Alpha-2-Macroglobulin: an Abundant Extracellular Chaperone
University of Wollongong Theses Collection University of Wollongong Theses Collection
University of Wollongong Year
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, 2008. http://ro.uow.edu.au/theses/114
This paper is posted at Research Online. http://ro.uow.edu.au/theses/114
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
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
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 α2M and shared the many trials
and tribulations of the α2M 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 2006- 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 α2M 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! Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone iv
ABSTRACT
Alpha-2-macroglobulin (α2M) 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. α2M is best known for its ability to inhibit a broad spectrum of proteases which it accomplishes using a unique
“trapping” method. Protease trapping induces α2M 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. α2M 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. α2M 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 α2M 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, α2M was found to inhibit the oxidation- induced precipitation of lysozyme (lys). In a preliminary study, it was shown that α2M forms stable, soluble complexes with heat-stressed proteins. In the current study, native agarose gel electrophoresis and immunoprecipitation analyses were used to demonstrate that α2M also forms stable, soluble complexes with oxidised proteins. Removal of α2M 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
α2M from human plasma (following incubation at either 43 °C or room temperature for 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 α2M. It was also shown that protease-mediated activation of α2M abolishes the chaperone activity, but that native α2M is able to form soluble complexes with heat stressed proteins and then subsequently become activated by protease trapping. Oxidation of (chaperone-inactive) protease bound α2M was shown to restore chaperone activity but not the protease inhibitor function. These behaviours provide an alternative means for generating α2M/stressed protein/protease complexes which could be cleared in vivo by LRP-mediated cellular uptake and degradation.
The ability of α2M/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. α2M/CS complexes had limited ability to bind to LRP expressed on the surface of JEG-3 cells. However, preliminary results indicated that activation of
α2M (α2M*) and α2M/stressed protein complexes (α2M*/CS) with trypsin resulted in subsequent binding to the surface of JEG-3 cells. Native α2M/CS complexes were found to bind to granulocytes and Hep-G2 cells via unidentified, non-LRP receptors.
Collectively, the results presented here further establish α2M as a potent extracellular chaperone with the ability to protect proteins from heat and oxidation-induced stress.
α2M 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 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone vi
proteins. This thesis provides evidence supporting the hypothesis that α2M 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.
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 ...... 1 1.1 PROTEIN FOLDING ...... 1
1.2 PROTEIN MISFOLDING, AGGREGATION AND DISEASE...... 3
1.3 MECHANISMS OF PROTEIN QUALITY CONTROL ...... 6
1.3.1 Molecular Chaperones - The Saviours of Protein Folding ...... 7
1.4 EXTRACELLULAR CHAPERONES ...... 9
1.4.1 Clusterin ...... 10
1.4.2 Haptoglobin...... 11
1.4.3 Serum Amyloid P Component ...... 12
1.4.4 Alpha-2-macroglobulin...... 12
1.5 ALPHA-2-MACROGLOBULIN...... 14
1.5.1 Synthesis, Structure and Protease Inhibitor Action of α2M ...... 14
1.5.2 Other Functions of α2M...... 18
1.5.3 Binding of α2M to Ligands...... 18
1.5.4 Receptor Binding and Internalisation of α2M ...... 22
1.5.5 The Effects of Oxidative Stress on the Structure, Function and Receptor
Recognition of α2M...... 25
Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone viii
1.6 AIMS ...... 29
CHAPTER 2: MATERIALS AND METHODS ...... 30 2.1 MATERIALS...... 30
2.2 PURIFICATION OF α2M...... 31
2.3 PREPARATION OF ACTIVATED α2M AND ACTIVATED (α2M/CS)*
COMPLEXES...... 31
2.4 PREPARATION OF OXIDISED α2M ...... 32
2.4.1 Size Exclusion Chromatography...... 32
2.5 FORMATION AND PURIFICATION OF COMPLEXES BETWEEN
α2M ANS STRESSED PROTEIN...... 33
2.6 ELECTROPHORESIS...... 33
2.6.1 SDS PAGE...... 33
2.6.2 Immunodetection ...... 34
2.6.3 Native Gel Electrophoresis ...... 35
2.6.4 Native PAGE...... 35
2.7 TRYPSIN BINDING ASSAY ...... 36
2.8 PROTEIN PRECIPITATION ASSAYS...... 36
2.9 PRECIPITATION OF PROTEINS IN WHOLE HUMAN PLASMA...... 37
2.9.1 Determination of Protein Concentration using BCA Assay ...... 38
2.9.2 Immunoprecipitations ...... 39
2.10 IDENTIFICATION OF ENDOGENOUS SUBSTRATES USING MASS SPECTROMETRY ...... 39
2.10.1 Spot Excision ...... 39
2.10.2 Trypsin Digestion...... 40 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone ix
2.10.3 MALDI-TOFF Mass Spectrometry...... 40
2.11 CELL CULTURE AND FLOW CYTOMETRY ...... 41
2.11.1 Culture of Cell Lines...... 41
2.11.2 Binding Assays using JEG-3,Hep-G2 and Activated U937 Cells ...... 42
2.11.3 Binding Assays using Granulocytes Isolated from Whole Blood...... 42
2.11.4 Binding Analysis using Flow Cytometry...... 43
CHAPTER 3: CHARACTERISING THE CHAPERONE
FUNCTION OF α2M...... 44 3.1 INTRODUCTION ...... 44
3.2 METHODS ...... 45
3.3 RESULTS ...... 45
3.3.1 Within α2M/heat Stressed Protein Complexes, α2M Remains
in its Native Conformation...... 45
3.3.2 Protease Activation Abolishes the Chaperone Activity of α2M...... 48
3.3.3 α2M Inhibits the Heat Induced Precipitaion of Proteins
in Whole Human Serum ...... 52
3.3.4 Identifying Endogenous Chaperone Substrates for α2M...... 55
3.4 DISCUSSION ...... 59
CHAPTER 4: BINDING OF α2M/STRESSED PROTEIN COMPLEXES TO CELL SURFACE RECEPTORS . 62 4.1 INTRODUCTION ...... 62
4.2 METHODS ...... 64
4.3 RESULTS ...... 64
4.3.1 Binding of α2M/Stressed Protein Complexes to JEG-3 cells...... 64 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone x
4.3.2 Binding of α2M/Stressed Protein Complexes to Hep-G2 cells ...... 66
4.3.3 Binding of α2M/Stressed Protein Complexes to Activated U937 cells..... 69
4.3.4 Binding of α2M/Stressed Protein Complexes to Neutrophils...... 70
4.4 DISCUSSION ...... 73
CHAPTER 5: OXIDATIVE STRESS AND THE CHAPERONE
ACTION OF α2M...... 77 5.1 INTRODUCTION ...... 77
5.2 METHODS ...... 78
5.3 RESULTS ...... 78
5.3.1 α2M Undergoes Conformational Changes under Oxidative Stress...... 78
5.3.2 α2M Functions as a Chaperone under Oxidative Conditions ...... 82
5.3.3 Oxidation of activated α2M Re-establishes Chaperone Action...... 89
5.4 DISCUSSION ...... 92
CHAPTER 6: DISCUSSION ...... 96
6.1 ADVANCES IN UNDERSTANDING THE CHAPERONE ACTION OF α2M ... 96
6.1.1 Dual Chaperone and Protease Inhibitory Roles of α2M ...... 97
6.2 OXIDATIVE STRESS AND α2M ...... 98
6.2.1 Implications for Inflammatory Response Regulation and Chaperone
Functionality ...... 99
6.3 FIBRINOGEN IS AN ENDOGENOUS CHAPERONE SUBSTRATE OF α2M 101
6.4 Cell Surface Receptor Binding of α2M and α2M/Stressed Protein Complexes ... 102
6.5 CONCLUSIONS...... 106
CHAPTER 7: REFERENCES ...... 107
Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone xi
ABBREVIATIONS
A360 Absorbance at 360 nm
A405 Absorbance at 405 nm
Aβ Amyloid-beta peptide
Alexa 488 Alexa fluor® 488
α2M Alpha-2-macroglobulin
α2M* Activated alpha-2-macroglobulin
α2M/CS Complex formed between alpha-2-macroglobulin and stressed
(unfolded) citrate synthase
α2M/CSb Complex formed between alpha-2-macroglobulin and
biotinylated, stressed (unfolded) citrate synthase
α2M*/CSb Complex formed between alpha-2-macroglobulin and
biotinylated, stressed (unfolded) citrate synthase which has been
activated.
α2M/CPK Complex formed between alpha-2-macroglobulin and stressed
(unfolded) creatine phosphokinase
α2M/CPKb Complex formed between alpha-2-macroglobulin and
biotinylated, stressed (unfolded) creatine phosphokinase
α2M/lys Complex formed between alpha-2-macroglobulin and lysozyme
α2MR Alpha-2-macroglobulin receptor
α2MR/LRP Alpha-2-macroglobulin/ Low density lipoprotein receptor-related
protein (the same receptor).
ASGP Asialoglycoprotein
ATP Adenosine triphosphate
Az Azide
BCA Bicinchoninic acid Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone xii
bisANS 4, 4’-dianilino- 1, 1’-binaphthyl-5, 5’-disulfonic acid
CPK Creatine phosphokinase
CPKb Biotinylated creatine phosphokinase
CS Citrate synthase
CSb Biotinylated citrate synthase
CNS Central nervous system dH2O Distilled water
Da Dalton
DMEM: F-12 Dulbecco’s modified eagle medium: F-12
DMSO Deoxymethylsulphoxide
ECL Enhanced chemiluminescence detection
EDTA Ethylenediamine tetracetic acid
FCS Foetal calf serum
FITC Fluoresein Isothiocyanate
FPLC Fast protein liquid chromatography
Geomean Geometric mean
GST-RAP Fusion protein containing glutathione-S-transferase and receptor
associated protein
GST-RAPb Biotinylated fusion protein containing glutathione-S-transferase
and receptor associated protein
HDC Heat denatured casein
HEPES N-(hydroxyethyl) piperazine-N’-(2-ethanesulfonic acid)
HRP Horse radish peroxidase g G- force
GST Glutathione-S-transferase
Hsp Heat shock protein
Hsp70 Heat shock protein 70 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone xiii
HMW High molecular weight
HPLC High pressure liquid chromatography
Ig-HRP Immunoglobulin conjugated to horse radish peroxidase
IPTG Isopropyl-1-thio-β-D-galactopyranoside
KD Constant of dissociation kDa Kilo Dalton
LB Luria Bertani
LDL Low density lipoprotein
LDLR Low density lipoprotein receptor
LRP Low density lipoprotein receptor-related protein lys Lysozyme
M Molar (moles/litre) mg Milligram (1 x 10-3 grams)
μg Microgram (1 x 10-6 grams) ml Millilitre (1 x 10-3 litres)
μl Microlitre (1 x 10-6 litres) mM Millimolar (1 x 10-3 moles/litre)
μM Micromolar (1 x 10-6 moles/litres)
NGE Native agarose gel electrophoresis
OSB Oxidative stress buffer
OVO Ovotransferrin
PBL Peripheral blood Leukocytes
PBS Phosphate buffered saline
PCDs Protein conformational disorders
PI Propidium iodide pI Isoelectric point Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone xiv
PMSF Phenylmethylsulphonylfluoride
RAP Receptor-associated protein
SA Streptavidin
SaRIg-FITC Sheep-anti-rabbit-immunoglobulin conjugated to FITC
SD Standard deviation
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SEC Size exclusion chromatography sHsp Small heat shock protein
TAE Tris-acetate-EDTA
TEMED N, N, N’, N- tetramethyl-ethylenediamine
TRIS Tri (Hydroxymethyl) aminomethane
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 ...... 15
Figure 1.5 Steric trapping of protease molecules by α2M ...... 17
Figure 1.6 Locations of binding sites within the α2M subunit ...... 21
Figure 1.7 Receptors mediate binding, internalisation an cell signalling of α2M ... 25
Figure 3.1 Native PAGE showing conformation of α2M within complexes...... 46
Figure 3.2 Trypsin binding ability of α2M within α2M/stressed protein complexes47
Figure 3.3 Activation abolishes the chaperone activity of α2M ...... 48
Figure 3.4 Native PAGE showing activation of α2M within α2M/CS complexes... 49
Figure 3.5 α2M/stressed proteins can trap trypsin ...... 50
Figure 3.6 SDS PAGE showing the effects of trypsin on α2M
and α2M/CS complexes...... 52
Figure 3.7 Depletion of α2M from normal human plasma ...... 53
Figure 3.8 α2M inhibits heat stress-induced precipitation in whole human plasma 54
Figure 3.9 α2M inhibits precipitation in whole human plasma at 37°C ...... 55
Figure 3.10 SDS PAGE identifing putative endogenous substrates for α2M...... 56
Figure 3.11 Mass spectra of trypsin digested endogenous substrates of α2M...... 57
Figure 3.12 Fibrinogen is an endogenous substrate of α2M under heat stress ...... 58
Figure 4.1 Binding of α2M and α2M/stressed protein complexes to JEG-3 cells... 65 Figure 4.2 Inhibition of JEG-3 cell binding using RAP and anti-LRP antibody ..... 66
Figure 4.3 Binding of α2M and α2M/stressed protein complexes to Hep-G2 cells 67 Figure 4.4 Inhibition of Hep-G2 cell binding using RAP and galactose ...... 68
Figure 4.5 Binding of α2M and α2M/stressed protein complexes to U937 cells .... 69 Figure 4.6 Inhibition of U937 cell binding using RAP and asialofetuin ...... 70 Figure 4.7 Detection of LDLR family members on granulocytes ...... 71
Figure 4.8 Binding of α2M and α2M/stressed protein complexes to granulocytes . 72 Figure 4.9 Inhibition of granulocyte cell binding using RAP ...... 73
Figure 5.1 SDS PAGE showing fragmentation of α2M under oxidative stress..... 79
Figure 5.2 SEC of native and oxidised α2M ...... 80
Figure 5.3 NGE of α2M under oxidative stress ...... 81 Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone xvi
Figure 5.4 The effect of α2M on oxidation-induced precipitation of lysozyme ...... 83 Figure 5.5 The effect of SOD and BSA on lys precipitation...... 84
Figure 5.6 Detection of putative α2M/lys complexes using NGE ...... 85
Figure 5.7 SDS PAGE showing α2M/lys complexes ...... 86
Figure 5.8 Effects of complex formation on the trypsin binding activity of α2M .. 87
Figure 5.9 α2M inhibits oxidation-induced precipitation in whole human serum... 88
Figure 5.10 The effect of α2M on the oxidation induced precipitation of lys ...... 89
Figure 5.11 Effects of pre-oxidised α2M and α2M* on protein precipitation...... 90
Figure 5.12 Effect of oxidation on the protease inhibitor function of α2M ...... 91
Figure 5.13 Proposed structural and functional changes to oxidised α2M ...... 95
Figure 6.1 Proposed model for the chaperone action of α2M ...... 105
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 α2M ...... 45 Table 4.1 Characteristics of the cell lines used in the study ...... 62
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 (60-100 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 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).
