Association of the aggrecan keratan sulfate-rich region with collagen in bovine articular cartilage

Aggrecan, the predominant large proteoglycan of cartilage, is a multidomain macromolecule with each domain contributing specific functional properties. One of the domains contains the majority of the keratan sulfate (KS) chain substituents and a protein segment with a proline-rich hexapeptide repeat sequence. The function of this domain is unknown but the primary structure suggests a potential for binding to collagen fibrils. We have examined binding of aggrecan fragments encompassing the KS-rich region in a solid-phase assay. A moderate affinity (apparent Kd = 1.1 microM) for isolated collagen II, as well as collagen I, was demonstrated. Enzymatic digestion of the KS chains did not alter the capacity of the peptide to bind to collagen, whereas cleavage of the protein core abolished the interaction. The distribution of the aggrecan KS-rich region in bovine tarsometatarsal joint cartilage was investigated using immunoelectron microscopy. Immunoreactivity was relatively low in the superficial zone and higher in the intermediate and deep zones of the uncalcified cartilage. Within the pericellular and territorial matrix compartments the epitopes representing the aggrecan KS-rich region were detected preferentially near or at collagen fibrils. Along the fibrils, epitope reactivity was non-randomly distributed, showing preference for the gap region within the D-period. Our data suggest that collagen fibrils interact with the KS-rich regions of several aggrecan monomers aligned within a proteoglycan aggregate. The fibril could therefore serve as a backbone in at least some of the aggrecan complexes.     

Immunological studies of sheep brain keratan sulphate proteoglycans

Recently, we reported the isolation and partial characterization of keratan sulphate (KS) from sheep brain. In this study, a panel of monoclonal antibodies (Mab) recognizing epitopes within KS chains and core proteins of KS-containing proteoglycans were used to detect, by immunoblotting, antigenically related molecules extracted from cerebrum, cerebellum and brainstem, respectively. Although the intensity of labelling varied with each of the antibodies, the brain KSPGs were recognized by all the monoclonals used, confirming the presence of KS side chains, which react with the Mabs: 5-D-4, EFG-11, EFG-4, I22, as also the presence of KSPGs related to phosphacan-KS (3H1 proteoglycan). Extracts of all the three brain areas could bind both anti-KS and anti-core protein Mabs, as also anti-HNK-1 monoclonal antibody. Binding was sensitive to keratanases degradation in the cerebrum and brainstem except cerebellum where the presence of a large molecular size hybrid CS/KSPG bearing KS chains partially resistant to keratanases was identified. This population reacts only with 5-D-4, EFG-11 and EFG-4 antibodies. Furthermore, the presence of HNK-1 epitope in CSPGs was detected in the cerebellum and brainstem. In contrast, in the cerebrum the coexistence of HNK-1 epitope and KS in KSPGs was identified. These data suggest that the KSs of sheep brain are part of proteoglycans containing protein and KS antigenic sites related to those of corneal and cartilage KSPG, as also of the brain proteoglycan phosphacan-KS.     

Structural studies of two populations of keratan sulphate chains from mature bovine articular cartilage

Two discrete peptido-keratan sulphate fragments were isolatedvia chondroitinase ABC and trypsin digestion of a proteoglycan aggregate fraction prepared from bovine femoral head cartilage (six year old animals). The larger fragments (K_av=0.07, CL-6B) contained peptides substituted with several keratan sulphate (KS) chains from the KS-rich region of the proteoglycan and the smaller fragments (K_av=0.5, CL-6B) contained peptides with, perhaps, only one KS chain and the stubs of post-chondroitinase-treated chondroitin sulphate chains.The two peptido-KS samples and the KS chains derived from these by alkaline borohydride reduction were characterised by¹³C-NMR spectroscopy. The two populations of KS chains were also examined by chromatography (Sephadex G-75), and keratanase digestion followed by chromatography on Bio-Gel P-10. From the results it was concluded that the KS chains from the two major trypsin-derived peptido-KS fragments had similar sulphation levels, distributions of hydrodynamic sizes and susceptibilities to keratanase.Abbreviations KS keratan sulphate - A1 proteoglycan aggregate - T diphenyl carbamyl chloride (DPCC)-trypsintreated - CB chondroitinase ABC-treated - C chymotrypsin-treated - P papain-treated - R alkaline borohydride-reduced - TSP sodium 3-trimethylsilylpropionate     

