Proteinase activity in chondroitin lyase (chondroitinase) and endo-β-<scp>d</scp>-galactosidase (keratanase) preparations and a method to abolish their proteolytic effect on proteoglycan

Oike, Y; Kimata, Koji; Shinomura, Tamayuki; Suzuki, Suguru

doi:10.1042/bj1910203

Cited by 88 publications

(35 citation statements)

References 10 publications

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“…Aggrecan (2 mg) or G1-link protein-hyaluronan ternary complex was dissolved in 2 ml of 0.1M Tris acetate buffer, pH 7.0, containing protease inhibitors (24), and 100 pl chondroitinase ABC (2.5 units/ml) and 100 pI keratanase (5 unitdml) were added and the solution incubated overnight at 37°C. The sample was desalted on a Sephadex G-50 column equilibrated with 0.05M NH,HCO,, pH 8.0, v d freeze dried.…”

Section: Methodsmentioning

confidence: 99%

Presence of antibodies to native G1 domain of aggrecan core protein in synovial fluids from patients with various joint diseases

Karopoulos

Rowley

Ilic

et al. 1996

Arthritis & Rheumatism

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Objective. To investigate the occurrence of IgG antibodies to aggrecan in synovial fluids (SF) from patients with arthritis and various articular diseases, and to determine the nature of epitopes present within aggrecan that react with these antibodies. Methods. SF samples were reacted with native aggrecan, reduced and alkylated aggrecan, chondroitin sulfate, and keratan sulfate, using dot‐blots and a novel enzyme‐linked immunosorbent assay (ELISA). The nature of the epitopes present on aggrecan was elucidated using Western blots and a competitive inhibition ELISA. Results. IgG antibodies to aggrecan were found in >50% of the SF samples tested. No IgG antibody reactivity was observed in serum from the same patients. The antibodies appeared to react predominantly with native aggrecan, and there was no disease specificity. It was shown that the epitopes to these antibodies were located within the N‐terminal region of the core protein. Conclusion. This study demonstrates the frequent occurrence of IgG antibodies to aggrecan in human SF. The major epitope is located in the G1 domain of the aggrecan core protein. These IgG antibodies appear to be produced locally within the synovial cavity, probably in response to various articular diseases, resulting in the loss of native aggrecan from articular cartilage.

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Section: Methodsmentioning

confidence: 99%

Presence of antibodies to native G1 domain of aggrecan core protein in synovial fluids from patients with various joint diseases

Karopoulos

Rowley

Ilic

et al. 1996

Arthritis & Rheumatism

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“…It has been reported that the GAG digestion enzyme preparations may contain low levels of contaminating proteases, and the use of a protease inhibitor mixture is recommended to eliminate any effect by contaminants on sample integrity (30). The protease inhibitor mixture included during deglycosylation of samples following enzymatic assay could not be used in the preparation of the deglycosylated substrate because the presence of the mixture would interfere with aggrecanase and tMMP-3 enzyme activities.…”

