Apolipoprotein (Apo) A-IV Is a Protein Synthesized by the Small Intestine In
Total Page：16
File Type：pdf, Size：1020Kb
UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ THE STRUCTURE AND FUNCTION OF APOLIPOPROTEIN A-IV By Kevin Joseph Pearson B.S., University of Pittsburgh, 1999 May 2005 A dissertation presented to the faculty of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pathobiology and Molecular Medicine Committee Members W. Sean Davidson-Chair Patrick Tso Ronald J. Jandacek Min Liu Simon L. Newman Randall R. Sakai THESIS ABSTRACT Apolipoprotein (apo) A-IV is a protein synthesized by the small intestine in response to lipid absorption. It has been proposed to play a role in cholesterol efflux, lipoprotein metabolism and food intake. Unfortunately, little information on its structure/function relationship is known. Therefore, it was important to establish a recombinant expression system for apoA-IV so deletion mutagenesis could be performed to achieve the specific aims. The following aims address the hypothesis that specific regions of apoA-IV are involved in its ability to interact with lipid and inhibit food intake. Aim 1: Determine the region of apoA-IV that is responsible for its ability to bind lipid as well as to identify the generalities of its structure. Initially, it was found that removing the C-terminal 44 amino acids from human apoA-IV caused a significant increase in lipid binding ability as compared to WT. Eventually, a smaller region from amino acid 333-343 was established as the ‘inhibitory’ region of apoA-IV at the C- terminus. Using the ∆333-343 as a template, the N-terminus was also mutated and it was found that the N-terminal amino acids from 11-20 were required for the mutants to be fast lipid binders. Therefore, we propose there is an interaction between the ‘inhibitory’ region from 333-343 and the N-terminus of the protein that does not allow the proper conformation for accelerated lipid binding. Aim 2: Determine the region of apoA-IV that is responsible for its role in the inhibition of food intake. Originally, several C-terminal deletion mutants were made in rat apoA-IV. The mutants were injected into the 3rd ventricle of rat brains and their food intake was determined. It was found that the food intake region was located in the N- terminal 116 amino acids. Next a 3rd mutant was made that removed the N-terminal 61 amino acids. This mutant did not inhibit food intake as compared to WT suggesting the region involved in food intake is located in the first 61 amino acids. In separate studies, a 14-residue peptide from amino acid 17-30 also inhibited food intake in rats suggesting this is the active region. ACKNOWLEDGEMENTS This work was supported by a pre-doctoral fellowship to K. Pearson from the American Heart Association, Southern and Ohio Valley Affiliate. I would like to thank Patrick Tso for funding several years of this project. I would also like to thank my advisor, W. Sean Davidson, for his constant support throughout this long and arduous journey. He has taught me how to become a successful scientist in many ways. He also made a pretty good target for me to fire at (or mostly complain to) when I was stressed or frustrated. When I leave UC, I will leave having had a great thesis advisor, but also a close friend. As for the rest of the lab…thanks - only one personal shout-out and he probably won’t ever read this…Nick, you’re the dude and thanks for helping out a scared kid from Pennsylvania when he didn’t know anything! Outside the lab…Thanks mom and dad for helping out so I could be a student until I was 27 (the car/insurance)…Skot, Jake, Wael, Kara and Kimberly – Those first years could have been so much harder…1214 McKeone - enough said…and finally three things I couldn’t have made it without (I know you’re thinking my girlfriend here but that’s too easy) 1. Cherry, my 1992 Dodge Spirit. 2. Pretty blue bullet, my 1997 Chevrolet Cavalier. 3. Cincinnati style chili and currently Subway. TABLE OF CONTENTS LIST OF TABLES 7 LIST OF FIGURES 8 ABBREVIATIONS 12 CHAPTER 1: GENERAL INTRODUCTION 13 OVERVIEW OF APOA-IV METABOLISM 13 GENETIC ORGANIZATION OF APOA-IV 15 PRIMARY SEQUENCE OF APOA-IV 16 SECONDARY STRUCTURE OF APOA-IV 17 TERTIARY STRUCTURE OF APOA-IV 18 LIPID ASSOCIATED FORMS OF APOA-IV 19 ROLE IN LIPOPROTEIN METABOLISM 20 ROLE OF APOA-IV IN INFLAMMATION 23 ROLE OF APOA-IV IN FOOD INTAKE 24 GENETIC MANIPULATIONS 25 THE AIMS OF THIS THESIS 27 THE ORGANIZATION OF THIS THESIS 28 REFERENCES 30 FIGURES 39 CHAPTER 2: BACTERIAL EXPRESSION AND CHARACTERIZ- ATION OF RAT APOLIPOPROTEIN A-IV AND APOLIPOPROTEIN E 41 ABSTRACT/SUMMARY 41 INTRODUCTION 42 EXPERIMENTAL PROCEDURES 44 Construction of the expression vector for apoA-IV 45 Cloning and mutagenesis of rat apoE 46 Protein expression