-
An Investigation of Porphyromonas gingivalis
Peptidylarginine Deiminase: A Putative Virulence Factor in
an
Animal Model of Inflammation
SYATIRAH NAJMI ABDULLAH
BSc.Hons(Biomedical Sc.)
Thesis submitted for the degree of Master of Science in
Dentistry
School of Dentistry
The University of Adelaide
November 2010
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ii
TABLE OF CONTENTS Table of Contents
...........................................................................................................................................
ii
List of Figures
................................................................................................................................................
ix
List of Tables
.................................................................................................................................................
xi
Abbreviations
................................................................................................................................................
xii
Abstract
........................................................................................................................................................
xvi
Declaration
.................................................................................................................................................
xviii
Acknowledgment
.........................................................................................................................................
xix
Chapter 1
.......................................................................................................................................................
1
1 A Review of the Literature
...................................................................................................................
2
1.1 Introduction
.....................................................................................................................
2
1.2 Periodontal Disease
.......................................................................................................
4
1.2.1 Introduction
..............................................................................................................
4
1.2.2 Types of periodontal disease
..................................................................................
7
1.2.3 Microorganisms linked to the aetiology of periodontal
disease............................. 8
1.3 Porphyromonas gingivalis and Periodontal Disease
..................................................10
1.3.1 Porphyromonas gingivalis virulence factors
.........................................................11
1.3.2 Peptidylarginine deiminases
.................................................................................13
1.3.3 Peptide metabolism by Porphyromonas gingivalis
..............................................15
1.4 Citrullination
..................................................................................................................16
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1.4.1 Citrullination and the immune system
..................................................................17
1.4.2 Citrullinated human proteins
.................................................................................18
1.5 Rheumatoid
Arthritis.....................................................................................................20
1.5.1 Introduction and history
.........................................................................................20
1.5.2 Features of rheumatoid arthritis
............................................................................24
1.6 Rheumatoid Arthritis and Citrullination
........................................................................25
1.6.1 Citrullination in RA patients and clinical markers
.................................................26
1.7 Research
Questions.....................................................................................................27
Chapter 2
.....................................................................................................................................................
28
2 Investigation of Porphyromonas gingivalis Peptidylarginine
Deiminase ......................................... 29
2.1 Citrulline Assay
.............................................................................................................30
2.1.1 Colour detection reagent
.......................................................................................30
2.1.2 Standard curve to determine citrulline concentration
..........................................31
2.2 Citrullination by Mammalian Peptidylarginine Deiminase
..........................................33
2.2.1 Citrullination of BAEE by mPAD
...........................................................................33
2.2.2 Citrullination of free arginine by mPAD
................................................................36
2.2.3 Discussion
.............................................................................................................38
2.3 Cultivation of Porphyromonas gingivalis
.....................................................................38
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2.4 Cell Harvesting and Preparation
.................................................................................40
2.5 Protein Assay
...............................................................................................................40
2.5.1 Method
...................................................................................................................40
2.5.2 Results
...................................................................................................................40
2.6 Citrullination of BAEE by Porphyromonas gingivalis
..................................................43
2.6.1 Method
...................................................................................................................43
2.6.2 Results and discussion
.........................................................................................44
2.7 Characterisation of P. gingivalis Peptidylarginine Deiminase
....................................46
2.7.1 Effect of environmental pH on PgPAD activity
.....................................................46
2.7.2 Effect of elevated temperature and enzyme localisation
.....................................48
2.7.3 Peptidylarginine deiminase specificity for
peptidylarginine position ...................51
2.7.4 Arginine analogues as substrates and competitive
inhibitors for citrullination ...53
2.8 Citrullination of Arginine-containing Proteins
..............................................................56
2.8.1 Citrullination of yeast enolase by mPAD
..............................................................56
2.8.2 Citrullination of arginine containing proteins by PgPAD
......................................59
2.9 Effect of Gingipains on PgPAD Activity
.......................................................................60
2.9.1 Gingipain activity
assay.........................................................................................61
2.9.2 Effect of gingipains inhibitors using azoalbumin assay
.......................................63
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2.9.3 Effect of gingipain inhibitors on citrullination of
albumin by PgPAD....................65
2.9.4 Effect of gingipain inhibitors on citrullination of BAEE
by PgPAD .......................67
Chapter 3
.....................................................................................................................................................
68
3 A Histological Survey of Infiltrated Cells in Selected Tissues
of Adjuvant Arthritis induced rats Pre-treated with Heat Killed
Porphyromonas gingivalis.
....................................................................
69
3.1 Introduction
...................................................................................................................69
3.2 Hypotheses
...................................................................................................................71
3.3 Experimental Animal Model for Chronic Inflammation and
Adjuvant Arthritis ...........71
3.3.1 Animal model
.........................................................................................................71
3.3.2 Preparation of periodontogenic stimulus
..............................................................72
3.3.3 Preparation of arthritogenic stimulus
....................................................................73
3.3.4 Assessment of clinical polyarthritis
.......................................................................73
3.4 Collection and Processing of Tissue Samples and Sponges
....................................73
3.4.1 Decalcification of the heads
..................................................................................73
3.4.2 Collection and processing of sponges and spleen for
routine histology .............74
3.4.3 Dissection of decalcified heads and sponges for routine
histology ....................74
3.4.4 Tissue processing
.................................................................................................74
3.5 Staining
.........................................................................................................................75
3.5.1 Routine haematoxylin and eosin
staining.............................................................75
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3.5.2 Histochemical staining for identification of osteoclasts
.......................................75
3.5.3 TRAP control
tissue...............................................................................................76
3.5.4 Cell counting
..........................................................................................................79
3.5.5 Immunohistochemical detection of citrullinated protein in
sponge inplants ........79
3.6 Results
..........................................................................................................................81
3.6.1 Histological Survey
................................................................................................81
3.6.2 Polymorphonuclear cells in bone marrow spaces as a measure
of proliferation ...
89
3.6.3 Cellular infiltrate in implanted sponges
................................................................92
3.6.4 In situ detection of citrullinated protein in the sponge
infiltrate ...........................96
3.6.5 Identification of osteoclasts
.................................................................................100
Chapter 4
...................................................................................................................................................
104
4 Discussion
........................................................................................................................................
105
4.1 Introduction
.................................................................................................................105
4.2 Characterisation of P. gingivalis PAD
.......................................................................106
4.2.1 PgPAD Specificity
...............................................................................................108
4.2.2 Citrullination of proteins by PgPAD
....................................................................109
4.2.3 Influence of gingipains on citrullination
..............................................................111
4.2.4 Role of PgPAD in arginine metabolism
..............................................................112
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4.2.5 Exacerbation of the inflammatory response in AA by prior
exposure to P.
gingivalis 114
4.3 Conclusion
..................................................................................................................115
Bibliography
...............................................................................................................................................
117
Appendices
................................................................................................................................................
130
Appendix 1.1 Protein Assay
..............................................................................................131
Appendix 1.2: Citrullination of BAEE by rabbit muscle PAD (Sigma
no.: p1584) ............133
Appendix 1.3: Tissue processing
.......................................................................................134
Appendix 1.4 Hematoxylin and eosin
staining..................................................................136
Appendix 1.5 Preparation of stock solution for TRAP staining
........................................137
Appendix 1.6 Immunohistochemistry – Citrullinated
protein............................................138
Appendix 2 Results
.............................................................................................................140
Appendix 2.1 Citrullination activity of rabbit muscle PAD at
absorbance of 530nm .......140
Appendix 2.2 Citrullination activity of Porphyromonas gingivalis
PAD at absorbance of
530nm 140
Appendix 2.3 Effect of environmental pH
.........................................................................141
Appendix 2.4 Peptidylarginine deiminase specificity for arginine
position......................141
Appendix 2.5 Arginine analogues as potential substrates and
potential competitive
inhibitors for citrullination
.........................................................................................................142
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Appendix 2.6 Gingipain assay absorbance reading at 440nm
........................................142
Appendix 2.7 Citrullination of yeast enolase by mPAD at
absorbance of 530nm ..........143
Appendix 2.8 Effect of gingipains inhibitors using azoalbumin
assay absorbance reading
at 440nm 143
Appendix 2.9 Effect of gingipain on citrullination of BAEE by
PgPAD at absorbance of
530nm 144
Appendix 2.10 Effect of gingipain on citrullination of albumin
by PgPAD at absorbance of
530nm 144
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LIST OF FIGURES Figure 1.1: Surgical exposure of bone loss
(arrow) resulting from periodontitis as adapted from
(Pihlstrom et al. 2005).
.....................................................................................................................
5
Figure 1.2: Electron microscopic image of Porphyromonas
gingivalis taken from
http://www.microbiologybytes.com.
...............................................................................................11
Figure 1.3: Citrullination activity by P. gingivalis PAD.
