An amino acid sequence common to both cartilage proteoglycan and link protein

Cartilage proteoglycan monomers associate with hyaluronic acid to form proteoglycan aggregates. Link protein, interacting with both hyaluronic acid and proteoglycan, serves to stabilize the aggregate structure. In the course of determining the primary structure of link protein, two peptides produced by digestion of rat chondrosarcoma link protein with trypsin or chymotrypsin have been selectively purified by immunoaffinity chromatography on a column of monoclonal anti-link protein antibody (8A4) immobilized to Sepharose 4B. These peptides have been sequenced using the double-coupling dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate procedure. A consensus sequence, Cys-X-Ala-Gly-Trp-Leu-X-Asp-Gly-Ser-Val-X-Tyr-Pro-Ile-X-X-Pro, obtained by comparing the affinity-isolated tryptic peptide with the affinity-isolated chymotryptic peptide and an overlapping tryptic peptide, shows homology with a sequence obtained from the NH2-terminal of a CNBr peptide from proteo glycan core protein of bovine nasal cartilage: Ser-Ser-Ala-Gly-Trp-Leu-Ala-Asp-Arg-Ser-Val-Arg-Tyr-Pro-Ile-Ser-. We suggest that the common sequence is structurally important to the function of these proteins and may be involved in the binding of both link protein and proteoglycan to hyaluronic acid.     

The N-terminal sequence of the large proteoglycan of articular cartilage

A peptide with hyaluronic acid-binding properties was isolated from trypsin digests of bovine articular cartilage proteoglycan aggregate. This peptide originated from the N-terminus of the proteoglycan core protein, retained its function of forming complexes with hyaluronate and link protein and contained at least one keratan sulfate chain. Amino acid sequence data demonstrated that the first six amino acid residues of the N-terminus of bovine articular cartilage proteoglycan core protein differed from the same region from the rat chondrosarcoma proteoglycan. Further sequence data indicate areas of considerable sequence homology in the hyaluronic acid-binding regions of proteoglycans from the two species.     

Degradation of human proteoglycan aggregate induced by hydrogen peroxide. Protein fragmentation, amino acid modification and hyaluronic acid cleavage.

We have previously shown that treatment of neonatal human articular-cartilage proteoglycan aggregates with H2O2 results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid and in fragmentation of the link proteins [Roberts, Mort & Roughley (1987) Biochem. J. 247, 349-357]. We now show the following. (1) Hyaluronic acid in proteoglycan aggregates is also fragmented by treatment with H2O2. (2) Although H2O2 treatment results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid, the loss of this function is not attributable to substantial cleavage of the hyaluronic acid-binding region of the proteoglycan subunits. (3) In contrast, link proteins retain the ability to bind to hyaluronic acid following treatment with H2O2. (4) The interaction between the proteoglycan subunit and link protein is, however, abolished. (5) N-Terminal sequence analysis of the first eight residues of the major product of link protein resulting from H2O2 treatment revealed that cleavage occurred between residues 13 and 14, so that the new N-terminal amino acid is alanine. (6) In addition, a histidine (residue 16) is converted into alanine and an asparagine (residue 21) is converted into aspartate by the action of H2O2. (7) Rat link protein showed no cleavage or modifications in similar positions under identical conditions. (8) This species variation may be related to the different availability of histidine residues required for the co-ordination of the transition metal ion involved in hydroxyl-radical generation from H2O2. (9) Changes in function of these structural macromolecules as a result of the action of H2O2 may be consequences of both fragmentation and chemical modification.     

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.     

The hyaluronic acid binding region as a specific probe for the localization of hyaluronic acid in tissue sections. Application to chick embryo and rat brain

The hyaluronic acid binding region was prepared by clostripain digestion of chondroitin sulfate proteoglycan isolated from the Swarm rat chondrosarcoma, and biotinylated in the presence of associated hyaluronic acid and link protein. After removal of hyaluronic acid by gel filtration in 4 M guanidine HCl, the biotinylated binding region-link protein complex was used as a specific histochemical probe in conjunction with avidin-peroxidase. Its utility was initially evaluated by comparison with Alcian blue staining of the axial region of 2 to 5 day chick embryos, where staining was seen in the dorsolateral area between the neural tube and the ectoderm, in the perichordal mesenchyme, and in developing limb buds. Light and electron microscopic studies of early postnatal rat cerebellum indicate that hyaluronic acid is primarily localized in the extracellular space of immature brain. Staining specificity was demonstrated by the ability of hyaluronic acid oligosaccharides of appropriate size to block the staining reaction, and by the absence of staining after treatment of tissue sections with protease-free Streptomyces hyaluronidase, which degrades only this glycosaminoglycan.     