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). CHAPTER 1: INTRODUCTION 4
Non-native proteins inevitably expose hydrophobic residues to the solvent, leading to self- association 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- 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).
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 (non- native; NN), which may progress to an unfolded conformation (U) via intermediate species (I1, I2). Non- native 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 post- translational 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 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).
1.3.1 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 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. 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 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).
1.4.1 Clusterin
Clusterin is an abundant extracellular protein present at concentrations ranging from 50-
370 μg/ml in human serum and 2.1-15 mg/ml in seminal fluid (O'Bryan et al., 1990).
Structurally, clusterin is a 75-80 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 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.
1.4.2 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
-15 with high affinity (KD ~ 10 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 0.3-2 mg/ml, with the basal concentration increased as part of the 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).
1.4.3 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.
1.4.4 Alpha-2-Macroglobulin (α2M)
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 (α2M), which was found to CHAPTER 1: INTRODUCTION 13
exhibit chaperone-like characteristics (French, 2005; French et al., 2008). Unlike the previously identified extracellular chaperones, α2M has numerous well described functions and established physiological roles, most importantly as a protease inhibitor.
The ability of α2M 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 α2M and the previously identified extracellular chaperones clusterin (Wilson and Easterbrook-Smith,
2000) (1.4.1) and haptoglobin (1.4.2) (Yerbury et al., 2005). α2M, 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 (α2M 2 - 4 mg/ml (Sottrup-Jensen, 1989), clusterin 50-370
µg/ml (O'Bryan et al., 1990) and haptoglobin 0.3-1.9 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).
CHAPTER 1: INTRODUCTION 14
Recent work showed that α2M 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 α2M 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 α2M 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 α2M, 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
1.5.1 Synthesis, Structure and Protease Inhibitor Action of α2M
α2M 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). α2M 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). α2M is encoded by a single copy gene, found within a cluster comprising an authentic α2M gene, an α2M pseudogene and the
PZP gene on the human chromosome 12p 12-13 (Matthijs et al., 1992). α2M is synthesised by several cell types including lung fibroblasts, monocytes-macrophages, hepatocytes and CHAPTER 1: INTRODUCTION 15
astrocytes (Sottrup-Jensen, 1989). Human neuronal cells may also be stimulated to produce
α2M 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 α2M in vitro and in vivo (Bizik et al., 1989).
α2M is a major human blood glycoprotein comprised of ~ 10% carbohydrate by mass
(Borth, 1992). In humans, α2M 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 α2M is formed by the non-covalent interaction of two identical dimers (green and red) (B) Structure of the two subunits of dimeric α2M. 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). CHAPTER 1: INTRODUCTION 16
α2M 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, α2M 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 α2M by reacting with an intramolecular thiol-ester bond to yield “activated” α2M
(α2M*), which exposes a receptor recognition site for LRP; in vivo, the α2M*/protease complex is cleared by LRP-mediated endocytosis and subsequently degraded (Sottrup-
Jensen, 1989). The “trapped” protease retains 80-100% of its hydrolytic activity against low molecular weight substrates (Travis and Salvesen, 1983). Therefore, the ‘trapping’ mechanism used by α2M 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).
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 α2M in native polyacrylamide gel electrophoresis (Barratt et al., 1979; Barratt, 1981). The circulating form of α2M which is reactive with proteases runs ‘slow’ on the gel, while the activated form of α2M (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 α2M to methylamine at alkaline pH results in the covalent incorporation of 4 molecules of methylamine per α2M tetramer, inducing a conformational change identical to that which occurs during interaction with proteases (Imber and Pizzo,
1981).
CHAPTER 1: INTRODUCTION 18
1.5.2 Other Functions of α2M
Aside from its role as a protease inhibitor, α2M has been shown to bind to and promote clearance of other endogenous and exogenous molecules, consistent with a broader protective function. α2M is known to bind to cytokines and growth factors (without converting to α2M*), 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)). α2M has also been shown to interact with endogenous disease-associated proteins, including the Aβ peptide associated with Alzheimer’s disease (Narita et al., 1997), β2-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, α2M 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 α2M-peptide complexes are immunogenic (Binder et al., 2001;
Binder et al., 2002; Binder, 2004). α2M bound peptides are internalised by LRP (also known as the α2M receptor and CD91) and fragments of the peptide are subsequently re- presented on the cell surface. This response is identical to the one elicited by peptides chaperoned by intracellular heat shock proteins (Srivastava, 2002).
1.5.3 Binding of α2M to Ligands
As described in section 1.5.2, α2M can form complexes with a wide range of molecules.
Many studies have attempted to elucidate the binding mechanisms used by α2M in its interactions with various ligands. Historically, α2M is known to have three distinct mechanisms of ligand binding (i) the steric “trapping” reaction specific for proteases 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 α2M, 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 non- proteolytic proteins when then go on to facilitate antigenic presentation by macrophages to
T-cells (Chu and Pizzo, 1993). As discussed previously, α2M can be converted to α2M* 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 α2M molecule is reversible, with the reverse reaction having two intermediate states between α2M* (the receptor recognised form) and native α2M (with 4 intact thiol ester bonds) (Grøn et al., 1996). In addition to the structural reformation, the
“reversed” α2M also regains its protease inhibitor function.
Proteins can also be covalently incorporated into synthetically activated, nucleophile treated α2M, removing the need for protease activation (Gron and Pizzo, 1998). The ability to covalently link a wide range of molecules to α2M* using a non-proteolytic method of activation, raised the possibility of using α2M as a novel drug delivery system. Preliminary CHAPTER 1: INTRODUCTION 20
investigations have successfully incorporated nucleosides and nucleobases into α2M*(Bond et al., 2007). Synthetic nucleosides such as guanosine have immuno-modulatory and immuno-stimulatory properties and when incorporated into α2M* 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 α2M molecule. In this mechanism, native
α2M 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 α2M 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 α2M 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
α2M to hold PDGF in a reversible, non-covalent bond has led to suggestions that α2M 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 α2M subunit has recently been identified by Gonias and associates. The growth factor binding site is contained within amino acids
718-733 near the C-terminal flank of the bait region of a mature α2M subunit. The binding site is a 16-amino acid sequence, composed mainly of hydrophobic amino acids with two 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 α2M
(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 α2M 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 α2M binding mechanisms. Thus, in addition to the bait region, there is now evidence to suggest that the
α2M 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 α2M subunit.
Figure 1.6 Locations of binding sites within the α2M 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 α2M 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).
CHAPTER 1: INTRODUCTION 22
1.5.4 Receptor Binding and Internalisation of α2M
The receptor binding site is comprised of a COOH-terminal 138 residue domain within each of the four α2M 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 α2M subunit have been identified as the specific binding sites for
LRP (Nielsen et al., 1996). α2M The conformational change and subsequent availability of the receptor binding domain allows activated α2M 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 α2M. Interestingly, some polypeptides have been shown to bind significantly to native α2M (carboxypeptidase B)
(Valnickova et al., 1996), to both native and activated α2M (TGFβ) (Crookston et al.,
1994), or to only α2M* (growth hormone) (Kratzsch et al., 1995). This suggests that the species of α2M 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 α2M 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 α2M* and α2M*/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 CHAPTER 1: INTRODUCTION 23
this process, specific cell surface receptors act to internalise complexes and dispatch them into endocytic and lysosomal pathways (Borth, 1992). α2M is known to bind to at least two different cell-surface receptors, the α2M receptor (α2MR, also known as LRP) and Grp 78
(the α2M 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 α2M (Horn et al., 1997).
α2MR/LRP is multifunctional, it is able to bind ligands from an array of classes including
α2M-protease complexes (Kristensen et al., 1990), plasminogen activator inhibitor- plasminogen activator complexes (Nykjaer et al., 1992), lipoprotein lipase (Beisiegel et al.,
1991) and apolipoprotein E (Kowal et al., 1989). The in vitro binding of α2M-protease complexes to LRP is suggested to occur when two domains of α2M recognise adjacent CHAPTER 1: INTRODUCTION 24
binding sites on LRP (Moestrup and Gliemann, 1991). Following association with LRP,
α2M-protease complexes may follow various routes including: intracellular sequestration, endocytosis or degradation (Borth, 1992). The common path taken by α2M-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 α2M to LRP, the existence of a second α2M receptor was first suggested in 1994 by Misra and associates (Misra et al., 1994). It was initially believed that α2M*-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 α2M* 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 α2M*- mediated signal transduction, acting in conjunction with LRP (Misra et al., 2002) (Figure
1.7 B). The existence of this second receptor suggests that α2M has additional roles beyond that of a protease inhibitor, including acting as a signalling molecule that triggers cellular responses (Misra et al., 1994).
CHAPTER 1: INTRODUCTION 25
Figure 1.7 Receptors mediate the binding, internalisation and cell signalling of α2M-protease complexes. (A) Many α2M complexes are bound by the α2M receptor (LRP). LRP consists of 5 structural units, two of which are bound by subunits of the α2M molecule. Following internalisation via clathrin coated pits, α2M 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 α2M, which in association with 2+ LRP, acts via a G-protein coupled mechanism (G) to initiate an IP3/Ca 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).
1.5.5 The Effects of Oxidative Stress on the Structure, Function and Receptor Recognition of α2M
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 (O2-), hydroxyl radical (OH-), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) (Martínez-Cayuela, 1995). Neutrophil- derived reactive oxygen species (ROS) have an important role in the inflammatory process 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 α2M may contribute to the increased level of proteolysis during the inflammatory process. Hypochlorite may be an oxidant responsible for α2M inactivation in vivo, directly contributing to enhanced tissue destruction during inflammation (Wu and Pizzo, 1999). It has also been proposed that the oxidation of α2M may act as a physiologically relevant switch mechanism to regulate the binding of cytokines and growth factors to α2M (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 α2M (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 α2M (thus the level of cytokine/growth factor-
α2M complexes) in plasma is high and is substantially increased during inflammation (Wu et al., 1998). α2M 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 α2M 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 α2M 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 α2M molecule
(Reddy et al., 1989). These physiological halogenated oxidants are released from activated leukocytes and act to shear the native α2M 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 α2M 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 α2M* 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 α2M exposes the receptor recognition site to LRP, to facilitate uptake and degradation, whilst interestingly the signalling function of α2M is lost (Wu et al., 1997). The exposure of the
LRP binding site of α2M 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 α2M. Overall, oxidised protease bound α2M loses its ability to bind to LRP but still retains its signalling capacity. While conversely, oxidation of native
α2M results in exposure of the receptor binding site and abolishment of its signalling and protease inhibitor activities. These characteristics indicate that α2M is likely to play a significant role in the inflammatory cascade, and highlight several important structural and CHAPTER 1: INTRODUCTION 28
functional variations which are likely to have implications for the chaperone function of
α2M.
