A novel keratanase-generated keratan sulfate antibody and its application to structural analysis of skeletal and corneal keratan sulfate

Introduction The objective of this study was to make monoclonal antibodies specific for keratanase‐generated neoepitopes in keratan sulfate (KS) and to use them along with existing KS monoclonal antibodies (e.g. 5D4, IB4) to investigate KS sulfation pattern motifs in connective tissue proteoglycans during development, ageing and disease. Methods Bovine nasal cartilage aggrecan (BNC A1D1) was trypsin digested, generating a range of GAG‐peptide fragments. The sample was then subjected to anion‐exchange and size exclusion chromatography to separate KS peptides from CS attachment domain fragments. Fractions were analysed by Western blotting for positive immunoreactivity for KS, then pooled and keratanase digested to generate ‘KS stub’ antigens. Immunization and fusions were carried out as previously described ( Caterson et al. 1983 ; Hughes et al. 1992 ). Screenings involved the use of a range of antigens; including keratanase vs. keratanase II‐digested bovine cartilage aggrecan and bovine corneal KS‐PGs. A new monoclonal antibody, BKS‐I, was identified that specifically recognized a keratanase‐generated neoepitope on both skeletal and corneal KS. This novel monoclonal antibody was used along with existing KS monoclonal antibodies 5D4 and 1B4 to investigate KS structure. Results and discussion Bovine trypsin‐generated aggrecan KS‐peptides were chondroitinase ABC treated and either keratanase or keratanase II treated. The digests were run on SDS‐PAGE and immunolocated with monoclonal antibody 5D4 (that recognizes linear disulfated N‐acetyl lactosamine disaccharide‐containing segments in KS) and the new ‘KS‐stub’ monoclonal antibody BKS‐I. Our results indicated that there was reduced monoclonal antibody 5D4 immunostaining after keratanase pretreatment. However, keratanase II digestion completely removed all 5D4 structural epitopes. In contrast, BKS‐I showed no immunostaining on the untreated KS‐peptides but strong staining on keratanase treated samples and no staining after keratanase II digestion. Similar patterns of immunoreactivity were observed with Western blot analysis of untreated, keratanase treated and keratanase II treated corneal KS‐PGs. Conclusion These data indicate that monoclonal antibody BKS‐I recognizes a nonreducing terminal neoepitope‐containing sulfated N‐acetylglucosamine adjacent to a nonsulfated lactosamine disaccharide. We also conclude that skeletal KS must have a structure with four possible variations opposed to the generic structures, proposed as being made of disulfated disaccharides at the nonreducing end, followed by a series of monosulfated disaccharides at the middle and nonsulfated disaccharides nearer the linkage region. 5D4 staining, observed after keratanase digestion, indicates that there must be a minimum structure of a pentasulfated hexasaccharide remaining on the KS chain ‘stubs’ near the linkage region of skeletal and corneal KS. The BKS‐I monoclonal antibody can be used to demonstrate differential substitution of KS GAG chains in the CS attachment region of cartilage aggrecan with ageing. It has also proven useful for immunohistochemical analyses identifying the sites of KS–PG association with collagen lamellae of cornea.     

Age-related changes in the glycosaminoglycans of human meniscal aggrecan

Introduction Meniscal damage and degradation, which are strongly correlated with subsequent OA, have been identified in approximately 60% of people over 60 years of age. Age‐related changes in articular cartilage glycosaminoglycans (GAGs) have been described, and used to facilitate the study of pathology‐related changes ( Plass et al. 1998 ). However, such data do not yet exist for the meniscus. Materials and methods Undamaged human menisci were obtained following leg amputations, and the vascular and avascular zones of each lateral and medial meniscus were extracted into 4 m GuHCl. Aggrecan was recovered in the A1 fraction following CsCl density gradient centrifugation, and the relative abundance of chondroitin, dermatan and keratan sulphates (CS, DS and KS) was examined by NMR spectroscopy at 400 MHz and 43 °C. Results Human meniscal aggrecan was shown to contain CS, DS and KS, and our data show age‐related changes in the relative abundance of these GAGs. The change was similar for medial and lateral menisci and for the vascular and avascular zones within these. The KS abundance in aggrecan from young menisci (<15 years) was found to be 15–20% of the total GAGs. However, in older samples, it comprised only 7–12% of the GAGs. We have confirmed the presence of DS in human meniscal aggrecan and show that the abundance of DS gradually falls from approximately 16% at 10 years to 2–4% at 75 years. There is some variability between humans, although the trend is clear and for each human there is good agreement between medial and lateral menisci and vascular and avascular locations. The levels of CS comprise the remainder of the GAG attached to aggrecan and contribute the remainder of the GAG abundance. This can be seen to increase from 67 to 72% at 10 years to approximately 90% at 75 years. Discussion Our data show a clear age‐related change in the relative abundance CS, DS and KS from human meniscal aggrecan. The data show a decrease in the abundance of KS and DS and a concomitant increase in CS levels. These observations differ from those widely seen for articular cartilage, in which the levels of CS are seen to fall with age. We have confirmed that DS is a component of human meniscal aggrecan in agreement with previous work ( McNicol & Roughley 1980 ). However, previously reported levels of DS, approximately 20%, are those found only in younger menisci. Absolute levels of these GAGs have not yet been determined, and hence the mechanisms which bring about this relative increase in CS with age may include either changes in biosynthetic output and/or widespread GAG loss in which KS and DS loss increases with age.     