.................................................................14
Figure 1.4: The process of citrullination of arginine to
citrulline catalysed by mammalian PAD.
........................................................................................................................................................17
Figure 1.5: La Familia de Jordaens en un Jardín Jacob Jordaens
..............................................21
Figure 1.6 Changes that occur in the synovial joint as a result
of RA. ........................................24
Figure 2.1: A standard curve for the coulorimetric determination
of citrulline concentration. .....32
Figure 2.2: The citrullination of BAEE by rabbit muscle PAD.
.....................................................35
Figure 2.3: The citrullination activity of mPAD against free
arginine. ..........................................37
Figure 2.4: A Gram stain of P. gingivalis W50 under light
microscopy oil immersion at 1,000 x
magnification.
.................................................................................................................................39
Figure 2.5: The standard curve of protein concentration at a
wavelength of 595 nm. ................42
Figure 2.6: The microtitre plate colourimetric method used to
detect citrulline. ..........................44
Figure 2.7: Rate of citrullination of BAEE by P. gingivalis W50
cells...........................................45
Figure 2.8: The effect of environmental pH on citrullination of
BAEE by PgPAD. ......................47
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Figure 2.9: The effect of heat and sonication on PgPAD
activity.................................................50
Figure 2.10: Citrullination of yeast enolase by mPAD.
.................................................................58
Figure 2.11: The proteolytic activity of P. gingivalis washed
cells using the azoalbumin assay.
........................................................................................................................................................62
Figure 3.1: Tartrate-resistant acid phosphatase (TRAP)
staining................................................77
Figure 3.2: Histology of the normal tissue sections of
rats...........................................................82
Figure 3.3: Lateral sagittal section from normal rat's head
stained by routine H&E (Low power)
........................................................................................................................................................84
Figure 3.4: Lateral sagittal section of normal rat head showing
maxillary region (Low power). .86
Figure 3.5: Lateral sagittal section of lower jaw from a normal
rat. .............................................88
Figure 3.6: Cross section of bone marrow taken from area of
interest of the head (High power).
........................................................................................................................................................90
Figure 3.7: Sections taken from sponges and tissue surroundings
stained by routine H&E ......93
Figure 3.8: Polyclonal anti-citrulline antibody staining of
sponges and tissues taken from the rat
flank.................................................................................................................................................97
Figure 3.9: The TRAP staining of rats from three different
groups. ...........................................101
Figure 4.1: Role of peptidylarginine deiminase in energy
production of P. gingivalis. ..............113
Figure 4.2: Shandon Citadel 2000 automatic tissue processor
.................................................134
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LIST OF TABLES Table 1.1: The 2010 American College of
Rheumatology/ European League against
rheumatism classification criteria for rheumatoid arthritis.
...........................................................23
Table 2.1: Comparison of PAD activity in intact heat killed and
sonicated cells. ........................49
Table 2.2: Peptides used as substrates for PgPAD.
....................................................................52
Table 2.3: PgPAD activity against arginine-containing peptides
and free arginine. ...................53
Table 2.4: The citrullination of various arginine analogues and
their ability to act as competitive
inhibitors of the reaction.
................................................................................................................55
Table 2.5: Arginine-containing proteins and their rates of
citrullination by PgPAD .....................60
Table 2.6: The inhibition of cellular proteolytic activity.
................................................................64
Table 2.7: The effect of gingipain inhibitors on PgPAD activity
...................................................66
Table 2.8: The direct effect of gingipain inhibitors on PgPAD
.....................................................67
Table 3.1: PMN proliferation in bone marrow (section stained by
H&E). ....................................92
Table 3.2: PMN cells observed within the infiltrate of implanted
sponges with and without
HKPg.
..............................................................................................................................................96
Table 4.1 Reagents used for the experiment and their
concentration and volume ..................133
Table 4.2: The step for Shandon Citadel 2000 automatic tissue
processor for impregnation of
tissue
.............................................................................................................................................135
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ABBREVIATIONS ACPA Anti-citrullinated protein antibodies
AKA Anti-keratin antibodies
anti-CCP Anti-citrullinated cyclic peptide antibodies
APF Anti-perinuclear factor
Arg-Xaa Arginine carboxy terminal peptide bond
ATP Adenosine triphosphatase
AU Absorbance unit
BAEE Benzoyl-arginine ethyl ester
BHI Brain-heart infusion
BSA Bovine serum albumin
Ca2+ Calcium ion
CaCl2 Calcium chloride
CO2 Carbon dioxide
C-terminal Carboxy terminal
DNA Deoxyribonucleic acid
EDTA Ethylenediaminetetraacetic acid
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EGTA ethylene glycol tetraacetic acid
et al. et alia
FeCl3 Iron (III) Chloride
FMN Flavin mononucleotide
g Gravitational force
GCF Gingival crevicular fluid
H2 Hydrogen
HCl Acid hydrochloric
IgA Immunoglobulin A
IgG Immunoglobulin G
kDa kilo Dalton
Kgp Lysine gingipain
Lys-Xaa Lysine carboxy terminal peptide bond
M Molar
mg cell protein-1.min-1 Milligram per cell protein per
minutes
mM milliMolar
mPAD Rabbit muscle/ mammalian PAD
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N2 Nitrogen
nm nanometre
nmoles nanomoles
nmoles citrulline.unit-1.min-1 nanomoles citrulline per unit per
minute
N-terminal Amino terminal
oC Degree Celcius
OD560 Optical Density at 560nm
PAD Peptidylarginine deiminase
PD Periodontal disease
Pg Porphyromonas gingivalis
PgPAD Porphyromonas gingivalis peptidylarginine deiminase
PMN Polymorphonuclear
RA Rheumatoid arthritis
RF Rheumatoid factor
Rgp Arginine gingipain
R-group Amino acid functional group
Sp. Species
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TNF- Tumor necrosis factor alpha
TPCK Tosyl phenylalanyl chloromethyl ketone
Tris-HCl Tri sulphate – hydrochloric acid
v/v Volume per volume
w/v Weight per volume
μL microlitre
μm micrometre
met-arg-phe Methionine-arginine-phenylalanine
H2O2 Hydrogen peroxide
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ABSTRACT Porphyromonas gingivalis, an oral periodontopathogen
linked to chronic periodontitis
expresses peptidylarginine deiminase (PAD), an enzyme that
converts peptide-bound arginine
to citrulline. A relationship between human PADs and chronic
inflammatory diseases has been
proposed. Citrullinated -enolase is a candidate auto-antigen in
rheumatoid arthritis. Vimentin
and fibrin are also likely target proteins in disease
development. This study partially
characterised the enzyme and the ability of P. gingivalis cells
to citrullinate peptides and these
rheumatoid arthritis relevant proteins. In addition, the
influence of gingipains, key P. gingivalis
virulence factors, on PgPAD activity was investigated. A limited
histological survey was
performed on selected tissues to investigate the effect of P.
gingivalis in an animal model of
adjuvant arthritis.
A colourimetric assay to quantify citrulline was developed and
used to determine the effect of
environmental pH and temperature on enzyme activity. Enzyme
localization was investigated
by comparing reaction rates of whole cells to cell sonicates.
Enzyme specificity was determined
by incubation of cells with a range of arginine analogues and
arginine-containing peptides. The
rates of citrullination of enolase, vimentin and fibrin by P.
gingivalis cells were calculated. The
influence of the gingipains on citrullination was measured by
comparing the rate of citrullination
of albumin in the presence and absence of the proteolytic
inhibitors tosyl phenylalanyl
chloromethyl ketone and leupeptin. Tissue sections from three
regions of the animal heads
were stained for polymorphonuclear cells and osteoclasts. In
addition sponge samples were
surveyed for polymorphonuclear cells and citrullinated proteins
detected using
immunohistochemical technique.
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xvii
PgPAD activity was heat stable, predominantly cell-surface
expressed and exhibited optimal
activity between pH 7.5 and 8. The enzyme was highly specific
for arginine and citrullinated
arginine residues in all positions in the peptides tested. PgPAD
was able to citrullinate all
rheumatoid arthritis relevant proteins, at rates slower than
peptides. Inhibition of the gingipains
failed to influence the rate of citrullination of albumin. In
the adjuvant arthritis animal study, pre-
treatment with P. gingivalis produced increased inflammatory
cellular infiltrate at the site of
exposure but no similar affect in the head tissue. There was a
significant increase numbers of
polymorphonuclear cells in the bone marrow from the head region
and in the implanted sponge
infiltrate from rats with prior exposure to P. gingivalis.
Although citrullinated proteins were
detected in sponge sections from both adjuvant arthritis-induced
rat groups, no difference
between them was observed. A similar result was seen with
osteoclasts, as both groups
exhibited increased numbers over the control group.
This study has shown that P. gingivalis peptidylarginine
deiminase has potential to influence
the inflammatory process by citrullinating arginine containing
peptides and rheumatoid arthritis
relevant proteins. An examination of rats exposed to the
bacterium in an animal model of
rheumatoid arthritis did not appear to exacerbate inflammation
in selected tissues.
