Alpha-2-Macroglobulin: an abundant extracellular chaperone

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1 University of Wollongong Theses Collection University of Wollongong Theses Collection University of Wollongong Year 2008 Alpha-2-Macroglobulin: an abundant extracellular chaperone Katie French University of Wollongong French, Katie, Alpha-2-Macroglobulin: an abundant extracellular chaperone, MSc thesis, School of Biological Sciences, University of Wollongong, This paper is posted at Research Online.

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3 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone i Alpha-2-Macroglobulin: An abundant extracellular chaperone A thesis submitted in fulfilment of the requirements for the award of the degree of MASTER OF SCIENCE from The University of Wollongong By KATIE FRENCH Supervisor: Professor Mark Wilson School of Biological Sciences April 2008

4 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone ii DECLARATION This thesis is submitted in accordance with the regulations of the University of Wollongong in partial fulfilment of the degree of Master of Science. It does not include any material published by another person except where due reference is made in the text. The experimental work described in this thesis is original work and has not been submitted for a degree or diploma at any other university. Katie French April 2008

5 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone iii ACKNOWLEDGEMENTS Firstly, I would like to say a huge thank-you to Professor Mark Wilson for all the patience, time and knowledge he has shared with me over the last three years. Despite thinking I am crazy for the decision I have made, he never once turned his back and still encouraged me to write this thesis. I learnt so much in the time I spent Lab 120, it gave me an appreciation of research and taught me the value of persistence and dedication. Thanks also to Justin Yerbury who introduced me to α 2 M and shared the many trials and tribulations of the α 2 M project. To all the members of lab 120 during my time in the lab, to Elise and Amy, thanks for the friendship and sharing your great knowledge of the lab. The 120B crew of Russ, Susie and Chris, you guys made that year a lot of fun! You put up with the med school saga- exams, interviews and all the nervous waits, I couldn t have got through that year without you. To the Shoalies from the Graduate School of Medicine, thanks for keeping me sane and putting up with my nerd babble about a serum protein called α 2 M that I tend to bring up at any given opportunity, you guys have made the decision to do medicine even more worthwhile. Shoalhaven represent. Most importantly to my family, Mum, Dad, Leah, Paul and Steve, thank you for your never-ending support, encouragement and advice. Without you I would not be where I am today, you made it possible for me to achieve my goals and to follow my dreams. 6 years down, two degrees and only one to go, I promise in three years time I ll finally get a job!

6 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone iv ABSTRACT Alpha-2-macroglobulin (α 2 M) is a 720 kda glycoprotein consisting of four identical (180 kda) subunits and is the major representative of the α-macroglobulin group of plasma proteins, present at high concentrations in human plasma. α 2 M is best known for its ability to inhibit a broad spectrum of proteases which it accomplishes using a unique trapping method. Protease trapping induces α 2 M to adopt an activated conformation which exposes a binding site for the low density lipoprotein receptor (LRP), facilitating clearance of the complexes from the body. α 2 M has been ascribed many biological roles which extend beyond simple protease inhibition including immune regulation, mediation of the inflammatory response via cytokine binding and more recently chaperone activity. α 2 M has been shown to inhibit the heat-induced precipitation of proteins in vitro through the formation of stable complexes. The work outlined in this study further characterises the chaperone activity of α 2 M under conditions of heat and oxidative stress and establishes the relationship between this and its role as a protease inhibitor. When present at physiological concentrations, α 2 M was found to inhibit the oxidationinduced precipitation of lysozyme (lys). In a preliminary study, it was shown that α 2 M forms stable, soluble complexes with heat-stressed proteins. In the current study, native agarose gel electrophoresis and immunoprecipitation analyses were used to demonstrate that α 2 M also forms stable, soluble complexes with oxidised proteins. Removal of α 2 M from human plasma was found to significantly increase the level of plasma protein precipitation under conditions of heat and oxidative stress. Proteins co-purifying with α 2 M from human plasma (following incubation at either 43 C or room temperature for

7 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone v 72 h) were analysed by mass spectrometry; this identified fibrinogen as a putative endogenous chaperone client protein of α 2 M. It was also shown that protease-mediated activation of α 2 M abolishes the chaperone activity, but that native α 2 M is able to form soluble complexes with heat stressed proteins and then subsequently become activated by protease trapping. Oxidation of (chaperone-inactive) protease bound α 2 M was shown to restore chaperone activity but not the protease inhibitor function. These behaviours provide an alternative means for generating α 2 M/stressed protein/protease complexes which could be cleared in vivo by LRP-mediated cellular uptake and degradation. The ability of α 2 M/stressed protein complexes to bind to cell surface receptors was investigated using JEG-3, Hep-G2, and U937 cell lines and granulocytes derived from whole human blood. α 2 M/CS complexes had limited ability to bind to LRP expressed on the surface of JEG-3 cells. However, preliminary results indicated that activation of α 2 M (α 2 M*) and α 2 M/stressed protein complexes (α 2 M*/CS) with trypsin resulted in subsequent binding to the surface of JEG-3 cells. Native α 2 M/CS complexes were found to bind to granulocytes and Hep-G2 cells via unidentified, non-lrp receptors. Collectively, the results presented here further establish α 2 M as a potent extracellular chaperone with the ability to protect proteins from heat and oxidation-induced stress. α 2 M appears likely to have a dual role in vivo, as a protease inhibitor and as an extracellular chaperone, the first identified mammalian protein with both activities. The evidence suggests that it may function as part of an extracellular quality control system for protein folding important in the control of inflammation and protein conformational disorders (PCDs) such as Alzheimer's disease and type II diabetes. The pathology of PCDs has been linked to the development of extracellular deposits of misfolded

8 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone vi proteins. This thesis provides evidence supporting the hypothesis that α 2 M binds to misfolded extracellular proteins to keep them soluble and mediates their cellular uptake and subsequent degradation. Future advances in understanding of extracellular protein folding quality control are likely to provide novel insights into the mechanisms underpinning the development of serious human diseases and identify opportunities for the development of new therapies.

9 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone vii TABLE OF CONTENTS TITLE PAGE...i DECLARATION... ii ACKNOWLEDGEMENTS...iii ABSTRACT... iv TABLE OF CONTENTS... vii ABBREVIATIONS... xi LIST OF FIGURES... xv LIST OF TABLES... xvi CHAPTER 1: INTRODUCTION PROTEIN FOLDING PROTEIN MISFOLDING, AGGREGATION AND DISEASE MECHANISMS OF PROTEIN QUALITY CONTROL Molecular Chaperones - The Saviours of Protein Folding EXTRACELLULAR CHAPERONES Clusterin Haptoglobin Serum Amyloid P Component Alpha-2-macroglobulin ALPHA-2-MACROGLOBULIN Synthesis, Structure and Protease Inhibitor Action of α 2 M Other Functions of α 2 M Binding of α 2 M to Ligands Receptor Binding and Internalisation of α 2 M The Effects of Oxidative Stress on the Structure, Function and Receptor Recognition of α 2 M... 25

10 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone viii 1.6 AIMS CHAPTER 2: MATERIALS AND METHODS MATERIALS PURIFICATION OF α 2 M PREPARATION OF ACTIVATED α 2 M AND ACTIVATED (α2m/cs)* COMPLEXES PREPARATION OF OXIDISED α 2 M Size Exclusion Chromatography FORMATION AND PURIFICATION OF COMPLEXES BETWEEN α2m ANS STRESSED PROTEIN ELECTROPHORESIS SDS PAGE Immunodetection Native Gel Electrophoresis Native PAGE TRYPSIN BINDING ASSAY PROTEIN PRECIPITATION ASSAYS PRECIPITATION OF PROTEINS IN WHOLE HUMAN PLASMA Determination of Protein Concentration using BCA Assay Immunoprecipitations IDENTIFICATION OF ENDOGENOUS SUBSTRATES USING MASS SPECTROMETRY Spot Excision Trypsin Digestion... 40

11 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone ix MALDI-TOFF Mass Spectrometry CELL CULTURE AND FLOW CYTOMETRY Culture of Cell Lines Binding Assays using JEG-3,Hep-G2 and Activated U937 Cells Binding Assays using Granulocytes Isolated from Whole Blood Binding Analysis using Flow Cytometry CHAPTER 3: CHARACTERISING THE CHAPERONE FUNCTION OF α 2 M INTRODUCTION METHODS RESULTS Within α 2 M/heat Stressed Protein Complexes, α 2 M Remains in its Native Conformation Protease Activation Abolishes the Chaperone Activity of α 2 M α 2 M Inhibits the Heat Induced Precipitaion of Proteins in Whole Human Serum Identifying Endogenous Chaperone Substrates for α 2 M DISCUSSION CHAPTER 4: BINDING OF α 2 M/STRESSED PROTEIN COMPLEXES TO CELL SURFACE RECEPTORS INTRODUCTION METHODS RESULTS Binding of α 2 M/Stressed Protein Complexes to JEG-3 cells... 64

12 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone x Binding of α 2 M/Stressed Protein Complexes to Hep-G2 cells Binding of α 2 M/Stressed Protein Complexes to Activated U937 cells Binding of α 2 M/Stressed Protein Complexes to Neutrophils DISCUSSION CHAPTER 5: OXIDATIVE STRESS AND THE CHAPERONE ACTION OF α 2 M INTRODUCTION METHODS RESULTS α 2 M Undergoes Conformational Changes under Oxidative Stress α 2 M Functions as a Chaperone under Oxidative Conditions Oxidation of activated α 2 M Re-establishes Chaperone Action DISCUSSION CHAPTER 6: DISCUSSION ADVANCES IN UNDERSTANDING THE CHAPERONE ACTION OF α 2 M Dual Chaperone and Protease Inhibitory Roles of α 2 M OXIDATIVE STRESS AND α 2 M Implications for Inflammatory Response Regulation and Chaperone Functionality FIBRINOGEN IS AN ENDOGENOUS CHAPERONE SUBSTRATE OF α 2 M Cell Surface Receptor Binding of α 2 M and α 2 M/Stressed Protein Complexes CONCLUSIONS CHAPTER 7: REFERENCES

13 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone xi ABBREVIATIONS A 360 A 405 Aβ Absorbance at 360 nm Absorbance at 405 nm Amyloid-beta peptide Alexa 488 Alexa fluor 488 α 2 M α 2 M* α 2 M/CS Alpha-2-macroglobulin Activated alpha-2-macroglobulin Complex formed between alpha-2-macroglobulin and stressed (unfolded) citrate synthase α 2 M/CSb Complex formed between alpha-2-macroglobulin and biotinylated, stressed (unfolded) citrate synthase α 2 M*/CSb Complex formed between alpha-2-macroglobulin and biotinylated, stressed (unfolded) citrate synthase which has been activated. α 2 M/CPK Complex formed between alpha-2-macroglobulin and stressed (unfolded) creatine phosphokinase α 2 M/CPKb Complex formed between alpha-2-macroglobulin and biotinylated, stressed (unfolded) creatine phosphokinase α 2 M/lys α 2 MR α 2 MR/LRP Complex formed between alpha-2-macroglobulin and lysozyme Alpha-2-macroglobulin receptor Alpha-2-macroglobulin/ Low density lipoprotein receptor-related protein (the same receptor). ASGP ATP Az BCA Asialoglycoprotein Adenosine triphosphate Azide Bicinchoninic acid

14 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone xii bisans CPK CPKb CS CSb CNS dh 2 O Da 4, 4 -dianilino- 1, 1 -binaphthyl-5, 5 -disulfonic acid Creatine phosphokinase Biotinylated creatine phosphokinase Citrate synthase Biotinylated citrate synthase Central nervous system Distilled water Dalton DMEM: F-12 Dulbecco s modified eagle medium: F-12 DMSO ECL EDTA FCS FITC FPLC Geomean GST-RAP Deoxymethylsulphoxide Enhanced chemiluminescence detection Ethylenediamine tetracetic acid Foetal calf serum Fluoresein Isothiocyanate Fast protein liquid chromatography Geometric mean Fusion protein containing glutathione-s-transferase and receptor associated protein GST-RAPb Biotinylated fusion protein containing glutathione-s-transferase and receptor associated protein HDC HEPES HRP g GST Hsp Heat denatured casein N-(hydroxyethyl) piperazine-n -(2-ethanesulfonic acid) Horse radish peroxidase G- force Glutathione-S-transferase Heat shock protein Hsp70 Heat shock protein 70

15 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone xiii HMW HPLC Ig-HRP IPTG K D kda LB LDL LDLR LRP lys M mg μg ml μl mm μm NGE OSB OVO PBL PBS PCDs PI pi High molecular weight High pressure liquid chromatography Immunoglobulin conjugated to horse radish peroxidase Isopropyl-1-thio-β-D-galactopyranoside Constant of dissociation Kilo Dalton Luria Bertani Low density lipoprotein Low density lipoprotein receptor Low density lipoprotein receptor-related protein Lysozyme Molar (moles/litre) Milligram (1 x 10-3 grams) Microgram (1 x 10-6 grams) Millilitre (1 x 10-3 litres) Microlitre (1 x 10-6 litres) Millimolar (1 x 10-3 moles/litre) Micromolar (1 x 10-6 moles/litres) Native agarose gel electrophoresis Oxidative stress buffer Ovotransferrin Peripheral blood Leukocytes Phosphate buffered saline Protein conformational disorders Propidium iodide Isoelectric point

16 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone xiv PMSF RAP SA SaRIg-FITC SD SDS-PAGE SEC shsp TAE TEMED TRIS Phenylmethylsulphonylfluoride Receptor-associated protein Streptavidin Sheep-anti-rabbit-immunoglobulin conjugated to FITC Standard deviation Sodium dodecyl sulfate polyacrylamide gel electrophoresis Size exclusion chromatography Small heat shock protein Tris-acetate-EDTA N, N, N, N- tetramethyl-ethylenediamine Tri (Hydroxymethyl) aminomethane

17 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone xv LIST OF FIGURES Figure 1.1 The mechanism of protein folding... 3 Figure 1.2 Pathways of protein aggregation... 6 Figure 1.3 Intracellular rotein quality control mechanisms... 9 Figure 1.4 The structure of alpha-2-macroglobulin Figure 1.5 Steric trapping of protease molecules by α 2 M Figure 1.6 Locations of binding sites within the α 2 M subunit Figure 1.7 Receptors mediate binding, internalisation an cell signalling of α 2 M Figure 3.1 Native PAGE showing conformation of α 2 M within complexes Figure 3.2 Trypsin binding ability of α 2 M within α 2 M/stressed protein complexes47 Figure 3.3 Activation abolishes the chaperone activity of α 2 M Figure 3.4 Native PAGE showing activation of α 2 M within α 2 M/CS complexes Figure 3.5 α 2 M/stressed proteins can trap trypsin Figure 3.6 SDS PAGE showing the effects of trypsin on α 2 M and α 2 M/CS complexes Figure 3.7 Depletion of α 2 M from normal human plasma Figure 3.8 α 2 M inhibits heat stress-induced precipitation in whole human plasma 54 Figure 3.9 α 2 M inhibits precipitation in whole human plasma at 37 C Figure 3.10 SDS PAGE identifing putative endogenous substrates for α 2 M Figure 3.11 Mass spectra of trypsin digested endogenous substrates of α 2 M Figure 3.12 Fibrinogen is an endogenous substrate of α 2 M under heat stress Figure 4.1 Binding of α 2 M and α 2 M/stressed protein complexes to JEG-3 cells Figure 4.2 Inhibition of JEG-3 cell binding using RAP and anti-lrp antibody Figure 4.3 Binding of α 2 M and α 2 M/stressed protein complexes to Hep-G2 cells 67 Figure 4.4 Inhibition of Hep-G2 cell binding using RAP and galactose Figure 4.5 Binding of α 2 M and α 2 M/stressed protein complexes to U937 cells Figure 4.6 Inhibition of U937 cell binding using RAP and asialofetuin Figure 4.7 Detection of LDLR family members on granulocytes Figure 4.8 Binding of α 2 M and α 2 M/stressed protein complexes to granulocytes. 72 Figure 4.9 Inhibition of granulocyte cell binding using RAP Figure 5.1 SDS PAGE showing fragmentation of α 2 M under oxidative stress Figure 5.2 SEC of native and oxidised α 2 M Figure 5.3 NGE of α 2 M under oxidative stress... 81

18 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone xvi Figure 5.4 The effect of α 2 M on oxidation-induced precipitation of lysozyme Figure 5.5 The effect of SOD and BSA on lys precipitation Figure 5.6 Detection of putative α 2 M/lys complexes using NGE Figure 5.7 SDS PAGE showing α 2 M/lys complexes Figure 5.8 Effects of complex formation on the trypsin binding activity of α 2 M.. 87 Figure 5.9 α 2 M inhibits oxidation-induced precipitation in whole human serum Figure 5.10 The effect of α 2 M on the oxidation induced precipitation of lys Figure 5.11 Effects of pre-oxidised α 2 M and α 2 M* on protein precipitation Figure 5.12 Effect of oxidation on the protease inhibitor function of α 2 M Figure 5.13 Proposed structural and functional changes to oxidised α 2 M Figure 6.1 Proposed model for the chaperone action of α 2 M LIST OF TABLES Table 1.1 Examples of protein conformational disorders (PCDs)... 4 Table 3.1 Characteristics of substrate proteins used to investigate the chaperone properties of α 2 M Table 4.1 Characteristics of the cell lines used in the study... 62

19 CHAPTER 1: INTRODUCTION 1 CHAPTER 1: INTRODUCTION 1.1 Protein Folding Despite the diversity of proteins they all share one essential property, rapid folding into a unique three dimensional structure Proteins which have successfully folded into their correct, native conformation have long term stability in the crowded environment of the cell and thus the potential to interact selectively with the environment (Dobson, 2003). Although the acquisition of a folded state is often required for optimal biological activity, it should be noted that intrinsically unfolded proteins also have a significant role in cell function and importantly are implicated in several disease processes (Tompa, 2002; Chiti et al., 2003). The process of protein folding must occur within a limited and biologically feasible timescale, excluding the possibility of a trial and error based mechanism to achieve a natively folded protein (Jahn and Radford, 2007). Native-like interactions between amino acid residues are more stable than non-native contacts the persistence of the former acts to direct the polypeptide towards its lowest-energy structure (Dobson, 2003). Folding in small polypeptides ( residues) occurs in two simple state transitions involving cooperative native interactions. However, most larger proteins (greater then 100 residues) fold via three state transitions involving a populated intermediate state or molten globule with exposed hydrophobic domains (Radford, 2000). The multi-step process involved in the folding of large polypeptides has been implicated in both productive folding (the on pathway ) and in the non-specific collapse and accumulation of proteins with non-native interactions (the off pathway ) (Brockwell, 2000). The energetics of protein folding can be described by a multidimensional energy landscape or folding funnel that represents the energy of interaction between protein

20 CHAPTER 1: INTRODUCTION 2 atoms as a function of their positions and highlights the numerous routes that ultimately lead to a native folded protein (Figure 1a) (Dinner et al., 2000; Radford, 2000; Dobson, 2001; Dobson, 2003). The funnel represents a conceptual mechanism for understanding the self organization of a protein; a progressive collection of geometrically similar collapsed structures, with native-like contacts found to be more thermodynamically stable than the incorrect, non-native interactions (Dobson and Karplus, 1999; Ellis and Hartl, 1999; Schultz, 2000; Dobson, 2001; Slavotinek and Biesecker, 2001). Although the folding funnel provides visual representation of the protein folding process, the overall mechanism is likely to be further complicated within the living cell where high cellular concentrations and resulting intermolecular collisions and interactions will greatly affect the thermodynamic landscape (Jahn and Radford, 2007). Additional off pathway folding is represented in Figure 1b and is likely to represent a more physiologically relevant depiction of protein folding kinetics. At the top of the funnel the protein exists in a large number of random, unfolded states with high enthalpy and entropy. As the proteins collapse and reconfigure they progress into lower energy conformations, ultimately forming the most thermodynamically favourable state. A deviation off the funnel results in a non-native protein, which fails to reach the optimal, energetically favourable state and is responsible for numerous complications within the cell. Those protein species with structures most distant from the native structure have the least ordered and therefore the most highly dynamic conformations (Jahn and Radford, 2007).

