Development of a high-throughput screening assay for inhibitors of aggrecan cleavage using luminescent oxygen channeling (AlphaScreen (TM)) (original) (raw)
Related papers
Biochemical Journal, 1999
The importance of aggrecanase versus matrix metalloproteinase (MMP) enzymic activities in the degradation of aggrecan in normal and osteoarthritic (OA) articular cartilage in itro was studied in order to further our understanding of the potential role of these two enzyme activities in aggrecan catabolism during the pathogenesis of cartilage degeneration. Porcine and bovine articular cartilage was maintained in explant culture for up to 20 days in the presence or absence of the catabolic stimuli retinoic acid, interleukin-1 or tumour necrosis factor-α. Release of proteoglycan from cartilage was measured as glycosaminoglycan (GAG) release using a colorimetric assay. Analysis of proteoglycan degradation products, both released into culture media and retained within the cartilage matrix, was performed by Western blotting using antibodies specific for the N-and Cterminal neoepitopes generated by aggrecanase-and MMPrelated catabolism of the interglobular domain of the aggrecan core protein (IGD). In addition, studies determining the mRNA expression for MMP-3 and MMP-13 in these same cultures were undertaken. These analyses indicated that all three catabolic agents stimulated the release of 80 % of the GAG from the articular cartilage over 4 days. The degree of GAG release corresponded to an increase in aggrecanase-generated aggrecan catabolites released into the media and retained within the cartilage. Importantly, there was no evidence for the release of
Aggrecanase: A Target for the Design of Inhibitors of Cartilage Degradation
Annals of the New York Academy of Sciences, 1999
In arthritic diseases there is a gradual erosion of cartilage that leads to a loss of joint function. Aggrecan, which provides cartilage with its properties of compressibility and elasticity, is the first matrix component to undergo measurable loss in arthritis. This loss of aggrecan appears to be due to an increased rate of degradation, that can be attributed to proteolytic cleavage of the core protein within the interglobular domain (IGD). Two major sites of cleavage have been identified within the IGD. One, between the amino acids Asn 341 -Phe 342 , where the matrix metalloproteinases (MMPs) have been shown to clip; and the other, between Glu 373 -Ala 374 , which is attributed to a novel protease, "aggrecanase." We have generated aggrecanase in conditioned media from IL-1-stimulated bovine nasal cartilage and have used an enzymatic assay to evaluate this proteinase activity. In these studies we follow the generation of aggrecanase and MMPs in response to IL-1 in this system and examine the contribution of these enzymes in aggrecan degredation. Our data suggest that aggrecanase is a key enzyme in cartilage aggrecan degradation that represents a novel target for cartilage protection therapy in arthritis.
Osteoarthritis and Cartilage, 2006
To identify the major aggrecanase- and matrix metalloproteinase (MMP)-generated aggrecan fragments in human osteoarthritis (OA) synovial fluid and in human OA joint cartilage. Aggrecan fragments were prepared by CsCl gradient centrifugation. Fragment distributions were compared with aggrecanase-1 (ADAMTS-4) and MMP-3 digested human aggrecan by analysis with neoepitope antibodies and an anti-G1 domain antibody, using Western immuno-blots. The overall fragment pattern of OA synovial fluid aggrecan was similar to the fragment pattern of cartilage aggrecan cleaved in vitro by ADAMTS-4. However, multiple glycosaminoglycan (GAG) containing aggrecanase and MMP-generated aggrecan fragments were identified in OA synovial fluid and some of these fragments were produced by the action of both types of proteinases. The synovial fluid content of large size aggrecan fragments with (374)ARGS- and (342)FFGV- N-terminals was about 107 and 40 pmoles per ml, respectively, out of a total concentration of aggrecan fragments of about 185 pmoles per ml. OA synovial fluid contained insignificant amounts of the G1-IPEN(341) fragment as compared to the G1-TEGE(373) fragment, while OA cartilage contained significant amounts of both fragments. OA cartilage contained several GAG-containing aggrecan fragments with N-terminals of G1- or (342)FFGV- but no fragments with an N-terminal of (374)ARGS-. The overall pattern of aggrecan fragments in human OA synovial fluid and cartilage supports an important role for aggrecanase in aggrecan degradation. However, the fragment patterns and their differential distribution between cartilage and synovial fluid are consistent with the existence of at least two proteolytic pathways for aggrecan degradation in human OA, generating both (342)FFGV- and (374)ARGS-fragments.
Osteoarthritis and Cartilage, 2008
Objective: In a previous study, we identified a 50-kDa G3-containing aggrecan degradation product in bovine cartilage, released from the tissue after interleukin-1 (IL-1) stimulation in the presence of oncostatin M (OSM). Our objective was to purify, determine the N-terminal sequence of this fragment and verify whether this cleavage could be attributed to a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 action in vitro.
Journal of Biological Chemistry, 1999
A recombinant human aggrecan G1-G2 fragment comprising amino acids Val 1-Arg 656 has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M r 90,000-95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. Neoepitope antibodies recognizing the N-terminal F 342 FGVG or C-terminal DIPEN 341 sequences were used to confirm MMP cleavage at the Asn 341 2 Phe bond, while neoepitope antibodies recognizing the N-terminal A 374 RGSV or C-terminal ITEGE 373 sequences were used to confirm aggrecanase cleavage at the Glu 373 2 Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1␣ or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.