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DECLARATION
This work contains no material which has been accepted for the
award of any other degree or
diploma in any university or other tertiary institution to
Syatirah Najmi Abdullah and, to the best
of my knowledge and belief, contains no material previously
published or written by another
person, except where due reference has been made in the
text.
I give consent to this copy of my thesis, when deposited in the
University Library, being made
available for loan and photocopying, subject to the provisions
of the Copyright Act 1968. I also
give permission for the digital version of my thesis to be made
available on the web, via the
University’s digital research repository, the Library catalogue,
the Australasian Digital Theses
Program (ADTP) and also through web search engines, unless
permission has been granted
by the University to restrict access for a period of time.
Signed:
Date:
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xix
ACKNOWLEDGMENT I would like to express my deepest sense of
gratitude to my Principal supervisor Dr. Neville
Gully, a friend and an excellent mentor, for his patient
guidance, constant encouragement and
invaluable suggestions throughout this study. He has been
everything that one could ask for as
an advisor.
My Co-supervisors, Dr Elizabeth-Anne Farmer, A/Prof Dr. Richard
Logan and Mr. Llew Spargo
for their countenance, valuable contributions and guidance in
making this happen.
I am deeply indebted to Mr Victor Marino and Prof Mark Bartold
of Colgate Australian Clinical
Dental Research Centre, Dr David Haynes, Ms. Melissa Cantley and
Dr Kencana Dharmapatni
of Discipline of Pathology, School of Medical Sciences, The
University of Adelaide for their
collaboration and valuable assistance in the AA induced HKPg
model research.
My sincere thanks go to Ms. Sandy Hughes and Ms. Marjorie Quinn
for their generous
assistance in histology and for being great friends in that
quiet yet wonderful time of tissue
sectioning.
To my friends Atika, Jactty, Yi, Arnida, Zati, Fauziah, Nadiyah,
Liyana, Yanti, Rafisah, Awan,
Nurul, Suhaiza, Azlina, Aini, Aida, Saidatul, Fiona, Nikki,
Kazu, Anh, Judy and all those people
who made this research possible, for sharing experiences and
knowledge and an enjoyable
experience for me.
I would like to express my gratitude to my Scholarship sponsor
from the Ministry of Higher
Education Malaysia, Universiti Sains Islam Malaysia, and to the
Australia Dental Research
Foundation and School of Dentistry, The University of Adelaide,
for their financial support.
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xx
Finally, I would like to express my deepest gratitude for the
constant support, understanding
and love that I received from my parents; Ayah Abdullah and Mak
Wan Nasrah, my incredible
sisters; Ainul and Atiqah, brother Shaharen and family during
the past years.
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CHAPTER 1
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1 A Review of the Literature
1.1 Introduction
Microbial pathogens often interact in complex ways within their
mammalian hosts. In human
disease, there are many well-understood examples of microbes
colonising bodily fluids and
tissues to the detriment of their host by producing destructive
toxins or enzymes, such as in
infections of environmentally accessible mucosal surfaces or in
opportunistic infections. Less
well-understood are periodontal diseases (PD) such as
periodontitis, where, in susceptible
individuals, a chronic inflammatory response is associated with
persistent microbial
colonisation of specific sites. Other examples of microbe-linked
chronic inflammation include
rheumatic fever, gastric ulcers, Whipples disease and reactive
arthritis. The reasons that the
microbial pathogens are able to colonise tissues successfully in
these cases are not clear but
are thought to include strategies to evade the immune response
while exploiting a nutrient
advantage.
PDs affect a large proportion of the world population (15%) and
have been linked to a consortia
of oral microbes of endogenous origin, such as Actinobacillus
actinomycetemcomitans,
Prevotella intermedia, Tannerella forsythia, Fusobacterium
nucleatum, Peptostreptococcus
micros and most commonly, Porphyromonas gingivalis
(P.gingivalis) (Petersen 2003; Shiloah
et al. 2000; van Winkelhoff et al. 2002). P. gingivalis is
thought to be able to successfully
colonise the gingival tissues because the microorganism
possesses numerous virulence
factors that facilitate its growth and survival (McGraw et al.
1999; Travis et al. 1997). P.
gingivalis is able to evade the host immune system, manipulate
the local environment in order
to supply nutrition for its own continuity and these activities
lead to the destruction of host
tissues. The microorganism is highly proteolytic and utilizes
environmental peptides and free
-
3
amino acids such as serine and threonine as sources of energy
(Dashper et al. 2001;
Takahashi et al. 2000). In addition, P. gingivalis expresses an
enzyme that modifies
peptidylarginine, a unique property among prokaryotes (McGraw et
al. 1999).
Periodontal diseases are relatively widespread and in recent
years have also been associated
with the development of several other chronic inflammatory
diseases, such as, coronary heart
disease, diabetes mellitus and rheumatoid arthritis (RA)
(Southerland et al. 2006). The basis of
these associations is unknown, but may be due to non-specific
positive feedback loops
involving inflammatory mediators, or to more specific effects,
from a common aetiological
agent. The ability of P. gingivalis to citrullinate arginine
containing peptides is of special interest
when investigating the potential link between PD and RA, because
auto-antibodies against
citrullinated peptides have recently been shown to be both
highly specific and sensitive for the
diagnosis of RA (Schellekens et al. 1998). Citrullinated
peptides have been suggested to play a
role in the pathogenesis of RA; however, the exact nature of
this role is unclear. Whether the
instigator of citrullination in RA is of host or prokaryotic
origin is also unknown. It is interesting
to note that P. gingivalis DNA has been found in the synovial
fluid from RA patients (Moen et al.
2006). It seems plausible that, in patients with chronic PD, the
increased levels of P. gingivalis,
frequently detected at diseased sites, might play a triggering
or amplification role in RA, via its
ability to promote peptide citrullination. This mechanism might
explain the over representation
of RA patients in the population suffering from PD.
It is for these reasons, it was decided to investigate the
conditions under which P. gingivalis
modifies peptide-linked arginine residues to citrulline. A
preliminary investigation of the links
between PD and polyarthritis in an animal model was also
undertaken, with special reference
to the presence of inflammatory cells and citrulline in areas of
inflammation.
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1.2 Periodontal Disease
1.2.1 Introduction
Periodontal diseases are a group of inflammatory disorders of
the hard and soft tissues
surrounding the teeth and are associated with plaque
accumulation. The uncontrolled
inflammatory response leads to the destruction of both the
periodontal ligament attachment and
the adjacent alveolar bone (Page and Schroeder 1976) as shown in
Figure 1.1. Chronic
periodontitis, one of the more severe forms of PD, is a major
cause of tooth loss in adults
(Niessen and Weyant 1989; Page and Schroeder 1976; Petersen
2003).
In the diseased state, inflammation of the gingival tissues
involves the clinical signs of redness,
warmth, swelling and pain as its normal response to infection,
trauma, or non-microbial
irritation. As disease progresses, epithelial attachment to the
tooth surface is lost, leading to
migration of the junctional epithelium and the development of a
gingival pocket (Loesche et al.
1985). Moreover, if left untreated, the destruction of
supporting tissue will eventually result in
tooth loss. In recent years, it has been suggested that the
inflammation associated with
periodontitis affects not only the dentition but may impact on
general health. PD has been
suggested as a possible risk factor in systemic diseases such as
myocardial infarction,
cardiovascular disease (Genco et al. 2002), pneumonia, pre-term
low birth weight (Lopez et al.
2002) and RA (Mercado et al. 2003; Ribeiro et al. 2005).
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5
Figure 1.1: Surgical exposure of bone loss (arrow) resulting
from periodontitis as adapted from
(Pihlstrom et al. 2005).
It is now widely recognised that PD has a multi-factorial
aetiology that can be genetically
related and environmentally involved. Bacterial colonization of
the gingival tissues and the
host’s response to this challenge are thought to contribute to
the disease severity (Pihlstrom et
al. 2005). Genetic disorders such as Chédiak-Higashi,
Ehlers-Danlos, Kindlers, Cohen
syndromes, Haim-Munk and Papillon-Lefèvre syndromes have been
linked to the onset of PD.
The disease prevalence is seen to increase with age (Horning et
al. 1992), although loss of
periodontal attachment and alveolar bone with age is dependent
on the presence of plaque as
shown on the presence of sub-gingival calculus, over-hanging
restorations (Lang et al. 1983)
and/or crowded teeth are also factors thought to contribute to
the disease process.
NOTE: This figure is included on page 5 of the print copy of the
thesis held in the University of Adelaide Library.
-
6
A report from the United States 3rd National Health and
Nutrition Examination Survey revealed
that approximately 42% of periodontitis cases in the adult
population were associated with
cigarette smoking. The study indicated that more than one-half
of the periodontitis affecting
adult cases maybe promoted by this habit (Tomar and Asma 2000).