A small proteoglycan isolated from human cartilage containing a nonfunctional hyaluronic acid binding region.

A low buoyant density fraction (A4) was isolated from human cartilage by CsCl density gradient ultracentrifugation. This fraction contained a hydrodynamically small proteoglycan (Kav, 0.74 on Sepharose CL-2B) that reacted with monoclonal antibody 12/20/1C6 specific for the hyaluronic acid binding region (G1 globe) of the large aggregating high-density proteoglycan isolated from many animal cartilages. Despite the presence of the hyaluronic acid binding region, this small proteoglycan did not form proteoglycan aggregates with hyaluronan, not even in the presence of link protein.     

Cartilage proteoglycan aggregate formation. Role of link protein.

Cartilage proteoglycan aggregate formation was studied by zonal rate centrifugation in sucrose gradients. Proteoglycan aggregates, monomers and proteins could be resolved. It was shown that the optimal proportion of hyaluronic acid for proteoglycan aggregate formation was about 1% of proteoglycan dry weight. The reaggregation of dissociated proteoglycan aggregate A1 fraction was markedly concentration-dependent and even at 9 mg/ml only about 90% of the aggregates were reformed. The lowest proportion of link protein required for maximal formation of link-stabilized proteoglycan aggregates was 1.5% of proteoglycan dry weight. It was separately shown that link protein co-sedimented with the proteoglycan monomer. By competition with isolated hyaluronic acid-binding-region fragments, a proportion of the link proteins was removed from the proteoglycan monomers, indicating that the link protein binds to the hyaluronic acid-binding region of the proteoglycan monomer.     

Correction of abnormal matrix formed by cmd/cmd chondrocytes in culture by exogenously added cartilage proteoglycan

The cartilage matrix deficiency (cmd/cmd) mouse fails to synthesize the core protein of cartilage-characteristic proteoglycan (cartilage PG). Chondrocytes from the cmd/cmd cartilage cultured in vitro produced nodules with greatly reduced extracellular matrix. Immunofluorescence staining revealed that the nodules of mutant cells differed from the normal in lacking cartilage PG and in uneven and reduced deposition of type II collagen. Exogenously added cartilage PG prepared from either normal mouse cartilage or Swarm rat chondrosarcoma to the culture medium was incorporated exclusively into the extracellular matrices of the nodules, with a concurrent correction of the abnormal distribution pattern of type II collagen. The incorporation of cartilage PG into the matrix was disturbed by hyaluronic acid or decasaccharide derived therefrom, suggesting that the incorporation process involves the interaction of added proteoglycan with hyaluronic acid. Both the hyaluronic acid-binding region and the protein-enriched core molecule prepared from rat chondrosarcoma cartilage PG could also be incorporated but, unlike the intact cartilage PG, they were distributed equally in the surrounding zones where fibroblast-like cells predominate. The results indicate that the intact form of cartilage PG is required for specific incorporation into the chondrocyte nodules, and further suggest that cartilage PG plays a regulatory role in the assembly of the matrix macromolecules.     

