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

Jacob Lund, Linda Troeberg, Henrik Kjeldal, Ole H Olsen, Hideaki Nagase, Esben S Sørensen, Henning R Stennicke, Helle H Petersen, Michael T Overgaard

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)6620-6629
Number of pages10
JournalThe Journal of Biological Chemistry
Issue number10
Early online date6 Jan 2015
Publication statusPublished - 6 Mar 2015


  • ADAM Proteins/antagonists & inhibitors
  • Amino Acid Sequence/genetics
  • Catalytic Domain
  • Crystallography, X-Ray
  • Gene Expression Regulation, Enzymologic
  • Humans
  • Metalloproteases/antagonists & inhibitors
  • Protein Structure, Tertiary
  • Proteolysis
  • Substrate Specificity
  • Tissue Inhibitor of Metalloproteinases/chemistry
  • Zinc/chemistry

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