Age-related changes in human meniscal glycosaminoglycans

Introduction With an increased human lifespan, a major challenge is now to ensure a concomitant increase in healthspan. Meniscal damage and degradation are common and are strongly correlated with subsequent osteoarthritis. Indeed, meniscal damage has been identified in about 60% of people over 60. Markers of pathology will facilitate intervention but first require normal age‐related changes to be established. Methods Undamaged vascular and avascular regions of medial and lateral human menisci were comminuted and the tissue extracted into 4‐m GuHCl and subject to associative CsCl density gradient centrifugation. Aggrecan and the small leucine rich PGs (SLRPs) were isolated and their GAG profiles examined by HPAEC fingerprinting, following enzyme depolymerization, and by an NMR spectroscopy. Results and discussion Analysis of aggrecan and the SLRPs show that there is a complex and dynamic pattern of KS, CS and DS abundance and distribution within human menisci, which changes with age. The abundance of SLRPs is higher in the avascular than vascular tissues, however, this is not reflected in the abundance of aggrecan which is present at similar levels in both tissue regions. The data show no other significant differences between medial and lateral and between vascular and avascular tissue regions. Analysis of the sulfation pattern of CS following digestion by ACII lyase, shows that in both aggrecan and SLRPs the 4‐sulfation level falls with age from 20 to 35% in young tissues to 10–20% in older. Subsequent analyses following ABC lyase depolymerization, to include DS, shows very significant change with age from CS + DS 4‐sulfation levels of ca. 40–55% in young tissue to ca. 15–30% in older. The difference between these datasets represents the contribution made by 4‐sulfated DS. Thus, analysis of the difference suggests that DS makes a decreasing contribution to the CS/DS profile with age. Indeed, this is confirmed by an NMR analysis of these samples. Analysis of the resonances in the region 1.95–2.2 p.p.m. (ref to TSP) allows the estimation of the contribution made by DS, CS and KS. These data show that, in aggrecan, the contribution made by DS chains falls from ca. 10% in younger tissues to ca. 2–4% in older tissues. NMR analysis also shows that KS levels fall with age from ca. 15–20% in younger tissues to 5–10% in older tissues. Analysis of the structure of the KS chains shows chains with a structure similar to that of in articular cartilage but that at all ages there are very low levels of fucosylation (ca. 1–5%). Previous studies of age‐related changes in CS/DS and KS structures have shown significant changes in the first 17 years of life, with only modest nonpathological changes after that time. These data from meniscal tissues do not show such a dramatic halting of normal age‐related changes. Indeed, the data show gradual age‐related changes in DS, CS and KS abundance and structure throughout life. These baseline age‐related changes data will now allow the analysis of pathology‐related changes.     