Characteristics of the core protein of the aggregating proteoglycan from the Swarm rat chondrosarcoma

A ternary complex of hyaluronic acid-binding region and link protein bound to hyaluronic acid was isolated from limit clostripain digests of proteoglycan aggregates isolated from the Swarm rat chondrosarcoma. Under these conditions, the hyaluronic acid-binding region has a molecular weight of ? 65,000 (HA-BR₆₅). N-terminal amino acids in the complex were selectively ^l4C-carbamylated. The resulting derivatized HA-BR₆₅ was isolated, and tryptic peptide maps were prepared and developed on two-dimensional TLC sheets. A single, labeled peptide was obtained which gave a M_r by ? 8,000 by SDS-PAGE. Chymotrypsin digestion of the ternary complex reduced the molecular weight of HA-BR₆₅ to a polypeptide of ? 55,000 (HA-BR₅₅) which still retains the same N-terminal tryptic peptide. Partial digestion of proteoglycan aggregates with clostripain generated a series of larger intermediates with the hyaluronic acid-binding region. Direct SDS-PAGE analysis revealed one major intermediate with M_r ? 109,000 (HA-BR₁₀₉) as well as HA-BR₆₅. After chondroitinase digestion, two additional prominent intermediates were observed on a SDS-PAGE gel at M_r ? 120,000 (HA-BR₁₂₀) and ? 140,000 (HA-BR₁₄₀). All the intermediates were recognized by a monoclonal antibody specific for the hyaluronic acid-binding region, and all of them contained the same N-terminal tryptic peptide. The results indicate that the N terminus of the core protein is at the hyaluronic acid-binding end of the proteoglycan and that the chondroitin sulfate chains are first present on the core protein in a region between 109,000 and 120,000 molecular weight away from the N terminus.     

Partial cDNA sequence encoding a globular domain at the C terminus of the rat cartilage proteoglycan

We have isolated and sequenced a cDNA clone of 872 base pairs from the 3' end of the mRNA for the large cartilage specific proteoglycan from rat. Identification was confirmed by a comparison with published protein sequence. Hybridization analysis shows the presence of an 8-9-kilobase mRNA for this proteoglycan in rat and chick sternal chondrocytes and rat chondrosarcoma cells, but not in RNA from rat fibroblasts, vitamin A-treated chick chondrocytes, chick crop, or bone. The carboxyl portion of the proteoglycan is deduced to terminate in a globular domain, which includes a region homologous to a chick hepatic lectin, and is possibly involved in binding to N-acetylglucosamine. The clone extends into a region where serines are clustered, probably the start of the chondroitin sulfate-rich region.     

Light and electron microscopic studies on the localization of hyaluronic acid in developing rat cerebellum

The hyaluronic acid-binding region was prepared by trypsin digestion of chondroitin sulfate proteoglycan aggregate from the Swarm rat chondrosarcoma, and biotinylated in the presence of hyaluronic acid and link protein. After isolation by gel filtration and HPLC in 4 M guanidine HCl, the biotinylated hyaluronic acid-binding region was used, in conjunction with avidin-peroxidase, as a specific probe for the light and electron microscopic localization of hyaluronic acid in developing and mature rat cerebellum. At 1 w postnatal, there is strong staining of extracellular hyaluronic acid in the presumptive white matter, in the internal granule cell layer, and as a dense band at the base of the molecular layer, surrounding the parallel fibers. This staining moves progressively towards the pial surface during the second postnatal week, and extracellular staining remains predominant through postnatal week three. In adult brain, there is no significant extracellular staining of hyaluronic acid, which is most apparent in the granule cell cytoplasm, and intra-axonally in parallel fibers and some myelinated axons. The white matter is also unstained in adult brain, and no staining was seen in Purkinje cell bodies or dendrites at any age. The localization of hyaluronic acid and its developmental changes are very similar to that previously found in immunocytochemical studies of the chondroitin sulfate proteoglycan in nervous tissue (Aquino, D. A., R. U. Margolis, and R. K. Margolis. 1984. J. Cell Biol. 99:1117-1129; Aquino, D. A., R. U. Margolis, and R. K. Margolis. J. Cell Biol. 99:1130-1139), and to recent results from studies using monoclonal antibodies to the hyaluronic acid-binding region and link protein. The presence of brain hyaluronic acid in the form of aggregates with chondroitin sulfate proteoglycans would be consistent with their similar localizations and coordinate developmental changes.     

