Interaction of cartilage proteoglycans with hyaluronic acid. The role of the hyaluronic acid carboxyl groups.

Hyaluronic acid-derived oligomers of five to fifteen repeat dissaccharides effectively bind to bovine nasal-cartilage proteoglycan and inhibit the interaction between proteoglycans and high-molecular-weight hyaluronic acid. If, however, the hyaluronic acid oligosaccharides are modified by reaction with diazomethane to form the carboxyl methyl esters of the glucuronic acid residues, their inhibitory activity is abolished. The binding capacity can be fully restored by saponification. The amide derivative, which is formed by condensation of the oligosaccharide carboxyl groups with glycine methyl ester, is also ineffective in blocking the proteoglycan-hyaluronic acid interaction. In this case, binding activity is not restored when the amidated oligomers are subjected to saponification to yield the free carboxylate groups on the glycine residues. Thus the displacement of the carboxylate groups on the polysaccharide chain by the interposition of a glycine residue blocks the interaction between the proteoglycans and the hyaluronic acid oligomers. When the oligosaccharide methyl ester is reduced with NaBH4, the resultant glucose-containing oligomers exhibit decreased binding to proteoglycans. Thus it appears that the hyaluronic acid carboxylate anion in a specific spatial orientation is required for hyaluronic acid-proteoglycan interaction.     

Biosynthesis of hyaluronic acid by Streptococcus.

Synthesis of hyaluronic acid was investigated in a cell-free system derived from a strain of Group A streptococci. Preparative procedures were improved so that an enzyme system 70 times more active than that previously reported was obtained. The hyaluronic acid synthesized could be separated into trichloroacetic acid-soluble and -insoluble fractions. On the basis of pulse-chase experiments, it was shown that the trichloroacetic acid-insoluble fraction is a precursor of the soluble fraction. The release of the trichloroacetic acid-insoluble hyaluronic acid is specifically blocked with p-chloromercuribenzoate, without inhibition of chain elongation. The addition of butanol to trichloroacetic acid resulted in solubilization of all of the hyaluronic acid. No detectable difference in molecular size was observed between the two hyaluronic acid fractions, both of which were estimated to be more than one million daltons in size. Testicular hyaluronidase digestion of either one of the two types of hyaluronic acid yielded no high molecular weight fragments, indicating that hyaluronic acid is not bound covalently to protein. However, following incubation of enzyme assay mixtures with UDP-[14C]GlcUA, even in the absence of UDP-GlcNAc, radioactive high molecular weight hyaluronic acid was obtained which suggests that the enzyme system elongates rather than initiates hyaluronic acid chains. Tunicamycin did not inhibit hyaluronic acid synthesis, indicating lack of participation of an intermediate of pyrophosphorylpolyisoprenol type. The results obtained are consistent with the hypothesis that chain elongation of hyaluronic acid proceeds by alternate addition of monosaccharides from UDP-sugars by a membrane-bound synthesizing system followed by release of completed hyaluronic acid chains.     

Fractionation of a hyaluronic acid preparation in a density gradient. Some properties of the hyaluronic acid

1. Hyaluronic acid was isolated from ox synovial fluid by sedimentation equilibrium in a caesium chloride density gradient (Silpananta, Dunstone & Ogston, 1967). The product was almost free from chondroitin sulphate and from protein. 2. Its composition did not differ significantly from that of the carbohydrate part of the protein-containing material isolated by filtration. Its physicochemical properties and molecular configuration were similar, except for its viscosity, which showed markedly reduced concentration-dependence and shear-dependence. This suggests that the associated protein tends to form links between molecules of hyaluronic acid. 3. The accurate measurement of viscosity at very low velocity gradient, by use of the damping of oscillations in a Couette viscometer, is described. 4. A method is described for measuring, approximately, the thermodynamic non-ideality of a solute from the shape of its schlieren curve at sedimentation equilibrium in a density gradient. 5. A value for the partial specific volume of hyaluronic acid in dilute salt solution was calculated from its isopycnic density in a caesium chloride gradient.     

Chemical modification of hyaluronic acid by carbodiimides.

Hyaluronic acid (HA) is a linear polysaccharide with repeating disaccharide units of glucuronic acid and N-acetylglucosamine and is found in the extracellular matrix of connective tissues. Reaction of high molecular weight sodium hyaluronate (NaHA, MW approximately 2 x 10(6] with EDC at pH 4.75, either in the presence or absence of a primary diamine, gave the N-acylurea and O-acylisourea as NaHA-carbodiimide adducts. None of the expected intermolecular coupling with the amine component was observed. On the basis of this new observation, this method for chemical modification of HA was used in conjunction with new synthetic carbodiimides to prepare HA derivatives bearing lipophilic, aromatic, cross-linked, and tethered functional groups. The degree of conversion to NaHA-acylurea products appears to depend upon both the characteristics of various carbodiimides and the conformational structure of NaHA.     

Selective aggregation of proteoglycans with hyaluronic acid.