Similar findings were
reported in a recent survey of the Australian population, where
23% of adults were classified as
having moderate or severe periodontitis, with more than half the
sufferers are former or current
cigarette smokers. In the same study, Do et al. (2008) estimated
that one third of the
periodontitis cases can be prevented by elimination of cigarette
smoking (Do et al. 2008).
Smoking has been suggested as a potential risk factor as it
promotes the accumulation of oral
pathogens and progression of PD. Intensive exposure to tobacco
smoke was found to affect
the rate of colonization by pathogenic bacteria in
periodontitis-free young smokers (Shiloah et
al. 2000). Shiloah and co-workers also reported that seven of
eight subjects with elevated
levels of PD associated pathogens were cigarette smokers. In
addition to these findings,
smokers also have increased attachment loss, deeper periodontal
pockets and more missing
teeth than non-smokers (Haffajee and Socransky 2001). Smoking
may influence PD
progression by affecting the host control of bacteria as well as
facilitating a more favourable
habitat for the establishment of pathogens such as P. gingivalis
and A.
actinomycetemcomitans, often isolated from the shallow sites of
gingival crevice (Eggert et al.
2001). Other potential effects of smoking on PD include the
increased levels of carbon
monoxide on gingival tissues. This toxic by-product damages
cells, such as neutrophils,
involved in the protection of the periodontal environment. It is
thought that this cell damage, in
turn, provides nutrition for the pathogenic Gram-negative
anaerobes, enhancing the growth of
these bacteria.
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7
1.2.2 Types of periodontal disease
Gingivitis and periodontitis are recognised as the most
prominent of the PDs. Gingivitis is an
inflammatory response to the presence of the dental plaque
biofilm that accumulates at the
gingival margin. It is often associated with poor oral hygiene
and can be reversed when good
oral hygiene is applied (Loe et al. 1965). Gingivitis has been
reported to be highly prevalent
throughout the developing world (Pilot 1998). It is thought that
prolonged chronic gingivitis may
lead to periodontitis (Criswell et al. 2002; Marc et al. 2003;
Page et al. 1997).
During early stages of gingival inflammation, redness of the
gingival tissues can be observed,
resulting from the enlargement of blood vessels in
sub-epithelial connective tissue and during
disease progression swelling increases. If left untreated,
chronic periodontitis may lead to
progressive loss of collagen attachment of the tooth to the
underlying alveolar (jaw) bone and
the teeth become loose. Bleeding of gingival tissue can be
triggered upon probing with blunt
instruments. These changes are rather vague, and usually
painless.
Numerous bacterial species, such as Fusobacterium nucleatum and
Eikenella corrodens, have
been isolated in elevated numbers in plaque from individuals
suffering from gingivitis. However,
pathogenic species, such as P. gingivalis, commonly isolated in
increased numbers and
proportions in plaque from periodontitis sufferers, were absent
or not significantly increased
when compared with plague from healthy individuals (Moore et al.
1987).
Periodontitis is characterised by the formation of a significant
gingival pocket, varying degrees
of gingival inflammation (swelling and redness) and bleeding
upon probing (Lang et al. 1996).
As the periodontal pocket deepens, the environmental redox
potential decreases and the
composition of the microbial community shifts, as the changed
environment encourage the
growth of anaerobic Gram-negatives. In turn the host response
becomes more destructive and
chronic disease ensues.
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8
Host defence mechanisms against microbial infection are thought
to contribute significantly to
the symptoms and severity of periodontitis. The oral pathogens
involved are thought to promote
host tissue damage and activate inflammatory and immune
responses. Various inflammatory
molecules such as proteases, cytokines, prostaglandins and
enzymes are released from
leukocytes and fibroblasts once inflammation is initiated. The
periodontal tissues become
loose, swollen and inflamed, there will be tissue inflammatory
infiltrate present and moreover,
osteocytes begin the destruction of bone. The destruction of
deeper tissues results, leading to
the loss of alveolar bone and periodontal ligament. Eventually,
the connective tissue
attachment to the tooth may be destroyed leading to tooth
loss.
1.2.3 Microorganisms linked to the aetiology of periodontal
disease
In a recent study, the most frequently detected species in
gingival crevicular epithelial cells
from chronic periodontitis lesions were P. gingivalis (42%),
Treponema denticola (38%),
Prevotella intermedia (37%), Streptococcus intermedius (36%),
Campylobacter rectus (35%),
Streptococcus sanguinis (35%) and Streptococcus oralis (34%)
(Colombo et al. 2006).
The presence of elevated numbers of microbes is believed to be
an important factor in the
progression of PD. However, the increased proportions of
specific microbial pathogens that is
likely to be the crucial factor in the progression of PD.
Several Gram-negative anaerobes,
including A. actinomycetemcomitans, P. gingivalis, Prevotella
intermedia, Tannerella forsythia,
Fusobacterium nucleatum and Peptostreptococcus micros, have been
associated with the
onset of periodontitis (Carlsson et al. 1984; van Winkelhoff et
al. 2002). Most of these species
are found in very low numbers in plaque collected from the
healthy gingival crevice (Griffen et
al. 1998). However, in the onset and progression of disease,
these Gram-negatives are
frequently found in significantly higher numbers and proportions
in sub-gingival plaque from the
gingival pockets that develop in PD sufferers.
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9
Many species of bacteria isolated from diseased plaque are
thought to generate much of their
energy from amino acid metabolism and some are highly
proteolytic. This activity enables the
bacterial community in the subgingival plaque biofilm to break
down host proteins and
glycoproteins, supplying nutrition for their growth. The battery
of proteases liberates a mixture
of peptides and amino acids that are able to be assimilated and
metabolised by the bacterial
cell to produce ATP. The ammonia that arises as a product of
amino acid metabolism, leads to
the environment in the pocket becoming increasingly alkaline,
compared to that of the healthy
gingival crevice. It is thought that the significant change in
the sub-gingival ecology facilitates
the shift in dominant organisms present, by providing a more
favourable environment for the
growth of the significant periodontopathogens, all of which have
optimal growth pH above
neutrality.
The gingival crevice differs from the other environments in the
mouth. It provides a relatively
anaerobic environment and the organisms present rely on the
supply of gingival crevicular fluid
(GCF) for their nutrition. At diseased sites, the secretion of
GCF will increase to continuously
flush the gingival sulcus. The increased flow of GCF is an
inflammatory response to the
increased microbial load, facilitating the removal of
non-adherent microbial cells and also
contains host defence components. GCF is a serum-like fluid,
containing neutrophils and also
immunoglobulins, predominantly IgG at levels higher than
salivary IgA. In addition, GCF
provides a range of proteins, including haem-containing proteins
and nutrients such as iron that
nourish the oral microbes present, especially the
black-pigmented anaerobic bacteria.
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10
1.3 Porphyromonas gingivalis and Periodontal Disease
P. gingivalis is a key member of the group of microbes described
above, releasing peptides
and other nutrients via the expression of multiple proteases
that target host proteins in GCF
and epithelial tissues. In addition, P. gingivalis is also able
to obtain essential factors for growth
such as haemin by producing haemolysin toxin. Interestingly,
McGraw and colleagues
suggested that this microbe has the ability to stimulate an
increased flow of GCF in order to
maintain their existence in a periodontal pocket (McGraw et al.
1999). Numerous studies have
proposed this species to be a predominant pathogen in PD
(Colombo et al. 2006; Lamont et al.
1995; Shiloah et al. 2000). P. gingivalis survives in
subgingival plague because it produces
numbers of virulence factors that play a role in tissue
colonization and destruction in addition to
the perturbation of host defences (Holt et al. 1999). Cell
surface structures such as fimbriae
and lectin-type adhesions, a polysaccharide capsule,
lipopolysaccharide and outer membrane
vesicles as shown in Figure 1.2 enable the organism to adhere to
the oral epithelium at buccal
and sub-gingival sites as well as co-aggregate with other oral
bacteria. P. gingivalis and
Fusobacterium sp. have the ability to adhere to each other and
also to crevicular epithelial cells
(Kolenbrander 2000).
P. gingivalis also possesses other factors associated with
tissue damage such as
haemagglutinating factors that have also been identified as
important adhesion molecules
(DeCarlo et al. 1999). These allow P. gingivalis cells to adhere
to gingival tissue cells in
addition to attachment and lysis of erythrocytes, facilitating
the uptake of essential iron (Olczak
et al. 2005). Metabolic end-products, such as an array of
volatile fatty acids that include
butanoate, propionate, and isobutanoate may also be cytotoxic to
the gingival tissue (Jeng et
al. 1999) in addition to the release of numerous enzymes (Cutler
et al. 1995; Mayrand and Holt
1988; Sundqvist 1993). These virulence factors lead to chronic
inflammation and ultimately to
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11
degradation of host periodontal tissues and, in turn, result in
the release of nutrient molecules
for their growth. These factors are also likely to play
important roles in manipulating the host
defence system for its own advantage.