The keratan sulfate-enriched region of bovine cartilage proteoglycan consists of a consecutively repeated hexapeptide motif

We have determined the sequence of a cDNA clone encoding the keratan sulfate-rich domain of the large aggregating cartilage proteoglycan core protein. The C-terminal portion of the deduced amino acid sequence is homologous to the chondroitin sulfate-rich region (domain CS1) of the rat chondrosarcoma proteoglycan, and the N-terminal portion is homologous to the second globular domain (G2) of the rat proteoglycan (Doege, K., Sasaki, M., Horigan, E., Hassell, J. R., and Yamada, Y. (1987) J. Biol. Chem. 262, 17757-17767). We could identify, inserted between these regions, a region absent in the rat proteoglycan. This domain corresponds to the keratan sulfate-enriched region of the bovine proteoglycan. It consists of a highly conserved hexapeptide motif consecutively repeated 23 times. Transfer blot analysis of genomic DNA indicated a single gene. The coding region for the keratan sulfate-enriched region was present both in human and bovine DNA, whereas the coding region for this domain appears to be absent in the rat genome. Transfer blot analysis of RNA showed that the keratan sulfate-rich region is present in proteoglycans from fetal as well as adult sources. Furthermore, RNA protection assays of RNA isolated from adult and fetal bovine articular cartilage showed that no alternative splicing occurs within this keratan sulfate-enriched region. These experiments show that the fetal bovine cartilage proteoglycan contains the keratan sulfate attachment domain, although it lacks the keratan sulfate side chains.     

Multiple domains of the large fibroblast proteoglycan, versican.

The primary structure of a large chondroitin sulfate proteoglycan expressed by human fibroblasts has been determined. Overlapping cDNA clones code for the entire 2389 amino acid long core protein and the 20-residue signal peptide. The sequence predicts a potential hyaluronic acid-binding domain in the amino-terminal portion. This domain contains sequences virtually identical to partial peptide sequences from a glial hyaluronate-binding protein. Putative glycosaminoglycan attachment sites are located in the middle of the protein. The carboxy-terminal portion includes two epidermal growth factor (EGF)-like repeats, a lectin-like sequence and a complement regulatory protein-like domain. The same set of binding elements has also been identified in a new class of cell adhesion molecules. Amino- and carboxy-terminal portions of the fibroblast core protein are closely related to the core protein of a large chondroitin sulfate proteoglycan of chondrosarcoma cells. However, the glycosaminoglycan attachment regions in the middle of the core proteins are different and only the fibroblast core protein contains EGF-like repeats. Based on the similarities of its domains with various binding elements of other proteins, we suggest that the large fibroblast proteoglycan, herein referred to as versican, may function in cell recognition, possibly by connecting extracellular matrix components and cell surface glycoproteins.     

Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage.

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.     

Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.     

Isolation and characterization of proteoglycans from the swarm rat chondrosarcoma.

Proteoglycan monomer (D1) and aggregate (A1) preparations were isolated from 4 M guanidinium chloride extracts of the Swarm rat chondrosarcoma. When EDTA, 6-aminohexanoic acid, and benzamidine were present in the solutions, the D1 preparation contained a single component (SO = 23 S), and the A1 preparation contained 30% monomer (SO = 23 S) and 70 percent aggregate (SO = 111 S). In the absence of EDTA, 6-aminohexanoic acid, and benzamidine, the A1 preparations contained only small proteoglycan fragments, indicating that extensive enzymatic degradation had occurred. The composition of the proteoglycan monomer was different from that of proteoglycan monomer preparations from normal hyaline cartilages in that it did not contain keratan sulfate and chondroitin 6-sulfate; only chondroitin 4-sulfate was found. The A1 preparation from the chondrosarcoma contained only one link protein, which was like the smaller (molecular weight of 40,000) of the two link proteins present in A1 preparations from bovine nasal cartilage. When the A1 preparation from the chondrosarcoma was treated with chondroitinase ABC and trypsin and the digest was chromatographed on Sepharose 2B, a complex was isolated which contained the link protein and the segments of the protein core from the hyaluronic acid-binding region of the proteoglycan molecules.     