α2M 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 α2M 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 α2M/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 α2M within α2M/stressed protein complexes, removing the need for prior protease activation. This would remove the need for α2M to first interact with a protease to expose
LRP binding sites and facilitate the rapid uptake and degradation of the complexes.
CHAPTER 1: INTRODUCTION 29
1.6 Aims
In order to further characterise the chaperone action of α2M and to determine how this relates to its known in vivo functions, this study aimed to:
(1) Determine the effects of the formation α2M/stressed protein complexes on the structure
and protease inhibitor function of α2M.
(2) Identify potential endogenous chaperone substrates for α2M in human plasma.
(3) Investigate the effects of oxidative stress on the structure of α2M and its ability to
function as a molecular chaperone.
(4) Investigate the interactions of α2M/stressed protein complexes with receptors on the
surface of cultured cell lines and human peripheral blood cells.
CHAPTER 1: INTRODUCTION 30
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protein potently inhibit hippocampal long-term potentiation in vivo." Nature, 416: 535-539. Webb, D. J. and S. L. Gonias (1997). "Chemical modification of alpha-2-macroglobulin to generate derivatives that bind transforming growth factor-beta with increased affinity." FEBS Letters, 410: 249-253. Webb, D. J., D. W. Roadcap, A. Dhakephalkar and S. L. Gonias (2000). "A 16-amino acid peptide from human alpha2-macroglobulin binds transforming growth factor-beta and platelet-derived growth factor-bb [in process citation]." Protein Sci, 9: 1986- 1992. Weiss, S., J. (1989). "Tissue destruction by neutrophils." The New England Journal of Medicine, 320: 365-376. Welsh, M. J. and M. Gaestel (1998). "Small heat-shock protein family: Function in health and disease." Annals of the New York academy of science, 851: 28-35. Wickner, S., M. R. Maurizi and S. Gottesman (1999). "Posttranslational quality control: Folding, refolding, and degrading proteins." Science, 286: 1888-1893. Williams, S. E., J. D. Ashcom, W. S. Argraves and D. K. Strickland (1992). "A novel mechanism for controlling the activity of alpha-2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha-2-macroglobulin receptor) in human brain." Journal of biological chemistry, 267: 9035-9040. Wilson, M. R. and S. B. Easterbrook-Smith (2000). "Clusterin is a secreted mammalian chaperone." Trends in Biochemical Sciences, 25: 95-8. Wu, S., M. and S. V. Pizzo (1999). "Mechanism of hypochlorite-mediated inactivation of proteinase inhibition by alpha-2-macroglobulin." Biochemistry, 38: 13983-13990. Wu, S. M., C. M. Boyer and S. V. Pizzo (1997). "The binding of receptor-recognized alpha 2-macroglobulin to the low density lipoprotein receptor-related protein and the alpha 2m signaling receptor is decoupled by oxidation." J. Biol. Chem. %R 10.1074/jbc.272.33.20627, 272: 20627-20635. Wu, S. M., D. D. Patel and S. V. Pizzo (1998). "Oxidized {alpha}2-macroglobulin ({alpha}2m) differentially regulates receptor binding by cytokines/growth factors: Implications for tissue injury and repair mechanisms in inflammation." J Immunol, 161: 4356-4365. 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: 1131-1136. Yerbury, J., M. S. Rybchyn, S. B. Easterbrook-Smith, C. Henriques and M. R. Wilson (2005). "The acute phase protein haptoglobin is a mammalian extracellular chaperone with an action similar to clusterin." Biochemistry, 44: 10914-10925. Yerbury, J. J., M. S. Rybchyn, S. B. Easterbrook-Smith, C. Henriques and M. R. Wilson (2005). "The acute phase protein haptoglobin is a mammalian extracellular chaperone with an action similar to clusterin." Biochemistry, 44: 10914-25. Yura, T. and K. Nakahigashi (1999). "Regulation of the heat-shock response." Current Opinion in Microbiology, 2: 153-158.
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-α2M 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).
CHAPTER 2: METHODS 31
2.2 Purification of α2M
Native α2M 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 Zn2+ HiTrapTM chelate-affinity column equilibrated with binding buffer
(0.02 M HEPES, 1 M NaCl, pH 7.2), using an ÄKTAdesignTM 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 Zn2+ binding proteins were eluted with 20 mM imidazole, 20 mM HEPES, 20 mM Na2HPO4, 0.5 M NaCl, pH 7.4, and
α2M was subsequently eluted with 500 mM imidazole, 20 mM HEPES, 20 mM Na2HPO4,
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 KH2PO4, 8 mM Na2HPO4, pH 7.4) containing 0.1%
(w/v) NaN3 (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 α2M was determined by absorbance at 280 nm (extinction coefficient 0.893 M-1 ; Hall, 1978).
2.3 Preparation of Activated α2M and Activated (α2M/CS)* Complexes
Native α2M was converted to activated α2M (α2M*) by incubation with either methylamine or trypsin. Native α2M (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. α2M-trypsin and α2M-trypsin/CS complexes were formed by incubating either native α2M (9.7 μM) or native α2M/CS complexes (9.7 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. α2M-trypsin and α2M-trypsin/CS ((α2M/CS)*) complexes were purified by size exclusion chromatography using a Biosep©-SEC-S4000 column equilibrated in PBS. Successful transformation of native α2M and native α2M/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). α2M* species were stored in PBS, pH 7.4 at -20 °C.
2.4 Preparation of Oxidized α2M
Native α2M was oxidized by incubation in oxidative stress buffer (OSB; 100 μM CuSO4, 4 mM H2O2 in PBS) at 37 °C for 2 hours.
2.4.1 Size Exclusion Chromatography of Oxidised α2M
In order to observe changes in structure following oxidation, samples of native α2M and oxidised α2M 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 α2M 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 ÄKTAdesignTM FPLC system (GE Healthcare, Australia).
CHAPTER 2: METHODS 33
2.5 Formation and Purification of Complexes Between α2M and Stressed Protein
Mixtures of α2M (2.5 mg/ml) and biotinylated CS (CS-b; 0.3 mg/ml) or α2M (0.5 mg/ml) and CPK-b (0.5 mg/ml) in PBS were incubated for 3 h at 43 °C. In similar experiments
α2M (2.5 mg/ml) and lys-b (1 mg/ml) were incubated for 10 h at 37 °C in OSB. Samples were centrifuged at 10000 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 α2M and substrate protein from complex. The presence of complex in fractions shown by SDS-PAGE to contain both α2M and the substrate protein was verified by immunoprecipitation, as described below. Complex-containing fractions were pooled, concentrated and stored frozen at - 20 oC in PBS.
2.6 Electrophoresis
2.6.1 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, 0.006 ml TEMED) was transferred to the chamber and overlayed with 1 ml of ethanol to avoid air-bubble formation. When set, ethanol was 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, 0.006 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).
2.6.2 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-α2M 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 CHAPTER 2: METHODS 35
washes, and a secondary antibody solution (sheep-anti rabbit Ig-HRP conjugate or sheep- anti 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.
2.6.3 Native Gel Elecrophoresis
Samples (30 μg total protein) of native α2M and oxidised α2M 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 α2M (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.
2.6.4 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 0.095 TRIS pH 5.7, 0.0075ml TEMED, 0.15 ml ammonium persulfate), 4% stacking gel (9.3 ml distilled water, 2 ml 30% (v/v) acrylamide, 3.7 ml
0.054 Tris, 0.03 M HCl, pH 6.1, 0.0015 ml TEMED, 0.05 ml 10% (w/v) ammonium persulfate), and the reservoir buffer 0.041 M Tris, 0.04 M boric acid, pH 8.6. Samples were CHAPTER 2: METHODS 36
electrophoresed for 2.5 h, gels were stained with Coomassie blue and destained using destain solution as in 2.6.1.
2.7 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 α2M or α2M/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 α2M), 80 μl of 0.055 M Tris-HCl,
5.55 μM CaCl2, 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 Plus384 microplate reader (Molecular Devices, USA).
2.8 Protein Precipitation Assays
Individual solutions of trypsin activated α2M* (0.6-13.9 μM) and CS (6 μM; in 50 mM
Tris, 2 mM EDTA, pH 8) or mixtures of CS and α2M* (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 CHAPTER 2: METHODS 37
of CPK and α2M* (0.6-6.95 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 α2M (1.39-13.9 μM) or α2M* (trypsin activated; 1.39-13.9
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 A360 in a SpectraMax Plus384 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
α2M was selectively depleted from normal human plasma (NHP) by repeated passes over a
2+ TM Zn HiTrap chelate-affinity column (GE Healthcare). Approximately 2.0-2.5 mg of α2M 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 α2M 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 α2M depleted plasma (α2MDP) to reconstitute them to approximately their original concentrations in plasma. A sample of double depleted plasma (DDP) was also prepared; clusterin was removed from α2MDP by immunoafinity chromatography as previously described (Poon et al., 2000). Depletion of
α2M and clusterin from plasma was confirmed by immunodetection (as described in section CHAPTER 2: METHODS 38
2.6.2). To allow for small dilution effects unavoidable during the depletion steps, A280 measurements of α2MDP, 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, α2MDP, (α2MDP + 2.5 mg/ml α2M), DDP and (DDP + 2.5 mg/ml α2M + 100
μg/ml clusterin), were diluted 1:2 with PBS, supplemented with 7.5 mM NaN3 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, α2MDP, (α2MDP + 2.5 mg/ml α2M), (DDP and
DDP + 2.5 mg/ml α2M + 100 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).
2.9.1 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) Na2CO3.H2O, 1.6 % (w/v)
Na2 tartrate, pH 11.25; 100 volumes of reagent B: 4% BCA. Na2; 4 volumes of reagent C: CHAPTER 2: METHODS 39
4% (w/v) CuSO4.5H2O) 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.
2.9.2 Immunoprecipitations
α2M (6.95 μM), biotinylated lys (lys-b; 69 μM), or mixtures of α2M 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 10000 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.
2.10 Identification of Endogenous Substrates using Mass Spectrometry
2.10.1 Spot Excision
Plasma samples were incubated at either 43 °C or room temperature for 72 h. α2M was purified from both samples using zinc chelate affinity chromatography (as described in section 2.2). Proteins co-purifed with α2M in the heated, but not the non-heated sample 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 α2M); instruments were washed with 100 % methanol between excisions to ensure that individual samples were not cross-contaminated.
2.10.2 Trypsin Digestion
Excised bands were destained by incubation with 100 μl of Coomassie Destain Buffer (60
% (v/v), 50 mM NH4HCO3, 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 NH4HCO3 (pH 7.8), overnight at 37 °C with gentle shaking (400 rpm). Samples were stored at 4 °C before analysis by mass spectrometry.
2.10.3 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-DETM STR Biospectometry WorkstationTM with Delayed
ExtractionTM (PerSeptive Biosystems, USA) at the Australian Proteomic Analysis Facility,
Maquarie University, Sydney, NSW, Australia. The spectrum obtained was analysed using CHAPTER 2: METHODS 41
VoyagerTM software with Data ExplorerTM. 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.
2.11 Cell Culture and Flow Cytometry
2.11.1 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) CO2. 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 KH2PO4, 0.3 mM Na2HPO4, 20 mM HEPES, 1 g/l BSA, 1 mM CaCl2, 2 mM MgCl2, 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.
CHAPTER 2: METHODS 42
2.11.2 Binding Assays Using JEG-3, Hep-G2 and Activated U937 Cells
Different cell lines were incubated for 30 min on ice with either α2M, trypsin activated
α2M* or trypsin activated α2M*/CS, all at a concentration of 200 µg/ml. Following washing with HBB the cells were incubated with rabbit anti-α2M 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
(α2M/CS) or typsin activated α2M*. Binding of α2M and α2M/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.
