Arnold Philipp, Schmidt Frederike, Prox Johannes, Zunke Friederike, Pietrzik Claus, Lucius Ralph, Becker-Pauly Christoph
*Anatomical Institute and Biochemical Institute, Christian-Albrecht's University, Kiel, Germany; and Institute of Pathobiochemistry, University Medical Centre of the Johannes Gutenberg University of Mainz, Mainz, Germany
*Anatomical Institute and Biochemical Institute, Christian-Albrecht's University, Kiel, Germany; and Institute of Pathobiochemistry, University Medical Centre of the Johannes Gutenberg University of Mainz, Mainz, Germany.
FASEB J. 2015 Aug;29(8):3549-57. doi: 10.1096/fj.15-272310. Epub 2015 May 8.
The meprin β metalloproteinase is an important enzyme in extracellular matrix turnover, inflammation, and neurodegeneration in humans and mice. Previous studies showed a diminished cleavage of certain meprin β substrates in the presence of calcium, although the mechanism was not clear. With the help of a specific fluorogenic peptide assay and the human amyloid precursor protein as substrate, we demonstrated that the influence of calcium is most likely a direct effect on human meprin β itself. Analyzing the crystal structures of pro- and mature meprin β helped to identify a cluster of negatively charged amino acids forming a potential calcium binding site. Mutation of 2 of these residues (D204A and D245A) led to severe differences in proteolytic activity and cellular localization of meprin β. D245A was almost completely inactive and largely stored into intracellular vesicles, indicating severe misfolding of the protein. Astonishingly, D204A was not transported to the cell surface, but exhibited strong β-secretase activity, resulting in massive accumulation of Aβ-peptides. This could be explained by constitutive maturation of this meprin β mutant already in the early secretory pathway. We hypothesize that lacking D204 abrogates the capability of binding calcium in the catalytic domain, an important step for proper folding of the propeptide and subsequent inhibition of the protease. This is supported by the inhibition constant of calcium for meprin β (inhibitory constant 50 = 11 mM), which resembles the physiologic concentrations found in the endoplasmic reticulum. For instance, it was shown for amyotrophic lateral sclerosis that a loss of calcium in the endoplasmic reticulum leads to the misfolding of calcium-dependent proteins, which might also be relevant for proper function of meprin β.
膜型基质金属蛋白酶β是人和小鼠细胞外基质周转、炎症及神经退行性变过程中的一种重要酶。以往研究表明，在有钙存在的情况下，某些膜型基质金属蛋白酶β底物的裂解作用减弱，但其机制尚不清楚。借助特异性荧光肽检测法并以人淀粉样前体蛋白为底物，我们证明钙的影响很可能是对人膜型基质金属蛋白酶β自身的直接作用。对前体膜型基质金属蛋白酶β和成熟膜型基质金属蛋白酶β的晶体结构进行分析，有助于确定形成潜在钙结合位点的一组带负电荷的氨基酸。其中2个残基（D204A和D245A）发生突变，导致膜型基质金属蛋白酶β的蛋白水解活性和细胞定位出现严重差异。D245A几乎完全无活性，大量储存在细胞内囊泡中，表明该蛋白发生了严重错误折叠。令人惊讶的是，D204A没有转运到细胞表面，但表现出很强的β-分泌酶活性，导致Aβ肽大量积累。这可以通过该膜型基质金属蛋白酶β突变体在早期分泌途径中就发生组成型成熟来解释。我们推测，缺少D204会消除催化结构域结合钙的能力，而这是前肽正确折叠及随后抑制蛋白酶的重要步骤。钙对膜型基质金属蛋白酶β的抑制常数（抑制常数50 = 11 mM）支持了这一推测，该常数与内质网中发现的生理浓度相似。例如，对于肌萎缩侧索硬化症，已表明内质网中钙的缺失会导致钙依赖性蛋白错误折叠，这可能也与膜型基质金属蛋白酶β的正常功能有关。
