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Introduction The evolution of multicellular organisms made necessary the development of an extracellular matrix to protect cells and to bind them together in a spatial arrangement required for specialized anatomical and physiological functions. Thus the extent of the matrix and how it is organized in different parts of the body depend on the specialized functions of cells. The matrix in its turn influences the sorting and organization of cells during embryonic development of animals (see Hay, 198 1). Collagen is the principal structural protein of animals, a primitive form of which was evolved by Porifera (Mathews, 1967; Adams, 1978). It has proved so suitable that it is highly conserved and remains the prime structural protein of higher animals, providing the fibrous framework for the body. The extracellular matrix also contains polyanionic macromolecules which fill the interfibrillar space and complement the role of collagen by retaining water in the tissue and controlling its flow (see below). In Nature, polyanions predominate in the pericellular environment, and it has been suggested (Scott, 1975, 1979) that this predominance arose because the Donnan effects of polyanions would exclude from their domains the extremely reactive hydrated electrons produced by ionizing radiations on water. Pericellular polyanions would thus have protected primitive organisms from damage by hydrated electrons and their reactive products generated by the intense radiation that reached the biosphere when there was a thinner protective ozone layer because there was less oxygen in the atmosphere. The acidic polysaccharides of the extracellular matrix of vertebrates are much less diverse than those of invertebrates (Mathews, 1967). In mammals there are a limited number, based on differences in repeating disaccharide units of which they are composed. They are collectively known as glycosaminoglycans and are long unbranched chains with many carboxy and/or sulphate groups made up of disaccharide units of hexosamine and uronic acid or hexosamine and galactose (for reviews, see Muir & Hardingham, 1975: Hardingham, 198 1). However, with the probable exception of hyaluronic acid, all glycosaminoglycans are attached at the reducing end to specific proteins to form large macromolecules known as proteoglycans, a term introduced in 1967 (see Balazs & Gibbs, 1970). Several glycosaminoglycan chains are attached laterally to the protein, from which they extend outwards like a bottle brush owing to electrostatic repulsion of their negatively charged groups. The physical properties of different tissues of the body depend mainly on differences in the proportion of collagen to proteoglycan. which varies widely, and also on the type of proteoglycan and collagen and how these are organized in the matrix. The specific anatomical distribution of different proteoglycans and collagen implies that they have different functions, although these are not yet known precisely. The organizing functions of proteoglycans can be roughly separated into space-filling functions and specific interactions; however, much more is known about the former than the latter. The space-filling role of proteoglycans is very important in cartilage. where the concentration of proteoglycans is higher than in any other tissue. Most progress has therefore been made in elucidating structure and function of proteoglycans of cartilage, which have particular features necessary for this space-filling role, which will be discussed in detail below.

Proteoglycans of cartilage when extracted by mild methods contain both aggregated and non-aggregated molecules (Sajdera & Hascall, 1969). In the presence of 4~-guanidinium chloride aggregates were dissociated and could be separated by equilibrium densitygradient centrifugation into a glycoprotein-like fraction containing little uronic acid and a proteoglycan fraction of lower protein content (Hascall & Sajdera, 1969). On mixing of both fractions in 4~-guanidinium chloride followed by dialysis to low ionic strength, proteoglycan aggregates were re-formed. When proteoglycans from pig laryngeal cartilage were fractionated in a similar way most of the proteoglycan separated from a glycoprotein-like fraction (Tsiganos et al., 1971), but when they were mixed the proteoglycans did not reaggregate (Tsiganos et a[., 1972). In contrast, the fraction from the middle of the density gradient, which contained only 5 % of the total uronic acid and 7.2 % of the protein, when mixed with the proteoglycan produced an increase in hydrodynamic size on gel chromatography, although there was no marked change in sedimentation rate in the ultracentrifuge (R. Pain, personal communication). The effect was thus similar to but not identical with the reaggregation of subunits (Hascall & Sajdera, 1969). The component responsible for the increase in hydrodynamic size of proteoglycans has now been purified and characterized. Proteoglycans were extracted from fresh pig laryngeal cartilage with 4~-guanidinium chloride in 50m~-sodium acetate buffer, pH4.5, and purified by equilibrium densitygradient centrifugation (Tsiganos et al., 1971). The proteoglycans were fractionated in a second CsCl gradient (po = 1.5) in 4~-guanidinium chloride, in an angle rotor (8 x 25ml) in an MSE65 centrifuge, at 95000g,,. for 48h at 20°C. Three fractions, obtained from each tube by freezing and cutting, corresponded to the bottom 4nd, middle lOml and top 4ml of the gradient. The distributions of total uronic acid and protein respectively were: bottom, 92.7 and 61 7;; middle, 5.0 and 7.2%; top, 2.3 and 31.8%. The ability of samples to produce an increase in hydrodynamic size of disaggregated proteoglycans was assessed as follows. Samples were added to proteoglycans in 4 ~ guanidinium chloride, dialysed against 0.5 M-sodium acetate buffer, pH 6.8, and chroniatographed in this buffer on a column (165cm x 1.1 cm) of Sepharose 2B. The uronic acid content (Bitter & Muir, 1962) of the eluate fractions was determined by an automated method (D. Heinegard, personal communication). Since little of the disaggregated proteoglycans was eluted in the void volume before the mixing, the extent of interaction was calculated from the proportion that did so after the mixing. When the bottom and middle fractions were mixed in the same relative proportions in which they occurred in the extract, the hydrodynamic size of about half the proteoglycans increased, whereas mixing of the top and bottom fractions did not produce this effect (Tsiganos et al., 1972). A sample of the middle fraction containing 0.93 mg of uronic acid was dialysed exhaustively against distilled water, conc. HCI was added to give a concentration of 20nm and the sample was applied to a column (1 6.0cm x 1.1 cm) of ECTEOLA-cellulose (Serva, Heidelberg, Germany) previously washed with 4M-HCI followed by distilled water until the eluate was pH5. The sample was washed in with 20ml of ~ O ~ M H C I and then eluted with 20ml each of 0 .5~-NacI , 2 .5~-NaCl and ~M-HCI. Acidic fractions were neutralized as soon as they cmerged from the column, and the last two fractions were dialysed against a large excess of 0.5 M-sodium acetate. Uronic acid contents of a11 fractions were determined, and their interactions with proteoglycan were tested. Fig. I shows the distribution of uronic acid of the fractions and the ability of each to interact with proteoglycans. Most of thc uronic acid (83 %)was eluted by 2 .5~-NaCl and ~ M H C I , whereas most of the capacity to interact witfi proteoglycan (73.2%) was eluted by 0.5 r~i-NaCl. This fraction contained equimolar amounts of hexuronic acid and hexosaniine, and the glucosaiiiiiie/galactosamine molar ratio was 25 : 1.

We acknowledge the excellent technical assistance of Miss Diane Orton and Mr. S. Brown, and thank the Medical Research Council for a grant to M. F. D., the Arthritis and Rheumatism Council, the Wellcome Trust and the Spastics Society for financial support. The clinical work described, for which the informed consent of the patients was obtained, was carried out with the full approval of the Ethical Committee of Guy's Hospital Medical School.