Mammalian expression of full-length bovine aggrecan and link protein: formation of recombinant proteoglycan aggregates and analysis of proteolytic cleavage by ADAMTS-4 and MMP-13

Aggrecan, a large chondroitin sulfate (CS) and keratan sulfate (KS) proteoglycan, has not previously been expressed as a full-length recombinant molecule. To facilitate structure/function analysis, we have characterized recombinant bovine aggrecan (rbAgg) and link protein expressed in COS-7 cells. We demonstrate that C-terminally truncated rbAgg was not secreted. Gel filtration chromatography of rbAgg and isolated glycosaminoglycan (GAG) chains, and their susceptibility to chondroitinase ABC digestion indicate that the GAG chains are predominantly CS, which likely occupy fewer serine residues than native aggrecan. To confirm functionality, we determined that rbAgg bound hyaluronan and recombinant link protein to form proteoglycan aggregates. In addition, cleavage of rbAgg by ADAMTS-4 revealed that the p68 form of ADAMTS-4 preferentially cleaves within the CS-2 domain, whereas the p40 form only effectively cleaves within the interglobular domain (IGD). MMP-13 cleaved rbAgg within the IGD, but cleaved more rapidly at a site within the CS domains, suggesting a role in C-terminal processing of aggrecan. Our results demonstrate that recombinant aggrecan can be used for in vitro analyses of matrix protease-dependent degradation of aggrecan in the IGD and CS domains, and both recombinant aggrecan and link protein can be used to study the assembly of proteoglycan aggregates with hyaluronan.     

Studies of a monoclonal antibody to skeletal keratan sulphate. Importance of antibody valency.

A mouse monoclonal antibody (AN9P1) to keratan sulphate is described. In a competitive-inhibition solution-phase radioimmunoassay employing 125I-labelled intact proteoglycan, it reacts preferentially with keratan sulphate bound to the core protein of adult human articular-cartilage proteoglycan and to a much lesser degree with keratan sulphate purified from this proteoglycan. Proteolytic cleavage of the proteoglycan by pepsin and trypsin has little effect on antibody binding, but treatment with papain decreases binding considerably and more than does treatment with keratanase. An even greater decrease in binding is observed after treatment with alkaline borohydride. A comparison of binding of antibody AN9P1 with that of another previously described monoclonal antibody, 1/20/5-D-4, to keratan sulphate [Caterson, Christner & Baker (1983) J. Biol. Chem. 258, 8848-8854] revealed similar binding characteristics, both showing much diminished binding after papain digestion of proteoglycan and even less with purified skeletal keratan sulphate. Removal of the Fc piece of antibody AN9P1 had no significant effect on the differential binding of divalent F(ab')2 fragment to proteoglycan, to papain-digested proteoglycan and to keratan sulphate, although there was a small decrease in binding to papain-digested proteoglycan. Conversion of the antibody into univalent Fab fragment with removal of the Fc piece resulted in diminished binding to proteoglycan, compared with that observed with IgG, and in enhanced binding to free keratan sulphate and to papain-digested proteoglycan. These results suggest that close proximity of keratan sulphate chains on the core protein of proteoglycans favours preferential reactivity of bivalent antibody with these species through cross-bridging of chains by antibody. Conversely, much decreased binding to keratan sulphate on proteoglycan core-protein fragments and to free keratan sulphate results from a lack of close proximity of keratan sulphate. By using univalent Fab fragment in these assays these differences in binding are minimized by preventing cross-bridging and thereby enhancing detection of smaller fragments without sacrificing too much sensitivity of detection of larger proteoglycan species. The persistent preferential binding of Fab fragment to proteoglycan is probably in part the result of the increased epitope density in the intact molecule compared with keratan sulphate in a more disperse form.     