2.11.3 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 NH4Cl,
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.
CHAPTER 2: METHODS 43
The white blood cell pellets obtained were first incubated for 30 minutes with 500 µg/ml of: native α2M, typsin activated α2M*, or α2M/CS complexes. Following a wash with HBB, the cells were incubated with polyclonal rabbit anti-α2M (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.
2.11.4 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.
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: 12837-12844. 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: 323-338. Hall, P., K., Roberts, R., C. (1978 .). "Physical and chemical properties of human plasma alpha2-macroglobulin." The biochemical journal, 173: 27–38. 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: 9069-75. 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: 8134- 8139. 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: 15953- 15960. 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: 15932-15937. 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: 76-85. 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: 9016-9022. 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: 319-326.
CHAPTER 3: RESULTS 44
CHAPTER 3: CHARACTERISING THE MECHANISM OF α2M CHAPERONE ACTION
3.1 Introduction
It has previously been shown, under conditions of heat stress, that α2M 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 α2M (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 α2M as an extracellular chaperone. Specifically, the effect of interactions with stressed proteins on the conformational structure of α2M, and how these impact on its protease inhibitor activity were unknown. The conformation of α2M when complexed with a stressed protein was investigated using native gel electrophoresis and sensitive trypsin binding assays.
When investigating the chaperone role of α2M 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 (α2M*) is present following interaction with a protease, and has exposed receptor recognition sites. Although native α2M has chaperone activity, it is unknown whether α2M* has a similar activity. In order to determine the ability of α2M* 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 CHAPTER 3: RESULTS 45
temperature (43°C) (Table 3.1). Furthermore, in an attempt to identify putative endogenous chaperone substrates for α2M in human plasma, samples were incubated at 43 °C for 72 h and any proteins co-purifying with α2M following these incubations were identified using
MALDI-TOF mass spectrometry.
Table 3.1 Characteristics of substrate proteins used to investigate the chaperone properties of α2M.
Substrate Mass Isoelectric Subunit Disulfide Secondary Protein (kDa) Point (Pi) Subunits Mass (kda) Bonds Structure Citrate 98 7 2 49 No Predominantly Synthase (CS) α-helix, small section of β- sheets Creatine Phosphokinase α-helix, β- (CPK) 81 8 2 40 No 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 2.1-2.10.
3.3 Results:
3.3.1 Within α2M/heat Stressed Protein Complexes, α2M Remains in its Native Conformation
In the protease-bound, activated form, α2M* has a more compact structure which may be detected using native tris/borate PAGE (Figure 3.1). Native α2M runs as a single band with limited migration on the gel, however activated α2M migrates significantly further. α2M within complexes formed with either heat stressed CS or CPK displays a migration pattern similar to that of native α2M, suggesting that interaction with a stressed protein may not activate α2M. The formation of complexes with either of the two substrate proteins CS and CHAPTER 3: RESULTS 46
CPK also appears to have little impact on the electrophoretic mobility of α2M. This may be explained by the fact that each α2M associates with only one or two substrate molecules.
(A) α2M α2M* α2M/CS ( (B) α2M α2M* α2M/CPK
◄α2M►
◄α2M*►
Figure 3.1 Image of Coomassie stained native PAGE gel showing migration of α2M, α2M*, α2M/CS and α2M/CPK complexes. (A) 6 % Tris/borate native PAGE gel showing native α2M, trypsin activated α2M (α2M*) and α2M/CS complex (B) 6 % Tris/borate native PAGE gel showing native α2M, trypsin activated α2M (α2M*) and α2M/CPK complex. Relative positions of α2M and α2M* are indicated. Each experiment was conducted in duplicate.
A classical trypsin binding assay was used to examine the ability of α2M incorporated into purified α2M/stressed protein complexes to carry out the protease trapping reaction. In the assay, trypsin (provided in excess) complexed with any native α2M present; any unbound trypsin was subsequently inactivated using soybean trypsin inhibitor (which is sterically unable to access and inactivate trypsin bound to α2M). Any residual trypsin activity
(corresponding to α2M-trapped trypsin) was measured using the low molecular weight substrate BAPNA. Purified α2M/CS and α2M/CPK complexes showed dose-dependent trypsin binding activity which was similar to that measured for native α2M (Figure 3.2).
Collectively, these results suggest that α2M may remain in its native form when complexed CHAPTER 3: RESULTS 47
with heat-stressed proteins and the similar trypsin-trapping ability of pure native α2M and
α2M/stressed protein complexes suggests that a majority of the mass of the complexes consists of α2M. This result is consistent with past findings which indicated that
α2M/stressed protein complexes could not be discriminated from α2M alone using size- exclusion chromatography (French, 2005). Thus, one large α2M tetramer (720 kDa) is likely to associate with a small number of lower molecular weight chaperone substrate molecules. The exact stoichiometry of α2M/stressed protein complexes may be determined in the future by using SDS PAGE and scanning densitometry, or mass spectrometry.
(A) CS (B) CPK
1.2 0.8 0.9 0.6 nm
405 0.6 0.4 A 0.3 0.2 0 0 0204060 0204060
Protein (μg)
Figure 3.2 When complexed with stressed proteins, α2M retains protease trapping activity. Native α2M (■), α2M*( ) (activated by methylamine) and (A) α2M/CS and (B) α2M/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.
CHAPTER 3: RESULTS 48
3.3.2 Protease Activation Abolishes the Chaperone Activity of α2M
α2M 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, α2M "activated" by incubation with trypsin (α2M*) had
limited effect on substrate protein precipitation (Figure 3.3). When α2M at a concentration
of 5 mg/ml was incubated with CS at 43 °C, precipitation was almost totally suppressed. In
comparison, addition of α2M* 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 α2M effectively abolishes its chaperone
activity.
(A) CS (B) CPK
0.8 0.8
0.6 0.6
A360 0.4 0.4
0.2 0.2
0.0 0.0
0 50 100 150 200 0 40 80 120 160 200 Time (min)
Figure 3.3 Activation abolishes the chaperone activity of α2M. Time-dependent changes in turbidity 360 (measured as A ) of heat-stressed CS (A) and CPK (B), either alone ( or in the presence of α2M (5 mg/ml) (▲) or trypsin activated α2M* (5 mg/ml) (▲). The data points are individual measurements; the results shown are representative of at least three independent experiments. CHAPTER 3: RESULTS 49
As described above, α2M* lacks the ability to inhibit protein precipitation. It was also suggested earlier in this chapter (section 3.3.1) that α2M within α2M/stressed protein complexes may remain in the native conformation. Further experiments were undertaken to investigate whether α2M within stressed protein complexes can subsequently undergo protease activation. Following incubation of α2M/CS complexes with a three fold molar excess of trypsin for 2 h at 37 °C, a similar migration pattern to α2M* was observed using native PAGE (Figure 3.4). This result suggests that α2M complexed with heat-stressed protein retains the ability to trap proteases and therefore the ability to subsequently expose the LRP binding site.
α2M α2M* α2M/CS (α2M/CS)*
α2M ►
α2M*►
Figure 3.4 Image of Coomassie stained native PAGE gel of α2M, α2M*, α2M/CS and (α2M/CS)*. 6 % Tris/borate native PAGE gel showing the native form of α2M (α2M), trypsin activated α2M (α2M*), α2M/CS complex (α2M/CS), and trypsin-activated α2M/CS complex ((α2M/CS)*). Relative positions of α2M and α2M* are indicated. The result shown is representative of two independent experiments.
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 α2M 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 (α2M, heat-stressed protein and trypsin).
(A) (B)
Figure 3.5 α2M/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 α2M alone (all at room temperature), or mixtures of α2M and either CS-b or CPK-b, which had been heated at 43 °C before being mixed with trypsin (i.e., α2M/CS and α2M/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 α2M (B). The results shown are representative of two independent experiments. CHAPTER 3: RESULTS 51
In order to further confirm that following the formation of complexes with heat-stressed protein, α2M can still interact with proteases to become activated, and that this activation does not involve proteolysis of heat-stressed protein, a purified α2M/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 α2M/CS sample contained bands at 180 kDa
(indicative of a reduced α2M subunit), and at about 95 kDa and 85 kDa which are likely to represent fragments of α2M generated by spontaneous bait region cleavage, attributable to freeze/thawing of the sample. Unexpectedly, the α2M/CS sample also contained a band at about 70 kDa. This band may represent the autolytic cleavage of thiol-ester bonds within
α2M subunits, produced as a result of heating during sample preparation. Faint bands at 55 and 45 kDa are present in both native and activated α2M/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 α2M/CS and (α2M/CS)* lanes, suggesting that trypsin activation is not associated with significant proteolysis of the α2M-bound CS substrate (Figure 3.6).
CHAPTER 3: RESULTS 52
α2M α2M* CS Trypsin α2M/CS (α2M/CS)*
200 150
120 ) 100
kDa 85 (
70 60
50
40 Molecular Mass Molecular
30
25
Figure 3.6 Image of Imperial Blue stained SDS PAGE showing the effects of trypsin on α2M and α2M/CS complexes. 12% SDS PAGE gel (under reducing conditions) showing α2M, α2M*, CS, trypsin, α2M/CS and trypsin-activated (α2M/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).
3.3.3 α2M Inhibits the Heat Induced Precipitation of Proteins in Whole Human Plasma
Zinc chelate affinity chromatography was used to selectively deplete virtually all α2M from an aliquot of normal human plasma (NHP). NHP was repeatedly
2+ TM passed over a 5 ml Zn HiTrap chelate-affinity column until negligible α2M could be detected by immunoblotting (Figure 3.7).
CHAPTER 3: RESULTS 53
(A) ▼ ▼
(B) NHP α2MDP
((
α2M
Figure 3.7 Depletion of α2M from normal human plasma (NHP). (A) α2M 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 α2M. The broad peak eluted first (not labeled) is the unbound protein fraction (B) Successful depletion of α2M from NHP was confirmed by immuno-blotting under reducing conditions as described in section 2.9. The position of α2M 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 α2M-depleted plasma (α2MDP) prepared from the same original batch of plasma (diluted 1 in 2 in PBS and supplemented with azide), were
o incubated at 43 C for 72 h, the α2MDP 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 α2M and its relationship with other known CHAPTER 3: RESULTS 54
extracellular chaperones, a sample of plasma depleted of both α2M and clusterin (double depleted plasma; DDP) was prepared (diluted 1:4 in PBS) and incubated at 43°C for 72 h.
The α2MDP was found to contain more precipitated protein than the NHS but less than that observed in the DDP (Figure 3.8).
0.20
0.15
0.10
Protein (mg) 0.05
0.00 123 NHP α2MDP DDP
Figure 3.8 α2M 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), α2M-depleted plasma (α2MDP) 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 α2M to protect proteins in whole human plasma from aggregation resulting from extended incubation at 37 °C. α2MDP, 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 CHAPTER 3: RESULTS 55
resulted from the removal of α2M (α2MDP) or α2M and clusterin (DDP) from the respective samples, physiological concentrations of α2M (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 α2M has a chaperone action that can suppress the stress-induced aggregation and precipitation of endogenous protein(s) in human plasma.
0.15 **
0.1 *
Protein (mg) 0.05
0 NHP α2MDP DDP α2MDP DDP+ α2M + α2M + Clusterin
Figure 3.9 α2M 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), α2M- depleted plasma (α2MDP) and plasma depleted of both α2M and clusterin (double depleted
plasma; DDP) heated at 37 °C for 7 days. Samples of α2MDP and DDP were supplemented
with 2.5 mg/ml α2M and (2.5 mg/ml α2M + 100 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 α2MDP (**). The results are representative of two independent experiments.
3.3.4 Identifying Endogenous Chaperone Substrates for α2M in Human Plasma
CHAPTER 3: RESULTS 56
The identification of specific proteins chaperoned by α2M within the body will contribute to a better understanding of the physiological importance of the in vivo chaperone functions of α2M. Preliminary investigations were undertaken in this study with this aim in mind.
α2M 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
α2M analysed by SDS PAGE. Any proteins co-purifying with α2M from the heat-stressed sample, but not from the control (room temperature) sample, represent putative chaperone substrates for α2M. Two significant bands of approximately ~ 50 kDa and ~ 55 kDa were co-eluted with α2M 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 α2M
) 150 kDa
( 120
100
85
Molecular Mass 70
60 ◄ X
◄ Y 50
3.10 Image of Coomassie-Blue stained SDS PAGE gel identifying putative endogenous chaperone substrates for α2M. 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 α2M 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.
