Molecular mechanisms and genetics of hyaluronan biosynthesis

Hyaluronan is an extremely important polysaccharide from both the biological and commercial points of view. This review summarizes the present state of the art concerning the polymer and our understanding of the molecular mechanisms of its synthesis with emphasis on the implications of this understanding for polysaccharide engineering of hyaluronan.     

The hyaluronan synthase catalyzes the synthesis and membrane translocation of hyaluronan

Hyaluronan (HA), an extracellular linear polysaccharide of alternating N-acetyl-glucosamine and glucuronic acid residues, is ubiquitously expressed in vertebrates, where it affects a broad spectrum of physiological processes, including cell adhesion, migration and differentiation. The HA polymer is synthesized on the cytosolic side of the cell membrane by the membrane-embedded hyaluronan synthase (HAS). However, the process by which the extremely hydrophilic HA polymer is translocated across the membrane is unknown to date. The bacterial HAS from Streptococcus equisimilis (Se) shares a similar transmembrane topology and significant sequence identity with human HASs and likely synthesizes HA by the same mechanism. We demonstrate that the Se-HAS is both necessary and sufficient to translocate HA in a reaction that is tightly coupled to HA elongation. The purified Se-HAS is reconstituted into proteoliposomes (PLs) where it synthesizes and translocates HA. In vitro synthesized, high-molecular-weight HA remains tightly associated with the intact PLs in sedimentation experiments. Most importantly, the newly formed HA is protected from enzymatic degradation by hyaluronidase unless the PLs are solubilized with detergent, thereby demonstrating that HA is translocated into the lumen of the vesicle. In addition, we show that HA synthesis and translocation are spatially coupled events, which allow HA synthesis even in the presence of a large excess of HA-degrading enzyme. The coupled synthesis and membrane translocation of a biopolymer represents a novel membrane translocation mechanism and is likely applicable to the synthesis of some of the most abundant biopolymers, including chitin and cellulose.     

Inhibition of Hyaluronan Synthesis Protects against Central Nervous System (CNS) Autoimmunity and Increases CXCL12 Expression in the Inflamed CNS

Hyaluronan (HA) may have proinflammatory roles in the context of CNS autoimmunity. It accumulates in demyelinated multiple sclerosis (MS) lesions, promotes antigen presentation, and enhances T-cell activation and proliferation. HA facilitates lymphocyte binding to vessels and CNS infiltration at the CNS vascular endothelium. Furthermore, HA signals through Toll-like receptors 2 and 4 to stimulate inflammatory gene expression. We assessed the role of HA in experimental autoimmune encephalomyelitis (EAE), an animal model of MS by administration of 4-methylumbelliferone (4MU), a well established inhibitor of HA synthesis. 4MU decreased hyaluronan synthesis in vitro and in vivo. It was protective in active EAE of C57Bl/6 mice, decreased spinal inflammatory infiltrates and spinal infiltration of Th1 cells, and increased differentiation of regulatory T-cells. In adoptive transfer EAE, feeding of 4MU to donor mice significantly decreased the encephalitogenicity of lymph node cells. The transfer of proteolipid protein (PLP)-stimulated lymph node cells to 4MU-fed mice resulted in a delayed EAE onset and delayed spinal T-cell infiltration. Expression of CXCL12, an anti-inflammatory chemokine, is reduced in MS patients in CSF cells and in spinal cord tissue during EAE. Hyaluronan suppressed production of CXCL12, whereas 4MU increased spinal CXCL12 in naive animals and during neuroinflammation. Neutralization of CXCR4, the most prominent receptor of CXCL12, by administration of AMD3100 diminished the protective impact of 4MU in adoptive transfer EAE. In conclusion, hyaluronan exacerbates CNS autoimmunity, enhances encephalitogenic T-cell responses, and suppresses the protective chemokine CXCL12 in CNS tissue. Inhibition of hyaluronan synthesis with 4MU protects against an animal model of MS and may represent an important therapeutic option in MS and other neuroinflammatory diseases.     