MMP proteolysis of the human extracellular matrix protein aggrecan is mainly a process of normal turnover

Although it has been shown that aggrecanases are involved in aggrecan degradation, the role of MMP (matrix metalloproteinase) aggrecanolysis is less well studied. To investigate MMP proteolysis of human aggrecan, in the present study we used neoepitope antibodies against MMP cleavage sites and Western blot analysis to identify MMP-generated fragments in normal and OA (osteoarthritis/osteoarthritic) cartilage, and in normal, knee injury and OA and SF (synovial fluid) samples. MMP-3 in vitro digestion showed that aggrecan contains six MMP cleavage sites, in the IGD (interglobular domain), the KS (keratan sulfate) region, the border between the KS region and CS (chondroitin sulfate) region 1, the CS1 region, and the border between the CS2 and the G3 domain, and kinetic studies showed a specific order of digestion where the cleavage between CS2 and the G3 domain was the most preferred. In vivo studies showed that OA cartilage contained (per dry weight) 3.4-fold more MMP-generated FFGV fragments compared with normal cartilage, and although aggrecanase-generated SF-ARGS concentrations were increased 14-fold in OA and knee-injured patients compared with levels in knee-healthy reference subjects, the SF-FFGV concentrations did not notably change. The results of the present study suggest that MMPs are mainly involved in normal aggrecan turnover and might have a less-active role in aggrecan degradation during knee injury and OA.     

Expression of highly sulfated keratan sulfate synthesized in human glioblastoma cells

Keratan sulfate (KS) proteoglycan is expressed in the extracellular matrix or cell surface in numerous tissues, predominantly in those of the cornea, cartilage, and brain. However, its structure, function, and regulation remain poorly understood. Our investigation of KS expression in glioblastoma cell lines using Western-blot and flow cytometry with anti-KS antibody (5D4) revealed that LN229 glioblastoma cell highly expresses KS on a cell surface. Real-time PCR analysis showed that LN229 expresses a high level of keratan sulfate Gal-6-sulfotransferase. Results of this study also demonstrate that recombinant 5D4-reactive aggrecan is produced in LN229. Taken together, these results suggest that LN229 produces 5D4-reactive highly sulfated KS and is useful to investigate the KS structure and function in glioblastoma.     

Detection of Sendai virus antibody in mouse and guinea pig sera by an enzyme-linked immunosorbent assay with protein A

An enzyme-linked immunosorbent assay using horseradish peroxidase (HRPO)-labeled protein A (P-ELISA) was established for detection of Sendai virus (SV) antibody in mouse and guinea pig sera. Sensitivity and specificity of P-ELISA were compared with those of ordinary ELISA using HRPO-labeled immunoglobulin G (IgG-ELISA) and the hemagglutination inhibition (HI) test. P-ELISA was 100 to 1,000 times more sensitive than the HI test for detection of the antibody in SV-naturally infected mice. P-ELISA and IgG-ELISA showed similar sensitivities for detection of the antibody in naturally infected mouse and guinea pig sera. A high specificity was demonstrated in P-ELISA with a cut-off optical density value of 0.2 (492 nm), while a non-specific reaction was observed when IgG-ELISA was used to both mouse and guinea pig sera at a low dilution (1:10-20). The antibody in rat sera was not detected by P-ELISA although it was realized by IgG-ELISA.     

Keratan sulfate suppresses cartilage damage and ameliorates inflammation in an experimental mice arthritis model

Proteoglycans bearing keratan sulfate (KS), such as aggrecan, are components of the human cartilage extracellular matrix (ECM). However, the role of KS in influencing cartilage degradation associated with arthritis remains to be completely understood. KS side chains of the length found in human cartilage are not found in murine skeletal tissues. Using a murine model of inflammatory polyarthritis and cartilage explants exposed to interleukin-1α (IL-1α), we examined whether administering KS could influence intraarticular inflammation and cartilage degradation. Acute arthritis was induced by intravenous administration of an anti-type II collagen antibody cocktail, followed by an intraperitoneal injection of lipopolysaccharide. This treatment was followed by an intraperitoneal KS administration in half of the total mice to evaluate the therapeutic potential of KS for ameliorating arthritis. To investigate the therapeutic potential ex vivo, we examined cartilage fragility by measuring IL-1α-induced aggrecan release from cartilage explants treated with or without KS. Intraperitoneal KS administration ameliorated arthritis in DBA/1J mice. The aggrecan release induced by IL-1α was less in cartilage explants containing media with KS than in those without KS. Our data indicate that exogenous KS ameliorated arthritis in vivo and suppressed cartilage degradation ex vivo. KS may have important therapeutic potential in the treatment of inflammatory arthritis. The mechanism responsible for this requires further investigation, but KS may become a novel therapeutic agent for treating inflammatory diseases such as rheumatoid arthritis.     

