T. E. Hardingham

The specific interaction of hyaluronic acid with cartilage proteoglycans

Abstract The addition of small amounts of hyaluronic acid (0.01–1.0%) to disaggregated cartilage proteoglyans produced a large increase in hydrodynamic size on gel chromatography. Re-chromatography of the excluded and retarded fractions showed that there was no equilibrium between them. The increase in hydrodynamic size, which was unaffected by the presence of EDTA, was maximal when the proportion of hyaluronic acid was rather less than 1% and decreased with higher proportions up to 50%. There was no such interaction when proteoglycans were mixed with other polyanions in comparable proportions. The interaction was accompanied by a large increase in viscosity which was eliminated in concentrated solutions of guanidine·HCl and at low pH. From the optimal proportions of proteoglycan and hyaluronic acid and estimates of their molecular weights, it was calculated that 10–30 proteoglycans were associated with each hyaluronic acid chain.

Proteoglycans of cartilage: An assessment of their structure

Abstract Purified proteoglycans from pig laryngeal cartilage were fractionated by equilibrium density gradient centrifugation in the presence of 4 M guanidinium chloride. A continuous distribution of both protein and uronic acid was observed in the gradient extending from a fraction containing most of the uronic acid to one containing one third of the protein but little uronic acid. Gel chromatography on Sepharose 2B showed all fractions to be polydisperse in size and heterogeneous in chemical composition. The chondroitin sulphate chains, however, appeared to be of the same average size in all fractions. The results imply that there are several core proteins differing in length and also in the type distribution and number of carbohydrate chains attached.

The influence of link protein stabilization on the viscometric properties of proteoglycan aggregate solutions

The dynamic, steady-shear and transient shear flow properties of precisely prepared link-stable (s0 136, 66% aggregate) and link-free (s0 93, 59% aggregate) proteoglycan aggregate solutions at concentrations ranging from 10 to 50 mg/ml were determined using a cone-on-plate viscometer in a mechanical spectrometer. All proteoglycan solutions tested possessed: (1) linear viscoelastic properties - as measured by the dynamic complex modulus under small amplitude steady oscillatory conditions (1 less than or equal to omega less than or equal to 100 rad/s) - and (2) nonlinear shear-rate dependent apparent viscosities and primary normal stress difference under steady shearing conditions (0.25 less than or equal to gamma less than or equal to 250 s-1). Our transient flow data show that all proteoglycan aggregate solutions exhibited transient stress overshoot effects in shear stress and normal stress. From these steady and transient flow data, we conclude that link protein stabilized aggregates have significant effects on their dynamic and steady-shear properties as well as transient flow properties. The transient stress overshoot data provide a measure of the energy per unit volume of fluid required to overcome the proteoglycan networks in solution from a resting state. Thus we found that link-stable aggregates form much stronger networks than link-free aggregates. This is corroborated by the fact that link-stable aggregates form more elastic (lower than delta) and stiffer (higher [G*]) networks than link-free aggregates. The complete spectrum of viscometric flow data is entirely compatible with the proposed role of link protein in adding structural stability to the proteoglycan-hyaluronate bond. In cartilage, the enhanced strength of the networks formed by link-stable aggregates may play an important role in determining the material properties of the tissue and thereby contribute to the functional capacity of cartilage in diarthrodial joints.

The effect of temperature on the biosynthesis of chondroitin 4-sulphate in cartilage slices in vitro.

The incorporation of 35S-sulphate into slices of cartilage in vitro has been used extensively in the study of the metabolism of acid glycosaminoglycans [l-4]. Measurement of radioactivity in the isolated glycosaminoglycan chains after fractionation with CPC on cellulose [3,4] or ion exchange chromatography on Ecteola cellulose [l] gives evidence for a marked incorporation into the longest chains. The apparent existence of two populations of chains of different size with different rates of metabolism was further investigated in this study.

Some Aspects of the Organization and Interaction of Connective Tissue Proteoglycans

Arterial mesenchyme, because of its micro-architecture and the variety of macromolecules it contains, is more complex than other types of connective tissue. In addition to collagen and elastin, it contains several glycosaminoglycans, including hyaluronic acid, dermatan sulphate, chondroitin-4-sulphate, chondroitin-6-sulphate and heparan sulphate, as well as glycoproteins that have yet to be fully characterized. These constituents are not distributed evenly through the arterial wall, but are localized in particular regions as shown by histological methods (reviewed by Muir, 1965). Presumably, therefore, each has a specific function related to the biomechanical and biochemical properties of the tissue.

Chemical and Molecular Heterogeneity of Cartilage Proteoglycans

The extracellular phase of cartilage, as in all connective tissues, is composed of collagen fibers and a polysaccharide-rich ground substance. The polysaccharide constituents have been characterized as proteoglycans containing chains of chondroitin-4-sulfate, chondroitin-6-sulfate and keratan sulfate covalently linked to a central protein core (see Muir, 1969). Early analyses of proteoglycan preparations from various cartilages showed that the relative proportions of these three constituents and protein were different in different sites in the same animal (Rosenberg et al., 1965; Gower and Pedrini, 1969; Tsiganos and Muir, 1970). Moreover, the composition of proteoglycan from any one site also varied with age (Goh and Lowther, 1966; Gower and Pedrini, 1969; Kroz and Buddecke, 1967; Brandt and Muir, 1969b; Rosenberg et al., 1969). These findings outlined large variations in composition and suggested that cartilage proteoglycan was not a single molecular entity, but a varying population of several molecular types. Although techniques have been perfected for the complete fractionation of the constituent glycosaminoglycan chains after proteolysis, fractionation of the intact proteoglycans has proved much more difficult due to their polyanionic macromolecular nature. However, the advent of both mild methods of preparation and new techniques of fractionation has now made it possible to demonstrate the possible extent of heterogeneity of proteoglycans under conditions where the possibility of any degradative changes has been largely eliminated.

Hyaluronic Acid in Proteoglycan Aggregation

Reversible aggregation is not uncommon amongst proteins, but amongst highly charged polyanionic proteoglycans it is unexpected. When extracted by comparatively mild methods, proteoglycans consist of aggregated and non-aggregated molecules (SAJDERA & HASCALL, 1969). Aggregates may be dissociated in 4M guanidinium chloride and the proteoglycans separated from other components of the aggregate by equilibrium density gradient centrifugation in caesium chloride when a protein-rich fraction separates at the top of the gradient (HASCALL & SAJDERA, 1969). This fraction was named ‘glycoprotein link’ because when it was recombined with the proteoglycans aggregates were re-formed as shown by sedimentation.