INTERNATIONAL GRADUATE SCHOOL OF NEUROSCIENCES (IGSN) RUHR UNIVERSITÄT BOCHUM THE OLFACTORY RECEPTOR ASSOCIATED PROTEOME Doctoral Dissertation David Jonathan Barbour Department of Cell Physiology Thesis advisor: Prof. Dr. Dr. Dr. Hanns Hatt Bochum, Germany (30.12.05) ABSTRACT Olfactory receptors (OR) are G-protein-coupled membrane receptors (GPCRs) that comprise the largest vertebrate multigene family (~1,000 ORs in mouse and rat, ~350 in human); they are expressed individually in the sensory neurons of the nose and have also been identified in human testis and sperm. In order to gain further insight into the underlying molecular mechanisms of OR regulation, a bifurcate proteomic strategy was employed. Firstly, the question of stimulus induced plasticity of the olfactory sensory neuron was addressed. Juvenile mice were exposed to either a pulsed or continuous application of an aldehyde odorant, octanal, for 20 days. This was followed by behavioural, electrophysiological and proteomic investigations. Both treated groups displayed peripheral desensitization to octanal as determined by electro-olfactogram recordings. This was not due to anosmia as they were on average faster than the control group in a behavioural food discovery task. To elucidate differentially regulated proteins between the control and treated mice, fluorescent Difference Gel Electrophoresis (DIGE) was used. Seven significantly up-regulated and ten significantly down-regulated gel spots were identified in the continuously treated mice; four and twenty-four significantly up- and down-regulated spots were identified for the pulsed mice, respectively. The spots were excised and proteins were identified using mass spectrometry. Several promising candidate proteins were identified including potential transcription factors, cytoskeletal proteins as well as calcium binding and odorant binding proteins. We propose that the dominant desensitizing factor in the continuously treated mice was down-regulation of odorant binding proteins. In the pulsed group no principle factor was evident, however, in terms of the number of proteins and degree of post-translational modifications, the pulsed group displayed greater plasticity. Secondly, in order to reveal which olfactory receptors are expressed in human spermatozoa, and alternative proteomic strategy was developed. The optimised method employs an ‘affinity two phase partition’ system in conjunction with multi- enzymatic digestion in the presence of an organic solvent. The resultant peptides were identified using Multidimensional Protein Identification Technology (MudPIT). 222 integral membrane proteins were identified including 57 GPCRs, 35 of which were chemoreceptors, consisting of 32 ORs and 3 taste receptors. Notably, most of the peptides which resulted in GPCR identification were cleaved from transmembrane domains, thus demonstrating the efficacy of this strategy in membrane proteomics. In addition to the chemoreceptors, 23 neuronal proteins were also detected suggesting that both cell types may have more in common than usually perceived. Both proteomic strategies afford a powerful means whereby novel protein candidates can be elucidated and thereby provide greater insight into plasticity of the olfactory receptor, its associated proteins, and the role of olfactory receptors in reproduction. TABLE OF CONTENTS 1 Introduction ____________________________________________1 1.1 The Olfactory System _____________________________________ 1 1.1.1 The olfactory epithelium _______________________________________ 3 1.1.2 The olfactory receptor protein (OR) ______________________________ 3 1.2 Project Aims_____________________________________________ 7 2 Proteomic investigation of the olfactory epithelium ___________ 7 2.1 Olfactory stimulus induced plasticity – a background __________ 7 2.2 Proteomics: an introduction_______________________________ 11 2.2.1 Technologies for large scale proteomics___________________________ 12 2.2.1.1 2D-electrophoresis _____________________________________ 12 2.2.1.2 Multi-dimensional protein identification technology MudPIT _____ 14 2.2.2 Neuro-proteomics___________________________________________ 16 2.3 Conclusion _____________________________________________ 18 2.4 Materials and Methods ___________________________________ 18 2.4.1 Animal and Tissue Preparation _________________________________ 18 2.4.2 Behavioural study ___________________________________________ 19 2.4.3 Microdissection of olfactory epithelium ___________________________ 20 2.4.4 Electro-olfactogram (EOG) recording____________________________ 20 2.4.5 Sample preparation for DIGE__________________________________ 21 2.4.6 Protein Labelling - Fluorescent DIGE minimal labelling ______________ 21 2.4.7 IEF and 