ADAMTS-4 and ADAMTS-5 sequestration and activity in chondrocyte-agarose cultures

Introduction A primary event in the destruction of cartilage in arthritic diseases is the loss of aggrecan from the extracellular matrix of articular cartilage. During aggrecan breakdown, cleavage sites are utilized, which reside within the IGD of the aggrecan core protein. The Asn³⁴¹–Phe³⁴² bond is cleaved by members of the MMP family, whereas the second of the two cleavage sites, the Glu³⁷³–Ala³⁷⁴ bond, is cleaved by the aggrecanases which are all members of the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family. Both ADAMTS‐4 and ‐5 have been shown to readily cleave aggrecan at this so‐called aggrecanase site ( Tortorella et al. 1999 ; Abbaszade et al. 1999 ; Sandy et al. 2000 ). An in vitro model of cartilage degradation has also shown that these enzymes are responsible for the loss of aggrecan from explant cultures of articular cartilage stimulated with IL‐1‐a or TNF‐a ( Tortorella et al. 2001 ). Both ADAMTS‐4 and ‐5 are thought to be synthesized as inactive zymogens that are activated via removal of their propeptide domain by the Golgi enzyme furin. The secreted active ADAMTS‐4 and ‐5 have predicted molecular weights of 67.9 and 73.6 kDa, respectively. The objective of this study was to monitor ADAMTS‐4 and ‐5 secretion and sequestration in the extracellular matrix of chondrocyte‐agarose cultures. Methods Porcine articular chondrocytes were isolated and embedded in agarose ( Hughes et al. 1997 ) before preculture in DMEM + 50 µg/ml gent. with 10% FBS and 25 µg/ml Phos.C for 21 days. The plates were washed, then cultured in serum‐free DMEM with or without IL‐1‐α for 96 h. GAG release to the medium was measured using the DMMB assay. Media samples were analysed by Western blotting for aggrecan metabolites using mAb BC‐3 to recognize the aggrecanase‐generated neoepitope ARGSV. The presence of ADAMTS‐4 in the media was analysed using the mAb anti‐TS‐4N, which recognizes the metalloproteinase domain, and commercially available polyclonal antibodies to the pro‐ and spacer domains of ADAMTS‐4. The presence of ADAMTS‐5 was detected using commercially available polyclonal antibodies to the pro‐ and spacer domains of ADAMTS‐5. Agarose plugs were extracted in detergent buffer and analysed by Western blotting for ADAMTS‐4 and ‐5 using the same monoclonal and polyclonal antibodies. Results In control cultures, only 20–30% of the total GAG was released into the medium after 96 h of culture. In contrast, 80–90% of the total GAG was released in cultures exposed to IL‐1. Western blot analysis showed aggrecanase‐generated aggrecan metabolites in the IL‐1‐treated cultures but none in the control cultures. Sequestered forms of both ADAMTS‐4 and ‐5 are present in the matrix prior to treatment in serum‐free conditions, and following treatment with or without IL‐1 for 96 h, there are no differences in the high molecular weight isoforms of the enzymes sequestered in the matrix. Western blots of partially purified media samples showed no differences in the zinc chelator‐bound isoforms of either ADAMTS‐4 or ‐5 between control and IL‐1‐treated cultures. However, the predominant heparin sepharose‐bound isoforms of ADAMTS‐4 and ‐5 co‐migrate at approximately 37 kDa. Each of the heparin‐bound 37‐kDa isoforms of ADAMTS‐4 and ‐5 are detected in increased amounts in IL‐1α‐treated cultures compared to controls. Discussion The increased amounts of the 37‐kDa isoforms of both ADAMTS‐4 and ‐5 in the IL‐1‐treated cultures suggest a role for these smaller isoforms in the increased aggrecanase activity seen in the IL‐1‐treated cultures compared to controls. This study has identified multiple isoforms of putative aggrecanase activity that could be responsible for increased aggrecan catabolism that leads to cartilage degradation in arthritis.     

Increased turnover of proteoglycan aggregates in tendon vs. cartilage

Introduction Although the function of proteoglycans (PGs) within the tendon extracellular matrix are not fully understood, changes in their turnover have been associated with tendinopathies ( Riley et al. 1994 ). In contrast to cartilage, aggrecanases are constitutively expressed and active in tendon ( Rees et al. 2000 ), indicative of a high rate of aggrecan turnover. Clinical trials investigating the use of active site MMP inhibitors have been confounded by side effects, which involve tendonitis and ‘musculoskeletal syndrome’. Such side effects may relate to nonspecific inhibition of tendon aggrecanases required to maintain normal metabolic homeostasis. The purpose of this study, therefore, was to compare the rate of turnover of tendon and cartilage PGs derived from the same joint and to determine the effect of MMP inhibitors (actinonin and marimastat) on aggrecan catabolism. Materials and methods Deep digital flexor tendon explants from compressed and tensional regions were dissected from young and mature bovine. Explants were precultured and then cultured for a further 4 days with or without marimastat (0–2 µm ) or actinonin (0–200 µm ). PG and lactate quantification, Western blot analysis of degradation products and RT‐PCR analyses were performed. In a separate experiment for measurement of PG turnover, explants were set up as described above and then pulse chase labelled with [³⁵S] sulfate. The rate of turnover of ³⁵S‐labelled PGs from the matrix of tendon (and articular cartilage obtained from the same animal) was subsequently calculated from the amount of ³⁵S‐labelled macromolecules appearing in the medium each day and that remaining in the matrix of explants at the termination of culture. Results PG turnover (presumably predominantly aggrecan) was markedly higher in tendon vs. cartilage. This difference was apparent in tendons from all regions and ages. Both marimastat and actinonin inhibited aggrecanase‐mediated PG catabolism in both tendon and cartilage explants. As expected, mRNA expression for the aggrecanases, MMPs and TIMPs was unaffected by the addition of these inhibitors to the culture medium. Discussion Aggrecan turnover in tendon is higher than that of articular cartilage, which may be attributed to distinct physiological properties of this PG in tendon. Importantly, immunohistochemical staining for aggrecan in tendon indicates its presence in between collagen fibres and fibril bundles ( Vogel et al. 1999 ), and thus aggrecan aggregates may dissipate resultant compressive loads by resisting the flow of water in these locations. In addition, aggrecan may facilitate the sliding of fibrils during the small amount of elongation of the tendon whilst under tension. Thus, the half‐life of tendon aggrecan is significantly reduced because it constantly participates in repeated resistance to compression. Our data also demonstrates that both marimastat and actinonin can inhibit aggrecanase‐mediated PG catabolism in tendon cultures. This suggests that the occurrence of ‘musculoskeletal syndrome’ in clinical trial patients may be due to the fact that these inhibitors affect the activity of aggrecanases in tendon, thus preventing them from playing their normal role in tendon aggrecan turnover and consequently perturbing normal physiological function.     

