Topographic localization of a 116,000-dalton protein in cartilage

A disulfide-bonded greater than 400,000-dalton (greater than 400-kD) protein with 116-kD subunits in hyaline cartilage from several species has recently been described. It constitutes 2-4% of the total noncollagenous protein in 4 M guanidinium chloride extracts of normal articular cartilage and accounts for most of the total noncollagen, nonproteoglycan protein synthesized in short-term organ cultures of canine articular cartilage. In the present study, immunofluorescence techniques were used to examine the topographic distribution of the 116-kD subunit protein in normal cartilage. In specimens of normal adult articular cartilage from several species, the protein was located throughout the matrix. More intense staining was observed at the articular surface than in the remainder of the uncalcified cartilage. In contrast, in fetal cartilage, the protein was uniformly distributed throughout the matrix without a marked increase in surface staining. Normal canine menisci and annulus fibrosus also demonstrated moderate fluorescence after incubation with the antiserum to the 116-kD subunit protein. Normal canine nucleus pulposus, synovium, aorta, and monolayer cultures of canine synovial cells exhibited only weak immunofluorescence after incubation with the antiserum. Therefore, the 116-kD subunit protein appears to be a ubiquitous matrix protein in cartilage.     

Identification of a high-molecular-weight (> 400 000) protein in hyaline cartilage

A high-molecular-weight (> 400 000) non-collagenous protein has been identified in normal articular cartilage from several mammalian species and in bovine tracheal cartilage. This protein is reduced by 2-mercaptoethanol to subunits with a molecular weight of 116 000, which appear to constitute approx. 2–4% of the total protein detectable by the Lowry assay in 4 M guanidinium chloride extracts of normal bovine and canine articular cartilage. Antiserum to the 116 kDa subunit protein from bovine articular cartilage cross-reacts with the intact and subunit proteins from bovine trachea and from normal canine, porcine and human articular cartilage. This protein is not found in non-cartilagenous tissues, suggesting that it is a cartilage-specific protein. We conclude that the > 400 kDa protein and its subunit are ubiquitous and quantitatively significant proteins in hyaline cartilage.     

Identification of a high-molecular-weight (greater than 400 000) protein in hyaline cartilage

A high-molecular-weight (greater than 400 000) non-collagenous protein has been identified in normal articular cartilage from several mammalian species and in bovine tracheal cartilage. This protein is reduced by 2-mercaptoethanol to subunits with a molecular weight of 116 000, which appear to constitute approx. 2-4% of the total protein detectable by the Lowry assay in 4 M guanidinium chloride extracts of normal bovine and canine articular cartilage. Antiserum to the 116 kDa subunit protein from bovine articular cartilage cross-reacts with the intact and subunit proteins from bovine trachea and from normal canine, porcine and human articular cartilage. This protein is not found in non-cartilagenous tissues, suggesting that it is a cartilage-specific protein. We conclude that the greater than 400 kDa protein and its subunit are ubiquitous and quantitatively significant proteins in hyaline cartilage.     

Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues

Introduction

The small leucine-rich proteoglycans (SLRPs) modulate tissue organization, cellular proliferation, matrix adhesion, growth factor and cytokine responses, and sterically protect the surface of collagen type I and II fibrils from proteolysis. Catabolism of SLRPs has important consequences for the integrity of articular cartilage and meniscus by interfering with their tissue homeostatic functions.

Methods

SLRPs were dissociatively extracted from articular cartilage from total knee and hip replacements, menisci from total knee replacements, macroscopically normal and fibrillated knee articular cartilage from mature age-matched donors, and normal young articular cartilage. The tissue extracts were digested with chondroitinase ABC and keratanase-I before identification of SLRP core protein species by Western blotting using antibodies to the carboxyl-termini of the SLRPs.

