Group 2 allergens from dust mite epitope mapping and functional characterization of der p 2, and identification of a paralogue of der f 2

GROUP 2 ALLERGENS FROM DUST MITE:
EPITOPE MAPPING AND FUNCTIONAL
CHARACTERIZATION OF DER P 2, AND
IDENTIFICATION OF A PARALOGUE OF DER F 2

KAVITA REGINALD
(B.Sc. (Hons.), UPM)

A THESIS SUMBITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
DEPARTMENT OF BIOLOGICAL SCIENCE
NATIONAL UNIVERSITY OF SINGAPORE
2006


Acknowledgements

It is close to impossible to do good scientific research in graduate school without help.
As most other students who have taken the same path will agree, it is a long and
arduous journey, with short bursts of satisfactions when discoveries are made. There
have been many people who have helped me in my journey as a young scientist in
graduate school, and I wish to extend my thanks to them in this section.
My supervisor, Dr Chew for the research opportunity and guidance in the last 5 years.
My lab mates, for assistance in laboratory techniques and continually giving me
constructive suggestions. A big thanks goes out to Tan Ching for the immunological
experiment techniques, and Siew Leong for advice and assistance in the protein
studies.
The allergic patient volunteers, for your time and cooperation throughout this study.
My friends in university. Special thanks goes out to Sai Mun and Souvik for always
giving me timely assistance and advice in so many areas of research. Also to Shruthi,
who has been of great assistance for databasing, and analysis of some sections. To

Vaane, Grace, and Dr. Tan for patiently reading and editing the thesis. To Siva, for
lending technical help.
My collaborator, Dr. Markus, for being an inspirational scientist, and giving me the
opportunity to venture into the study of lipids. Also to members of his research lab,
especially Guanghou and Gek Huey whom I have worked closely with.
My friends outside university. I thank all of them for making my stay in Singapore a
fulfilling one. To Radhi, Shion, Ken, Shih Lene and Wan Yee, needless to say, I have
thoroughly enjoyed all our house parties, cook outs and chill out sessions. To Shashi,
Harveen, Jam and Punam, you have evolved from friends to my family. I have no
words to express my gratitude.
The Sahaja Yogis. How would you thank those who have helped you to connect to
your spirit? There are no words, just bliss. You have helped me discover the true
meaning of life, and made it possible for me to go through the difficult moments. A
huge hug for the ‘world collective’ especially to Geethanjali and Michalis for your
assistance.
My family. Without their blessing and continual support, none of this would be
possible.
The Divine.

i


Disclaimer

Some parts of the experiments were done together with other lab members, and are
listed here.
In chapter 3, IgE inhibitions were done together with Aaron Chen.
In chapters 3-8, screening of IgE reactions was done together with Yap Kwong Hsia.
In chapter 4, cloning of Blo t 2 was done together with Kway Kwee Theng.
Quantification of allergen concentration in dust samples was done with Kelly Goh.

Extraction of native Blo t 2 was done with Alvin. Histamine release assay was done
with Gavin. Study of isoforms was done with Jia Yi.
In chapter 5, immmunostaining and southern blot analysis was done together with Dr.
Tan Chye Ling. Quantification of allergen concentration in dust samples was done
with Chen Simin. Phylogenetic analysis was done with Shruthi and Dr. Yap Von Bing.
In chapter 6, mass spectrometry was done together with Shui Guanghou. Docking
analysis was done with Chua Gek Huey.
In chapter 8, the cytokine assay was done together with Dr. Ong Tan Ching.

ii


List of conference abstracts and book chapters

International Conference Abstracts
Reginald K, L Haroon-Rashid, YS Sew, SH Tan, FT Chew (2002). Identification of
putative Tyrophagus putrescentiae allergens with sequence homology to other known
allergens by expressed sequence tagging. In: XXIth European Academy of
Allergology and Clinical Immunology Annual Meeting (EAACI), June 2002, Naples,
Italy. Allergy 57 (Suppl. 73): 286-7.
Loo AHB, SPL Tan, AC Angus, KT Kuay, K Reginald, YF Gao, FT Chew (2003).
Genetic Relationship Between Allergy-Causing Dust Mites: Phylogenetic Inference
From Random Amplified Polymorphic DNA (RAPD) Markers, Housekeeping Gene
(18S rDNA) And Group 2 Allergens. In: The 60th American Academy of Allergy and
Immunology Annual Meeting, 7 - 12 March 2003, Denver, USA. J Allergy Clin
Immunol 111 (2): S162.
K Reginald, XZ Bi, ST Ong, FT Chew (2003). Profiling of Crude Allergen Extracts
Using SELDI Mass Spectrometry for Rapid Standardization. In: The 60th American
Academy of Allergy and Immunology Annual Meeting, 7 - 12 March 2003, Denver,
USA. J Allergy Clin Immunol 111 (2): S242.

