Keratan sulphate metabolism in connective tissue proteoglycans

The generally accepted structure for KS type I has been proposed to be composed of a pattern of non-sulphated N-acetyl-lactosamine disaccharides located next to the linkage region oligosaccharide, with mono-sulphated N-acetyl-lactosamine in the mid-regions of the KS GAG chain and disulphated N-acetyl-lactosamine disaccharides concentrated towards the non-reducing end of the KS chain (Oeben et al., 1987 Stuhlsatz et al., 1989). The generic structure for KS type II has been reported to occur with a similar pattern but with a predominance of disulphated and occasional interdispersed monosulphated N-acetyl-lactosamine residues present throughout the KS glycosaminoglycan chain (Stuhlsatz et al., 1989). A novel KS monoclonal antibody (mAb) recognising a keratanase-generated KS 'stub' neoepitope was developed and used in conjunction with mAb 5D4 (recognising linear sulphated motifs) to analyse the sulphation pattern of KS from cornea and cartilage. The results of these analyses have demonstrated that KS types I and II chains both contain a combination of differentially sulphated (mono- versus di- versus unsulphated) N-acetyl-lactosamine domains. In the bovine model different tissue sources of KS most likely produce similar varieties of KS disaccharide structures, but that sulphation patterns are more randomly located along the KS chain than previously reported. Two mAbs were also generated to the small leucine rich KS proteoglycans lumican and keratocan. They were used in the identification of low and increased levels of these 'corneal associated'; PGs in musculoskeletal tissue locations with pathology. Collectively the data challenges the generic structure for KS and proposes a range of KS chain structures that are more heterogeneous in nature, even from within the same tissue sources, than previously thought. The work also suggests that the expression of the KSPGs, lumican and keratocan, may be useful biomarkers to detect cellular changes in metabolism that lead to the onset of degenerative joint disease

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

Atypical composition and ultrastructure of proteoglycans in the mouse corneal stroma

PURPOSE: Recently, gene-targeted strains of mice with null mutations for specific proteoglycans (PGs) have been used for investigations of the functional role of these molecules. In the present study, the corneal stroma of the mouse was examined to provide some baseline PG morphologies in this species. METHODS: Monoclonal antibodies to specific glycosaminoglycan (GAG) chain sulfation patterns were used to characterize PG composition in corneal extracts by SDS-PAGE and Western blot analysis and to identify their tissue distribution by immunofluorescence microscopy. PGs were also visualized by transmission electron microscopy after contrast enhancement with cationic dye fixation. RESULTS: Western blot analysis of pooled corneal extracts and immunofluorescence of tissue sections identified 4-sulfated, but not 6-sulfated, chondroitin sulfate/dermatan sulfate (CS/DS). Keratan sulfate (KS) was present only as a low-sulfated moiety. Electron microscopic histochemistry disclosed a complex array of corneal PGs present as (1) fine filaments radiating from collagen fibrils, and (2) elongate, straplike structures, running either along the fibril axis or weaving across the primary fibril orientation. These large structures were digested by chondroitinase ABC, but not by keratanase. CONCLUSIONS: KS in the mouse is predominantly undersulfated and generates an immunostaining pattern that differs from that observed in corneas of other mammalian species thus far investigated. The mouse cornea resembles other mammalian corneas in the presence of filamentous arrays of small, collagen-associated stromal PGs visualized by cationic dye staining. However, large dye-positive structures with a CS/DS component are also present and appear to be unique to the cornea of this species

Differential relative sulfation of keratan sulfate glycosaminoglycan in the chick cornea during embryonic development

Purpose. To investigate structural remodeling of the developing corneal stroma concomitant with changing sulfation patterns of keratan sulfate (KS) glycosaminoglycan (GAG) epitopes during embryogenesis and the onset of corneal transparency. Methods. Developing chick corneas were obtained from embryonic day (E)12 to E18 of incubation. Extracellular matrix composition and collagen fibril spacing were evaluated by synchrotron x-ray diffraction, hydroxyproline assay, ELISA (with antibodies against lesser and more highly sulfated KS), and transmission electron microscopy with specific proteoglycan staining. Results. A significant relative increase in highly sulfated KS epitope labeling occurred with respect to hydroxyproline content in the final week of chick development, as mean collagen interfibrillar distance decreased. Small KS PG filaments increased in frequency with development and were predominantly fibril associated. Conclusions. The accumulation of highly sulfated KS during the E12 to E18 timeframe could serve to fine tune local matrix hydration and collagen fibril spacing during corneal growth, as gross dehydration and compaction of the stroma progress through the action of the nascent endothelial pump

Differential immunogold localisation of sulphated and unsulphated keratan sulphate proteoglycans in normal and macular dystrophy cornea using sulphation motif-specific antibodies

Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the corneal stroma for tissue organisation and transparency. Macular corneal dystrophy (MCD) is a rare, autosomal recessive disease characterised by disturbances in KS expression. MCD is caused by mutations in CHST6, a gene encoding the enzyme responsible for KS sulphation. Sulphated KS is absent in type I disease causing corneal opacity and loss of vision. Genetic studies have highlighted the mutational heterogeneity in MCD, but supportive immunohistochemical studies on corneal KS have previously been limited by the availability of antibodies mostly reactive only with highly sulphated KS epitopes. In this study, we employed four antibodies against specific KS sulphation patterns, including one against unsulphated KS, to investigate their reactivity in a case of MCD compared with normal cornea using high-resolution immunogold electron microscopy. Mutation analysis indicated type I MCD with deletion of the entire open reading frame of CHST6. Contrast enhanced fixation revealed larger PG structures in MCD than normal. Unlike normal cornea, MCD cornea showed positive labelling with antibody to unsulphated KSPG, but was negative with antibodies to sulphated KSPG. These antibodies will thus facilitate high-resolution investigations of phenotypic heterogeneity in support of genetic studies in this disease

The role of human HtrA1 in arthritic disease

Human HtrA1 belongs to a widely conserved family of serine proteases involved in various aspects of protein quality control and cell fate. Although HtrA1 has been implicated in the pathology of several diseases, its precise biological functions remain to be established. Through identification of potential HtrA1 targets, studies presented herein propose that within the context of arthritis pathology HtrA1 contributes to cartilage degradation. Elevated synovial HtrA1 levels were detected in fluids obtained from rheumatoid and osteoarthritis patients, with synovial fibroblasts identified as a major source of secreted HtrA1. Mass spectrometry analysis of potential HtrA1 substrates within synovial fluids identified fibronectin as a candidate target, and treatment of fibronectin with recombinant HtrA1 led to the generation of fibronectin-degradation products that may be involved in cartilage catabolism. Consistently, treatment of synovial fibroblasts with HtrA1 or HtrA1-generated fibronectin fragments resulted in the specific induction of matrix metalloprotease 1 and matrix metalloprotease 3 expression, suggesting that HtrA1 contributes to the destruction of extracellular matrix through both direct and indirect mechanisms