Hyaluronan Structures in Solution Relevance to Tissue Biologic Functions and Aging

A. Extracellular HA

Extracellular HA is a polydisperse molecule whose molecular weight ranges from 300 to >103 kDa (37,38). It displays both flexible and stiff domains in solution, suggesting deviation of its primary structure from that elucidated by Meyer and co-workers, which is an unbranched chain of alternating D-GlcA and D-GlcNAc in a 1:1 molar ratio (1,2,39). The secondary and tertiary structures of HA may be determined by its primary structure (sugar-sequence) like in proteins (40). If this is the case, the flexible and stiff domains that HA forms in solution are functions of its primary structure. Therefore, the stiff segments, which comprise 55-70% of the molecule and are not affected by changes in temperature, ionic strength, denaturing reagents like urea or pH, may originate from a primary structure different from the one indicated above (41). It is worth noting that some reports suggest that basic reagents relieve stiffness (42).

Stiffness can be relieved by base, whether it originates from hydrogen bonding or hydrophobic interactions. The hydrogen bond network would be disrupted as the base (e.g., OH") replaces the hydrogen acceptor of the existing hydrogen bonds. Thus, the size needed for stiffness is altered and relaxation occurs (42). The intramolecular hydrogen bond pattern of HA proposed by Scott and co-workers (3,4) appears in Fig. 1. If stiffness originates from hydrophobic interactions of the acetamido methyl of D-GlcNAc, a basic reagent may introduce relaxation, as it abstracts methyl protons of the acetamido moiety. Such process would break the van der Waals interactions responsible for the stiffness (43). Methyl protons of the acetamido moieties are acidic, because they are a to the carbonyl (C=O) group (44).

Hydrophobic (stiff) regions of about eight sequential CH groups have been reported in HA solutions of dimethylsulfoxide (DMSO) (3). They would form if the acetamido moieties of D-GlcNAc positioned themselves in such a way as to accommodate DMSO in between as shown in Fig. 3. Such an arrangement would be facilitated if HA coiled to bring acetamido moieties closer, with at least one DMSO holding their methyl groups in a van der Waals association. The helical structures proposed for HA support this hypothesis (45). Hydrophobic patches may also result from sequences of D-GlcNAc only as shown in Fig. 4. These patches might hinder the spectroscopic detection of carboxylate moieties (3), but

GlcNAc

GlcNAc

DMSO

Figure 3 Hyaluronan hexasaccharide (1) in DMSO showing the hypothetical location of DMSO between D-GlcNAc moieties.

DMSO

Figure 3 Hyaluronan hexasaccharide (1) in DMSO showing the hypothetical location of DMSO between D-GlcNAc moieties.

should facilitate the association of HA with the hydrophobic regions of lipids (46), proteins (32,47) and other HA molecules (48).

HA-stiff regions of at least 60 disaccharides, each distributed along the polymer and separated by flexible regions, have been proposed based on their susceptibility to hyaluronidase (41). It has also been reported that HA-stiff segments with fewer than 60 disaccharides form in solution, suggesting that the fine structures responsible for this 2D-arrangement occur along the entire HA chain (41).

Scott and co-workers (3,4) have proposed a model for high molecular weight HA in aqueous solutions, based on NMR spectroscopy, X-ray crystallography, rotary shadowing and electron microscopy data. In this model they suggest that HA forms "2-fold helices with gentle curves in the polymer backbone in two planes at right angles, with hydrophobic patches on alternate sides of the polymer" (4). This model is facilitated by an anti-parallel arrangement of the HA chains that provides the greatest proximity for groups on adjacent chains to interact. Thus, hydrogen bonds of the polar and ionic groups, and van der Waals interactions of the methyl groups of the acetamido moieties could hold many HA chains together in extensive networks of sizes

GlcNAc

GlcNAc

DMSO

Figure 4 A hypothetical tetrasaccharide of hyaluronan showing three adjacent D-GlcNAcs where two of them are connected through DMSO by van der Waals bonds.

DMSO

Figure 4 A hypothetical tetrasaccharide of hyaluronan showing three adjacent D-GlcNAcs where two of them are connected through DMSO by van der Waals bonds.

limited mainly by HA concentration (3,4). Such macromolecular aggregates should affect the biologic functions of the tissues containing HA.

1. Highly Hydrophilic Hyaluronan Sequences

In aqueous, HA solutions, basic (D-GlcN) (Fig. 5) and acidic (D-GlcA) (Fig. 6) sugar-sequences should form stronger hydrogen bond networks with the solvent than the alternating sequence of D-GlcNAc and D-GlcA, where the methyl moieties of the acetamido groups would interrupt the hydrogen bond meshwork (43). Water would form cages around the basic and acidic regions making them appear stiff. This dense, hydrogen bond web may not be disrupted by low concentrations of urea, since this reagent may hydrogen bond with the water present in the medium without disturbing the HA-water bonds. The presence of basic or acidic regions on the HA chain would support the hypothesis that HA stiffness in aqueous solution originates from highly dense, hydrogen bond webs that resist cleavage by low concentrations of urea (41).