21 CHAPTER 1: INTRODUCTION 3 Entropy Figure 1.1 The mechanism of protein folding. a) Schematic diagram of the folding energy landscape of a protein molecule. The unfolded protein located at the top of the funnel has numerous possible conformations and may take several different routes to reach the native state, many of which contain transient folding intermediates. Some of these intermediates retain a more stable structure such as the molten globule, whereas others become involved in folding traps, where the protein is irreversibly captured in a misfolded state. From (Radford, 2000). b) Illustration of a combined energy landscape for protein folding and aggregation. The diagram shows the undulating level of the protein energy landscape. The simple funnel shown in a) is indicated by the light grey; intermolecular protein association dramatically increases the ruggedness of the landscape (dark grey). From (Jahn and Radford, 2007). 1.2 Protein Misfolding, Aggregation and Disease As described in section 1.1, not all proteins go on to adopt their ideal, native conformation. Small proteins which become unfolded may rectify the problem and convert to the correct fold almost instantaneously (milliseconds) (Hartl and Hayer-Hartl, 2002), however in larger, multi-domain proteins, refolding is not as efficient. Misfolding of the polypeptide chain occurs when regions normally separated in the native conformation interact to form stable structures. Misfolding of protein is believed to be triggered by several cellular stressors including: mutations, mis-processing, interactions with metal ions, changes in environmental conditions (such as temperature and ph), oxidation (Wickner et al., 1999; Stefani and Dobson, 2003) and molecular crowding (Bross et al., 1999; Barral et al., 2004).

22 CHAPTER 1: INTRODUCTION 4 Non-native proteins inevitably expose hydrophobic residues to the solvent, leading to selfassociation and the formation of disordered aggregates stabilised by hydrophobic interactions and hydrogen bonding (Barral et al., 2004). Partially unfolded or misfolded proteins produced as a result of mutation or stress have been implicated in the formation of toxic pathological aggregates. Aggregated protein may be linked to the malfunctioning of living systems and is responsible for a family of diseases, termed protein conformational disorders (PCDs) (Dobson, 2001). PCDs are characterised by the occurrence of lesions associated with toxic intra- or extracellular accumulations of unfolded, aggregated protein (Muchowski, 2002) (Table 1). Table 1 Examples of protein conformational disorders (PCDs). The aggregation process is initiated by unfolding of the native structure and the formation of a stable, partially collapsed, intermediate state (Dobson, 2001). These intermediate states can give rise to highly ordered, hydrogen bonded, fibrillar structures called amyloid. Amyloid is a general term describing protein aggregates which share common features including: fibrillar morphology, a mostly β-sheet secondary structure, birefringence when stained with Congo red, insolubility in common solvents and detergents, and protease-

23 CHAPTER 1: INTRODUCTION 5 resistance (Murphy, 2002). The amyloid fold consists of continuous β-sheets with β-strands perpendicular to the fibril long axis - the resulting structure is very stable (Guijarro et al., 1998; Jahn and Radford, 2007). While amyloid poses a threat to human health as previously described, it offers great potential in the field of nano-biotechnology where research is exploiting the well-ordered structure, regularity and helical periodicity of amyloid fibrils for use in extracellular matrices to facilitate cell adhesion during the in vitro differentiation of functional tissues (Gras et al., 2008). The mechanism by which β-amyloid peptide (Aβ) exerts its neurodegenerative effects is of great importance to the understanding of Alzheimer s disease and potentially many others. Until recently it was assumed that the toxicity of extracellular, mature amyloid fibrils was responsible for the pathogenic features observable in amyloid disorders, including Alzheimer s disease (Dobson, 2001). However, due to the poor correlation between the location of β-amyloid plaques and sites of neurodegenerative damage, it is now generally accepted that the early pre-fibrillar aggregates of proteins associated with neurodegenerative disorders are in fact highly toxic and damaging to cells, whereas mature amyloid fibrils are relatively benign (Muchowski, 2002; Walsh et al., 2002; Dobson, 2003) (Figure 1.2).

24 CHAPTER 1: INTRODUCTION 6 Figure 1.2 Pathways of protein aggregation. From its synthesis on the ribosome, the protein may progress to its native fold via transition through intermediate states. Native proteins (N) may become unfolded (nonnative; NN), which may progress to an unfolded conformation (U) via intermediate species (I 1, I 2 ). Nonnative proteins have an increased likelihood of association and aggregation into oligomeric intermediates (which are the toxic precursors of amyloid fibrils), amorphous aggregates and fibres, the latter two exhibiting limited solubility. Native proteins are less likely to form fibres but do however have the ability to do so. From (Yerbury et al., 2005). 1.3 Mechanisms of Protein Quality Control In order to regulate the level of aberrantly folded protein, the cell has developed several mechanisms which act to distinguish between native and non-native conformations, facilitating degradation or the refolding of non-native polypeptides. Intracellular posttranslational quality control systems are found in prokaryotes and eukaryotes, and both involve proteases and molecular chaperones which patrol the cell for non-native protein species. Overall there are three possible fates for a misfolded protein; degradation, refolding or aggregation. The function of the quality control system is to minimise the occurrence of toxic protein aggregates in an attempt to reduce the onset of disease thus

25 CHAPTER 1: INTRODUCTION 7 maintaining optimal biological functioning and efficiency (Hartl and Hayer-Hartl, 2002). The ubiquitin-proteasome system plays a role in the degradation of unfolded and misfolded proteins, inhibiting their potential to form toxic aggregates. In this elaborate system the unfolded protein is tagged by several ubiquitin molecules which serve to deliver the potentially hazardous protein to the proteasome for subsequent degradation and recycling (Berke and Paulson, 2003). Intracellular misfolded proteins may also be degraded by hydrolytic enzymes contained within the lysosome (Kopito, 2000) Molecular Chaperones - The Saviours of Protein Folding Molecular chaperones play a vital role in the process of protein folding. Not only do they function as catalysts, assisting in the de novo adoption of stable, native conformations, they also act as protein saviours, detecting non-native conformations, and assisting in subsequent refolding or acting to prevent the formation of aggregates (Wickner et al., 1999; Barral et al., 2004). Chaperones are a diverse group of proteins with the ability to detect and bind to exposed hydrophobic regions of non-native proteins using stable, non-covalent interactions (Yerbury et al., 2005). Molecular chaperones may be divided into two categories: folding helper or holding type, based on their role in the protein quality control system (Bross et al., 1999). Folding helper molecular chaperones (Hsp70, chaperonins) assist in the correct folding of nascent chains during translation and prevent the aggregation of newly synthesised chains during protein biosynthesis (Braig, 1998; Barral et al., 2004). Many folding helper chaperones use an ATP-regulated cycle of substrate binding and release; however, a small number use an ATP-independent mechanism (e.g. calnexin and calreticulin) (Hartl and Hayer-Hartl, 2002). Holding type chaperones (e.g. small heat shock proteins (shsps), α-crystallin), on the other hand, bind (ATP-independently) to hydrophobic regions of partially folded or misfolded proteins, protecting them from

26 CHAPTER 1: INTRODUCTION 8 aggregation. Holding-type chaperones facilitate the stabilisation of non-native proteins until the arrival of folding helper chaperones (Barral et al., 2004). Under normal conditions, molecular chaperones effectively deal with aberrantly folded proteins, minimizing the threat of protein aggregation. However ideal conditions are not always available. Situations such as heat stress and chemical damage involving reactive oxygen species (ROS) favour the generation of unfolded and mutated proteins which places an increased strain on the quality control system. Synthesis of chaperones and proteases is induced as part of the heat shock response, up-regulated by signalling from the accumulation of unfolded proteins, thus maintaining the conditions necessary for the regulation of protein folding (Yura and Nakahigashi, 1999). When the balance between protein unfolding and quality control is disturbed by either the mutational loss of chaperones or reduced levels of proteases, insoluble protein aggregates result, exacerbating the sensitivity of the cell to heat shock and other stresses (Wickner et al., 1999). Neurodegenerative conditions related to protein misfolding tend to develop later in life when the imbalance between cellular chaperone capacity and the pool of misfolded protein species significantly increases (Sherman and Goldberg, 2001; Barral et al., 2004). An overview of the intracellular protein quality control system is presented in Figure 1.3.

27 CHAPTER 1: INTRODUCTION 9 Figure 1.3 Overview of the intracellular protein quality control mechanisms. Chaperones function to assist in protein folding within the endoplasmic reticulum (ER) and bind to non-native (NN) proteins in the cytosol to facilitate refolding, lysosomal degradation or ubiquitination and proteosomal degradation. Persistance of the non-native conformation within the ER can result in proteolytic degradation, retrotranslocation to the cytosol or transportation to the lysosome for degradation. In the absence of quality control mechanisms NN protein may form insoluble aggregates in the cytosol or within the ER. From (Yerbury et al., 2005). 1.4 Extracellular Chaperones Extracellular fluid has a protein concentration of 7% (w/v) which is substantially lower than that of the intracellular environment (30 %, w/v) (Costanzo, 2006). Unlike intracellular fluid, the extracellular fluid is constantly subjected to shear stress, particularly from blood turbulence - which is known to induce protein unfolding and inflammation (Kerr and Chen, 1998; Yerbury et al., 2005). Given the high level of stress in the extracellular space it is no surprise that many toxic aggregates responsible for PCD s are

28 CHAPTER 1: INTRODUCTION 10 located outside of the cell (Table 1.1). Unlike the well-characterised protein quality control mechanisms of the intracellular space, analogous mechanisms in the extracellular environment are yet to be characterised. Although intracellular molecular chaperones such as Hsp60, Hsp70 and Hsp90 have been attributed extracellular roles in processes such as antigen presentation and immune stimulation (Milani et al., 2002), they are present in plasma only at ng/ml levels (Yerbury et al., 2005). Thus, they are unlikely to have a major role in dealing with bulk extracellular misfolded proteins. However, recent studies have identified several abundant plasma proteins as having chaperone-like properties: clusterin, haptoglobin, serum amyloid P component and most recently alpha-2-macroglobulin. Each of these proteins can bind to misfolded protein substrates in vitro to protect them from further interaction and aggregation (Coker et al., 2000; Poon et al., 2000; Yerbury et al., 2005; French et al., 2008) Clusterin Clusterin is an abundant extracellular protein present at concentrations ranging from μg/ml in human serum and mg/ml in seminal fluid (O'Bryan et al., 1990). Structurally, clusterin is a kda disulfide-linked heterodimeric protein with 30% of its mass comprised of N-linked carbohydrate (Jenne and Tschopp, 1992). Clusterin is highly conserved, maintaining a 70-80% homology between mammalian species (Jenne and Tschopp, 1992). There have been numerous postulated roles for clusterin including the regulation of apoptosis (Buttyan et al., 1989), protection from complement attack (Jenne and Tschopp, 1989), lipid transportation (de Silva et al., 1990) and membrane remodeling (Fritz and Murphy, 1993), however none of these suggestions has been confirmed as a genuine physiological function. The increased expression of clusterin in times of

29 CHAPTER 1: INTRODUCTION 11 pathological stress and disease including Alzheimer s disease (Jenne and Tschopp, 1992), suggests that clusterin may be a stress-response protein. The ability of clusterin to protect proteins from heat-induced precipitation via the formation of high molecular weight complexes indicates its ability to function in an analogous manner to the intracellular group of shsps (Humphreys et al., 1999). As introduced in the previous section, shsps are a unique group of chaperone molecules with the ability to protect cells from heat stress by forming stable complexes with the exposed hydrophobic regions of non-native protein molecules (Welsh and Gaestel, 1998). Clusterin functions as a holding type chaperone it binds to non-native proteins, holding them in a stable, soluble complex which, in vitro, can be acted on by Hsp70 to refold the protein (Poon et al., 2002). Clusterin is currently the best characterised extracellular chaperone Haptoglobin Haptoglobin (Hp) is an acute-phase, acidic glycoprotein produced by the liver and is present in most human body fluids including serum, bile, synovial fluid, cerebrospinal fluid and milk (Dobryszycka, 1997). In humans, there are three major phenotypic forms of Hp (Hp1-1, Hp2-1 and Hp2-2); each individual expresses only one of these (Bowman and Kurosky, 1982). The main physiological function of Hp is the binding and clearance of vascular haemoglobin (released following damage to red blood cells), to which Hp binds with high affinity (K D ~ M) (Bowman and Kurosky, 1982). Other functions ascribed to haptoglobin include an anti-inflammatory action mediated by binding to haemoglobin (preventing oxidative damage) (Melamed-Frank et al., 2001), immune regulation (Louagie et al., 1993) and pro-angiogenic effects (Cid et al., 1993). Hp is abundant in human plasma at a concentration of mg/ml, with the basal concentration increased as part of the

30 CHAPTER 1: INTRODUCTION 12 acute phase stress response (Bowman and Kurosky, 1982), and during numerous disease states (Dobryszycka, 1997). Recent findings have also indicated that like clusterin, haptoglobin potently inhibits stress-induced protein aggregation (Yerbury et al., 2005). Hp carries out this function via an ATP-independent binding mechanism which acts to stabilise unfolded proteins in a high molecular weight complex (Yerbury et al., 2005) Serum Amyloid P Component (SAP) SAP is a non-fibrillar plasma glycoprotein, structurally comprised of an oligomer of five identical subunits, non-covalently associated in a disc-like particle (Coker et al., 2000). SAP has been shown to bind to and stabilise all types of amyloid fibrils, however it does not bind to the same proteins when they are in their native conformation (Tennent et al., 1995). The inherent ability of SAP to recognise misfolded proteins suggests that it may indeed have a molecular chaperone action. Unlike clusterin and haptoglobin, SAP has the ability to refold non-native proteins by interacting with intermediates on the refolding pathway, increasing the passage through productive, native protein forming routes (Coker et al., 2000). However, previous in vitro studies demonstrating SAP mediated protein refolding have required at least a ten-fold molar excess of SAP to enzyme substrate. Therefore, the in vivo relevance of findings produced in such a non-physiological environment is very questionable Alpha-2-Macroglobulin (α 2 M) The discovery of chaperone-like proteins in the extracellular space is a major step toward determining the fate of extracellular unfolded/misfolded proteins. Recently, attention has turned to the abundant plasma protein, alpha-2-macroglobulin (α 2 M), which was found to

31 CHAPTER 1: INTRODUCTION 13 exhibit chaperone-like characteristics (French, 2005; French et al., 2008). Unlike the previously identified extracellular chaperones, α 2 M has numerous well described functions and established physiological roles, most importantly as a protease inhibitor. The ability of α 2 M to bind to many diverse ligands, inhibit Aβ aggregation, and influence the immune response to peptides prompted investigation into the possibility that it might be a novel member of a small group of extracellular chaperones that have been proposed as major elements of a quality control system for the folding state of proteins in extracellular body fluids (Yerbury et al., 2005). There are notable similarities between α 2 M and the previously identified extracellular chaperones clusterin (Wilson and Easterbrook-Smith, 2000) (1.4.1) and haptoglobin (1.4.2) (Yerbury et al., 2005). α 2 M, clusterin and haptoglobin are all secreted glycoproteins with distant evolutionary relationships to complement (Bowman and Kurosky, 1982; Kirszbaum et al., 1989; Dodds and Law, 1998). In addition, all three are: (i) structurally comprised of disulfide linked subunits (Bowman and Kurosky, 1982; Jensen and Sottrup-Jensen, 1986; Wilson and Easterbrook-Smith, 2000), (ii) abundant in human plasma (α 2 M 2-4 mg/ml (Sottrup-Jensen, 1989), clusterin µg/ml (O'Bryan et al., 1990) and haptoglobin mg/ml (Bowman and Kurosky, 1982), (iii) mediate ligand degradation by receptor mediated endocytosis (Ashcom et al., 1990; Hammad et al., 1997; Kristiansen et al., 2001), and (iv) are known to co-localise with Aβ deposits in Alzheimer s disease (Powers et al., 1981; Calero et al., 2000; Fabrizi et al., 2001).

