Hyaluronan
Is Bigger Better?

Todd D. Camenisch and John A. McDonald

Departments of Biochemistry and Molecular Biology, and Internal Medicine, Mayo Clinic Scottsdale, Scottsdale, Arizona

	Hyaluronan Structure and Function

Top Hyaluronan Structure and... HA Biosynthesis HA Interactions: Motifs and... HA Receptors HA Degradation Biologic Activities Attributed... Caveat Emptor and Caveat... References

Hyaluronan (HA) is a polymer of molecular mass from ~ 2 × 10⁵ to ~ 10 × 10⁶ comprising repeating disaccharide units of glucuronic acid and N-acetyl-D-glucosamine. HA is abundant, present in virtually all biologic fluids. Moreover, there is a brisk metabolism of HA in humans, with approximately one-third (~ 5 g) of total HA degraded and replaced per day predominantly by the reticuloendothelial system (1). HA performs three functions: It expands the extracellular space by binding salt and water, interacts with a variety of extracellular molecules to form a composite extracellular matrix, and is recognized by several cell surface receptors that activate intracellular signaling pathways in response to HA (and likely, costimulatory signals) or function in HA internalization (reviewed in Reference 2). HA is especially prominent during embryonic development and at sites of wound healing. In the lung, HA accumulates as part of the fibroproliferative response to injury and in tissue remodeling.

	HA Biosynthesis

Top Hyaluronan Structure and... HA Biosynthesis HA Interactions: Motifs and... HA Receptors HA Degradation Biologic Activities Attributed... Caveat Emptor and Caveat... References

HA is synthesized by a family of plasma membrane associated glycosyltransferases in mammals (3). Gene targeting reveals that one of these, Has2, is the major source of HA in midgestation in the mouse. Mice lacking Has2 die around embryonic day (E) 9.5 with profound defects in the cardiovascular system and a general paucity of extracellular matrix (4). In contrast, mice harboring null mutations in Has1 and/or Has3 are viable and fertile (J. McDonald and T. Camenisch, unpublished data). Has2-deficient mice bear a striking resemblance to hdf mice lacking the HA-binding proteoglycan versican (5), demonstrating the biologic importance of a composite matrix containing HA. Interestingly, versican and HA are also coexpressed in lung fibroproliferative disorders (6) and during vascular injury and repair (7, 8). Most data suggest that Has1 and Has2 synthesize high molecular weight HA, whereas Has3, a more catalytically active enzyme, makes somewhat shorter chains (3, 9). Thus, differential expression of Has genes offers one potential mechanism for the generation of smaller HA chains. One patient with overproduction of HA, resulting in abundant lax skin similar to that of the Shar-Pei breed of dog, has been reported (10), but the genetic basis of this disorder is unknown.

	HA Interactions: Motifs and Proteins

Top Hyaluronan Structure and... HA Biosynthesis HA Interactions: Motifs and... HA Receptors HA Degradation Biologic Activities Attributed... Caveat Emptor and Caveat... References

Most HA-binding proteins contain one or two copies of the so-called link module (also known as the proteoglycan tandem repeat or PTR) (11). This module consists of two alpha helices and two triple-stranded antiparallel -sheets arranged around a large hydrophobic core, and resembles the C-type lectin module (12). Although one link module is sufficient for HA binding, as exemplified by the tumor necrosis factor-stimulated gene 6 (TSG-6), many proteins contain two copies of the link module (e.g., link protein, the proteoglycans aggrecan, versican, brevican, and neurocan). In aggrecan, two link modules and adjacent sequences in the G1 globular domain are required for high avidity HA binding (13). The identification of conserved residues in the link module has resulted in isolation of additional cDNAs encoding a number of related molecules by in silico cloning (e.g., 14, 15). The receptor for hyaluronan-mediated motility (RHAMM) has another HA binding motif of BX₇B, where B is a basic residue and X is any amino acid that is not negatively charged.

	HA Receptors

Top Hyaluronan Structure and... HA Biosynthesis HA Interactions: Motifs and... HA Receptors HA Degradation Biologic Activities Attributed... Caveat Emptor and Caveat... References

CD44 and RHAMM represent the best characterized molecules that activate intracellular signaling in response to HA. CD44 binding to HA stimulates signaling mediated via Rac (16, 17) and Ras (18). Given its ubiquitous nature, a biologic requirement for a costimulatory signal would be anticipated for a cellular response to HA. HA binding to CD44 expressed on ovarian carcinoma cells has been shown to engage the ErbB family of receptors, resulting in Ras activation (19). This is consistent with a costimulatory mechanism requiring HA engagement of CD44 (or other HA receptors), as well as signaling through receptor tyrosine kinases (4). The report of Ohkawara and colleagues (31) extends this paradigm. RHAMM is also thought to signal via Ras, although the situation is complicated by the presence of RHAMM both on the cell surface and intracellularly (20). HA has been found intracellularly, raising the possibility of an autocrine signaling mechanism involving RHAMM (23).

HA binding by CD44 is not constitutive and is subject to complex regulation. Sialic acid modification of N-linked oligosaccharides, as well as sulfation, are implicated in regulation of HA binding (24). The cytoplasmic domain of CD44 plays no role (25). Binding of high molecular weight HA to CD44 is inhibited by HA-derived oligosaccharides of between six and 18 sugars. This finding agrees with the predicted interaction of HA with the link module, suggesting that a hexamer occupies a link module binding site (11, 12). Inhibition increases with chain lengths of ~ 20 to 30 sugars. These data indicate that more than one CD44 molecule can bind to an oligosaccharide of 20 to 30 sugars (26). Thus, highly purified HA oligosaccharides can serve as probes for HA-dependent interactions by specifically inhibiting the interaction of HA with HA-binding proteins such as CD44. However, such binding studies do not shed light on potential mechanisms that can distinguish between HA of average mass in hundreds of thousands versus millions of daltons.