Results

Multiple core-protein species were detected for all of the SLRPs (except fibromodulin) in the degenerate osteoarthritic articular cartilage and menisci. Fibromodulin had markedly less fragments detected with the carboxyl-terminal antibody compared with other SLRPs. There were fewer SLRP catabolites in osteoarthritic hip than in knee articular cartilage. Fragmentation of all SLRPs in normal age-matched, nonfibrillated knee articular cartilage was less than in fibrillated articular cartilage from the same knee joint or total knee replacement articular cartilage specimens of similar age. There was little fragmentation of SLRPs in normal control knee articular cartilage. Only decorin exhibited a consistent increase in fragmentation in menisci in association with osteoarthritis. There were no fragments of decorin, biglycan, lumican, or keratocan that were unique to any tissue. A single fibromodulin fragment was detected in osteoarthritic articular cartilage but not meniscus. All SLRPs showed a modest age-related increase in fragmentation in knee articular and meniscal cartilage but not in other tissues.

Conclusion

Enhanced fragmentation of SLRPs is evident in degenerate articular cartilage and meniscus. Specific decorin and fibromodulin core protein fragments in degenerate meniscus and/or human articular cartilage may be of value as biomarkers of disease. Once the enzymes responsible for their generation have been identified, further research may identify them as therapeutic targets.     

A comparison of isometallothionein synthesis in rat liver after partial hepatectomy and parenteral zinc injection.

Proteoglycan aggregates free of non-aggregating proteoglycan have been prepared from the annuli fibrosi and nuclei pulposi of intervertebral discs of three human lumbar spines by extraction with 4M-guanidinium chloride, associative density gradient centrifugation, and chromatography on Sepharose CL-2B. The aggregate (A1-2B.V0) was subjected to dissociative density-gradient ultracentrifugation. Three proteins of Mr 38 900, 44 200 and 50 100 found in the fraction of low buoyant density (A1-2B.V0-D4) reacted with antibodies to link protein from newborn human articular cartilage. After reduction with mercaptoethanol, two proteins of Mr 43 000 and two of Mr 20 000 and 14 000 were seen. The A1-2B.V0-D4 fraction, labelled with 125I, coeluted with both hyaluronate and a hyaluronate oligosaccharide (HA14) on a Sepharose CL-2B column. HA10 and HA14 reduced the viscosity of A1 fractions; HA4, HA6 and HA8 did not. HA14 decreased the viscosity of disc proteoglycans less than it did that of bovine cartilage proteoglycans. Thus, although a link protein was present in human intervertebral disc, it stabilized proteoglycan aggregates less well than did the link protein from bovine nasal cartilage.     

Identification of link proteins in canine synovial cell cultures and canine articular cartilage

Link proteins are glycoproteins in cartilage that are involved in the stabilization of aggregates of proteoglycans and hyaluronic acid. We have identified link proteins in synovial cell cultures form normal canine synovium using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunofluorescence, and immunolocation with specific antibodies by electrophoretic transfer. We have also found evidence for the synthesis of link proteins in these cultures by fluorography of radiolabeled synovial cell extracts. We have identified a 70,000 mol-wt protein in canine synovial cell culture extracts that has antigenic cross-reactivity with the 48,000-mol-wt link protein. Three link proteins were identified in normal canine articular cartilage. These results indicate that link proteins are more widely distributed in connective tissues than previously recognized and may have biological functions other than aggregate stabilization.     

Biochemical analysis of normal and osteoarthritic human cartilage

Non-collagenous proteins from the articular cartilage of normal subjects and patients with degenerative joint disease were extracted sequentially. Proteoglycans and the other glycoproteins were more extractable from the osteoarthritic cartilage at lower ionic strength than those from the normal cartilage. A 50-kD protein which seems specific to osteoarthritic cartilage was identified. Three different populations of proteoglycans were purified from normal and only two from osteoarthritic cartilage. Moreover, greater amounts of albumin and fibronectin were found in the pathological cartilage. No differences were observed between link proteins from normal and osteoarthritic cartilage, nor in their molecular weight or the amounts extracted.     