32 CHAPTER 1: INTRODUCTION 14 Recent work showed that α 2 M does indeed have chaperone activity in vitro. It binds to a broad range of partly unfolded stressed proteins to form soluble, stable complexes and inhibit their aggregation and precipitation (French, 2005). Preliminary evidence suggests that α 2 M remains in its native conformation when bound to stressed proteins in this state it retains its protease inhibitor function and lacks the ability to bind to cell surface expressed low density lipoprotein receptor related protein (LRP) (French, 2005). It is yet to be determined how the function of α 2 M as a chaperone impacts upon its activity as a protease inhibitor, and whether there is any synergy between the two roles. The remainder of this thesis will focus on α 2 M, further exploring its role as an extracellular chaperone, and investigating the relationship between this activity and its protease inhibitor function. How these two potentially complementary activities might fit into a system for the control of extracellular protein folding will also be considered. 1.5 Alpha-2-Macroglobulin Synthesis, Structure and Protease Inhibitor Action of α 2 M α 2 M is the major representative of the α-macroglobulin group of plasma proteins which also contains pregnancy zone protein (PZP) and the complement components C3 and C4 (Sottrup-Jensen, 1989; Borth, 1992). α 2 M is best known for its protease inhibitor function - it has the ability to bind to and inhibit a wide range of plasma proteases including cathepsin, plasmin, thrombin and trypsin (Borth, 1992). α 2 M is encoded by a single copy gene, found within a cluster comprising an authentic α 2 M gene, an α 2 M pseudogene and the PZP gene on the human chromosome 12p (Matthijs et al., 1992). α 2 M is synthesised by several cell types including lung fibroblasts, monocytes-macrophages, hepatocytes and

33 CHAPTER 1: INTRODUCTION 15 astrocytes (Sottrup-Jensen, 1989). Human neuronal cells may also be stimulated to produce α 2 M by the cytokine interleukin-6, which is likely to contribute to the development of diseases targeting the central nervous system (Strauss et al., 1992). Some tumour lines such as melanoma cells are also capable of producing significant amounts of α 2 M in vitro and in vivo (Bizik et al., 1989). α 2 M is a major human blood glycoprotein comprised of ~ 10% carbohydrate by mass (Borth, 1992). In humans, α 2 M is assembled from four identical 180 kda subunits into a 720 kda tetramer; the 180 kda subunits are covalently linked by two disulphide bonds into dimers, which non-covalently interact to yield the final tetrameric quaternary structure which encloses a central cavity (Jensen and Sottrup-Jensen, 1986) (Figure 1.4). Figure 1.4 The structure of alpha-2-macroglobulin. (A) Native α 2 M is formed by the non-covalent interaction of two identical dimers (green and red) (B) Structure of the two subunits of dimeric α 2 M. The two polypeptide chains are cross-linked by two disulfide bonds in an anti-parallel manner near the centre of the dimer. The bait domains and thiol ester moieties are found near the central cavity, while the receptor binding domain (RBD) is located in the C-terminal region of the subunits (Kolodziej et al., 2002).

34 CHAPTER 1: INTRODUCTION 16 α 2 M subunits contain three major regions that are essential for efficient functioning: the bait region, the internal thiol-ester bond and the receptor recognition site (Van Leuven et al., 1986). Within each of the four subunits, the bait region is found as an exposed stretch of 25 amino acids that contains several cleavage sites for a variety of proteases that originate from the host or from foreign sources (Figure 1.4 B) (Barratt, 1981; Sottrup- Jensen et al., 1984; Borth, 1992). When exposed to a protease, α 2 M undergoes limited proteolysis at its bait region which leads to a large conformational change, physically trapping the protease within a steric "cage" (Sottrup-Jensen, 1989) (Figure 1.5). The trapped protease forms a covalent linkage with α 2 M by reacting with an intramolecular thiol-ester bond to yield activated α 2 M (α 2 M*), which exposes a receptor recognition site for LRP; in vivo, the α 2 M*/protease complex is cleared by LRP-mediated endocytosis and subsequently degraded (Sottrup- Jensen, 1989). The trapped protease retains % of its hydrolytic activity against low molecular weight substrates (Travis and Salvesen, 1983). Therefore, the trapping mechanism used by α 2 M differs from other protease inhibitors in that the enzyme binding site remains unblocked, thus allowing the retention of its activity (Borth, 1992). However, since only molecules less than 10 kda are able to freely diffuse in and out of the closed trap, the trapped enzyme is unable to react with protease substrates greater than 10 kda in mass (Barratt, 1981; Travis and Salvesen, 1983).

35 CHAPTER 1: INTRODUCTION 17 The cleavage of the internal thiol-ester bond and the subsequent conformational change is manifested by a shift in the electrophoretic mobility of α 2 M in native polyacrylamide gel electrophoresis (Barratt et al., 1979; Barratt, 1981). The circulating form of α 2 M which is reactive with proteases runs slow on the gel, while the activated form of α 2 M (produced irreversibly from the slow form by reaction with proteases) is more compact and hence has greater mobility (Barratt et al., 1979; Barratt, 1981). The cleavage of the bait region by proteases that results in the subsequent cleavage of the thiol-ester bond can be by-passed in vitro by the use of amines which directly cleave the bond by nucleophilic attack (Borth, 1992). Exposure of native α 2 M to methylamine at alkaline ph results in the covalent incorporation of 4 molecules of methylamine per α 2 M tetramer, inducing a conformational change identical to that which occurs during interaction with proteases (Imber and Pizzo, 1981).

36 CHAPTER 1: INTRODUCTION Other Functions of α 2 M Aside from its role as a protease inhibitor, α 2 M has been shown to bind to and promote clearance of other endogenous and exogenous molecules, consistent with a broader protective function. α 2 M is known to bind to cytokines and growth factors (without converting to α 2 M*), including transforming growth factor-β (TGF-β), tumour necrosis factor-α (TNF-α), interleukin 1β (IL-1β), interleukin 8 (IL-8), platelet-derived growth factor-bb (PDGF-BB), nerve growth factor-β (NGF-β), and vascular endothelial growth factor (VEGF) (reviewed in (LaMarre et al., 1991; Feige et al., 1996)). α 2 M has also been shown to interact with endogenous disease-associated proteins, including the Aβ peptide associated with Alzheimer s disease (Narita et al., 1997), β 2 -microglobulin which forms insoluble deposits in dialysis related amyloidosis (Motomiya et al., 2003), and prion protein which is associated with plaques in Creutzfeldt-Jakob disease (Adler and Kryukov, 2007). Interestingly, α 2 M has been shown to suppress the aggregation of Aβ and protect cells from its toxicity (Du et al., 1997; Hughes et al., 1998; Fabrizi et al., 1999). Recent work has indicated that α 2 M-peptide complexes are immunogenic (Binder et al., 2001; Binder et al., 2002; Binder, 2004). α 2 M bound peptides are internalised by LRP (also known as the α 2 M receptor and CD91) and fragments of the peptide are subsequently represented on the cell surface. This response is identical to the one elicited by peptides chaperoned by intracellular heat shock proteins (Srivastava, 2002) Binding of α 2 M to Ligands As described in section 1.5.2, α 2 M can form complexes with a wide range of molecules. Many studies have attempted to elucidate the binding mechanisms used by α 2 M in its interactions with various ligands. Historically, α 2 M is known to have three distinct mechanisms of ligand binding (i) the steric trapping reaction specific for proteases

37 CHAPTER 1: INTRODUCTION 19 (discussed in section 1.5.1), (ii) covalent linking of proteases or other proteins present during the trapping reaction, and (iii) an adherence reaction based on ionic and hydrophobic interactions with basic proteins (Barratt, 1981; Webb and Gonias, 1997; Mathew et al., 2003) The linking reaction occurs at the thiol ester bond and involves the covalent and irreversible binding of ligands. The thiol ester is stable in native α 2 M, but is revealed as a highly reactive group following proteolytic cleavage (Travis and Salvesen, 1983). If proteases or other proteins are in the immediate vicinity at the time of proteolytic activation, they can become linked or covalently co-trapped, and compete to bind to thiol-esters (LaMarre et al., 1991; Borth, 1992; Gron and Pizzo, 1998). Many growth factors use this method of attachment, binding covalently via thiol esters, as do other nonproteolytic proteins when then go on to facilitate antigenic presentation by macrophages to T-cells (Chu and Pizzo, 1993). As discussed previously, α 2 M can be converted to α 2 M* by reaction with small nucleophiles which directly attack the thiolester bond, omitting the proteolytic step (Howard et al., 1980). It has been found that cleavage of the thiol ester bonds within the α 2 M molecule is reversible, with the reverse reaction having two intermediate states between α 2 M* (the receptor recognised form) and native α 2 M (with 4 intact thiol ester bonds) (Grøn et al., 1996). In addition to the structural reformation, the reversed α 2 M also regains its protease inhibitor function. Proteins can also be covalently incorporated into synthetically activated, nucleophile treated α 2 M, removing the need for protease activation (Gron and Pizzo, 1998). The ability to covalently link a wide range of molecules to α 2 M* using a non-proteolytic method of activation, raised the possibility of using α 2 M as a novel drug delivery system. Preliminary

38 CHAPTER 1: INTRODUCTION 20 investigations have successfully incorporated nucleosides and nucleobases into α 2 M*(Bond et al., 2007). Synthetic nucleosides such as guanosine have immuno-modulatory and immuno-stimulatory properties and when incorporated into α 2 M* have the potential to provide anti-tumour and anti-viral activity through the stimulation of cytokines (Nagahara et al., 1990; Lee et al., 2003; Bond et al., 2007). The adherence reaction is quite distinct from the trapping and linking mechanisms and is likely to be controlled by different parts of the α 2 M molecule. In this mechanism, native α 2 M behaves as if it is sticky, mediating the binding of various proteins and cationic molecules such as aspartate aminotransferase and myelin basic protein (Barratt, 1981; Travis and Salvesen, 1983). Unlike other binding interactions, the adherence reaction does not lead to a conformation change in the α 2 M molecule, or the exposure of the receptor recognition site. However, the binding of ligands via the adherence reaction does not prevent protease trapping and subsequent activation (Barratt, 1981; Travis and Salvesen, 1983; Gron and Pizzo, 1998). Platelet derived growth factor (PGDF) binds to α 2 M near the centre of the molecule partly via non-covalent, adherence-type mechanisms, and may be subsequently released by incubation at low ph (4.0) (Bonner et al., 1992). The ability of α 2 M to hold PDGF in a reversible, non-covalent bond has led to suggestions that α 2 M plays a major role in the transport of growth factors in the circulation and furthermore as a positive or negative regulator of growth factor activity (Bonner et al., 1992). The precise binding site of growth factors on the α 2 M subunit has recently been identified by Gonias and associates. The growth factor binding site is contained within amino acids near the C-terminal flank of the bait region of a mature α 2 M subunit. The binding site is a 16-amino acid sequence, composed mainly of hydrophobic amino acids with two

39 CHAPTER 1: INTRODUCTION 21 potentially important acidic residues (Webb et al., 2000). TGF-β, platelet-derived growth factor-bb (PDGF-BB), and NGF-β all interact with the growth factor-binding site in α 2 M (Gonias et al., 2000; Webb et al., 2000). In addition to the growth factor binding site, a specific binding site for Aβ has also been identified (Mettenburg et al., 2002). Aβ binds to a single sequence located near the C terminus of the α 2 M subunit, a sequence entirely distinct from the growth factor-binding site (Mettenburg et al., 2002). Identification of precise binding sites for growth factors and Aβ, in addition to determining the exact site of LRP recognition (see section 1.5.4), has further expanded the knowledge of α 2 M binding mechanisms. Thus, in addition to the bait region, there is now evidence to suggest that the α 2 M subunit has at least three distinct "protein interaction sites" with distinct binding specificities that mediate interactions with growth factors, Aβ and LRP (Mettenburg et al., 2002). Figure 1.6 indicates the positions of the known binding sites within an α 2 M subunit. Figure 1.6 Locations of binding sites within the α 2 M subunit. The precise location of the binding sites for growth factors, amyloid-beta (Aβ) and the low-density lipoprotein receptor related protein (LRP) are indicated in relation to the bait domain, thiol ester bond receptor binding fragment (RBF). The figure represents a 180 kda α 2 M subunit, numbers on the diagram indicate the position of binding sites in the amino acid sequence of the protein. Modified from (Mettenburg et al., 2002).

40 CHAPTER 1: INTRODUCTION Receptor Binding and Internalisation of α 2 M The receptor binding site is comprised of a COOH-terminal 138 residue domain within each of the four α 2 M subunits. Each individual binding site may be exposed following interaction with a protease and consequent cleavage of the thiol ester bond (Van Leuven et al., 1986; Sottrup-Jensen, 1989). Two lysine residues (1370 and 1374) within the receptor binding domain of each α 2 M subunit have been identified as the specific binding sites for LRP (Nielsen et al., 1996). α 2 M The conformational change and subsequent availability of the receptor binding domain allows activated α 2 M to bind to LRP and facilitates the cell uptake of a variety of ligands (Moestrup and Gliemann, 1991; Binder et al., 2001). As described in section 1.5.2, proteases, cytokines and growth factors such as interleukin 1β, interleukin 6, transforming factor-β (TGFβ), β amyloid peptide (Aβ) (Fabrizi et al., 1999) and fibroblast growth factor (James, 1990) all bind to α 2 M. Interestingly, some polypeptides have been shown to bind significantly to native α 2 M (carboxypeptidase B) (Valnickova et al., 1996), to both native and activated α 2 M (TGFβ) (Crookston et al., 1994), or to only α 2 M* (growth hormone) (Kratzsch et al., 1995). This suggests that the species of α 2 M to which the ligands bind will depend largely on whether the complex is intended to be stable in the circulation (native) or destined for degradation (activated) (Phillips et al., 1997). Furthermore, the tendency of proteins such as carboxypeptidase B to bind to the form of α 2 M with the longest plasma half-life, without affecting its catalytic activity, may represent a mechanism important to prolong the availability of the active enzyme for processes such as fibrinolysis and coagulation (Valnickova et al., 1996). In vivo clearance studies in mice demonstrate that α 2 M* and α 2 M*/ligand complexes are cleared in less than 10 minutes, with greater than 90% of clearance mediated by hepatocytes and Kupffer cells of the liver (Imber and Pizzo, 1981; Feldman et al., 1983). In

41 CHAPTER 1: INTRODUCTION 23 this process, specific cell surface receptors act to internalise complexes and dispatch them into endocytic and lysosomal pathways (Borth, 1992). α 2 M is known to bind to at least two different cell-surface receptors, the α 2 M receptor (α 2 MR, also known as LRP) and Grp 78 (the α 2 M signalling receptor). The most well characterised is LRP, (Moestrup and Gliemann, 1991; Williams et al., 1992; Hussain et al., 1999) which is an endocytic membrane-bound receptor that belongs to the low density lipoprotein receptor (LDLR) family (Kristensen et al., 1990). The LDLR family consists of cell-surface receptors able to recognise extracellular ligands and facilitate their cellular uptake and subsequent degradation by lysosomes (Hussain et al., 1999). Members of this family include the prototype low density lipoprotein receptor (LDL-R), very low density lipoprotein receptor (VLDLR), megalin and the LDL-R related protein (LRP). LRP is a hetero-dimeric protein with non-covalently associated 515 and 85 kda subunits (Borth, 1992; Hussain et al., 1999). Receptor-associated protein (RAP), a 39 kda glycoprotein, acts intracellularly as a chaperone within the ER and Golgi to regulate the binding of ligands to LRP (Bu and Schwartz, 1998; Li et al., 1998). RAP also provides assistance in the process of LRP folding (Bu and Schwartz, 1998). LRP contains multiple RAP binding sites which gives RAP the potential to either competitively or sterically hinder the binding of LRP ligands such as α 2 M (Horn et al., 1997). α 2 MR/LRP is multifunctional, it is able to bind ligands from an array of classes including α 2 M-protease complexes (Kristensen et al., 1990), plasminogen activator inhibitorplasminogen activator complexes (Nykjaer et al., 1992), lipoprotein lipase (Beisiegel et al., 1991) and apolipoprotein E (Kowal et al., 1989). The in vitro binding of α 2 M-protease complexes to LRP is suggested to occur when two domains of α 2 M recognise adjacent

42 CHAPTER 1: INTRODUCTION 24 binding sites on LRP (Moestrup and Gliemann, 1991). Following association with LRP, α 2 M-protease complexes may follow various routes including: intracellular sequestration, endocytosis or degradation (Borth, 1992). The common path taken by α 2 M-protease complexes that enter the cell via receptor mediated endocytosis is delivery to the lysosome accompanied by recycling of the LRP receptor back to the cell surface (Brown et al., 1983) (Figure 1.7 A). In addition to the well characterised binding of α 2 M to LRP, the existence of a second α 2 M receptor was first suggested in 1994 by Misra and associates (Misra et al., 1994). It was initially believed that α 2 M*-induced signal transduction was mediated by LRP, however further studies showing the inability to block signalling with saturating concentrations of RAP indicated the existence of a unique signalling receptor (Misra et al., 1994). The second receptor, initially termed the α 2 M* signalling receptor, was later isolated from murine peritoneal macrophages and 1-LN human prostate cancer cells and identified as cell surface-associated glucose-regulated protein 78 (Grp78) (Misra et al., 2002). Interestingly, Grp 78 is a member of the HSP70 superfamily of heat shock proteins, found in the endoplasmic reticulum (Gething, 1999). Grp 78 is now known to be essential for α 2 M*- mediated signal transduction, acting in conjunction with LRP (Misra et al., 2002) (Figure 1.7 B). The existence of this second receptor suggests that α 2 M has additional roles beyond that of a protease inhibitor, including acting as a signalling molecule that triggers cellular responses (Misra et al., 1994).