	HA Degradation

Top Hyaluronan Structure and... HA Biosynthesis HA Interactions: Motifs and... HA Receptors HA Degradation Biologic Activities Attributed... Caveat Emptor and Caveat... References

Four hyaluronidases have been identified, one of which is associated with mucopolysaccharidosis IX (27). On the basis of isotope studies, it appears that between 10 to 100 mg of HA are turned over each day via the circulation (28). HA in plasma is rapidly removed via the lymphatics and liver via specific endocytic receptors (14, 29, 30) and catabolized.

	Biologic Activities Attributed to HA Vary Depending upon Molecular Mass

Top Hyaluronan Structure and... HA Biosynthesis HA Interactions: Motifs and... HA Receptors HA Degradation Biologic Activities Attributed... Caveat Emptor and Caveat... References

The biologic effects of HA appear to vary depending upon its average mass. Ohkawara and colleagues (31) demonstrate that HA of average mass 0.2 × 10⁶ daltons improves survival of peripheral blood eosinophils in vitro. Surprisingly, HA of 3 to 6 × 10⁶ daltons has much less effect. The mechanism involves increased expression of granulocyte macrophage colony-stimulating factor (GM-CSF), known to enhance eosinophil survival in vitro. The increased expression of GM-CSF is partially, but not completely inhibited, by antibodies to CD44. In addition, HA increases production of transforming growth factor 1 by eosinophils. The authors suggest that local production of lower molecular weight HA may contribute to the airway fibrosis associated with chronic asthma.

This provocative observation follows a number of previous reports suggesting distinct angiogenic (32, 33) and proinflammatory biologic activities of lower molecular weight HA or HA oligomers (34). Lower molecular weight HA, but not high molecular weight HA, stimulates the production of metalloelastase in MH-S cells (41) and expression of inducible nitric oxide synthase in rat liver endothelial and Kupffer cells (40). In some cases, the stimulatory effects of low molecular weight HA were shown to be mediated by activation of nuclear factor (NF)-B (35). Conversely, interleukin (IL)-10 and interferon- were found to inhibit low molecular weight HA-induced cytokine production in mouse bone marrow-derived macrophages (39). McKee and associates (36) showed that HA of average relative molecular mass (M_r) 0.47 × 10⁶ obtained by sonication, but not intact, high molecular weight HA increased the expression of a number of chemokines (macrophage inflammatory protein [MIP]-1, MIP-1, crg-2, regulated on activation, normal T cell expressed and secreted [RANTES], and monocyte chemotactic protein-1 [MCP-1]) in the murine alveolar macrophage cell line MH-S. This effect was mimicked by HA hexamers and by HA of average molecular mass 0.28 × 10⁶. The HA effect was clearly not due to endotoxin contamination. Binding of HA to MH-S cells was shown to be mediated by CD44. However, similar to results in the study by Ohkawara and associates (31), antibody to CD44 only partially inhibited chemokine expression, suggesting involvement of additional receptors (or stimuli).

	Caveat Emptor and Caveat Lector

Top Hyaluronan Structure and... HA Biosynthesis HA Interactions: Motifs and... HA Receptors HA Degradation Biologic Activities Attributed... Caveat Emptor and Caveat... References

The work of Ohkawara and coworkers and numerous other observations suggest that there is something fundamentally different about the biologic response to high (megadaltons) and lower molecular weight HA. Currently, no compelling mechanistic explanation for this difference (apart from physical characteristics, i.e., rheologic properties) exists. Oligomers could function by inhibiting HA-receptor interactions without clustering receptors (26), but it is more difficult to conceptualize how higher (e.g., 200,000 daltons) HA fragments stimulate gene expression whereas high molecular weight HA does not. It is essential to identify the receptor(s) involved in this interesting biologic response to HA to better understand its proinflammatory and angiogenic properties. The availability of CD44-null mice should help elucidate its role in the response to HA (42).

HA is widely available from commercial suppliers. Common sources include rooster comb, human umbilical cord, and gram-positive streptococci. In the case of the current work, two different suppliers provided HA of differing average molecular masses and quantitative differences in biologic activity. In previously published work, several methods have been used to create HA of differing molecular weights, including sonication and incomplete digestion with hyaluronidases (typically from bovine testes or Streptomyces hyaluronilyticus). Because HA is found at high concentration in biologic fluids and tissues (µg to mg/ml), it is often used at a relatively high concentration in vitro when assaying for a biologic response. This raises the specter of contamination with substances active in the nanogram range. Accordingly, it would be prudent to consider including several controls in experiments using HA (Table 1). In particular, one should be suspicious of differences arising between lots of HA or between HA samples provided by different suppliers. In special cases, transfection with a cDNA encoding HA synthase offers an alternative control (4).

	Footnotes

Address correspondence to: John A. McDonald, Departments of Biochemistry and Molecular Biology and Internal Medicine, Samuel C. Johnson Medical Research Building, Mayo Clinic Scottsdale, 13400 East Shea Blvd., Scottsdale, AZ 85259. E-mail: mcdonald.john{at}mayo.edu

(Received in original form August 11, 2000).

Acknowledgments: The authors acknowledge valuable discussions with Dr. Vincent Hascall of the Department of Biomedical Engineering of the Cleveland Clinic.

Abbreviations HA, hyaluronan; RHAMM, receptor for hyaluronan-mediated motility.

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Top Hyaluronan Structure and... HA Biosynthesis HA Interactions: Motifs and... HA Receptors HA Degradation Biologic Activities Attributed... Caveat Emptor and Caveat... References

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