Partial characterization of matrix components interacting with cartilage proteoglycans

The charge content of aqueous suspensions of milled cartilage samples was determined by a colloid titration technique using a particle charge detector, and the data were compared with estimates from chemical analyses. Results indicated a close correlation between charge content determined by titration and that estimated by chemical analyses for samples of nasal septa only (a nonarticular cartilage). Such correlation did not hold for articular cartilages (metacarpalphalangeal joint and patella); extraction of these tissues with 0.1 or 1.2 M NaCl markedly increased the availability of the negative groups. Protein analysis, by SDS--PAGE, of the 1.2 M extracts indicated the presence of basic proteins, some of collagenous origin, such as chondrocalcin and proline-arginine-rich protein, and some of noncollagenous proteins such as pleiotrophin and histone-H2b. These data thus suggest electrostatic interactions between these basic proteins and the negative groups of proteoglycans. Such interactions would have an important effect on the osmotic properties and in the organization of cartilage.     

Immunochemistry of cartilage proteoglycan. Immunodiffusion and gel-electrophoretic studies

Cartilage proteoglycan is thought to be composed of subunits, core proteins with covalently attached sulphated polysaccharide side chains, which form aggregates by non-covalent association with a link protein. The new technique of non-disruptive extraction followed by fractionation in caesium chloride gradients provides a useful means of preparing relatively pure proteoglycan aggregate, subunit and link fractions. Immunological studies of these fractions led to the identification of an antigen associated with the proteoglycan subunit which was common to several species and to the demonstration of additional species-specific antigens in aggregate and link fractions derived from bovine nasal cartilage. Polyacrylamide-gel electrophoresis with sodium dodecyl sulphate of bovine proteoglycan aggregate and link fractions gave two protein bands in the gels and a protein-polysaccharide band at the origin; subunit fractions gave only the band at the origin. These results are consistent with the current concept of cartilage proteoglycan structure.     

Degradation of proteoglycan in articular cartilage.

Adult rabbit articular cartilage was labelled in vivo over 48 h with [35S]sulphate and was then incubated in organ culture at pH 7.2. Approx. 65% of the tissue content of [35S]proteoglycan was released into the culture medium during the first 48 h of incubation. The average molecular size of the released proteoglycans, as assessed by fractionation on Sepharose 2B/CL and 4B/Cl, was only slightly smaller than that of the proteoglycans extracted from non-cultured cartilage with 4 M guanidine HCl. The percentage of released proteoglycans and extracted proteoglycans which formed aggregates with hyaluronic acid was approx. 25% and 75%, respectively. The results indicate that proteoglycan degradation in adult articular cartilage is initiated by a limited proteolysis of subunit core protein, with the production of non-aggregating species which diffuse readily from the tissue.     

The macromolecular characteristics of cartilage proteoglycans do not change when synthesis is up-regulated by link protein peptide.

Previous studies have shown that a synthetic, unglycosylated analogue of the N-terminal peptide from link protein can function as a growth factor and up-regulate proteoglycan biosynthesis in explant cultures of normal human articular cartilage from a wide age range of subjects (McKenna et al., Arthritis Rheum. 41 (1998) 157-162). The present work further shows that link peptide increased proteoglycan synthesis by cartilage cultured in both the presence and absence of serum, suggesting that the mechanism of up-regulation may be different from that of insulin-like growth factors. The proteoglycans synthesised during stimulation with link peptide were of normal hydrodynamic size and the ratio of core protein to glycosaminoglycan side chains and the proportions of the large proteoglycan aggrecan to the small proteoglycans, decorin and biglycan, remained constant. Aggrecan molecules were equally capable of forming aggregates as those from control tissues and the relative proportions of decorin and biglycan were unchanged showing that both were co-ordinately up-regulated. These results confirmed that this novel peptide is a potent stimulator of proteoglycan synthesis by articular cartilage and showed that the newly synthesised proteoglycans were of normal composition.     

