Large scale analysis of olfactory receptors with highly genetically variations in relation with specific anosmia Dissertation to obtain the degree Doctor Philosophiae (Doctor of Philosophy, PhD) At the Faculty of Biology and Biotechnology Ruhr-University Bochum International Graduate School of Biosciences Ruhr-University Bochum (Department of Cell Physiology) Submitted by Kaveh Ashti Baghaei From (Tehran, Iran) Bochum, Germany (June, 2013) First supervisor: Prof. Dr. Dr. Dr. Hanns Hatt Second supervisor: Prof. Dr. Stefan Wiese Umfassende Analyse Olfaktorischer Rezeptoren in Bezug auf genetische Varianten im Zusammenhang mit spezifischer Anosmie Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie und Biotechnologie an der Internationalen Graduiertenschule Biowissenschaften der Ruhr-Universität Bochum angefertigt im Lehrstuhl für Zellphysiologie vorgelegt von Kaveh Ashti Baghaei Aus Tehran, Iran Bochum Juni, 2013 Referent: Prof. Dr. Dr. Dr. Hanns Hatt Korreferent: Prof. Dr. Stefan Wiese To my parents, brother and wife whose love and support sustained me throughout. Erklärung Hiermit erkläre ich, dass ich die Arbeit selbständig verfasst und bei keiner anderen Fakultät eingereicht und dass ich keine anderen als die angegebenen Hilfsmittel verwendet habe. Es handelt sich bei der heute von mir eingereichten Dissertation um sechs in Wort und Bildvöllig übereinstimmende Exemplare. Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten und in keinem Fall inhaltsverändernde Bildverarbeitung vorgenommen wurde. Kaveh Ashti Baghaei Bochum Jun.2013 Contents 1. Introduction ............................................................................................................................ -1- 1.1 Olfactory system .................................................................................................................... -1- 1.2 Olfactory receptors (ORs) ...................................................................................................... -2- 1.2.1 Segregating pseudogenes (SPGs) ........................................................................................ -4- 1.2.2 Copy number variations (CNVs) ........................................................................................ -6- 1.3 Olfactory signal transduction pathways ............................................................................. -8- 1.4 Functional expression of olfactory receptors in recombinant systems ............................. -10- 1.5 Odourant perception ........................................................................................................... -12- 1.5.1 Chemical structure basis of odourant ............................................................................ -13- 1.5.2 Olfactory Dysfunction ................................................................................................... -16- 1.5.3 Specific anosmia ........................................................................................................... -18- Aim of study .............................................................................................................................. -21- 2. Materials and Methods ....................................................................................................... -23- 2.1 DNA constructs and plasmids .......................................................................................... -23- 2.2 Mutagenesis by Overlap Extension PCR .......................................................................... -26- 2.3 Odourant library ................................................................................................................ -27- 2.4 Cell culture and transient DNA transfection .................................................................... -30- 2.5 Ca-imaging ........................................................................................................................ -31- 2.6 Stimulation and screening with odourants ....................................................................... -33- 2.7 Strategy of screening ........................................................................................................ -34- 2.8 CRE-Luciferase ................................................................................................................ -35- 2.9 CRE-SEAP assay ............................................................................................................. -37- 2.10 Immunohistochemistry ................................................................................................... -39- 2.11 Data analysis ................................................................................................................... -40- 3. Results ................................................................................................................................... -41- 3.1 Identification of SPG and CNV OR candidates ................................................................. -41- 3.2 Large scale investigation of olfactory receptors ................................................................ -44- 3.3 Deorphanization of olfactory receptors ............................................................................. -49- 3.4 Ca-imaging and CRE-luciferase: similarities and differences ........................................... -55- 3.5 Cell surface expression of olfactory receptors .................................................................. -57- 3.6 Broadly-narrowly tune of olfactory receptors ................................................................... -58- 3.7 Distance between odourants and response variability in the olfactory system ................. -59- 3.8 Similar odourant structure, similar response? .................................................................... -62- 3.9 Relation between odour specificity and receptor sequence .............................................. -64- 3.10 Single nucleotide polymorphism and odourant responses: Functional analysis of OR1B and OR10Q1-mutants ................................................................. -66- 4. Discussion .............................................................................................................................. -73- 4.1 Large scale investigation of ORs including SPGs and CNVs .......................................... -73- 4.2 Broadly and narrowly tuned olfactory receptors .............................................................. -81- 4.3 Scent and Chemistry .......................................................................................................... -82- 4.4 5a-androst-16-en-3one (urinous) and ω-pentadecalactone (musky) molecules ................. -85- Summary ................................................................................................................................... -87- Zusammenfassung .................................................................................................................... -90- Publication bibliography ......................................................................................................... -93- List of Figures ......................................................................................................................... -108- List of Tables .......................................................................................................................... -110- Abbreviations ......................................................................................................................... -111- Publications ............................................................................................................................ -113- Curriculum Vitae ................................................................................................................. -115- Acknowledgements ................................................................................................................ -117- Introduction 1. Introduction: 1.1 Olfactory system The human olfactory system has the remarkable capacity to recognise and discriminate a large number of different chemical components and odour molecules. As a chemical sensor, the olfactory system is effective on social and sexual behaviour and quality of life since odourants can evoke memories and emotions or influence our mood. Odourant perception starts with the chemical interaction between an odourant molecule and an olfactory receptor expressed in an olfactory sensory neuron (OSN). These neurons are located in the olfactory epithelium (OE), which is covered with a mucous layer which supplies lipid/protein-rich secretions for the OE surface. Most parts of the human nasal cavity consist of ciliated columnar respiratory epithelial tissue. The upper section of the nasal cavity, lining the cribriform plate, consists of OSNs (Morrison & Costanzo 1990). OSNs are bipolar cells with a single unbranched dendrite with axons penetrating through the cribriform plate to the olfactory bulb of the brain (Fig. 1.1). Axons of mitral cell then leave the olfactory bulb (OB) to higher brain structures including the piriform cortex, hippocampus and amygdale. About 10–25 cilia, each ~5 μm long, extend from the knob of each OSN (Menco 1980; Morrison & Costanzo 1992). Each OSN expresses just one type of olfactory receptor (OR) protein which is the first level for the primary events of chemo-sensory transduction. - 1 - Introduction Figure 1.1: Different