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 1000-2500 m/z were used to search the SwissProt database using the Mascot PMF query option (http://www.matrixscience.com). 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 CHAPTER 3: RESULTS 58
y Intensit
Y
y Intensit
Figure 3.11 Mass spectra of trypsin digested putative endogenous substrates of α2M. 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 α2M 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). CHAPTER 3: RESULTS 59
1. 2.
200
α M ) 150 2 120 kDa
(
100 85
70
60 Molecular Mass Molecular ◄ β
50 ◄ γ
40
3.12 Fibrinogen is an endogenous human plasma chaperone substrate for α2M under heat stress conditions. Image of 10% Coomassie-Blue stained SDS PAGE gel comparing proteins purified by Zn2+ 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 α2M 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 α2M 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 α2M to inhibit heat induced protein precipitation is abolished when α2M is in its activated conformation (α2M*). Results presented here confirm and extend these findings. CHAPTER 3: RESULTS 60
In this study it was determined that interaction of α2M with a stressed protein does not lead to its activation, thus α2M 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 α2M to α2M*.
Immunodetection provided direct evidence of covalent association between trypsin and
α2M within preformed A2M-(stressed) protein complexes. It follows that if one important function of α2M 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 α2M/stressed protein complexes.
α2M* 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 α2M at least in part via hydrophobic interactions (French et al., 2008). Previous work has shown that, overall, α2M 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 α2M important in the chaperone-like action are sterically more accessible to stressed proteins before protease activation. It is also possible that there are 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 α2M 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 α2M 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 α2M 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 CHAPTER 3: RESULTS 62
preliminary insight into the link between the roles of α2M as an inflammatory response mediator, protease inhibitor and extracellular chaperone.
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: 239-243. 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: 323-338. 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: 1176-1185. Lowe, G. D. O., A. Rumley and I. J. Mackie (2004). "Plasma fibrinogen." Annals of Clinical Biochemistry, 41: 430-440. 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: 1131-1136.
CHAPTER 4: RESULTS 62
CHAPTER 4: BINDING OF α2M/STRESSED PROTEIN COMPLEXES TO CELL SURFACE RECEPTORS
4.1 Introduction:
It is well established that protease bound α2M* 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 α2M-protease complexes, only very limited previous studies of the interaction of α2M/stressed protein complexes with LRP have been carried out. Preliminary investigations (French, 2005) indicated that complexes formed between α2M 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
α2M/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 Human hepatocellular LRP (Grimsley et al., 1998), carcinoma Scavenger Receptors (Schwartz et al., 1982) U937 Human leukemic LRP (Elsas et al., 1990) monocyte lymphoma CHAPTER 4: RESULTS 63
A functional property of all LDL family receptors, including LRP, is the requirement of
Ca2+ 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 α2M-protease complexes
(Feldman et al., 1983), investigations were undertaken to determine whether α2M/stressed protein bound to the ASGP receptor. The specificity of the binding was determined by pre- incubating cells with saturating concentrations of galactose or asialofetuin, both ASGP receptor ligands.
The binding of α2M-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 α2M to bind to other immunological cell subsets such as granulocytes and lymphocytes remains largely CHAPTER 4: RESULTS 64
uncharacterized. Preliminary studies have indicated that α2M* binds significantly to the surface of granulocytes and monocyte populations (French, 2005). Interestingly, preliminary data also suggested that α2M/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 α2M/CS complexes to granulocytes, using cells isolated from human blood and flow cytometric analysis.
4.2 Methods: Refer to materials and methods sections 2.1-2.3, 2.5 and 2.11.
4.3 Results:
4.3.1 Binding of α2M/Stressed Protein Complexes to JEG-3 Cells
As shown in Figure 4.1, native α2M has only limited binding to JEG-3 cells. Following typsin activation however, α2M* 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, α2M remains in the native conformation, retaining the ability to function as a protease inhibitor. To investigate the effect of activating α2M in α2M/stressed protein complexes on interactions with LRP,
α2M/CS complexes were incubated with a three-fold molar excess of trypsin, and the ability of this activated complex (α2M/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 α2M and heat-stressed CS showed little binding to JEG-3 cells; in contrast, following activation with CHAPTER 4: RESULTS 65
trypsin, (α2M/CS)* showed substantial binding (Figure 4.1). Incubation of cells with
(α2M/CS)* resulted in a significantly greater increase in fluorescence than that obtained for cells incubated with α2M/CS (t = 9.68, p<0.05, df =4) (Figure 4.1).
LRP α2M α2M*
α2M/CS (α2M/CS)* Number of Cells
FITC Fluorescence
Figure 4.1 Expression of LRP and binding of native α2M, α2M*, α2M/CS and (α2M/CS)* to JEG-3 cells. Histograms of 10 x 103 cells incubated with either rabbit anti-LRP antibody and subsequently sheep-anti- rabbit Ig-FITC conjugate (top left panel), or with native α2M, α2M*, α2M/CS or (α2M/CS)* (indicated in the corresponding panels) followed by anti-α2M antibody which was subsequently detected with sheep-anti- rabbit Ig-FITC conjugate. Other cells were separately treated with anti-DNP antibody followed by sheep-anti- rabbit Ig-FITC conjugate (background fluorescence; red peak). Results are indicative of two independent experiments.
In order to confirm that the binding observed for (α2M/CS)* and α2M* 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. Pre- incubation with the ligand GST-RAP (100 µg/ml) significantly reduced the binding of
α2M* (t = 5.87, p <0.05, df = 4) and (α2M/CS)* complexes (t = 4.7, p <0.05, df = 4) (Figure CHAPTER 4: RESULTS 66
4.2). Pre-incubation with anti-LRP antibody was also found to significantly reduce the binding of α2M* (t = 16.62, p <0.05, df = 4) and (α2M/CS)* (t = 19.87, p <0.05, df = 4) to the surface of JEG-3 cells.
50
40
30
20 # # 10 * * # geomean fluorescence * 0 1 2 α α α α α α α α 2 M 2 M 2 M 2 M 2 M 2 M 2 M 2 M / * * * * * * C / / / S C C C + + S S S a R n + + t A a R i- P n A L t R i P P -LR P
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 α2M, α2M*, and native α2M/CS or activated (α2M/CS)* complexes. In all cases, α2M was trypsin activated. In some cases, cells were pre- incubated with an inhibitory anti-LRP antibody or GST-RAP (indicated below the x-axis; see methods section 2.11.2 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. α2M*/CS, left) and # (vs. α2M*, right).
4.3.2 Binding of α2M/Stressed Protein Complexes to Hep-G2 Cells
Preliminary experiments were undertaken to determine the ability of α2M, α2M* and native
α2M/CS (α2M/CS) complexes to bind to the surface of Hep-G2 cells. Cells were incubated with 200 μg/ml of each sample, followed by anti-α2M antibody and subsequently detected CHAPTER 4: RESULTS 67
using sheep-anti-rabbit IgG-FITC conjugate. In contrast to the results obtained with JEG-3 cells, native α2M bound significantly to the surface of Hep-G2 cells (t = 29.88, p< 0.05, df
=2) (Figure 4.3). α2M* 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 α2M. Lastly,
α2M/CS also showed significant binding to the surface of Hep-G2 cells (t = 23.49, p< 0.05, df =2) (Figure 4.3).
100 100
LRP α2M 80 80
60 60
40 40
20 20
0 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 515/20-A 515/20-A Number of Cells
100 100
α2M* α2M/CS
80 80
60 60
40 40
20 20
0 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 515/20-A 515/20-A FITC Fluorescence
Figure 4.3 Expression of LRP and binding of native α2M, α2M*, α2M/CS complexes to Hep-G2 cells. Histograms of Hep-G2 cells incubated with either rabbit anti-LRP antibody and subsequently sheep-anti-
rabbit Ig-FITC conjugate (top left panel) or with native α2M, α2M*or α2M/CS (indicated in corresponding panels) followed by anti-α2M 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. 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 α2M and α2M/CS complexes. Pre-incubation of the cells with RAP significantly reduced the binding of α2M* to the cell surface, while pre-incubation with 1 mg/ml galactose significantly reduced the cell surface binding of α2M and to a lesser extent α2M/CS, but not the binding of α2M* (Figure 4.4).
40
35 ■ No Inhibition
30 ■ GST RAP 25
20 Galactose (1 mg/ml) 15
Geomean Fluorescence 10 5 0 α2M α2M* α2M/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 α2M, α2M* and α2M/CS. Where appropriate, α2M was trypsin activated. In some cases, cells were pre-incubated with GST-RAP (see methods section 2.11.2 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. CHAPTER 4: RESULTS 69
4.3.3 Binding of α2M/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 α2M and α2M/CS complexes had limited binding to activated U937 cells. Following trypsin activation, α2M* showed increased and significant binding (t = 17.53, p< 0.05, df =2) (Figure 4.5).
LRP α2M
α2M* α2M/CS Number of Cells
FITC Fluorescence
Figure 4.5 Expression of LRP and binding of native α2M, α2M* and α2M/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 α2M, α2M* or α2M/CS (yellow peaks), followed by anti-α2M 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. 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 α2M* cells with the asialoglycoprotein receptor ligand asialofetuin (1 mg/ml) had little effect on α2M* binding.
60
50 ■ No Inhibition
40 ■ Asialofetuin (1 mg/ml)
30 GST RAP
20 Geomean Fluorescence 10
0
Figure 4.6 Histogram plot showing the average geometric mean fluorescence (n = 2, ± range, arbitrary units) for immunochemical detection of the binding of α2M* to activated U937 cells. α2M 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.
4.3.4 Binding of α2M/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).
CHAPTER 4: RESULTS 71
100
80
60 % ofMax 40
20
0 10 0 10 1 10 2 10 3 10 4 515/20-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 2.11.3 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 α2M, α2M* and purified α2M/CS complexes to bind to any receptors
present on the granulocyte population was investigated using flow cytometry. The
incubation of cells with native α2M (200 µg/ml), followed by subsequent incubations
with rabbit-anti-human-α2M 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, α2M* showed increased binding to the surface of granulocytes. The α2M/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. CHAPTER 4: RESULTS 72 Number of Cells
FITC Fluorescence
Figure 4.8 Binding of native α2M, α2M*, CS and CS/α2M complexes to granulocytes. Histograms of cells incubated with either native α2M, α2M*, CS or CS/α2M (red peaks) followed by anti-α2M 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 α2M* and α2M/CS
complexes, cells were pre-incubated with GST-RAP (100 μg/ml). It was found that GST-
RAP significantly inhibited the binding of α2M* but had only a small effect on the
binding of α2M/CS (Figure 4.9). These results suggest the possible existence of a novel
receptor on the surface of granulocytes that is able to bind α2M within α2M/CS
complexes. CHAPTER 4: RESULTS 73
20
16 ■ No Inhibitor
12 GST RAP 8
Geomean Fluorescence 4
0 α2M* α2M/CS
Figure 4.9 Histogram plot showing the average geometric mean fluorescence (n = 2, ± range, arbitrary units) for immunochemical detection of the binding of α2M* and α2M/CS to granulocytes. α2M was trypsin activated. Cells were pre-incubated with GST-RAP (see methods section 2.11.3 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 α2M/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). α2M and α2M/CS complexes showed only limited binding to JEG-3 cells, however after incubation with trypsin, activated α2M* and
(α2M/CS)* showed an increased level of binding. This binding was RAP inhibitable, providing evidence that α2M* and (α2M/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. CHAPTER 4: RESULTS 74
The results presented for JEG-3 cells support those found in an earlier study which showed that complexes formed between α2M 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, α2M remains in a native-like conformation. However, α2M 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 α2M/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 α2M and α2M/CS complexes bound significantly to Hep-G2 cells, without the need to first undergo activation. Binding was also observed with α2M* but to a lesser extent.
Whereas the binding of α2M* could be significantly reduced by pre-incubation with GST-
RAP, this had little effect on the binding of α2M and α2M/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 α2M and α2M/CS to Hep-G2 cells, but not the binding of α2M*. These preliminary results suggest that a receptor other than LRP CHAPTER 4: RESULTS 75
may play a role in the binding and uptake of α2M/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 α2M – antigen complexes in vivo (Hart et al.,
2004). Results presented in this chapter indicate that α2M and α2M/CS complexes are unable to bind to the surface of PMA-differentiated U937 cells, however α2M* 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 α2M* 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 α2M/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 α2M, α2M* and α2M/CS determined. α2M* showed significant RAP-inhibitable binding, indicating the involvement of the LDLR family which is consistent with the results of past investigations (French, 2005). α2M/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 CHAPTER 4: RESULTS 76
responsible, at least in part, for the binding of α2M/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 α2M/CS complexes is required in order to determine whether ASGPR is involved in the binding of α2M/stressed protein complexes to granulocytes.
