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Objective: To obtain recombinant mouse collagenase ADAMTS2 (ADAM metallopeptidase with thrombospondin type 1 motif, 2) C terminal (1109-1213), and prepare the corresponding rabbit anti-ADAMTS2 polyclonal antibodies., Methods: The recombinant expression plasmid pGEX-6p-1-ADAMTS2 (1109-1213) was transformed into E.coli. The target protein was induced by IPTG and identified by mass spectrometry following affinity purification. The expressed and purified ADAMTS2 (1109-1213) protein was used to immunize New Zealand rabbits to prepare anti-ADAMTS2 polyclonal antibodies. The antibody titers were detected by ELISA and the antibody specificity by Western blotting., Results: The protein ADAMTS2 (1109-1213) was expressed in E.coli after IPTG induction, and with the purified protein, we prepared antiserum in the immunized rabbits. The titer of the antiserum reached over 1:160 000. The antiserum showed a good specificity., Conclusion: The high titer and specific rabbit anti-ADAMTS2 antibody has been prepared successfully.

ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2), members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, are considered to play a key role in aggrecan degradation of articular cartilage in human osteoarthritis. Here, we developed a neutralizing antibody to these aggrecanases by screening human combinatorial antibody library. Among the five candidate antibodies, one antibody was immunoreactive with both ADAMTS4 and ADAMTS5, showing no or negligible cross-reactivity with 10 different related metalloproteinases of the ADAMTS, ADAM (a disintegrin and metalloproteinase) and MMP (matrix metalloproteinase) gene families. This antibody almost completely and partially inhibited aggrecanase activity of ADAMTS4 and ADAMTS5, respectively. It also suppressed the aggrecanase activity derived from interleukin-1-stimulated osteoarthritic chondrocytes. These data demonstrate that the antibody is specific to ADAMTS4 and ADAMTS5 and inhibits their aggrecanase activity at molecular and cellular levels, and suggest that this antibody may be useful for treatment of pathological conditions such as osteoarthritis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Temporomandibular joint (TMJ) internal derangement (ID) is one of the most common form of temporomandibular disorders. There is evidence showing the increased expression of matrix metalloproteinases (MMPs) in the cells from degenerated TMJ disc. ADAMTS are a large family of metalloproteases which are responsible for proteoglycans degradation. The present study aimed to evaluate ADAMTS-4 and ADAMTS-5 immunohistochemical expression in human TMJ discs from patients affected by ID, and to find out if there is any correlation with the degree of histopathological changes. Eighteen temporomandibular displaced disc specimens and sixteen TMJ disc control were used for the present study. Specimens were immunohistochemically processed and ADAMTS-4 and ADAMTS-5 expression were obtained respectively for the anterior (AB), intermediate (IB) and posterior (PB) bands and compared to the histopathological degeneration score (HDS). Immunoreactivity for ADAMTS-4 and -5, was observed in both not degenerated and degenerated human TMJ discs. Both the percentage of ADAMTS-4 and -5 immunostained cells (ES) and the intensity of staining (IS) were significantly greater in affected specimens compared with those in control discs. The ADAMTS-5 ES and IS of the 3 bands of the disc correlated to the TMJ disc HDS (0.001 < P < 0.05), on the other hand only AB and IB, ADAMTS-4 immunostaining scores correlated to HDS. According to these findings it can be assumed in that the more histopathological changes in the disc are detected, the higher levels of ADAMTS are produced. This in turn can lead to ECM breakdown and in turn to a more advanced disc displacement., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: We previously described a messenger RNA variant of ADAMTS4 (ADAMTS4&#95;v1) in human synovial cell cocultures obtained from patients with osteoarthritis (OA). This RNA message has been found only in OA synovium and, if translated, would result in a protein identical to ADAMTS-4, except that the C-terminal spacer domain would be different. The purpose of this study was to determine whether ADAMTS4&#95;v1 is translated into a protein, is expressed in vivo, and acts as a functional aggrecanase., Methods: Polyclonal antibodies were raised against unique C-terminal sequences of ADAMTS-4&#95;v1. An immunohistochemical study of human OA synovium was performed. A mammalian expression vector coding for FLAG-tagged human ADAMTS4 was mutated to contain the different sequences of ADAMTS4&#95;v1, and the resultant plasmid was used to transfect HEK 293 cells. ADAMTS-4&#95;v1 produced by these cells was purified via the FLAG epitope, and the ability of this recombinant protein to cleave aggrecan, biglycan, and decorin was investigated., Results: An antibody specific for ADAMTS-4&#95;v1 was found to bind to the synovial membrane surface on cryosections, and the protein was detected in cell lysates from synovium obtained from OA patients. The recombinant ADAMTS-4&#95;v1 demonstrated enzyme activity toward the target substrate in a commercial aggrecanase 1 enzyme-linked immunosorbent assay and was also found to cleave aggrecan at the pathologically important Glu(373↓374) Ala aggrecanase site., Conclusion: ADAMTS-4&#95;v1 is expressed as a protein in vivo in human OA synovium, functions as an aggrecanase, and cleaves other proteoglycan substrates. This splice variant may be a major contributor to loss of aggrecan from the superficial zone of OA cartilage., (© The Authors. Arthritis & Rheumatism is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

ADAMTS-4 (aggrecanase1) is believed to play an important role in the degradation of aggrecan during the progression of joint diseases. ADAMTS-4 is synthesized as a latent pro-enzyme that requires the removal of the pro-domain, exposing the N-terminal neoepitope, to achieve activity. We developed a monoclonal antibody against this neoepitope of active ADAMTS-4. Furthermore, we established and characterized a competitive ELISA for measuring active ADAMTS-4 form applying the specific antibody. We used this assay to profile the presence of active ADAMTS-4 and its aggrecan degradation product (NITEGE(373)) in a bovine cartilage ex vivo model. We found that after stimulation with catabolic factors, the cartilage initially released high levels of aggrecanase-derived aggrecan fragments into supernatant but subsequently decreased to background levels. The level of active ADAMTS-4 released into the supernatant and retained in the cartilage matrix increased continuously throughout the 21days of the study. The activity of ADAMTS-4 on the last day of catabolic stimulation was verified in vitro by adding deglycosylated or native aggrecan to the conditioned medium. Samples of human cartilage affected by varying degrees of osteoarthritis stained strongly for active ADAMTS-4 where surface fibrillation and clustered chondrocytes were observed. This assay could be an effective tool for studying ADAMTS-4 activity and for screening drugs regulating ADAMTS-4 activation. Moreover, it could be a potential biomarker for degenerative joint disease., (Copyright © 2012 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)