Evidence for restricted reactivity of ADAMDEC1 with protein substrates and endogenous inhibitors

Lund, Jacob, Troeberg, Linda ORCID: https://orcid.org/0000-0003-0939-4651, Kjeldal, Henrik, Olsen, Ole H, Nagase, Hideaki, Sørensen, Esben S, Stennicke, Henning R, Petersen, Helle H and Overgaard, Michael T (2015) Evidence for restricted reactivity of ADAMDEC1 with protein substrates and endogenous inhibitors. The Journal of Biological Chemistry, 290 (10). pp. 6620-6629. ISSN 0021-9258

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ADAMDEC1 is a proteolytically active metzincin metalloprotease displaying rare active site architecture with a zinc-binding Asp residue (Asp-362). We previously demonstrated that substitution of Asp-362 for a His residue, thereby reconstituting the canonical metzincin zinc-binding environment with three His zinc ligands, increases the proteolytic activity. The protease also has an atypically short domain structure with an odd number of Cys residues in the metalloprotease domain. Here, we investigated how these rare structural features in the ADAMDEC1 metalloprotease domain impact the proteolytic activity, the substrate specificity, and the effect of inhibitors. We identified carboxymethylated transferrin (Cm-Tf) as a new ADAMDEC1 substrate and determined the primary and secondary cleavage sites, which suggests a strong preference for Leu in the P1' position. Cys(392), present in humans but only partially conserved within sequenced ADAMDEC1 orthologs, was found to be unpaired, and substitution of Cys(392) for a Ser increased the reactivity with α2-macroglobulin but not with casein or Cm-Tf. Substitution of Asp(362) for His resulted in a general increase in proteolytic activity and a change in substrate specificity was observed with Cm-Tf. ADAMDEC1 was inhibited by the small molecule inhibitor batimastat but not by tissue inhibitor of metalloproteases (TIMP)-1, TIMP-2, or the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3). However, N-TIMP-3 displayed profound inhibitory activity against the D362H variants with a reconstituted consensus metzincin zinc-binding environment. We hypothesize that these unique features of ADAMDEC1 may have evolved to escape from inhibition by endogenous metalloprotease inhibitors.

Item Type: Article
Additional Information: © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Uncontrolled Keywords: antagonists & inhibitors,genetics,catalytic domain,crystallography, x-ray,gene expression regulation, enzymologic,humans,antagonists & inhibitors,protein structure, tertiary,proteolysis,substrate specificity,chemistry,chemistry
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
UEA Research Groups: Faculty of Medicine and Health Sciences > Research Groups > Musculoskeletal Medicine
Faculty of Medicine and Health Sciences > Research Centres > Metabolic Health
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Depositing User: LivePure Connector
Date Deposited: 09 Jan 2019 12:30
Last Modified: 19 Oct 2023 02:20
URI: https://ueaeprints.uea.ac.uk/id/eprint/69505
DOI: 10.1074/jbc.M114.601724

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