Autoantibodies against aggrecan in systemic rheumatic diseases

OBJECTIVE: This study was undertaken to investigate the presence of autoantibodies against the main cartilage proteoglycan, aggrecan, in systemic rheumatic disease sera, and to identify substructure(s) responsible for the autoimmune response. METHODS: Sera were obtained from 86 patients with various systemic rheumatic diseases, 14 with osteoarthritis (OA), 18 with cancer and 40 healthy individuals. The presence of autoantibodies against aggrecan was examined by a solid phase assay and by Western blotting, using proteoglycan aggregates treated with proteolytic enzymes. The positive bands were subjected to nanohigh performance liquid chromatography (nanoHPLC)-MS, in order to identify the aggrecan substructures involved in the autoimmune response. RESULTS: Autoantibodies against aggrecan were identified in all systemic rheumatic disease sera at a high titre, almost three times that observed in healthy controls. OA and cancer sera produced a reaction equal to that of the healthy. Western blotting analysis of aggrecan proteolytic fragments revealed the presence of a triple band, reacting with the patients' sera, of about 37 kDa, which also reacted with a polyclonal antibody against hyaluronan-binding region. NanoHPLC-MS analysis suggested that this band belonged to the G2 domain of aggrecan. CONCLUSION: At least a part of the autoimmune reaction to aggrecan, displayed by the systemic disease sera, involves the G2 domain. The significant difference observed between these sera and those from other diseases, especially cancer, may suggest a possible discriminatory role of anti-aggrecan antibodies. This may help in the differential diagnosis in complicated clinical cases. However, for this to be confirmed, studies in larger cohorts of patients should be performed.     

Chondroitin Sulfate and Chondroitin/Keratan Sulfate Proteoglycans of Nervous Tissue: Developmental Changes of Neurocan and Phosphacan

Abstract: We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached ～1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-β-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.     

Age-related changes in aggrecan glycosylation affect cleavage by aggrecanase

Aggrecan degradation involves proteolytic cleavage of the core protein within the interglobular domain. Because aggrecan is highly glycosylated with chondroitin sulfate (CS) and keratan sulfate (KS), we investigated whether glycosylation affects digestion by aggrecanase at the Glu(373)-Ala(374) bond. Treatment of bovine aggrecan monomers to remove CS and KS resulted in loss of cleavage at this site, suggesting that glycosaminoglycans (GAGs) play a role in cleavage at the Glu(373)-Ala(374) bond. In contrast, MMP-3 cleavage at the Ser(341)-Phe(342) bond was not affected by glycosidase treatment of aggrecan. Removal of KS, but not CS, prevented cleavage at the Glu(373)-Ala(374) bond. Thus, KS residues may be important for recognition of this cleavage site by aggrecanase. KS glycosylation has been observed at sites adjacent to the Glu(373)-Ala(374) bond in steer aggrecan, but not in calf aggrecan (Barry, F. P., Rosenberg, L. C., Gaw, J. U., Gaw, J. U., Koob, T. J., and Neame, P. J. (1995) J. Biol. Chem. 270, 20516-20524). Interestingly, although we found that aggrecanase degraded both calf and steer cartilage aggrecan, the proportion of fragments generated by cleavage at the Glu(373)-Ala(374) bond was higher in steer than in calf, consistent with our observations using aggrecan treated to remove KS. We conclude that the GAG content of aggrecan influences the specificity of aggrecanase for cleavage at the Glu(373)-Ala(374) bond and suggest that age may be a factor in aggrecanase degradation of cartilage.     

