.

A partial list of HA fragments and their assigned biological functions is presented in Table 2, correlated with molecular size.

Angiogenesis, and wound healing

Wound healing is an example of the precise regulation required of HA fragmentation. In the earliest phase of wound healing, there is a sharp increase in HA after injury, a result of a combination of increased synthesis and impaired clearance. In these very first stages, high-molecular-size HA accumulates with the ability to bind fibrinogen, a reaction intrinsic to clot formation (Frost and Weigel, 1990). This initial HA is a product of platelets (de la Motte et al., submitted), with

Cancer biology

HA deposition is up-regulated in most malignancies. While high-molecular-mass HA is found in most normal biological processes, much lower weight material is readily detected in cancers (Kumar et al., 1989; Lokeshwar et al., 1997), where it facilitates tumor cell motility and invasion.

HA oligosaccharides of a certain size range induce proteolytic cleavage of CD44 on the surface of cancer cells, and promote tumor cell migration in a CD44- and dose-dependent manner (Sugahara et al., 2003, Sugahara

Chondrogenesis

HA oligosaccharides have a key role in chondrogenesis, in the expression of the cartilage-specific phenotype from mesenchymal precursors. Understanding such interactions is pivotal in tissue engineering.

Hexasaccharides of HA induce activation of a specific profile of transcription factors in chondrocytes that is not observed in other tissues (Ohno et al., 2005a). Enhanced expression of key genes involved in cartilage remodeling occurs, including MMP-3 and type II collagen. By contrast,

Infection

Very little investigation has been carried out on the role of HA polymers on infectious processes. Just as malignancies have commandeered normal HA functions for their own progression, similar techniques may have evolved by organisms for successful infections.

Aspects of HA metabolism have been shown to participate in infection with bacteria (Markowitz et al., 1959), mycobacteria (Aoki et al., 2004), Leishmania (Rao et al., 1999), sheep retroviruses (Rai et al., 2001), a porcine virus (Bratanich

Signal transduction

Several signal transduction pathways are initiated by various sizes of HA fragments binding to cell surface HA receptors such as CD44 and RHAMM. Table 3 provides a sample of only some of the transduction pathways that have been documented.

Angiogenic oligosaccharides of HA induce tyrosine kinases in endothelial cells, and activate several cytoplasmic signaling transduction pathways such as Raf-1 kinase, MAP kinase, and extracellular signaling kinases such as ERK-1, all of which result in

HA fragments modulate effects of the HA receptor, CD44

Fragments of HA can function at the cell surface by multiple mechanisms. High-molecular-mass HA chains inhibit their own elongation when bound to the plasma membrane-associated HAS. However, activation of the synthase can occur by HA oligosaccharides displacing these large nascent chains (Lueke and Prehm, 1999). CD44 appears to be involved in this process, presumably by keeping the growing chain in the vicinity of the synthase. An HA fragment of 20–30 saccharide size can bind to a variant of

Size-specific binding of HA fragments to hyaladherins

HA fragments can bind to HA-binding proteins or hyaladherins (Toole, 1990; Knudson and Knudson, 1993). Such binding has an array of functions, from intracellular effects, such as regulators of the cell cycle (Grammatikakis et al., 1995) or as splicing factors (Deb and Datta, 1996). Extracellular effects are provided by binding to cell surface receptors such as RHAMM and CD44, or to extracellular proteoglycans such as aggrecan and versican. Association with specific HA-binding proteins provides

Anomalies among the smallest HA oligosaccharides

Hexasaccharides of HA inhibit endothelial migration and formation of capillary-like tubules (Banerjee and Toole, 1992), indicating that subsequent products of HA degradation have the capacity to inhibit earlier stages in angiogenesis and wound healing.

