Institute of Veterinary Pathology, Department of Veterinary Medicine, Freie Universität Berlin Are CLCA Proteins Metalloproteases? Comparison of the Cleavage Processes of the Secreted mCLCA3 and the Transmembrane mCLCA6 Proteins Thesis submitted for the fulfillment of a Doctor of Philosophy (Ph.D.) degree in Veterinary Medicine at the Freie Universität Berlin submitted by Melanie Kathrin Bothe Veterinarian from Braunschweig Berlin 2011 Journal-No.: 3553 Printed with permission of the Department of Veterinary Medicine of the Freie Universität Berlin Dean: Univ.-Prof. Dr. Leo Brunnberg Supervision: Univ.-Prof. Dr. Achim D. Gruber, Ph.D.(Cornell Univ.) First Reviewer: Univ.-Prof. Dr. Achim D. Gruber, Ph.D.(Cornell Univ.) Second Reviewer: Univ.-Prof. Dr. Dr. Ralf Einspanier Third Reviewer: PD Dr. Michael Veit Descriptors (according to CAB-Thesaurus): cystic fibrosis; asthma; chronic obstructive pulmonary disease; mice; metalloproteinases; zinc; protein transport Day of Doctorate: 23.11.2012 This document is protected by copyright law. No part of this document may be reproduced in any form by any means without prior written authorization of the publisher. All rights reserved © Mensch und Buch Verlag 2012 Choriner Str. 85 - 10119 Berlin [email protected] – www.menschundbuch.de Aus dem Institut für Tierpathologie des Fachbereichs Veterinärmedizin der Freien Universität Berlin Sind CLCA-Proteine Metalloproteasen? Vergleich der Spaltungsprozesse des sezernierten mCLCA3- und des transmembranären mCLCA6-Proteins Inaugural-Dissertation zur Erlangung des Grades eines Doctor of Philosophy (Ph.D.) in Veterinärmedizin an der Freie Universität Berlin vorgelegt von Melanie Kathrin Bothe Tierärztin aus Braunschweig Berlin 2011 Journal-Nr.: 3553 Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freie Universität Berlin Dekan: Univ.-Prof. Dr. Leo Brunnberg Betreuung: Univ.-Prof. Dr. Achim D. Gruber, Ph.D.(Cornell Univ.) Erster Gutachter: Univ.-Prof. Dr. Achim D. Gruber, Ph.D.(Cornell Univ.) Zweiter Gutachter: Univ.-Prof. Dr. Dr. Ralf Einspanier Dritter Gutachter: PD Dr. Michael Veit Deskriptoren (nach CAB-Thesaurus): cystic fibrosis; asthma; chronic obstructive pulmonary disease; mice; metalloproteinases; zinc; protein transport Tag der Promotion: 23.11.2012 Bibliografische Information der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über <http://dnb.ddb.de> abrufbar. ISBN: 978-3-86387-252-6 Zugl.: Berlin, Freie Univ., Diss., 2011 Dissertation, Freie Universität Berlin D 188 Dieses Werk ist urheberrechtlich geschützt. Alle Rechte, auch die der Übersetzung, des Nachdruckes und der Vervielfältigung des Buches, oder Teilen daraus, vorbehalten. Kein Teil des Werkes darf ohne schriftliche Genehmigung des Verlages in irgendeiner Form reproduziert oder unter Verwendung elektronischer Systeme verarbeitet, vervielfältigt oder verbreitet werden. Die Wiedergabe von Gebrauchsnamen, Warenbezeichnungen, usw. in diesem Werk berechtigt auch ohne besondere Kennzeichnung nicht zu der Annahme, dass solche Namen im Sinne der Warenzeichen- und Markenschutz-Gesetzgebung als frei zu betrachten wären und daher von jedermann benutzt werden dürfen. Alle Rechte vorbehalten © Mensch und Buch Verlag 2012 Choriner Str. 85 - 10119 Berlin [email protected] – www.menschundbuch.de Für Verena Contents Contents Contents ................................................................................................................................ VII List of abbreviations ................................................................................................................ X 1 INTRODUCTION ...................................................................................................... 11 2 LITERATURE ........................................................................................................... 13 2.1 Tissue expression patterns of CLCA proteins .......................................................... 13 2.1.1 CLCA cluster 1 ......................................................................................................... 14 2.1.2 CLCA cluster 2 ......................................................................................................... 15 2.1.3 CLCA cluster 3 ......................................................................................................... 16 2.1.4 CLCA cluster 4 ......................................................................................................... 18 2.2 Biomedical relevance of CLCA proteins ................................................................... 19 2.2.1 Diseases with secretory dysfunction ........................................................................ 