2-DE______________________________________________ 22 2.4.8 Gel scanning, digitising and analysis______________________________ 22 2.4.9 2D-SDS PAGE Gel total protein staining _________________________ 23 2.4.10 Trypsin digestion _________________________________________ 23 2.4.11 Mass Spectrometry ________________________________________ 24 2.4.12 Statistics ________________________________________________ 25 2.5 Results ________________________________________________ 25 2.5.1 Regions of interest___________________________________________ 29 2.5.2 Pulsed Gel Analysis __________________________________________ 33 2.5.3 Continuous ________________________________________________ 39 2.5.4 Protein identification _________________________________________ 43 2.5.5 Behavioural study ___________________________________________ 49 2.5.6 EOG_____________________________________________________ 51 2.6 Discussion _____________________________________________ 54 2.6.1 Mice physiology_____________________________________________ 54 2.6.2 Regulation of proteins: functional categorisation & comparison_________ 55 2.6.2.1 The cytoskeleton _______________________________________ 57 2.6.2.2 Intermediate early genes / transcription ______________________ 60 2.6.2.3 Calcium binding proteins _________________________________ 62 2.6.2.4 Chaperones ___________________________________________ 68 2.6.2.5 Lipocalins ____________________________________________ 70 2.6.2.6 Xenobiotic & anti-oxidant metabolism_______________________ 76 2.6.2.7 Energy metabolism _____________________________________ 77 2.6.3 Consolidating the findings: Protein to Phenotype ___________________ 78 2.6.3.1 Protein regulation in the continuously treated mice _____________ 79 2.6.3.2 Protein regulation in the pulsed treated mice __________________ 79 2.6.4 Conclusion ________________________________________________ 80 3 Proteomic investigation of the human sperm membrane_______82 3.1 A correlation between chemosenses and reproduction? ________ 82 3.2 The challenge of membrane proteomics ____________________ 83 3.2.1 In-gel methods _____________________________________________ 84 3.2.2 In-Solution shotgun approach __________________________________ 85 3.2.3 Fractionation_______________________________________________ 86 3.3 Sperm proteomics: a background __________________________ 87 3.4 Conclusion _____________________________________________ 89 3.5 Materials and Methods ___________________________________ 90 3.5.1 LC/LC-MS/MS and Protein Identification ________________________ 90 3.5.2 Sperm preparation___________________________________________ 91 3.5.3 Ca2+ imaging _______________________________________________ 91 3.5.4 Immunocytochemistry________________________________________ 91 3.5.5 Sample optimisation - main strategies ____________________________ 92 3.5.5.1 Gel Based Approach ____________________________________ 92 3.5.5.2 In Solution Strategy _____________________________________ 94 3.6 Results ________________________________________________ 97 3.6.1 Gel Based Strategy___________________________________________ 97 3.6.2 In Solution Strategy _________________________________________ 100 3.6.2.1 Solubilisation using organic acid___________________________ 100 3.6.2.2 Solubilisation using organic solvent ________________________ 101 3.6.2.3 Vectorial labelling _____________________________________ 102 3.6.2.4 Lipase & Affinity Enrichment ____________________________ 102 3.6.3 Validation of data __________________________________________ 107 3.6.3.1 Functional validation - calcium imaging _____________________ 108 3.6.3.2 Immunocytochemistry__________________________________ 109 3.7 Discussion ____________________________________________ 110 3.7.1 Strategy development _______________________________________ 110 3.7.1.1 Gel Based Strategy_____________________________________ 110 3.7.1.2 ‘In-solution’ based strategies _____________________________ 113 3.7.2 The Solution: A final strategy__________________________________ 117 3.8 Identified membrane proteins ____________________________ 118 3.8.1 A technical discussion on the identified proteins ___________________ 119 3.8.2 The identified proteins from a biological perspective ________________ 120 4 Final Conclusion ______________________________________124 4.1.1 Olfactory receptor plasticity___________________________________ 124 4.1.2 Spermatozoa membrane proteome _____________________________ 125 4.1.3 The olfactory receptor proteome _______________________________ 125 ii LIST OF FIGURES Figure 1.1 Diagramatic representation of the rodent olfactory system Error! Bookmark not defined. Figure 1.2 Diagram depicting initial olfactory receptor signal transduction _________ 5 Figure 2.1 Schematic representation