43 CHAPTER 1: INTRODUCTION 25 Figure 1.7 Receptors mediate the binding, internalisation and cell signalling of α 2 M-protease complexes. (A) Many α 2 M complexes are bound by the α 2 M receptor (LRP). LRP consists of 5 structural units, two of which are bound by subunits of the α 2 M molecule. Following internalisation via clathrin coated pits, α 2 M complexes may be dispatched into various routes of the endocytic/lysosomal pathways. The most common route is delivery to the lysosome and recycling of the receptor back to the surface (Borth, 1992; Mikhailenko et al., 2001). (B) Grp78 is the second identified receptor for α 2 M, which in association with LRP, acts via a G-protein coupled mechanism (G) to initiate an IP 3 /Ca 2+ mediated MAPK (mitogen-activated protein kinase) cell signalling cascade triggered by the signalling element RAS (isolated from RAt Sarcoma) (Adapted from (Misra et al., 2002) The Effects of Oxidative Stress on the Structure, Function and Receptor Recognition of α 2 M One of the hallmarks of the inflammatory response is the marked increase in oxidation and resultant oxidation products (such as free radicals) produced in response to cellular injury and bacterial invasion. Both acute and chronic inflammation is characterised by a high level of oxidants including superoxide anion (O 2- ), hydroxyl radical (OH - ), hydrogen peroxide (H 2 O 2 ) and hypochlorous acid (HOCl) (Martínez-Cayuela, 1995). Neutrophilderived reactive oxygen species (ROS) have an important role in the inflammatory process

44 CHAPTER 1: INTRODUCTION 26 - they act to neutralise bacteria and accelerate tissue destruction either directly by apoptosis and tissue necrosis, or indirectly by altering the protease/protease inhibitor balance. ROS have been detected at levels as high as the millimolar range during an oxidative burst (Weiss, 1989). Free radicals have adverse effects on many important biomolecules including DNA and protein. As a result, oxidants are associated with a variety of pathological disorders, many of which are also linked to errors in protein folding including rheumatoid arthritis and neurological conditions (Stadtman, 1993). The oxidation of α 2 M may contribute to the increased level of proteolysis during the inflammatory process. Hypochlorite may be an oxidant responsible for α 2 M inactivation in vivo, directly contributing to enhanced tissue destruction during inflammation (Wu and Pizzo, 1999). It has also been proposed that the oxidation of α 2 M may act as a physiologically relevant switch mechanism to regulate the binding of cytokines and growth factors to α 2 M (Wu et al., 1998). Very few of the inflammatory response mediators and growth factors, including TNF-α, IL-6, TGF-β and PGDF are found free in circulation. Rather, they exist as complexes with α 2 M (85-90%) (Raines et al., 1984). It is yet to be understood how these mediators are able to respond to cellular injury when the concentration of α 2 M (thus the level of cytokine/growth factorα 2 M complexes) in plasma is high and is substantially increased during inflammation (Wu et al., 1998). α 2 M oxidation results in its increased binding to acute inflammatory mediators (TNF-α, IL-G), thus playing an anti-inflammatory role by inhibiting the progression of the pro-inflammatory cascade. On the other hand, oxidation of α 2 M abolishes its binding to growth factors which are generally considered to be the mediators of cell repair (Wu et al., 1998). Therefore it has been proposed that oxidation of α 2 M

45 CHAPTER 1: INTRODUCTION 27 facilitates the transition from early phase cellular destruction to the remodelling phase of inflammation (Wu et al., 1998). Physiologically relevant concentrations of the oxidants HOCl and hypobromous acid (HOBr) have been shown to cause significant structural changes to the α 2 M molecule (Reddy et al., 1989). These physiological halogenated oxidants are released from activated leukocytes and act to shear the native α 2 M tetramer across its non-covalent axis into covalently linked dimers. The oxidised dimers display normal bait regions, thus retain the ability to undergo thiol ester cleavage, however the oxidised α 2 M molecules are unable to covalently trap or bind proteases (Reddy et al., 1994). The structural changes that occur in the native form upon oxidation are also observed in the activated, protease-bound form of the molecule. Oxidation of α 2 M* abolishes its ability to bind to the receptor LRP, without affecting its ability to bind to the signalling receptor Grp78 (Wu et al., 1997). Furthermore, hypochlorite-mediated oxidation of native α 2 M exposes the receptor recognition site to LRP, to facilitate uptake and degradation, whilst interestingly the signalling function of α 2 M is lost (Wu et al., 1997). The exposure of the LRP binding site of α 2 M upon oxidation is concentration dependant, with as low as 5 μm hypochlorite sufficient for site exposure, with a peak observed at 25 μm (Wu et al., 1997). The exposure of the LRP binding site is believed to result from an oxidation-induced conformational change in α 2 M. Overall, oxidised protease bound α 2 M loses its ability to bind to LRP but still retains its signalling capacity. While conversely, oxidation of native α 2 M results in exposure of the receptor binding site and abolishment of its signalling and protease inhibitor activities. These characteristics indicate that α 2 M is likely to play a significant role in the inflammatory cascade, and highlight several important structural and

46 CHAPTER 1: INTRODUCTION 28 functional variations which are likely to have implications for the chaperone function of α 2 M. α 2 M is able to inhibit oxidation-induced precipitation of substrate proteins in a dose dependent manner (French et al., 2008). The mechanism by which it does this is yet to be determined. The chaperone role of α 2 M is likely to be of greater importance in times of stress and inflammation, when non-native proteins will be more abundant. It was previously hypothesised that α 2 M/stressed protein complexes must subsequently undergo protease activation in order to achieve receptor recognition and binding. However, it is conceivable that oxidation may serve as a convenient method to expose the LRP binding sites of α 2 M within α 2 M/stressed protein complexes, removing the need for prior protease activation. This would remove the need for α 2 M to first interact with a protease to expose LRP binding sites and facilitate the rapid uptake and degradation of the complexes.

47 CHAPTER 1: INTRODUCTION Aims In order to further characterise the chaperone action of α 2 M and to determine how this relates to its known in vivo functions, this study aimed to: (1) Determine the effects of the formation α 2 M/stressed protein complexes on the structure and protease inhibitor function of α 2 M. (2) Identify potential endogenous chaperone substrates for α 2 M in human plasma. (3) Investigate the effects of oxidative stress on the structure of α 2 M and its ability to function as a molecular chaperone. (4) Investigate the interactions of α 2 M/stressed protein complexes with receptors on the surface of cultured cell lines and human peripheral blood cells.

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57 CHAPTER 2: METHODS 30 CHAPTER 2: METHODS 2.1 Materials Bovine serum albumin (BSA), citrate synthase (CS, from porcine heart), 4,4'-dianilino-1,1'- binaphthyl-5,5'-disulfonic acid (bisans), lysozyme (Lys, from chicken egg white), creatine phosphokinase (CPK, from rabbit muscle), superoxide dismutase (SOD, from bovine erythrocytes), trypsin (type 1, bovine), soybean trypsin inhibitor (type 1), N-alpha-benzoyl- DL-arginine-p-nitroaniline (BAPNA), phenylmethylsulfonyl-fluoride (PMSF), propidium iodide (PI) and methylamine hydrochloride were purchased from Sigma (MO, USA). Triton X-100 was from Ajax Chemicals (Sydney, Australia). Glutathione-S-transferase (GST) from Schistosoma japonicum was prepared by thrombin cleavage of recombinant Jun leucine zipper-gst fusion protein and purified by GSH-agarose-affinity chromatography as previously described (Heuer et al., 1996). A plasmid encoding a fusion protein comprised of GST and receptor associated protein (RAP, an inhibitor of ligand binding to low density lipoprotein family receptors) was obtained as a kind gift from Dr Y. Li (Washington University School of Medicine, MO, USA); GST-RAP was purified as described above for GST. CS, CPK and lys were biotinylated using NHS-LC-biotin (Pierce, Sydney, Australia) as per the manufacturer's instructions (the efficiency of labelling is typically > 90% when using a protein concentration of 10 mg/ml). Streptavidin-agarose was purchased from Calbiochem (Sydney, Australia). Rabbit polyclonal anti-α 2 M and anti-dnp antibodies (IgG fractions) were obtained from Dako Cytomation (CA, USA). Horseradish peroxidase and fluorescein conjugates of sheep-anti-rabbit IgG antibody (SaRIgG-HRP and SaRIgG-FITC, respectively) were from Chemicon (Melbourne, Australia).

58 CHAPTER 2: METHODS Purification of α 2 M Native α 2 M was purified from human serum by zinc chelate affinity chromatography using a method modified from (Imber and Pizzo, 1981). Briefly, fresh heparinized human plasma (100 ml, containing 0.2 mm PMSF, 1 M NaCl and 20 mm HEPES, adjusted to ph 7.2) was passed over a 5 ml Zn 2+ HiTrap TM chelate-affinity column equilibrated with binding buffer (0.02 M HEPES, 1 M NaCl, ph 7.2), using an ÄKTAdesign TM Explorer system (GE Healthcare, Australia). Plasma was loaded onto the column and then washed with binding buffer at 5 ml/min to remove unbound proteins. Unwanted Zn 2+ binding proteins were eluted with 20 mm imidazole, 20 mm HEPES, 20 mm Na 2 HPO 4, 0.5 M NaCl, ph 7.4, and α 2 M was subsequently eluted with 500 mm imidazole, 20 mm HEPES, 20 mm Na 2 HPO 4, 0.5 M NaCl, ph 7.4. The eluate was dialysed against phosphate buffered saline (PBS; 137 mm NaCl, 2.7 mm KCl, 1.5 mm KH 2 PO 4, 8 mm Na 2 HPO 4, ph 7.4) containing 0.1% (w/v) NaN 3 (i.e. PBS/Az). Any remaining contaminant protein was removed by size exclusion chromatography using a Biosep -SEC-S4000 column (Phenomenex, Sydney Australia) equilibrated in PBS/Az. The concentration of α 2 M was determined by absorbance at 280 nm (extinction coefficient M -1 ; Hall, 1978). 2.3 Preparation of Activated α 2 M and Activated (α 2 M/CS)* Complexes Native α 2 M was converted to activated α 2 M (α 2 M*) by incubation with either methylamine or trypsin. Native α 2 M (9.7 μm) was incubated overnight at room temperature with 0.15 M methylamine hydrochloride in 0.5 M Tris, ph 8.2. The mixture was subsequently dialysed against PBS to remove excess methylamine. α 2 M-trypsin and α 2 M-trypsin/CS complexes were formed by incubating either native α 2 M (9.7 μm) or native α 2 M/CS complexes (9.7

59 CHAPTER 2: METHODS 32 μm total concentration) in 25 mm Tris, ph 8.0, with a 3-fold molar excess of trypsin at 37 C for 2 h. Any unbound trypsin was inactivated by addition of an equimolar amount of soybean trypsin inhibitor. α 2 M-trypsin and α 2 M-trypsin/CS ((α 2 M/CS)*) complexes were purified by size exclusion chromatography using a Biosep -SEC-S4000 column equilibrated in PBS. Successful transformation of native α 2 M and native α 2 M/CS into the activated species was confirmed using native PAGE (performed on discontinuous 6 % gels using an adaptation of the previously described tris/borate method (Van Leuven et al., 1981)), a trypsin binding assay (section 2.7 (Bonner et al., 1992)) and immunodetection (section 2.6.2). α 2 M* species were stored in PBS, ph 7.4 at -20 C. 2.4 Preparation of Oxidized α 2 M Native α 2 M was oxidized by incubation in oxidative stress buffer (OSB; 100 μm CuSO 4, 4 mm H 2 O 2 in PBS) at 37 C for 2 hours Size Exclusion Chromatography of Oxidised α 2 M In order to observe changes in structure following oxidation, samples of native α 2 M and oxidised α 2 M were subjected to size exclusion chromatography using a Superose 6 column (24 ml bed volume) (GE Healthcare, Australia) with a size exclusion limit of 5,000,000 Da. Samples of native and oxidised α 2 M were prepared at a final concentration of 1 mg/ml and a sample volume of 100 μl was injected onto the column which was equilibrated in PBS/Az and run at 0.5 ml/min using an ÄKTAdesign TM FPLC system (GE Healthcare, Australia).

60 CHAPTER 2: METHODS Formation and Purification of Complexes Between α 2 M and Stressed Protein Mixtures of α 2 M (2.5 mg/ml) and biotinylated CS (CS-b; 0.3 mg/ml) or α 2 M (0.5 mg/ml) and CPK-b (0.5 mg/ml) in PBS were incubated for 3 h at 43 C. In similar experiments α 2 M (2.5 mg/ml) and lys-b (1 mg/ml) were incubated for 10 h at 37 C in OSB. Samples were centrifuged at g for 5 min to remove any insolubles, and subsequently dialysed against 20 mm TAPS buffer, ph 9, overnight at 4 C. The samples were then applied to a 1 ml HiTrap Q fast flow Sepharose column (GE Healthcare, Australia) previously equilibrated with 20 mm TAPS buffer, ph 9, and eluted with a linear gradient of 0-1 M NaCl in the same buffer. Analysis of eluted fractions by SDS-PAGE identified that this method separated uncomplexed α 2 M and substrate protein from complex. The presence of complex in fractions shown by SDS-PAGE to contain both α 2 M and the substrate protein was verified by immunoprecipitation, as described below. Complex-containing fractions were pooled, concentrated and stored frozen at - 20 o C in PBS. 2.6 Electrophoresis SDS PAGE Electrophoresis was conducted using a Tall-Mighty-Small vertical slab gel (Hoeffer Scientific Instruments, USA) connected to a Bio-Rad power pack 300 power supply (Bio- Rad, USA). The components of the SDS-PAGE unit were assembled and melted agar (1%) used to seal the gel chamber. 10% acrylamide separating gel (5.9 ml distilled water, 5.0 ml 30% (w/v) acrylamide mix, 3.8 ml 1.5 M Tris ph 8.8, 0.15 ml 10% (w/v) SDS, 0.15 ml 10% (w/v) ammonium persulfate, ml TEMED) was transferred to the chamber and overlayed with 1 ml of ethanol to avoid air-bubble formation. When set, ethanol was

61 CHAPTER 2: METHODS 34 removed and a 5% stacking gel (4.1 ml distilled water, 1.0 ml 30% (w/v) acrylamide mix, 0.75 ml 1.0 M Tris ph 8.8, 0.06 ml 10% (w/v) SDS, 0.06 ml 10% (w/v) ammonium persulfate, ml TEMED) was loaded into the chamber with a comb immediately inserted to form wells. After the stacking gel set, the comb was removed and running buffer (0.192 M glycine, 0.1% (w/v) SDS, 0.25 M Tris Buffer, ph 8.3) was poured into the appropriate chambers. Samples (generally containing 10 µg protein) were diluted with an equal volume of sample buffer (0.005% (w/v) Bromophenol Blue, 20% (v/v) glycerol, 5% SDS, 0.5 M Tris HCl, ph 8.8) and reduced by the addition of 2% (v/v) β-mercaptoethanol and boiling for 1 minute. Once samples were loaded, the gel was electrophoresed at a constant 120 V until the dye front reached the bottom of the gel. The gel was then stained overnight using Coomassie blue staining solution (0.25% (w/v) Coomassie brilliant blue (R250), 10% (v/v) glacial acetic acid, 45% (v/v) methanol, 45% (v/v) RO water) and destained using destain solution (10% (v/v) glacial acetic acid, 45% (v/v) methanol, 45% (v/v) RO water) Immunodetection Samples were separated under reducing conditions using SDS PAGE, and electrophoretically transferred to nitrocellulose membrane (Osmonics, USA) using a Western Transfer Unit (BioRad, USA). The membrane was then blocked overnight at 4 C with 1% (w/v) heat-denatured casein in PBS (HDC/PBS). The membrane was then washed with PBS, before being incubated in polyclonal rabbit anti-α 2 M antibody (DakoCytomation, Denmark; diluted 1:500 in HDC/PBS), G7 monoclonal anti-clusterin antibody (diluted to 10 μg/ml in HDC/PBS) (Wilson and Easterbrook-Smith, 1992) or rabbit anti-trypsin antibody (DakoCytomation, Denmark; diluted 1:500 in HDC/PBS), for two hours at room temperature. Any unbound antibody was then removed with PBS

62 CHAPTER 2: METHODS 35 washes, and a secondary antibody solution (sheep-anti rabbit Ig-HRP conjugate or sheepanti mouse Ig-HRP conjugate; Silenus, Australia), diluted 1:500 in HDC/PBS, was added to the membrane and incubated for two hours at room temperature. The membrane was then washed with PBS, followed by 0.1% (v/v) Triton X-100 in PBS, and developed using enhanced chemiluminescence detection (ECL). Supersignal substrate solution (Pierce, Sydney, Australia) was used to detect bound HRP following the manufacturer s instructions Native Gel Elecrophoresis Samples (30 μg total protein) of native α 2 M and oxidised α 2 M were electrophoresed on a 1% native agarose gel in TAE buffer (40 mm Tris, 20 mm acetic acid, 5 mm EDTA, ph 7.5). The gel was run for approximately 2.5 h at a constant 60 V before being washed twice with milli-q water and stained with Imperial protein stain (Pierce, USA). The gel was then destained with washes of milli-q water until the protein bands became visible. In similar experiments, mixtures of lys (1 mg/ml) and α 2 M (2.5 mg/ml) were incubated in the presence (or absence) of OSB for 2 h at 37 C. Samples were then analysed by native gel electrophoresis as described above Native PAGE Samples of protein (10 μg) were run on discontinuous polyacylamide gels, using a modified version to that of (Van Leuven et al., 1981); 4% resolving gel (9.3 ml distilled water, 2 ml 30% (v/v) acrylamide, 3.7 ml TRIS ph 5.7, ml TEMED, 0.15 ml ammonium persulfate), 4% stacking gel (9.3 ml distilled water, 2 ml 30% (v/v) acrylamide, 3.7 ml Tris, 0.03 M HCl, ph 6.1, ml TEMED, 0.05 ml 10% (w/v) ammonium persulfate), and the reservoir buffer M Tris, 0.04 M boric acid, ph 8.6. Samples were

63 CHAPTER 2: METHODS 36 electrophoresed for 2.5 h, gels were stained with Coomassie blue and destained using destain solution as in Trypsin Binding Assay Trypsin binding assays were performed using an adaptation of a previously described method (Bonner et al., 1992). Briefly, in the wells of a 96 well plate, 5 μl of a stock solution of trypsin (in 1 mm HCl) was added to 50 μl aliquots of α 2 M or α 2 M/stressed protein complexes (at 0-50 μg/ml total protein) in 25 mm tris-hcl, 150 mm NaCl, ph 7.4, to give a final concentration of 3.8 μm trypsin and the mixture incubated at room temperature for 10 min. Unbound trypsin was then inhibited by adding 10 μl of a stock solution of soybean trypsin inhibitor (in PBS) to give a final concentration of 7.7 μm. To assay the remaining trypsin activity (i.e. trypsin bound to α 2 M), 80 μl of M Tris-HCl, 5.55 μm CaCl 2, ph 8.0, was added to each well, immediately followed by 100 μl of 5 mm BAPNA (dissolved in DMSO and diluted in water). After incubation at 37 C for 30 min, the reaction was stopped with the addition of 10 μl of 10 M glacial acetic acid and the conversion of BAPNA to its product, p-nitroaniline, was measured at 405 nm using a SpectraMax Plus 384 microplate reader (Molecular Devices, USA). 2.8 Protein Precipitation Assays Individual solutions of trypsin activated α 2 M* ( μm) and CS (6 μm; in 50 mm Tris, 2 mm EDTA, ph 8) or mixtures of CS and α 2 M* (at the same final concentrations) were heated at 43 C for 4 h in a 384 well plate and precipitation measured as turbidity (absorbance at 360 nm). Absorbance readings were acquired every 4 min using a FLUOstar plate reader (BMG Labtech, Germany). In similar experiments, CPK (25 μm) or mixtures

