Cartilage Proteoglycan Aggregates Research Articles

Extracellular Processing of the Cartilage Proteoglycan Aggregate and Its Effect on CD44-mediated Internalization of Hyaluronan

In many cells hyaluronan receptor CD44 mediates the endocytosis of hyaluronan and its delivery to endosomes/lysosomes. The regulation of this process remains largely unknown. In most extracellular matrices hyaluronan is not present as a free polysaccharide but often is found in complex with other small proteins and macromolecules such as proteoglycans. This is especially true in cartilage, where hyaluronan assembles into an aggregate structure with the large proteoglycan termed aggrecan. In this study when purified aggrecan was added to FITC-conjugated hyaluronan, no internalization of hyaluronan was detected. This suggested that the overall size of the aggregate prevented hyaluronan endocytosis and furthermore that proteolysis of the aggrecan was a required prerequisite for local, cell-based turnover of hyaluronan. To test this hypothesis, limited C-terminal digestion of aggrecan was performed to determine whether a size range of aggrecan exists that permits hyaluronan endocytosis. Our data demonstrate that only limited degradation of the aggrecan monomer was required to allow for hyaluronan internalization. When hyaluronan was combined with partially degraded, dansyl chloride-labeled aggrecan, blue fluorescent aggrecan was also visualized within intracellular vesicles. It was also determined that sonicated hyaluronan of smaller molecular size was internalized more readily than high molecular mass hyaluronan. However, the addition of intact aggrecan to hyaluronan chains sonicated for 5 and 10 s reblocked their endocytosis, whereas aggregates containing 15-s sonicated hyaluronan were internalized. These data suggest that hyaluronan endocytosis is regulated in large part by the extracellular proteolytic processing of hyaluronan-bound proteoglycan.

Link peptide cartilage growth factor is degraded by membrane proteinases.

The peptide DHLSDNYTLDHDRAIH (Link N), cleaved from the N-terminus of the link protein component of cartilage proteoglycan aggregates by the action of stromelysin, can act as a growth factor and stimulate synthesis of proteoglycans and collagen in articular cartilage [McKenna, Liu, Sansom and Dean (1998) Arthritis Rheum. 41, 157-161]. The mechanism by which this biologically active peptide is degraded and inactivated was investigated using U937 monocytes as a model cell. Time-course experiments showed that two major proteases, an initial serine proteinase followed by a metalloproteinase, acted in sequence. Analysis of the resulting fragments showed that the serine endopeptidase cleavage was at the Leu(3)-Ser(4) bond to produce the peptide SDNYTLDHDRAIH. The terminal serine could then be removed from the resulting peptide by an aminopeptidase. A second metallopeptidase liberated the peptides SDNYTL or DNYTL from DHDRAIH by cleavage at the Leu(9)-Asp(10) bond. The DNYTL peptide intermediate was degraded too rapidly to allow sequencing and sequential aminopeptidase cleavages removed further amino acids from the N-terminus of the remaining DHDRAIH peptide. The identical patterns of breakdown that occurred when either whole cells or purified plasma membranes were used indicated that proteolysis and inactivation of Link N was carried out entirely by membrane-associated enzymes.

Link peptide cartilage growth factor is degraded by membrane proteinases

The peptide DHLSDNYTLDHDRAIH (Link N), cleaved from the N-terminus of the link protein component of cartilage proteoglycan aggregates by the action of stromelysin, can act as a growth factor and stimulate synthesis of proteoglycans and collagen in articular cartilage [McKenna, Liu, Sansom and Dean (1998) Arthritis Rheum. 41, 157-161]. The mechanism by which this biologically active peptide is degraded and inactivated was investigated using U937 monocytes as a model cell. Time-course experiments showed that two major proteases, an initial serine proteinase followed by a metalloproteinase, acted in sequence. Analysis of the resulting fragments showed that the serine endopeptidase cleavage was at the Leu3-Ser4 bond to produce the peptide SDNYTLDHDRAIH. The terminal serine could then be removed from the resulting peptide by an aminopeptidase. A second metallopeptidase liberated the peptides SDNYTL or DNYTL from DHDRAIH by cleavage at the Leu9-Asp10 bond. The DNYTL peptide intermediate was degraded too rapidly to allow sequencing and sequential aminopeptidase cleavages removed further amino acids from the N-terminus of the remaining DHDRAIH peptide. The identical patterns of breakdown that occurred when either whole cells or purified plasma membranes were used indicated that proteolysis and inactivation of Link N was carried out entirely by membrane-associated enzymes.

