A Study of Type-3 Copper Proteins from Arthropods
Total Page：16
File Type：pdf, Size：1020Kb
A Study of Type-3 Copper Proteins from Arthropods By Sharon Baird Submitted to: The Faculty of Natural Sciences University of Stirling September 2007 For the degree of Doctor of Philosophy This research was conducted in the School of Biological and Environmental Sciences, University of Stirling, Stirling, UK. ACKNOWLEDGEMENTS Firstly I would like to dedicate this thesis to my now husband, Craig McFadyen, who has helped keep me connected with the real world over the past few years, and has always been there with a reassuring hug in times of need! I also wish to dedicate this thesis to my mum and dad for their financial support and pride and belief in my ability. This research was funded by the University of Stirling. My primary supervision was provided by Dr Jacqueline Nairn whose knowledge of an array of biological and biophysical techniques helped me tremendously in undertaking this research. Many thanks also to Jacqueline for never failing to find the time amongst her hectic schedule to sit down and discuss my work and make sure I kept on track. Secondary supervision was provided by Dr Kenneth Wilson (University of Lancaster, formerly University of Stirling). The biophysical research would have been much less fruitful without the invaluable guidance and thoughts from my many collaborators: Thomas Jess, Dr Sharon Kelly and Professor Nicholas Price of the BBSRC funded Circular Dichroism Facility, University of Glasgow, Margaret Nutley and Professor Alan Cooper of the BBSRC/EPSRCMicrocalorimetry Service, University of Glasgow, who obtained and interpreted the ITC data and Dr Elmar Jaenicke and Professor Heinz Decker of the Institute of Molecular Biophysics, University of Mainz, Germany. I Further thanks extend to: Dr Peter Dominy of the University of Glasgow for guiding the ICP OES experimental design, Professor Thorsten Burmester of the Institute of Zoology, University of Mainz, Germany for assistance with oligomeric primer design, Dr Michael Wyman for his useful input on various aspects of the the molecular biology based research, Mary Everhart and Dr. Michael Kanost of the Department of Biochemistry, Kansas State University for generously providing the Manduca sexta prophenoloxidase cDNA clones, Beatrice Lanzrein of the Institute of Cell Biology, University of Berne, Switzerland for kindly gifting two Spodoptera littoralis cDNA libraries and Dr Kenneth Wilson and Susan Williamson for providing live Spodoptera littoralis specimens and training in bleeding of larvae. II STATEMENT OF ORIGINALITY I hereby confirm that the work contained within is original and written by the undersigned, and that all research material has been duly referenced and cited. ……………………………………………………. Sharon Baird September 2007 III ABSTRACT Arthropod hemocyanin and phenoloxidase are members of a group of proteins called the Type-3 copper oxygen-binding proteins, both possessing a highly conserved oxygen- binding site containing two copper atoms each coordinated by three histidine residues (Decker and Tuczek, 2000). Despite similarities in their active site, these proteins have very different physiological functions. Phenoloxidase possesses both tyrosinase and o- diphenoloxidase activity, and is predominantly involved in reactions which protect insects from infection (Kopàcek et al., 1995). Hemocyanin is a large multi-subunit protein with a primary function as a respiratory protein, reversibly binding and transporting molecular O2 (Decker and Rimke, 1998; Decker and Tuczek, 2000). Recently, it has been demonstrated in vitro that arthropod hemocyanin possesses an inducible phenoloxidase activity when incubated with denaturants, detergents, phospholipids or proteolytic enzymes. This activity appears to be restricted to only a few subunit types, and it has been hypothesised that it may be accompanied by conformational change which opens the active site increasing access for larger phenolic substrates (Decker and Jaenicke, 2004; Decker et al., 2001; Decker and Tuczek, 2000). This possibly suggests a dual role of hemocyanin in arthropods. The presented thesis deals with two distinct aims. The first was to isolate and sequence a phenoloxidase gene from the insect Spodoptera littoralis (Egyptian Cottonleaf Worm). Despite efforts, progress was hindered by a number of experimental problems which are outlined within the relevant chapters. The second aim was to characterise the mode of SDS induced phenoloxidase activity in arthropod hemocyanin from the ancient chelicerates Limulus polyphemus (horseshoe crab) and Eurypelma californicum (tarantula) and the more modern chelicerate Pandinus imperator (scorpion), using a number of biophysical techniques. The results indicated that the SDS induced phenoloxidase activity is associated