Hyaluronan fragments: an information-rich system

Hyaluronan is a straight chain, glycosaminoglycan polymer of the extracellular matrix composed of repeating units of the disaccharide [-D-glucuronic acid-beta1,3-N-acetyl-D-glucosamine-beta1,4-]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 x 10(4) 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.     

Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis

Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4 pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.     

The functional molecular mass of the Pasteurella hyaluronan synthase is a monomer

Hyaluronan (HA), a linear polysaccharide composed of beta1,3-GlcNAc-beta1,4-GlcUA repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. All known HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The bacterial hyaluronan synthase, PmHAS from Gram-negative Pasteurella multocida, is a 972-residue membrane-associated protein. Previously, the Gram-positive Streptococcus pyogenes enzyme, SpHAS (419 residues), and the vertebrate enzyme, XlHAS1 (588 residues), were found to function as monomers of protein, but the PmHAS is not similar at the protein sequence level and has quite different enzymological properties. We have utilized radiation inactivation to measure the target size of recombinant full-length and truncated PmHAS. The target size of HAS activity was confirmed using internal enzyme standards of known molecular weight. We found that the Pasteurella HA synthase protein functions catalytically as a monomer. Functional truncated soluble PmHAS also behaves as a polypeptide monomer as assessed by gel filtration chromatography and light scattering.     

A truncated RHAMM protein for discovering novel therapeutic peptides

The receptor for hyaluronan mediated motility (RHAMM, gene name HMMR) belongs to a group of proteins that bind to hyaluronan (HA), a high-molecular weight anionic polysaccharide that has pro-angiogenic and inflammatory properties when fragmented. We propose to use a chemically synthesized, truncated version of the protein (706–767), 7?kDa RHAMM, as a target receptor in the screening of novel peptide-based therapeutic agents. Chemical synthesis by Fmoc-based solid-phase peptide synthesis, and optimization using pseudoprolines, results in RHAMM protein of higher purity and yield than synthesis by recombinant protein production. 7?kDa RHAMM was evaluated for its secondary structure, ability to bind the native ligand, HA, and its bioactivity. This 62-amino acid polypeptide replicates the HA binding properties of both native and recombinant RHAMM protein. Furthermore, tubulin-derived HA peptide analogues that bind to recombinant RHAMM and were previously reported to compete with HA for interactions with RHAMM, bind with a similar affinity and specificity to the 7?kDa RHAMM. Therefore, in terms of its key binding properties, the 7?kDa RHAMM mini-protein is a suitable replacement for the full-length recombinant protein.     

Biological properties of hyaluronan in aqueous solution are controlled and sequestered by reversible tertiary structures,defined by NMR spectroscopy

Hyaluronan (HA) is a ubiquitous polysaccharide of (predominantly) animal origin that has important medical applications in joint, skin, and eye conditions. Biological activities shown by HA fragments in angiogenesis, inflammation, etc. are absent from highly polymerized HA. We propose that HA physiological properties are controlled by molecular mass dependent transitions between tertiary structures (e.g., beta sheets) and 2-fold helices, - reversible "denaturation", which is central to HA solution behavior. We demonstrate this phenomenon by 13C NMR. Four different acetamido C=O resonances, assigned to secondary, tertiary, and disordered HA structures, monitored "denaturation" by (a) warming, (b) alkalinizing to pH >12.0, (c) hyaluronidase digestion, and (d) methylation of carboxylates. (a) and (b) acted reversibly but (c) and (d) are irreversible. 1H NMR implicated H-bonded acetamido NH in (b). Temperature dependencies of other 13C chemical shifts were small and unspecific. Arrhenius plots indicate that hyaluronan tertiary structures are on the edge of instability under physiological conditions. The results help to explain the appearance of biological activities on "denaturation" or degradation of HA.     

