Utilization of a Recombinant Substrate rAgg1 to Study the Biochemical Properties of Aggrecanase in Cell Culture Systems

Hughes, Claire E.; Büttner, Frank; Eidenmüller, Bernd; Caterson, Bruce; Bartnik, Eckart

doi:10.1074/jbc.272.32.20269

Cited by 39 publications

(42 citation statements)

References 29 publications

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“…Other studies comparing the presence or absence of endogenous keratan sulfate chains have suggested that aggrecanase cleavage in the IGD may be increased in the presence of keratan sulfate and reduced when keratan sulfate is enzymatically removed (36,37). However, recombinant substrates that lack keratan sulfate are not totally resistant to cleavage by aggrecanases (38,39). Collectively, these studies suggest that keratan sulfate in the IGD may influence aggrecanolysis.…”

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confidence: 66%

N-Linked Keratan Sulfate in the Aggrecan Interglobular Domain Potentiates Aggrecanase Activity

Poon

Plaas

Keene

et al. 2005

Journal of Biological Chemistry

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Keratan sulfate is thought to influence the cleavage of aggrecan by metalloenzymes. We have therefore produced a recombinant substrate, substituted with keratan sulfate, suitable for the study of aggrecanolysis in vitro. Recombinant human G1-G2 was produced in primary bovine keratocytes using a vaccinia virus expression system. Following purification and digestion with specific hydrolases, fluorophore-assisted carbohydrate electrophoresis was used to confirm the presence of the monosulfated Gal-GlcNAc6S and GlcNAc6s-Gal disaccharides and the disulfated Gal6S-GlcNAc6S disaccharides of keratan sulfate. Negligible amounts of fucose or sialic acid were detected, and the level of unsulfated disaccharides was minimal. Treatment with keratanases reduced the size of the recombinant G1-G2 by ϳ5 kDa on SDS-PAGE. Treatment with N-glycosidase F also reduced the size of G1-G2 by ϳ5 kDa and substantially reduced G1-G2 immunoreactivity with monoclonal antibody 5-D-4, indicating that keratan sulfate on the recombinant protein is Nlinked. Cleavage of G1-G2 by aggrecanase was markedly reduced when keratan sulfate chains were removed by treatment with keratanase, keratanase II, endo-␤-galactosidase, or N-glycosidase F. These results indicate that modification of oligosaccharides in the aggrecan interglobular domain with keratan sulfate, most likely at asparagine residue 368, potentiates aggrecanase activity in this part of the core protein.Aggrecan is a major structural component of cartilage and together with type II collagen it enables this tissue to bear load and resist compression. Aggrecan has three globular domains, G1 and G2 at the N terminus and G3 at the C terminus. An extended sequence between the G2 and G3 domains is heavily substituted with chondroitin sulfate and keratan sulfate chains, organized into distinct chondroitin sulfate-1, chondroitin sulfate-2, and keratan sulfate-rich regions. An interglobular domain (IGD) 1 of ϳ150 amino acids separates G1 from G2 and is substituted with keratan sulfate chains as well as O-linked and N-linked oligosaccharides.The IGD is highly sensitive to proteinases. Cleavage in the IGD releases the entire chondroitin sulfate and keratan sulfate-rich regions essential for the biomechanical properties of aggrecan and is therefore the most detrimental for cartilage function. In pathology, proteolysis is driven by aggrecanases and, to a lesser extent, by matrix metalloproteinases. Aggrecanase was first identified as a novel activity that cleaved the aggrecan core protein at the E 373 2 374 A bond in the IGD (1-3); the products of this cleavage were found in synovial fluids from patients with osteoarthritis, joint injury, and inflammatory joint disease (4, 5). Subsequently, cartilage enzymes with aggrecanase activity were revealed as members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family (6, 7), and ADAMTS5 has recently been identified as the major aggrecanase in mouse cartilage (8,9).Proteolysis by matrix metalloproteinases at the N 341 2 342 F bond i...

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confidence: 66%

N-Linked Keratan Sulfate in the Aggrecan Interglobular Domain Potentiates Aggrecanase Activity

Poon

Plaas

Keene

et al. 2005

Journal of Biological Chemistry

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“…One caveat of studies with monolayer chondrocytes is that, to our knowledge, no one has been able to demonstrate production of soluble aggrecanase activity from these cells. For example, Hughes et al (37) have shown that an activity representing aggrecanase could be de- Figure 4. Effect of interleukin-1 (IL-1) and chondroitinase ABC (C-ABC) on immunolocalization of aggrecanase 1 (ADAM-TS4) in bovine articular chondrocytes.…”

Section: Resultsmentioning

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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“…The total amount of aggrecan loaded in each lane was 12.5 g. Native represents aggrecan substrate that has been pretreated with heat-denatured GAG digestion enzymes; Treated represents aggrecan substrate treated with keratanases (A) or chondroitinase ABC (B). Arrowheads in B indicate BC-3-reactive products with a higher molecular mass than in the control at 4 h. cleavage at the Glu 373 -Ala 374 bond by aggrecanase was detected using recombinant substrates lacking KS (28,34). These data suggest that the presence of KS is not an absolute requirement for cleavage at the Glu 373 -Ala 374 bond, but rather that the presence of KS may increase the efficiency of cleavage at this site.…”

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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Utilization of a Recombinant Substrate rAgg1 to Study the Biochemical Properties of Aggrecanase in Cell Culture Systems

Cited by 39 publications

References 29 publications

N-Linked Keratan Sulfate in the Aggrecan Interglobular Domain Potentiates Aggrecanase Activity

N-Linked Keratan Sulfate in the Aggrecan Interglobular Domain Potentiates Aggrecanase Activity

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

Age-related Changes in Aggrecan Glycosylation Affect Cleavage by Aggrecanase

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