Figure 1.2: Electron microscopic image of Porphyromonas
gingivalis taken from http://www.microbiologybytes.com.
This rod-like Gram negative bacterium has vesicles at its outer
membrane.
1.3.1 Porphyromonas gingivalis virulence factors
1.3.1.1 Fimbriae
The invasion of epithelial cells by oral bacteria is facilitated
by the expression of surface
proteins known as fimbriae. In advanced periodontitis, 5% of
plasma cells taken from lesion
sites formed antibody to the fimbriae of P.gingivalis (Ogawa et
al. 1991). These structures act
as adhesion molecules, capable of binding specifically to and
activating various host cells such
NOTE: This figure is included on page 11 of the print copy of
the thesis held in the University of Adelaide Library.
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12
as human epithelial cells, endothelial cells, spleen cells and
peripheral blood monocytes
(reviewed in Amano et al. 2004). A unique class of Gram-negative
fimbriae, they consist of
protein subunits called fimbrillins with sizes varying between
41 to 49 kDa (Dickinson et al.
1988; Lee et al. 1991). These proteinaceous hair-like appendages
are 2 to 8 nm in diameter
and range between 0.3 and 3 μm in length. The fimbrillin
polypeptide binds to proline-rich
proteins, statherin, lactoferrin, fibrinogen and fibronectin,
oral epithelial cells and other oral
bacterial species cells (e.g. A. naeslundii), (Amano et al.
1996; Lamont and Jenkinson 1998;
Murakami et al. 1996). A 48 kDa surface protein on human
gingival epithelial cells has also
been reported to interact with fimbriae of P. gingivalis
(Weinberg et al. 1997).
The expression of P. gingivalis fimbriae appears environmentally
influenced. Changes in
temperature altered the regulation of fimbriae gene expression
and fimbriae mediated
adherence to oral epithelium and to other oral microbes (Amano
et al. 1994). The production of
fimbriae is reduced by 54% when subgingival temperature is
elevated from 37oC to 39oC
(Amano et al. 1994).
Recent studies have shown that a fimbriae-deficient P.
gingivalis mutant had decreased
binding to whole saliva-coated oral surfaces and caused
periodontal bone loss in an animal
model of PD when compared with wild type P. gingivalis (Malek et
al. 1994). Hamada et al.
also reported the inability of this P. gingivalis mutant to bind
human gingival fibroblasts and
epithelial cells (Hamada et al. 1994). These findings provide
evidence that suggest P. gingivalis
fimbriae are important virulence factors, aiding adherence and
play a role in the pathogenesis
of human periodontal disease.
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13
1.3.1.2 The gingipain proteinases
The gingipains are a group of cysteine endo-proteinases that
have long been associated with
P. gingivalis virulence (Imamura 2003; Potempa et al. 1995). The
gingipains are classified
according to their specificity of peptide bond cleavage; the
R-gingipains (Rgp) hydrolyse
peptide bonds C-terminal to arginine residues and K-gingipain
(Kgp) C-terminal to lysine
residues (Imamura 2003). The gingipains can degrade collagens,
important structural
components of periodontal connective tissue and other
extracellular matrix proteins, including
fibronectin and laminin, are also targeted by these enzymes. The
gingipains are resistant to
host proteinase inhibitors such as cystatins, serpins and tissue
inhibitors of metalloproteinases
as they exhibit significant proteolytic activity in their
presence (Abe et al. 1998; Kadowaki et al.
1994). This suggests that these enzymes are significant
virulence factors in P. gingivalis.
There are compounds that inhibit the activity of gingipains,
such as the serine proteinase
inhibitor, tosyl-L-phenylalanine that inhibits both Kgp and Rgp,
metal chelators (EDTA, EGTA)
which inhibit Rgp but not Kgp. Leupeptin can also be used as an
Rgp specific inhibitor (Abe et
al. 1998; Holzhausen et al. 2006).
1.3.2 Peptidylarginine deiminases
Peptidylarginine deiminase (PAD) activity is possessed by
P.gingivalis and also by many
mammalian cells. The first mammalian PAD was identified in 1977
(Rogers et al. 1977) and the
PAD enzymes were eventually grouped as guanidino-group modifying
enzymes by Shirai et al.
(Shirai et al. 2001). Subsequently mammalian PADs have been
extracted from human, rat,
rabbit, guinea pig, chicken and sheep tissues. There are five
PAD isotypes produced by a
variety of human cells. PAD-1 is mainly expressed in the uterus
and epidermal tissue (Guerrin
et al. 2003; Rus'd et al. 1999; Terakawa et al. 1991). PAD-2 is
a widely expressed PAD, and is
most abundant in the cells of tissues such as skeletal muscle,
brain, uterus, salivary glands and
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14
pancreas (Foulquier et al. 2007; Terakawa et al. 1991). PAD-3 is
located in hair follicles and
epidermis (Kanno et al. 2000; Rogers et al. 1997; Rus'd et al.
1999; Terakawa et al. 1991).
PAD-4, formerly known as PAD-5, is found primarily in
haematopoietic cells such as
eosinophils, neutrophils, lymphocytes and monocytic cells (Asaga
et al. 2001; Foulquier et al.
2007). The most recently discovered PAD enzyme is PAD-6
expressed in ovary and testis
tissues, small intestine, spleen, lung, liver and skeletal
muscle cells (Chavanas et al. 2004;
Zhang et al. 2004).
The first purification of P. gingivalis PAD (PgPAD) was reported
in 1999 by McGraw et al.
(McGraw et al. 1999). The enzyme, although not evolutionarily
related to the mammalian
PADs, catalyses the same chemical reaction, the modification of
the guanidino group of
arginine residues from various peptides to produce ammonia
(shown in Figure 1.3).
Figure 1.3: Citrullination activity by P. gingivalis PAD.
This was modified from Shirai et al., 2001
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15
Although catalysing the same reaction, P. gingivalis and
mammalian PADs do not share the
same catalytic mechanism. Mammalian PADs are metalloenzymes and
require calcium ions for
activation, in contrast to PgPAD, which can modify arginine
residues in the absence of calcium
(McGraw et al. 1999; Takahara et al. 1986). Furthermore,
mammalian PADs are unable to
convert free arginine to citrulline, unlike PgPAD, that has been
reported to catalyze conversion
of both peptide-bound arginine and free arginine to citrulline
containing peptides and free
citrulline, respectively. As with all enzymes, PADs are
influenced by environmental pH. The
activity of purified PgPAD was optimal at pH 9.3 (McGraw et al.
1999). Interestingly,
Nakayama-Hamada and colleagues reported that PAD-2 had an
optimal activity between pH 6
and 10 while PAD-4 is active between pH 6.5 and 9
(Nakayama-Hamada et al. 2005)
suggesting that all PADs prefer an alkaline pH environment for
maximal activity. Mammalian
PADs are located intracellularly whereas 90% of PgPAD activity
has been reported to be cell or
membrane vesicle associated (McGraw et al. 1999).
1.3.3 Peptide metabolism by Porphyromonas gingivalis
The metabolism of energy-yielding peptides by P. gingivalis is
vital for growth of the
microorganism as it is unable to utilize glucose as source of
energy (Masuda et al. 2001; Shah
and Gharbia 1989; Shah and Williams 1987). Evidence of the
asaccharolytic nature of P.
gingivalis is provided from a study showing that growth yields
of the organism were constant in
the presence or absence of glucose (Takahashi and Schachtele
1990). A number of studies
have shown that P. gingivalis prefers to utilise peptides over
free amino acids (Milner et al.
1996; Takahashi et al. 2000; Wyss 1992). P. gingivalis is
reported to be able to generate ATP
via catabolism of amino acids and releases the ammonia it
produces as a result of its
proteolytic metabolism to the environment (Shah and Williams
1987). This end product alters
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16
the environment by elevating the local pH to a more favourable
alkaline growth condition
(Takahashi and Schachtele 1990) as the bacterium showed a stable
growth over a pH range
from 6.7 to 9 when cultured in haemin-excess conditions in a
chemostat (McDermid et al.
1988).
The metabolism of arginine via the arginine deiminase pathway
has been suggested as a
mechanism to produce energy from free arginine in P. gingivalis
(Masuda et al. 2001). In
addition to arginine, glutamate- and aspartate-containing
peptides are metabolised by P.
gingivalis and result in the production of a mixture of
cytotoxic fatty acid metabolic end
products. Of these, butanoate is considered one of the most
toxic to host tissues (Niederman et
al. 1997).