with localised tertiary and secondary conformational changes in hemocyanin, most likely in the vicinity of the dicopper centre, thus enhancing access for larger phenolic substrates. Experiments indicate that copper remains associated with the protein during these structural changes; however the nature of the association is unclear. SDS concentrations approximating the CMC appeared critical in causing the necessary structural changes required for a significant increase in the detectable phenoloxidase activity to be exhibited. IV ABBREVIATIONS βGRP β – glucan recognition protein CD Circular dichroism CMC Critical micelle concentration CPC Cetyl pyridium chloride Cu Copper ddNTP Dideoxy nucleoside triphosphate DDP Density dependant prophylaxis DLS Dynamic light scattering DOPA Dihydroxyphenylalanine EtBr Ethidium bromide EuryHc Eurypelma californicum hemocyanin Hc Hemocyanin ICP OES Inductively coupled plasma optical emission spectroscopy IL-1 Interleukin-1 ITC Isothermal titration calorimetry LimHc Limulus polyphemus hemocyanin LPS Lipopolysaccharide MAP kinase Mitogen activated protein kinase NADA N – acetyldopamine NBAD N – β - alanyldopamine NBDG n – nonyl – β – D - glucopyranoside PanHc Pandinus imperator hemocyanin PAP Prophenoloxidase activating proteinase PC Phosphatidylcholine PE Phosphatidylethanolamine PGRP Peptidoglycan recognition protein Phe Phenylalanine PO Phenoloxidase PPAE Prophenoloxidase activating enzyme PPO Prophenoloxidase PRP Pattern recognition protein SDS Sodium dodecyl sulphate SPH Serine protein homologue SUV Small unilamellar vesicle TBE Tris borate EDTA Trp Tryptophan Tyr Tyrosine V TABLE OF CONTENTS Acknowledgements___________________________________________I Statement of Originality_____________________________________III Abstract__________________________________________________IV Abbreviations______________________________________________V Table of Contents__________________________________________VI List of Figures_____________________________________________XI List of Tables___________________________________________XXVI Chapter 1 : Thesis Overview ...................................................................... 1 1.1 Insect phenoloxidase ............................................................................... 1 1.2 Arthropod hemocyanin........................................................................... 2 1.3 Research Objectives................................................................................ 3 Chapter 2 : Phenoloxidase in Insect Immunity – A Review of the Literature................................................................................. 5 2.1 Introduction to Insect Immunity ........................................................... 5 2.2 Insect Phenoloxidase ............................................................................... 8 2.2.1 Phenoloxidase Types and their Functions................................................... 9 2.2.2 Site of Synthesis of Phenoloxidase........................................................... 10 2.2.3 Structural Features of Phenoloxidase........................................................ 10 2.3 Phenoloxidase is a Type-3 Copper Protein Similar to Arthropod Hemocyanin ........................................................................................... 15 2.4 The Prophenoloxidase Activation Cascade......................................... 20 2.4.1 Introduction............................................................................................... 20 2.4.2 Pattern Recognition Proteins..................................................................... 23 2.4.3 PPAE Activation and its Activation of Phenoloxidase............................. 25 2.5 Physiological Roles of Active Phenoloxidase in Insects ..................... 29 2.5.1 Melanisation – Melanotic Encapsulation and Wound Healing................. 29 2.5.2 Sclerotisation............................................................................................. 31 2.6 Control of Phenoloxidase Activity in vivo ........................................... 34 2.6.1 Introduction............................................................................................... 34 2.6.2 Metabolon Formation................................................................................ 36 2.6.3 Hemocyte Surface Phenoloxidase............................................................. 37 VI 2.7 Phenoloxidase as an Indicator of Immune Function in the Lepidoptera............................................................................................ 39 Chapter 3 : Sequencing and analysis of a putative 600 bp Spodoptera littoralis phenoloxidase gene fragment ................................ 43 3.1 Introduction ..........................................................................................