An N-terminal peptide from link protein can stimulate biosynthesis of collagen by human articular cartilage

Previous studies have shown that a peptide identical in sequence to the N-terminal of link protein can function as a growth factor and up-regulate proteoglycan synthesis by human articular cartilage in explant culture (L. A. McKenna et al., Arthritis Rheum. 41, 157-162, 1998). The present study has extended these investigations to determine the effects of this peptide on the synthesis of collagen, another essential component of normal cartilage matrix. Explants from normal adult knee cartilage were maintained for periods of up to 8 days in medium with or without serum. Peptides were added during each day of culture. Synthesis of collagen was determined by the incorporation of [3H]proline into hydroxyproline and proteoglycans by incorporation of [35S]sulfate. The type of newly synthesized collagen was measured by SDS-polyacrylamide gel electrophoresis, fluorography, and immunoblotting. The link protein peptide stimulated synthesis of type II collagen in cartilage from a number of different subjects. Maximum up-regulation of synthesis was attained at a concentration of 100 ng/ml, similar to that observed previously for up-regulation of proteoglycan. Synthesis was up-regulated in both the presence and the absence of serum, although the overall rate of synthesis was greater when serum was added. The findings that this link peptide growth factor stimulated synthesis of proteins, including collagen, in a manner analogous to that shown previously for proteoglycans support the hypothesis that this peptide may have an important role in the feedback control of cartilage matrix synthesis.     

The dermatan sulfate proteoglycans of bovine sclera and their relationship to those of articular cartilage. An immunological and biochemical study

Dermatan sulfate proteoglycans were isolated from adult bovine sclera and adult bovine articular cartilage. Their immunological relationships were studied by enzyme-linked immunosorbent assays using polyclonal antibodies raised against the large and small dermatan sulfate proteoglycans from sclera and a polyclonal and monoclonal antibody directed against the small dermatan sulfate proteoglycans from cartilage. The small dermatan sulfate proteoglycans from sclera and cartilage displayed immunological cross-reactivity while there was no convincing evidence of shared epitope(s) with the larger dermatan sulfate proteoglycans, nor did these larger proteoglycans share any common epitopes with each other. A hyaluronic acid binding region was detected immunologically on the larger scleral dermatan sulfate proteoglycan but was absent from the larger dermatan sulfate proteoglycan of cartilage and both the small dermatan sulfate proteoglycans. These antibodies were used in immunofluorescence microscopy to localize the scleral proteoglycans and molecules containing these epitopes in the eye. The large scleral dermatan sulfate proteoglycan was restricted to sclera while molecules related to the small scleral and cartilage proteoglycans were found in the sclera, anterior uveal tract, iris, and cornea. Amino acid sequencing of the amino-terminal regions of the core proteins of the small dermatan sulfate proteoglycans from sclera and articular cartilage showed that all the first 14 amino acids analyzed were identical and the same as reported earlier for the small bovine skin and tendon dermatan sulfate proteoglycans. These studies demonstrate that the larger dermatan sulfate proteoglycans of sclera and cartilage are chemically unrelated to each other and to the smaller dermatan sulfate proteoglycans isolated from these tissues. The latter have closely related core proteins and probably represent a molecule with a widespread distribution in which the degree of epimerization of glucuronic acid and iduronic acid varies between tissues.     

Degradation of proteoglycan in articular cartilage

Adult rabbit articular cartilage was labelled in vivo over 48 h with [³⁵S]sulphate and was then incubated in organ culture at pH 7.2. Approx. 65% of the tissue content of [³⁵S]proteoglycan was released into the culture medium during the first 48 h of incubation. The average molecular size of the released proteoglycans, as assessed by fractionation on Sepharose 2B/CL and 4B/Cl, was only slightly smaller than that of the proteoglycans extracted from non-cultured cartilage with 4 M guanidine HCl. The percentage of released proteoglycans and extracted proteoglycans which formed aggregates with hyaluronic acid was approx. 25% and 75%, respectively. The results indicate that proteoglycan degradation in adult articular cartilage is initiated by a limited proteolysis of subunit core protein, with the [roduction of non-aggregating species which diffuse readily from the tissue.     