Identification of a protein in squid cranial cartilage with link protein properties

Squid cranial cartilage extracts were found to contain a protein with a molecular mass of 35 kDa immunoreacting with an antiserum against sheep link protein. Because hyaluronan is not detected in this tissue and the structure of proteoglycans is different to that of aggreean or versican, this observation was studied further. The 35 kDa protein was purified from cartilage extracts and immunolocalised in Western blots by both the polyclonal antibody and the mAb 8A4. It was found that it was able to bind to hyaluronan and to aggrecan. Direct and competitive microplate binding experiments showed that the squid protein binds to G1 domain of aggrecan, similarly to cartilage link protein and, therefore, it could be a link-like protein molecule of squid cranial cartilage. The 35 kDa protein was also able to bind to squid proteoglycan and this suggested that it might participate in squid cartilage proteoglycan aggregate formation.

Quantification of aggrecan and link-protein mRNA in human articular cartilage of different ages by competitive reverse transcriptase-PCR.

A competitive reverse transcriptase-PCR (RT-PCR) assay has been developed for the quantification of particular mRNA species in human articular cartilage. Competitor RNA species were synthesized that differed from the amplified target sequence only by the central insertion of an EcoRI restriction site. By using known amounts of synthetic target and competitor RNA, it was shown that competitor RNA molecules designed in this way are reverse-transcribed and amplified with equal efficiency to the target of interest. Furthermore quantification could be performed during the plateau phase of the PCR, which was necessary when using ethidium bromide fluorescence as a detection system. The inhibition of aggrecan and link-protein mRNA expression by interleukin 1 or tumour necrosis factor in monolayers of human articular chondrocytes quantified by this competitive RT-PCR method compared favourably with Northern hybridization studies. The main advantage of this technique is that it can be used to quantify levels of mRNA with RNA extracted directly from 100 mg wet weight of human articular cartilage. Age-related changes in aggrecan and link-protein mRNA were therefore quantified in human articular cartilage directly after dissection from the joint. The concentration of link-protein mRNA was higher in immature cartilage than in mature cartilage when expressed relative to the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA, but no age-related changes were observed in aggrecan mRNA expression. The ratio of aggrecan to link-protein mRNA was higher in mature cartilage than in immature tissue. These age-related differences in the molecular stoichiometry of aggrecan and link-protein mRNA might have implications with respect to the regulation of the formation and the stability of the proteoglycan aggregates in cartilage.

Effect of link protein concentration on articular cartilage proteoglycan aggregation.

Previous work has shown that alterations in proteoglycan aggregates are among the first changes detected with aging, disuse, and degeneration of articular cartilage, yet the cause or causes of these alterations remain unknown. To determine if differences in link protein concentration can explain alterations in the assembly, size, and stability of articular cartilage proteoglycan aggregates, we isolated proteoglycan monomer (aggrecan) and link protein from adult bovine articular cartilage and then assembled proteoglycan aggregates from aggrecan and 0.8% hyaluronan relative to aggrecan weight, in the presence of 0, 2, 4, 6, 8, 10, 15, and 20% concentrations of link protein relative to aggrecan weight. We determined the amount, sedimentation coefficient, and stability of the aggregates by analytical ultracentrifugation and measured their dimensions by electron microscopy with use of the monolayer technique. Increased aggregate size, as determined by ultracentrifugation, was directly correlated with an increased number of aggrecans per aggregate and with increased hyaluronan length, as determined by electron microscopy. The concentration of link protein significantly influenced aggregation: concentrations of 6-8% produced maximum aggregation, aggregate stability, and uniformity of aggrecan spacing; concentrations greater than 10% led to the formation of superaggregates (aggregates with sedimentation velocities greater than 100 S that may result from linking two or more hyaluronan filaments) but decreased aggregate stability; and concentrations of less than 4% link protein significantly decreased aggregation, the size and stability of aggregates, and the regularity of aggrecan spacing. The latter observations suggest that a decline in the concentration of link protein could decrease the organization and stability of the articular cartilage matrix.