Age-related nanostructural and nanomechanical changes of individual human cartilage aggrecan monomers and their glycosaminoglycan side chains

The nanostructure and nanomechanical properties of aggrecan monomers extracted and purified from human articular cartilage from donors of different ages (newborn, 29 and 38 year old) were directly visualized and quantified via atomic force microscopy (AFM)-based imaging and force spectroscopy. AFM imaging enabled direct comparison of full length monomers at different ages. The higher proportion of aggrecan fragments observed in adult versus newborn populations is consistent with the cumulative proteolysis of aggrecan known to occur in vivo. The decreased dimensions of adult full length aggrecan (including core protein and glycosaminoglycan (GAG) chain trace length, end-to-end distance and extension ratio) reflect altered aggrecan biosynthesis. The demonstrably shorter GAG chains observed in adult full length aggrecan monomers, compared to newborn monomers, also reflects markedly altered biosynthesis with age. Direct visualization of aggrecan subjected to chondroitinase and/or keratanase treatment revealed conformational properties of aggrecan monomers associated with chondroitin sulfate (CS) and keratan sulfate (KS) GAG chains. Furthermore, compressive stiffness of chemically end-attached layers of adult and newborn aggrecan was measured in various ionic strength aqueous solutions. Adult aggrecan was significantly weaker in compression than newborn aggrecan even at the same total GAG density and bath ionic strength, suggesting the importance of both electrostatic and non-electrostatic interactions in nanomechanical stiffness. These results provide molecular-level evidence of the effects of age on the conformational and nanomechanical properties of aggrecan, with direct implications for the effects of aggrecan nanostructure on the age-dependence of cartilage tissue biomechanical and osmotic properties.     

Perlecan from human epithelial cells is a hybrid heparan/chondroitin/keratan sulfate proteoglycan

Perlecan is a multidomain proteoglycan, usually substituted with heparan sulphate (HS), and sometimes substituted with both HS and chondroitin sulphate (CS). In this paper, we describe perlecan purified from HEK-293 cells substituted with HS, CS and keratan sulphate (KS). KS substitution was confirmed by immunoreactivity with antibody 5D4, sensitivity to keratanase treatment, and fluorophore-assisted carbohydrate electrophoresis. HEK-293 perlecan failed to promote FGF-dependent cell growth in an in vitro assay. This study is the first to report perlecan containing KS, and makes perlecan one of only a very few proteoglycans substituted with three distinct types of glycosaminoglycan chains.     

Structural and immunological studies of keratan sulphates from mature bovine articular cartilage.

Two populations of alkaline-borohydride-reduced keratan sulphate (KS) chains were prepared from the two peptido-keratan sulphate trypsin fragments of proteoglycan aggregates isolated from bovine femoral head cartilage (6-year-old animals). Each population was separated by high-performance ion-exchange chromatography on a Pharmacia Mono-Q column into eight pools, Q1-Q8. These were analysed by gel permeation chromatography, radioimmunoassay with the monoclonal antibody MZ15, and 500 MHz 1H n.m.r. spectroscopy. Upon chromatography on Sephadex G-75 the Mono-Q fractions were shown to increase in hydrodynamic size progressively from Q1 to Q8 for both KS populations. For each population the strongest antigenic response with the anti-KS monoclonal antibody MZ15 was expressed by the two fractions of greatest size and charge density, Q7 and Q8. Proton n.m.r. spectroscopic studies on the two series of fractions demonstrated: (i) a progressive increase in the level of galactose sulphation from Q1 to Q8, (ii) the presence of approximately one alpha(1-3)-linked fucose residue per chain in every sample, and (iii) the presence of N-acetylneuraminic acids in three discrete environments, two alpha(2-3)- and one alpha(2-6)-linked in every sample. The results are discussed in terms of a possible heterogeneity in the carbohydrate-protein linkage region of keratan sulphates from bovine articular cartilage.     