ADAMTS-4 activity on a proteoglycan vs. a polypeptide is controlled by the ancillary domains

Introduction In combination with the catalytic domain, the ancillary domains of the ADAMTS' are proposed to regulate activity via interactions with sulfated GAGs, the extracellular matrix (ECM) and cell surface. Interactions with both GAGs and the ECM have been attributed to the thrombospondin (TSP) type 1 motifs and spacer region ( Kuno and Matsushima 1998 ; Flannery et al. 2002 ). ADAMTS‐1, ‐4 and ‐5, all undergo cleavage within their respective spacer regions ( Flannery et al. 2002 ; Rodriguez‐Manzaneque et al. 2000 ; Georgiadis et al. 2002 ), an event which has been reported to increase activity towards the interglobular domain of aggrecan and decrease the heparin affinity of ADAMTS‐4 ( Flannery et al. 2002 ; Gao et al. 2002 ). Materials and methods V5‐ and His‐tagged recombinant human ADAMTS‐4 constructs terminating after the catalytic (?DTS), disintegrin‐like (?TS), TSP (?S) or spacer region (Full) were expressed in High‐Five cells. Proteoglycanase activities of the resultant proteins were assayed with solution digests of aggrecan and a polyacrylamide‐entrapped aggrecan particle assay. Proteolytic activity was measured using a novel, nonglycosylated, reporter substrate assay. Results All forms of ADAMTS‐4 were active to varying degrees in the reporter substrate assay. Digestion of aggrecan in solution digests was apparent in all proteins with the exception of the catalytic domain in isolation (?DTS). Activity towards aggrecan decreased with increasing truncation of the protein. Discussion Removal of the cysteine‐rich‐spacer domain and further C‐terminal truncations decrease the activity of ADAMTS‐4 towards aggrecan, whilst the proteolytic activity remains intact. Cleavages releasing the ancillary domains of ADAMTS' may therefore alter the catalytic activity of these enzymes against proteoglycans and also nonglycosylated polypeptides. More information is required about potential substrates for the processed forms of ADAMTS‐4.     

The role of cartilage glycosaminoglycan structure in gagCEST

Glycosaminoglycan (GAG) chemical exchange saturation transfer (gagCEST) is a potential method for cartilage quality assessment. The aim of this study was to investigate how the gagCEST effect depends on the types and molecular organization of GAG typically found in articular cartilage. gagCEST was performed on different concentrations of GAG in various forms: free chains of chondroitin sulfate (CS) of different types (‐A and ‐C) and GAG bound to protein in aggregated and nonaggregated aggrecan extracted from calf articular cartilage. The measured magnetization transfer ratio asymmetry (MTR_asym) was compared with known GAG concentrations or GAG concentrations determined through biochemical analysis. The gagCEST effect was assessed through the linear regression coefficient with 95% confidence interval of MTR_asym per GAG concentration. We observed a lower gagCEST effect in phantoms containing a mixture of CS‐A and CS‐C compared with phantoms containing mainly CS‐A. The difference in response corresponds well to the difference in CS‐A concentration. GAG bound in aggrecan from calf articular cartilage, where CS‐A is assumed to be the major type of GAG, produed a similar gagCEST effect as that observed for free CS‐A. The effect was also similar for aggregated (ie, bound to hyaluronic acid) and nonaggregated aggrecan. In conclusion, our results indicate that the aggrecan structure in itself does not impact the gagCEST effect, but that the effect is strongly dependent on GAG type. In phantoms, the current implementation of gagCEST is sensitive to CS‐A while for CS‐C, the main GAG component in mature human articular cartilage, the sensitivity is limited. This difference in gagCEST sensitivity between GAG types detected in phantoms is a strong motivation to also explore the possibility of a similar effect in vivo.     

Plasma biomarkers of oxidative and AGE-mediated damage of proteins and glycosaminoglycans during healthy ageing: A possible association with ECM metabolism

The aim of this study was to examine whether oxidative and AGE-mediated processes correlates with the metabolic changes of proteoglycans (PGs) and proteins during physiological ageing. The age and gender-associated changes of PGs metabolism were evaluated by plasma chondroitin sulfates (CS), dermatan sulfates (DS) and heparan sulfates and heparin (HS/H). We found a linear age-related decline in CS, DS and HS/H, the first one being the predominant plasma GAG during ageing. The possible deleterious effect of oxidative phenomenon on proteins’ and proteoglycans’ metabolism during ageing process was analyzed by plasma carbonyls (PCO) and thiols (PSH) as well as by total antioxidant capacity (TAS). An age-dependent increase in PCO and decrease in PSH concentrations were found, both strongly correlated with decreasing with age plasma TAS. Intensity of glycation was assessed by circulating N^?-(carboxymethyl)lysine (CML) and endogenous secretory receptor for AGE (esRAGE), both of them founding associated with ageing. Moreover, all markers of oxidative and AGE-mediated damage correlated with CS and DS level and could be contributing factors to age-related changes of these GAG types. Thus, plasma CS and DS could become promising biomarkers of human ageing to date, owning to its close association with oxidative status and glycation processes.     