AHB Loo, SY Goh, K Reginald, YF Gao, H Jethanand, HS Shang, FT Chew (2004).
Validation of the Purity of Acarid Mite Cultures Used for Allergen Extract
Preparation and Identification of Contaminants by Ribosomal DNA Sequencing via a
PCR-Cloning- and Sequence Homology-Based Approach. In: The 61th American
Academy of Allergy and Immunology Annual Meeting, 19 - 24 March 2004, San
Francisco, USA. J Allergy Clin Immunol 113 (2): S140.
K Reginald, YF Gao, YS Siew, HS Shang, FT Chew (2004). Cross Comparison of
the IgE Binding Profiles to Recombinant Allergens from Suidasia medanensis,
Blomia tropicalis and Dermatophagoides farinae using Sera from Blomia- and
Dermatophagoides-Predominant Environments. In: The 61th American Academy of
Allergy and Immunology Annual Meeting, 19 - 24 March 2004, San Francisco, USA.
J Allergy Clin Immunol 113 (2): S228-9.
Reginald K, Gao YF, Lim YP, Chew FT (2004). The expressed sequence tag
catalogue and allergens of dust mite, Suidasia medanensis. In: XXIIIth European
Academy of Allergology and Clinical Immunology Annual Meeting (EAACI), June
2004, Amsterdam, The Netherlands.
Tay ASL, Shang HS, Bi XZ, Reginald K, Gao YF, Angus AC, Ong ST, Wang WL,
Kuay KT, Wang DY, Mari A, Chew FT (2005). Component-Resolved Diagnosis Of
House Dust Mite Allergy With A Large Repertoire Of Purified Natural And
Recombinant Allergens From The Major Species Of Mites Worldwide. In: The 62th
American Academy of Allergy and Immunology Annual Meeting, March 2005, San
Antonio, USA. J Allergy Clin Immunology, 115 (2): S164
iii


Reginald K, Wenk MR, Chew FT (2005). The major mite allergen from
Dermatophagoides pteronyssinus, Der p 2, is a sterol binding protein. In: The 62th
American Academy of Allergy and Immunology Annual Meeting, March 2005, San
Antonio, USA. J Allergy Clin Immunology, 115 (2): S88
Tan CL, Reginald K, Chew FT (2006). Genomic organization and characterization of

group 2 allergen paralogs from Dermatophagoides farinae. In: The 63th American
Academy of Allergy and Immunology Annual Meeting, March 2006, Miami, Florida,
USA. J Allergy Clin Immunology, 117 (2): S120
Reginald K, Chew FT (2006). Epitope mapping of Der p 2 by site directed
mutagenesis: Differential IgE binding epitope profile among individuals sensitized to
only Dermatophagoides spp. and those with non-pyroglyphid mite responses. In: The
63th American Academy of Allergy and Immunology Annual Meeting, March 2006,
Miami, Florida, USA. J Allergy Clin Immunology, 117 (2): S118

Book Chapter
Reginald K, Sew YS, Haroon-Rashid L, Kulaveerasingam H, Tan SH, Chew FT.
Chapter 49. Identification of putative Tyrophagus putrescentiae allergens with
sequence homology to other known allergens by Expressed Sequence Tagging.
Progress in Clinical Immunology and Allergy in Medicine. Edited by Gianni Marone.
(invited Book Chapter)

