A multifunctional protein : Phosphoglucose isomerase / autocrine motility factor / neuroleukin by Nathalie Y B.Sc, Universite de Montreal, 2004 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Anatomy) THE UNIVERSITY OF BRITISH COLUMBIA April 2007 © Nathalie Y, 2007 II ABSTRACT Phosphoglucose isomerase (PGI) is a glycolytic enzyme that moonlights as a cellular cytokine. The protein is also known as autocrine motility factor (AMF), neuroleukin and maturation factor. PGI/AMF interaction with its receptor interaction is pH-dependent. Indeed, at neutral pH, PGI/AMF binds its receptor AMFR at the cell surface and can be endocytosed via two different pathways: caveolae/raft-dependent endocytosis to the smooth ER or clathrin-dependent endocytosis to multivesicular bodies (MVBs). Internalized PGI/AMF can recycle from MVBs to the plasma membrane where it can undergo further rounds of endocytosis and recycling. Recycling receptor-ligand complexes can also be sequestered via stable association with FN fibrils. Recent data show that, at acid pH, endocytosis is inhibited and PGI/AMF binds directly to FN fibrils or to HS. Heparan sulfate proteoglycans, when expressed on the surface of cells, modulate the actions of a large number of extracellular ligands while fibronectin is involved in many cellular processes such as tissue repair and cell migration/adhesion. However, the mechanisms that regulate PGI/AMF binding to its receptors still remain unclear. PGI/AMF cytokine activity, associated with several diseases, has been reported in rheumatoid synovial fluid and its deposition on synovial surfaces and ability to induce an autoimmune response in rheumatoid arthritis (RA) identified it as a possible autoantigen different from normal circulating PGI/AMF. However, more recent manuscripts have questioned the prevalence of an autoimmune response to PGI in RA. Ill In this study, recombinant PGI constructs were used to characterize PGI interactions and functions. We demonstrate that PGI behaves differently after N or C-terminal residue additions. Our data also suggest that monomerization but not enzymatic activity is necessary to induce cell motility at neutral pH. The putative function of PGI in RA was assessed and using the recombinant PGI constructs and PGI autoantibodies was found to be species and conformation-dependant. IV TABLE OF CONTENTS pages Abstract II Table of contents IV List of tables VII List of figures VIII List of symbols and abbreviations X 1. Introduction 1 1.1 Historic 1 1.2 Molecular Biology of PGI 4 Gene structure of PGI 4 Gene 4 Minisatellites 4 Protein structure of PGI 5 Backbone structure 5 Active site 7 Interspecies homology 7 Mutations 7 1.3 Functions 8 Catalytic function of PGI 8 Glycolysis 8 Active sites 9 Moonlighting functions of PGI 13 PGI/AMF/ neuroleukin secretion 13 Neuroleukin 13 Autocrine motility factor and maturation factor 15 Involvement in mineralization during osteoblast differentiation. 19 Embryo implantation 20 1.4 Receptors 21 AMFR / gp78 22 Protein motifs implicated in AMF/PGI cytokine activity and receptor binding 24 Fibronectin 25 Heparan sulfate 26 IGFPB-3 27 Another receptor 27 1.5 PGI/AMF implication in diseases 28 Non-spherocytic haemolytic anaemia 28 Cancer 29 Rheumatoid Arthritis 30 V 2. Hypothesis 32 3. Material and methods 33 3.1 Protein purification 33 3.2 SDS-page and western blots 33 3.3 Enzymatic activity assay 34 3.4 Glutaraldehyde cross-linking assay 35 3.5 Circular dichroism 35 3.6 Fibronectin binding assay..... 35 3.7 Cell motility assay 36 3.8 Sera and synovial fluids 37 3.9 Human RA antisera ELISA screening 37 3.10 Human RA antisera Western Blot screening 37 4. Results 39 4.1 Analysis of recombinant AMF/PGI expression and purification 39 4.2 Enzymatic activity of recombinant PGI/AMF 40 4.3 Recombinant PGI/AMF glutaraldehyde cross-linking 41 4.4 Circular dichroism of recombinant PGI/AMF 42 a. Far UV 42 b. NearUV 43 4.5 Binding to fibronectin 43 4.6 Recombinant cell-induced motility 44 4.7 Implication of AMF/PGI in Rheumatoid Arthritis 45 a. ELISA essay 45 b. Western blot essay 46 5. Discussion 48 5.1 Conformational effects of residue additions to C-terminus and N-terminus. 48 5.2 Cell-induced motility and cell interaction of AMF/PGI 49 5.3 Implications for the Role of PGI in Rheumatoid Arthritis 51 6. Conclusion 53 VI 7. Figures, tables and legends 54 8. Bibliography 79 VII LIST OF TABLES Table I Summary: Recombinant AMF/PGI properties 70 Table II Analysis of densitometry 75 VIII LIST OF FIGURES 1. INTRODUCTION Figure 1 Structural organization of the human glucose phosphate isomerase gene 4 Figure 2 Ribbon representation of PGI from different species 6 Figure 3 Glycolysis pathway 8 Figure 4 Interconversion between glucose 6-phosphate and fructose 6-phosphate 9 Figure 5 Proposed catalytic mechanism for PGI 11 Figure 6 Molecular signaling in AMF/PGI motility stimulation 16 Figure 7 Interaction between AMF-AMFR and VEGF-VEGFR signal in tumor and host endothelial cells 17 Figure 8 Aptosis-related signal pathways induced by AMF/PGI overexpression. 