Section: Discussionmentioning

confidence: 99%

Age-related Changes in Aggrecan Glycosylation Affect Cleavage by Aggrecanase

Pratta

Tortorella²,

Arner³

2000

Journal of Biological Chemistry

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. 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.The articular cartilage matrix consists primarily of collagen, which provides strength and support, and proteoglycan, which contributes qualities of compressibility and elasticity. The major type of proteoglycan present in cartilage is aggrecan, which is composed of a protein core that contains a high level of sulfated glycosaminoglycans (GAGs), 1 both CS and KS. The sulfate groups of these sugar molecules impart a net negative charge to aggrecan thus providing the attractive forces that incorporate water into the matrix and endow the tissue with its shock-absorbing quality. The aggrecan core protein contains three globular domains; G1, through which the molecule binds to hyaluronic acid, G2, and G3 (1, 2). In diseased tissue, the matrix is lost, and this loss is associated with degradation of the aggrecan monomers (3). The interglobular domain (IGD) of the aggrecan core protein, which is located between G1 and G2, contains proteolytic cleavage sites that are believed to be critical to the overall loss of aggrecan function. Two major sites of digestion have been identified within the IGD between residues Asn 341 and Phe 342and between Glu 373 and Ala 374 . The first site has been shown to be cleaved by a variety of matrix metalloproteinases (MMPs) (4 -8), whereas cleavage at the second site is catalyzed by aggrecanase. The contribution of aggrecanase to aggrecan cleavage has been investigated based on the generation of products that terminate with Glu 373 or begin with Ala 374. Several reports have shown that the majority of the aggrecan fragments found both in vitro in response to stimulated cartilage degradation (9 -13) and in vivo in arthritic synovial fluids (14, 15) are generated by cleavage at the aggrecanase site.Recent in vitro studies from our laboratory (16) show that there is a strong correlation between specific cleavage at the Glu 373 -Ala 374 bond and the release of aggrecan catabolites in response to cytokine stimulation. In addition, the ability of inhibitors to block the release of aggrecan catabolites correlates with their ability to block specific cleavage at the aggrecanase site. Taken together these data suggest that aggrecanase plays a key role in aggrecan degradation. Thus, identifying factors that influence the ability of aggrecanase to cleave cartilage aggrecan is important for understanding the regulation of aggrecan catabolism by this enzyme in both normal matrix turnover and in arthritic disease.Because the aggrecan core protein is heavily glycosylated, it is possible that the glycosaminoglycans on the a...

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“…Studies demonstrating an interaction of a protein with the extracellular matrix (ECM) using bacterial glycosidases are often compromised by the potential contribution of protease contaminants that have been detected in the commercially available preparations (24). To eliminate the contribution of the contaminating proteases, all digestions using the glycosidases were done in the presence of a broad-spectrum protease inhibitor cocktail (Complete), which was purchased from Boehringer Mannheim (Indianapolis, IN).…”

Section: Methodsmentioning

confidence: 99%

Induction of aggrecanase 1 (ADAM‐TS4) by interleukin‐1 occurs through activation of constitutively produced protein

Pratta

Scherle

Yang

et al. 2003

Arthritis & Rheumatism

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Objective. To study the production of aggrecanase 1 (ADAM-TS4) in monolayer chondrocytes, capsular fibroblasts, and cartilage.Methods. Bovine nasal and articular cartilage, monolayer chondrocytes, and capsular fibroblasts were incubated in the absence and presence of interleukin-1 (IL-1). ADAM-TS4 production was evaluated by immunofluorescence or by Western blot analysis. Aggrecanase activity was measured in cells grown on an immobilized peptide substrate, and peptide cleavage was monitored by enzyme-linked immunosorbent assay.Results. There was constitutive production of ADAM-TS4 in both cells and tissue. The protein was associated with the extracellular matrix based on the observation that the staining could be reduced following treatment of chondrocytes with heparin or exposure to chondroitinase ABC. Interestingly, there was no detectable change in the abundance of ADAM-TS4 in response to IL-1. Western blot analysis of cell lysates from IL-1-stimulated chondrocytes showed no evidence of increased ADAM-TS4 production, but resulted in activation of ADAM-TS4. The activation was associated with an increased generation in the aggrecanase neoepitope NITEGE in nasal cartilage in response to IL-1. These data suggest that induction of aggrecanase activity both in cells and in cartilage by IL-1 may involve the stimulation of an activator of ADAM-TS4. Consistent with this observation, culture of chondrocytes on a solid support containing a peptide substrate resulted in the generation of aggrecanase-mediated cleavage that could be blocked by selective inhibitors of ADAM-TS4.Conclusion. These data support the hypothesis that ADAM-TS4 is constitutively produced in these cells and tissue, and that stimulation by IL-1 results in aggrecanase activation. Thus, the activator could be a potential target by which to control aggrecanasemediated degradation in arthritic diseases.

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Proteinase activity in chondroitin lyase (chondroitinase) and endo-β-d-galactosidase (keratanase) preparations and a method to abolish their proteolytic effect on proteoglycan

Cited by 88 publications

References 10 publications

Presence of antibodies to native G1 domain of aggrecan core protein in synovial fluids from patients with various joint diseases

Presence of antibodies to native G1 domain of aggrecan core protein in synovial fluids from patients with various joint diseases

Age-related Changes in Aggrecan Glycosylation Affect Cleavage by Aggrecanase

Induction of aggrecanase 1 (ADAM‐TS4) by interleukin‐1 occurs through activation of constitutively produced protein

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