iv


Table of contents
Page

Acknowledgements

i

Disclaimer

ii

List of conference abstracts and book chapters

iii

Table of contents

v

List of figures

xi

List of tables

xv

List of abbreviations

xvi

Summary

xx

Chapter 1: Introduction

1

1.1 Literature Review

1

1.1.1 Allergy

1

1.1.2 Dust mites

4

1.1.3 Dust mite allergens

7

1.1.4 Immunotherapy as a treatment for allergic diseases

12

1.2 Aims

14

Chapter 2: Materials and methods

16

2.1 Cloning, mutagenesis, DNA sequencing and gene characterization

16

2.1.1 Sub-cloning and site-directed mutagenesis

16

2.1.2 RT-PCR of putative Blo t 2 using degenerate primers

16

2.1.3 DNA sequencing

17
v


2.1.4 Isolation of Blo t 2 isoforms

18

2.1.5 Isolation of the genomic DNA encoding for Der f 2 and Der f 22

18

2.1.6 Genomic DNA extraction, Southern Blot analysis and hybridization

19

2.2 Protein expression, purification, CD analysis and antibody generation

20

2.2.1 Expression and purification of wild type and mutant allergens

20

2.2.2 Isolation of native Blo t 2

21

2.2.3 Circular dichroism (CD) spectropolarimetry

21

2.2.4 Gel Filtration

21

2.2.5 Generation of rabbit polyclonal antibodies

22

2.3 Serum samples

22

2.4 Immunological assays

22

2.4.1 Immuno dot blot

22

2.4.2 Specific IgE binding ELISA

23

2.4.3 Inhibition ELISA

24

2.4.4 Histamine release assay

25

2.4.5 Dust sample collection, processing and quantification.

25

2.4.6 Staining and immunoprobing

26

2.4.7 Skin prick test

26

2.4.8 Isolation of PBMC and measurement of proliferation upon stimulation with
wild type or mutant allergen

27

2.4.9 Measurement of excreted cytokines

28

2.4.10 Mouse immunization

28

2.4.11 Inhibition of human IgE binding to Der p 2 by specific mouse IgG
antibodies
2.5 Computer based characterization and analysis

28
29

vi


2.5.1 Analysis of DNA and protein sequences

29

2.5.2 Three dimensional protein structure predictions

29

2.5.3 Phylogenetic sequence analysis

30

2.5.4 Docking of cholesterol to Der p 2

31

2.6 Lipid assays
2.6.1 Liposome preparation

33
33

2.6.2 Detection of liposome binding to Der p 2 by liposome sedimentation and
SDS-PAGE

33

2.6.3 Lipid ELISA

34

2.6.4 Extraction of lipid fraction from Der p 2

34

2.6.5 HPLC/APCI/MS/MS analysis of cholesterol

35

2.7 Statistical analyses

36

2.8 Approval

36

Chapter 3: IgE reactivity and cross reactivity profiles of group 2 allergens

37

3.1 Introduction

37

3.2 Cloning and sequence analysis of Ale o 2, Sui m 2 and Blo t 2

39

3.3 IgE reactivity to group 2 allergens

50

3.3.1 IgE reactivity to group 2 allergens in the Singaporean population

50

3.3.2 IgE reactivity to group 2 allergens in the Italian population

56

3.4 Further characterization of Blo t 2

61

3.4.1 Isolation of native Blo t 2

61

3.4.2 Histamine release of Blo t 2

61

3.4.3 Blo t 2 is present in the environmental dust samples

64

3.4.4 Isoforms of Blo t 2

66

vii


3.5 Discussion

69

Chapter 4: Identification and characterization of Der f 22, a novel allergen from
Dermatophagoides farinae: a paralogue of Der f 2?