64 CHAPTER 2: METHODS 37 of CPK and α 2 M* ( μm) (at the same final concentrations; all in PBS) were heated at 43 C for 3 h and precipitation measured as described above. In similar experiments, lys (69 μm) and mixtures of lys and α 2 M ( μm) or α 2 M* (trypsin activated; μm) were incubated in wells of a 96-well plate (100 μl/well) at 37 C in OSB. Changes in solution turbidity were measured as A 360 in a SpectraMax Plus 384 microplate reader (Molecular Devices, USA). As a control, the effects of superoxide dismutase (SOD) and bovine serum albumin (BSA) on protein precipitation induced by oxidative and heat stress were tested in similar assays. SOD and BSA were chosen as appropriate control proteins because of their known physical stability and lack of chaperone activity (Buchner et al., 1998; Rodriguez et al., 2002). 2.9 Precipitation of Proteins in Whole Human Plasma α 2 M was selectively depleted from normal human plasma (NHP) by repeated passes over a Zn 2+ HiTrap TM chelate-affinity column (GE Healthcare). Approximately mg of α 2 M was recovered per ml of plasma, together with about 0.5 mg of "contaminant proteins" (comprised of 4 proteins). The contaminant proteins were separated from α 2 M by size exclusion chromatography using a Superose 6 column operated by an FPLC system (GE Healthcare), concentrated by ultrafiltration using a 30 kda cut-off microconcentrator (Millipore, Sydney, Australia), and then added back to the α 2 M depleted plasma (α 2 MDP) to reconstitute them to approximately their original concentrations in plasma. A sample of double depleted plasma (DDP) was also prepared; clusterin was removed from α 2 MDP by immunoafinity chromatography as previously described (Poon et al., 2000). Depletion of α 2 M and clusterin from plasma was confirmed by immunodetection (as described in section

65 CHAPTER 2: METHODS ). To allow for small dilution effects unavoidable during the depletion steps, A 280 measurements of α 2 MDP, DDP and a "matched" sample of NHP (the same batch of plasma from which the depleted plasma had been prepared) were taken and used to "normalize" the total protein concentration of each sample; the maximum dilution factor was less than 7% and adjustments to the total protein concentration were made by adding PBS. Aliquots (100 μl) of NHP, α 2 MDP, (α 2 MDP mg/ml α 2 M), DDP and (DDP mg/ml α 2 M μg/ml clusterin), were diluted 1:2 with PBS, supplemented with 7.5 mm NaN 3 and 1 mm PMSF, and incubated with gentle shaking at 43 C for 72 h or 37 C for 1 week. In similar experiments, aliquots (100 μl) of NHP, α 2 MDP, (α 2 MDP mg/ml α 2 M), (DDP and DDP mg/ml α 2 M μg/ml clusterin), were diluted 1:2 in OSB, supplemented with 7.5 mm sodium azide and complete protease inhibitor (EDTA free), and incubated with gentle shaking at 37 C for 72 h. Precipitated proteins were recovered by filtering samples through 0.45 μm ULTRAFREE centrifugal filtration units (Millipore, Sydney, Australia). Each filter was washed with PBS (3 x 500 μl) before the precipitate was solubilised by incubation with 2 M guanidine hydrochloride in PBS for 2 h at 60 C. The protein content of each sample was determined using a bicinchoninic acid (BCA) microprotein assay (Smith, 1985) Determination of Protein Concentration using BCA Assay To determine the concentration of the proteins used in this study BCA micro protein assays were carried out. Duplicate samples (100 µl) of BSA standards (0-80 µg/ml) and binary dilutions of the sample protein were added to the wells of a 96 well plate. Freshly prepared BCA working solution (104 volumes of reagent A: 8 % (w/v) Na 2 CO 3.H 2 O, 1.6 % (w/v) Na 2 tartrate, ph 11.25; 100 volumes of reagent B: 4% BCA. Na 2 ; 4 volumes of reagent C:

66 CHAPTER 2: METHODS 39 4% (w/v) CuSO 4.5H 2 O) was added in 100 µl aliquots to each well and the plate incubated at 60 C for 30 minutes or until sufficient colour had developed. The absorbance at 590 nm was measured using a Labstar Spectrophotometer (BMG Labtechnologies, USA) and the concentration of the unknowns determined by interpolation using the standard curve. Values for protein concentration reported in this study are calculated based on the assumption that the protein(s) of interest had the same colorimetric reaction with BCA as the BSA standards Immunoprecipitations α 2 M (6.95 μm), biotinylated lys (lys-b; 69 μm), or mixtures of α 2 M and lys-b at the same final concentrations in OSB were incubated for 13 h at 37 C. All samples were then centrifuged (5 min at g) to remove insoluble material and incubated on an end over end shaker for 1 h at room temperature with streptavidin-agarose (50 µl packed volume; Calbiochem, USA). The streptavidin-agarose was washed twice with PBS by centrifugation followed by 2 washes with 0.1% (v/v) Triton X-100 in PBS, and then boiled in SDS-PAGE sample buffer for 5 min; eluted proteins were subsequently analysed by SDS-PAGE under reducing conditions Identification of Endogenous Substrates using Mass Spectrometry Spot Excision Plasma samples were incubated at either 43 C or room temperature for 72 h. α 2 M was purified from both samples using zinc chelate affinity chromatography (as described in section 2.2). Proteins co-purifed with α 2 M in the heated, but not the non-heated sample

67 CHAPTER 2: METHODS 40 represent putative chaperone substrates and were detected using reducing SDS PAGE. Following staining with Coomassie Blue and subsequent destaining, a clean scalpel was used to excise the protein bands of interest (putative chaperone substrates for α 2 M); instruments were washed with 100 % methanol between excisions to ensure that individual samples were not cross-contaminated Trypsin Digestion Excised bands were destained by incubation with 100 μl of Coomassie Destain Buffer (60 % (v/v), 50 mm NH 4 HCO 3, 40 % (v/v) 100% acetonitrile, ph 7.8) for 1 h at room temperature with shaking (700 rpm). Destain was removed by aspiration and samples placed in a vacuum desiccator for 1.5 h. Each sample was then incubated with 140 ng of sequencing grade modified trypsin (Promega, USA) for 1 h at 4 C with gentle shaking (400 rpm). Following removal of excess trypsin, the digested protein was released from the gel by incubation with 20 μl of 50 mm NH 4 HCO 3 (ph 7.8), overnight at 37 C with gentle shaking (400 rpm). Samples were stored at 4 C before analysis by mass spectrometry MALDI-TOF Mass Spectrometry All mass spectrometry and data analysis was kindly undertaken by Dr Andrew Aquilina (University of Wollongong). Briefly, 1.2 μl of tryptic digest was placed on a mass spectrometry plate and covered with Mass Spectrometry Matrix (10 mg α-cyano-4- hydroxycinnamic acid, 1 ml 70% acetonitrile). The plate was dried and MALDI-TOF MS was performed using a Voyager-DE TM STR Biospectometry Workstation TM with Delayed Extraction TM (PerSeptive Biosystems, USA) at the Australian Proteomic Analysis Facility, Maquarie University, Sydney, NSW, Australia. The spectrum obtained was analysed using

68 CHAPTER 2: METHODS 41 Voyager TM software with Data Explorer TM. The unique fingerprint of the protein was compared to the theoretical masses predicted using the Swiss-Prot and TrEMBL databases located at ExPASy and any protein containing a high number of identical fragments was selected as a possible match Cell Culture and Flow Cytometry Culture of Cell Lines JEG-3 (a human placental adenocarcinoma cell line expressing LRP), Hep-G2 (a human hepatocellular cell line) and U937 (a human lymphoma cell line), all obtained from the American Type Culture Collection (ATCC, VA, USA), were routinely cultured in Dulbecco s Modified Eagle Medium: F-12 (DMEM: F-12) (Invitrogen, USA) supplemented with 5% (v/v) foetal calf serum (FCS; Thermotrace, Australia), incubated at 37 C and 5% (v/v) CO 2. Cells were cultured for approximately 48 h without a change of media before they were detached using 5 mm EDTA in PBS and then washed by centrifugation at 300 g for 10 min in Hank s binding buffer (HBB; 0.14 M NaCl, 5 mm KCl, 6 mm glucose, 0.4 mm KH 2 PO 4, 0.3 mm Na 2 HPO 4, 20 mm HEPES, 1 g/l BSA, 1 mm CaCl 2, 2 mm MgCl 2, ph 7.4). In order to differentiate U937 cells into cells with monocyte-like characteristics, cells were grown to confluence and then incubated with 20 nm phorbol myristate acetate (PMA) in growth medium for 72 h.

69 CHAPTER 2: METHODS Binding Assays Using JEG-3, Hep-G2 and Activated U937 Cells Different cell lines were incubated for 30 min on ice with either α 2 M, trypsin activated α 2 M* or trypsin activated α 2 M*/CS, all at a concentration of 200 µg/ml. Following washing with HBB the cells were incubated with rabbit anti-α 2 M or (control) anti-dnp antibodies (Dako; diluted 1:500 in HBB), and finally with SaRIgG-FITC (diluted 1:200 in HBB). In order to confirm that the binding observed was specific to LRP, similar experiments were undertaken in which cells were first pre-incubated with either an inhibitory rabbit polyclonal anti-lrp antibody (200 μg/ml; kindly donated by S. K. Moestrup, University of Aarhus, Denmark) or GST-RAP (100 μg/ml; RAP is a known LRP binding protein and inhibitor, here expressed as a fusion protein with GST; see 2.1) before incubation with (α 2 M/CS) or typsin activated α 2 M*. Binding of α 2 M and α 2 M/stressed protein complexes to the asialoglycoprotein receptor expressed on U937 cells was also investigated. Following washing in HBB, U937 cells were pre-incubated with asialofetuin (1 mg/ml), a known ligand of the asialoglycoprotein receptor family Binding Assays Using Granulocytes Isolated from Whole Blood Fresh human blood (supplemented with 5 mm EDTA) was centrifuged at 600 g for 20 minutes. The plasma was then removed and freshly prepared lysis buffer (0.144 M NH 4 Cl, 17 mm Tris, ph 7.2, 37 C) added to the blood cells (packed volume 20 ml) at a ratio of 3:1 (buffer volume: blood volume). The sample was incubated at 37 C until the red blood cells were completely lysed, detected by a change in colour and turbidity. Chilled HBB was then added to the sample which was centrifuged at 300 g for 10 minutes. The pellet was then resuspended and washed twice with PBS.

70 CHAPTER 2: METHODS 43 The white blood cell pellets obtained were first incubated for 30 minutes with 500 µg/ml of: native α 2 M, typsin activated α 2 M*, or α 2 M/CS complexes. Following a wash with HBB, the cells were incubated with polyclonal rabbit anti-α 2 M (DakoCytomation, Denmark; diluted 1:500 in 1% in HBB), washed in HBB, and then sheep-anti rabbit Ig-FITC conjugate (DakoCytomation, Denmark; diluted 1:50 in HBB). In order to measure background fluorescence, cells were incubated with an equal concentration of species matched, irrelevant control antibody (anti-dnp antibody) followed by sheep-anti rabbit Ig- FITC conjugate. In cases where binding was shown to be significant, in some samples, cells were incubated with GST-RAP (100 μg/ml) prior to incubation with the respective protein in order to determine if LDLR were responsible for the binding measured. Specific binding to granulocytes was assessed by gating on this population on the basis of known forward and side scatter characteristics during flow cytometric analysis Binding Analysis Using Flow Cytometry An LSR II flow cytometer (Becton Dickinson, Sydney, Australia) was used to analyse cells which were incubated with 1 μg/ml propidium iodide (PI) immediately before analysis to stain the nuclei of dead cells. Cells were excited at 488 nm and fluorescence emissions collected at 515 ± 10 nm (FITC) and 695 ± 20 nm (PI). Electronic gating was used to exclude dead cells from the analyses. Data was collected using FACS Diva software (v4.0; Becton Dickinson) and analysed using FloJo v6.4.1 (Treestar Inc., USA). Where relevant, the significance of differences in binding were assessed using the Student s t-test.

71 CHAPTER 2: METHODS 44 references Bonner, J., A. L. Goodell, J. A. Laskey and M. R. Hoffman (1992). "Reversible binding of platelet-derived growth factor-aa, -ab and -bb isoforms to a similar site on the "Slow" And "Fast" Conformations of alpha-2-macroglobulin." Journal of Biological Chemistry, 262: Buchner, J., H. Grallert and U. Jakob (1998). "Analysis of chaperone function using citrate synthase as a non-native substrate protein." Methods in enzymology, 290: Hall, P., K., Roberts, R., C. (1978.). "Physical and chemical properties of human plasma alpha2-macroglobulin." The biochemical journal, 173: Heuer, K. H., J. P. Mackay, P. Podzebenko, N. P. Bains, A. S. Weiss, G. F. King and S. B. Easterbrook-Smith (1996). "Development of a sensitive peptide-based immunoassay: Application to detection of the jun and fos oncoproteins." Biochemistry, 35: Imber, M. J. and S. V. Pizzo (1981). "Clearance and binding of two electrophoretic forms of human alpha-2-macroglobulin." The Journal of Biological Chemistry, 256: Poon, S., S. B. Easterbrook-Smith, M. S. Rybchyn, J. A. Carver and M. R. Wilson (2000). "Clusterin is an atp-independent chaperone with very broad substrate specificity that stabilises stressed proteins in a folding- competent state." Biochemistry, 39: Rodriguez, J. A., J. S. Valentine, D. K. Eggers, J. A. Roe, A. Tiwari, R. H. Brown, Jr., and L. J. Hayward (2002). "Familial amyotrophic lateral sclerosis-associated mutations decrease the thermal stability of distinctly metallated species of human copper/zinc superoxide dismutase." Journal of Biological Chemistry, 277: Smith, P. K., Krohn, R. I, Hermanson, G.T, Mallia,A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson B. J., and Klenk, D. C. (1985). "Measurement of protein using bicinchoninic acid." Analytical Biochemistry, 150: Van Leuven, F., J. J. Cassiman and H. Van den Berghe (1981). "Functional modifications of alpha 2-macroglobulin by primary amines. I. Characterization of alpha 2 m after derivatization by methylamine and by factor xiii." J. Biol. Chem., 256: Wilson, M. R. and S. B. Easterbrook-Smith (1992). "Clusterin binds by a multivalent mechanism to the fc and fab regions of igg." Biochim Biophys Acta., 1159:

72 CHAPTER 3: RESULTS 44 CHAPTER 3: CHARACTERISING THE MECHANISM OF α 2 M CHAPERONE ACTION 3.1 Introduction It has previously been shown, under conditions of heat stress, that α 2 M has the ability to form complexes with the substrate proteins citrate synthase and creatine phosphokinase, protecting them from precipitation and aggregation (French, 2005). Data from this previous study and results presented in this chapter (along with other supporting results) were included in a recent publication reporting the chaperone abilities of α 2 M (French et al., 2008). Unless otherwise indicated, all results shown in this Chapter were produced by the author. Other than the studies mentioned, limited research has been undertaken to examine the role of α 2 M as an extracellular chaperone. Specifically, the effect of interactions with stressed proteins on the conformational structure of α 2 M, and how these impact on its protease inhibitor activity were unknown. The conformation of α 2 M when complexed with a stressed protein was investigated using native gel electrophoresis and sensitive trypsin binding assays. When investigating the chaperone role of α 2 M it is essential to consider the two fundamentally different conformations present in human plasma. The native form has the ability to trap proteases, while the active form (α 2 M*) is present following interaction with a protease, and has exposed receptor recognition sites. Although native α 2 M has chaperone activity, it is unknown whether α 2 M* has a similar activity. In order to determine the ability of α 2 M* to inhibit heat-induced protein precipitation, two substrate proteins were chosen, CS and CPK. These were selected due to their ability to precipitate at a relatively low

73 CHAPTER 3: RESULTS 45 temperature (43 C) (Table 3.1). Furthermore, in an attempt to identify putative endogenous chaperone substrates for α 2 M in human plasma, samples were incubated at 43 C for 72 h and any proteins co-purifying with α 2 M following these incubations were identified using MALDI-TOF mass spectrometry. Table 3.1 Characteristics of substrate proteins used to investigate the chaperone properties of α 2 M. Substrate Protein Citrate Synthase (CS) Mass (kda) Isoelectric Point (Pi) Subunits Subunit Mass (kda) Disulfide Bonds Secondary Structure No Predominantly α-helix, small section of β- sheets Creatine Phosphokinase (CPK) No α-helix, β- sheet Data shown was taken from information available on the Swiss-Prot database and (Buchner et al., 1998) 3.2 Methods: Refer to materials and methods sections Results: Within α 2 M/heat Stressed Protein Complexes, α 2 M Remains in its Native Conformation In the protease-bound, activated form, α 2 M* has a more compact structure which may be detected using native tris/borate PAGE (Figure 3.1). Native α 2 M runs as a single band with limited migration on the gel, however activated α 2 M migrates significantly further. α 2 M within complexes formed with either heat stressed CS or CPK displays a migration pattern similar to that of native α 2 M, suggesting that interaction with a stressed protein may not activate α 2 M. The formation of complexes with either of the two substrate proteins CS and

74 CHAPTER 3: RESULTS 46 CPK also appears to have little impact on the electrophoretic mobility of α 2 M. This may be explained by the fact that each α 2 M associates with only one or two substrate molecules. (A) α 2 M α 2 M* α 2 M/CS ((B) α 2 M α 2 M* α 2 M/CPK α 2 M α 2 M* Figure 3.1 Image of Coomassie stained native PAGE gel showing migration of α 2 M, α 2 M*, α 2 M/CS and α 2 M/CPK complexes. (A) 6 % Tris/borate native PAGE gel showing native α 2 M, trypsin activated α 2 M (α 2 M*) and α 2 M/CS complex (B) 6 % Tris/borate native PAGE gel showing native α 2 M, trypsin activated α 2 M (α 2 M*) and α 2 M/CPK complex. Relative positions of α 2 M and α 2 M* are indicated. Each experiment was conducted in duplicate. A classical trypsin binding assay was used to examine the ability of α 2 M incorporated into purified α 2 M/stressed protein complexes to carry out the protease trapping reaction. In the assay, trypsin (provided in excess) complexed with any native α 2 M present; any unbound trypsin was subsequently inactivated using soybean trypsin inhibitor (which is sterically unable to access and inactivate trypsin bound to α 2 M). Any residual trypsin activity (corresponding to α 2 M-trapped trypsin) was measured using the low molecular weight substrate BAPNA. Purified α 2 M/CS and α 2 M/CPK complexes showed dose-dependent trypsin binding activity which was similar to that measured for native α 2 M (Figure 3.2). Collectively, these results suggest that α 2 M may remain in its native form when complexed

75 CHAPTER 3: RESULTS 47 with heat-stressed proteins and the similar trypsin-trapping ability of pure native α 2 M and α 2 M/stressed protein complexes suggests that a majority of the mass of the complexes consists of α 2 M. This result is consistent with past findings which indicated that α 2 M/stressed protein complexes could not be discriminated from α 2 M alone using sizeexclusion chromatography (French, 2005). Thus, one large α 2 M tetramer (720 kda) is likely to associate with a small number of lower molecular weight chaperone substrate molecules. The exact stoichiometry of α 2 M/stressed protein complexes may be determined in the future by using SDS PAGE and scanning densitometry, or mass spectrometry. (A) CS (B) CPK A 405 nm Protein (μg) Figure 3.2 When complexed with stressed proteins, α 2 M retains protease trapping activity. Native α 2 M ( ), α 2 M*( ) (activated by methylamine) and (A) α 2 M/CS and (B) α 2 M/CPK complexes ( ) were assayed for trypsin binding activity, as described in methods section 2.7. Data points represent means of triplicate measurements and the error bars represent SE of the means; the results shown are representative of three independent experiments.

