title: Calcium negatively regulates meprin β activity and attenuates substrate cleavage.
abstract: 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.
authors.affiliation: *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 p.arnold@anat.uni-kiel.de.
authors.forename: Philipp
authors.initials: P
authors.lastname: Arnold
authors.affiliation: *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.
authors.forename: Frederike
authors.initials: F
authors.lastname: Schmidt
authors.affiliation: *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.
authors.forename: Johannes
authors.initials: J
authors.lastname: Prox
authors.affiliation: *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.
authors.forename: Friederike
authors.initials: F
authors.lastname: Zunke
authors.affiliation: *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.
authors.forename: Claus
authors.initials: C
authors.lastname: Pietrzik
authors.affiliation: *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.
authors.forename: Ralph
authors.initials: R
authors.lastname: Lucius
authors.affiliation: *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.
authors.forename: Christoph
authors.initials: C
authors.lastname: Becker-Pauly
mesh_terms: D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D045726:Metalloproteases / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D016415:Sequence Alignment; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D053829:Amyloid Precursor Protein Secretases; D045726:Metalloproteases; D008666:Metalloendopeptidases; C095663:meprin B; D002118:Calcium
keywords: APP; amyloid β; protease; protein folding