75

4.1 Introduction

75

4.2 Identification, isolation and characterization of Der f 22

76

4.3 Genomic organization of Der f 22 and Der f 2

83

4.4 Southern blot analysis

86

4.5 IgE binding capacities of Der f 22 and Der f 2

88

4.6 Localization of Der f 22 and Der f 2 on sectioned D. farinae

92

4.7 Concentration of Der f 22 and Der f 2 in the indoor environment

94

4.8 Der f 2 and Der f 22 binds to cholesterol

96

4.9 Presence of paralogues of group 2 allergens

98

4.10 Discussion

101

Chapter 5: Der p 2 is a cholesterol binding protein

105

5.1 Introduction

105

5.2 Der p 2 binding to liposomes

107

5.3 Binding of Der p 2 to purified lipids

109

5.4 Analysis of lipid extracts from nDer p 2 and rDer p 2

112

5.5 Characterization of potential cholesterol binding sites in Der p 2 via site
directed mutagenesis

114

5.6 Native Der p 2 and two other allergens of the ML domain family bind to
cholesterol

117

5.7 Docking of cholesterol on Der p 2

119

viii


5.8 Discussion

121

Chapter 6: IgE epitope mapping of Der p 2

125

6.1 Introduction

125

6.2 Design and production of Der p 2 alanine mutants with single amino acid
substitution.

127

6.3 Reaction profile of dust mite allergic patients from Singapore and Italy

133

6.4 IgE epitope mapping of Der p 2 in the Singaporean and Italian populations
based on sensitization profiles.

139

6.5 Evaluation of the changes in secondary structures of mutant E102A and
unfolded Der p 2

151

6.6 Discussion

155

Chapter 7: Evaluation of hypoallergen vaccine candidates for Der p 2

158

7.1 Introduction

158

7.2 Specific IgE binding to site directed mutants of Der p 2 in five allergic
individuals

159

7.3 Skin Prick Test

167

7.4 Mouse IgG antibodies raised against mutant E102A and unfolded Der p 2 are
able to block allergic individuals’ IgE binding to WT Der p 2

169

7.5 T cell reactivity and cytokine profile

172

7.6 Discussion

175

ix


Chapter 8: Conclusion and future direction

181

8.1 Conclusion

181

8.2 Future directions

186

References

190

Appendix I

210

Appendix II

213

x


List of figures
Figure 1.1

A simplified overview of the allergic reaction

3

Figure 1.2

Taxonomic classification of common dust mites

5

Figure 3.1

Nucleotide and translated amino acid sequence of Ale o 2

40

Figure 3.2

Nucleotide and translated amino acid sequence of Sui m 2

41

Figure 3.3

Nucleotide and translated amino acid sequence of Blo t 2

43

Figure 3.4

Predicted three dimensional structures of recombinant
allergens

45

Figure 3.5

Far UV circular dichroism spectra of recombinant Ale o 2,
Sui m 2, Blo t 2 and Der p 2

46

Figure 3.6

Multiple alignments of the mature protein sequences of group
2 allergens

48

Figure 3.7

Phylogenetic tree of group 2 allergens from nine mite species

49

Figure 3.8

IgE binding of Singaporean dust mite positive individuals’
sera to eight group 2 allergens

51

Figure 3.9

Correlation between IgE binding of dust mite allergic
individuals from Singapore

53

Figure 3.10 IgE inhibitions of selected group 2 allergens using ELISA in
three allergic individuals from the Singaporean population

55

Figure 3.11 IgE binding of Italian dust mite positive individuals sera to
eight group 2 allergens

57

Figure 3.12 Correlation between IgE binding of dust mite allergic patients
from Italy

58

Figure 3.13 IgE inhibitions of selected group 2 allergens using ELISA in
three allergic individuals from the Italian population

60

Figure 3.14 Correlation between the amount of IgE binding of 20 dust
mite allergic individuals sera to native Blo t 2 (nBlot 2) and
recombinant Blo t 2 (rBlo t 2)

62

Figure 3.15 In vitro histamine release from whole blood of two dust mite
allergic individuals

63

Figure 3.16 Concentration of Blo t 2 and Der f 2 in dust samples from 101 65
xi


homes
Figure 3.17 Location of the 8 polymorphic residues on the predicted three
dimensional structure of Blo t 2

68

Figure 4.1

Nucleotide and translated amino acid sequence of Der f 22

77

Figure 4.2

Alignment between the mature protein sequences of Der f 2
and Der f 22

79

Figure 4.3

Cystein pairing of Der f 22 and Der f 2

80

Figure 4.4

Ribbon structures of Der f 22 and Der f 2

80

Figure 4.5

CD spectra of Der f 22 (solid line) and Der f 2 (dashed line)

82

Figure 4.6

Location of intron sequences of Der f 22 and Der f 2

84-85

Figure 4.7

Genomic Southern blot analysis of Der f 2 and Der f 22

87

Figure 4.8

IgE binding capacities of Der f 22 and Der f 2

89

Figure 4.9

Correlation between IgE binding of dust mite allergic
individuals’ sera to Der 22 and Der f 2

