Biol. Chem. 2016; 397(1): 45–55 a Andreas Becker and Rico Eichentopf: These authors contributed equally to this work. b Present address: Fraunhofer Institute for Cell Therapy and Immunology IZI-MWT, Weinbergweg 22, D-06120 Halle/Saale, Germany *Corresponding author: Stephan Schilling, Probiodrug AG, Weinbergweg 22, D-06120 Halle/Saale, Germany, e-mail: stephan.schilling@izi.fraunhofer.de Andreas Becker, Reinhard Sedlmeier, Christoph Bäuscher, Stephanie Kohlmann and Sigrid Graubner: Ingenium GmbH, Fraunhoferstrasse 13, D-82152 Martinsried, Germany Rico Eichentopf, Holger Cynis, Birgit Koch, Anett Stephan, Torsten Hoffmann and Astrid Kehlen: Probiodrug AG, Weinbergweg 22, D-06120 Halle/Saale, Germany Alexander Waniek and Steffen Roßner: Paul Flechsig Institute for Brain Research, University of Leipzig, Liebigstraße 19, 04103 Leipzig, Germany Sabine Berg and Ernst-Joachim Freyse: Institute of Diabetes “Gerhardt Katsch”, Greifswalder Str. 11e, D-17495 Karlsburg, Germany Alexander Osmand: Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, 1414 Cumberland Avenue, Knoxville, TN 37996, USA Anne-Christine Plank and Stephan von Hörsten: Department of Experimental Therapy, University of Erlangen-Nürnberg, Franz- Penzoldt-Center, Palmsanlage 5, D-91054 Erlangen, Germany Hans-Ulrich Demuth: Ingenium GmbH, Fraunhoferstrasse 13, D-82152 Martinsried, Germany; and Probiodrug AG, Weinbergweg 22, D-06120 Halle/Saale, Germany Andreas Becker a , Rico Eichentopf a , Reinhard Sedlmeier, Alexander Waniek, Holger Cynis b , Birgit Koch, Anett Stephan, Christoph Bäuscher, Stephanie Kohlmann, Torsten Hoffmann, Astrid Kehlen, Sabine Berg, Ernst-Joachim Freyse, Alexander Osmand, Anne-Christine Plank, Steffen Roßner, Stephan von Hörsten, Sigrid Graubner, Hans-Ulrich Demuth b and Stephan Schilling b, * IsoQC (QPCTL) knock-out mice suggest differential substrate conversion by glutaminyl cyclase isoenzymes DOI 10.1515/hsz-2015-0192 Received June 10, 2015; accepted August 12, 2015; previously published online August 19, 2015 Abstract: Secretory peptides and proteins are fre- quently modified by pyroglutamic acid (pE, pGlu) at their N-terminus. This modification is catalyzed by the glutaminyl cyclases QC and isoQC. Here, we decipher the roles of the isoenzymes by characterization of IsoQC -/- mice. These mice show a significant reduction of glu- taminyl cyclase activity in brain and peripheral tissue, suggesting ubiquitous expression of the isoQC enzyme. An assay of substrate conversion in vivo reveals impaired generation of the pGlu-modified C-C chemokine ligand 2 (CCL2, MCP-1) in isoQC -/- mice. The pGlu-formation was also impaired in primary neurons, which express sig- nificant levels of QC. Interestingly, however, the forma- tion of the neuropeptide hormone thyrotropin-releasing hormone (TRH), assessed by immunohistochemistry and hormonal analysis of hypothalamic-pituitary-thyroid axis, was not affected in isoQC -/- , which contrasts to QC -/- . Thus, the results reveal differential functions of isoQC and QC in the formation of the pGlu-peptides CCL2 and TRH. Substrates requiring extensive prohormone processing in secretory granules, such as TRH, are primarily converted by QC. In contrast, protein substrates such as CCL2 appear to be primarily converted by isoQC. The results provide a new example, how subtle differences in subcellular local- ization of enzymes and substrate precursor maturation might influence pGlu-product formation. Keywords: 5-oxo-L-proline; posttranslational modifica- tion; pyroglutamate; TRH. Introduction Among the diverse posttranslational modifications, the N-terminal formation of pyroglutamate (pGlu, pE) from glutaminyl or glutamyl precursors is prominent among secretory peptides and proteins. Due to this modification, the peptides are stabilized towards degradation by amin- opeptidases and may obtain their receptor-active confor- mation. N-terminally modified peptides such as monocyte chemoattractant protein 1 (CCL2; MCP-1) and pGlu-Aβ(3-42) are assumed to exert important roles in pathophysiolo- gical conditions such as atherosclerosis and Alzheimer’s disease (AD), respectively (Saido et al., 1995; Iwatsubo Brought to you by | Colorado State University Authenticated Download Date | 12/16/15 11:52 AM