Conditions were established to separate proteoglycan aggregate (AH1) from a bovine nasal septum extract by associative rate zonal sedimentation on a NaCl gradient. The AH1 has a higher protein content than the mixed aggregate-monomer (A1) isolated by conventional associative CsCl density gradient centrifugation from a portion of the same extract. The same associative rate zonal conditions separated the A1 fraction into aggregated AH1 containing hyaluronic acid and nonaggregated proteoglycan monomer (N1) essentially free of hyaluronic acid. The AH1 fraction is richer in protein and keratin sulfate than is N1. Dissociative rate zonal sedimentation of A1 under conditions which totally sedimented most of the disaggregated monomer (AH1-D1) and the nonaggregated monomer N1 separated a less sedimentable protein and keratan sulfate-rich proteoglycan monomer (AH1-D2). Chromatography on Sepharose 2B under dissociative conditions demonstrated that the nonaggregated N1 monomer is intermediate in size between the disaggregated monomers AH1-D1 and AH1-D2. N1 has a buoyant density higher than AH1 and is practically equivalent to AH1-D1. All are dense fractions so that separation by CsCl density gradient equilibration is not feasible.     

Rheology of hyaluronic acid

The dynamic viscoelastic properties of hyaluronic acid solutions have been measured over the frequency range 0.02–1.67 cps. The effects of varying temperature, hyaluronic acid concentration, pH, and ionic strength on the dynamic shear moduli were studied. The solutions exhibited a sharp transition from viscous to elastic behavior as the strain frequency increased. No entanglement coupling of the hyaluronic acid molecules was evident over the concentration range 2.0–4.0 mg./ml. Solutions at pH 2.5 showed a pronounced elastic behavior relative to both higher and lower pH's. This effect was attributed to a stiffening of the hyaluronic acid molecule at this pH.     

Microbial hyaluronic acid production

Hyaluronic acid (HA) is a commercially valuable medical biopolymer increasingly produced through microbial fermentation. Viscosity limits product yield and the focus of research and development has been on improving the key quality parameters, purity and molecular weight. Traditional strain and process optimisation has yielded significant improvements, but appears to have reached a limit. Metabolic engineering is providing new opportunities and HA produced in a heterologous host is about to enter the market. In order to realise the full potential of metabolic engineering, however, greater understanding of the mechanisms underlying chain termination is required.     

Studies on the structure of hyaluronic acid. Characterization of the product formed when hyaluronic acid is treated with ascorbic acid

Physical and chemical methods were used to characterize hyaluronic acid before (fraction HAIIBI) and after (fraction HA-AA) treatment with ascorbic acid. Fraction HA-AA was recovered with an almost quantitative yield and was shown to be chemically identical with fraction HAIIBI by all the methods used. These two materials, however, differed markedly in their molecular sizes and degree of polydispersity. By using sedimentation, diffusion and sedimentation-equilibrium analyses, weight-average molecular weights of about 1.2x10(6) and 6.5x10(4) respectively were obtained for fractions HAIIBI and HA-AA. It is concluded from these results that hyaluronic acid has a molecular weight of about 65000 and that the polysaccharide chain of this molecule is not depolymerized by ascorbic acid. It is further proposed that hyaluronic acid molecules in the matrix of connective tissues are present either in an aggregated form or as subunits of heterogeneous macromolecules, and that it is the linkages responsible for the organization of these structures which are broken by ascorbic acid.     

Cell-free synthesis of hyaluronic acid in Marfan syndrome.

The cell-free synthesis of hyaluronic acid has been demonstrated in extracts of cultured human fibroblasts. Preparations from fibroblasts of normal individuals as well as those from patients with Marfan syndrome incorporate glucuronic acid and N-acetylglucosamine from their UDP derivatives into hyaluronic acid. Extracts from Marfan fibroblasts demonstrate 3 to 10 times more total and specific hyaluronic acid synthetase activity than do preparations from normal fibroblasts. All synthetic activity was found in particulate fractions with the bulk of activity localized in material sedimenting as large membrane fragments. Marfan and normal preparations exhibited similar properties with respect to substrate, cofactor, pH requirements, and heat stability. Neither the Marfan nor normal enzyme systems could be stimulated by exogenous acceptors, nor did either preparation contain a soluble factor which stimulated or inhibited the enzymic activity of the other. The genetic defect in Marfan syndrome appears to result in increased activity of hyaluronic acid synthetase without demonstrable changes in properties of the particulate enzymes involved.     

Study of hyaluronic acid flexibility by electric birefringence.

Transient-electric-birefringence experiments were conducted on four samples of hyaluronic acid over the molecular-mass (M) range 5 X 10(4)-4 X 10(6) in dilute aqueous solution. The geometrical, optical and electrical characteristics were monitored via the rotary relaxation times, optical-polarizability antisotropies and electrical polarizabilities respectively. Each indicates the molecular conformation to be consistent with some degree of rigidity at low M but that this does not persist at high M. The molecules do not become true random coils, but are best characterized in terms of a persistence length of 20 nm or 20 disaccharide units.