Collectively, the results presented in this chapter indicate that α2M may play a vital role in the disposal of misfolded extracellular proteins. The recognition of activated α2M/stressed protein complexes by LRP and the possible existence of a receptor for native α2M/stressed protein complexes are just two mechanisms by which misfolded extracellular proteins may be internalised for subsequent removal and degradation.
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: 29874-29882. Bu, G. and A. L. Schwartz (1998). "Rap, a novel type of er chaperone." Trends in Cell Biology, 8: 272-276. 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: 2427-2433. 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: 757-762. 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: 485-498. 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: 70-78. 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: 21232-21238. 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: 14011-14017. 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: 199-207. 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: 4230-4237. CHAPTER 4: RESULTS 78
Spiess, M. (1990). "The asialoglycoprotein receptor: A model for endocytic transport receptors." Biochemistry, 29: 10009-10022.
CHAPTER 5: RESULTS 77
CHAPTER 5: OXIDATIVE STRESS AND THE CHAPERONE FUNCTION OF α2M
5.1 Introduction:
Previous investigations have identified that α2M has the ability to protect substrate proteins from heat stress at 43°C by forming stable complexes with them (French 2005). The ability of α2M 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 (H2O2), 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
α2M. Halogen oxidized α2M 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 α2M* 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 CHAPTER 5: RESULTS 78
assume that the molecular mass for an intact α2M tetramer is 720 kDa, with a homodimer subunit mass of 360 kDa.
The ability of α2M to chaperone proteins undergoing oxidative stress and the effects of oxidation on the chaperone activity of α2M* (which is chaperone inactive under heat stress conditions) had not been tested. In order to investigate the effects of α2M on the oxidation- induced 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 α2M. Considering the high concentration of oxidative species present during the inflammatory response, the ability of α2M and α2M* 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 α2M and α2M*, 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 2.1-2.9.
5.3 Results:
5.3.1 α2M Undergoes Conformational Changes when Exposed to Oxidative Stress
α2M is a tetrameric molecule, consisting of two identical, disulfide-linked dimers, non- covalently associated to form the native 720 kDa molecule. When analysed by reducing
SDS PAGE, α2M migrated as a single band at 180 kDa, representing a single α2M subunit CHAPTER 5: RESULTS 79
(Figure 5.1, lane 1). Upon interaction with a protease, α2M 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, α2M 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
α2M (Figure 5.1, lane 2). When native α2M 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
α2M 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).
1. 2. 3. 4. 200 150
) 120 KDa ( 100 85
70 60 Molecular Mass Molecular 50
Figure 5.1 Image of Coomassie Blue stained 10% SDS PAGE gel showing the fragmentation of α2M when exposed to oxidative stress. Molecular mass standards (left-most lane) were used to estimate the mass of proteins present. Native α2M (lane 1), α2M + methylamine (lane 2), α2M + trypsin (3-fold molar excess) (lane 3), α2M + oxidative stress buffer (OSB) (lane 4). Results shown are representative of two independent experiments. CHAPTER 5: RESULTS 80
In order to further investigate the effect of oxidation on the structure of α2M, samples of native and oxidized α2M were analysed using size exclusion chromatography. When
© injected onto a Superose 6 column, native α2M eluted as a dominant, symmetrical peak
(Figure 5.2, purple line). However, when similarly analysed, oxidized α2M 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 α2M had occurred.
2000 kDa 669 kDa 158 kDa 67 kDa
) nm 280 A ( Absorbance
Elution volume (ml)
280 Figure 5.2 Plot showing the A as a function of elution volume for SEC of native and oxidised α2M. 500 µl samples of α2M (1 mg/ml) and oxidized α2M (1 mg/ml; pre-incubated in OSB as per methods section) were passed over a Superose 6© column, equilibrated in PBS. α2M 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).
CHAPTER 5: RESULTS 81
In order to further characterize the effects of oxidation on the structural integrity of α2M, samples were subjected to native gel electrophoresis (NGE). NGE separates proteins on the basis of mass and overall charge. Alterations to the structure of α2M in response to oxidative stress are likely to alter its size and charge. Therefore it was expected that the migration of oxidized α2M on the native gel would differ somewhat from that of the native form. Solutions of α2M (2.5 mg/ml), trypsin activated α2M* (2.5 mg/ml) or oxidized α2M
(2.5 mg/ml) were loaded onto a native agarose gel. As expected, α2M* displayed a migration pattern different to that of the native species. α2M* migrated further and in a more compact band compared to the native α2M species. The mobility of oxidized α2M also differed markedly from the native form (Figure 5.3). In comparison to native α2M, the oxidized form migrated further on the gel, and also exhibited a broader pattern of migration. This result confirms the interpretation that α2M undergoes major structural modifications in response to oxidative stress, resulting in alterations to either the size and/or overall charge of the molecule.
α2M
α2M*
α2M (oxidized)
Figure 5.3 Analysis of various forms of α2M by NGE. Samples (30 μg) of α2M, α2M* or oxidised α2M, 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. CHAPTER 5: RESULTS 82
5.3.2 α2M 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 α2M is able to inhibit the precipitation and aggregation of heat stressed proteins by forming stable complexes with them (French,
2005), however the ability of α2M 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 α2M and an oxidatively stressed substrate protein, lysozyme.
The effect of α2M on the oxidation-induced precipitation of lysozyme was investigated. In the absence of α2M, lysozyme (lys; 1 mg/ml) gradually precipitates in OSB, indicated by an increase in absorbance at 360 nm. α2M showed dose-dependant inhibition of oxidation- induced precipitation of lys (Figure 5.4). In the presence of substoichiometric SMRs of
α2M (see section 3.1), the level of lys precipitation was markedly reduced. At SMRs of
α2M:lys of 1:50 and 1:5, the lys precipitation was reduced by 40% and 80%, respectively
(Figure 5.4).
CHAPTER 5: RESULTS 83
0.8
0.6
nm 0.4 360 A 0.2
0.0
0 200 400 600 800
Time (min)
Figure 5.4 The effect of α2M on the oxidation-induced precipitation of lysozyme. Lysozyme (69 μM) was incubated in OSB in the presence or absence of α2M (1.4-14 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 α2M (SMRs of α2M: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 α2M 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 α2M and were found to have negligible effects on the precipitation of lys (Figure 5.5).
CHAPTER 5: RESULTS 84 nm 360 A
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 (A360). Data points shown are individual measurements and are representative of two independent experiments.
Once α2M was established as an inhibitor of the oxidation-induced precipitation of lysozyme, tests were then carried out to determine if α2M 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 α2M and lys. When analysed alone, α2M and lys were found to migrate to distinct positions on the gel according to their mass and charge. Oxidised α2M migrated slightly further on the gel than
α2M, whereas oxidized lys was barely detected on the gel (this was due to it largely precipitating from solution). Mixtures containing α2M (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 α2M/lys complex (Figure
5.6; black arrowhead). CHAPTER 5: RESULTS 85
Putative complex ►
(α2M + lys) (OSB)
α2M + lys (PBS)
α2M (OSB)
α2M (PBS)
lys (OSB)
lys (PBS)
Figure 5.6 Detection of putative α2M/lysozyme (lys) complexes by NGE. Samples of lys (1 mg/ml), α2M (2.5 mg/ml) or a mixture of lys and α2M (at the same final concentrations) were incubated at 37 °C in OSB (lys (ox) and α2M (ox) or α2M + lys (ox) respectively) or PBS (lys, α2M or lys + α2M respectively) and then analysed on a 1% native agarose gel. The arrowhead indicates a band of unique electrophoretic mobility which represents a putative α2M/lys complex.
The results obtained from NGE suggested that α2M 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 α2M and biotinylated lysozyme (lys- b). 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,
α2M alone did not bind to the streptavidin-agarose beads. However, both lys-b and α2M 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 α2M forms soluble complexes with lysozyme under conditions of oxidative stress, but not under normal physiological conditions. CHAPTER 5: RESULTS 86
α2M+ lys-b α2M lys-b non-OX OX
200 150 ◄ α2M
) 120
kDa ( 85
50
30
Molecular Mass Molecular
20 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 α2M, or mixtures of α2M 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 α2M 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 α2M within α2M/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 α2M within α2M/oxidized-protein complexes. In the classical trypsin binding assay a molar excess of trypsin is incubated with
α2M. Trypsin interacts with α2M to produce α2M*, 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 α2M* 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 α2M). Residual trypsin activity, attributable to α2M-trapped trypsin, was measured using the low molecular weight CHAPTER 5: RESULTS 87
substrate BAPNA. As expected, native α2M was able to bind trypsin in a dose-dependant manner, whereas methylamine-activated α2M did not bind trypsin (Figure 5.8).
Interestingly, the α2M in complexes formed with lysozyme under oxidative conditions acted in a similar fashion to α2M*, showing very limited binding to trypsin. In addition, samples of oxidized α2M alone did not bind to trypsin (Figure 5.8).
1
0.8
0.6 nm
405 405 0.4 A
0.2
0 0204060 Amount of protein (μg)
Figure 5.8 The effects of oxidation on the trypsin binding activity of α2M. Native α2M (♦), trypsin- activated α2M*( ), oxidised α2M (●) and α2M/lys complexes (■). The assay was performed 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.
α2M was found to protect a range of proteins in plasma from heat stress
(Chapter 3). Experiments were undertaken to investigate the ability of
α2M to protect proteins in whole human plasma during oxidative stress. When aliquots of NHP and α2M-depleted plasma (α2MDP) prepared from the same original batch of plasma (diluted 1 in 2 in OSB) and supplemented with azide, were incubated at 37 oC CHAPTER 5: RESULTS 88
for 48 h, the α2MDP contained significantly more aggregated protein than that detected in the NHP sample (Figure 5.9; Students t-test, p < 0.05). It was also found that when a physiological concentration of α2M (2.5 mg/ml) was added back into the α2MDP the level of protein precipitation returned to that observed in the NHP sample, significantly less than that observed in α2MDP (Figure 5.9; Students t-test, p < 0.05). Collectively, these results indicate that α2M has a chaperone action that can suppress the oxidation- induced aggregation and precipitation of unfractionated proteins in whole human plasma.
0.8 # * 0.6
g)
m ( 0.4
Protein 0.2
0
NHP α2MDP α2MP + α2M
Figure 5.9 α2M inhibits oxidation-induced protein precipitation in whole human plasma. Histogram showing the total protein precipitated from 100 μl aliquots of normal human plasma
(NHP), α2M-depleted plasma (α2MDP) and α2M-depleted plasma + 2 mg/ml α2M (α2MDP +α2M) (all diluted 1:2 in OSB) incubated at 37 °C for 48 h. Data points represent the means of triplicate measurements and error bars indicate the standard errors (SE) of the CHAPTER 5: RESULTS 89
means (* and # denote p < 0.05, Student's t-test). The results are representative of two independent experiments.
5.3.3 Oxidation of Protease Bound (Activated) α2M Re-establishes Chaperone Activity, but not Protease Inhibitor Function
The effect of oxidation on protease-activated α2M* was investigated. In contrast to heat- stress models (where α2M* shows negligible chaperone activity), under oxidative conditions, α2M* inhibited the precipitation of lys in a dose-dependant manner and at substoichiometric ratios (Figure 5.10). At an SMR of α2M*:lys of 1:10, precipitation was reduced by ~ 40%. At an SMR of α2M*:lys of 1:4, precipitation was reduced by approximately 75%.
0.8
0.6 nm 0.4 360 360 A 0.2
0.0 0 200 400 600 800 Time (min)
Figure 5.10 The effect of α2M* on the oxidation-induced precipitation of lysozyme. Lysozyme (69 μM in OSB) was incubated in the presence or absence of trypsin-activated α2M* (1.4-14 μM) and the turbidity associated with precipitation detected as an increase in absorbance at 360 nm, as described in methods section
2.8. Lys was stressed in the presence of various concentrations of α2M* (SMRs of α2M: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; X)]. Data points shown are individual measurements and are representative of three independent experiments.