18 Figure 9 The complex biology of PGI/AMF and its receptor 21 Figure 10 Structure of AMFR/gp78 22 Figure 11 Increased association of AMF/PGI to fibronectin at acid pH 26 4. RESULTS Figure 12 Vector and Constructs 55 Figure 13 Western blot and SDS-page analysis of recombinant AMF/PGI 57 Figure 14 Enzymatic activity of recombinant AMF/PGI 59 Figure 15 Glutaraldehyde cross-linking and semi-log graph analysis 1 61 Figure 15 Glutaraldehyde cross-linking and semi-log graph analysis II 62 Figure 16 Circular dichroism of recombinant AMF/PGI 64 Figure 17 Binding of PGI/AMF to dimeric FN at neutral and acid pH 66 IX Figure 18 Recombinant AMF/PGI cell-induced motility 68 Figure 19 Human RA antisera ELISA screening 71 Figure 20 Western Blot control 73 Figure 21 Western Blot screening: species-specific recognition of human RA anti-sera to PGI/AMF 75 Figure 22 Western Blot screening: conformation-specific recognition of human RA anti-sera to recombinant PGI/AMF 77 LIST OF SYMBOLS AND ABBREVIATIONS AMF Autocrine motility factor AMFR Autocrine motility factor receptor ATP Adenosine triphosphate BSA Bovine Serum Albumine CD Circular Dichroism cDNA Complementary DNA ECM Extracellular matrix ER Endoplasmic reticulum ERAD Endoplasmic reticulum associated degradation F6P Fructose-6-phosphate FN Fibronectin G6P Glucose-6-phosphate GDI GDP-dissociation inhibitor GTP Guanosine triphosphate HS Heparan sulfate IGF Insulin growth factor IGFBP Insulin-like growth factor binding protein kDa Kilo Daltons KDR kinase domain region mpCD methyl beta-cyclodextrin mRNA messenger RNA MVB Multivesicular bodies NAD Nicotamine adenine dinucleotide PGI Phosphoglucose isomerase RA Rheumatoid arthritis RBC Red blood cells SDS Sodium dodecyl sulfate Tfr Transferrin UV Ultraviolet VEGF Vascular endothelial growth factor 1 1. INTRODUCTION 1.1 - HISTORY Phosphoglucose isomerase is a glycolytic enzyme present in all types of human cells. It had been studied for decades, but its molecular structure and the biological understanding of its numerous functions have emerged only during the past 20 years. Significant technical advances have led to the discovery of its structure, its identity as an extracellular cytokine and its receptor. The human enzyme is of medical interest because PGI is believed to be involved in several diseases. The interest of PGI/AMF and its receptor is growing not only because of its biological significance, but also because of its practical importance as a major target in the post-genomic era for developing therapeutics for diverse diseases. Glycolysis Glycolysis has been studied for over a century. This degradation pathway of sugar molecules leads to the formation of pyruvate releasing energy in the form of ATP and molecules with reduction potential (NAD) [1]. Initially described by Lohman in 1933, phosphoglucose isomerase (PGI) is known to be a ubiquitous cytosolic enzyme that catalyzes the interconversion between glucose-6-phosphate and fructose-6-phosphate during the second step of glycolysis [2]. Neuroleukin, lymphokin and phosphoglucose isomerase In response to partial denervation or paralysis, new neurotic processes, called terminal sprouts, can appear from the remaining motor axon terminal,[3]. In their attempt to identify factors produced by denervated or inactive muscle that might be necessary for 2 motor axon terminal sprouting, Gurney and colleagues purified in 1986 a ~56 kD protein, neuroleukin. Neuroleukin was subsequently shown to be capable of increasing survival of cultured sensory neurons [4] and, as they found later that year, to act as a lymphokine. Following secretion by lectin-stimulated T cells, neuroleukin was able to induce the maturation of B-cells into antibody secreting cells but was not necessary involved in the continued production of immunoglobulin by differentiated antibody-secreting cells [5]. Two years later, in 1988, the mouse phosphoglucose isomerase (PGI) cDNA was isolated and sequenced. The investigators, a group working on the molecular genetics of carbohydrate metabolism, surprisingly found a whole sequence identity between the 759 nucleotides at the 3' end of the mouse PGI clone and the sequence of mouse neuroleukin [6]. Moreover a second group showed that same year a 90% homology between PGI and neuroleukin [7]. Hence, the question as to how a ubiquitous, cytosolic enzyme could also function as an extracellular cytokine had been raised. Gurney et al. immediately reacted to this discovery and subsequently confirmed that both mouse and human neuroleukin expressed PGI enzymatic activity. However, they found that PGI activity was not blocked with monoclonal antibodies that were able to block neuroleukin activities. Thus, they hypothesized for the first time the existence of a PGI/neuroleukin receptor [8]. Autocrine motility factor, maturation factor and phosphoglucose isomerase Several steps are involved in the progression of a tumor, which include unrestrained growth and invasive behaviour or active locomotion of tumor cells is also one of its major properties.