19 2.2.1.1 Modulation of calcium activated chloride channels in cystic fibrosis ........................ 19 2.2.1.2 Mucus hypersecretion in cystic fibrosis, asthma and chronic obstructive pulmonary disease ................................................................................................... 22 2.2.2 Cell adhesion under physiologic conditions and in metastasizing tumor cells ......... 24 2.2.3 Role in apoptosis and tumor growth ......................................................................... 25 2.3 Structure and biochemical characteristics of CLCA proteins .................................... 26 2.3.1 n-CLCA domain ........................................................................................................ 26 2.3.2 Von Willebrand factor A domain ............................................................................... 27 2.3.3 Fibronectin type III domain ....................................................................................... 27 2.4 Cellular processing of CLCA proteins ...................................................................... 28 2.4.1 Signal sequence ....................................................................................................... 28 2.4.2 Glycosylation ............................................................................................................ 28 2.4.3 Cleavage of CLCA proteins ...................................................................................... 29 2.4.4 Secretion of whole protein versus ectodomain shedding of transmembrane CLCA proteins .......................................................................................................... 30 VII Contents 3 AIMS OF THIS PH.D. PROJECT ............................................................................. 32 3.1 Aim no. 1: Characterization of the cleavage process of the secreted mCLCA3 protein ...................................................................................................................... 32 3.2 Aim no. 2: Identification of a murine transmembrane CLCA protein in tissues relevant for cystic fibrosis ......................................................................................... 33 3.2.1 Expression pattern analysis and biochemical characterization of mCLCA5 ............. 33 3.2.2 Expression pattern analysis and biochemical characterization of mCLCA6 ............. 34 3.3 Aim no. 3: Comparison of the cleavage processes of a secreted CLCA protein versus a transmembrane CLCA protein ................................................................... 34 4 RESEARCH PUBLICATIONS IN PEER-REVIEWED JOURNALS ......................... 35 4.1 Murine mCLCA6 is an Integral Apical Membrane Protein of Non-goblet Cell Enterocytes and Co-localizes with the Cystic Fibrosis Transmembrane Conductance Regulator ............................................................................................ 35 4.2 Murine mCLCA5 is Expressed in Granular Layer Keratinocytes of Stratified Epithelia .................................................................................................................... 36 4.3 The Murine mCLCA3 is a Zinc-dependent Metalloprotease with Autoproteolytic Activity ...................................................................................................................... 37 4.4 Impaired Autoproteolytic Cleavage of mCLCA6, a Murine Integral Membrane Protein Expressed in Enterocytes, Leads to Cleavage at the Plasma Membrane Instead of Cleavage in the Endoplasmic Reticulum ................................................. 38 5 CONCLUDING DISCUSSION .................................................................................. 83 5.1 Hypothesis I: CLCA proteins show characteristics of metalloproteases ................... 83 5.1.1 Aim no. 1: Characterization of the cleavage process of the secreted mCLCA3 protein ...................................................................................................................... 83 5.1.2 Are CLCA proteins metalloproteases of the zincin family?....................................... 85 5.1.2.1 HEXXH motif of metalloproteases ............................................................................ 85 5.1.2.2 Zinc-dependent cleavage mechanism ...................................................................... 86 5.1.2.3 (Auto-)proteolytic activity .......................................................................................... 87 VIII Contents 5.2 Hypothesis II: The cleavage processes of transmembrane and secreted CLCA proteins differ in consequence of the transmembrane domain................................. 87 5.2.1 Aim no. 2: Identification of a murine transmembrane