Evidence of a defined spatial arrangement of hyaluronate in the central filament of cartilage proteoglycan aggregates.

Aggregates of proteoglycans from the Swarm rat chondrosarcoma reassembled in vitro have been studied by rotary-shadowing electron microscopy, and shown to be similar to native structures that have never been dissociated [Mörgelin, Engel, Heinegård and Paulsson (1992) J. Biol. Chem. 267, 14275-14284]. A hyaluronate with defined chain length (HAshort) has now been prepared by autoclaving high-Mr hyaluronate and fractionation to a narrow size distribution by gel filtration. Proteoglycan monomers, core protein, hyaluronate-binding region and link protein were combined with HAshort. Free chains of HAshort and reconstituted complexes with proteoglycan, link protein and aggrecan fragments were examined by electron microscopy after rotary shadowing. Length measurements showed that the hyaluronate was condensed to about half of its original length on binding intact aggrecan monomers, any aggrecan fragment or link protein alone. This strongly implies that hyaluronate adopts a defined spatial arrangement within the central filament of the aggregate, probably different from its secondary structure in solution. No differences in length were observed between link-free and link-stabilized aggregates.

The cartilage proteoglycan aggregate: assembly through combined protein—carbohydrate and protein—protein interactions

In vitro reassembled aggregates of cartilage proteoglycan (aggrecan) were studied by glycerol spraying/rotary shadowing electron microscopy and compared to the corresponding native (i.e. never dissociated) structures. In both cases a tightly packed central filament structure was observed consisting of the hyaluronate binding region (HABR) of the proteoglycan, link protein (LP) and hyaluronate (HA). This differs from earlier results where a discontinuous central filament structure was seen after spreading proteoglycan aggregates at a water/air interphase. Binding of isolated HABR to HA is random but upon addition of link protein a clustering of the HA-binding proteins is observed, indicating a cooperativity. In a fully saturated aggregate the HA is covered by a continuous protein shell consisting of HABR and LP. When added in amounts below saturation HABR and LP bind to the HA in clusters which are interrupted by free strands of HA. The proteoglycan aggregate is thus an example for a structure where a polysaccharide forms a template for a supramolecular assembly largely stabilized by protein-protein interactions.

Structural differences between two populations of articular cartilage proteoglycan aggregates

To determine if articular cartilage contains structurally distinct populations of proteoglycan aggregates, we extracted and purified proteoglycans from canine knee cartilage under associative conditions. Equilibrium density gradient centrifugation separated three proteoglycan populations, on the basis of differences in sedimentation velocity, into groups of 21, 106, and 270 S. Electron microscopic examination showed that the 21 S samples contained free aggrecan molecules and clusters of aggrecan molecules, with a mean of five aggrecan molecules per cluster. The 106 and 270 S samples contained proteoglycan aggregates consisting of central hyaluronan filaments with multiple attached aggrecan molecules. The two populations of aggregates did not differ in mean aggrecan length or in the spacing of aggrecan molecules along the hyaluronan filaments, but the slower sedimenting aggregates (106 S) had significantly shorter hyaluronan filaments as measured by electron microscopy (mean hyaluronan length, 400 compared with 1,162 nm) and one-third as many aggrecan molecules per aggregate (mean number of aggrecan molecules per aggregate, 15 compared with 44). This study shows that articular cartilage contains aggrecan clusters and two structurally distinct populations of proteoglycan aggregates. The differences between the two types of aggregate, in particular the number of aggrecan molecules per aggregate, may reflect differences in their assembly, stability, or turnover and give them different mechanical and biological properties.

