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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.

An electric field causes partial alignment of macromolecules in a dilute solution. The accompanying changes in the solution birefringence offer a sensitive and quick means of monitoring the rates of particle orientation and hence the size of the solute molecules. Such measurements are reported for dilute solutions of proteoglycans in the absence and presence of added hyaluronic acid. The proteoglycan molecules are shown to be some 580 nm long. In the presence of hyaluronic acid they form aggregates that appear to be consistent with the model previously proposed in which the proteoglycans attach radially to the extended hyaluronic acid chain. The electric-birefringence relaxation rates indicate aggregates of similar length to that of the extended hyaluronic acid chain, with the proteoglycans spaced on average at 29nm intervals. A proteoglycan sample the cystine residues of which had been reduced and alkylated showed no evidence of aggregation with hyaluronic acid up to the concentrations of the acid corresponding to 1% of the total uronic acid content. The electric-birefringence method is shown to have a large potential in the study of associating polysaccharide solutions.

Low molecular mass proteoglycans (PG) were isolated from human articular cartilage and from pig laryngeal cartilage, which contained protein cores of similar size (Mr 40-44 kDa). However, the PG from human articular cartilage contained dermatan sulphate (DS) chains (50% chondroitinase AC resistant), whereas chains from pig laryngeal PG were longer and contained only chondroitin sulphate (CS). Disaccharide analysis after chondroitinase ABC digestion showed that the human DS-PG contained more 6-sulphated residues (34%) than the pig CS-PG (6%) and both contained fewer 6-sulphated residues than the corresponding high Mr aggregating CS-PGs from these tissues (86% and 20% from human and pig respectively). Cross-reaction of both proteoglycans with antibodies to bovine bone and skin DS-PG-II and human fibroblasts DS-PG suggested that the isolated proteoglycans were the humans DS-PG-II and pigs CS-PG-II homologues of the cloned and sequenced bovine proteoglycan. Polyclonal antibodies raised against the pig CS-PG-II were shown to cross-react with human DS-PG-II. SDS/polyacrylamide-gel analysis and immunoblotting of pig and human cartilage extracts showed that some free core protein was present in the tissues in addition to the intact proteoglycan. The antibodies were used in a competitive radioimmunoassay to determine the content of this low Mr proteoglycan in human cartilage extracts. Analysis of samples from 5-80 year-old humans showed highest content (approximately 4 mg/g wet wt.) in those from 15-25 year-olds and lower content (approximately 1 mg/g wet wt.) in older tissue (greater than 55 years). These changes in content may be related to the deposition and maintenance of the collagen fibre network with which this class of small proteoglycan has been shown to interact.

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.

Analysis of the knee-joint cartilage of pigs at five ages (namely foetuses from the second half of pregnancy and animals 10 weeks, 25 weeks, 3 years and 5 years old) showed that the composition approached that of adult cartilage by 25 weeks of age, the most marked differences being between foetal and 10 week-old cartilage. Protein–polysaccharides were extracted sequentially, first by brief low-speed homogenization with iso-osmotic sodium acetate, then by two extractions with 2m-CaCl2 for 24h with gentle agitation interspersed with brief low-speed homogenization and agitation for another 24h. About half of the protein–polysaccharides were removed from foetal cartilage by the first extraction and the remainder by the second. The proportion in the first extract declined sharply with the age of the animal, but that in the first CaCl2 extract was similar at all ages other than 10 weeks. The amount left in the residue increased approximately with the collagen content from about one-fifth at 10 weeks of age to one-third in adult and old cartilage. The proportion of medium-sized protein–polysaccharides in the extracts changed little with age after birth, but the glucosamine content increased about fivefold and the protein content almost doubled between 10 weeks and 5 years of age. Other analytical values changed little. These results cannot be explained solely by changes in the proportion of ‘link-glycoprotein’ in the protein–polysaccharides. Since major changes in most parameters had taken place by 25 weeks of age, the first weeks after birth may be a critical period for cartilage development in the pig.

The kinetics of incorporation of [35S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ‘pulse–chase’ radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ‘pulse–chase’ experiments over 5h did not demonstrate any precursor–product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ‘pulse–chase’ experiments there was again no evidence for precursor–product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.

