Dynamic role of plasma ferritin during pseudomonas infection insights from the limulus

DYNAMIC ROLE OF PLASMA FERRITIN
DURING PSEUDOMONAS INFECTION:
INSIGHTS FROM THE LIMULUS

ONG SEK TONG DERRICK

DEPARTMENT OF BIOLOGICAL SCIENCES
NATIONAL UNIVERSITY OF SINGAPORE
2003/2004


DYNAMIC ROLE OF PLASMA FERRITIN DURING
PSEUDOMONAS INFECTION: INSIGHTS FROM
THE LIMULUS

ONG SEK TONG DERRICK
(Bachelor of Science (Hon))

A THESIS SUBMITTED TO THE
FOR THE DEGREE OF MASTER OF SCIENCES
DEPARTMENT OF BIOLOGICAL SCIENCES
THE NATIONAL UNIVERSITY OF SINGAPORE
2004


ACKNOWLEDGEMENTS

I would like to express my heartfelt gratitude to:
Professors Ding and Ho for their constant support, patience and guidance in this
project. I really thank them for their understanding and assistance for my years in
their labs;

Dr Zhu, Lihui, Patricia and Sean, for imparting me their wealth of knowledge,
giving me uncountable precious advices and directing me when I am lost. In
particular, Lihui for helping me with the preparation of the huge amount of RNA,
teaching me of the electrophoretic mobility shift assay and in the preparation of my
manuscript;

Nicole, who has assisted me and will be furthering some other aspects in this project;

My family members, for their unfailing support;

Han Chong, Sook Yin and Bee Ling, for helping me with the arrangement of the
ultracentrifuge facility;

Last but not least, all my lab-mates for their kind help extended from time to time.

i


TABLE OF CONTENTS
Acknowledgements
Table of Contents
List of Abbreviations
List of Figures
List of Tables

Page
i
ii
vi

vii
x

Summary

xi

1.

INTRODUCTION

1

1.1

Iron in biological systems and its toxic effects

1

1.2

Iron in host-pathogen interaction during infections

2

1.2.1

Iron, microbial pathogens and sepsis

2

1.2.2

Adaptive immunity and its iron-dependent nature

3

1.2.3

Innate immunity

5

1.2.3.1 The iron-withholding strategy as a component
of innate immunity

6

1.3

The iron-binding proteins involved during infection and
inflammation

9

1.3.1

The transferrin family

9

1.3.2

The vertebrate ferritins

10

1.3.2.1 Cytosolic ferritins

11

1.3.2.2 Secreted ferritins

13

Invertebrate ferritins

14

1.3.3.1 Cytosolic ferritins

14

1.3.3.2 Secreted ferritins

15

1.3.3

1.4

Model for host-pathogen competition for iron

16

1.4.1

The horseshoe crab

16

1.4.2

Pseudomonas aeruginosa- a model pathogen for iron piracy

ii


study

20

1.5

The Rationale and Aims of this Thesis

23

2.

MATERIALS AND METHODS

25

2.1

Materials

25

2.2

Infection of horseshoe crab and preparation of cell-free haemolymph/
plasma

25

Oxidative activity of plasma during P. aeruginosa infection

25

2.3.1

25

2.3

2.4

2.5

Purification and identification of plasma ferritin

26

2.4.1

Purification and resolution of plasma ferritin

26

2.4.2

Two dimensional gel electrophoresis

27

2.4.3

Edman degradation for N-terminal sequencing of ferritin

29

2.4.4

Mass Spectrometry analysis of proteins

29

Cloning of horseshoe crab ferritin cDNA

29

2.5.1

Degenerate RT-PCR of CrFer-H1

30

2.5.2

5’ and 3’ Rapid Amplification of cDNA ends (RACE) of
CrFer-H1

30

PCR amplification and cloning of CrFer-H2 cDNA

31

2.5.3
2.6

2.7

2.8

Supercoil relaxation assay

Regulation of ferritin during P. aeruginosa infection

32

2.6.1

Northern analysis of CrFer-H2

32

2.6.2

Western analysis

32

Strategy employed by P. aeruginosa to ‘steal’ host iron

33

2.7.1

Reaction between plasma ferritin and P. aeruginosa

33

2.7.2

Quantification of total and labile iron pool in the plasma

33

2.7.3

Measurement of redox potential and pH

34

Plasma ferritin-DNA interaction

34

iii


2.8.1

Electrophoretic mobility shift of DNA by ferritin

34

2.8.2

Fluorescence measurement of ferritin complex-DNA interaction

35

2.9

Statistical analysis

35

2.10

Homology modeling methods

36

3.