76 CHAPTER 3: RESULTS Protease Activation Abolishes the Chaperone Activity of α 2 M α 2 M has been shown to potently inhibit the heat-induced precipitation of a range of substrate proteins including CS, CPK and collagenase (French, 2005). However, under similar heat-stress conditions, α 2 M "activated" by incubation with trypsin (α 2 M*) had limited effect on substrate protein precipitation (Figure 3.3). When α 2 M at a concentration of 5 mg/ml was incubated with CS at 43 C, precipitation was almost totally suppressed. In comparison, addition of α 2 M* at a concentration of 5 mg/ml to CS during heat stress did not offer any protection from precipitation (Figure 3.3 A). Similar results were observed when using CPK as the substrate protein (Figure 3.3 B). Collectively, these results demonstrate that protease-mediated activation of α 2 M effectively abolishes its chaperone activity. (A) CS (B) CPK A Time (min) Figure 3.3 Activation abolishes the chaperone activity of α 2 M. Time-dependent changes in turbidity (measured as A 360 ) of heat-stressed CS (A) and CPK (B), either alone ( or in the presence of α 2 M (5 mg/ml) ( ) or trypsin activated α 2 M* (5 mg/ml) ( ). The data points are individual measurements; the results shown are representative of at least three independent experiments.

77 CHAPTER 3: RESULTS 49 As described above, α 2 M* lacks the ability to inhibit protein precipitation. It was also suggested earlier in this chapter (section 3.3.1) that α 2 M within α 2 M/stressed protein complexes may remain in the native conformation. Further experiments were undertaken to investigate whether α 2 M within stressed protein complexes can subsequently undergo protease activation. Following incubation of α 2 M/CS complexes with a three fold molar excess of trypsin for 2 h at 37 C, a similar migration pattern to α 2 M* was observed using native PAGE (Figure 3.4). This result suggests that α 2 M complexed with heat-stressed protein retains the ability to trap proteases and therefore the ability to subsequently expose the LRP binding site. α 2 M α 2 M* α 2 M/CS (α 2 M/CS)* α 2 M α 2 M* Figure 3.4 Image of Coomassie stained native PAGE gel of α 2 M, α 2 M*, α 2 M/CS and (α 2 M/CS)*. 6 % Tris/borate native PAGE gel showing the native form of α 2 M (α 2 M), trypsin activated α 2 M (α 2 M*), α 2 M/CS complex (α 2 M/CS), and trypsin-activated α 2 M/CS complex ((α 2 M/CS)*). Relative positions of α 2 M and α 2 M* are indicated. The result shown is representative of two independent experiments.

78 CHAPTER 3: RESULTS 50 Immunoprecipitation analysis was used to confirm that trypsin was bound by A2M to form complexes containing A2M, trypsin and chaperone substrate protein. Results indicated that the only conditions tested under which the immunoprecipitate contained trypsin was when a heated mixture of α 2 M and substrate protein had been subsequently incubated with trypsin (Figure 3.5). This establishes that a complex is formed that contains all three molecular species (α 2 M, heat-stressed protein and trypsin). (A) (B) Figure 3.5 α 2 M/stressed protein complexes retain the ability to trap trypsin. Image of sections of nonreduced Coomassie blue stained 10% SDS-PAGE gels (A) and corresponding immunoblots probed with an anti-trypsin antibody (B), showing proteins affinity absorbed by streptavidin-agarose from samples incubated with trypsin containing CS-b or CPK-b alone, or α 2 M alone (all at room temperature), or mixtures of α 2 M and either CS-b or CPK-b, which had been heated at 43 C before being mixed with trypsin (i.e., α 2 M/CS and α 2 M/CPK). On the SDS-PAGE gels, the identity of bands was established by comparison with molecular mass standards (not shown); where detected on the immunoblots, trypsin migrated to the same position as α 2 M (B). The results shown are representative of two independent experiments.

79 CHAPTER 3: RESULTS 51 In order to further confirm that following the formation of complexes with heat-stressed protein, α 2 M can still interact with proteases to become activated, and that this activation does not involve proteolysis of heat-stressed protein, a purified α 2 M/CS complex was incubated with a 3 fold molar excess of trypsin at 37 C for 2 h. Unexpectedly, there were additional bands present in the reduced, commercially purchased CS. These bands may indicate incomplete reduction of disulfide bonds or the presence of impurities. When analysed using reducing SDS PAGE, the α 2 M/CS sample contained bands at 180 kda (indicative of a reduced α 2 M subunit), and at about 95 kda and 85 kda which are likely to represent fragments of α 2 M generated by spontaneous bait region cleavage, attributable to freeze/thawing of the sample. Unexpectedly, the α 2 M/CS sample also contained a band at about 70 kda. This band may represent the autolytic cleavage of thiol-ester bonds within α 2 M subunits, produced as a result of heating during sample preparation. Faint bands at 55 and 45 kda are present in both native and activated α 2 M/CS samples and are probably minor contaminant proteins. Regardless of these complications, the SDS PAGE analysis indicates that the major CS band detected (at about 38 kda) is very similar in the α 2 M/CS and (α 2 M/CS)* lanes, suggesting that trypsin activation is not associated with significant proteolysis of the α 2 M-bound CS substrate (Figure 3.6).

80 CHAPTER 3: RESULTS 52 α 2 M α 2 M* CS Trypsin α 2 M/CS (α 2 M/CS)* Molecular Mass (kda) Figure 3.6 Image of Imperial Blue stained SDS PAGE showing the effects of trypsin on α 2 M and α 2 M/CS complexes. 12% SDS PAGE gel (under reducing conditions) showing α 2 M, α 2 M*, CS, trypsin, α 2 M/CS and trypsin-activated (α 2 M/CS)*. The identity of each sample is indicated above the corresponding wells. The identity of the bands was established by comparison with molecular mass standards (shown at the left of the image) α 2 M Inhibits the Heat Induced Precipitation of Proteins in Whole Human Plasma Zinc chelate affinity chromatography was used to selectively deplete virtually all α 2 M from an aliquot of normal human plasma (NHP). NHP was repeatedly passed over a 5 ml Zn 2+ HiTrap TM chelate-affinity column until negligible α 2 M could be detected by immunoblotting (Figure 3.7).

81 CHAPTER 3: RESULTS 53 (A) (B) NHP α 2 MDP (( α 2 M Figure 3.7 Depletion of α 2 M from normal human plasma (NHP). (A) α 2 M was depleted from normal human plasma by three successive passes over a zinc chelate column. The first elution peak in each run (indicated by the black arrowheads) represents the 20 mm imidazole elution step and contains contaminating proteins. The second peak (500 mm) imidazole (shown by the empty arrowheads) contains pure α 2 M. The broad peak eluted first (not labeled) is the unbound protein fraction (B) Successful depletion of α 2 M from NHP was confirmed by immuno-blotting under reducing conditions as described in section 2.9. The position of α 2 M is indicated by the empty arrowhead and corresponds to a mass of 180 kda (expected under reducing conditions), estimated by comparison with molecular mass standards (not shown). When aliquots of NHP and α 2 M-depleted plasma (α 2 MDP) prepared from the same original batch of plasma (diluted 1 in 2 in PBS and supplemented with azide), were incubated at 43 o C for 72 h, the α 2 MDP contained significantly more aggregated protein than that detected in the NHP sample (Figure 3.8; Students t-test, p < 0.05). In order to further investigate the protective ability of α 2 M and its relationship with other known

82 CHAPTER 3: RESULTS 54 extracellular chaperones, a sample of plasma depleted of both α 2 M and clusterin (double depleted plasma; DDP) was prepared (diluted 1:4 in PBS) and incubated at 43 C for 72 h. The α 2 MDP was found to contain more precipitated protein than the NHS but less than that observed in the DDP (Figure 3.8) Protein (mg) NHP α 2 MDP DDP Figure 3.8 α 2 M inhibits heat stress-induced protein precipitation in whole human plasma. Histogram showing the total protein precipitated from 100 μl aliquots of normal human plasma (NHP), α 2 M-depleted plasma (α 2 MDP) and double depleted plasma (DDP) heated at 43 C for 72 h. Data points represent the means of triplicate measurements and error bars indicate the standard errors (SE) of the means (* denotes p < 0.05, Student's t-test). The results are representative of two independent experiments. Experiments were carried out to determine the ability of α 2 M to protect proteins in whole human plasma from aggregation resulting from extended incubation at 37 C. α 2 MDP, when incubated for 7 days at 37 C, showed a significantly greater level of precipitation compared to NHP (Figure 3.9). The level of protein precipitation was greater again in plasma also depleted of clusterin. In order to confirm that the increases in precipitation

83 CHAPTER 3: RESULTS 55 resulted from the removal of α 2 M (α 2 MDP) or α 2 M and clusterin (DDP) from the respective samples, physiological concentrations of α 2 M (2.5 mg/ml) and clusterin (100 μg/ml) were added back to aliquots of the depleted samples. As expected this returned the level of precipitation to that of NHP. These results indicate that α 2 M has a chaperone action that can suppress the stress-induced aggregation and precipitation of endogenous protein(s) in human plasma ** Protein (mg) * 0 NHP α 2 MDP DDP α 2 MDP DDP+ α 2 M + α 2 M + Clusterin Figure 3.9 α 2 M inhibits protein precipitation in whole human plasma at 37 C. Histograms showing the total protein precipitated from 100 μl aliquots of normal human plasma (NHP), α 2 M- depleted plasma (α 2 MDP) and plasma depleted of both α 2 M and clusterin (double depleted plasma; DDP) heated at 37 C for 7 days. Samples of α 2 MDP and DDP were supplemented with 2.5 mg/ml α 2 M and (2.5 mg/ml α 2 M μg/ml) clusterin, respectively. Data points represent the means of triplicate measurements and error bars indicate the standard errors (SE) of the means. Asterisks denote significant differences (p < 0.05, Student s t test) when compared with NHP (*) or both NHP and α 2 MDP (**). The results are representative of two independent experiments Identifying Endogenous Chaperone Substrates for α 2 M in Human Plasma

84 CHAPTER 3: RESULTS 56 The identification of specific proteins chaperoned by α 2 M within the body will contribute to a better understanding of the physiological importance of the in vivo chaperone functions of α 2 M. Preliminary investigations were undertaken in this study with this aim in mind. α 2 M was purified by Zn chelate affinity chromatography from NHP which had been incubated at either room temperature or 43 C for 72 h and the proteins co-purified with α 2 M analysed by SDS PAGE. Any proteins co-purifying with α 2 M from the heat-stressed sample, but not from the control (room temperature) sample, represent putative chaperone substrates for α 2 M. Two significant bands of approximately ~ 50 kda and ~ 55 kda were co-eluted with α 2 M in the fraction prepared from heated plasma, but not in the fraction prepared from plasma held at room temperature (Figure 3.10). 43 C RT 200 α 2 M Molecular Mass (kda) X Y 3.10 Image of Coomassie-Blue stained SDS PAGE gel identifying putative endogenous chaperone substrates for α 2 M. 10% SDS PAGE gel (under reducing conditions) of proteins co-purifying from human plasma incubated for 72 h at either 43 C or at room temperature (RT). See methods section for more detailed description of methods used. The gel was stained using Coomassie Blue and the identity of the bands established by comparison with molecular mass standards (as shown). The position of α 2 M is shown by the empty arrowhead and the putative endogenous substrates (X and Y) are indicated by the black arrowheads. The results shown are representative of two independent experiments.

85 CHAPTER 3: RESULTS 57 In order to determine the identity of the putative endogenous substrates the two bands labeled X and Y (Figure 3.10) were excised from the SDS PAGE gel, digested with trypsin and analysed by MALDI-TOF mass-spectrometry (kindly performed by Dr Andrew Aquilina, University of Wollongong). For each of the band digests a unique spectrum of tryptic fragments was obtained, termed a peptide mass fingerprint (PMF). Figure 3.11 shows the spectra derived from band X (upper) and band Y (lower). Peptides corresponding to peaks within the range of m/z were used to search the SwissProt database using the Mascot PMF query option ( For the upper spectrum, 7 peaks were found to match peptide masses for a theoretical tryptic digest of the beta subunit of human fibrinogen. Similarly, 7 peaks in the lower spectrum were found to match the gamma subunit of human fibrinogen. X

86 CHAPTER 3: RESULTS 58 Y Intensity Intensity Figure 3.11 Mass spectra of trypsin digested putative endogenous substrates of α 2 M. The upper spectrum, derived from band X, was found to have 7 peptides matching the beta subunit of human fibrinogen. The lower spectrum, derived from band Y, contained 7 peaks matching the gamma subunit of human fibrinogen. The x axis represents mass/charge and the y axis represents the absolute intensity (number of ions of each species that reach the detector). SDS PAGE was then used to confirm the identity of the putative endogenous substrate samples as the beta and gamma subunits of fibrinogen. Reduced samples of the proteins purified from heated, purified plasma were compared with purified human fibrinogen (Figure 3.12). The unknown proteins co-purified with α 2 M from heat-stressed plasma (lane 1) were found to migrate to positions consistent with the β and γ chains of purified fibrinogen (lane 2). The band(s) located at ~70 kda represent the α chain of fibrinogen (not analysed using mass spectrometry in this study).

87 CHAPTER 3: RESULTS Molecular Mass (kda) β γ α 2 M Fibrinogen is an endogenous human plasma chaperone substrate for α 2 M under heat stress conditions. Image of 10% Coomassie-Blue stained SDS PAGE gel comparing proteins purified by Zn 2+ affinity chromatography from heat-stressed human plasma (lane 1) with purified fibrinogen (lane 2). The gel was stained using Coomassie Blue and the identity of the bands established by comparison with molecular mass standards (as shown). The position of α 2 M is shown by the empty arrowhead and the beta and gamma chains of fibrinogen are indicated by the black and grey arrowheads, respectively. The results shown are representative of two independent experiments. 3.4 Discussion Previous studies have indicated that α 2 M inhibits heat induced precipitation of a broad range of protein substrates including CS, CPK, GST, and ovotransferrin by forming stable complexes with them (French, 2005; French et al., 2008). Earlier investigations have also indicated that the ability of α 2 M to inhibit heat induced protein precipitation is abolished when α 2 M is in its activated conformation (α 2 M*). Results presented here confirm and extend these findings.

88 CHAPTER 3: RESULTS 60 In this study it was determined that interaction of α 2 M with a stressed protein does not lead to its activation, thus α 2 M retains the ability to function as a protease inhibitor following its binding to a chaperone substrate. Native PAGE and trypsin binding assays suggested that, whilst complexed with a stressed protein, interaction with trypsin converted α 2 M to α 2 M*. Immunodetection provided direct evidence of covalent association between trypsin and α 2 M within preformed A2M-(stressed) protein complexes. It follows that if one important function of α 2 M is to bind to and solubilize extracellular proteins with non-native conformations, and subsequently mediate their clearance by LRP, then interaction with a protease may act as an in vivo switch to trigger LRP-mediated uptake of α 2 M/stressed protein complexes. α 2 M* was unable to inhibit the heat-induced precipitation of CS and CPK. This result supports previous findings (French, 2005) and suggests that following the conformational change induced by interaction with a protease, the sites used for binding to chaperone substrates may become nonfunctional. Although the exact mechanism for chaperone substrate binding is yet to be determined, investigations using the hydrophobic probe bisans have indicated that stressed proteins bind to α 2 M at least in part via hydrophobic interactions (French et al., 2008). Previous work has shown that, overall, α 2 M contains more surface-exposed hydrophobicity after it has been activated (Birkenmeier et al., 1989). However, this does not exclude the possibility that specific region(s) of exposed hydrophobicity on α 2 M important in the chaperone-like action are sterically more accessible to stressed proteins before protease activation. It is also possible that there are

89 CHAPTER 3: RESULTS 61 other unknown determinants required for binding to stressed proteins that are affected by the conformational changes associated with protease activation. Endogenous α 2 M and clusterin were found to significantly inhibit the spontaneous precipitation of proteins in unfractionated human plasma incubated at 37 C. This finding may have important medical implications. Abundant extracellular proteins with this type of chaperone property may act as an important line of defense against inappropriate extracellular protein aggregation, which underpins a variety of serious human diseases (Yerbury et al., 2005). The effects of α 2 M and clusterin on plasma protein precipitation are additive, suggesting that even though they are promiscuous in their interactions with different substrate proteins, they may provide complementarity with respect to the endogenous extracellular proteins they protect. Preliminary investigations were also undertaken to identify putative endogenous chaperone substrates for α 2 M in human plasma. Using MALDI-TOF mass spectrometry, the plasma protein fibrinogen was identified as one such putative substrate. Fibrinogen is an abundant acute phase protein with roles in inflammation and the stress response (Lowe et al., 2004). Fibrinogen is the main acute phase protein responsible for blood coagulation, with deficiencies linked to impaired homeostasis and increased risk of bleeding (Lowe et al., 2004). The role of fibrinogen is therefore vital in the inflammatory response, and precipitation under conditions of heat stress would markedly reduce the fibrinogen pool available for anti-inflammatory functionality. The results presented here provide a

90 CHAPTER 3: RESULTS 62 preliminary insight into the link between the roles of α 2 M as an inflammatory response mediator, protease inhibitor and extracellular chaperone.

