title: Purification of an insect derived recombinant human ADAMTS-1 reveals novel gelatin (type I collagen) degrading activities.
abstract: ADAMTS-1 (A Disintegrin And Metalloprotease with ThromboSpondin repeats) is a member of a family of secreted proteolytic enzymes with a complex modular structure. These enzymes are characterised by an N-terminal metalloproteinase domain, a disintegrin-like domain and a carboxyl terminal region containing variable numbers of a repeat sequence with homology to thrombospondin-1. The expression of the gene for ADAMTS-1 has been associated with inflammation, ovulation, angiogenesis, cellular proliferation and bone formation. ADAMTS-1 can proteolytically process large proteoglycans indicating a potential role in extracellular matrix turnover. In this study, we have tested ADAMTS-1 activity in gelatin zymogram assays. Since previous data demonstrate that ADAMTS-1 is a matrix metalloproteinase (MMP) substrate and is highly unstable in conditioned medium from eukaryotic cell types, we created an insect cell line expressing human ADAMTS-1. We isolated an epitope tagged full-length recombinant ADAMTS-1 from serum free insect cell conditioned medium. The purified protein had aggrecanase activity and appears as two major bands on the silver stained SDS-PAGE corresponding well to a pro-domain on form of 115 kDa and a pro-domain off form of 90 kDa. Using denatured type I collagen in zymographic analysis we demonstrate that ADAMTS-1 has a previously unreported gelatinolytic activity. Also, we notice that processing of its C-terminal region by an apparently autocatalytic process reveals a 27 kDa species with gelatinolytic activity. Furthermore, we show that MMP2 but not MMP13 remove ADAMTS-1 specific gelatin zymopraphic zones.
authors.affiliation: Musculoskeletal Research Group (SCMS), University of Newcastle Medical School, Newcastle, UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lind
authors.forename: Mark A
authors.initials: MA
authors.lastname: Birch
authors.forename: Norman
authors.initials: N
authors.lastname: McKie
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D000071097:ADAMTS1 Protein; D000818:Animals; D002417:Cattle; D024042:Collagen Type I / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism*; D006801:Humans; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D009036:Moths; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D024042:Collagen Type I; D011994:Recombinant Proteins; D005780:Gelatin; D051722:ADAM Proteins; D000071097:ADAMTS1 Protein; C103859:ADAMTS1 protein, human; D053509:Matrix Metalloproteinase 13; D020778:Matrix Metalloproteinase 2