Generation and novel distribution of matrix metalloproteinase-derived aggrecan fragments in porcine cartilage explants

We have studied aggrecan catabolism mediated by matrix metalloproteinases (MMPs) in a porcine cartilage culture system. Using antibodies specific for DIPEN(341) and (342)FFGVG neoepitopes, we have detected MMP-derived fragments in conditioned medium and cultured cartilage, by radioimmunoassay, Western blotting, and immunolocalization. Radioimmunoassay revealed that the amount (pmol of epitope/mg of total glycosaminoglycan) of (342)FFGVG epitope released from cartilage remained constant over a 5-day culture period and was not increased by IL-1alpha or retinoate. However, the proportion (pmol of epitope/mg of released glycosaminoglycan) of (342)FFGVG epitope released was decreased upon stimulation, consistent with the involvement of a non-MMP proteinase, such as aggrecanase. The data suggest that in vitro MMPs may be involved in the base-line catabolism of aggrecan. Immunolocalization experiments showed that DIPEN(341) and ITEGE(373) epitopes were increased by treatment with IL-1alpha and retinoate. Confocal microscopy revealed that ITEGE(373) epitope was largely intracellular but with matrix staining in the superficial zone, whereas DIPEN(341) epitope was cell-associated and widely distributed in the matrix. Surprisingly, the majority of (342)FFGVG epitope, determined by radioimmunoassay and Western blotting, was retained in the tissue despite the absence of a G1 domain anchor. Interleukin-1alpha stimulation caused a marked increase in tissue DIPEN(341) and (342)FFGVG epitope, and the (342)FFGVG fragments retained in the tissue were larger than those released into the medium. Active porcine aggrecanase was unable to cleave (342)FFGVG fragments at the downward arrowGlu(373) downward arrowAla(374) bond but cleaved intact aggrecan at this site, suggesting that (342)FFGVG fragments are not substrates for aggrecanase. The apparent retention of large (342)FFGVG fragments within cartilage, and their resistance to N-terminal cleavage by aggrecanase suggests that (342)FF6V6 fragments may have a role in cartilage homeostasis.     

Investigating ADAMTS-mediated aggrecanolysis in mouse cartilage

Proteolysis of the cartilage proteoglycan aggrecan is a feature of arthritis. We present a method for analyzing aggrecanolysis in in vitro cultures of 3-week-old mouse femoral head cartilage based on traditional methods developed for large animal species. Investigators can choose either a simple analysis that detects several aggrecan fragments released into culture medium only or a more comprehensive study that detects all fragments present in both the medium and the cartilage matrix. The protocol comprises (i) cartilage culture and optional cartilage extraction, (ii) a quick and simple colorimetric assay for quantitating aggrecan and (iii) neoepitope western blotting to identify specific aggrecan fragments partitioning to the medium or cartilage compartments. The crucial difference between the methods for mice and larger animals is that the proportion of aggrecan in a given sample is normalized to total aggrecan rather than to tissue wet weight. This necessary break from tradition arises because tiny volumes of liquid clinging to mouse cartilage can increase the apparent tissue wet weight, causing unacceptable errors. The protocol has broad application for the in vitro analysis of transgenic mice, particularly those with mutations that affect cartilage remodeling, arthritic disease and skeletal development. The protocol is robust, reliable and takes 7-11 d to complete.     

An enzyme-linked immunosorbent assay (ELISA) of denatured cartilage link protein

Antiserum (MB007) was raised in rabbits to SDS-denatured cartilage link protein in order to develop an enzyme-linked immunosorbent assay (ELISA) to quantify link protein in cartilage extracts. The antibodies were characterized by using native and denatured link protein, either as the immobilised or the inhibiting antigen in the assay, and shown to bind more effectively to denatured link protein. At low concentrations, neither hyaluronate (0-30 micrograms/ml), proteoglycan (0-50 micrograms/ml) nor hyaluronate-binding region (0-3 micrograms/ml) competitively inhibited the link protein assay. However, at higher concentrations of proteoglycan (50 micrograms/ml-4 mg/ml) and hyaluronate-binding region (3-40 micrograms/ml) inhibition was observed. A more highly purified proteoglycan and a further purified hyaluronate-binding region preparation showed identical behaviour. The inhibition produced by proteoglycan and hyaluronate-binding region occurred at approximately equivalent molar concentrations (assuming Mr of 10(6) and 7 X 10(4), respectively). These results suggest that a significant proportion of these polyclonal antibodies recognize an epitope common to link protein and hyaluronate-binding region. However, the possibility that these effects are due to contamination with covalently bound link protein cannot be excluded. Trypsinated aggregates (0-10 micrograms/ml) produced no inhibition in the link protein ELISA, but as higher concentrations the inhibition was approximately 2000-fold lower than might have been expected from the link protein concentration present. Thus, the accessibility and/or binding of the antibodies to link protein was substantially decreased, illustrating masking of the link protein antigenic sites, as found by A. Ratcliffe and T.E. Hardingham (Biochem. J. 213 (1983) 371-378). These studies indicate that link protein in tissue extracts may be quantified in the concentration range 30-200 ng/ml and in the presence of hyaluronate, proteoglycan and hyaluronate-binding region, provided that both the immobilised and extracted link proteins are denatured.     