1. Protein–polysaccharides of chondroitin 4-sulphate were extracted with neutral calcium chloride from pig laryngeal cartilage that was not completely homogenized. The protein–polysaccharides were purified by precipitation with 9-aminoacridine. On zone electrophoresis in compressed glass fibre at pH7·2 it was separated into two fractions, although two distinct zones were not obtained. These fractions, which had already been shown to differ in their antigenic determinants, also differed considerably in amino acid composition, total protein, hexose and glucosamine contents. 2. The fraction of higher mobility contained approx. 2% of protein and only traces of glucosamine. Serine and glycine accounted for over half the total amino acid residues, but aromatic, basic and sulphur-containing amino acids were not detected. The weight-average molecular weight, determined by sedimentation, was 230000. 3. Assuming that there was the same sequence of neutral sugars at the linkage points as in PP-L fraction (protein–polysaccharide light fraction), the approximate molar ratio of hexose to serine suggested that most of the serine residues were linked to chondroitin sulphate chains. Support for this was derived from the agreement between the weight-average molecular weight of the chondroitin sulphate–peptide after proteolysis, and the chain weight calculated from its serine content. The chain weight based on the serine content of the fraction of higher electrophoretic mobility was approximately similar. 4. In contrast, the fraction of lower electrophoretic mobility resembled PP-L fraction in its amino acid composition, protein and glucosamine contents. The presence of glucosamine, together with the higher hexose content, suggested that this fraction contained some keratan sulphate. 5. The relatively low molecular weight of the fraction of higher mobility enabled it to be extracted without complete disintegration of the cartilage. The unlikelihood of its being produced by autolytic enzymes is discussed.

1. Protein–polysaccharides from pig laryngeal cartilage extracted by two procedures described in the preceding paper (Tsiganos & Muir, 1969) were shown to consist of macromolecules of various sizes as assessed by gel filtration in 4% and 6% agarose. 2. A larger proportion of the smaller molecules was present in the preparation obtained by brief extraction in iso-osmotic sodium acetate (procedure I) than in that obtained by more prolonged extraction in 10% (w/v) calcium chloride (procedure II). 3. Two fractions were separated by gel filtration in 6% agarose and by electrophoresis in compressed glass fibre. These fractions differed in chemical composition and in antigenic determinants. The gel-retarded fraction R and that of higher electrophoretic mobility possessed the same single antigen, whereas the gel-excluded fraction E and the slower electrophoretic fraction contained all the antigens of the starting material including that of fraction R. 4. Five N-terminal amino acid residues were identified in preparation I and fraction E, only two of which were present in fraction R. 5. The relative proportions of gel-excluded and gel-retarded fractions did not change when solutions of high ionic strength, urea or guanidine hydrochloride were used for elution. 6. The differences in chemical and amino acid composition between fractions R and E showed that the latter was not a simple aggregate of the former. Fraction E contained more basic and aromatic amino acids, and some methionine and cystine; the last two were absent from fraction R. Hydroxyproline was not detected in either fraction. 7. The number of glycosidic linkages in both fractions was estimated by alkaline β-elimination. Appreciable amounts of threonine as well as serine were destroyed in both fractions. An average chain length for chondroitin sulphate was calculated from the galactosamine content of both fractions and the amounts of hydroxy amino acid destroyed. Average chain lengths were also calculated from the xylose and galactosamine content of each fraction. Each independent method gave a value of approximately 28 disaccharide units for the chain length in both fractions and hence their difference in size could not be explained by differences in the length of carbohydrate chains. 8. All fractions contained glucosamine, which was attributed to keratan sulphate. Content of both protein and keratan sulphate increased with the size of the macromolecules. 9. It is suggested, from these results, that chondroitin sulphate–protein complexes normally exist as a heterogeneous population of macromolecules in cartilage, and that keratan sulphate is involved in the formation of larger molecules.

