详细信息:ADAMTS-5, recombinant human truncated.ADAMTS's (a disintegrin and metalloproteinase with thrombospondin motif) are multidomain extracellular proteinases, containing at least one thrombospondin type-1 repeat (Tang, 2001). The enzymes are involved in processing of extracellular matrix proteins and proteins in the circulation. ADAMTS-5 was first purified from bovine nasal cartilage conditioned media and human ADAMTS-5 cDNA was cloned from a human liver cDNA library (Abbaszade, I. et al, 1999). ADAMTS-5 consists of a prodomain, a catalytic domain, a disintegrin domain, a thrombospondin type-1 motif, a Cys-rich region followed by a spacer sequence and thrombospondin submotifs. The prodomain is most likely cleaved-off by a furin-type enzyme before ADAMTS-5 is released from cells. ADAMTS-5 is expressed constitutively in synovium (Vankemmelbeke, MN. et al, 2001) and it degrades aggrecan, the major proteoglycan of articular cartilage. Like ADAMTS4, another member of the ADAMTS-family, ADAMTS-5 cleaves aggrecan at 5 sites (Tortorella, MD. et al, 2002). Four cleavage sites are located in the chondroitin sulfate-rich region between aggrecan globular domains G2 and G3, while one site is contained in the rod-shaped polypeptide between globular domains G1 and G2. In addition, ADAMTS-5 cleaves one more site in the chondroitin sulfate-rich region that is not cleaved by ADAMTS4 (Tortorella, MD. et al, 2002). ADAMTS-5 is inhibited by TIMP 3 (Kashigawa, M. et al, 2001) and a2-macroglobulin (Tortorella, MD. et al, 2004).
Molecular form: Recombinant human ADAMTS-5 D625-930 is produced with the baculo-virus expression system and purified from insect cell culture supernatants. The protein contains the catalytic domain, the disintegrin domain and the thrombospondin type-1 motif of full-length ADAMTS-5 followed by a C-terminal His6-tag. The calculated Mr of the amino acid sequence is 40.8 kDa. In SDS-PAGE the protein exhibits a Mr of about 50 kDa.
ACTIVITY:
Aggrecanase activity of the ADAMTS-5 preparation is determined with recombinant aggrecan interglobular domain (Aggrecan-IGD from Chemicon). ADAMTS-5 hydrolyzes the "aggrecanase" site within this domain (peptide bond E373 -A374 in human aggrecan). The recombinant substrate is incubated at a concentration of 0.1 μM with ADAMTS-5 in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 1 μM leupeptin, 1 μM pepstatin, 1 mM Pefabloc, 0.05 % Brij 35 for 15 min at 37 °C. Substrate cleavage at the "aggrecanase"-site is estimated from the appearance of the hydrolysis fragment with the novel N-terminus ARGSVIL. The fragment is quantified with two monoclonal antibodies. Under the specified conditions the hydrolysis rate is > 0.5 nmoles hydrolyzed substrate/ min . ml ADAMTS-5 preparation or > 5 nmoles hydrolyzed substrate/ min . mg.
Inhibitors: ADAMTS-5 is inhibited by tissue inhibitor of matrix metalloproteinase 3 (TIMP3) and by alpha2-macroglobulin. Enzyme activity is also suppressed by chelators of divalent cations such as EDTA and by synthetic metalloproteinase inhibitors.