91 CHAPTER 3: RESULTS 63 Birkenmeier, G., L. Carlsson-Bostedt, V. Shanbhag, T. Kriegel, G. Kopperschlager, L. Sottrup-Jensen and T. Stigbrand (1989). "Differences in hydrophobic properties for human α2-macroglobulin and pregnancy zone protein as studied by affinity phase partitioning." European Journal of Biochemistry, 183: Buchner, J., H. Grallert and U. Jakob (1998). "Analysis of chaperone function using citrate synthase as a non-native substrate protein." Methods in enzymology, 290: French, K. (2005). Alpha-2-macroglobulin: A putative extracellular chaperone. School of Biological Sciences. Wollongong, University of Wollongong: 93. French, K., J. J. Yerbury and M. R. Wilson (2008). "Protease activation of alpha-2- macroglobulin modulates a chaperone-like action with broad specificity." Biochemistry, 47: Lowe, G. D. O., A. Rumley and I. J. Mackie (2004). "Plasma fibrinogen." Annals of Clinical Biochemistry, 41: Yerbury, J., J., E. Stewart, M., A. Wyatt, R. and M. R. Wilson (2005). "Quality control of protein folding in extracellular space " EMBO Rep., 6:

92 CHAPTER 4: RESULTS 62 CHAPTER 4: BINDING OF α 2 M/STRESSED PROTEIN COMPLEXES TO CELL SURFACE RECEPTORS 4.1 Introduction: It is well established that protease bound α 2 M* is recognised by LRP and undergoes receptor mediated endocytosis in hepatocytes (Moestrup and Gliemann, 1991). Although several studies have investigated the binding and internalization of α 2 M-protease complexes, only very limited previous studies of the interaction of α 2 M/stressed protein complexes with LRP have been carried out. Preliminary investigations (French, 2005) indicated that complexes formed between α 2 M and heat stressed CS or CPK did not bind to the surface of cultured U87 cells, a human glioblastoma cell line known to express LRP (Bu et al., 1994). However, if the complexes were subsequently activated with methylamine, the activated complex showed significant binding to the cell surface (French, 2005). The focus of this study was to further investigate the interaction of α 2 M/stressed protein complexes with cell surface receptors on a range of human cell lines and on granulocytes derived from whole human blood. The cell lines used in this study are presented in Table 4.1. Table 4.1 Characteristics of cell lines used in the study. Cell Line Cell Type Receptors References (Studied in this project) JEG-3 Human adenocarcinoma LRP (Sarti et al., 2000) Hep-G2 U937 Human hepatocellular carcinoma Human leukemic monocyte lymphoma LRP (Grimsley et al., 1998), Scavenger Receptors (Schwartz et al., 1982) LRP (Elsas et al., 1990)

93 CHAPTER 4: RESULTS 63 A functional property of all LDL family receptors, including LRP, is the requirement of Ca 2+ for ligand binding. Therefore, to facilitate ligand binding, all experiments were performed in Hank s binding buffer, a solution containing calcium. Another common property shared by all LDL receptors is their binding to receptor associated protein (RAP). RAP is a 39 kda protein that binds to multiple sites of LRP with high affinity, thereby successfully inhibiting binding of all other known ligands (Hertz et al., 1991; Bu and Schwartz, 1998). Scavenger receptors located on the surface of liver cells represent a common route for the uptake and removal of many glycoproteins within human serum. One such receptor is the asialoglycoprotein (ASGP) receptor, expressed on Hep-G2 cells, a human hepatoma cell line. The ASGP receptor is a 46 kda protein with binding sites specific for galactose, N- acetylgalactosamine and related galactosides (Spiess, 1990). Considering the abundance of the ASGP receptors on liver cells, the site for disposal of α 2 M-protease complexes (Feldman et al., 1983), investigations were undertaken to determine whether α 2 M/stressed protein bound to the ASGP receptor. The specificity of the binding was determined by preincubating cells with saturating concentrations of galactose or asialofetuin, both ASGP receptor ligands. The binding of α 2 M-antigen complexes to monocytes is well-characterised, and is thought to function in the removal of toxic antigenic peptides from sites of infection and their subsequent degradation (Hart et al., 2004). The ability of α 2 M to bind to other immunological cell subsets such as granulocytes and lymphocytes remains largely

94 CHAPTER 4: RESULTS 64 uncharacterized. Preliminary studies have indicated that α 2 M* binds significantly to the surface of granulocytes and monocyte populations (French, 2005). Interestingly, preliminary data also suggested that α 2 M/CS complexes bound significantly to granulocytes without first becoming activated. No other binding to other white cell types was detected (French, 2005). The current investigation aims to further investigate binding of α 2 M/CS complexes to granulocytes, using cells isolated from human blood and flow cytometric analysis. 4.2 Methods: Refer to materials and methods sections , 2.5 and Results: Binding of α 2 M/Stressed Protein Complexes to JEG-3 Cells As shown in Figure 4.1, native α 2 M has only limited binding to JEG-3 cells. Following typsin activation however, α 2 M* showed significantly more binding than the native form (t = 16.62, p< 0.05, df =4). The results presented in chapter 3 demonstrate that following interaction and complex formation with heat stressed CS and CPK, α 2 M remains in the native conformation, retaining the ability to function as a protease inhibitor. To investigate the effect of activating α 2 M in α 2 M/stressed protein complexes on interactions with LRP, α 2 M/CS complexes were incubated with a three-fold molar excess of trypsin, and the ability of this activated complex (α 2 M/CS)* to bind to JEG-3 cells measured. The background fluorescence was detected by incubating cells with a species-matched control antibody, polyclonal rabbit anti-dnp antibody. Complexes formed between native α 2 M and heat-stressed CS showed little binding to JEG-3 cells; in contrast, following activation with

95 CHAPTER 4: RESULTS 65 trypsin, (α 2 M/CS)* showed substantial binding (Figure 4.1). Incubation of cells with (α 2 M/CS)* resulted in a significantly greater increase in fluorescence than that obtained for cells incubated with α 2 M/CS (t = 9.68, p<0.05, df =4) (Figure 4.1). LRP α 2 M α 2 M* Number of Cells α 2 M/CS (α 2 M/CS)* FITC Fluorescence Figure 4.1 Expression of LRP and binding of native α 2 M, α 2 M*, α 2 M/CS and (α 2 M/CS)* to JEG-3 cells. Histograms of 10 x 10 3 cells incubated with either rabbit anti-lrp antibody and subsequently sheep-antirabbit Ig-FITC conjugate (top left panel), or with native α 2 M, α 2 M*, α 2 M/CS or (α 2 M/CS)* (indicated in the corresponding panels) followed by anti-α 2 M antibody which was subsequently detected with sheep-antirabbit Ig-FITC conjugate. Other cells were separately treated with anti-dnp antibody followed by sheep-antirabbit Ig-FITC conjugate (background fluorescence; red peak). Results are indicative of two independent experiments. In order to confirm that the binding observed for (α 2 M/CS)* and α 2 M* was via LDL receptors on the surface of JEG-3 cells, specifically LRP, cells were first incubated with either GST-RAP (binds to all members of the LDLR family) or anti-lrp antibody. Preincubation with the ligand GST-RAP (100 µg/ml) significantly reduced the binding of α 2 M* (t = 5.87, p <0.05, df = 4) and (α 2 M/CS)* complexes (t = 4.7, p <0.05, df = 4) (Figure

96 CHAPTER 4: RESULTS ). Pre-incubation with anti-lrp antibody was also found to significantly reduce the binding of α 2 M* (t = 16.62, p <0.05, df = 4) and (α 2 M/CS)* (t = 19.87, p <0.05, df = 4) to the surface of JEG-3 cells. geomean fluorescence α2m/cs * * # * α2m*/cs + anti-lrp α2m*/cs 1 2 α2m*/cs + RAP α2m α2m* # # α2m* + anti-lrp α2m* + RAP Figure 4.2 Histogram plot showing the average geometric mean fluorescence (n = 3, ± SE, arbitrary units) for immunochemical detection of the binding to JEG-3 cells of α 2 M, α 2 M*, and native α 2 M/CS or activated (α 2 M/CS)* complexes. In all cases, α 2 M was trypsin activated. In some cases, cells were preincubated with an inhibitory anti-lrp antibody or GST-RAP (indicated below the x-axis; see methods section for details). The values shown have been corrected for the fluorescence associated with cells stained with negative control antibody (rabbit polyclonal anti-dnp antibody). The results shown are representative of several independent experiments. Significant differences (p < 0.05) are indicated by * (vs. α 2 M*/CS, left) and # (vs. α 2 M*, right) Binding of α 2 M/Stressed Protein Complexes to Hep-G2 Cells Preliminary experiments were undertaken to determine the ability of α 2 M, α 2 M* and native α 2 M/CS (α 2 M/CS) complexes to bind to the surface of Hep-G2 cells. Cells were incubated with 200 μg/ml of each sample, followed by anti-α 2 M antibody and subsequently detected

97 CHAPTER 4: RESULTS 67 using sheep-anti-rabbit IgG-FITC conjugate. In contrast to the results obtained with JEG-3 cells, native α 2 M bound significantly to the surface of Hep-G2 cells (t = 29.88, p< 0.05, df =2) (Figure 4.3). α 2 M* also showed significant cell surface binding (t = 21.03, p< 0.05, df =2), however, the level of binding was slightly less than that of native α 2 M. Lastly, α 2 M/CS also showed significant binding to the surface of Hep-G2 cells (t = 23.49, p< 0.05, df =2) (Figure 4.3) LRP 80 α 2 M Number of Cells /20-A /20-A 100 α 2 M* 100 α 2 M/CS /20-A FITC Fluorescence /20-A Figure 4.3 Expression of LRP and binding of native α 2 M, α 2 M*, α 2 M/CS complexes to Hep-G2 cells. Histograms of Hep-G2 cells incubated with either rabbit anti-lrp antibody and subsequently sheep-antirabbit Ig-FITC conjugate (top left panel) or with native α 2 M, α 2 M*or α 2 M/CS (indicated in corresponding panels) followed by anti-α 2 M antibody and then sheep-anti-rabbit Ig-FITC conjugate. Other cells were separately incubated with anti-dnp antibody followed by sheep-anti-rabbit Ig-FITC conjugate (background fluorescence) which is shown as the red peak. Results are indicative of two independent experiments.

98 CHAPTER 4: RESULTS 68 In order investigate the possible receptor(s) responsible for the measured binding, cells were pre-incubated with saturating concentrations of either receptor associated protein (RAP) or galactose (a ligand of the asialoglycoprotein receptor family). It was found that preincubation of Hep-G2 cells with 100 μg/ml RAP had little effect on the binding of α 2 M and α 2 M/CS complexes. Pre-incubation of the cells with RAP significantly reduced the binding of α 2 M* to the cell surface, while pre-incubation with 1 mg/ml galactose significantly reduced the cell surface binding of α 2 M and to a lesser extent α 2 M/CS, but not the binding of α 2 M* (Figure 4.4). 40 Geomean Fluorescence No Inhibition GST RAP Galactose (1 mg/ml) 0 α 2 M α 2 M* α 2 M/CS Figure 4.4 Histogram plot showing the average geometric mean fluorescence (n = 2, ± range, arbitrary units) for immunochemical detection of the binding to Hep-G2 cells of α 2 M, α 2 M* and α 2 M/CS. Where appropriate, α 2 M was trypsin activated. In some cases, cells were pre-incubated with GST-RAP (see methods section for details) or 5 mm galactose (1 mg/ml). The values shown have been corrected for the fluorescence associated with cells stained with negative control (anti-dnp) antibody. The results shown are representative of two independent experiments.

99 CHAPTER 4: RESULTS Binding of α 2 M/Stressed Protein Complexes to Activated U937 Cells Following a 72 h exposure to 20 nm PMA (which is known to induce differentiation into monocyte-like cells), U937 cells expressed significant levels of LRP at their surface (Figure 4.5). Subsequent binding analysis determined that α 2 M and α 2 M/CS complexes had limited binding to activated U937 cells. Following trypsin activation, α 2 M* showed increased and significant binding (t = 17.53, p< 0.05, df =2) (Figure 4.5). LRP α 2 M Number of Cells α 2 M* α 2 M/CS FITC Fluorescence Figure 4.5 Expression of LRP and binding of native α 2 M, α 2 M* and α 2 M/CS complexes to PMA activated U937 cells. Histograms of U937 cells incubated with either rabbit anti-lrp antibody and subsequently detected with sheep-anti-rabbit Ig-FITC conjugate or with native α 2 M, α 2 M* or α 2 M/CS (yellow peaks), followed by anti-α 2 M antibody which was subsequently detected with sheep-anti-rabbit Ig- FITC conjugate. Other cells were separately incubated with sheep-anti-rabbit Ig-FITC conjugate alone (background fluorescence, red peak). Results are representative of two independent experiments.

100 CHAPTER 4: RESULTS 70 Preincubation of activated U937 cells with RAP resulted in a significant reduction in binding (t = 11.48, p< 0.05, df =2) (Figure 4.6). Preincubation of α 2 M* cells with the asialoglycoprotein receptor ligand asialofetuin (1 mg/ml) had little effect on α 2 M* binding. 60 Geomean Fluorescence No Inhibition Asialofetuin (1 mg/ml) GST RAP Figure 4.6 Histogram plot showing the average geometric mean fluorescence (n = 2, ± range, arbitrary units) for immunochemical detection of the binding of α 2 M* to activated U937 cells. α 2 M was trypsin activated. Cells were pre-incubated with GST-RAP (see methods section for details) or 1 mg/ml asialofetuin. The values shown have been corrected for the fluorescence associated with cells stained with negative control anti-dnp antibody. The results shown are representative of two independent experiments Binding of α 2 M/Stressed Protein Complexes to Granulocytes Granulocytes are the most abundant population of white blood cells and consist of three sub-populations; neutrophils, eosinophils and basophils. Preliminary data showed that granulocytes express a high level of LRP, amongst other receptors (Figure 4.7).

101 CHAPTER 4: RESULTS % of Max /20-A Figure 4.7 Detection of low density lipoprotein receptor family members on granulocytes. White blood cells were isolated from whole blood as per methods section and were incubated with (i) GST-RAPb followed by SA-Alexa fluor 488 (blue peak) or (ii) SA-Alexa fluor 488 alone (background fluorescence, red peak). The neutrophil population was selected for analysis by electronic gating on the basis of forward and side scatter. Results are representative of two independent experiments. The ability of native α 2 M, α 2 M* and purified α 2 M/CS complexes to bind to any receptors present on the granulocyte population was investigated using flow cytometry. The incubation of cells with native α 2 M (200 µg/ml), followed by subsequent incubations with rabbit-anti-human-α 2 M antibody and sheep-anti-rabbit Ig-FITC (SaRIg-FITC) conjugate detected only low levels of binding, not significantly greater than the background fluorescence (t = 5.07, p <0.05, df = 2). When activated by incubation with trypsin, α 2 M* showed increased binding to the surface of granulocytes. The α 2 M/CS complex also showed significant binding to granulocytes (t = 27.5, p <0.05, df = 2) (Figure 4.8). In order to establish if the substrate protein was responsible for the significant binding observed, CS was incubated with the cells alone. CS alone showed insignificant levels of binding to granulocytes.

102 CHAPTER 4: RESULTS 72 Number of Cells FITC Fluorescence Figure 4.8 Binding of native α 2 M, α 2 M*, CS and CS/α 2 M complexes to granulocytes. Histograms of cells incubated with either native α 2 M, α 2 M*, CS or CS/α 2 M (red peaks) followed by anti-α 2 M antibody which was subsequently detected by sheep-anti-rabbit Ig-FITC conjugate. Other cells were separately treated with sheep-anti-rabbit Ig-FITC conjugate alone (background fluorescence; red peaks). Results are indicative of two independent experiments. In order to characterize the receptor(s) responsible for the binding of α 2 M* and α 2 M/CS complexes, cells were pre-incubated with GST-RAP (100 μg/ml). It was found that GST- RAP significantly inhibited the binding of α 2 M* but had only a small effect on the binding of α 2 M/CS (Figure 4.9). These results suggest the possible existence of a novel receptor on the surface of granulocytes that is able to bind α 2 M within α 2 M/CS complexes.

103 CHAPTER 4: RESULTS Geomean Fluorescence No Inhibitor GST RAP 0 α 2 M* α 2 M/CS Figure 4.9 Histogram plot showing the average geometric mean fluorescence (n = 2, ± range, arbitrary units) for immunochemical detection of the binding of α 2 M* and α 2 M/CS to granulocytes. α 2 M was trypsin activated. Cells were pre-incubated with GST-RAP (see methods section for details). The values shown have been corrected for the fluorescence associated with cells stained with negative control (anti-dnp) antibody. The results shown are representative of two independent experiments. 4.4 Discussion The binding of α 2 M/stressed protein complexes to the surfaces of JEG-3, Hep-G2 and PMA-activated U937 cells was investigated. JEG-3, a human adenocarcinoma cell line, was found to express a high level of LRP detected by an anti-lrp antibody, supporting previous findings (Sarti et al., 2000). α 2 M and α 2 M/CS complexes showed only limited binding to JEG-3 cells, however after incubation with trypsin, activated α 2 M* and (α 2 M/CS)* showed an increased level of binding. This binding was RAP inhibitable, providing evidence that α 2 M* and (α 2 M/CS)* bind specifically to a member of the LDL receptor family. Furthermore, binding was also inhibitable with an anti-lrp antibody, establishing that the receptor responsible for the binding was LRP.