Stimulation of hyaluronan synthesis by interleukin-1beta involves activation of protein kinase C betaII in fibroblasts from patients with Graves' ophthalmopathy

Hyaluronan accumulation in the retroorbital connective tissue is one of the pathological features of Graves' ophthalmopathy. Interleukin-1beta (IL-1beta) is known to stimulate hyaluronan synthesis in orbital fibroblasts. In the present study, the intracellular signal transduction pathways involved in this stimulatory effect were investigated in cultured human retroorbital fibroblasts from patients with Graves' ophthalmopathy. IL-1beta-induced hyaluronan synthesis was significantly inhibited by pretreatment of the cells with two protein kinase C (PKC) inhibitors, chlerythrine chloride and H-7. In addition, treatment with phorbol 12-myristate 13-acetate (PMA), a direct PKC activator, also resulted in increased hyaluronan production. IL-1beta- or PMA-stimulated hyaluronan synthesis was blocked by the protein synthesis inhibitor, cycloheximide. Moreover, the intracellular Ca(2+) concentration of the orbital fibroblasts was also involved in the IL-1beta induced transduction pathway, the effect being completely inhibited by BAPTA, an internal calcium chelator. In addition, A23187, a calcium ionophore, increased hyaluronan synthesis in unstimulated cells. These results suggest that the Ca(2+)-dependent PKC signal transduction pathway plays an important role in the IL-1beta-induced hyaluronan synthesis. Moreover, IL-1beta treatment resulted in increased PKC activity and the rapid translocation of PKC betaII from the cytoplasm to the plasma membrane. These results indicate that cytosolic Ca(2+) and PKC betaII are involved in IL-1beta-induced hyaluronan synthesis in cultured orbital fibroblasts from patients with Graves' ophthalmopathy.     

Hyaluronan oligosaccharides induce matrix metalloproteinase 13 via transcriptional activation of NFkappaB and p38 MAP kinase in articular chondrocytes

Hyaluronan exerts a variety of biological effects on cells including changes in cell migration, proliferation, and matrix metabolism. However, the signaling pathways associated with the action of hyaluronan on cells have not been clearly defined. In some cells, signaling is induced by the loss of cell-hyaluronan interactions. The goal of this study was to use hyaluronan oligosaccharides as a molecular tool to explore the effects of changes in cell-hyaluronan interactions and determine the underlying molecular events that become activated. In this study, hyaluronan oligosaccharides induced the loss of extracellular matrix proteoglycan and collagen from cultured slices of normal adult human articular cartilage. This loss was coincident with an increased expression of matrix metalloproteinase (MMP)-13. MMP-13 expression was also induced in articular chondrocytes by hyaluronan (HA) hexasaccharides but not by HA tetrasaccharides nor high molecular weight hyaluronan. MMP-13 promoter-reporter constructs in CD44-null COS-7 cells revealed that both CD44-dependent and CD44-independent events mediate the induction of MMP-13 by hyaluronan oligosaccharides. Electromobility gel shift assays demonstrated the activation of chondrocyte NFkappaB by hyaluronan oligosaccharides. NFkappaB activation was also documented in C-28/I2 immortalized human chondrocytes by luciferase promoter assays and phosphorylation of IKK-alpha/beta. The link between activation of NFkappaB and MMP-13 induction by HA oligosaccharides was further confirmed through the use of the NFkappaB inhibitor helenalin. Inhibition of MAP kinases also demonstrated the involvement of p38 MAP kinase in the hyaluronan oligosaccharide induction of MMP-13. Our findings suggest that hyaluronan-CD44 interactions affect matrix metabolism via activation of NFkappaB and p38 MAP kinase.     

The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence recovery after photobleaching.