Passive loss of proteoglycan from articular cartilage explants

The addition of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 0.25 mM benzamidine hydrochloride, 6.25 mM EDTA, 12.5 mM 6-aminohexanoic acid and 2 mM iodoacetic acid) to explant cultures of adult bovine articular cartilage inhibits proteoglycan synthesis as well as the loss of the macromolecule from the tissue. Those proteoglycans lost to the medium of explant cultures treated with proteinase inhibitors were either aggregates or monomers with functional hyaluronic acid-binding regions, whereas proteoglycans lost from metabolically active tissue also included a population of monomers that were unable to aggregate with hyaluronate. Analysis of the core protein from proteoglycans lost into the medium of inhibitor-treated cultures showed the same size distribution as the core proteins of proteoglycans present in the extracellular matrix of metabolically active cultures. The core proteins of proteoglycans appearing in the medium of metabolically active cultures showed that proteolytic cleavage of these macromolecules occurred as a result of their loss from the tissue. Explant cultures of articular cartilage maintained in medium with proteinase inhibitors were used to investigate the passive loss of proteoglycan from the tissue. The rate of passive loss of proteoglycan from the tissue was dependent on surface area, but no difference in the proportion of proteoglycan aggregate to monomer appearing in the medium was observed. Furthermore, proteoglycans were lost at the same rate from the articular and cut surfaces of cartilage. Proteoglycan aggregates and monomer were lost from articular cartilage over a period of time, which indicates that proteoglycans are free to move through the extracellular matrix of cartilage. The movement of proteoglycans out of the tissue was shown to be temperature dependent, but was different from the change of the viscosity of water with temperature, which indicates that the loss of proteoglycan was not solely due to diffusion. The activation energy for the loss of proteoglycans from articular cartilage was found to be similar to the binding energies for electrostatic and hydrogen bonds.     

Characterization of the dermatan sulfate proteoglycans, DS-PGI and DS-PGII, from bovine articular cartilage and skin isolated by octyl-sepharose chromatography

Two forms of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from both bovine fetal skin and calf articular cartilage and characterized. The proteoglycans were isolated using either (a) molecular sieve chromatography under conditions where DS-PGI selectively self-associates or (b) chromatography on octyl-Sepharose, which separates DS-PGI from DS-PGII based on differences in the hydrophobic properties of their core proteins. The NH2-terminal amino acid sequence of DS-PGI from skin and cartilage is identical. The NH2-terminal amino acid sequence of DS-PGII from skin and cartilage is identical. However, the amino acid sequence data and tryptic peptide maps demonstrate that the core proteins of DS-PGI and DS-PGII differ in primary structure. In DS-PGI from bovine fetal skin, 81-84% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4) disaccharide repeating units. In DS-PGI from calf articular cartilage, only 25-29% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4). In DS-PGII from bovine fetal skin, 85-93% of the glycosaminoglycan was IdoA-GalNAc(SO4), whereas in DS-PGII from calf articular cartilage, only 40-44% of the glycosaminoglycan was IdoA-GalNAc(SO4). Thus, analogous proteoglycans from two different tissues, such as DS-PGI from skin and cartilage, possess a core protein with the same primary structure, yet contain glycosaminoglycan chains which differ greatly in iduronic acid content. These differences in the composition of the glycosaminoglycan chains must be determined by tissue-specific mechanisms which regulate the degree of epimerization of GlcA-GalNAc(SO4) into IdoA-GalNAc(SO4) and not by the primary structure of the core protein.     

Articular cartilage cultured with interleukin 1. Increased release of link protein, hyaluronate-binding region and other proteoglycan fragments.