Intramolecular polyspecificity in CD4 T‐cell recognition of Ad‐restricted epitopes of proteoglycan aggrecan

T‐cell recognition of MHC–peptide complexes shows a high degree of polyspecificity extending to recognition of a large number of structurally unrelated peptides. Examples of polyspecificity reported to date are confined to recognition of epitopes from distinct proteins or synthetic peptide libraries. Here we describe intramolecular polyspecificity of CD4 T cells specific for several epitopes within proteoglycan aggrecan, a structural glycoprotein of cartilage and candidate autoantigen in rheumatoid arthritis. T‐cell hybridomas from aggrecan‐immunized mice recognized four structurally unrelated epitopes from the G1 domain of aggrecan, but not other aggrecan epitopes or a variety of other peptide epitopes restricted by the same MHC class II allele. We also showed that the hierarchy of cross‐reactivity broadly correlated with the strength of peptide binding to MHC class II. Similar polyspecificity was observed in responses of lymph node cells from peptide‐immunized mice, suggesting polyspecificity of a significant proportion of the in vivo aggrecan specific T‐cell repertoire. Polyspecific recognition of several epitopes within the same autoantigen may provide a novel mechanism to reach the activation threshold of low‐affinity autoreactive T cells in the initiation of autoimmune diseases.     

Analytical method to determine keratan sulfate in the serum using HPLC

There is an established method to assess the extent of articular cartilage damage that is based on the determination of keratan sulfate (KS) in synovial fluid by high performance liquid chromatography (HPLC). However, since it is easier to collect serum than synovial fluid, and the serum concentration of KS might reflect the extent of degeneration and injury of the articular cartilage, we aimed at developing a method to determine KS in serum by HPLC. Human serum was spiked with low-sulfated KS or poly-sulfated KS. After digestion of serum protein with a protease, the KS fraction was extracted by anion exchange column chromatography and then desalted. The KS fraction was digested with keratanase II and determined by HPLC. The recovery of KS added to the serum was 98% or more and did not depend on the degree of KS sulfation. A good recovery rate was obtained within a range of 50-100,000 ng/ml. The inter-day assay variation was 6.0% for 10 days. This method enables the determination of serum KS concentration without being influenced by the degree of KS sulfation.     

Molecular recognition and modulation of hepatocyte growth factor activity by heparan and dermatan sulfates

Introduction Hepatocyte growth factor/scatter factor (HGF/SF) is an unusual growth factor in that it binds both heparan sulfate (HS) ( Lyon et al. 1994 ) and dermatan sulfate (DS) ( Lyon et al. 1998 ) glycosaminoglycans (GAGs) with similar high affinities. Both these GAGs act as co‐receptors for HGF/SF in the activation of the Met receptor ( Lyon et al. 2002 ). Our aim was to determine the sequences in HS and DS that specifically interact with and modulate HGF/SF activity. Materials and methods A structurally unique DS, which possesses O‐sulfation at carbon‐6 of the hexosamine residue (and not carbon‐4 as in mammalian DS), was obtained from the sea cucumber, Ascidia nigra. A variety of HS‐ and DS‐like structures were also generated using various chemical modification procedures (specific desulfations and carboxyl reductions). The ability of these various GAG species to compete with cell surface GAGs for HGF/SF binding was tested using radiolabelled HGF/SF and MDCK cells. The modified GAG structures and the A. nigra DS are currently being tested for their ability to act as co‐receptors for the interaction between HGF/SF and Met by studying cell signalling and cellular response assays, using the sulfated GAG‐deficient CHO‐745 cell line. Results Unexpectedly, A. nigra DS was found to bind HGF/SF strongly with a KD of around 1 nm . This interaction is 20‐fold stronger than that of between HGF/SF and mammalian DS, but similar to that of with HS. A. nigra DS also stimulated HGF/SF‐mediated Erk activation and migration in CHO‐745 cells. Studies using the modified GAG species showed that, in the case of HS, 6‐O‐sulfate and N‐sulfate groups are most important for HGF/SF binding. For HGF/SF binding to DS, hexosamine O‐sulfate is most important. HGF/SF was also found to bind 6‐O‐sulfated GAGs more strongly than 4‐O‐sulfated ones. Discussion The data show that there is flexibility in the structures recognized by HGF/SF, and this explains the ability of the growth factor to bind both HS and DS. However, there are still observable preferences in GAG structure, such as 6‐O‐sulfation over 4‐O‐sulfation. Information on HGF/SF‐binding GAG structures is valuable for the design of HGF/SF antagonists that could be useful therapeutically in the treatment of solid tumours where HGF/SF‐Met activity is up‐regulated.     

