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.

Functional Studies

Degradation of extracellular matrix proteoglycans

Screening and characterisation of inhibitors

Standard in enzymatic and immunological assays

Optimal working dilutions must be determined by the end user.

Solubilized in 50mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 50mM imidazol 0.05 % Brij-35.

Maintain at -70°C for 12 months from date of receipt. The enzyme can be kept at -20°C for several weeks and ice for several days. Avoid repeated freeze/thaw cycles.

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