RESULTS

38

3.1

Iron contributes to the high oxidative activity of plasma.

38

3.2

Horseshoe crab plasma ferritin is made up of subunits of 21 kDa.

42

3.3

CrFer-H1 has 2 possible transcripts that are translated to plasma ferritin.

45

3.4

Another ferritin gene, CrFer-H2, codes for a secretory protein
that is apparently absent in the plasma

52

3.5

Common features of the 3 ferritin cDNAs

55

3.6

CrFer-H2 is ubiquitously expressed and its transcription is responsive
to LPS and bacterial infection.

61

LPS and iron can regulate ferritin protein synthesis during
Pseudomonas infection.

64

3.8

P. aeruginosa ‘steals’ host iron by degrading plasma ferritin.

64

3.9

Ferritin switches from a DNA-binding to non-DNA-binding conformer
during infection.

69

Uninfected and infected plasma ferritin complexes contain different
ferritin isoforms.

69

4.

DISCUSSIONS

73

4.1

Plasma ferritin is directly involved in innate immune response.

73

3.7

3.10

4.1.1
4.1.2
4.2

The horseshoe crab plasma ferritin evades degradation by
P. aeruginosa to prevent iron loss.

73

Regulation of plasma ferritin may contribute to iron
homeostasis and constant free radical level.

75

A dynamic role of ferritin during Pseudomonas infection.

76

iv


4.3

5.

Insights into the role of plasma ferritin: from horseshoe crab to
mammalian plasma ferritin

80

CONCLUSION AND FUTURE PERSPECTIVES

82

v


LIST OF ABBREVIATIONS
2DE
Ame
CFH
CFU
CrFer-H
DTT
ES-Q-TOF
EST
Fur
Hep
HT
Hpi
IAA
INT
IRE
LIP
LPS
MALDI-TOF
MS-BLAST
MUS
ORF

PCR
PMF
PVDF
RACE
RT-PCR
SDS-PAGE
STC
TPTZ
TSB
UTR

Two dimensional gel electrophoresis
Amebocytes
Cell-free hemolymph
Colony forming unit
Carcinoscorpius rotundicauda ferritin heavy chain
Dithiothreitol
Electrospray-Quadrupole-Time-of-flight
Expressed sequence tags
Ferric uptake regulator
Hepatopancreas
Heart
Hour post-infection
Iodoacetamide
Intestine
Iron responsive element
Labile iron pool
Lipopolysaccharides
Matrix-Assisted Laser Desorption Ionization Time-of-flight
Mass spectrometry-Basic Local Alignment Search Tools

Muscles
Open-reading frame
Polymerase chain reaction
Peptide mass fingerprint
Polyvinylidene fluoride
Rapid Amplification cDNA end
Reverse Transcription-Polymerase Chain Reaction
Sodium Dodecylsulphate-Polyacrylamide Gel Electrophoresis
Stomach
2,4,6-tripyridyl-s-triazine
Tryptone soy broth
Untranslated region

vi


LIST OF FIGURES
Fig. No.
1.1

1.2

1.3

1.4
2.1
2.2
3.1

3.2

3.3

Title
Confocal microscopic images of GFP-labeled P.
aeruginosa in biofilm flow cells perfused with lactoferrin
free (a-d) and lactoferrin-containing (20 µg/ml) (e-h)
media.
Human H-chain and human L-chain both have 5 α-helices
and the heavy chain subunits then assemble into a
apoferritin complex of 24 subunits in 432 symmetry
viewed down the a four-fold axis.
(A) A hypothetical scenario to the coagulation-based
clotting mechanism and containment of foreign
invaders.
(B) The LPS- and glucan-mediated pathways require the
serine protease Factor C and Factor G respectively.
Gram staining of Pseudomonas aeruginosa and colony
morphology on agar plates.
Schematic view of the partial purification and enrichment
of horseshoe crab plasma ferritin.
Diagrammatic representation of the iron assay to measure
total plasma iron and LIP.
Plasma iron regulates free radical-induced DNA nicking.
(A) The role of iron as a catalyst in the Haber-Weiss
reaction.
(B) Concentration dependent DNA nicking activity by
naïve plasma and the effect of nicking buffer.
(C) Effect of glycerol as a free radical scavenger in the
highly oxidative plasma.