2. Biological Importance of Highly Dense, Hyaluronan-Water, Hydrogen Bond Networks

Densely hydrogen bonded HA in water should be relevant in numerous physiologic functions, as the hydrogen bonding contributes to its viscosity (42). In synovial fluid, a highly viscous HA-water medium offers the efficient support required for healthy joints (49). In the corneal endothelium, viscous solutions of Na-HA in water are known to provide protection from mechanical damage (50). The viscosity of the vitreous body of the eye should, at least partially, be a function of the degree of hydrogen bonding provided by HA in water (38). Fetal skin contains highly hydrated HA in gel-like structures that may result mainly from the HA-water hydrogen bonds and may be needed for cell differentiation (51). The 3D-structural support that HA provides in the extracellular space of connective tissue should, at least partly, be a function of its degree of hydration and hydrogen bonding (27-32,42). The SHAP-HA complex has been found in rheumatoid arthritis, not in normal joints, suggesting its involvement in this

GkA GlcN GJcN GlcN

Figure 5 Hypothetic, basic tetrasaccharide of hyaluronan showing one D-GlcA followed by a sequence of three D-GlcN moieties.

GkA GlcN GJcN GlcN

Figure 5 Hypothetic, basic tetrasaccharide of hyaluronan showing one D-GlcA followed by a sequence of three D-GlcN moieties.

GlcA GicA GlcA GlcNAc

Figure 6 Hypothetic, acidic tetrasaccharide of hyaluronan showing three adjacent D-GlcA moieties with a terminal d-GlcNAc.

GlcA GicA GlcA GlcNAc

Figure 6 Hypothetic, acidic tetrasaccharide of hyaluronan showing three adjacent D-GlcA moieties with a terminal d-GlcNAc.

disorder (52). In addition, the number of therapeutic and medical uses of HA is rapidly growing (42).

The interaction of extracellular HA with cells in vivo should be affected by its degree of hydration and the density of the hydrogen bond meshwork it forms. A highly dense, hydrogen bond network would hinder HA interactions with the polar groups on the cell surface. The degree of hydrogen bonding HA undergoes in vivo should also affect the nature of the classical proteoglycan aggregates it is known to form (Fig. 2) (12-15).

Proton-NMR spectroscopy data indicate that not all hexosamine in HA is N-acetylated (Longas et al., unpublished work). Therefore, the universally accepted, alternating sequence of equimolar amounts of D-GlcA and D-GlcNAc, shown in Fig. 1 (1-5), may incorporate some D-GlcN. Other regions may have sequences of D-GlcNAc only followed by at least one D-GlcA (Fig. 4); basic (Fig. 5), acidic (Fig. 6) and D-GlcNAc segments alternating with D-GlcN (Fig. 7) are also possible.

In HA covalently bonded to protein (SHAP-HA), the specific sugar-sequences under consideration may or may not occur in vivo, since the covalent binding and the protein should give different senses to the secondary and tertiary structures of the GAG. Deviations from the alternate sugar-sequence (1) are possible in HA, since dermatan sulfate, which is related to HA, has been reported to display sequential D-GlcA (53).

GkA GcNAc GlcN GcNAc

Figure 7 Hypothetic, hyaluronan tetrasaccharide where the D-GlcNAc segments are interrupted by one D-GlcN.

GkA GcNAc GlcN GcNAc

Figure 7 Hypothetic, hyaluronan tetrasaccharide where the D-GlcNAc segments are interrupted by one D-GlcN.

3. Relevance in Aging

The highly hydrophilic HA segments (Figs. 5 and 6) should render HA more hydrophilic than the alternating sequence of D-GlcA and D-GlcNAc (Fig. 1). This should be important in aging, because human skin, for example, loses 77% (w/w) of its HA content at 75 years (Table 1) (54). Besides, HA becomes N-deacetylated at this age (Fig. 8) (54,55). Perlish and his collaborators (56) found a significant loss of GAGs in the salt-soluble extracts of human dermis, but the quantitation of HA in human skin extracts carried out by other workers showed no significant age-related changes (57). In the dermis of ISh rats, HA does not appear to change with age either (58). Fasted rats lose the GAGs from their skin, suggesting that fasting fatigues the system and makes it function as an aged one (59). The discrepancy in the data may originate from the different species analyzed and from the purity of the GAGs used. The work of Longas et al. (54,55) was performed with highly purified molecules.

The reason (or reasons) for the loss of HA with aging has not been elucidated. Some studies suggest that it is due to depolymerization caused by natural free radicals produced during metabolism (62). The finding that free radical scavengers inhibit HA fragmentation in vitro suggests that its depolymerization in vivo follows a free radical pathway (63). HA has also been described as a free radical scavenger and antioxidant (64,65). Its cleavage in vitro upon exposure to irradiation (66 -68) also supports the free radical pathway for its depolymerization.

In vivo, the UV irradiation of hairless mice resulted in an increase of their skin GAGs including HA (69). Also, the UVA irradiation of albino rats yielded abnormally elevated GAG composition in their skin (70). Because the latter effects were reversed by adding vitamin E, a free radical scavenger, to the rat's diet, the data suggest a free radical involvement (70). Overall, UV-light irradiation stimulates new GAG biosynthesis, while destroying the old ones.