1.4 Citrullination
More than 70 years ago Fearon was the first to describe the
conversion of arginine to citrulline
(Fearon 1939). Citrullination involves modification of the
guanidino group of arginine,
specifically by the replacement of a nitrogen-containing group
by an oxygen atom in the side
chain of the amino acid. This conversion also results in the
loss of a positive charge, therefore
citrullinated proteins lose charge, are unfolded and
interference in organized protein structure is
promoted (Tarcsa et al. 1996). The citrullination reaction is
catalysed by PADs and occurs
when protein-bound arginine or free arginine are converted into
protein-bound citrulline or free
citrulline, respectively. Ammonia is produced as by-product of
the reaction.
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17
Figure 1.4: The process of citrullination of arginine to
citrulline catalysed by mammalian PAD.
The diagram was modified from Masson-Bessi`ere et al., 2001.
1.4.1 Citrullination and the immune system
The human immune response is highly complex and functions to
protect the individual from
foreign cells and molecules detected in the body. Occasionally,
this response can cause
damage to host tissues when the immune system misinterprets host
tissue components as
foreign and initiates an inflammatory response. This type of
response against host tissue is
known as an autoimmune disease.
Citrulline is classified as a non-coded amino acid that is not
incorporated during the translation
of protein. Citrullination leads to post-translationally
modified proteins and peptides as their
formation occurs subsequent to protein synthesis. Enzymatic
citrullination abolishes positive
changes contained within native proteins, inevitably causing
significant alterations in their
tertiary structures and loss of function. Although the
conversion results in a relatively small
NOTE: This figure is included on page 17 of the print copy of
the thesis held in the University of Adelaide Library.
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18
chemical alteration to the protein involved, the reactivity of
auto-antibodies specific for citrulline-
containing epitopes seems to be critically dependent on the
presence of a citrulline residues.
Peptides that are post-translationally modified to contain
citrulline will exhibit different epitopes
when compared to those of containing normal peptide-bound
arginine residues (Masson-
Bessiere et al. 2001). It is possible that these modified
proteins and peptides can be detected
as foreign antigens by the host’s immune system (Doyle and
Mamula 2002).
The reaction catalysed by PADs, the modification of arginine
residues to citrulline, has been
reported to trigger the host immune response (Girbal-Neuhauser
et al. 1999; Schellekens et al.
1998). Accordingly, PAD activity has been linked to the
initiation of a number of inflammatory
diseases, with citrullinated proteins proposed as a linking
autoantigen (Masson-Bessiere et al.
2000; Nienhuis et al. 1964). The reactivity of auto-antibodies
towards citrulline-containing
epitopes is critically dependent on the presence of a citrulline
residue.
1.4.2 Citrullinated human proteins
Previous studies have shown the effect of citrullination on the
immune response (Lundberg et
al. 2005; van Boekel et al. 2002). Recently, the presence of
citrullinated peptides has been
reported to be associated with a number of inflammatory
disorders (Chapuy-Regaud et al.
2005; Makrygiannakis et al. 2006; Wanchu et al. 2001). The
presence of citrullinated peptides
in inflamed tissues has been proposed as a general sign of
inflammation (Kinloch et al. 2008)
as they can be detected in synovial membranes in patients with
rheumatoid arthritis (Chapuy-
Regaud et al. 2005), systemic lupus erythematosus (SLE)(Wanchu
et al. 2001), and Sjögren
syndrome (Nissinen et al. 2003).
Anti-citrullinated cyclic protein (anti-CCP) antibodies are
produced by rheumatoid arthritis
patients against citrulline-containing epitopes
(Girbal-Neuhauser et al. 1999; Schellekens et al.
1998). Masson-Bessi`ere and colleagues reported the presence of
citrulline in joint synovium
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19
and suggested it becomes a potential target for IgG antibodies
(Masson-Bessiere et al. 2001).
Citrullinated proteins can be detected in the leukocyte
infiltrate in inflamed synovium and fibrin,
along with other proteins, is citrullinated during the
inflammation of mouse synovium
(Vossenaar et al. 2003). Interestingly, the antibody response to
citrullinated proteins is highly
specific for RA and may be involved in the perpetuation of the
human disease. Their detection
has also provided hope for better diagnosis of the disease.
There are several citrullinated proteins that are present
naturally in the body. Filaggrin is a 40
kDa protein that is found the in human epidermis and was
identified as a neutral/acidic isoform
of pro-filaggrin (Simon et al. 1993). Tarcsa reported that
filaggrin is mostly β-turn in structure,
but after complete citrullination, the shape became flat, which
is indicative of the loss of
organized structure that can arise following modification
mentioned previously (Tarcsa et al.
1996). The presence of filaggrin in humans can be detected by
anti-perinuclear factor and anti-
keratin antibody tests (Vincent et al. 1999). In addition to
filaggrin, vimentin, a structural
component of the intermediate filaments, is also citrullinated
in synovial sites (Bang et al.
2007). The antigenic properties of vimentin are substantially
activated by citrullination and are
thought to be the target of anti-Sa antibodies (Vossenaar et al.
2004).
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20
1.5 Rheumatoid Arthritis
1.5.1 Introduction and history
Rheumatoid arthritis is a well-known autoimmune chronic
inflammatory disorder that affects
humans of all ages and races with a prevalence of 1% in the
world population (Silman and
Pearson 2002). The first clinical description of RA was reported
by Augustin-Jacob Landre-
Beauvais in 1800 (Landre-Beauvais 2001). RA is believed to have
existed more than 3,000
years ago with the finding of ancient skeletons that’s showed
evident of RA in North America.
There are certain ethnic groups that suffer increased prevalence
of RA, for example Pima
(5.3%) and Chippewa (6.8%) Indians; two Native American tribes
exhibit the highest rates of
disease compared to other groups. In Europe, the evidence of
RA-like symptoms can be seen
in 17th century art as shown in a family portrait of a Dutch
artist dated 1622 (Figure 1.5).
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21
Figure 1.5: La Familia de Jordaens en un Jardín Jacob
Jordaens
Note from this portrait the maid’s fingers showed the evident of
features of RA; swelling of the metacarpal-phalangeal and proximal
interphalangeal joints (Firestein 2003).
A review of literature shows that, the incidence of RA is
two-fold higher in women than men,
with Gabriel reporting that females had a prevalence of RA of
1.37% compared to 0.74% in
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22
males (Gabriel 2001). In addition, there is approximately double
the risk of RA for
postmenopausal women (Criswell et al. 2002). This finding has
led some to suggest sex
hormones play a role in the development of RA.
Genetic inherence has been proposed as an important factor, as
the disease tends to cluster in
families. There are several environmental factors associated
with an increased incidence of
RA, including smoking (Heliovaara et al. 1993; Klareskog et al.
2006) and obesity (Voigt et al.
1994). In contrast, alcohol consumption, which was previously
reported as one of the causes of
RA (Hazes et al. 1990), has now been shown to be assiociated
with decreased risk for the
disease (Kallberg et al. 2009; Voigt et al. 1994).
Smoking has been suggested as an environmental trigger and a
risk factor for numerous
illnesses such as myocardial infarction (Prescott et al. 1998),
diabetes mellitus (Will et al. 2001)
and PD. A Finnish study suggested that exposure to tobacco
smoke, or factors associated with
smoking, may trigger the production of rheumatoid factors in
males (Heliovaara et al. 1993) and
in women who had smoked more than 15 cigarettes per day with a
2.5-fold increase in the risk
of contracting RA (Vessey et al. 1987). Interestingly, there is
evidence that shows smoking
promote the production of citrullinated peptides which may
activate the host immune system
(Klareskog et al. 2006). Although these findings demonstrate the
increased risk of RA in
individuals exposed to cigarette smoke, further investigation is
needed to determine its cause
as RA is a multi-factorial disease.
No aetiological agent has been identified and there are no
unique clinical or laboratory features
that can be used to define this disease clearly. In order to
more accurately estimate the
prevalence of RA, American Rheumatoid Association provided
classification criteria in 1956
and revised them in 1987, 1988 and 2010 (Aletaha et al. 2010;
Arnett et al. 1988).
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23
Table 1.1: The 2010 American College of Rheumatology/ European
League against rheumatism classification criteria for rheumatoid
arthritis.
NOTE: This table is included on page 23 of the print copy of the
thesis held in the University of Adelaide Library.
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24
1.5.2 Features of rheumatoid arthritis
RA presents as a series of symptoms including redness;
symmetrical joint swelling that is warm to
touch (Arnett et al. 1988), joint deformity and weight loss
(Munro and Capell 1997). The sufferer may
commonly experience fatigue, malaise, joint pain and morning
stiffness. This debilitating chronic
inflammatory disorder primarily has a focus on the synovial
joints, leading to joint swelling,
progressive joint erosions eventually leading to disability and
changes to the synovium, as shown in
Figure 1.6. The synovial membrane in RA patients is
characterized by hyperplasia, increased
vascularity, and an infiltrate of inflammatory cells.
Individuals with RA also demonstrate moderate to
severe periodontal bone loss (Mercado et al. 2001).