(D) Effect of metal chelators on oxidative activity of
plasma using EDTA, potassium ferrocyanide and
ferricyanide.
(E) Oxidative activity of plasma during P. aeruginosa
infection of the horseshoe crab.
Identification of limulus plasma ferritin complex and its
subunits in the plasma.
(A) The native state of limulus plasma ferritin was
detected by Prussian blue staining.
(B) The ferritin complex is made up of 21 kDa subunits.
(C) Protein sequencing of 21 kDa band.
(A) PCR products of the same size (~ 240 and 330 bp)
were obtained from degenerate RT-PCR using naïve
heart, intestine and stomach cDNA as template.
(B) Alignment of the deduced amino acid sequence of the
240 and 330 bp PCR products show that they may be
encoded by the same gene.
(C) Design of 5’ and 3’ RACE primers using the partial
ferritin DNA sequence.
(D) 5’ and 3’ RACE products of ferritin gene.
(E) Screening of positive clones by EcoRI digestion of

Page
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12

17
17
21

28
37
39

41

43

46
47
48
49
50
vii


3.4

3.5

3.6

3.7
3.8

3.9

3.10
4.1

pGEM-T Easy vector.
(F) Nucleotide sequence and deduced amino acid
sequence of CrFer-H1a and -H1b.
Cloning of CrFer-H2.
(A) Screening of clones that harbor the 3’ fragment of
CrFer-H2 after EcoRI digestion of pGEM-T Easy
vector.
(B) 5’ RACE product of CrFer-H2 at various annealing
temperatures using naïve cardiac cDNA as template.
(C) Nucleotide sequence and deduced amino acid
sequence of CrFer-H2.
(A) Predicted secondary structures of CrFer IRE at the 5’
UTR and the alignment of CrFer IRE sequence with
that from other organisms.
(B) Both CrFer-H1 and –H2 are predicted to possess the
typical 5 α-helices of ferritins.
(C) Phylogenetic analysis of CrFer-H1 and –H2.
(D) Central region of subunits of human H-ferritin.
(E) CrFer-H1 and –H2 share ~ 72 % identity (top) and
there are likely to be other isoforms of plasma ferritin
(bottom).
(A) Northern analysis to study differential expression of
CrFer-H2 in various naïve,
3h LPS-induced and 3h FeSO4 induced tissues of the
limulus.
(B) Quantitative analyses of CrFer-H expression using
ImageMaster software.
(C) Northern analysis to study kinetics of CrFer-H2
expression in various tissues after infection with P.
aeruginosa and (D) the change in fold of CrFer-H2

normalized against actin 3.
Regulation of limulus plasma ferritin at the protein level
during P. aeruginosa infection.
Strategy employed by P. aeruginosa to obtain host iron.
(A) P. aeruginosa can degrade ferritin in vitro.
(B) Pseudomonas infection does not result in
hypoferraemia of the limulus.
(C) Pseudomonas does not lower plasma redox potential
or pH to ‘steal’ host iron.
Uninfected plasma ferritin but not infected plasma ferritin
can bind to DNA in a sequence-independent manner.
(A) Using LDorThr and LkBCom as probes, plasma
ferritin from naïve, 3, 6 and 72 hpi individuals, as well
as 3 h iron-loaded individuals were incubated and run
on 4 % PAGE gel.
(B) Fluorescence measurement of ferritin complex-DNA
interaction.
Uninfected and infected plasma ferritins consist of
different 21 kDa isoforms.
Proposed model for the dynamic role of plasma ferritin

51
53

54
56
57
58
59
60

62

63

65
67

70

72
78
viii


during infection.

ix


LIST OF TABLES
Table No.
1
2
3

Title
Effect of iron deficiency and iron overload on various
immunological functions.
Defense molecules found in the hemoctyes and
haemolymph plasma of the horseshoe crab.

Summary of primers used in the cloning of ferritin genes.

