title: Evidence for restricted reactivity of ADAMDEC1 with protein substrates and endogenous inhibitors.
abstract: 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.
authors.affiliation: From the Department of Chemistry and Bioscience, Aalborg University, DK-9220 Aalborg Oest, Denmark, the Department of Haemophilia Biochemistry, Novo Nordisk A/S, DK-2760 Måløv, Denmark.
authors.forename: Jacob
authors.initials: J
authors.lastname: Lund
authors.affiliation: the Kennedy Institute of Rheumatology, University of Oxford, Oxford OX3 7FY, United Kingdom, and.
authors.forename: Linda
authors.initials: L
authors.lastname: Troeberg
authors.affiliation: From the Department of Chemistry and Bioscience, Aalborg University, DK-9220 Aalborg Oest, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Kjeldal
authors.affiliation: the Department of Haemophilia Biochemistry, Novo Nordisk A/S, DK-2760 Måløv, Denmark.
authors.forename: Ole H
authors.initials: OH
authors.lastname: Olsen
authors.affiliation: the Kennedy Institute of Rheumatology, University of Oxford, Oxford OX3 7FY, United Kingdom, and.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nagase
authors.affiliation: the Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark.
authors.forename: Esben S
authors.initials: ES
authors.lastname: Sørensen
authors.affiliation: the Department of Haemophilia Biochemistry, Novo Nordisk A/S, DK-2760 Måløv, Denmark.
authors.forename: Henning R
authors.initials: HR
authors.lastname: Stennicke
authors.affiliation: the Department of Haemophilia Biochemistry, Novo Nordisk A/S, DK-2760 Måløv, Denmark.
authors.forename: Helle H
authors.initials: HH
authors.lastname: Petersen
authors.affiliation: From the Department of Chemistry and Bioscience, Aalborg University, DK-9220 Aalborg Oest, Denmark, mto@bio.aau.dk.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Overgaard
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D013379:Substrate Specificity; D019714:Tissue Inhibitor of Metalloproteinases / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000737:chemistry
chemical_list: D019714:Tissue Inhibitor of Metalloproteinases; D045726:Metalloproteases; D051722:ADAM Proteins; C108169:decysin; D015032:Zinc
keywords: ADAM; ADAMTS; Metalloprotease; Metzincin; Substrate Specificity; Tissue Inhibitor of Metalloproteinase (TIMP)