An improved method for the structural profiling of keratan sulfates: analysis of keratan sulfates from brain and ovarian tumors.
A previously developed method for the structural fingerprinting of keratan sulfates (Brown et al., Glycobiology, 5, 311-317, 1995) has been adapted for use with oligosaccharides fluorescently labeled with 2-aminobenzoic acid following keratanase II digestion. The oligosaccharides are separated by high-pH anion-exchange chromatography on a Dionex AS4A-SC column. This methodology permits quantitative analysis of labeled oligosaccharides which can be detected at the sub-nanogram ( approximately 100 fmol) level. Satisfactory calibration of this method can be achieved using commercial keratan sulfate standards. Keratan sulfates from porcine brain phosphocan and human ovarian tumors have been examined using this methodology, and their structural features are discussed. (+info)
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. (+info)
Fibromodulin-null mice have abnormal collagen fibrils, tissue organization, and altered lumican deposition in tendon.
Fibromodulin is a member of a family of connective tissue glycoproteins/proteoglycans containing leucine-rich repeat motifs. Several members of this gene family bind to fibrillar collagens and are believed to function in the assembly of the collagen network in connective tissues. Here we show that mice lacking a functional fibromodulin gene exhibit an altered morphological phenotype in tail tendon with fewer and abnormal collagen fiber bundles. In fibromodulin-null animals virtually all collagen fiber bundles are disorganized and have an abnormal morphology. Also 10-20% of the bundles in heterozygous mice are similar to the abnormal bundles in fibromodulin-null tail tendon. Ultrastructural analysis of Achilles tendon from fibromodulin-null mice show collagen fibrils with irregular and rough outlines in cross-section. Morphometric analysis show that fibromodulin-null mice have on the average thinner fibrils than wild type animals as a result of a larger preponderance of very thin fibrils in an overall similar range of fibril diameters. Protein and RNA analyses show an approximately 4-fold increase in the content of lumican in fibromodulin-null as compared with wild type tail tendon, despite a decrease in lumican mRNA. These results demonstrate a role for fibromodulin in collagen fibrillogenesis and suggest that the orchestrated action of several leucine-rich repeat glycoproteins/proteoglycans influence the architecture of collagen matrices. (+info)
Longitudinal and cross-sectional variability in markers of joint metabolism in patients with knee pain and articular cartilage abnormalities.
OBJECTIVE: To determine the within- and between-patient variability in the concentrations of synovial fluid, serum and urine markers of joint tissue metabolism in a cohort of patients with knee pain and cartilage changes consistent with early-stage knee osteoarthritis. DESIGN: Samples of synovial fluid, serum, and urine were obtained from 52 patients on eight different occasions during 1 year, as part of a clinical trial in patients with cartilage abnormalities and knee pain. In joint fluid, aggrecan fragments were quantified by dye precipitation and enzyme-linked immunosorbent assay (ELISA), and matrix metalloproteinases-1 and -3, and tissue inhibitor of metalloproteinases-1 by sandwich ELISAs. In serum, keratan sulfate was quantified by ELISA. Type I collagen N-telopeptide cross-links in urine were determined by ELISA. RESULTS: The degree of cross-sectional variability in marker concentrations did not vary between the different sampling occasions, and did not differ between the periods of weeks 0 (baseline), 1-4 (treatment) and 13-26 (follow-up). Both between-patient and within-patient coefficients of variation varied for markers in different body fluid compartments, with the lowest variability for serum keratan sulfate, followed by urine type I collagen N-telopeptide crosslinks, and the highest for synovial fluid markers. For synovial fluid, aggrecan fragments showed the least variability, and matrix metalloproteinases the highest. One patient with septic arthritis showed a fivefold peak increase in joint fluid aggrecan fragment concentrations, while the concentration of matrix metalloproteinase-3 increased 100-fold. CONCLUSIONS: Molecular markers of joint tissue metabolism have been suggested as, for example, outcome measures for clinical trials of disease-modifying drugs in osteoarthritis. This report is the first to present data on between- and within-patient variability for such molecular markers in three different body fluid compartments in stable cohort of patients. The availability of such data enables calculations to determine the number of patients needed in prospective studies using these markers as outcome measures. (+info)
Resistance of small leucine-rich repeat proteoglycans to proteolytic degradation during interleukin-1-stimulated cartilage catabolism.
A bovine nasal-cartilage culture system has been utilized to analyse the catabolic events occurring in response to interleukin-1beta over a 14-day period. An early event following the start of interleukin-1 treatment was the release of glycosaminoglycan into the culture medium. This release was accompanied by the appearance in the tissue, and shortly thereafter also in the culture media, of a globular domain (G1)-containing aggrecan degradation product generated by the action of aggrecanase. Link protein was also released from the cartilage with a similar timeframe to that of the G1 fragment, although there was no evidence of its proteolytic degradation. By comparison with aggrecan, the small leucine-rich repeat proteoglycans decorin, biglycan and lumican showed a resistance to both proteolytic cleavage and release throughout the culture period. In contrast, fibromodulin exhibited a marked decrease in size after day 4, presumably due to proteolytic modification, but the major degradation product was retained throughout the culture period. Also in contrast with the early changes in the components of the proteoglycan aggregate, type II collagen did not display signs of extensive degradation until much later in the culture period. Collagen degradation products compatible with collagenase action first appeared in the medium by day 10 and increased thereafter. These data demonstrate that the leucine-rich repeat proteoglycans are resistant to proteolytic action during interleukin-1-stimulated cartilage catabolism, compared with aggrecan. This resistance and continued interaction with the surface of the collagen fibrils may help to stabilize the collagen fibrillar network and protect it from extensive proteolytic attack during the early phases of cartilage degeneration. (+info)
Genomic characterization of human DSPG3.