Page
4
19
31

x


Summary
Ferritin, normally found intracellularly in vertebrates, is responsible for iron
storage and detoxification, although it has been isolated from plasma in trace amount.
Plasma ferritins serve as extracellular iron storage molecules and loss of plasma iron
to pathogen is detrimental to the host during infection. Interestingly, the horseshoe
crab plasma iron level is 8-10-fold higher than human plasma. In this study, horseshoe
crab plasma ferritin complex was purified, characterized and its dynamic role in
innate immune defense was investigated using Pseudomonas aeruginosa as a model
pathogen. We demonstrate the interesting phenomenon that on one hand,
Pseudomonas attempts to degrade the host ferritin in order to usurp the host iron for
its survival.

On the other hand, the host maintains iron homeostasis by tightly

regulating its level of plasma ferritin, plasma redox potential and pH that keeps the
plasma free radicals in check. Between 6-48 h of infection, the host plasma ferritin
evades Pseudomonas-mediated degradation by transiting from extracellular to
intracellular space, during which different ferritin isoforms constitute the ferritin
complex. Our data show that the host recovers its level of plasma ferritin by 72 h.
Furthermore, we demonstrate that contrary to the naïve ferritin, which binds the host

DNA sequence-independently and probably protects the host genome, infection
somehow disables the ferritin complex from binding host DNA. We propose that the
plasma ferritin plays dual roles: (i) pathogen evasion and (ii) DNA protection or
chromatin remodeling after nuclear translocation.

xi


1.

Introduction

1.1

Iron in biological systems and its toxic effects
Iron is an abundant metal, being the fourth most plentiful element in the

earth’s crust. It can be found in the first row of transition metals in the periodic table.
It exists mainly in one of the two readily reversible redox states: the reduced Fe2+
ferrous form and the oxidized Fe3+ ferric form. Depending on its ligand environment,
both ferrous and ferric forms can adopt different spin states. As a result of these
properties, iron is an extremely attractive prosthetic component for incorporation into
proteins as a biocatalyst or electron carrier during evolution of early life (Andrews et
al., 2003). Iron plays an indispensable role in various physiological processes, such as
photosynthesis, nitrogen fixation, methanogenesis, hydrogen production and
consumption, respiration, the trichloroacetic acid cycle, oxygen transport, gene
regulation and DNA biosynthesis. The incorporation of iron into proteins allows its
local environment to be regulated such that iron can adopt the necessary redox
potential (-300 to +700 mV), geometry and spin state for realization of its prescribed
function (Andrews et al., 2003).

Unfortunately with the appearance of oxygen on earth ~ 2.2 to 2.7 billion
years ago, two major problems arose. One was the production of toxic oxygen species
and the other, a drastic decrease in iron availability (Touati, D., 2000). In its reduced
ferrous form, iron potentiates oxygen toxicity by converting the less reactive
hydrogen peroxide to the more reactive oxygen species, hydroxyl radical and ferryl
iron, via the Fenton reaction (O2- + H2O2 → HO + OH- + O2; iron as a catalyst).
Conversely, superoxide favours the Fenton reaction by releasing iron from ironcontaining molecules. It is widely accepted that tight regulation of iron assimilation
prevents an excess of free intracellular iron that could lead to oxidative stress.

1


1.2

Iron in host-pathogen interaction during infection
1.2.1

Iron, microbial pathogens and sepsis

Sepsis has been a challenge to humans and it has steadily worsened in recent
years. In the United States alone, there are ~ 500,000 incidents each year with a death
rate of 35-65 % (Dellinger et al., 1997; Bone et al., 1997). Amongst the numerous
complex interactions between host and pathogen, one common and essential factor is
the ability to invade and multiply successfully within host tissues. Proliferation of
pathogen is critical to establishing an infection and this mediates the pathogen to
produce the full arsenal of virulence determinants required for pathogenicity (Bullen
and Griffiths, 1999). The availability of iron in the host environment and its effects on
bacterial growth is one of the best studied aspects in pathogenicity. Humans are
equipped with a well-developed natural resistance against bacterial infection.
Currently, some of the understood mechanisms involved are the antibacterial

properties of tissue fluids and the phagocytic abilities of cells (Bullen et al., 2000).
However, research has revealed that these mechanisms require a virtual iron-free
environment for proper function (Ward et al., 1999). In normal human plasma, the
high affinity constant for Fe3+ (10-36 M) and the unsaturated state of the iron-binding
protein transferrin ensure that the amount of free ferric iron is ~ 10-18 M (Bullen, et al.,
1978). In vivo, bacterial growth is inhibited by strong bactericidal and bacteriostatic
mechanisms in the plasma. These include unsaturated transferrin, antibody, and
complement, which function in the virtual absence of freely available iron.
Even though freely available iron in normal body fluids is virtually absent,
pathogenic bacteria are able to multiply successfully in vivo to establish an infection.
The observation that all known bacterial pathogens require iron to multiply suggests
that they must adapt to the iron-free extracellular environment in vivo and develop