90

Figure 4.10 Competitive IgE ELISA assay between Der f 22 and Der f 2

91

Figure 4.11 Cross reactivity of polyclonal IgG antibodies raised against
Der f 22 and Der f 2, and immunolocalization on D. farinae
sections

93

Figure 4.12 Concentration of Der f 22 and Der f 2 in dust samples

95

Figure 4.13 Binding of Der f 22 and Der f 2 to cholesterol and POPG

97

Figure 4.14 Phylogenetic relationship of known and putative group 2
allergens

99

Figure 5.1

Liposome pull down assay

108

Figure 5.2

Binding of recombinant Der p 2 to five sterol as well as nonsterol lipids

110

Figure 5.3

Binding of recombinant Der p 2 to a selection of sterols,
phospholipids and sphingiolipids

111

Figure 5.4

LC-MSMS analysis of cholesterol

113

Figure 5.5

Multiple alignments of the amino acid sequences of Der p 2,
Der f 2 and Der f 22 coded by their mature proteins

115

xii


Figure 5.6

Binding of single site directed mutants of Der p 2 to
cholesterol

116

Figure 5.7

Binding of native or recombinant Der p 2 to cholesterol and
POPG

118

Figure 5.8

Predicted binding site of cholesterol in Der p 2

120

Figure 6.1

Amount of IgE binding to Der p 2 and hNPC2

128

Figure 6.2

Alignments of the mature protein sequence of Der p 2 and
hNPC2

130

Figure 6.3

Far UV circular dichroism spectra of wild type Der p 2, the
unfolded protein (K96A) and mutants E25A, and E102A

132

Figure 6.4

Biplots of IgE reaction between Der p 2, Blo t 2 and hNPC2
among dust mite allergic individuals in the Singaporean
population

135

Figure 6.5

Biplots of IgE reaction between Der p 2, Blo t 2 and hNPC2
among dust mite allergic individuals in the Italian population

136

Figure 6.6

Venn diagram of the number of allergic individuals with IgE
reactivity to Der p 2, Blo t 2 and hNPC2

138

Figure 6.7

IgE binding of the 21 single alanine mutants of Der p 2 in
individuals of group (i) sensitization profile

140

Figure 6.8

Location of important IgE binding residues which were
conserved in the Singaporean and Italian populations on Der
p 2 in the group of patients from the group (i) sensitization
profile

142

Figure 6.9

IgE binding of the 21 single alanine mutants of Der p 2 in
individuals of group (ii) sensitization profile

144

Figure 6.10 Location of important IgE binding residues which were
conserved in the Singaporean and Italian populations on Der
p 2 in patients from the groups (ii) and (iii) sensitization
profiles

145

Figure 6.11 IgE binding of the 21 single alanine mutants of Der p 2 in
individuals of group (iii) sensitization profile

146

Figure 6.12 IgE binding of the 21 single alanine mutants of Der p 2 in the
Singapore population in the group (iv) sensitization profile,
with patients who show IgE binding to Der p 2 and Blo t 2,
but not NPC2

148

xiii


Figure 6.13 Size exclusion chromatography elution profiles and predicted
structures of WT Der p 2 and E102A mutant of Der p 2

152

Figure 6.14 Circular dichroism (CD) spectra of WT Der p 2 and alanine
mutants of Der p 2

154

Figure 7.1

Amount of IgE binding of 5 allergic individuals to Der p 2,
hNPC2, Blo t 2 and selected site directed mutants of Der p 2

161

Figure 7.2

Inhibition of IgE antibody binding from allergic patients
between WT Der p 2 and mutant E102A

165

Figure 7.3

Inhibition of IgE antibody binding from allergic patients
between WT Der p 2 and unfolded Der p 2

166

Figure 7.4

Percentage of inhibition of human serum IgE binding to WT
Der p 2 after preincubation with immunized mouse serum

170171

Figure 7.5

PBMC proliferation induced by WT Der p 2, mutant E102A
or unfolded Der p 2

173

Figure 7.6

Profile of cytokine secretion induced by WT Der p 2, mutant
E102A or unfolded Der p 2