DSPG3, the human homolog to chick PG-Lb, is a mejrkp6of the small leucine-rich repeat proteoglycan (SLRP) family, including decorin, biglycan, fibromodulin, and lumican. In contrast to the tissue distribution of the other SLRPs, DSPG3 is predominantly expressed in cartilage. In this study, we have determined that the human DSPG3 gene is composed of seven exons: Exon 2 of DSPG3 includes the start codon, exons 4-7 code for the leucine-rich repeats, exons 3 and 7 contain the potential glycosaminoglycan attachment sites, and exon 7 contains the potential N-glycosylation sites and the stop codon. We have identified two polymorphic variations, an insertion/deletion composed of 19 nucleotides in intron 1 and a tetranucleotide (TATT)n repeat in intron 5. Analysis of 1.6 kb of upstream promoter sequence of DSPG3 reveals three TATA boxes, one of which is 20 nucleotides before the transcription start site. The transcription start site precedes the translation start site by 98 nucleotides. There are 14 potential binding sites for SOX9, a transcription factor present in cartilage, in the promoter, and in the first intron of DSPG3. We have examined the evolution of the SLRP gene family and found that gene products clustered together in the evolutionary tree are encoded by genes with similarities in genomic structure. Hence, it appears that the majority of the introns in the SLRP genes were inserted after the differentiation of the SLRP genes from an ancestral gene that was most likely composed of 2-3 exons. (+info)
Treatment with calcitonin suppresses the responses of bone, cartilage, and synovium in the early stages of canine experimental osteoarthritis and significantly reduces the severity of the cartilage lesions.
OBJECTIVE: To relate the rate of bone resorption to serum levels of both hyaluronan (HA) and antigenic keratan sulfate (KS) in canine experimental osteoarthritis (OA) and to evaluate the effects of calcitonin on these parameters and the OA lesions of the unstable knee. METHODS: Twenty-two dogs underwent anterior cruciate ligament transection (ACLT) and 6 dogs underwent sham operation. Urinary pyridinium crosslinks were quantified by high-performance liquid chromatography. Immunoassays quantified hyaluronan (HA) and antigenic KS. Macroscopic and histologic OA lesions were scored. Calcitonin treatment was started on day 14 postsurgery and stopped on either day 49 or day 104 postsurgery. Control dogs and all treated dogs were killed on day 105. RESULTS: All ACLT joints developed OA. In contrast to sham-operated animals, all operated dogs exhibited an early and sustained rise in the levels of their urinary and serum markers. Calcitonin markedly reduced the levels of these markers and the severity of OA lesions. Furthermore, the longer the period of calcitonin therapy, the lower the score of the OA lesions. CONCLUSION: Bone, synovium, and articular cartilage all appear to be involved in the state of hypermetabolism that develops in unstable joints. Furthermore, the rate of bone resorption increases markedly in the early stages of this OA model and is likely to contribute to cartilage breakdown. Since calcitonin reduced the severity of OA changes, this form of therapy may have benefits for humans who have recently experienced a traumatic knee injury. (+info)
Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: maintenance of the keratocyte phenotype in culture.
PURPOSE: To determine the effect of serum on morphology, growth, and proteoglycan synthesis by primary cultures of collagenase-isolated bovine keratocytes. METHODS: Keratocytes were isolated from bovine corneas using sequential collagenase digestion and cultured in Dulbecco's modified Eagle's medium (DMEM), with and without fetal bovine serum (FBS). Proteoglycans synthesized by the cells in culture and by keratocytes in intact cornea culture were metabolically radiolabeled with 35SO4. The proteoglycans were characterized by their sensitivity to keratanase, chondroitinase ABC, and heparatinase and by their size on Superose 6 HR. Cell number was determined by measuring DNA content of the culture dishes. RESULTS: Keratocytes cultured in 10% FBS proliferated, appeared fibroblastic, and synthesized only 9% of the total glycosaminoglycan as keratan sulfate (KS), whereas cells in serum-free media were quiescent, appeared dendritic, and synthesized 47% KS, a value similar to the 45% KS for corneas radiolabeled overnight in organ culture. This increased proportion of KS synthesis in serum-free media was caused by a moderate increase in KS synthesis combined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fractionation on Superose 6 High Resolution showed the size and relative amounts of the CS- and KS-containing proteoglycans synthesized by keratocytes in serum-free media also more closely resembled that of keratocytes in corneas in organ culture than keratocytes in media containing serum. CONCLUSIONS: A comparison of proteoglycan synthesis and cell morphology between keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearance when cultured in serum-free media. These results also suggest that culturing in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound healing. (+info)