Protein–polysaccharides of knee-joint cartilage of 9-month-old pigs were extracted sequentially with neutral iso-osmotic sodium acetate after five repeated homogenizations. One-third of the uronic acid originally present in the tissue was brought into solution, about half being in the first extract. The protein–polysaccharides, which were purified by precipitation with 9-aminoacridine, were heterogeneous in size on gel chromatography. The smallest (retarded by 6% agarose) were the most easily extracted since they were most prevalent in the initial extracts and absent from later ones, whereas the proportion of larger molecules increased progressively in successive extracts. Nevertheless a small proportion of the largest molecules (excluded from Sepharose 2B) was present even in the first extract. None of the protein–polysaccharide preparations contained hydroxyproline, and the analyses of their constituent sugars were the same, although there was a progressive increase in the protein content and in the glucosamine/galactosamine molar ratio of successive extracts. In each preparation this molar ratio was invariably greater in larger than in smaller molecules separated by gel filtration. From galactosamine/pentose molar ratios it appeared that the chondroitin sulphate chains were on average about 29 disaccharide units in length in the protein–polysaccharides of each extract, although gel-chromatography and cetylpyridinium chloride elution profiles showed that a somewhat higher proportion of shorter chondroitin sulphate chains occurred in the larger protein–polysaccharides. In the last extract, where the largest molecules predominated, about half could be reversibly dissociated by urea, whereas this had no effect on the protein–polysaccharides of earlier extracts even though these contained some large molecules.

Explants of cartilage from tibiae of 11–12 days chick embryos were grown in organ culture. To one group hyaluronidase was added to the medium during the first 2 days of culture; the treated tissue was then cultured in medium without enzyme for a further 4 days. Control explants grown in hyaluronidase-free medium for 6 days grew rapidly in size and the total hexosamine content more than doubled during this time. After exposure to hyaluronidase, much of the hexosamine was lost from treated cartilage and appeared in the culture medium, but it was mostly replaced in the tissue during the subsequent recovery period. Analysis of cartilage and medium showed that net synthesis of hexosamine increased greatly in treated cartilage. The proteoglycans were extracted by two procedures from control and treated cartilage after 2, 4 and 6 days in culture. The hydrodynamic sizes of the purified proteoglycans were compared by gel chromatography and the composition of the gel-chromatographic fractions was determined. The proteoglycans from controls did not change during culture, but after exposure to hyaluronidase the proteoglycans from treated cartilage were of much smaller size and lower chondroitin sulphate content. During recovery, even though new proteoglycans were formed, they were nevertheless of smaller size and lower chondroitin sulphate content than control proteoglycans. They gradually became more like control proteoglycans during recovery from treatment, but even after 4 days they were not yet the same. After 2 days of treatment with the enzyme, the chondroitin sulphate in the cartilage was of shorter chain length than in controls but during recovery after 4 and 6 days in culture, the chain lengths in control and treated cartilage were similar. It is concluded that the proteoglycans formed in embryo cartilage in response to their depletion by enzyme treatment contained fewer chondroitin sulphate chains attached to the protein moiety of proteoglycans. This may have resulted from a failure under stress to glycosylate the protein moiety to the usual extent; alternatively the synthesis of normal proteoglycans of low chondroitin sulphate content may have increased, thus changing the proteoglycan population.

Pig articular cartilage, from which protein–polysaccharides soluble in iso-osmotic sodium acetate had been removed, was extracted in three further stages with 8m-urea in 2m-sodium acetate and with tris–HCl buffer after bacterial collagenase digestion, followed by the same urea–sodium acetate solution, thus leaving only 2% of the original uronic acid in the tissue. The histological appearance of the cartilage was unaltered until after collagenase digestion. The collagenase used did not affect the viscosity or molecular size of a protein–polysaccharide preparation obtained previously. The protein–polysaccharides in each extract differed in size, amino acid composition and protein content, but protein and keratan sulphate contents were not related to hydrodynamic size, in contrast with protein–polysaccharides extracted previously before collagenase digestion. Hydroxyproline could not be removed from those obtained by the first urea–sodium acetate extraction until degraded by heat. The galactosamine/pentose molar ratio agreed closely with the galactosamine/serine molar ratio that was destroyed on treatment with 0.5m-sodium hydroxide, showing that chondroitin sulphate was attached only to serine residues. From these molar ratios the chondroitin sulphate chains were calculated to be of the same average length in protein–polysaccharides in all three extracts although somewhat shorter than in protein–polysaccharides extracted previously. Some threonine residues were also destroyed on alkali treatment suggesting that keratan sulphate may be attached to threonine. These findings together with previous results show that differences in size, composition and physical state extend to all the protein–polysaccharides in cartilage.

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.