and purification 47 SDS-PAGE and immunoblot 49 Electrospray mass spectrometry 50 N-terminal amino acid sequencing 50 Lipoprotein particle reconstitution 50 Biological activity of recombinant apoA-IV 51 Conditioned taste aversion 52 Native protein purification 53 Western blot analysis for apoE 53 Circular dichroism spectroscopy 54 DMPC liposome solubilization 54 Cholesterol efflux assay 54 RESULTS: RAT APOA-IV 55 DISCUSSION: RAT APOA-IV 57 2 RESULTS: RAT APOE 59 DISCUSSION: RAT APOE 62 REFERENCES 63 TABLES 67 FIGURES 68 CHAPTER 3: THE STRUCTURE OF HUMAN APOLIPOPROTEIN A-IV: A DISTINCT DOMAIN ARCHITECTURE AMONG EXCHANGEABLE APOLIPOPROTEINS WITH POTENTIAL FUNCTIONAL IMPLICATIONS 81 ABSTRACT 81 INTRODUCTION 82 EXPERIMENTAL PROCEDURES 86 Cloning and mutagenesis of human apoA-IV 86 Protein expression and purification 87 Circular dichroism spectroscopy 88 Fluorescence measurements 88 Self-association determination 89 DMPC liposome solubilization 89 Cholesterol efflux assay 90 RESULTS 90 DISCUSSION 97 3 REFERENCES 103 TABLES 106 FIGURES 108 CHAPTER 4: A POTENTIAL INTERACTION BETWEEN THE N- AND C-TERMINI OF APOLIPOPROTEIN A-IV AND ITS ROLE IN LIPID ASSOCIATION 117 ABSTRACT 117 INTRODUCTION 117 EXPERIMENTAL PROCEDURES 119 Mutagenesis of human apoA-IV 120 Protein expression and purification 120 DMPC liposome solubilization 121 Interfacial Behavior at the Oil/water Interface 122 Thermal denaturation and fluor. spectroscopy 122 RESULTS 123 DISCUSSION 127 REFERENCES 132 TABLES 135 FIGURES 137 4 CHAPTER 5: IDENTIFICATION OF AN APOLIPOPROTEIN A-IV SEQUENCE RESPONSIBLE FOR CENTRAL INHIBITION OF FOOD INTAKE 144 ABSTRACT 144 INTRODUCTION 145 EXPERIMENTAL PROCEDURES 147 Cloning and purification of rat apoA-IV mutants 148 Removal of LPS 149 Purification of native apoA-IV 150 Peptide synthesis 150 Food intake studies 151 BBB studies 152 RESULTS 153 DISCUSSION 157 REFERENCES 161 FIGURES 164 CHAPTER 6: GENERAL DISCUSSION 174 SUMMARY: AIM 1 174 DISCUSSION: AIM 1 175 FUTURE STUDIES: AIM 1 176 SUMMARY: AIM 2 177 5 DISCUSSION: AIM 2 178 FUTURE STUDIES: AIM 2 179 FINAL THOUGHTS 179 REFERENCES 181 6 TABLES: CHAPTER 2: BACTERIAL EXPRESSION AND CHARACTERIZ- ATION OF RAT APOLIPOPROTEIN A-IV AND APOLIPOPROTEIN E Table 2-1: Purification of recombinant rat apolipoprotein E. 67 CHAPTER 3: THE STRUCTURE OF HUMAN APOLIPOPROTEIN A-IV: A DISTINCT DOMAIN ARCHITECTURE AMONG EXCHANGEABLE APOLIPOPROTEINS WITH POTENTIAL FUNCTIONAL IMPLICATIONS Table 3-1: Conformation and stability properties of WT apoA-IV and the various deletion mutants. 106 Table 3-2: Kinetic parameters for cholesterol efflux from RAW264 macrophages to lipid-free apoA-IV and its deletion mutants. 107 CHAPTER 4: A POTENTIAL INTERACTION BETWEEN THE N- AND C-TERMINI OF APOLIPOPROTEIN A-IV AND ITS ROLE IN LIPID ASSOCIATION Table 4-1. Rate constants for WT and mutant apoA-IV in the DMPC clearance assay. 135 Table 4-2. Fluorescence and thermal denaturation parameters of WT and mutant apoA-IV 136 7 FIGURES: CHAPTER 1: GENERAL INTRODUCTION Figure 1-1: The primary sequence of human apoA-IV. 39 Figure 1-2: A general schematic of lipid metabolism and the potential roles of apoA-IV. 40 CHAPTER 2: BACTERIAL EXPRESSION AND CHARACTERIZ- ATION OF RAT APOLIPOPROTEIN A-IV AND APOLIPOPROTEIN E Figure 2-1: Schematic representation of the pET 30 vector. 68 Figure 2-2: SDS polyacrylamide gel of recombinant apoA-IV produced by E.Coli at different steps of the purification process. 69 Figure 2-3: Immunoblot analysis of recombinant rat apoA-IV expressed in E. coli. 70 Figure 2-4: Electrospray mass spectrum of recombinant apoA-IV. 71 Figure 2-5: Native gradient polyacrylamide gel of reconstituted particles made with recombinant apoA-IV. 72 Figure 2-6: Mean food intake following i3vt recombinant rat apoA-IV and natural rat apoA-IV. 73 Figure 2-7: Conditioned taste aversion. 74 Figure 2-8: Forward oligonucleotide primer sequence. 75 Figure 2-9: SDS-PAGE analysis of recombinant rat apoE at different stages of purification and western blot analysis following purification. 76 Figure 2-10: SDS-PAGE analysis of rat apoE under different storage conditions. 77 8 Figure 2-11: Far UV circular dichroism spectra of apoE. 78 Figure 2-12. Dimyristoylphosphatidylcholine (DMPC) liposome solubilization by apoE. 79 Figure 2-13: ABCA1-mediated cholesterol efflux from RAW264 macrophages. 80 CHAPTER 3: THE STRUCTURE OF HUMAN APOLIPOPROTEIN A-IV: A DISTINCT DOMAIN ARCHITECTURE AMONG EXCHANGEABLE APOLIPOPROTEINS WITH POTENTIAL FUNCTIONAL IMPLICATIONS Figure 3-1: Linear diagrams of WT apoA-IV and truncation mutants. 108 Figure 3-2: SDS-PAGE analysis of WT apoA-IV and representative mutant forms. 109 Figure 3-3: Thermal unfolding of N- and C-terminal mutants of apoA-IV. 110 Figure 3-4: Thermal unfolding of combined N- and C-terminal mutants.