The ovine newborn and human foetal intervertebral disc contain perlecan and aggrecan variably substituted with native 7D4 CS sulphation motif: spatiotemporal immunolocalisation and co-distribution with Notch-1 in the human foetal disc

Composite agarose (1.2 %) polyacrylamide (0.6 %) gel electrophoresis was used to separate discrete populations of native aggrecan and perlecan in newborn to 10 year old ovine intervertebral discs (IVDs). Semi-dry immunoblotting using core-protein and glycosaminoglycan (GAG) side chain specific monoclonal antibodies in combination with chondroitin ABC lyase demonstrated intra-chain native 7-D-4 chondroitin sulphate (CS) sulphation motifs and variable proportions of non-reducing terminal Δ4,5-unsaturated uronate-N-acetylgalactosamine-4-sulphate [2B6(+)] and Δ4,5-unsaturated glucuronate-N-acetylgalactosamine-6-sulphate [3B3(+)] disaccharides. The relative abundance of 2-B-6(+) aggrecan increased with advancing age of the IVD samples while the converse was true for the 3-B-3(+) aggrecan population. Relative 7D4 levels in aggrecan and perlecan were highest in the newborn IVD and significantly lower in the older IVD and other cartilage samples. Quantitation of 7D4 proteoglycan by enzyme linked immunosorbent inhibition assay confirmed the newborn ovine nucleus pulposus (NP) and inner annulus fibrosus (AF) contained higher levels (1.2-1.32 μg 7-D-4-proteoglycan/mg tissue wet weight) than the 2 (0.35-0.42 μg/mg wet weight tissue) and 10 year old IVD samples (0.16-0.22 μg/mg tissue wet weight) with the outer AF zones consistently containing lower levels of 7-D-4 epitope in all cases (P?<?0.001). Cell populations on the margins of the AF and cartilaginous vertebral rudiments in newborn ovine and human foetal IVD strongly expressed 7-D-4 CS epitope and perlecan, This was co-distributed with Notch-1 expression in human foetal IVDs consistent with the 7-D-4 CS sulphation motif representing a marker of tissue development expressed by disc progenitor cell populations.     

Perlecan,the multidomain HS-proteoglycan of basement membranes,is a prominent pericellular component of ovine hypertrophic vertebral growth plate and cartilaginous endplate chondrocytes

The aim of this study was to immunolocalise perlecan in ovine vertebral growth plate (VGP) and cartilaginous endplate (CEP) cartilages using a monoclonal antibody (MAb A76) directed to a core protein epitope in perlecan domain-I, and to compare and contrast its localisation patterns with known cartilage matrix components. Perlecan was a prominent pericellular component of mature hypertrophic chondrocytes in the VGP and CEP in newborn 2- to 5-day-old sheep. Type I, II, VI and X collagen, chondroitin-4 and 6-sulphate, 7-D-4 chondroitin sulphate isomer proteoglycan epitope, keratan sulphate, aggrecan core protein, hyaluronan (HA) and hyaluronan binding proteins (HABPs) each had distinct localisation patterns in the VGP and CEP. Type X collagen was a prominent component of the VGP but was undetectable in the CEP. Aggrecan was strongly localised extracellularly throughout the VGP and CEP but increased cell-associated staining was also evident. In contrast to the aforementioned matrix components, HA, HABPs and perlecan were localised strongly to the pericellular matrices of the hypertrophic VGP and CEP chondrocytes apparently indicating an important role for these components in terminal chondrocyte differentiation.