A brain extracellular matrix proteoglycan forms aggregates with hyaluronan

Unlike many tissues, the adult central nervous system extracellular matrix (ECM) has few known components. Previously, we characterized a large chondroitin sulfate proteoglycan, pgT1, from adult rat brain which has the properties of a general brain ECM component and is immunologically distinct from aggrecan and versican (Iwata, M., and Carlson, S.S. (1993) J. Neurosci. 13, 195-207). In this study we demonstrate that pgT1 binds hyaluronan with relatively high affinity. The pgT1 preparation isolated from rat brain aggregates in non-denaturing conditions. This aggregation is abolished by incubation of pgT1 with Streptomyces hyaluronidase. Examination of these aggregates by electron microscope reveals a structure in which an average of 18 subunits arise laterally from opposite sides of an elongated 350-nm filament. These pgT1 aggregates resemble the proteoglycan aggregates in cartilage which are composed of aggrecan and hyaluronan. Using affinity coelectrophoresis, we measure a dissociation constant (Kd) of 0.9 +/- 0.2 nM for the interaction of pgT1 and hyaluronan. These new findings, combined with the general distribution of pgT1 in brain, suggest that pgT1/hyaluronan aggregates are an extended general structure of the brain extracellular matrix network.

Hyaluronan interactions with Hydroxyapatite do not Alter In Vitro Hydroxyapatite Crystal Proliferation and Growth

The interaction of hyaluronan (Mr range 80–120 X 104) with poorly crystalline hydroxyapatite, such as is found in calcified cartilage and bone, was studied to challenge the hypothesis that free hyaluronan found in proteoglycan aggregate preparations could affect in vitro mineralization. Using a Langmuir adsorption isotherm, based on uronic acid content, hyaluronan was found to bind to hydroxyapatite with an affinity K of 0.12 ml/μg uronate and N = 6.8 μg uronate/m2 hydroxyapatite binding sites. This is contrasted with K = .047 ml/μg uronate and N = 9.0 μg uronate/m2 for a bovine nasal proteoglycan monomer preparation. Although the proteoglycan monomer and aggregate preparations have been reported to inhibit hydroxyapatite growth at concentrations of 1 mg/ml, using solution concentrations of 0, 0.01, 0.1 and 1 mg/ml hyaluronan there were no detectable alterations in the rate of seeded hydroxyapatite growth and proliferation. These data indicate that although in vitro hyaluronan may bind with weak affinity to hydroxyapatite, this interaction does not affect mineral growth, and the presence of hyaluronan would not contribute to the increased inhibitory potential of cartilage proteoglycan aggregate relative to monomer preparations.

Link protein as a monitor in situ of endogenous proteolysis in adult human articular cartilage

The link protein components of proteoglycan aggregates in adult human articular cartilage show heterogeneity due to proteolysis. Cleavages near the N-terminus of the intact link proteins, before residues 17, 19 and 24, generate three proteins of slightly diminished size (LP3). Cleavages within the N-terminal disulphide-bonded loop, before residues 66 and 73 of the intact link proteins, generate proteins that yield smaller degradation products upon reduction (LP fragments). In vitro, modified link protein components of a similar size to LP3 can be generated by a variety of proteinases, but of the physiologically relevant enzymes only stromelysin, cathepsin B and cathepsin G have the ability to yield modified link proteins with N-termini identical with those observed in situ. None of the proteolytic agents tested was able to produce LP fragments with N-termini identical with those observed in situ, and the majority of proteinases were not able to cleave within the disulphide-bonded loops. Cathepsin L and hydroxyl radicals can cleave within the N-terminal disulphide-bonded loop, and have the potential of initially opening the loop to allow further proteolytic processing by other agents to generate the native cleavage sites.

Proteoglycans of bovine articular cartilage. The effects of divalent cations on the biochemical properties of link protein.

