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Purification of an insect derived recombinant human ADAMTS-1 reveals novel gelatin (type I collagen) degrading activities

Lookup NU author(s): Dr Thomas Lind, Dr Mark Birch, Dr Norman Balfour-McKie


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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. © Springer Science + Business Media, Inc. 2006.

Publication metadata

Author(s): Lind T, Birch MA, McKie N

Publication type: Article

Publication status: Published

Journal: Molecular and Cellular Biochemistry

Year: 2006

Volume: 281

Issue: 1-2

Pages: 95-102

ISSN (print): 0300-8177

ISSN (electronic): 1573-4919

Publisher: Springer


DOI: 10.1007/s11010-006-0637-y


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