Osteoarthritis and Cartilage, 2010
Objective: To develop a short-term in vivo model in rats, with an enzyme-linked immunosorbent assay (ELISA) readout for specific aggrecanase-cleaved aggrecan fragments, to facilitate testing of aggrecanase inhibitors. Methods: Monosodium iodoacetate (MIA), a metabolic inhibitor, was injected into the right knee joint of male Lewis rats and the release of aggrecanase-cleaved fragments of aggrecan containing the NITEGE or ARGN neoepitope was measured in the synovial fluid at 7 days post MIA injection using novel ELISAs. The ELISAs utilize a commercial antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan, in combination with either an a-NITEGE antibody (NITEGE ELISA) or an a-ARGS/BC3 antibody (ARGS ELISA), to detect aggrecanase-cleavage of aggrecan within the interglobular domain (IGD). Aggrecan fragments present in in vitro digests, in cytokine-treated cartilage explant culture supernatants and in rat synovial fluid lavage samples were detected and quantified using the two ELISAs. Small molecule inhibitors of aggrecanase activity were dosed orally on days 3e7 to determine their ability to inhibit MIA-induced generation of the NITEGE and ARGN neoepitopes measured in the rat synovial fluid. Results: The NITEGE assay was shown to specifically detect the N-terminal fragment of aggrecan comprising the G1 domain and the NITEGE neoepitope sequence. This assay can readily measure aggrecanase-cleaved bovine, human and rat aggrecan without the need for deglycosylation. The ARGS assay specifically detects C-terminal fragments of aggrecan comprising the ARGS/ARGN neoepitope and the G2 domain. Keratan sulfate (KS) residues of aggrecan interfere with this ELISA, and hence this assay works well with native rat articular cartilage aggrecan (that lacks KS residues) and with deglycosylated bovine and human aggrecan. Injection of MIA into the rat knee joints resulted in a time-dependent increase in the release of aggrecanase-cleaved aggrecan fragments into the synovial fluid and treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of the generation of these neoepitopes. Conclusions: We have established a short-term in vivo model in rats that involves measurement of synovial fluid biomarkers that are dependent on aggrecanase activity in the joint. The short duration of the model combined with the mechanistic biomarker readout makes it very useful for the initial in vivo screening of aggrecanase inhibitors prior to testing them in time and resource-intensive disease models of osteoarthritis (OA).
The cleavage of biglycan by aggrecanases
Osteoarthritis and Cartilage, 2006
Objective: Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4] and aggrecanase-2 (ADAMTS-5) have been named for their ability to degrade the proteoglycan aggrecan. While this may be the preferred substrate for these enzymes, they are also able to degrade other proteins. The aim of this work was to determine whether the aggrecanases could degrade biglycan and decorin.
Arthritis & Rheumatism, 1993
Synovial fluid was collected from patients with recent knee injury and from patients with early or late stage osteoarthritis. Chondroitin sulfate-substituted aggrecan fragments present in these fluids, and in normal bovine synovial fluid, were purified by cesium chloride gradient centrifugation, enzymically deglycosylated and fractionated by gel filtration on Superose-12. Each sample contained two major aggrecan core protein populations with apparent molecular masses of 90 kD and 150 kD. For all samples, NH2-terminal analysis of both populations gave a single major sequence beginning ARGSV. This NH2 terminus results from cleavage ofthe human aggrecan core protein at the Glu 373-Ala 374 bond within the interglobular domain between the G1 and G2 domains. Cleavage at this site also occurs during control and interleukin-1 stimulated aggrecan catabolism in bovine cartilage explant cultures (Sandy, J., P. Neame, R. Boynton, and C. Flannery. 1991. J. Biol. Chem. 266:8683-8685).
2000
We have studied aggrecan catabolism mediated by matrix metalloproteinases (MMPs) in a porcine cartilage culture system. Using antibodies specific for DIPEN 341 and 342 FFGVG neoepitopes, we have detected MMP-derived fragments in conditioned medium and cultured cartilage, by radioimmunoassay, Western blotting, and immunolocalization. Radioimmunoassay revealed that the amount (pmol of epitope/mg of total glycosaminoglycan) of 342 FFGVG epitope released from cartilage remained constant over a 5-day culture period and was not increased by IL-1␣ or retinoate. However, the proportion (pmol of epitope/mg of released glycosaminoglycan) of 342 FFGVG epitope released was decreased upon stimulation, consistent with the involvement of a non-MMP proteinase, such as aggrecanase. The data suggest that in vitro MMPs may be involved in the base-line catabolism of aggrecan. Immunolocalization experiments showed that DIPEN 341 and ITEGE 373 epitopes were increased by treatment with IL-1␣ and retinoate. Confocal microscopy revealed that ITEGE 373 epitope was largely intracellular but with matrix staining in the superficial zone, whereas DIPEN 341 epitope was cell-associated and widely distributed in the matrix. Surprisingly, the majority of 342 FFGVG epitope, determined by radioimmunoassay and Western blotting, was retained in the tissue despite the absence of a G1 domain anchor. Interleukin-1␣ stimulation caused a marked increase in tissue DIPEN 341 and 342 FFGVG epitope, and the 342 FFGVG fragments retained in the tissue were larger than those released into the medium. Active porcine aggrecanase was unable to cleave 342 FF-GVG fragments at the 2Glu 373 2Ala 374 bond but cleaved intact aggrecan at this site, suggesting that 342 FFGVG fragments are not substrates for aggrecanase. The apparent retention of large 342 FFGVG fragments within cartilage, and their resistance to N-terminal cleavage by aggrecanase suggests that 342 FF6V6 fragments may have a role in cartilage homeostasis.