STUDIES OF THE STRUCTURE AND FUNCTION OF RECOMBINANT HUMAN HEPHAESTIN by Ganna Vashchenko B.Sc., Taras Shevchenko National University of Kyiv, 2007 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2012 © Ganna Vashchenko, 2012 ABSTRACT Hephaestin is a multicopper ferroxidase involved in iron absorption in the small intestine. The ferroxidase activity of hephaestin is thought to play an important role during iron export from intestinal enterocytes and the subsequent iron loading of the blood protein transferrin, which delivers iron to the tissues. Structurally, the ectodomain of hephaestin is predicted to resemble ceruloplasmin, the soluble ferroxidase of blood. In this work I investigated substrate specificity, copper loading and the ferroxidation mechanism of recombinantly expressed human hephaestin. The hephaestin ectodomain (Fet3Hp) was expressed in Pichia pastoris and purified to electrophoretic homogeneity by immunoaffinity chromatography. Recombinant hephaestin retained ferroxidase activity and showed an average copper content of 4.2 copper atoms per molecule. The Km values of Fet3Hp for such organic substrates as p-phenylenediamine and o- dianisidine were close to values determined for ceruloplasmin. However, in contrast to ceruloplasmin, recombinant hephaestin was incapable of direct oxidation of adrenaline and dopamine implying a difference in biological substrate specificities between these two homologous oxidases. I also expressed hephaestin ectodomain with the ceruloplasmin signal peptide (CpHp) using BHK cells as an expression system. Ion exchange chromatography of purified CpHp resulted in the production of a hephaestin fraction with improved catalytic and spectroscopic properties. Detailed kinetic analysis of CpHp ferroxidation rates revealed the presence of two types of iron-binding sites with different affinities towards ferrous iron. Michaelis constants for high- and low-affinity ferrous binding sites in CpHp were comparabale to the corresponding Km values in ceruloplasmin suggesting that both paralogs utilize similar amino acid residues for iron binding. To investigate the role of particular residues in iron specificity of hephaestin, mutations of putative iron ligands were introduced into CpHp using site-directed mutagenesis. Kinetic ii analysis of ferroxidation rates of wild-type CpHp and variants revealed the important roles of residues E960 and H965 in hephaestin ferroxidase activity. iii PREFACE The work outlined in chapter 3 has been published (Vashchenko G., Bleackley M.R., Griffiths T.A. & MacGillivray R.T. (2011) Oxidation of organic and biogenic amines by recombinant human hephaestin expressed in Pichia pastoris. Arch. Biochem. Biophys. 514, 50- 6). I was responsible for designing and carrying out the experiments. Initial cloning steps required for these experiments were performed by Dr. Mark Bleackley. Sepharose derivative with ethylamine used for purification of human ceruloplasmin was synthesized by Dr. Kay Yu. The work outlined in Chapter 5 along with the work described in Chapter 4 is being prepared for publication. I created all of the text, figures and tables in this thesis. iv TABLE OF CONTENTS ABSTRACT..................................................................................................................................................................... II PREFACE....................................................................................................................................................................... IV TABLE OF CONTENTS ............................................................................................................................................... V LIST OF TABLES........................................................................................................................................................ VII LIST OF FIGURES..................................................................................................................................................... VIII LIST OF ABBREVIATIONS....................................................................................................................................... IX ACKNOWLEDGEMENTS .......................................................................................................................................... XI 1. INTRODUCTION ......................................................................................................................................................1 1.1 IRON IN BIOLOGY ....................................................................................................................................................1 1.2 PROKARYOTIC IRON METABOLISM .........................................................................................................................2 1.3 YEAST IRON METABOLISM ......................................................................................................................................3 1.3 IRON METABOLISM IN HUMANS ..............................................................................................................................4 1.3.1 Iron absorption in small intestine..................................................................................................................4 1.3.2 Iron uptake in different cell types..................................................................................................................8 1.3.3 Regulation of iron homeostasis ...................................................................................................................10 1.3.4 Inherited disorders of iron metabolism.......................................................................................................12 1.4 MULTICOPPER OXIDASES ......................................................................................................................................14 1.4.1 Marcus theory of electron transfer..............................................................................................................15 1.4.2 Type 1 copper sites.......................................................................................................................................16 1.4.3 Transfer of electrons to the trinuclear cluster and dioxygen reduction.....................................................18 1.5 MULTICOPPER METALLOOXIDASES ......................................................................................................................18 1.5.1 Fet3p.............................................................................................................................................................20 1.5.2 Ceruloplasmin..............................................................................................................................................22 1.5.3 Hephaestin....................................................................................................................................................26 1.5.4 Zyklopen .......................................................................................................................................................27 1.5.5 Ferroxidases in human iron metabolism.....................................................................................................28 1.5 STATEMENT OF HYPOTHESES AND OBJECTIVES ....................................................................................................29 2. MATERIAL AND METHODS...............................................................................................................................31 2.1 MATERIALS...........................................................................................................................................................31 2.2 DNA MANIPULATIONS..........................................................................................................................................31 2.2.1 Production of the Fet3Hp expression cassette and its integration into the P. pastoris genome ...............32 2.2.2 Production of stably transfected BHK cells for expression of CpHp and its variants...............................34 2.3 RECOMBINANT PROTEIN EXPRESSION...................................................................................................................37 2.3.1 Expression of Fet3Hp in P. pastoris............................................................................................................37 2.3.2 Expression of CpHp and its variants in BHK cells.....................................................................................38 2.4 PROTEIN PURIFICATION AND STORAGE.................................................................................................................39 2.4.1 Purification of Fet3Hp.................................................................................................................................39 2.4.2 Purification of CpHp and variants..............................................................................................................40 2.4.3 Purification of human ceruloplasmin..........................................................................................................40 2.5 ANALYTICAL METHODS ........................................................................................................................................41