Sensitivity of monoclonal antibody, 5-D-4, for the detection of aggrecan,aggrecan fragments,and keratan sulfate

Bovine nasal septum aggrecan and selected proteinase-digested products of aggrecan were evaluated in an inhibition ELISA using the anti-keratan sulfate (KS) monoclonal antibody 5-D-4 (5D4). Undegraded aggrecan was recognized with an IC₅₀ of 0.27 μg/ml. When aggrecan was treated with human stromelysin (SLN), human leukocyte elastase (HLE), or papain, the degradation fragments had different hydrodynamic sizes. Treatment with SLN produced the largest fragments, HLE generated intermediate fragments, and papain the smallest fragments. Whereas degradation of aggrecan by SLN had little effect on recognition of proteoglycan in the ELISA (IC₅₀-0.5 μg/ml), degradation by both HLE and papain significantly decreased the sensitivity for detection of KS epitope (IC₅₀-170 and 215 μg/ml, respectively). In addition, 5D4 detected single chain costal and corneal KS with much less sensitivity (IC₅₀-21 and 469 μg/ml, respectively) than undegraded aggrecan (IC₅₀-0.27 μg/ml).

     

Reversible Secretion of Glycosaminoglycans and Proteoglycans by Cyclically Stretched Valvular Cells in 3D Culture

Mitral valve leaflets and chordae have been shown to contain different amounts and proportions of glycosaminoglycans (GAGs) and proteoglycans (PGs) corresponding to in vivo normal or diseased cyclic strain patterns. To understand the effect of cyclic strains on GAG/PG synthesis by valvular interstitial cells (VICs) isolated from valve leaflet and chordae separately, porcine VICs were seeded within collagen gels and alternately stretched or relaxed for 24 h periods for one week in a custom-designed tissue engineering bioreactor. We found cyclic-stretch-induced upregulation of total GAGs and of individual GAG classes secreted into the culture medium. Leaflet cells showed a delayed response to stretching compared to chordal cells, but altered the proportions of various GAG classes they secreted during the culture duration. Decorin and biglycan PGs were slightly responsive to stretch. We demonstrated that mechanical stretch and relaxation conditions reversibly regulate GAG and PG production in a novel 3D model of valve tissues. This is the first study using cyclic strains to modulate GAG/PG synthesis by valve cells and our results may have implications for the remodeling of the mitral valve as well as other tissues.     

Discovery of HLA-B*480102 in Taiwanese

Human leucocyte antigen (HLA)‐B48, an antigen within 7C CREG (cross‐reacting group) ( Steiner et al., 2001 ) that cross‐reacts frequently with HLA‐B40 (i.e. HLA‐B60 and ‐B61) group of antibodies serologically, can be found in Alaskan Natives ( Leffell et al., 2002 ), Amerindians ( Martinez‐Naves et al., 1997 ), African Americans, Caucasians, and Oriental ethnicities ( Mori et al., 1997 ; Schipper et al., 1997 ; Cao et al., 2001 ; Middleton et al., 2004 ; Hong et al., 2005 ; Itoh et al., 2005 ; Lee et al., 2005 ; Ogata et al., 2007 ). Sequencing investigations demonstrated that the common allele encoding the B48 antigen is B*4801 ( Belich et al., 1992 ). To date, there are at least 16 WHO recognized B*48 alleles according to the most recent report from the WHO nomenclature committee ( Marsh et al., 2005 ). Here we report a newly discovered allele B*480102, a variant of B*4801, detected in a 55‐year‐old Taiwanese patient of Minan origin (southern China).     

Unconventional T‐cell recognition of an arthritogenic epitope of proteoglycan aggrecan released from degrading cartilage

It has been proposed that peptide epitopes bind to MHC class II molecules to form distinct structural conformers of the same MHC II–peptide complex termed type A and type B, and that the two conformers of the same peptide–MHC II complex are recognized by distinct CD4 T cells, termed type A and type B T cells. Both types recognize short synthetic peptides but only type A recognize endosomally processed intact antigen. Type B T cells that recognize self peptides from exogenously degraded proteins have been shown to escape negative selection during thymic development and so have the potential to contribute to the pathogenesis of autoimmunity. We generated and characterized mouse CD4 T cells specific for an arthritogenic epitope of the candidate joint autoantigen proteoglycan aggrecan. Cloned T‐cell hybridomas specific for a synthetic peptide containing the aggrecan epitope showed two distinct response patterns based on whether they could recognize processed intact aggrecan. Fine mapping demonstrated that both types of T‐cell recognized the same core epitope. The results are consistent with the generation of aggrecan‐specific type A and type B T cells. Type B T cells were activated by supernatants released from degrading cartilage, indicating the presence of antigenic extracellular peptides or fragments of aggrecan. Type B T cells could play a role in the pathogenesis of proteoglycan‐induced arthritis in mice, a model for rheumatoid arthritis, by recognizing extracellular peptides or protein fragments of joint autoantigens released by inflamed cartilage.     