HA hexasaccharides inhibit the formation of pericellular coats on cultured chondrocytes (Knudson and Knudson, 1991; Knudson et al., 1996), while high-molecular-size HA stimulates chondrogenesis. Hexasaccharides of HA may actually induce

Conclusion

HA polymers occur that are of varying sizes. The metabolic pathways for HA synthesis and degradation are highly ordered, composed of carefully controlled reactions that rely on regulation of individual enzyme activities. How such regulation is accomplished, and whether or not these pathways are coordinated with the maintenance of size-specific HA fragments is unknown.

From the evolutionary perspective, it is postulated that the HA-rich glycocalyx (Hedman et al., 1979), or pericellular matrix,

Acknowledgments

The authors acknowledge informational conversations with Drs. Endre Balazs, Lilly Bourguignon, Vincent Hascall, Peter Prehm, Carl Verkoelen, and Mr. Leif Tellmann, and are grateful for their many useful comments, and encouragement.

References (178)

  • A.J. Day et al.

    Hyaluronan cross-linking: a protective mechanism in inflammation?

    Trends Immunol.

    (2005)
  • A.J. Day et al.

    Hyaluronan-binding proteins: tying up the giant

    J. Biol. Chem.

    (2002)
  • P.L. DeAngelis

    Molecular directionality of polysaccharide polymerization by the Pasteurella multocida hyaluronan synthase

    J. Biol. Chem.

    (1999)
  • T.B. Deb et al.

    Molecular cloning of human fibroblast hyaluronic acid-binding protein confirms its identity with P-32, a protein co-purified with splicing factor SF2. Hyaluronic acid-binding protein as P-32 protein, co-purified with splicing factor SF2

    J. Biol. Chem.

    (1996)
  • T.B. Deb et al.
  • V. Deguine et al.

    Free radical depolymerization of hyaluronan by Maillard reaction products: role in liquefaction of aging vitreous

    Int. J. Biol. Macromol.

    (1998)
  • C.A. de la Motte et al.

    Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid:polycytidylic acid: inter-alpha-trypsin inhibitor is crucial to structure and function

    Am. J. Pathol.

    (2003)
  • S.J. Frost et al.

    Binding of hyaluronic acid to mammalian fibrinogens

    Biochim. Biophys. Acta

    (1990)
  • Y. Fujita et al.

    CD44 signaling through focal adhesion kinase and its anti-apoptotic effect

    FEBS Lett.

    (2002)
  • S. Ghatak et al.

    Hyaluronan oligosaccharides inhibit anchorage-independent growth of tumor cells by suppressing the phosphoinositide 3-kinase/Akt cell survival pathway

    J. Biol. Chem.

    (2002)
  • S. Ghatak et al.

    Hyaluronan constitutively regulates ErbB2 phosphorylation and signaling complex formation in carcinoma cells

    J. Biol. Chem.

    (2005)
  • P. Ghosh et al.

    Potential mechanism of action of intra-articular hyaluronan therapy in osteoarthritis: are the effects molecular weight dependent?

    Semin. Arthritis Rheum.

    (2002)
  • N. Grammatikakis et al.

    A novel glycosaminoglycan-binding protein is the vertebrate homologue of the cell cycle control protein, Cdc37

    J. Biol. Chem.

    (1995)
  • V.C. Hascall et al.

    Aggregation of cartilage proteoglycans. II. Oligosaccharide competitors of the proteoglycan–hyaluronic acid interaction

    J. Biol. Chem.

    (1974)
  • V.C. Hascall et al.

    Intracellular hyaluronan: a new frontier for inflammation?

    Biochim. Biophys. Acta

    (2004)
  • M.R. Horton et al.

    Hyaluronan fragments synergize with interferon-γ to induce the C-X-C chemokines Mig and interferon-inducible protein-10 in mouse macrophages

    J. Biol. Chem.

    (1998)
  • M.R. Horton et al.

    Hyaluronan fragments induce plasminogen activator inhibitor-1 and inhibit urokinase activity in mouse alveolar macrophages: a potential mechanism for impaired fibrinolytic activity in acute lung injury

    Chest

    (1999)
  • S. Iacob et al.