2


mechanisms to acquire protein-bound iron. Thus, pathogens have evolved various
ways to compete for the host iron. The production of low molecular mass ironchelating compounds (siderophores), expression of transferrin and lactoferrin
receptors, proteolytic cleavage of iron-binding glycoproteins, disruption of ironbinding site, reduction of ferric to ferrous complex to effect ferrous iron release and
utilization of iron in haem compounds are some of the strategies developed during coevolution of host and pathogen (Bullen and Griffiths, 1999). The invading pathogens
could also migrate into local environments where iron is more readily available, such
as inside some cells. Low environmental iron levels can signal pathogens to induce
their virulence genes (Litwin et al., 1993) and this has been extensively demonstrated
in the opportunistic human pathogen, Pseudomonas aeruginosa, which employs a Fur
protein as an iron sensor to induce cytotoxic exotoxin A and extracellular proteases
under iron-depleted conditions (Bullen et al., 1978).

1.2.2

Adaptive immunity and its iron-dependent nature

Adaptive immunity can be classified as humoral immunity, mediated by
antibodies which are produced by activated B lymphocytes, and cell-mediated
immunity, mediated by T lymphocytes. The immune system is activated when an
antigen is recognized and processed by an antigen-presenting cell such as macrophage,
dendritic cell, or a B lymphocyte. Subsequently, the T and/or B lymphocytes are
activated and this leads to cell division, phenotypic changes and protein synthesis.
The cytokines activate the phagocytic cells and lymphocytes to exert increased
microbicidal and cytotoxic activities (Brock, 1999). Iron is critical for many
metabolic processes and since immunological activation involves various metabolic
events, iron bioavailability has been believed to influence the immune system. This

3


link has been supported by studies of various immune functions in humans and
experimental animals that reveal defects associated with abnormalities of iron
metabolism, as well as in vitro studies that illustrate the iron-dependent nature of the
immune system. Some of these effects are summarized in Table 1.

4


1.2.3

Innate immunity

The innate immune system is believed to predate the adaptive immune
response. The innate immune system represents a frontline defense that targets
microbial pathogens by recognizing molecular structures that are shared by large

groups of pathogens, the pathogen-associated molecular patterns via pattern
recognition proteins or the pattern recognition receptors. The pathogen-associated
molecular patterns are conserved products of microbial metabolism and they are
essential for the survival or pathogenicity of the microorganisms (Medzhitov and
Janeway, 1997). Examples of pathogen-associated molecular patterns include
lipopolysaccharides (LPS) of all gram-negative bacteria, lipoteichoic acids of all
gram-positive bacteria and the mannans of yeasts /fungi. A key feature of these
microbial patterns is their polysaccharide chains that vary in length and carbohydrate
composition (Franc and White, 2000).
The invertebrates have a defense system centered on both cellular and humoral
immune response. The former is known to include encapsulation, phagocytosis
(Foukas et al., 1998), and nodule formation, while the humoral response includes the
coagulation system of arthropods (Iwanaga et al., 1998), the synthesis of a broad
spectrum of potent antimicrobial proteins in many insects and crustaceans (Hoffman
et al., 1996), and the prophenoloxidase activating system (proPO system) (Soderhall
et al., 1998).
In the vertebrates, innate immunity provides a first line of host defense against
pathogens and the signals that are needed for the activation of the adaptive immunity
(Fearson and Locksley, 1996). The vertebrate innate immunity was suggested to
resemble a mosaic of invertebrate immune responses. For example, the effectors,
receptors and regulation of gene expression of insects in acute immune response are

5


similar to those of humans. Some antibacterial peptides and immune stimulators have
originated from the processing of neuropeptide precursors (Salzet, 2001). The
vertebrate pathogen recognition receptors are displayed by particular cell types, such
as macrophages, natural killer cells, and probably also epithelial and endothelial cells
in the lung, kidney, skin and gastrointestinal tract (Wright, 1991). Similar to the

invertebrate innate immune molecules, expression of the vertebrate innate immune
molecules works on a broad-based specificity targeted at broad classes of pathogens
and their corresponding pathogen-associated molecular patterns. A number of
mammalian pathogen recognition receptors have been characterized and these include
the macrophage mannose receptor, scavenger receptors, integrins, collectins, and
some clusters of differentiation antigens (Epstein et al., 1996; Wright et al., 1990).