Hyaluronan (HA) is a highly hydrated polyanion, which is a network-forming and space-filling component in the extracellular matrix of animal tissues. Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to investigate intramolecular hydrogen bonding and electrostatic interactions in hyaluronan solutions. Self and tracer lateral diffusion coefficients within hyaluronan solutions were measured over a wide range of concentrations (c), with varying electrolyte and at neutral and alkaline pH. The free diffusion coefficient of fluoresceinamine-labeled HA of 500 kDa in PBS was 7.9 x 10(-8) cm(2) s(-1) and of 830 kDa HA was 5.6 x 10(-8) cm(2) s(-1). Reductions in self- and tracer-diffusion with c followed a stretched exponential model. Electrolyte-induced polyanion coil contraction and destiffening resulted in a 2.8-fold increase in self-diffusion between 0 and 100 mM NaCl. Disruption of hydrogen bonds by strong alkali (0.5 M NaOH) resulted in further larger increases in self- and tracer-diffusion coefficients, consistent with a more dynamic and permeable network. Concentrated hyaluronan solution properties were attributed to hydrodynamic and entanglement interactions between domains. There was no evidence of chain-chain associations. At physiological electrolyte concentration and pH, the greatest contribution to the intrinsic stiffness of hyaluronan appeared to be due to hydrogen bonds between adjacent saccharides.     

Hyaluronan fragments induce endothelial cell differentiation in a CD44- and CXCL1/GRO1-dependent manner

Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12- or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.     

The dynamic metabolism of hyaluronan regulates the cytosolic concentration of UDP-GlcNAc

Hyaluronan, a macromolecular glycosaminoglycan, is normally synthesized by hyaluronan synthases at the plasma membrane using cytosolic UDP-GlcUA and UDP-GlcNAc substrates and extruding the elongating chain into the extracellular space. The cellular metabolism (synthesis and catabolism) of hyaluronan is dynamic. UDP-GlcNAc is also the substrate for O-GlcNAc transferase, which is central to the control of many cytosolic pathways. This Perspective outlines recent data for regulation of hyaluronan synthesis and catabolism that support a model that hyaluronan metabolism can be a rheostat for controlling an acceptable normal range of cytosolic UDP-GlcNAc concentrations in order to maintain normal cell functions.     

Hyaluronan in part mediates IL-1beta-induced inflammation in mouse chondrocytes by up-regulating CD44 receptors

Interleukin-1beta (IL-1beta) elicits the expression of inflammatory mediators through a mechanism involving the CD44 receptor. Hyaluronan (HA) depolymerization also contributes to CD44 activation. This study investigated the potential of HA fragments, obtained by hyaluronidase (HYAL) treatment, as mediators of CD44 activation on IL-1beta-induced inflammation in mouse chondrocytes.mRNA and related protein levels were measured for CD44, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), matrix metalloproteinase-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in chondrocytes, treated or untreated with IL-1beta, either with or without the addition of HYAL. The level of NF-kB activation was also assayed.CD44 mRNA expression was higher than controls in chondrocytes treated with IL-1beta. IL-1beta also induced NF-kB up-regulation and increased TNF-alpha, IL-6, MMP-13 and iNOS expression. Different effects resulted from HYAL treatment. Treatment of chondrocytes exposed to IL-1beta with HYAL synergistically increased the same parameters up-regulated by IL-1beta, while the same parameters were increased by HYAL in chondrocytes not exposed to IL-1beta but to a lesser extent. Specific CD44 blocking antibody and hyaluronan binding protein (HABP), which inhibit HA activity, were used to confirm CD44 to be the target of IL-1beta action through HA mediation. HA levels and molecular size further confirm the role of degraded HA.These findings suggest that IL-1beta exerts inflammatory activity via CD44 by the mediation of HA fragments derived from HA depolymerization.     

Hyaluronan.