In cartilage proteoglycan aggregates, link protein stabilizes the binding of proteoglycan monomers to hyaluronate by binding simultaneously to hyaluronate and to the G1 globular domain of proteoglycan monomer core protein. Studies reported here involving metal chelate affinity chromatography demonstrate that link protein is a metalloprotein that binds Zn2+, Ni2+, and Co2+. Zn2+ and Ni2+ decrease the solubility of link protein and result in its precipitation. However, link protein is readily soluble and functional in low ionic strength solvents from which divalent cations have been removed with Chelex 100. These observations make it possible to study the biochemical properties of link protein in low ionic strength, physiologic solvents. Studies were carried out to define the oligomeric state of link protein alone in physiologic solvents, and the transformation in oligomeric state that occurs when link protein binds hyaluronate. Sedimentation equilibrium studies demonstrate that in 0.15 M NaCl, 5 mM EDTA, 50 mM Tris, pH 7, link protein exists as a monomer-hexamer equilibrium controlled by a formation constant of 2 x 10(27) M-5, yielding a delta G' of -36 kcal/mol for the formation of the hexamer from six monomers. On binding hyaluronate oligosaccharides (HA10 or HA12), link protein dissociates to dimer. Link protein hexamer is rendered insoluble by Zn2+. Greater than 90% of the protein is precipitated by 2 mol of Zn2+/mol of link protein monomer. The binding of hyaluronate oligosaccharide by link protein strongly inhibits the precipitation of link protein by Zn2+. The link protein/hyaluronate oligosaccharide complex is completely soluble in the presence of 2 mol of Zn2+/mol of link protein. At higher molar ratios of Zn2+/link protein, the inhibitory effect of hyaluronate oligosaccharide on the precipitation of link protein is gradually overcome. Hyaluronate oligosaccharide is not dissociated from link protein by Zn2+. Hyaluronate remains bound to the link protein which is precipitated by Zn2+, or to the link protein which binds to Zn2(+)-charged iminodiacetate-Sepharose columns. Hyaluronate oligosaccharides and Zn2+ bind to different sites on link protein.

Isolation and primary structure of PARP, a 24-kDa proline- and arginine-rich protein from bovine cartilage closely related to the NH2-terminal domain in collagen alpha 1 (XI)

A protein rich in proline and arginine (proline/arginine-rich protein (PARP] has been isolated from dissociative extracts of bovine nasal and articular cartilage, and its primary structure has been determined. The protein has 218 amino acids, giving a calculated protein Mr of 24,075. In nasal cartilage, this protein is in molar concentrations equivalent to 1/20-1/10 that of the link protein of cartilage proteoglycan aggregates. PARP has also been isolated from bovine articular cartilage, bovine fetal epiphysis, and nonossified human tarsal bones. PARP is similar to various collagen NH2-terminal domains. It is 49% identical to the NH2-terminal end of collagen alpha 1 (XI), 17% identical to the NC4 domain of collagen alpha 1 (IX), and 14% identical to the NC3 domain of collagen alpha 1 (XII). Four cysteines are conserved between type XI collagen and PARP, and these form two disulfide bonds. Two of the cysteines are also conserved between PARP and collagens IX and XII. The homology between the collagens and PARP makes it possible to speculate on the likely disulfide bond pattern in the collagen NH2-terminal domains. It is probable that PARP is a collagen fragment removed during processing in a manner analogous to chondrocalcin (the C-terminal propeptide of type II collagen).

An 18-kDa glycoprotein from bovine nasal cartilage. Isolation and primary structure of small, cartilage-derived glycoprotein.

A glycosylated protein (small, cartilage-derived glycoprotein, SCGP) of approximately 18 kDa with unknown function has been isolated from dissociative extracts of bovine nasal cartilage and its primary structure determined. The protein has 121 amino acids, giving a calculated protein molecular weight of 13,878, four disulfide bonds, two N-linked oligosaccharides and one O-linked oligosaccharide. In nasal cartilage, this glycoprotein is in molar concentrations equivalent to 1/5-1/2 that of the link protein of cartilage proteoglycan aggregates, and it has also been isolated from bovine articular cartilage and from bovine fetal epiphysis. The N-terminal, glycosylated region of the molecule is relatively rich in arginine, proline, glycine, and threonine. The C-terminal 82 amino acids (which contains all four of the disulfide bonds and none of the carbohydrate) can be found as a discrete entity in cartilage extracts, indicating that the N-terminal domain is readily removed by extracellular proteolytic attack.