1.2.3.1 The iron-withholding strategy as a component of innate
immunity
Iron sequestration is recognized as an ancient host defense mechanism against
invading pathogens (Beck et al., 2002) and it is widespread in occurrence. Upon
infection, iron acquisition is critical for bacterial growth and pathogenicity (Bullen,
1981). However in the vertebrates, bacterial infection can drastically reduce plasma
iron level (Lauffer, 1992) as the host withholds iron within the cells and tissues
(Konijn and Hershko, 1977 ; Roeser, 1980; Brock, 1989). Some features of the ironwithholding defense system include constitutive components such as transferrin,
lactoferrin and ovotransferrin, as well as processes which are induced at the time of
microbial cell invasion. The suppression of iron efflux from macrophages hence,
reduction in plasma iron and increased synthesis of ferritin by macrophages to
accommodate iron from phagocytosed lactoferrin iron (Lauffer, R.B., 1992) is one
such example.

6


Currently, the iron-withholding strategy is accepted as a new component of the
innate immune system. (Singh, et al., 2002; Ganz, 2003). Singh et al. (2002)
demonstrated that lactoferrin stimulates twitching, a specialized form of surface
motility by chelating iron, causing the P. aeruginosa to wander around instead of
forming clusters & biofilms. Conalbumin was also shown to block biofilm formation
of P. aeruginosa through iron chelation, hence biofilm formation as well. Thus, iron

deprivation inhibits the formation of resistant bacterial biofilms, prevents recalcitrant
bacteria that survive initial defenses from forming squatters and favours the
vulnerable unicellular forms that are better equipped to reach alternative iron sources
(Singh et al., 2002) (Fig. 1.1).

7


4 hour

24 hour

3 days

7 days

Fig. 1.1 Confocal microscopic images of GFP-labeled P. aeruginosa in biofilm
flow cells perfused with lactoferrin-free (a-d) and lactoferrin-containing (20
µg/ml) (e-h) media. Images were obtained 4 h (a, e), 24 h (b, f), 3 days (c, g) and 7
days (d, f) after inoculating the flow cells. Images a, b, e and f are top views; scale
bar, 10 µm. Images c, d, g and h are side views; scale bar, 50 µm. Results are
representatives of 6 experiments. (Adapted from Singh et al., 2002).

8


1.3

The iron-binding proteins involved in infection and inflammation
To achieve an iron-free physiological environment, mammals employ iron-

binding proteins to reduce the level of extracellular iron to around 10-18 M (Bullen et
al., 1978) so as to stall bacterial growth (Jamroz et al., 1993). At least two classes of
iron-binding proteins, ferritin and transferrins, are present across phyla (Singh et al.,
2002).

1.3.1

The transferrin family

As a major iron transporter in the blood of vertebrates, transferrin absorbs iron
in the gut, shuttles between peripheral sites of storage and uses, and maintains iron
level sufficient to support cells having a particular demand for iron (Yoshiga et al.,
1997; Jamroz et al., 1993). Transferrins are serum glycoproteins (extracellular), with a
molecular weight of ~ 75-80 kDa. Each transferrin molecule is folded to give 2
globular domains. Each domain contains a specific binding site for a single Fe3+
(Caccova et al., 2002). Diferric iron is taken into cells by receptor-mediated
endocytosis. Dissociation of iron from transferrin then occurs in an acidic endosome,
after which the iron is transferred to the cytoplasm. Within cells, the iron is
subsequently incorporated into metalloproteins or stored in the cytoplasm either
within the iron storage protein, ferritin, or chelated to small molecules (Welch, 1992).
At physiological pH, the affinity of transferrin for Fe3+ (Kd ~ 10-20 M) is very high.
Lactoferrin, a member of the transferrin family, is a 78 kDa glycoprotein
present in various secretions (eg. milk, tears, saliva and pancreatic juice). In the
vertebrates, serum transferrin is an acute-phase protein as its concentration closely
mirrors conditions of stress or infection, although its rise or fall varies with the
infective microorganisms. Human lactoferrin is stored in specific granules of