Hyaluronan (hyaluronic acid) is a high-molecular-mass polysaccharide found in the extracellular matrix, especially of soft connective tissues. It is synthesized in the plasma membrane of fibroblasts and other cells by addition of sugars to the reducing end of the polymer, whereas the nonreducing end protrudes into the pericellular space. The polysaccharide is catabolized locally or carried by lymph to lymph nodes or the general circulation, from where it is cleared by the endothelial cells of the liver sinusoids. The overall turnover rate is surprisingly rapid for a connective tissue matrix component (t1/2 0.5 to a few days). Hyaluronan has been assigned various physiological functions in the intercellular matrix, e.g., in water and plasma protein homeostasis. Hyaluronan production increases in proliferating cells and the polymer may play a role in mitosis. Extensive hyaluronidase-sensitive coats have been identified around mesenchymal cells. They are either anchored firmly in the plasma membrane or bound via hyaluronan-specific binding proteins (receptors). Such receptors have now been identified on many different cells, e.g., the lymphocyte homing receptor CD 44. Interaction between a hyaluronan receptor and extracellular polysaccharide has been connected with locomotion and cell migration. Hyaluronan seems to play an important role during development and differentiation and has other cell regulatory activities. Hyaluronan has also been recognized in clinical medicine. A concentrated solution of hyaluronan (10 mg/ml) has, through its tissue protective and rheological properties, become a device in ophthalmic surgery. Analysis of serum hyaluronan is promising in the diagnosis of liver disease and various inflammatory conditions, e.g., rheumatoid arthritis. Interstitial edema caused by accumulation of hyaluronan may cause dysfunction in various organs.     

Modification of Hyaluronan by Heavy Chains of Inter-α-Inhibitor in Idiopathic Pulmonary Arterial Hypertension

We previously reported an altered hyaluronan (HA) metabolism in idiopathic pulmonary arterial hypertension (IPAH) lung tissue and cultured smooth muscle cells. Hyaluronan was present in the smooth muscle cell layer surrounding the pulmonary vasculature and in plexigenic lesions. Additionally, cultured pulmonary artery smooth muscle cells produced spontaneous HA “cable” structures, without additional stimuli, that were leukocyte-adhesive. We now present evidence that the HA that accumulates in IPAH plexigenic lesions is a pathological form of HA in which heavy chains (HCs) from the serum-derived proteoglycan inter-α-inhibitor are covalently attached to the HA backbone to form a pathological HC-HA complex. CD45-positive leukocytes were identified within these HC-HA matrices. Elevated mRNA levels of the enzyme that transfers HCs to HA, known as tumor necrosis factor-stimulated gene 6, were detected in IPAH lung tissue.     

The effect of mechanical strain on hyaluronan metabolism in embryonic fibrocartilage cells

The development of the synovial joint cavity between the cartilage anlagen of the long bones is thought to be mediated by differential matrix synthesis at the developing articular surfaces. In addition, many studies have shown that removal of movement-induced mechanical stimuli from developing diarthrodial joints prevents cavity formation or produces a secondary fusion of previously cavitated joints. Herein, we describe an inductive influence of mechanical strain on hyaluronan metabolism and the expression of hyaluronan-binding proteins in cultured cells isolated from the articular surface of the distal tibial condyles of 18-day chick embryos. The effect of 10 min of mechanical strain on hyaluronan release into culture media, intracellular uridine diphospho-glucose dehydrogenase activity (an enzyme required for hyaluronan saccharide precursor production), cell surface hyaluronan-binding protein expression and HA synthase mRNA expression were analysed up to 24 h later. Six hours after the application of strain, there was a significant increase in the accumulation of hyaluronan released into tissue culture media by strained fibrocartilage cells compared with controls, an effect still detectable after 24 h. Strained cells also showed increased activity for uridine diphospho-glucose dehydrogenase and expressed higher levels of the hyaluronan-binding protein CD44 at 24 h. In addition, at 24 h mRNA for HA synthase 2 was expressed in all samples whereas mRNA for HA synthase 3 was only expressed in strained cells. These results further highlight the role for movement-induced stimuli in differential extracellular matrix metabolism during joint development and also show that strain may facilitate differential HA synthase gene expression.