    Hyaluronan fragments activate nitric oxide synthase and the production of nitric oxide by articular chondrocytes

    Int. J. Biochem. Cell Biol.

    (2006)
  • N. Itano et al.

    Molecular cloning of human hyaluronan synthase

    Biochem. Biophys. Res. Commun.

    (1996)
  • N. Itano et al.

    Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties

    J. Biol. Chem.

    (1999)
  • N. Itano et al.

    Selective expression and functional characteristics of three mammalian hyaluronan synthases in oncogenic malignant transformation

    J. Biol. Chem.

    (2004)
  • W. Jing et al.

    Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers

    J. Biol. Chem.

    (2004)
  • J.D. Kahmann et al.

    Localization and characterization of the hyaluronan-binding site on the link module from human TSG-6

    Struct. Fold. Des.

    (2000)
  • C.I. Kennedy et al.

    Proinflammatory cytokines differentially regulate hyaluronan synthase isoforms in fetal and adult fibroblasts

    J. Pediatr. Surg.

    (2000)
  • J.H. Kimura et al.

    Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the Swarm rat chondrosarcoma

    J. Biol. Chem.

    (1979)
  • M. Kinoshita et al.

    Analysis of the interaction between hyaluronan and hyaluronan binding proteins by capillary affinity electrophoresis: significance of hyaluronan molecular size on binding reaction

    J. Chromatogr. B

    (2005)
  • W. Knudson et al.

    CD44-anchored hyaluronan-rich pericellular matrices: an ultrastructural and biochemical analysis

    Exp. Cell Res.

    (1996)
  • D. Kohda et al.

    Solution structure of the link module: a hyaluronan-binding domain involved in extracellular matrix stability and cell migration

    Cell

    (1996)
  • M.J. Kujawa et al.

    Hyaluronic acid bonded to cell-culture surfaces stimulates chondrogenesis in stage 24 limb mesenchyme cell cultures

    Dev. Biol.

    (1986)
  • M.J. Kujawa et al.

    Substrate-bonded hyaluronic acid exhibits a size-dependent stimulation of chondrogenic differentiation of stage 24 limb mesenchymal cells in culture

    Dev. Biol.

    (1986)
  • M.J. Kujawa et al.

    Hyaluronic acid bonded to cell culture surfaces inhibits the program of myogenesis

    Dev. Biol.

    (1986)
  • E. Lara-Pezzi et al.

    The hepatitis B virus X protein (HBx) induces a migratory phenotype in a CD44-dependent manner: possible role of HBx in invasion and metastasis

    Hepatology

    (2001)
  • H.G. Lee et al.

    An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution

    Anal. Biochem.

    (1994)
  • J.Y. Lee et al.

    Hyaluronan: a multifunctional, megaDalton, stealth molecule

    Curr. Opin. Cell Biol.

    (2000)
  • G. Lepperdinger et al.

    HYAL2, a human gene expressed in many cells, encodes a lysosomal hyaluronidase with a novel type of specificity

    J. Biol. Chem.

    (1998)
  • G. Lepperdinger et al.

    Hyal2—less active, but more versatile?

    Matrix Biol.

    (2001)
  • G. Lepperdinger et al.

    Biodegradation of hyaluronan

  • J. Lesley et al.

    Hyaluronan binding by cell surface CD44

    J. Biol. Chem.

    (2000)
  • V.B. Lokeshwar et al.

    Differences in hyaluronic acid-mediated functions and signaling in arterial, microvessel, and vein-derived human endothelial cells

    J. Biol. Chem.

    (2000)
  • V.B. Lokeshwar et al.

    The cell adhesion molecule, GP116, is a new CD44 variant (ex14/v10) involved in hyaluronic acid binding and endothelial cell proliferation

    J. Biol. Chem.

    (1996)

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    Present address: Burnham Institute for Medical Research, Cancer Research Center, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

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