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2008
Alpha-2-Macroglobulin: an abundant extracellular
chaperone
Katie French
University of Wollongong
Recommended Citation
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
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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
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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 oxidationinduced 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
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
xiii
Alpha-2-Macroglobulin: An Abundant Extracellular Chaperone
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
xiv
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