The extracellular matrix is composed of collagen fibers, fibronectins, glycoproteins, proteoglycans, cytokines and growth factors, and more. Among the vast complement of human glycoproteins and proteoglycans is a unique spicy of molecules called glycosaminoglycans (GAGs). Glycosaminoglycans are defined as a type of polysaccharide composed of repeating units of disaccharides. The GAG family includes chondroitin sulfate, heparan sulfate, heparin, dermatan sulfate, keratan sulfate, and hyaluronan.
Hyaluronan (HA) was discovered by Meyer in 1934 as a novel glycosaminoglycan from the vitreous humor of the bovine eye. The structure determination as repeating disaccharides of D-glucuronic acid-β-1,3-N-acetyl-D-glucosamine-β-1,4- was described by Weissman and Meyer in 1954. HA is a ubiquitous polysaccharide present in almost all tissues of vertebrates. The molecular mass of this GAG can go from several disaccharides to up to 25000 disaccharides repeats, corresponding to several kilo-Daltons (kDa) to 10 Million- Daltons (MDa). The intact HA in tissues exists in several millions in molecular mass, which is significantly longer in comparison with other GAGs which can only reach up to ~40 kDa. One disaccharide is approximately 1 nm in length, so a chain of 25000 repeating units will have a length of 25 μm, which is at the same scale of a eukaryotic cell. The huge molecular mass of this molecule leads to its unique solution properties. HA is very hydrophilic due to the hydroxyl, acetamido, and carboxylate groups on the side chains. In water, many previous studies such as viscometry, osmometry, sedimentation and light scattering of HA characterization have indicated a semi-rigid random coil conformation of the molecule. When crystalized or solubilized in water-ethanol or water DMSO mixtures, the HA chains form inter- or intra- molecular associations via forming 2-, 3-, 4-fold helices stabilized through hydrogen bonds between the carboxylate groups, N-acetyl groups and hydroxyl groups, as well as inter- or intra-chain hydrophobic hydrocarbon patch interactions. In aqueous solution, the proposal of inter or intra chain association or crosslinking has been questioned (the interaction has been shown not significant, as the chainchain hydrogen bonding exists only transiently).
The transient hydrogen bonding among the functional groups on HA chains help explain the semi-rigidity of the polymer. The semi-rigid chain together with the enormously high molecular mass leads to a very large hydrodynamic volume (a 500 kDa chain has a ~70 nm radius under physiological condition). The giant molecular size of HA leads to unique biophysical properties which results in several important physiological functions for HA, such as extracellular matrix stabilization, joint lubrication, shock absorption, water maintenance, and regulation of protein distribution.
All mammalian cells are able to synthesize HA. However, generally some types synthesize HA much more routinely than other cells, such as keratinocytes, chondrocytes, fibroblasts, etc. The HA distribution in the body is correlated with the location of these cell types. Unlike all other GAGs that are synthesized by glycosyltransferases and linked onto glycoproteins/proteoglycans in the Golgi apparatus, HA is synthesized by three integral membrane HA synthases (HAS) in the plasma membrane and is directly extruded out of the cellular membrane after generation. The reason for HA extrusion outside the plasma membrane during synthesis may be that the limited cell space cannot tolerate the transfer of such an enormously large molecule (several millions in M) as well as the high viscosity it causes. Three HASs, HAS1, 2 and 3 are responsible for HA synthesis. The catalytic site is in proposed to locate in the cytoplasmic side . The substrates, which are UDP (uridine diphosphate)-GlcNAc and UDP-GlcA, are copolymerized together in the HAS in alternating β1→3 and β1→4 glycosidic linkages. In HAS, the UDP at the non-reducing chain end is displaced with UDP-sugars, resulting in release of the UDP, glycosidic linkage of the incoming UDP-sugar, and HAS position adjustment to the next alternate UDP-sugar substrate. The cycling of the HAS conformations results in alternating GlcNAc and GlcA glycosyltransferase activity, and therefore, the consistent disaccharide repeating units elongation of HA. A HA template is not needed in HA synthesis, but the synthesis seems to depend on HAS-phospholipid interaction. Under the cell free HA synthesis, adding cardiolipin can increase the activity of solubilized recombinant HAS.
The general HA turnover rate is fast in vertebrates. The HA dynamic metabolism is strictly regulated during biological processes such as homoeostasis, wound healing and embryogenesis to balance synthesis rate in order to maintain constant HA concentration in tissues. For a 70 kg person, the estimated total HA content is 15 g, and 1/3 is renewed daily. The life time of HA varies depending on tissues type; for example, in skin, the time is less than a day, in serum, the metabolism rate is within several minutes, and in cartilage, the normal lifetime is within 2-3 weeks.
There is little evidence showing that an active HA digesting enzyme is present in extracellular matrix. Actually, most HA in the extracellular matrix is drained into the lymphatic system for turnover through endocytosis and cytoplasmic enzymatic digestion catabolism, although HA can also be degraded locally in tissues in a similar way. Lymph nodes are shown to have a large capacity for HA absorbance and degradation. The rest (~10%) of HA that is not extracted by lymph nodes enters into the circulation system and is degraded in liver and spleen. It is generally accepted that HA turnover in mammalian species is mainly accomplished via two mechanisms. One is through enzymatic degradation by hyaluronidase.
Hyaluronidase in mammalians belongs to β-endo- N-acetylglucosaminidase, which are hydrolases that cleave HA into fragments with GlcNAc ends. Most of the hyaluronidases also degrade chondroitin sulfates at a lower rate. The other mechanism is through chemical degradation by reactive oxygen species (ROS) or reactive nitrogen species (RNS). To date, the relative contribution of each mechanism is still largely unknown. As HA is stable at acidic pH and high temperature (up to 100 ⁰C), other physiological metabolism mechanisms such as heating and acidic breakdown in lysosome without hyaluronidase seems limited. However, HA is susceptible by alkaline hydrolysis, ultrasonic degradation and high shear force.