Hyaluronate‐Binding Proteins of Murine Brain

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.

Degradation of proteoglycan aggregate by a cartilage metalloproteinase. Evidence for the involvement of stromelysin in the generation of link protein heterogeneity in situ.

Cartilage proteoglycan aggregates were subjected to degradation by a metalloproteinase, capable of degrading proteoglycan, released from cartilage in culture. This proteinase was demonstrated to be immunologically identical with fibroblast stromelysin. An early release of hyaluronic acid-binding region and large glycosaminoglycan-attachment regions was observed. With increasing time the glycosaminoglycan-attachment regions were digested into smaller fragments and the hyaluronic acid-binding regions accumulated. The degradation of link proteins also occurred concomitantly with these events. Link proteins were converted into a component of similar size to that of the smallest native link protein component. N-Terminal sequence analysis of the three human link protein components indicated that they are all derived from the same protein core, which is closely homologous to that of the rat chondrosarcoma link protein. The two larger link proteins (Mr 48,000 and 44,000) contain the same N-terminal sequence, but they differ by the apparent presence of an N-linked oligosaccharide at residue 6 of the largest link protein component. The smallest link protein (Mr 41,000), however, has an N-terminal sequence equivalent to that commencing at residue 17 in the larger link proteins. It was found that the cartilage metalloproteinase cleaves link proteins in human neonatal cartilage proteoglycan aggregates at the His-16-Ile-17 bond, the same position at which the smallest link protein component appears to be derived naturally from the two larger link protein components. These results suggest that stromelysin secreted by chondrocytes can account for the increased accumulation of hyaluronic acid-binding regions and much of the degradation of link protein observed during aging within human articular cartilage.

Electron-microscopic study of various salts of proteoglycan aggregates

Chemically isolated preparations of acid and normal Mg2+, Ca2+-, and guanidine+ salts of cartilage proteoglycan aggregates were investigated by electron microscopy. Some differences between acid and normal salts depend on the amount, valency and distribution of basic ion as well as between carboxyl and sulphate groups.

Cartilage proteoglycan aggregate and fibronectin can modulate the expression of type X collagen by embryonic chick chondrocytes cultured in collagen gels.

Chick embryo sternal chondrocytes from the caudal and cephalic regions were cultured within type I collagen gels and type I collagen/proteoglycan aggregate composite gels in normal serum. Caudal region chondrocytes were also cultured within type I collagen gels in the presence of fibronectin-depleted serum. There was a marked stimulation of type X collagen synthesis by the caudal region chondrocytes after 9 days in the presence of fibronectin-depleted serum and after 14 days in the presence of proteoglycan aggregate. These results provide evidence for the ability of chondrocytes from a zone of permanent cartilage to synthesise type X collagen and for the involvement of extracellular matrix components in the control of type X collagen gene expression.

Cartilage proteoglycan aggregates. The link protein and proteoglycan amino-terminal globular domains have similar structures.

Cartilage proteoglycan aggregates contain two components (proteoglycan monomer and link protein) which interact with each other and with hyaluronic acid. Data from amino acid sequence analysis are presented that shows that a domain of the proteoglycan, the hyaluronic acid binding region, which interacts with link protein and hyaluronic acid is very similar to link protein in terms of its primary structure. However, the pattern of glycosylation in the hyaluronic acid binding region is different from that found in link protein. After removal of N-linked oligosaccharides, the tryptically prepared hyaluronic acid binding region from rat chondrosarcoma has a mass by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of 43 +/- 2 kDa. The COOH-terminal two-thirds of rat chondrosarcoma link protein, starting at residue 105, has 41.3% identity with a similar region in the hyaluronic acid binding region. We show that, in addition to the hyaluronic acid binding region, proteoglycan contains another region with similarity to the two repeating loop structures in the COOH-terminal two-thirds of link protein. This presumably corresponds to the second globular domain reported in rotary shadowing studies of cartilage proteoglycans. We have deduced the positions of all of the disulfide bonds in the hyaluronic acid binding region and find them to be in the same positions as would be expected from comparison of these sequences with link protein.