CHAPTER 5: RESULTS 90
The apparent ability of α2M* to regain its chaperone activity following oxidation was
further investigated by co-incubating pre-oxidised samples of α2M and α2M* with heat-
stressed substrate proteins. It was found that pre-oxidised α2M was unable to inhibit the
precipitation of CS under heat stress, whereas the native form significantly reduced the
level of precipitation (Figure 5.11 A). However, pre-oxidised α2M* markedly reduced the
amount of precipitation to a level comparable to that obtained with native α2M (Figure 5.11
A). Similar results were obtained using CPK as the substrate protein. Oxidised α2M* was
able to inhibit the precipitation of CPK at 43 °C to a similar extent as native α2M (Figure
5.11 B). These results support the findings in section 5.3.1 which suggested that oxidation
results in marked structural and functional changes to the α2M molecule. It is possible that
when oxidized, α2M* adopts a conformation similar to that of the native form, facilitating
chaperone activity.
(A) CS (B) CPK
1.0 0.5 0.8 0.4
nm 0.6 0.3
360 A 0.4 0.2 0.2 0.1 0.0 0.0 0 40 80 120 160 200 240 0 40 80 120 160 200 240 280
Time (min)
CHAPTER 5: RESULTS 91
Figure 5.11 The effect of pre-oxidised α2M and α2M* on the heat-induced precipitation of substrate proteins. (A) Citrate synthase (CS; 6 μM) or (B) creatine phosphokinase (CPK; 25 μM) were incubated at 43
°C for 4 h either alone (●) or in the presence of native α2M (▲), oxidised α2M (▲) or oxidized α2M* ( ) (all at 7 μM for CS, or at 1.4 μM for CPK). The turbidity associated with protein precipitation was detected as an increase in absorbance at 360 nm, as described in methods section 2.8. Data points shown are individual measurements and are representative of three independent experiments.
Oxidation of α2M appears to have significant effects on its conformation, switching the molecule between chaperone active (native-like) and chaperone inactive (protease- activated-like) conformations. Experiments were undertaken to determine if α2M* could not only regain its chaperone activity but also its protease inhibitor activity following oxidation. This was investigated using a trypsin binding assay.
As shown previously (Chapter 3), native α2M was able to bind trypsin in a dose-dependant manner, while α2M* had limited ability to bind trypsin. Oxidation of α2M* had no effect on its ability to bind typsin (Figure 5.12), indicating that oxidized α2M* remains unable to function as a protease inhibitor.
1.5
1.0
nm
405 A 0.5
0.0
0 5 10 15 20 α M (μg) 2 CHAPTER 5: RESULTS 92
Figure 5.12 The effect of oxidation on the protease inhibitor activity of α2M. Native α2M (▲), oxidised α2M (■) and oxidized α2M* (▲) were assayed for trypsin binding activity, as described in methods section 2.7. Data points represent means of triplicate measurements and the error bars (visible when larger than the data points) represent SE of the means. Results shown are representative of three independent experiments.
5.4 Discussion
Results presented in this study support previous investigations which suggested that oxidation of α2M results in structural and functional changes to the protein. However, these results vary somewhat to previous studies in which H2O2 was used as the oxidant, where it was reported that millimolar levels of H2O2 have negligible effects on the structure and function of α2M (Wu and Pizzo 1999; Khan and Khan 2004). When exposed to OSB, α2M was found to fragment, consistent with bait region cleavage as detected by reducing SDS
PAGE. NGE also indicated that following oxidation, α2M migrates to a position significantly different from that of native α2M and α2M*, indicative of a change in size and/or overall charge of the molecule. These results were further supported by size exclusion chromatography analyses, which detected an asymmetrical peak for oxidized
α2M, which had a slower elution time than that of native α2M, again suggesting fragmentation.
Results presented in this chapter indicate that α2M specifically inhibits the oxidation- induced precipitation of lys. It was also shown that α2M inhibits the oxidation-induced precipitation of endogenous protein(s) in whole human plasma. The formation of complexes with stressed protein is a common mechanism used by chaperones to stabilize CHAPTER 5: RESULTS 93
proteins, to prevent their precipitation and protect them from further stresses. The formation of such complexes has been previously described for the extracellular chaperones clusterin, haptoglobin and α2M (under heat-stress conditions) (Humphreys, Carver et al. 1999;
French 2005; Yerbury, Rybchyn et al. 2005). Results presented in this chapter show, for the first time, the formation of stable complexes between α2M and oxidized lys. NGE identified a band of unique electrophoretic mobility, which represents a putative α2M-lys complex. The existence of a complex between α2M and lys was confirmed using immunoprecipitation. This is the first study of the chaperone action of α2M under oxidative-stress conditions. Future investigations should use a range of chaperone substrate proteins to confirm that α2M protects a variety of purified proteins from oxidative stress.
The use of physiological oxidants, such as those released from neutrophils during oxidative burst (a hallmark of the early inflammatory response), would expand the results of the
- current study. Hypochlorite is produced by the neutrophil H2O2-myeloperoxidase-Cl system at millimolar concentrations during inflammation and has been associated with significant structural changes in α2M and a resulting loss of protease inhibitor activity. The effect of hypochlorite on the chaperone function of α2M may provide further insight into the role of α2M during oxidative burst in vivo.
The relationship between the chaperone and the protease inhibitor activities of α2M under oxidative stress conditions was also investigated. In chapter 3 it was revealed that α2M within complexes with heat stressed CS and CPK retained the ability to interact with proteases. However, results presented here suggest that when in a complex with lys formed under oxidative conditions, α2M has very limited ability to interact with trypsin. This CHAPTER 5: RESULTS 94
finding may be significant in the context of in vivo mechanisms for disposal of
α2M/stressed protein complexes. Whereas native α2M within heat-stressed complexes must undergo protease activation before recognition by LRP and subsequent internalization, the
“active-like” state of α2M within complexes formed with chaperone substrates during oxidative stress may facilitate simple, single-step receptor binding. Further studies examining LRP binding and internalization of α2M/lys complexes are required to verify this proposed mechanism.
α2M* is able to function as a chaperone under oxidative conditions. At substoichiometric
SMRs, oxidised α2M* was able to inhibit the aggregation of lys to a level similar to that obtained with native α2M. The apparent ability of α2M* to revert to a “native-like” form following oxidation was further investigated by incubating pre-oxidised samples of α2M and α2M* with CS and CPK during heat stress. Whereas pre-oxidised α2M showed limited chaperone activity, for both substrate proteins, pre-oxidised α2M* was able to inhibit heat- induced protein precipitation to a similar level to that obtained with native α2M.
Collectively, these results indicate that oxidation may act as a switch for the chaperone activity of α2M, initiating transformation between chaperone-active and -inactive forms.
The ability of oxidation to act as a switch may be of importance during inflammation. In the presence of oxidative species formed during inflammation, protease bound α2M* may be converted to a chaperone active form, whereas native α2M within complexes with heat- stressed protein may be converted to an “active-like” state, facilitating receptor recognition.
A hypothetical model incorporating results presented in this chapter, the previous chapter, CHAPTER 5: RESULTS 95
and in the literature is shown in Figure 5.13. Further investigation is necessary to support the proposed mechanisms.
α2
Figure 5.13 Proposed structural and functional changes to α2M under conditions of oxidative stress. At sites of inflammation there is a high level of chaperone active α2M and chaperone-inactive α2M/protease OX complex. During oxidative stress, α2M may undergo conformational changes to form α2M which lacks both chaperone and protease inhibitor activities but is able to bind to LRP and is rapidly cleared in vivo (Wu, CHAPTER 5: RESULTS 96
Boyer et al. 1997). Following interaction with a stressed protein (SP), α2M/SP complexes may interact with protease molecules or undergo oxidation to facilitate LRP mediated binding and uptake. In addition, they may bind directly to the cell surface via other currently unknown receptors. Alternatively (right side of diagram),
α2M may interact firstly with proteases abundant at inflammatory sites to form chaperone-inactive α2M- OX protease complexes, which can subsequently be oxidized to yield chaperone-active (α2M-protease) complexes (these do not bind to LRP (Wu, Boyer et al. 1997)). After interacting with a stressed protein, OX α2M*/SP complexes may be internalized by currently unknown receptor(s) distinct from LRP. The exact structure of oxidized α2M and oxidized α2M/SP complexes for the oxidative conditions used in this study is currently unknown and is represented by a question mark (?) in each case.
French, K. (2005). Alpha-2-Macroglobulin: a putative extracellular chaperone. School of Biological Sciences. Wollongong, University of Wollongong: 93. Humphreys, D. T., J. A. Carver, et al. (1999). "Clusterin has chaperone-like activity similar to that of small heat shock proteins." Journal of biological chemistry 274(11): 6875- 6881. Khan, S., A. and F. Khan, H. (2004). "Oxidized caprine alpha-2-macroglobulin: damaged but not completely dysfunctional." Biochim Biophys Acta. 1674(2): 139-48. Reddy, V. Y., P. E. Desorchers, et al. (1994). "Oxidative dissociation of human alpha 2- macroglobulin tetramers into dysfunctional dimers." J. Biol. Chem. 269(6): 4683- 4691. Wu, S., M. and S. V. Pizzo (1999). "Mechanism of Hypochlorite-mediated inactivation of proteinase inhibition by alpha-2-macroglobulin." Biochemistry 38(42): 13983- 13990. Wu, S. M., C. M. Boyer, et al. (1997). "The Binding of Receptor-recognized alpha 2- Macroglobulin to the Low Density Lipoprotein Receptor-related Protein and the alpha 2M Signaling Receptor Is Decoupled by Oxidation." J. Biol. Chem. %R 10.1074/jbc.272.33.20627 272(33): 20627-20635. Yerbury, J., M. S. Rybchyn, et al. (2005). "The acute phase protein haptoglobin is a mammalian extracellular chaperone with an action similar to clusterin." Biochemistry 44(32): 10914-10925.
CHAPTER 6: DISCUSSION 96
CHAPTER 6: DISCUSSION
6.1 Advances in Understanding the Chaperone Action of α2M
It is a fundamental property of chaperones to form complexes with non-native proteins. By this action, chaperones prevent aggregation of misfolded proteins by ensuring exposed hydrophobic surfaces are protected from aberrant interactions with surrounding proteins
(Hartl and Hayer-Hartl, 2002). The extracellular chaperones clusterin, haptoglobin and α2M
(under heat stress conditions) all form stable complexes with substrate protein to prevent aggregation (Humphreys et al., 1999; French, 2005; Yerbury et al., 2005). Results of this study indicate, for the first time, that under conditions of oxidative stress α2M forms stable complexes with lys. NGE of oxidized mixtures of α2M and lys detected a band of unique electrophoretic mobility, representing a putative α2M/lys complex. The identity of this species was later confirmed by immunoprecipitation analysis. In contrast to α2M within complexes formed with heat-stressed proteins, α2M incorporated into complexes with oxidatively stressed lys showed limited ability to bind trypsin. This result suggests that following interaction with oxidatively stressed lys, the α2M molecule undergoes a conformational change which inhibits subsequent binding of a protease, or that when bound to α2M, lys itself sterically obstructs access of trypsin to the α2M bait region. Further investigation is required to determine the exact mechanism responsible for the loss of protease inhibitory activity.
Holdase chaperones lack the ability to independently refold proteins (Fink, 1999). A defining characteristic of the small heat shock proteins (sHsps), and other “holdase” type CHAPTER 6: DISCUSSION 97
extracellular chaperones such as clusterin and haptoglobin, is their ability to selectively bind stressed unfolding proteins, usually by hydrophobic interactions, to form stable soluble complexes (Fink, 1999; Yerbury et al., 2005). Although the mechanism of stressed protein binding and the location of chaperone binding sites on the α2M molecule are yet to be revealed, studies using the hydrophobic probe bisANS have revealed that α2M binds to stressed proteins, at least in part, via hydrophobic interactions (French et al., 2008).
Furthermore, α2M is now known to form stable soluble complexes with proteins exposed to either heat or oxidative stress. Using the enzymes GST and CS, α2M alone was unable to protect enzymes from heat-induced loss of activity or to promote recovery of this activity following heat stress, regardless of the presence or absence of ATP (French et al., 2008). A motifscan (http://myhits.isb-sib.ch/cgi-bin/motif_scan) and a BLAST search failed to identify any known ATPase motifs in the primary structure of α2M or sequence similarity to any known ATPases, respectively. Collectively, these findings suggest that α2M is an extracellular "holdase" type chaperone, which acts to sequester non-native proteins into stable, soluble complexes which may subsequently become internalized by cells.
6.1.1 Dual Chaperone and Protease Inhibitory Roles of α2M
In this study it was revealed that α2M remains in a native-like conformation when incorporated into complexes with heat-stressed proteins, retaining its protease inhibitor activity. Once it has undergone the protease trapping reaction, α2M loses its chaperone activity - α2M* had limited ability to inhibit the precipitation of CS and CPK at 43 °C.