The primary structure of link protein from rat chondrosarcoma proteoglycan aggregate

Cartilage proteoglycan monomers associate with hyaluronic acid to form proteoglycan aggregates. Link protein, a glycoprotein interacting with both hyaluronic acid and proteoglycan, serves to stabilize the aggregate structure. The primary structure of the link protein has been determined with a view to defining its interaction with both hyaluronic acid and proteoglycan. Thus, the link protein has been digested with staphylococcal V8 protease, trypsin, and chymotrypsin and the resulting peptides characterized by amino acid composition and sequence. We have determined that the link protein is a single peptide with 339 amino acid residues. The protein core has a molecular weight of 38,564. There is one N-linked oligosaccharide at residue 41 with a molecular weight of approximately 2,500. There are five disulfide bonds which define three loops within the amino acid sequence. The loop nearest to the NH2-terminal contains 78 amino acids and is followed by a section of 42 amino acids between it and the second loop. The second and third loops display considerable homology with each other; they consist of 71 and 70 amino acids, respectively, each contain two disulfide bonds, and both loops possess, approximately centrally, an epitope for the species nonspecific anti-link protein monoclonal antibody, 8A4. These loops are separated by a short section of 27 amino acids. We speculate that these loops are functionally important in the interaction of link protein with hyaluronic acid, as they appear to be the most conserved regions of link protein between species.     

Biosynthesis and cell-free translation of Swarm rat chondrosarcoma and bovine cartilage link proteins

In cartilage, link protein(s) (LP) stabilize proteoglycan aggregates via their specific association with hyaluronic acid and proteoglycan monomers. Two major link glycoproteins are produced in bovine articular cartilage, designated LP1 (49.5 kDa) and LP2 (44.0 kDa), whereas rat chondrosarcoma produces a single link protein species similar in size to bovine LP2. Although multiple link proteins differ to a significant degree in carbohydrate content, it is not known whether they arise from variable glycosylation of a single common protein core or from complete glycosylation of different protein cores. Biosynthesis of these molecules has been studied under conditions where differences generated by N-linked glycosylation would not be evident. Link proteins were immunoprecipitated 1) from cell-free translation products of total cellular and size fractionated RNA and 2) from cell lysates and medium of cultured chondrocytes using short term radioactive labeling of the protein in the presence and absence of tunicamycin. A 42-kDa link protein precursor is synthesized by cell-free translation of either rat chondrosarcoma or bovine chondrocyte mRNa. An apparently single 41.5-kDa link protein is synthesized with inhibition of N-linked glycosylation by tunicamycin, whereas LP1 and LP2 are the mature products of cultured bovine chondrocytes. The size range of translatable rat chondrosarcoma LP mRNA is 4.0-5.5 kilobase pairs and bovine LP mRNA is 3.0-4.5 kilobase pairs, both much larger than required to encode the link protein molecule. These results suggest that a single link protein precursor gives rise to multiple fully glycosylated forms and that link protein is not synthesized as a significantly larger "pro" form.     

Isolation and characterization of a link protein from bovine aorta proteoglycan aggregate

Proteoglycan aggregates were isolated from bovine aorta by extraction with 0.5 M guanidine hydrochloride in the presence of proteinase inhibitors and purified by isopycnic CsCl centrifugation. The bottom two-fifths (A1) of the gradient contained 30% of proteoglycans in the aggregated form. The aggregate had 14.8% protein and 20.4% hexuronic acid with hyaluronic acid, dermatan sulfate and chondroitin sulfates in a proportion of 18:18:69. A link protein-containing fraction was isolated from the bottom two-fifths by dissociative CsCl isopycnic centrifugation. The link protein that floated to the top one-fifth of the gradient was purified by chromatography on Sephadex G-200 in the presence of 4 M guanidine hydrochloride. It moved as a single band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 49 000. The amino acid composition of link protein resembled that of link protein from cartilage, but was strikingly different from that of the protein core of the proteoglycan monomer. The neutral sugar content of link protein was 3.5% of dry weight. Galactose, mannose and fucose constituted 21, 62 and 16%, respectively of the total neutral sugars. In aggregation studies the link protein was found to interact with both proteoglycan monomer and hyaluronic acid. Oligosaccharides derived from hyaluronic acid decreased the viscosity of link protein-free aggregates of proteoglycan and hyaluronic acid but not of link-stabilized aggregates, demonstrating that the link protein increases the stability of proteoglycan aggregates.