Lund Jacob, Troeberg Linda, Kjeldal Henrik, Olsen Ole H, Nagase Hideaki, Sørensen Esben S, Stennicke Henning R, Petersen Helle H, Overgaard Michael T
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.
the Kennedy Institute of Rheumatology, University of Oxford, Oxford OX3 7FY, United Kingdom, and.
J Biol Chem. 2015 Mar 6;290(10):6620-9. doi: 10.1074/jbc.M114.601724. Epub 2015 Jan 6.
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.
ADAMDEC1是一种具有蛋白水解活性的金属锌蛋白酶，其活性位点结构罕见，含有一个与锌结合的天冬氨酸残基（Asp-362）。我们之前证明，将Asp-362替换为组氨酸残基，从而用三个组氨酸锌配体重建典型的金属锌蛋白酶锌结合环境，可提高蛋白水解活性。该蛋白酶还具有非典型的短结构域结构，金属蛋白酶结构域中的半胱氨酸残基数量为奇数。在此，我们研究了ADAMDEC1金属蛋白酶结构域中这些罕见的结构特征如何影响蛋白水解活性、底物特异性以及抑制剂的作用。我们鉴定出羧甲基化转铁蛋白（Cm-Tf）是一种新的ADAMDEC1底物，并确定了一级和二级切割位点，这表明其对P1'位置的亮氨酸有强烈偏好。发现存在于人类但在已测序的ADAMDEC1直系同源物中仅部分保守的半胱氨酸（Cys392）未配对，将Cys392替换为丝氨酸可增加其与α2-巨球蛋白的反应性，但与酪蛋白或Cm-Tf的反应性无增加。将Asp(362)替换为His导致蛋白水解活性普遍增加，并且观察到与Cm-Tf的底物特异性发生了变化。ADAMDEC1被小分子抑制剂batimastat抑制，但不被金属蛋白酶组织抑制剂（TIMP）-1、TIMP-2或TIMP-3的N端抑制结构域（N-TIMP-3）抑制。然而，N-TIMP-3对具有重建的金属锌蛋白酶锌结合环境的D362H变体显示出强烈的抑制活性。我们推测ADAMDEC1的这些独特特征可能是为了逃避内源性金属蛋白酶抑制剂的抑制而进化而来的。
