Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing

Kashiwagi, M., Enghild, J. J., Gendron, Christi, Hughes, Clare Elizabeth ORCID: https://orcid.org/0000-0003-4726-5877, Caterson, Bruce ORCID: https://orcid.org/0000-0001-6016-0661, Itoh, Y. and Nagase, H. 2004. Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing. Journal of Biological Chemistry 279 (11) , pp. 10109-10119. 10.1074/jbc.M312123200

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Abstract

ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu373-Ala374 bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu1480-Gly1481 bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the C-terminal spacer domain deletion mutant more effectively hydrolyzed both the Glu373-Ala374 and Glu1480-Gly1481 bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein. Finding of ADAMTS-4 in the interleukin-1α-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Publisher:	American Society for Biochemistry and Molecular Biology
ISSN:	0021-9258
Last Modified:	27 Oct 2022 08:45
URI:	https://orca.cardiff.ac.uk/id/eprint/63312

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