The effects of human intervertebral disc aggrecan on neuronal and endothelial cell growth

Introduction The development of ‘discogenic’ low back pain has been associated with increased nerve growth into degenerated intervertebral discs, particularly in neovascularized areas. A marked loss of proteoglycans, particularly from the disc's inner regions, is also a marked feature of the degenerative process. As proteoglycans from other tissues can have inhibitory effects on nerve growth, we have hypothesized that the alterations in proteoglycan content seen in disc degeneration may affect the growth of nerves and blood vessels into the disc. To test our hypothesis, we have established cell culture assays to determine the effects of human intervertebral disc aggrecan, which forms the major proteoglyan found in the disc, on neuronal and endothelial cell growth. Materials and methods Aggrecan (A1D1 preparations) isolated from the outer (anulus fibrosus, AF) and inner (nucleus pulposus, NP) regions of human lumbar intervertebral discs was incorporated into culture substrata, using methods previously described ( Snow et al. 1990 ). Chick dorsal root ganglia (DRG) and the cell line SH‐SY5Y were used as models of nerve growth. HMEC‐1 and Eahy‐926 cell lines were used as models of endothelial cell growth. Results Human disc aggrecan inhibited SHSY‐5Y cell attachment, SHSY5Y neurite outgrowth and induced sensory DRG neurite growth cone turning in a concentration‐dependent manner. Sensory neurites emanating from DRG across permissive substrates (collagen/laminin) were repelled by high (1 mg/ml), but not low (0.01 mg/ml) concentrations of disc aggrecan and became aligned to the aggrecan border. Disc aggrecan similarly inhibited endothelial cell adhesion, cell spreading and migration. HMEC‐1 and Eahy926 cells migrated over collagen substrates (type I) until they encountered disc aggrecan, where they either stopped migrating or, more commonly, changed their direction of movement and aligned to the aggrecan border. In general, aggrecan isolated from the AF was more inhibitory than that isolated from the NP. The inhibitory effects were partially abrogated following enzymic deglycosylation of the aggrecan, or if aggrecan was added in solution (i.e. was nonsubstrate bound). Conclusions This study provides evidence that disc aggrecan inhibits neuronal and endothelial growth and migration and therefore supports a hypothesis that a loss of aggrecan from degenerated discs predispose the tissue to vascular and neuronal invasion.     

An ultrastructural investigation of dermatopontin-knockout mouse corneas

Introduction The corneal stroma is composed of a network of heterotypic collagen fibrils, proteoglycans and matrix proteins. Transparency of the tissue principally requires the uniformity of fibril diameters and interfibrillar distances and the presence of a quasi‐hexagonal lattice arrangement of parallel fibrils. Keratan sulfate proteoglycans (KSPGs) have a crucial role and the KS chains are clearly required for the maintenance of transparency. Undersulfation of corneal KS results in tissue opacity and the lumican (a KSPG) knockout mouse shows corneal opacity and the disruption of collagen fibril diameters and interfibrillar distances ( Chakravarti et al. 1998 ). Biochemical analysis has shown that dermatopontin is an abundant component of the extracellular matrix and that it interacts with KSPGs via the KS chains. This study aims to determine whether dermatopontin has a direct role in corneal matrix organization by investigating the corneal ultrastructure of dermatopontin‐null (dpt^–/–) mouse corneas. Materials and methods Conventional light microscopy was used to compare the corneal thickness of dpt^–/– mice ( Takeda et al. 2002 ) with that of the wild‐type. Collagen fibril distribution was studied using transmission electron microscopy and the datasets analysed using SIS‐pro image analysis software to determine fibrillar volume, shape factor, fibril diameter and spacing. Results Light microscopy demonstrated that dpt^–/– corneas show a 24% reduction in average stromal thickness compared to wild‐type (P < 0.001). The epithelium and Descemet's membrane appear normal. Examination of dpt^–/– stroma by transmission electron microscopy indicates a significant disruption to lamellar organization in the posterior region while the central and anterior regions appear largely unaffected compared to wild‐type. The collagen fibrils in dpt^–/– stroma show a pronounced increase in interfibrillar spacing as well as exhibiting a lower fibril volume fraction. There is no apparent difference in fibril diameter between dpt^–/– and wild‐type mice. Discussion Collectively, these data suggest that dermatopontin plays a key role in collagen fibril organization and deposition. Like the cornea from lumican‐knockout mice ( Chakravarti et al. 1998 ), the defects in collagen organization in dpt^–/– cornea appear to be most severe in the posterior stroma. It is likely that dermatopontin interacts with the KS chains on lumican and this interaction is involved in the maintenance of stromal architecture.     