9

polymorphonuclear granulocytes from which it is released following activation. It
binds with high affinity to lipid A and may play an antibiotic role by depriving
invading microorganisms of iron, which is required for their proliferation and
(Yoshiga et al., 1997; Caccova et al., 2002). Owing to their bacteriostatic activity,
members of the transferrin family (ovotransferrin and lactoferrin) have been
considered major contributors to host iron sequestration.
Transferrins have also been isolated from cockroach, mosquito, Bombyx mori,
Drosophila and Manduca sexta at the genetic and protein level (Jamroz et al., 1993;
Yoshiga et al., 1997; Yun et al., 1997; Yoshiga et al., 1999; Hueber et al., 1988). The
involvement of transferrin in immune defense of mosquitoes has been shown by
Yoshiga et al. (1997) as transferrin synthesis and secretion are increased upon
exposure of mosquito cells (Aedes aegypti or Aedes albopictus) to bacteria.
Inoculation of adult Drosophila with E. coli also led to dramatic increase in
transferrin mRNA (Yoshiga et al., 1999). In the goldfish, transferrin serves as a
primary activating molecule of macrophage antimicrobial response (Stafford and
Belosevic, 2003). It was found that the products released by necrotic/damaged cells
can enzymatically cleave transferrin, and the cleavage products of transferrin were
able to induce nitric oxide response of macrophages. Addition of transferrin also
significantly enhanced the killing response of the goldfish macrophages exposed to
different pathogens or pathogen-associated molecular patterns.

1.3.2

The vertebrate ferritins

Another important iron sequestration protein, ferritin, has been extensively
investigated, showing pivotal roles in iron storage and detoxification. In the
vertebrates, ferritin is mainly intracellular although trace levels of plasma ferritin can

10


be found in ng/L quantity. In higher vertebrates, ferritin has been indirectly linked to
innate immune response since the synthesis of ferritin is regulated by proinflammatory cytokines at both transcriptional and translational levels (Torti et al.,
1988; Konijn and Hershko, 1989; Roger et al., 1990; Huang et al., 1999). More
recently, the ferroxidase sites of ferritin H subunit have been reported to be critical for
direct DNA binding, suggestive of a new important role of ferritin in the protection of
host cell genome by preventing DNA nicking due to free radical effects caused by
free iron in the nucleus (Surguladze et al., 2004). An overview of the current
understanding of both cytosolic and secreted ferritins in vertebrates is discussed
below.

1.3.2.1 Cytosolic ferritins
Ferritin is present in all types of mammalian cells, being most abundant in
macrophages and hepatocytes. The structure of cytosolic ferritin from various
organisms has been solved and they share a similar structure composed of 5 α-helices
(Fig. 1.2A and B). In the native state, the ferritin complex is a hollow sphere
(apoferritin) composed of 24 subunits (Fig. 1.2C), with very high iron binding
capacity (4500 iron atoms). There are 24 subunits of two types, H and L (each of ~20
kDa), which exist in different ratios, from different tissues and in various
physiological states (Nichol and Locke, 1999). With the completion of the human
genome project, it is now known that there are at least 15 genes encoding ferritin Hchain subunits (FTHL1-4, FTHL7-8 and FTHL10-18) and 1 gene for ferritin L-chain
subunit (FTL) (NCBI LocusLink, ).
The human H- and L-subunits are ~ 55 % homologous and are coded by genes on
various chromosomes. However, it is the H-chain that possesses ferroxidase activity.

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(A)

(B)

(C)

Fig. 1.2 Human H-chain (A) and human L-chain (B) both have 5 α-helices and
the heavy chain ferritin subunits then assemble into a apoferritin complex of 24
subunits in 432 symmetry viewed down a four-fold axis. (C). The structures were
obtained from the Protein Database. Human H-chain: pdb 2fha; human L-chain: pdb
1aew. The structure of the ferritin complex was adapted from Chasteen and Harrison,
1999.

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