104 CHAPTER 4: RESULTS 74 The results presented for JEG-3 cells support those found in an earlier study which showed that complexes formed between α 2 M and heat-stressed protein only bound to receptors on LRP expressing cells (the U87 cell line) following activation with methylamine (French, 2005). The results further reinforce the idea that following the formation of complexes with a heat-stressed protein, α 2 M remains in a native-like conformation. However, α 2 M within these complexes can interact with proteases (e.g. trypsin) to expose the LRP binding site. In vivo, this may provide one mechanism by which these complexes can be cleared from the extracellular space. Hep-G2, a human hepatoma cell line, was used to investigate binding of α 2 M/stressed protein complexes not only to LRP, but also to scavenger receptors which are abundant on the surface of liver cells. Unlike results obtained with U87 (French, 2005) and JEG-3 cells, native α 2 M and α 2 M/CS complexes bound significantly to Hep-G2 cells, without the need to first undergo activation. Binding was also observed with α 2 M* but to a lesser extent. Whereas the binding of α 2 M* could be significantly reduced by pre-incubation with GST- RAP, this had little effect on the binding of α 2 M and α 2 M/CS to the cell surface. This suggests that a receptor other than LRP is responsible for the binding. One such receptor is the asialoglycoprotein (ASGP) receptor, a member of the scavenger receptor family with a high affinity for galactose and other galactosides (Spiess, 1990). In order to investigate the possible involvement of the ASGP receptor, cells were pre-incubated with galactose (1 mg/ml). This resulted in a reduction in binding of α 2 M and α 2 M/CS to Hep-G2 cells, but not the binding of α 2 M*. These preliminary results suggest that a receptor other than LRP

105 CHAPTER 4: RESULTS 75 may play a role in the binding and uptake of α 2 M/stressed protein complexes, without the need for activation of the complexes by proteases. The incubation of confluent U937 cells with PMA results in their differentiation into monocyte-like cells. Monocytes are known to express a high level of LRP at their surface, and have been associated with the disposal of α 2 M antigen complexes in vivo (Hart et al., 2004). Results presented in this chapter indicate that α 2 M and α 2 M/CS complexes are unable to bind to the surface of PMA-differentiated U937 cells, however α 2 M* shows significant, RAP-inhibitable binding indicating the involvement of an LDL family receptor. The ASGP receptor ligand, asialofetuin (1mg/ml) had little impact on the level of α 2 M* binding, indicating that the binding observed was not mediated by scavenger receptors, which are also present on monocytes. Granulocytes are the most abundant population of white blood cells, consisting of three sub-populations; neutrophils, eosinophils and basophils. Previous investigations indicated that α 2 M/stressed protein complexes bound significantly to granulocytes without the need for protease activation (French, 2005). The granulocyte population was selected on the basis of cell characteristics of forward and side scatter using a flow cytometer, and the binding of α 2 M, α 2 M* and α 2 M/CS determined. α 2 M* showed significant RAP-inhibitable binding, indicating the involvement of the LDLR family which is consistent with the results of past investigations (French, 2005). α 2 M/CS complexes also bound significantly to the surface of granulocytes but this binding was not inhibited by RAP. This finding has also been reported in previous studies and suggests that a receptor distinct from LRP may be

106 CHAPTER 4: RESULTS 76 responsible, at least in part, for the binding of α 2 M/stressed protein complexes to granulocytes (French, 2005). Further studies examining firstly the presence of the ASGPR on the surface of granulocytes, and secondly, the ability of ASGP ligands to inhibit binding of α 2 M/CS complexes is required in order to determine whether ASGPR is involved in the binding of α 2 M/stressed protein complexes to granulocytes. Collectively, the results presented in this chapter indicate that α 2 M may play a vital role in the disposal of misfolded extracellular proteins. The recognition of activated α 2 M/stressed protein complexes by LRP and the possible existence of a receptor for native α 2 M/stressed protein complexes are just two mechanisms by which misfolded extracellular proteins may be internalised for subsequent removal and degradation.

107 CHAPTER 4: RESULTS 77 Bu, G., E. A. Maksymovitchi, H. Geuzen and A. L. Schwartzi (1994). "Subcellular localization and endocytic function of low density lipoprotein receptor-related protein in human glioblastoma cells." Journal of Biological Chemistry, 269: Bu, G. and A. L. Schwartz (1998). "Rap, a novel type of er chaperone." Trends in Cell Biology, 8: Elsas, M. I., A. J. Dessein and P. X. Elsas (1990). "Selection of u937 histiocytic lymphoma cells highly responsive to phorbol ester-induced differentiation using monoclonal antibody to the eosinophil cytotoxicity-enhancing factor." Blood, 75: Feldman, S. R., K. A. Ney, S. L. Gonias and S. V. Pizzo (1983). "In vitro binding and in vivo clearance of human [alpha]2-macroglobulin after reaction with endoproteases from four different classes." Biochemical and Biophysical Research Communications, 114: French, K. (2005). Alpha-2-macroglobulin: A putative extracellular chaperone. School of Biological Sciences. Wollongong, University of Wollongong: 93. Grimsley, P., G., K. Quinn, A., C. Chesterman, N. and D. Owensby, A. (1998). "Low density lipoprotein receptor-related protein (lrp) expression varies among hep g2 cell lines." Thrombosis Research, 88: Hart, J. P., M. D. Gunn and S. V. Pizzo (2004). "Acd91-positive subset of cd11c+ blood dendritic cells: Characterisation of the apc that functions to enhance adaptive immune responses against cd91-targeted antigens." The journal of Immunology, 172: Hertz, J., J. L. Goldstein, D. K. Strickland, Y. K. Ho and M. S. Brown (1991). "39 kda protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha-2-macroglobulin receptor." Journal of biological chemistry, 266: Moestrup, S. K. and J. Gliemann (1991). "Analysis of ligand recognition by the purified a2- macroglobulin receptor (low density lipoprotein receptor-related protein)." The Journal of Biological Chemistry, 266: Sarti, M., M. G. Farquhar and R. A. Orlando (2000). "The receptor-associated protein (rap) interacts with several resident proteins of the endoplasmic reticulum including a glycoprotein related to actin." Experimental Cell Research, 260: Schwartz, A. L., S. E. Fridovich and H. F. Lodish (1982). "Kinetics of internalization and recycling of the asialoglycoprotein receptor in a hepatoma cell line." J. Biol. Chem., 257:

108 CHAPTER 4: RESULTS 78 Spiess, M. (1990). "The asialoglycoprotein receptor: A model for endocytic transport receptors." Biochemistry, 29:

109 CHAPTER 5: RESULTS 77 CHAPTER 5: OXIDATIVE STRESS AND THE CHAPERONE FUNCTION OF α 2 M 5.1 Introduction: Previous investigations have identified that α 2 M has the ability to protect substrate proteins from heat stress at 43 C by forming stable complexes with them (French 2005). The ability of α 2 M to exert a similar effect with chemically stressed proteins was yet to be tested. During times of inflammation, in response to cellular injury and bacterial invasion there is not only a rise in temperature but also a marked increase in the level of oxidative products and free radicals (Khan and Khan 2004). Levels of neutrophil derived oxidants released during the oxidative burst, including hypochlorite (HOCl), hydroxyl radical (OH) and hydrogen peroxide (H 2 O 2 ), can be in the millimolar range (Wu and Pizzo 1999). Various oxidants are known to have an impact on the structure and protease inhibitor activity of α 2 M. Halogen oxidized α 2 M dimers display normal bait regions (which contain protease cleavage sites), however they are unable to covalently bind or trap proteases (Reddy, Desorchers et al. 1994). Studies have also identified that hypochlorite oxidation of α 2 M* completely destroys its ability to bind to LRP (Wu, Boyer et al. 1997). Many chaperones have been reported to exist in solution as aggregates of undefined size. In order to account for this, a convention has been devised, the subunit molar ratio (SMR), for dealing with the interactions between chaperones and other proteins. The SMR relates to the stoichiometry of the individual subunits of the chaperone and of the substrate protein with which it interacts (Humphreys, Carver et al. 1999). The calculations in this chapter

110 CHAPTER 5: RESULTS 78 assume that the molecular mass for an intact α 2 M tetramer is 720 kda, with a homodimer subunit mass of 360 kda. The ability of α 2 M to chaperone proteins undergoing oxidative stress and the effects of oxidation on the chaperone activity of α 2 M* (which is chaperone inactive under heat stress conditions) had not been tested. In order to investigate the effects of α 2 M on the oxidationinduced precipitation of protein, the substrate protein lysozyme (lys) was incubated in oxidative stress buffer (OSB). Investigations were also undertaken to investigate the effects of oxidative stress on the structure and protease inhibitor function of α 2 M. Considering the high concentration of oxidative species present during the inflammatory response, the ability of α 2 M and α 2 M* to bind and mediate removal of stressed proteins from the extracellular environment is likely to play a significant role in the process of inflammation. The concentrations of α 2 M and α 2 M*, especially the latter, are markedly increased during inflammation. This study was undertaken with the aim of increasing knowledge of the role(s) of A2M and its chaperone action in processes related to the inflammatory response. 5.2 Methods: Refer to materials and methods sections Results: α 2 M Undergoes Conformational Changes when Exposed to Oxidative Stress α 2 M is a tetrameric molecule, consisting of two identical, disulfide-linked dimers, noncovalently associated to form the native 720 kda molecule. When analysed by reducing SDS PAGE, α 2 M migrated as a single band at 180 kda, representing a single α 2 M subunit

111 CHAPTER 5: RESULTS 79 (Figure 5.1, lane 1). Upon interaction with a protease, α 2 M becomes activated, undergoing a characteristic conformational change, initiated by cleavage of the bait region and subsequent cleavage of the highly reactive thiolester bond. The cleavage of the bait region following interaction with trypsin resulted in the appearance of a single band at ~ 85 kda, representing the fragmented subunit (Figure 5.1, lane 3). In the presence of small nucleophiles such as methylamine, α 2 M also becomes activated, however in this scenario the thioester bond is cleaved but the bait region is not. When analysed by SDS PAGE, direct cleavage of the thioester bond resulted in a band with similar migration to native α 2 M (Figure 5.1, lane 2). When native α 2 M was incubated with OSB for 2 h at 37 C, the structure of the molecule was found to vary from the native conformation, adopting an active-like conformation. When analysed by reducing SDS PAGE, the oxidized form of α 2 M migrated as two distinct bands, the major one representing an 85 kda fragment of the subunit (analogous to the trypsin activated sample) and the other representing the intact 180 kda subunit (Figure 5.1, lane 4) Molecular Mass (KDa) Figure 5.1 Image of Coomassie Blue stained 10% SDS PAGE gel showing the fragmentation of α 2 M when exposed to oxidative stress. Molecular mass standards (left-most lane) were used to estimate the mass of proteins present. Native α 2 M (lane 1), α 2 M + methylamine (lane 2), α 2 M + trypsin (3-fold molar excess) (lane 3), α 2 M + oxidative stress buffer (OSB) (lane 4). Results shown are representative of two independent experiments.

112 CHAPTER 5: RESULTS 80 In order to further investigate the effect of oxidation on the structure of α 2 M, samples of native and oxidized α 2 M were analysed using size exclusion chromatography. When injected onto a Superose 6 column, native α 2 M eluted as a dominant, symmetrical peak (Figure 5.2, purple line). However, when similarly analysed, oxidized α 2 M showed an additional shoulder peak, eluting more slowly than the main peak (Figure 5.2, dark blue line). This suggests that some fragmentation of the α 2 M had occurred kda 669 kda 158 kda 67 kda Absorbance (A280 nm) Elution volume (ml) Figure 5.2 Plot showing the A 280 as a function of elution volume for SEC of native and oxidised α 2 M. 500 µl samples of α 2 M (1 mg/ml) and oxidized α 2 M (1 mg/ml; pre-incubated in OSB as per methods section) were passed over a Superose 6 column, equilibrated in PBS. α 2 M is shown as the purple line and the oxidized form is represented by the blue line. Molecular mass standards were used to confirm the identity of the eluted peaks (>2000 kda, blue dextran; 669 kda, thyroglobulin; 158 kda, aldolase; 67 kda bovine serum albumin).

113 CHAPTER 5: RESULTS 81 In order to further characterize the effects of oxidation on the structural integrity of α 2 M, samples were subjected to native gel electrophoresis (NGE). NGE separates proteins on the basis of mass and overall charge. Alterations to the structure of α 2 M in response to oxidative stress are likely to alter its size and charge. Therefore it was expected that the migration of oxidized α 2 M on the native gel would differ somewhat from that of the native form. Solutions of α 2 M (2.5 mg/ml), trypsin activated α 2 M* (2.5 mg/ml) or oxidized α 2 M (2.5 mg/ml) were loaded onto a native agarose gel. As expected, α 2 M* displayed a migration pattern different to that of the native species. α 2 M* migrated further and in a more compact band compared to the native α 2 M species. The mobility of oxidized α 2 M also differed markedly from the native form (Figure 5.3). In comparison to native α 2 M, the oxidized form migrated further on the gel, and also exhibited a broader pattern of migration. This result confirms the interpretation that α 2 M undergoes major structural modifications in response to oxidative stress, resulting in alterations to either the size and/or overall charge of the molecule. α 2 M α 2 M* α 2 M (oxidized) Figure 5.3 Analysis of various forms of α 2 M by NGE. Samples (30 μg) of α 2 M, α 2 M* or oxidised α 2 M, were analysed on a 1% native agarose gel. The identity of the samples loaded is indicated next to the corresponding wells. The gel was stained using Imperial protein stain. The results are representative of two independent experiments.

114 CHAPTER 5: RESULTS α 2 M Functions as a Chaperone Under Oxidative Conditions by Forming Stable, Soluble Complexes With the Chaperone Substrate Protein The ability to inhibit protein precipitation by forming complexes with the substrate protein is a characteristic feature of the shsps and the extracellular molecular chaperones clusterin and haptoglobin. It was previously shown that α 2 M is able to inhibit the precipitation and aggregation of heat stressed proteins by forming stable complexes with them (French, 2005), however the ability of α 2 M to function as a chaperone for oxidatively stressed substrates was untested. NGE and affinity absorption were used in an attempt to identify, for the first time, high molecular weight complexes formed between α 2 M and an oxidatively stressed substrate protein, lysozyme. The effect of α 2 M on the oxidation-induced precipitation of lysozyme was investigated. In the absence of α 2 M, lysozyme (lys; 1 mg/ml) gradually precipitates in OSB, indicated by an increase in absorbance at 360 nm. α 2 M showed dose-dependant inhibition of oxidationinduced precipitation of lys (Figure 5.4). In the presence of substoichiometric SMRs of α 2 M (see section 3.1), the level of lys precipitation was markedly reduced. At SMRs of α 2 M:lys of 1:50 and 1:5, the lys precipitation was reduced by 40% and 80%, respectively (Figure 5.4).

115 CHAPTER 5: RESULTS A 360 nm Time (min) Figure 5.4 The effect of α 2 M on the oxidation-induced precipitation of lysozyme. Lysozyme (69 μm) was incubated in OSB in the presence or absence of α 2 M ( μm) and the turbidity associated with precipitation detected as an increase in absorbance at 360 nm, as described in the methods section 2.8. Data points shown are individual measurements and are representative of at least three independent experiments. Lys was stressed in the presence of various concentrations of α 2 M (SMRs of α 2 M:lys are shown in brackets following the respective concentrations) [0 mg/ml ( ), 0.5 mg/ml (1: 50)( ), 2.5 mg/ml (1: 10) ( ) and 5 mg/ml (1:5) ( )]. In order to confirm that the chaperone activity was due to the presence of α 2 M alone and not a result of non-specific protein-protein interactions, assays were also conducted using the oxidation-stable control proteins superoxide dismutase (SOD) and bovine serum albumin (BSA). In all assays, SOD and BSA were used at the same maximum SMR used for α 2 M and were found to have negligible effects on the precipitation of lys (Figure 5.5).

116 CHAPTER 5: RESULTS 84 A 360 nm Time (min) Figure 5.5 The effect of superoxide dismutase (SOD) and bovine serum albumin (BSA) on the oxidative stress-induced precipitation of lysozyme. Lys (70 μm) ( ) was incubated in OSB at 37 C for 2 h alone or in the presence of SOD (7 μm) ( ) or BSA (7 μm) ( ) The turbidity associated with protein precipitation was detected as an increase in absorbance (A 360 ). Data points shown are individual measurements and are representative of two independent experiments. Once α 2 M was established as an inhibitor of the oxidation-induced precipitation of lysozyme, tests were then carried out to determine if α 2 M exerted this effect by forming stable complexes with oxidized lys, as shown previously for heat stressed proteins. Firstly, NGE was used in an attempt to identify a complex formed between α 2 M and lys. When analysed alone, α 2 M and lys were found to migrate to distinct positions on the gel according to their mass and charge. Oxidised α 2 M migrated slightly further on the gel than α 2 M, whereas oxidized lys was barely detected on the gel (this was due to it largely precipitating from solution). Mixtures containing α 2 M (2.5 mg/ml) and lys (1 mg/ml) under non-oxidising conditions showed bands corresponding to both constituents (Figure 5.6). However, when this mixture was exposed to oxidative stress, a band of unique electrophoretic mobility was observed, representing a putative α 2 M/lys complex (Figure 5.6; black arrowhead).

117 CHAPTER 5: RESULTS 85 Putative complex (α 2 M + lys) (OSB) α 2 M + lys (PBS) α 2 M (OSB) α 2 M (PBS) lys (OSB) lys (PBS) Figure 5.6 Detection of putative α 2 M/lysozyme (lys) complexes by NGE. Samples of lys (1 mg/ml), α 2 M (2.5 mg/ml) or a mixture of lys and α 2 M (at the same final concentrations) were incubated at 37 C in OSB (lys (ox) and α 2 M (ox) or α 2 M + lys (ox) respectively) or PBS (lys, α 2 M or lys + α 2 M respectively) and then analysed on a 1% native agarose gel. The arrowhead indicates a band of unique electrophoretic mobility which represents a putative α 2 M/lys complex. The results obtained from NGE suggested that α 2 M may form a complex with oxidatively stressed lys. This interpretation was confirmed using streptavidin-agarose to affinity adsorb proteins from oxidized and non-oxidised mixtures of α 2 M and biotinylated lysozyme (lysb). The protein adsorbed was analysed using reducing SDS-PAGE. In non-oxidised mixtures, only the reduced form of the lys-b was evident (Figure 5.7). Following oxidation, α 2 M alone did not bind to the streptavidin-agarose beads. However, both lys-b and α 2 M were eluted from streptavidin-agarose beads that had been incubated in an oxidized mixture of the two proteins (Figure 5.7). This result demonstrates that α 2 M forms soluble complexes with lysozyme under conditions of oxidative stress, but not under normal physiological conditions.

118 CHAPTER 5: RESULTS α 2 M+ lys-b α 2 M lys-b non-ox OX α 2 M Molecular Mass (kda) lys-b Figure 5.7 Image of a Coomassie Blue-stained 10% reducing SDS PAGE gel showing proteins affinity adsorbed by streptavidin-agarose from oxidized (OX) and non-oxidised (non-ox) solutions of lys-b, or α 2 M, or mixtures of α 2 M and lys-b. The identity of samples adsorbed by the streptavidin-agarose beads is indicated above the corresponding lanes. The identity of bands was established by comparison with molecular mass standards (as shown). The position of α 2 M is shown by the black arrowhead and the position of lys-b is indicated by the empty arrowhead. The results shown are representative of two independent experiments. It was previously shown that α 2 M within α 2 M/heat-stressed protein complexes retains the ability to bind proteases such as trypsin (Chapter 3). A trypsin binding assay was undertaken to investigate the protease binding ability of α 2 M within α 2 M/oxidized-protein complexes. In the classical trypsin binding assay a molar excess of trypsin is incubated with α 2 M. Trypsin interacts with α 2 M to produce α 2 M*, coincidently trapping the protease in the steric cage. In this situation trypsin remains able to cleave substrates small enough to diffuse into the cage (less than 10 kda). In contrast α 2 M* is unable to bind trypsin. Unbound trypsin may be subsequently inactivated using soybean trypsin inhibitor (which is sterically unable to access and inactivate trypsin bound to α 2 M). Residual trypsin activity, attributable to α 2 M-trapped trypsin, was measured using the low molecular weight

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