A method for fluorescent HPLC analysis of CS/DS glycosaminoglycans

Introduction Chondroitin and dermatan sulfate (CS/DS) show considerable species, tissue, age and pathology‐related structural heterogeneity. In addition within chains their sulfation patterns are not random. To elucidate their structure/function relationships, methods for complete characterization are required. A method for keratan sulfate (KS) fingerprinting ( Whitham et al. 1999 ) has been extended for the linkage, repeat and chain cap regions of CS/DS, including the acquisition of CS/DS ratios. Methods Chains were depolymerized by 1 U/100 mg of chondroitin ABC endolyase or ACII lyase at 50 mg/ml in 0.1 m ammonium acetate, pH 8 and for 15 h at 37 °C. Alternatively, chains were de‐N‐acetylated by hydrazinolysis at 98 °C for 24 h at 10 mg/1 ml in anhydrous hydrazine with 100 mg/ml hydrazine sulfate. Then, they were depolymerized by 3.9 m sodium nitrite/0.28 m acetic acid at 0 °C for 4 h. Unreduced chains were released from their protein core in 0.5 m LiOH at 4 °C for 12 h. Materials were fluorescently labelled with 2‐AA as previously described (Whitham et al. 1999) and characterized by HPAEC using a Dionex AS4‐SC column at 50 °C and 2 ml/min with constant 15% 1 m NaOH. A 5‐min isocratic period of 85% H₂O/0% 2 m NaCl was followed by a linear gradient of 0–30% 2 m NaCl over 60 min. The oligosaccharides were monitored using a λ_ex of 315 nm and a λ_em of 400 nm. Results and discussion This method resolves repeat region di‐, tri‐ and tetrasaccharides, capping oligosaccharides and linkage regions and can be used to profile known and unknown oligosaccharides. Unsulfated oligosaccharides elute between 2 and 10 min, monosulfated between 7 and 30 min, disulfated between 25 and 40 min and trisulfated between 49 and 54 min. Allied with data on size, oligosaccharide identification is facilitated. Hydrazinolysis/nitrous acid depolymerization of CS/DS chains results in disaccharides from CS with 4‐ or 6‐sulfation and from DS with 4‐sulfation which retain IdoA and GlcA structures and which can be distinguished chromatographically. The methodology was used to examine CS/DS from shark, whale, bovine and human tracheal, articular and meniscal cartilage and cornea. Tracheal cartilages show predominantly 4‐sulfation with porcine sources being more highly 4‐sulfated (ca. 75%) than bovine (ca. 65%). Articular cartilage comprises mainly 6‐sulfated GalNAc (ca. 95% in the adult), while adult meniscal cartilage shows only ca. 85%. Tracheal and articular cartilage aggrecan showed no IdoA; however, it represented ca. 20% of the uronic acids of bovine meniscal aggrecan, showing the presence of DS. Corneal CS/DS has a very low level of 6‐sulfation (<ca. 5%) but shows an equal abundance of unsulfated and 4‐sulfated residues and contains high levels, ca. 50%, of IdoA residues. Shark cartilage shows ca. 75% 6‐sulfation with significant levels of uronic acid 2‐sulfation found only between a 4‐sulfated residue and a 6‐sulfated residue, reflecting sulfotransferase specificity. Shark cartilage contains modest (ca. 1–5%) levels of DS that may be contaminants of preliminary isolation. This method extends a previous method to now allow the complete examination of KS, CS and DS chains by a single rapid chromatographic method.     

Qualitative changes in human osteoarthritic hip cartilage proteoglycan synthesis during long-term explant culture

Cartilage explants from 14 human osteoarthritic (OA) femoral heads synthesize 4 subpopulations of proteoglycan (PG) based on hydrodynamic size on Sepharose CL-2B (Kav: I, 0.05; II, 0.28; III, 0.68; IV, 0.9-1.0). A detailed analysis of newly synthesized PG monomer from each PG subpopulation was made during protracted (20 days vs. 1 day) explant culture of the cartilage specimens. Subpopulations I and II--high-density PG each eluted off Sepharose CL-2B as a unimodal peak, Kav, 0.2-0.25. High-density PG from subpopulation II appeared as a broad polydisperse symmetrical peak. Subpopulation IV eluted as 2 peaks; a minor peak of large size, and a major peak, Kav, 0.9. Large pore composite polyacrylamide gel electrophoresis (CAPAGE) of intact PG monomer (fraction D1D1) resolved at least 2 discrete PG subpopulations as constituents of both subpopulations I and II. Subpopulations III and IV PG monomer consisted of several heterogeneous subpopulations. The size of the high-density PG from subpopulations I and II decreased with time-in-culture. No significant differences were found in the average glycosaminoglycan (GAG) chain size (Kav, 0.65 on Sepharose CL-6B) or in susceptibility to chondroitinase ABC or AC-II (both 80%) in subpopulations I, II and III high density PG as a function of time-in-culture. The average GAG chain length of subpopulation IV high density PG (Kav, 0.75-0.9) was significantly shorter than the high-density PG of other subpopulations. Variations in the average GAG length and chondroitinase susceptibility did not appear to underly the smaller size of subpopulations I and II high-density PG with culture age. By contrast, keratanase susceptibility of subpopulations I and II high-density PG increased as a function of culture time.