Figure 1.6 Changes that occur in the synovial joint as a result
of RA.
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25
Early erosion of cartilage and bone is occupied predominantly by
activated macrophages and T
cells. The T cells stimulate monocytes, macrophages, and
synovial fibroblasts to produce the
cytokines such as interleukin-1, interleukin-6, and TNF- . The
cells also secrete matrix
metalloproteinases. Cytokines enhance the expression of adhesion
molecules on endothelial cells
and increase the recruitment of inflammatory cells such as
macrophages, lymphocytes, fibroblasts
and neutrophils into the joints. Neutrophils release proteases,
which degrade proteoglycan in the
superficial layer of cartilage. Vascularity is increased in the
synovium of patients with RA by means
of the stimulation of angiogenesis.
This disease shares similar features with adult periodontitis
(reviewed by (Bartold et al. 2005;
Mercado et al. 2000)). Mercado (2000) reported that RA patients
are more likely to experience more
significance periodontitis and vice versa. It is of interest to
note that the presence of bacterial DNA
and peptidoglycan has been reported in the joints of RA patients
(Van Der Heijden et al. 2000). As in
the PD, bacteria may play an important role in determination of
the disease severity. Mercado (2001)
has proposed an underpinning commonality of dysregulation of the
immune system between
periodontitis and RA (Mercado et al. 2001).
1.6 Rheumatoid Arthritis and Citrullination
RA affected individuals have been found to express PAD-4 and
citrullinated peptides can be
detected in their synovial tissue. Chang and colleagues reported
in their findings that PAD-4 is
expressed by T cells, B cells, macrophages, neutrophils,
fibroblast-like cells and endothelial cells in
the lining and sub-lining areas of the RA synovium (Asaga et al.
2001; Chang et al. 2005). The
production of post-translationally citrullinated peptides may
trigger the body’s immune system, to
attack what appear to be foreign epitopes and thus cause
inflammation of the synovium. Hence,
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26
detection of citrullinated peptides which are specifically
present in the sera of RA patients might help
in the early detection of RA.
Previous studies have shown the effect of citrullination on the
autoimmune response (Lundberg et al.
2005; van Boekel et al. 2002). Anti-citrullinated cyclic protein
(anti-CCP) antibodies are produced by
RA patients against citrulline-containing epitopes
(Girbal-Neuhauser et al. 1999; Schellekens et al.
1998). Masson-Bessi`ere and colleagues reported the presence of
citrullinated peptides in joint
synovium and their potential as targets for IgG antibodies
(Masson-Bessiere et al. 2001).
1.6.1 Citrullination in RA patients and clinical markers
Cytoplasmic PADs can be activated by stimulation of cells with a
calcium ionophore (Asaga et al.
1998). In the inflamed synovium, many cells undergo apoptosis or
necrosis and as the membrane
integrity is lost during cell death, Ca2+ can easily enter and
activate the PAD. Alternatively, PAD
enzymes may also be released from dying cells and become
activated as the extracellular Ca2+
concentration is approximately 1 mM, and thus induce the
citrullination of extracellular proteins such
as fibrin (Bongartz et al. 2007).
Rheumatoid factor (RF) is an antibody that binds to the Fc
portion of immunoglobulin and was first
identified by Eric Waaler in 1939. Historically, RF has been
used as a marker in the early detection
of RA. However, approximately 25% of RA patients are RF
negative, and reports have shown that
RF can also be found in other diseases in addition to 3-5% of
the healthy population (Mageed et al.
1997). Due to this lack of combined specificity and sensitivity,
many studies have been undertaken
in search of a more accurate indicator for RA.
Recently, the presence of autoantibodies against citrullinated
proteins have being used to aid in the
diagnosis of RA with 80% higher specificity and sensitivity than
diagnosis using RF (Suzuki et al.
2007; van Boekel et al. 2002). When using citrulline-containing
peptide variants in ELISA there are
autoantibodies detected in 76% of RA sera with a specificity of
96% (Schellekens et al. 1998). The
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antibodies showed reactivity towards citrullinated peptides and
grouped in anti-citrullinated
protein/peptide antibodies (ACPAs). There are several types of
ACPAs that have been determined,
such as anti-perinuclear factor (APF) (Nienhuis et al. 1964),
anti-keratin antibody (AKA) (Young et al.
1979), anti-Sa (Despres et al. 1994) and anti-CCP antibodies
(Schellekens et al. 1998). All of these
have a high specificity for citrullinated peptides.
1.7 Research Questions
There has been much recent interest in possible links between in
the inflammatory diseases PD and
RA. A review of the literature has shown that citrullination
appears to be an important factor in the
inflammatory process. As P. gingivalis is a significant pathogen
in the aetiology of PD and
possesses the ability to citrullinate via PAD activity the
following research questions were posed.
Is PgPAD able to citrullinate arginine containing peptides and
RA relevant host proteins?
Does exposure to PgPAD exacerbate inflammation in mammalian
tissues via host protein
citrullination?
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CHAPTER 2
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2 Investigation of Porphyromonas gingivalis
Peptidylarginine Deiminase
P. gingivalis is the only prokaryote known to express PAD
activity to date. PgPAD was investigated
by McGraw and colleagues at the end of last decade and this
group was the first to partially
characterise the purified enzyme (McGraw et al. 1999). As
discussed in the previous chapter the oral
anaerobe P. gingivalis has long been implicated as one of the
key pathogens in the aetiology of
human PD, however little work has been undertaken to investigate
the specific involvement of
PgPAD in the inflammatory disease process. Due to the recent
interest in citrullination in relation to
RA and the suggested links between this disease and PD, it was
decided to further investigate the
enzyme and its potential role as a virulence factor in
inflammatory disease, RA in particular.
As this study proposed to survey scavenged tissues obtained from
a parallel study investigating the
effect of P.gingivalis in the rat adjuvant arthritis model, the
main focus of this chapter was to further
characterise the activity of the enzyme peptidylarginine
deiminase in intact cells of the organism.
Accordingly, the following hypotheses were proposed;
� P. gingivalis cells express surface-associated PAD
activity
� The enzyme is active over a biologically relevant pH range and
is heat stable
� PgPAD is highly specific for arginine
� PgPAD preferentially citrullinates C-terminal arginine
residues in peptides
� PgPAD is able to citrullinate arginine residues in RA relevant
proteins; and
� The gingipains influence the rate of citrullination of
proteins by PgPAD.
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It should be noted that, to ensure reliability and consistency
of results, all analyses in this chapter
were performed at least in triplicate and the data points are
expressed as means of these
determinations. The primary results for each the following
experiments can be found in Appendix 2.
2.1 Citrulline Assay
In order to investigate the activity of PgPAD, a reliable method
for the detection and quantifying of
citrulline in assay samples was required. Therefore, the initial
work conducted in this study was to
develop such a method. Accordingly, a citrulline assay to detect
and measure citrulline in samples
was adapted and modified from that employed by McGraw (Boyde and
Rahmatullah 1980; McGraw
et al. 1999). This method was further developed to facilitate
the measurement of citrulline in the
small reaction volumes of microtitre plates.
2.1.1 Colour detection reagent
The carbamino detection reagent, used in this assay was prepared
daily, prior to use, for the
detection of citrulline. Briefly, one part of solution A,
consisting of 0.5% (v/v) diacetyl monoximine
(Sigma, USA) and 0.01% (w/v) thiosemicarbazide (Sigma, USA), was
added to two parts of solution
B, consisting of 0.25 mg.mL-1 of FeCl3 (Sigma, USA), in a
solution containing 24.5% (v/v) sulphuric
acid (Ajax Chemicals) and 17% (v/v) phosphoric acid (Ajax
Chemicals). During the method
development process it was discovered that the resulting colour
detection reagent was unstable,
changing from a colourless to a yellowish liquid over time and
so, it was necessary to use this
reagent within 1 hour of the preparation of Part A.
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2.1.2 Standard curve to determine citrulline concentration
In order to measure citrulline concentrations in unknown
samples, a standard curve was constructed
by the addition 100 μL of varying amounts of L-citrulline
(Sigma, USA), to microtitre plate wells using
2-fold dilution. The citrulline was dissolved in incubation
buffer, containing 1 mM EDTA (Sigma,
USA), 10 mM cysteine (Sigma, USA), 1 μM FMN (Sigma, USA) in 0.2
M Tris-HCl (Sigma, USA) at
pH 8.0. To each well 100 μL of the colour detection reagent was
added and the plates were
incubated at 100oC for 5 minutes to hasten colour development.
The samples were allowed to cool
for 10 minutes following incubation and the absorbance of
samples was determined at a wavelength
of 530 nm using a BIO-TEK Powerwave XS microtitre plate reader
(Crown Scientific, NSW).
2.1.2.1 Results
The absorbance of samples was linear over the range of
concentrations used, representing 0.5 to 16
nmoles of citrulline (Figure 2.1). Subsequently, this standard
curve was used to quantify the amount
of citrulline produced by PAD in samples.