The age-mediated depolymerization of HA has been demonstrated in rat skin (71), while other work has indicated no change in HA size as a function of age (57). Regardless of the discrepancies, there is enough evidence in support of the hypothesis that HA functions in vivo as a free radical scavenger that protects

Table 1 Effect of Age on the Concentration of Human Skin Hyaluronan

Age group (years)

Hyaluronana (%)

SDb (%)

Decrease

19 ± 2.5

0.030

0.005

35 ± 3.5

0.030

0.005

47 ± 1.7

0.030

0.006

60 ± 0.8

0.015

0.003

50

75 ± 5.0

0.007

0.001

77

aMean percent (w/w) based on whole, surgically defatted, wet mastectomy skin from four different people of every age group.

bSD, standard deviation of the mean (54).

aMean percent (w/w) based on whole, surgically defatted, wet mastectomy skin from four different people of every age group.

bSD, standard deviation of the mean (54).

Figure 8 Effect of age on the composition of reducing 2-acetamido-2-deoxyglucose generated from hyaluronan upon digestion by Streptomyces hyaluronidase. HA (100 mg) and 2 turbidity units of enzyme were used under the conditions described previously (54). D-GlcNAc was quantified by utilizing the Morgan-Elson method (60) as modified by Rissing et al. (61).

the skin from endogenous and exogenous, free radicals. Hydrolytic enzymes, whose catalytic activity is known to increase with aging, may cleave HA into small fragments that are then removed from the tissues (72).

4. In Vitro Aging and Hyaluronan

Fibroblast cells in culture have been studied with regard to the fate of HA concentration during cell aging (cell passage) with and without UVA irradiation. The results show that the biosynthesis of HA increases with the number of cell passages (73) in an UVA dose-dependent way (74), but the newly synthesized HA is rapidly degraded (73). These findings suggest that the biosynthesis of the respective hydrolytic enzymes also increases with cell passage (73,74). In the absence of UV irradiation, hyaluronidase stimulates cell proliferation during in vitro aging, and the biosynthesis of HA decreases with the number of cell passages (75,76).

Although fibroblast cultures from human skin of 75-year-old subjects synthesize HA with D-GlcNAc as its hexosamine component (Longas MO, unpublished observations), an enzyme that appears to be induced with aging and becomes highly active in the seventh decade of life is present in the skin of these individuals and cleaves the acetyl (-COCH3) group from N at position 2 of the hexosamine (77). These findings are relevant in aging, because N-deacetylated HA is more hydrophilic than the one found in young skin. The formation of wrinkles is believed to originate, at least partially, from the loss of GAG in the extracellular space and the water they retain (27,54). Highly hydrophilic HA may be needed in aged skin to retain water, if no other molecules are made to replace

Figure 8 Effect of age on the composition of reducing 2-acetamido-2-deoxyglucose generated from hyaluronan upon digestion by Streptomyces hyaluronidase. HA (100 mg) and 2 turbidity units of enzyme were used under the conditions described previously (54). D-GlcNAc was quantified by utilizing the Morgan-Elson method (60) as modified by Rissing et al. (61).

it, because about half the water content of the skin is apparently bonded to this GAG (54,55,78,79). The age-related loss of water has also been demonstrated in the skin of Sprague-Dawley rats (80).

5. Hyaluronan, Body Fluids and Aging

Data available on the effect of age on the HA concentration in body fluids indicate that it increases in human serum (81). Based on its concentration in human urine, HA has been utilized as a marker for aging (82).

6. Hyaluronan in Aging Disorders

Conclusive data on HA alterations in disorders that resemble premature aging like progeria and Down's syndrome are scarce. In progeria patients, urinary HA is abnormally elevated (83,84). In this disorder, HA has been postulated as the culprit for the lack of vasculogenesis, characteristic of these patients (84). In the serum of patients with Down's syndrome, HA is only slightly higher than in the normal serum (85). It would be of utmost importance to elucidate the exact chain sequence of HA from these patients. Chances are that the HA of senescent human skin, which lacks its N-acetyl moieties (54,55,76), plays a role in these pathologic states (83,84).

B. Intracellular Hyaluronan

In the intracellular space, HA finds nucleic acids, nuclear proteins and cellular organelles, among others, with which to associate. As in the extracellular space, the nature of the HA sugar-sequence(s), the degree of hydrogen bonding, and its secondary and tertiary structures should affect its interactions with other intracellular constituents and thus the biologic functions of the cells. High molecular weight HA appears to form highly dense, hydrogen bond networks in vitro (3,4) that may be needed by some cells, while the smaller fragments identified intracellularly may be needed by others (20-26). Cell functions that originate intracellularly like mitosis appear to be affected by the interactions of extracellular HA with the cell surface receptors: CD44, TSG-6, RHAMM and HABP1 (33-35). Obviously, intracellular and extracellular functions mediated by HA are interconnected.

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