174

xiv


List of tables

Table 1.1

House dust mite allergens

8

Table 2.1

Parameters used for Autodock

32

Table 3.1

The number of patiets showing IgE reaction to each
recombinant group 2 allergen from the Singaporean and
Italian dust mite positive patients

51

Table 3.2

Correlation of amount of IgE binding to group 2 allergens in 53
the 116 dust mite allergic patients from Singapore

Table 3.3

Correlation of amount of IgE binding to group 2 allergens in 58
the 85 dust mite allergic patients from Italy

Table 3.4

Nucleotide and amino acid changes in isoforms of Blo t 2

67

Table 6.1

List of amino acid residues selected for epitope mapping

130

Table 6.2

Summary of IgE reactions to dust mite allergic individuals
based on population and sensitization groups

150

Table 7.1

Site directed mutants with <75% IgE binding compared to
WT Der p 2 in 5 selected dust mite allergic individuals

163

Table 7.2

Inhibition of IgE binding to immobilized WT Der p 2, by
the addition of mutant E102A

165

Table 7.3

Inhibition of IgE binding to immobilized WT Der p 2, by
the addition of unfolded Der p 2

166

Table 7.4

Skin prick test for wild type Der p 2, mutant E102A or
unfolded Der p 2

168

xv


List of abbreviations

Chemical and reagents
BCIP

5-bromo-4-chloro-3-indolyl phosphate

BrdU

bromodeoxyuridine

BSA

bovine serum albumin

DIG

digitoxigenin

DTT

dithiothreitol

EDTA

ethylenediaminetetraacetic acid

FBS

fecal bovine serum

HRP

horse radish peroxidase

IPTG

isopropyl-β-thiogalactopyranoside

NBT

nitroblue tetrazolium

PBS

phosphate-buffered saline

POPC

palmitoyloleoylphosphatidylcholine

POPE

palmitoyloleoylphosphatidylethanolamine

POPG

palmitoyloleoylphosphatidylglycerol

SDS

Sodium dodecylsulphate

SDS-PAGE

Sodium dodecylsulphate polyacrylamide gel electrophoresis

Units and Measurements
bp

base pair

kb

kilo base pair

kDa

kilo Dalton

hr

hour
xvi


IU

international unit

M

Molar

min

minute

OD

optical density

pH

abbreviation of "potential of hydrogen"

rpm

revolutions per minute

sec

second

V

Volt

(w/w)

weight: weight ratio

Others
3D

three dimensional

APC

antigen presenting cell

A. ovatus

Aleuroglyphus ovatus

BLAST

Basic Local Alignment Search Tool

B. tropicalis

Blomia tropicalis

CD

circular dichroism

cDNA

complementary deoxyribonucleic acid

DNA

deoxyribonucleic acid

D. farinae

Dermatophagoides farinae

D. pteronyssinus

Dermatophagoides pteronyssinus

Ek/LIC

enterokinase/ ligation independent cloning

ELISA

enzyme linked immunosorbant assay

EST

expressed sequence tag

GM-CSF

granulocyte-macrophage colony-stimulating factor

xvii


GST

glutathione-S-transferase

G. domesticus

Glycyphagus domesticus

hNPC2

human NPC2

IFN

interferon

IgE

immunoglobulin E

IgG1

immunoglobulin G, class 1

IgG4

immunoglobulin G, class 4

IL

interleukin

L. destructor

Lepidoglyphus destructor

mRNA

messenger ribonucleic acid

MHC

major histocompatibility complex

ML

MD2- related lipid domain

MW

molecular weight

n

native

NCBI

National Center for Biotechnology Information

NMR

nuclear magnetic resonance

NPC2

Niemann Pick protein type C2

ORF

open reading frame

PBMC

peripheral blood mononuclear cells

PBS

phosphate buffered saline

PBS-T

phosphate buffered saline – Tween20

PCR

polymerase chain reaction

PDB

Protein Data Bank

pI

isoelectric point

r

recombinant

xviii

RACE

random amplification of cDNA ends

RAST

radioallergosorbent test

RNA

ribodeoxyribonucleic acid

RT-PCR

reverse transcription- polymerase chain reaction

spp.