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32
Figure 2.1: A standard curve for the colourimetric determination
of citrulline concentration.
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2.2 Citrullination by Mammalian Peptidylarginine Deiminase
2.2.1 Citrullination of BAEE by mPAD
Six mammalian PADs have been identified to date, and as with
PgPAD, this family of enzymes are
characterised by their ability to citrullinate peptide-bound
arginine residues (Raijmakers et al. 2007).
An early study by Takahara showed that rabbit muscle PAD (mPAD)
(Sigma, USA) is a calcium
dependant enzyme (Takahara et al. 1986) and others have shown
the enzyme is able to catalyze
the citrullination of the synthetic substrate benzoyl-arginine
ethyl ester (BAEE) (Nakayama-Hamada
et al. 2005; Raijmakers et al. 2007). To ensure that BAEE was a
valid substrate for the
measurement of PAD activity, mPAD, was used to confirm the
suitability of this substrate for
subsequent enzyme characterisation experiments.
2.2.1.1 Method
The mPAD was stored in a buffered aqueous glycerol solution
consisting of 20 mM Tris-HCl; pH 7.4
containing 10 mM 2-mercaptoethanol, 1 mM EDTA and 10% glycerol
(v/v). Tris-HCl was included to
protect the enzyme against adverse pH change and the other
ingredients conferred thermal stability,
which is important to prevent loss of activity from multiple
freezes thawing cycles, or elevated
storage temperature. The manufacturer’s instructions indicated
that the freezing and thawing
process would not normally adversely affect enzyme activity if
the solution was routinely stored at -
80oC degrees.
To measure citrullination of BAEE by mPAD, a modified method
from Takahara was used (Takahara
et al. 1986). Briefly, a working buffer was prepared containing
70 mM CaCl2 and 70 mM dithiothreitol
(Sigma, USA) in 350 mM Tris-HCl adjusted to pH 7.2. In order to
confirm the previously reported
calcium dependence of the enzyme an additional working buffer
was prepared excluding CaCl2.
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34
To prepare the enzyme solution for the citrullination assay,
immediately prior to commencing the
reaction, 2 μL of mPAD (0.3 Units) was mixed with cold 0.1%
(w/v) bovine serum albumin (BSA).
(Sigma, USA). The solution was then pre-incubated in working
buffer for 2 minutes at 55oC resulting
in a final volume of 900 μL. Subsequently, 100 μL of 50 mM BAEE
was added to the reaction
solution, prior to incubation at 55oC for 60 minutes. BAEE in
incubation buffer, in the absence of
enzyme, was used as a negative control. Over a one hour period,
25 μL aliquots were removed from
the reaction mixture at 10 minutes intervals and immediately
placed into the wells of a microtitre
plate. All wells contained 20 μl of 10 μM EDTA in order to stop
the reaction. To measure the
citrullination of BAEE, the colour detection reagent was
prepared as reported in Section 2.1.1 and
100 μL of this solution was added to samples and incubated for 5
minutes at 100oC. Following
incubation, the absorbance of samples at a wavelength of 530 nm
was measured using the
microtitre plate reader.
2.2.1.2 Results
The results of this experiment are displayed in Figure 2.2. As
the change in absorbance over time
was linear, the rate of reaction was constant for the period of
incubation. Of the two solutions used to
prepare mPAD for assay, only reaction mixtures containing
calcium ions in the working buffer
exhibited a significant increase in absorbance over the time. A
reaction mixture containing BAEE
without added enzyme also showed no increase in absorbance.
Accordingly, the rate of citrullination
of BAEE by mPAD in the presence of CaCl2 was calculated to be
0.072 nmoles citrulline.unit-1.min-1.
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35
Figure 2.2: The citrullination of BAEE by rabbit muscle PAD.
The solid blue line shows the reaction in the sample in the
presence of calcium and the dashed red line represents the reaction
in its absence.
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36
2.2.2 Citrullination of free arginine by mPAD
Mammalian PADs have also been reported to be unable to
citrullinate free arginine. In order to
confirm this finding, free L-arginine, dissolved in incubation
buffer, was used as a substrate for
mPAD, replacing BAEE.
2.2.2.1 Method
Free arginine at a final concentration of 5 mM was used as the
substrate for the reaction and
incubated with mPAD prepared in working buffer containing CaCl2.
Control samples containing
BAEE (5 mM) were incubated with the enzyme in the presence and
absence of calcium and the
method described in Section 2.2.1 was employed in order to
measure the rate of citrullination.
2.2.2.2 Results
The change in absorbance at 530 nm of samples over time, for
each test condition, is displayed in
Figure 2.3. A comparison of the rates of reaction of mPAD for
the substrates BAEE and arginine, in
the presence of calcium, revealed only samples containing the
former exhibited in significant rates of
citrullination. No activity was observed when mPAD was incubated
with free arginine, thus
confirming the previous report that mPAD was unable to
citrullinate the free amino acid, even in the
presence of calcium ions (Takahara et al. 1986).
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37
Figure 2.3: The citrullination activity of mPAD against free
arginine.
The blue solid line represents sample of arginine incubated with
calcium while the red dashed line is for sample of BAEE without
calcium (control), and BAEE incubated with calcium for green dashed
dot lines.
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38
2.2.3 Discussion
These preliminary experiments were carried out as confirmatory
steps, required for the future
investigation of PgPAD activity. The assay used to detect
citrulline was validated and it was also
determined that BAEE was a suitable substrate for measuring the
rate of citrullination by PAD. The
results also confirmed the previously reported calcium ion
dependency of mammalian enzymes and
that mPAD was unable to citrullinate free arginine (Kuhn et al.
2006; Raijmakers et al. 2007;
Vossenaar et al. 2003). These findings proved that the
citrulline assay used was able to detect
citrullinated residues and could therefore be used for future
experiments with P. gingivalis with
confidence.
2.3 Cultivation of Porphyromonas gingivalis
The Porphyromonas gingivalis strain W50 was employed in this
study of PgPAD. The bacterium was
maintained by weekly subculture on anaerobic blood agar (Oxoid,
Australia) by incubation at 37oC in
an anaerobic jar containing an atmosphere of 90% N2:5% H2:5%
CO2. Following growth on solid
medium, several bacterial colonies from the agar plate were
selected using a sterile wire loop and
cultured in brain heart infusion (BHI) broth(Oxoid, Australia)
enriched with 5 mg.L-1 haemin (Sigma,
USA) and 0.5 g.L-1 cysteine (Sigma, USA), at 37oC for 48 hours
in atmosphere of 90% N2:5% H2:5%
CO2. Following growth, the P. gingivalis culture was checked for
purity by Gram staining prior to cell
harvesting (Figure 2.4).
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39
Figure 2.4: A Gram stain of P. gingivalis W50 under light
microscopy oil immersion at 1,000 x magnification.
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40
2.4 Cell Harvesting and Preparation
P. gingivalis cells were prepared for the enzyme assay according
to the following protocol. The
optical density of the cells in BHI broth was determined at a
wavelength of 560 nm (OD560) using
Lambda 5 UV/Vis Spectrophotometer (Perkin-Elmer, USA) and
adjusted to an OD560 of 1
absorbance unit (AU). Tubes containing 10 mL of cells were then
centrifuged at 10,000 g for 10
minutes at 4oC and the resultant pellet suspended and washed in
the incubation buffer. The resulting
cell suspension was centrifuged once again, employing the same
conditions as described previously
and the washed cell pellet was suspended in 1 mL of fresh
incubation buffer prior to the assessment
of PAD activity. A protein assay was utilised to determine the
quantity of protein in cell samples.
2.5 Protein Assay
2.5.1 Method
An aliquot of cell suspension was sonicated using a SONIPROBE
sonicator (DAWE Instrument,
England) for five cycles of 10 seconds to disrupt the cells
prior to protein estimation. The samples
were kept on ice during processing and cell disruption was
confirmed by Gram staining the
sonicated sample. The protein concentrations of sonicated
samples were determined using a
Coomassie plus® protein assay kit (Thermo scientific, USA) using
BSA as a protein standard (refer
for detailed method to Appendix 1.1).
2.5.2 Results
The absorbance of the protein standard solutions was measured at
a wavelength of 595 nm and the
mean readings were calculated and a standard curve for protein
concentration was plotted (Figure
2.5).
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41
The absorbance of the concentrated P. gingivalis cell samples at
a wavelength of 595 nm was 0.711
AU, a value equating to 0.67 mg.mL-1 of protein when calculated
using the equation of the trend line
generated from the standard plot (Figure 2.5).
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42
Figure 2.5: The standard curve of protein concentration at a
wavelength of 595 nm.
The Coomassie protein assay is showing absorbance readings for a
range of BSA concentrations.
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43
2.6 Citrullination of