However, α2M may first interact with stressed proteins and later trap proteases to form a CHAPTER 6: DISCUSSION 98
stressed protein/α2M/protease complex. This establishes α2M as the first known mammalian protein with coexisting chaperone-like and protease inhibitor activities.
6.2 Oxidative Stress and α2M
Reactive oxygen species such as superoxide anion, hydrogen peroxide (H2O2), hydroxyl radical and hypochlorite play many important roles during acute and chronic inflammation including neutralization of bacteria, apoptosis and tissue destruction (Wu et al., 1998). The
-9 -10 steady state concentration of H2O2 in the cell ranges from 10 – 10 M (Boveris and
Chance, 1973), with the concentration markedly increasing at the inflammatory site.
2+ During inflammation and carcinoma, the in vivo levels of H2O2 and Cu can approach the millimolar level, similar to those used in this study to exert oxidative stress (Abdulla et al.,
1979; Weiss, 1989). When exposed to 4 mM H2O2 in OSB, α2M underwent significant structural changes. Reducing SDS PAGE, size exclusion chromatography and native PAGE all suggested fragmentation and conformational modification. These results differ somewhat from the results of Khan and Khan (2004), who suggested that at “modest levels” of H2O2 (500 mM H2O2 in 50 mM phosphate buffer pH 7.4 for 2 h, with the inclusion of free radical scavengers), the conformational status of α2M remained largely unaffected. However, these conditions differ considerably from those used in this study (4 mM H2O2 in OSB), providing a possible explanation for the variation in results.
α2M was found to significantly inhibit the oxidation-induced precipitation of lys in a dose dependent manner. This effect was specific because in the same system superoxide dismutase (SOD) and bovine serum albumin (BSA) had negligible effects on the level of CHAPTER 6: DISCUSSION 99
precipitation. Depletion of α2M from human plasma also resulted in an elevated level of protein precipitation following incubation under oxidative conditions for 48 h. This demonstration of the endogenous effects of α2M as a chaperone highlights its potential importance in the first line defense against oxidative stress in the extracellular environment.
It was determined in this study that under conditions of heat-stress, the abundant extracellular protein fibrinogen associates with α2M. Further investigations are needed to identify putative endogenous chaperone substrates of α2M under oxidative stress conditions. Considering the postulated role of α2M in the coordination and regulation of the inflammatory response (see below), identification of proteins chaperoned by α2M during oxidative conditions (a hallmark of early inflammation), may provide further insight into the role of α2M in inflammation and disease.
6.2.1 Implications for Inflammatory Response Regulation and Chaperone Functionality
An important observation in this study was the inability of α2M* to protect the heat stressed precipitation of CS and CPK. It was determined that the oxidative stress conditions used in this study (4 mM H2O2 in OSB) were responsible for significant structural changes in the
α2M molecule. Furthermore, under these oxidative stress conditions, α2M* retained the ability to protect lys from oxidation-induced precipitation. A possible explanation for this is the formation of a chaperone-active “native-like” α2M* structure under oxidative stress conditions. This was further investigated by incubation of oxidized and non-oxidised α2M* with CS and CPK under heat stress conditions (43˚C). Pre-oxidised α2M* had the ability to inhibit the heat-induced precipitation of substrate proteins CS and CPK. Collectively, the results presented here indicate that oxidation of α2M may act as a functional “switch”. CHAPTER 6: DISCUSSION 100
When the switch is turned on (i.e. in the presence of oxidative stress) chaperone inactive
α2M* is converted to a “chaperone active” with characteristics similar to that of native
α2M. Conversely, oxidation may convert α2M into an “active-like state”, ablating its chaperone activity but exposing an LRP binding site.
Previous studies have investigated the effect of hypochlorite oxidation on the abilities of
α2M and α2M* to bind to cell surface receptors. Hypochlorite oxidation of α2M* completely abolishes its ability to bind LRP, without affecting its ability to bind to the signaling receptor (responsible for triggering cell responses during inflammation) (Wu et al., 1997). Furthermore, studies have also revealed that oxidation of α2M results in the selective exposure of receptor recognition sites for LRP while destroying the capacity of the signaling receptor to trigger cellular responses (Wu et al., 1997). Oxidation inhibits the uptake of α2M* into the cell, allowing it to signal prostaglandins and platelet activating factor which are necessary for tissue repair (Wu et al., 1998). On the other hand, oxidation facilitates the uptake and removal of native α2M and importantly, α2M in complexes with acute phase cytokines, into the cell via LRP (TNF-α, IL-2 and IL-6). These cytokines function as pro-inflammatory mediators, responsible for the initial stages of inflammation.
Collectively, these findings suggest that oxidation controls the anti-inflammatory function of α2M. Oxidation acts to inhibit the removal of α2M* from plasma allowing it to signal the mediators of tissue repair via signaling receptors, whilst also inhibiting the progression of the pro-inflammatory cascade induced by acute phase reactants and cytokines by exposing the receptor binding site in native α2M, allowing the removal of α2M complexed with pro-infammatory cytokines from the circulation (Wu et al., 1998) (Wu et al., 1997). CHAPTER 6: DISCUSSION 101
It is possible that oxidation may also provide a means by which to control the chaperone action of α2M, a role that is linked to its protease inhibitor and inflammatory modulator functionalities. Oxidation provides a mechanism by which α2M can stay “chaperone active” in the protease-rich sites of inflammation, where high levels of protein misfolding are likely to occur and chaperones are needed. Oxidation also removes the need for
α2M/stressed protein complexes to interact with a protease before exposure of the LRP binding sites. Although this study is preliminary in nature, the results presented here not only confirm α2M as a chaperone for oxidatively stressed proteins, but importantly provide further insight into a possible mechanism for the removal of α2M/stressed complexes.
6.3 Fibrinogen is a Putative Endogenous Chaperone Substrate of α2M
Results presented in this study demonstrate that endogenous α2M significantly inhibits the spontaneous precipitation of proteins in unfractionated human plasma incubated at 37˚C.
Furthermore, it was found that the effects of α2M and clusterin are additive, with a greater level of precipitation observed when both extracellular chaperones were removed from plasma. This finding suggests complementarity with respect to the proteins they protect.
Abundant extracellular proteins with chaperone abilities may act as an important line of defense against inappropriate extracellular protein aggregation, which occurs at high levels during inflammation and is associated with many human diseases (Yerbury et al., 2005).
MALDI TOF mass spectrometry was used to determine the identity of putative endogenous substrates for α2M in human plasma. Fibrinogen (β and γ chains) was positively identified as an extracellular protein chaperoned by α2M. Human fibrinogen is a heterodimeric, CHAPTER 6: DISCUSSION 102
multichain, acute-phase protein, with each identical monomer containing three distinct polypeptides (Aα, Bβ and γ) which are linked in dimers by symmetrical disulfide bonds
(Roy et al., 1992). Fibrinogen, the precursor of fibrin, is the major plasma protein coagulation factor, playing an important role in platelet aggregation (Lowe et al., 2004).
The role of α2M in the protection of fibrinogen from extracellular stresses may have important medical implications. Loss of functional fibrinogen and resulting low concentrations of the coagulation factor have been associated with an increased risk of bleeding due to impaired haemostasis (Lowe et al., 2004). Further investigations characterising the interactions of α2M and fibrinogen, including protein precipitation assays and receptor recognition studies are required in order to confirm and further expand on the preliminary results presented in this study. Further studies are also required to identify other endogenous substrates for α2M.
6.4 Cell Surface Receptor Binding of α2M and α2M/Stressed Protein Complexes
This study demonstrated that following interaction with a protease (trypsin), but not otherwise, α2M incorporated into complexes with heat-stressed proteins exposed binding site(s) for LRP, consistent with it adopting an "activated" conformation. Conversion to
α2M* was also indicated by the demonstration of a covalent association between trypsin and α2M in these complexes. In support of this, “activated” (α2M/CS)* complexes showed significant, RAP inhibitable binding to LRP expressed on JEG-3 cells. It follows that if one important function of α2M 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 be one in vivo switch to trigger LRP-mediated uptake of α2M/stressed protein CHAPTER 6: DISCUSSION 103
complexes. At physiological locales such as sites of inflammation, this process would be facilitated by the relative abundance of proteases. Considering the effects of oxidation on the structure of α2M as detailed in this study (including the significant conformational change into an “active-like” form) and published reports indicating that α2M can to bind to
LRP following oxidation, further investigations focusing on cell surface binding of oxidized α2M/stressed protein complexes will provide further insight into the role of oxidation in the clearance of stressed extracellular proteins. If the receptor binding sites do indeed become exposed following oxidation of α2M within α2M/stressed protein complexes, it will provide a putative mechanism for α2M/stressed protein complex internalization and removal.
In this study it was determined that α2M and α2M/CS complexes bound significantly to the surface of Hep-G2 and granulocytes derived from whole human blood. Investigations with
Hep-G2 cells revealed that the binding was not affected by pre-incubation of cells with the
LRP ligand, RAP. However, pre-incubation of cells with galactose resulted in a significant reduction in binding. Galactose is a ligand of the asialoglycoprotein receptor, a scavenger- type receptor abundant on the surface of liver cells, such as the Hep-G2 cell line (Schwartz et al., 1982). α2M/CS complexes also showed significant binding to granulocytes. Similar to the results obtained for Hep-G2 cells, pre-incubation with RAP had little effect on the level of binding. The ability of α2M/stressed protein complexes to interact with receptors prior to interaction with proteases or other “activating events” (such as oxidation) provides a convenient alternative to LRP mediated binding, which may facilitate rapid uptake of stressed extracellular protein by cells. Results presented here indicate that non-activated CHAPTER 6: DISCUSSION 104
α2M/stressed protein complexes may be recognised and internalised via currently unknown cell surface receptors, distinct from LRP. The asialoglycoprotein receptor is one potential candidate for this role. Further investigations using other specific inhibitors, such as asialofetuin (a ligand specific for the asialoglycoprotein receptor), are necessary to determine the identity of the receptor(s) responsible for binding of α2M/stressed protein complexes and α2M in its native conformation.
A hypothetical model for the in vivo functions of α2M, incorporating findings from this thesis, is presented in Figure 6.1.
CHAPTER 6: DISCUSSION 105
Figure 6.1 Proposed model for the in vivo functions of 2M. At sites of inflammation, factors such as elevated temperature, reactive oxygen species (ROS), and lowered pH may cause damage to extracellular proteins inducing them to partially unfold. Host immune cells are known to secrete proteases in an attempt to destroy invading pathogens. In addition, a variety of cell types may locally secrete cytokines or growth factors. Native 2M may exert a broad anti-inflammatory action by binding to and promoting the clearance of endogenous or exogenous proteases and other ligands such as denatured proteins and cytokines. The α2M- mediated clearance of non-protease ligands can occur via LRP following the activation of 2M/ligand complexes by interaction with proteases, or possibly via conversion to an active-like state by oxidation, which are both likely occurrences at sites of inflammation. Clearance of native 2M/ligand complexes may also occur via other cell surface receptors, independently of protease activation. Adapted from (French et al., 2008).
CHAPTER 6: DISCUSSION 106
6.5 Conclusion
The results presented in this thesis further characterize the chaperone function of α2M, and provide insight into how the chaperone role may relate to the protease inhibitor and anti- inflammatory functions in vivo. It was determined that α2M remains in a native-like state following the formation of complexes with heat-stressed proteins and can subsequently become activated by interaction with proteases. This study also identified fibrinogen as a putative endogenous chaperone substrate of α2M in human plasma, under heat stress conditions. α2M was shown to also function as a chaperone under conditions of oxidative stress, inhibiting the oxidation-induced precipitation of lys by forming stable α2M/lys complexes. Oxidation was found to switch α2M* from a chaperone-inactive state to a chaperone-active state. Receptor binding studies of purified α2M/stressed protein complexes indicated that complexes formed between α2M and CS can be subsequently proteolytically activated and recognised by LRP. Furthermore, preliminary investigations identified significant non-LDLR-mediated binding of α2M/CS complexes to liver cells and granulocytes. This finding suggests the existence of receptors other than LRP for complexes formed between α2M and chaperone substrate proteins.
Overall, the results presented in this thesis establish α2M as the first known mammalian protein with both chaperone and protease inhibitor activities. These findings contribute to a greater understanding of protein quality control in the extracellular space, and provide information which may ultimately prove vital in the treatment and prevention of PCDs.
CHAPTER 6: DISCUSSION 107
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