Pig articular cartilage was maintained in culture for 3 days with and without porcine interleukin 1. The proteoglycans remaining in the cartilage and those released into the medium were analysed by using radioimmunoassays for the hyaluronate-binding region, link protein and keratan sulphate. In interleukin 1-treated cultures after 3 days there was 38% release of total glycosaminoglycans into the medium, 18% release of binding region, 14% release of link protein and 20% release of keratan sulphate epitope, whereas in control cultures the proportions released were much less (16, 9, 10 and 7% respectively). Characterization of the proteoglycans in the media after 1.5 days and 3 days of culture showed that interleukin 1 promoted the release of proteoglycan of large average size and also the release of link protein and of low-Mr binding region which was unattached to proteoglycan. Both the link protein and binding region released were able to bind to exogenously added hyaluronate, whereas the proteoglycan in the medium was not. The proteoglycans extracted from cultured cartilage were similar to those from fresh cartilage: they contained a high proportion of aggregating proteoglycans and some low-Mr binding region. The proportion of this binding region extracted from the interleukin 1-treated cartilage was increased. The presence of interleukin 1 in the cultures therefore appeared to increase the rate of proteolytic degradation of proteoglycan in the matrix and to lead to a more rapid loss of intact binding region, of link protein and of large proteoglycan fragments into the medium.     

Nonenzymatic glycation of cartilage proteoglycans: an in vivo and in vitro study

In this study we have investigated whether proteoglycans (aggrecan) are modified by nonenzymatic glycation as in collagen. Purified human aggrecan from osteoarthritic and normal human knee articular cartilage was assayed for pentosidine, a cross-link formed by nonenzymatic glycation, using reverse-phase HPLC. In addition, an in vitro study was done by incubation of purified bovine nasal cartilage aggrecan with ribose. Pentosidine was found in all the purified human aggrecan samples. 2-3% of the total articular cartilage pentosidine was found in aggrecan. Purified link protein also contained penosidine. The in vitro study led to pentosidine formation, but did not appear to increase the molecular size of the aggrecan suggesting that pentosidine was creating intramolecular cross-links. Similar amounts of glycation were found in osteoarthritic and normal cartilage. Like collagen, aggrecan and link proteins are crosslinked by nonenzymatic glycation in normal and osteoarthritic cartilage. Crosslinking could be reproduced, in vitro, by incubating aggrecan with ribose. This revised version was published online in August 2006 with corrections to the Cover Date.     

Establishment of a reliable method for direct proteome characterization of human articular cartilage

Articular cartilage consists mainly of extracellular matrix, mostly made of collagens and proteoglycans. These macromolecules have so far impaired the detailed two-dimensional electrophoresis-based proteomic analysis of articular cartilage. Here we describe a method for selective protein extraction from cartilage, which excludes proteoglycans and collagen species, thus allowing direct profiling of the protein content of cartilage by two-dimensional electrophoresis. Consistent electrophoretic patterns of more than 600 protein states were reproducibly obtained after silver staining from 500 mg of human articular cartilage from joints with diverse pathologies. The extraction yield increased when the method was applied to a chondrosarcoma sample, consistent with selective extraction of cellular components. Nearly 200 of the most intensely stained protein spots were analyzed by MALDI-TOF mass spectrometry after trypsin digestion. They represented 127 different proteins with diverse functions. Our method provides a rapid, efficient, and pertinent alternative to previously proposed approaches for proteomic characterization of cartilage phenotypes. It will be useful for detecting protein expression patterns that relate pathophysiological processes of cartilaginous tissues such as osteoarthritis and chondrosarcoma.     

Structure and biology of cartilage and bone matrix noncollagenous macromolecules

Over recent years a number of cartilage and bone matrix molecules have been identified and characterized. These include major constituents such as collagens and proteoglycans as well as a number of less-abundant matrix proteins. In several cases these proteins have been characterized by cloning and sequence analysis of the corresponding cDNA. Some properties of the macromolecules have been studied and an understanding of their functions in the structure, assembly, and breakdown of connective tissue matrix is emerging. It appears that some of these molecules have structural roles whereas others participate in the assembly of the tissue. In this paper we attempt to give a current picture of the organization and role of the noncollagenous matrix macromolecules in cartilage and bone.