species

S. medanensis

Suidasia medanensi

SIT

Specific immunotherapys

TCR

T cell receptor

Th

T-helper cell

TNF

tumor necrosis factor

T. putrescentiae

Tyrophagus putrescentiae

WHO/ IUIS

World Health Organization/ International Union of
Immunologic Societies Subcommittee

WT

wild type

xix


Summary

Group 2 allergens from dust mites cause allergies in >60% of dust mite
sensitized individuals. Der p 2 was the most allergenic of the eight group 2 allergens

tested in individuals from two populations (Singaporean and Italian). Following this
observation, the IgE epitopes of Der p 2 were characterized in the same populations
using single alanine site directed mutants of Der p 2. Three mutants (E25A, E102A
and K96A) showing consistent reduced IgE reactions compared to wild type Der p 2
were then evaluated for their efficacy as hypoallergen vaccine candidates. Mutants
E102A and K96A (which resulted in an unfolded protein) were potential hypoallergen
vaccine candidates as they demonstrated reduced IgE reaction, no skin prick reactivity,
the ability to elicit blocking IgG antibodies and stimulation of T cell proliferation.
The biochemical function(s) of group 2 allergens are unknown to date. To aid
the elucidation of their function, the ligand of Der p 2 was characterized. Structural
analysis of Der p 2 and close homologues indicate a hydrophobic cavity with potential
for lipid binding. Using biochemical assays, cholesterol is the preffered ligand of Der
p 2 among 11 structurally similar sterols and other lipids, including glycerolipids,
glycerophospholipids and sphingiolipids. Cholesterol was also found in the lipid
extract of native and recombinant Der p 2 using tandem mass spectrometry. Site
directed mutagenesis of selected amino acid residues lining the cavity of Der p 2 was
used to investigate their role in cholesterol binding. Alanine mutation of eleven amino
acid residues lining the cavity of Der p 2 did not show a significant change in
cholesterol binding when compared to wild type Der p 2. In silico docking studies
showed multiple binding orientations of cholesterol within the cavity of Der p 2,
suggesting promiscuity in lipid recognition.
xx


In addition, a new allergen, Der f 22 was isolated and characterized. This
allergen showed 32% amino acid sequence identity to the group 2 allergen from D.
farinae, Der f 2. The full length sequence of Der f 22 coded for 155 amino acids, with
a 20 amino acid signal peptide, and 6 cystein residues. Both Der f 2 and Der f 22
belong to the MD-2 related lipid recognition domain family, and was shown to bind to
cholesterol at equal intensities. Der f 22 and Der f 2 have similar gene organization

(one intron and two exons). Both Der f 22 and Der f 2 genes are present as single
copy genes as shown by Southern Blot analysis. Fifty percent of the dust mite allergic
individuals showed IgE reactivity to Der f 22, and these reactions were not cross
reactive with that of Der f 2. The low sequence identity, but functional similarities
(staining at gut region of D. farinae and cholesterol binding) between Der f 22 and
Der f 2 suggest that these allergens may be paralogous.

xxi


Chapter 1: Introduction

1.1 Literature Review

1.1.1 Allergy

Allergy affects more than 25% of the world population and the prevalence of
this disease increases annually (Casolaro et al., 1996; Walker and Zuany-Amorim,
2001). Allergic rhinitis, asthma, conjunctivitis, dermatitis and anaphylactic shock are
examples of immediate symptoms resulting from allergic reactions (Beaven and
Metzger, 1993; Turner and Kinet, 1999). An estimated 100 – 150 million people
suffer from allergic asthma, and 180 000 die from this disease annually (Sly, 1999). In
terms of economics, approximately US$12.7 billion is spent on medical expenditure
for treatment of asthma annually (Weiss and Sullivan, 2001).
Patients having allergies are characterized by increased IgE production upon
exposure to normally non-harmful antigens from dust mites, food, fungi, pollen grains
and animal dander (Kay, 1997). Although the causes of allergies are not known,
atopic individuals (persons with allergic heredity) are at a higher risk of sensitization
and allergic diseases (Casolaro et al., 1996). According to the classification by
Coombs and Gell allergy isa type I hypersensitivity reaction. Most allergens are

proteins (or glycoproteins), usually 5-80 kDa in size, and are highly immunogeic
(Valenta and Kraft, 2001). The immediate symptoms of allergies are caused by the
cross-linking of the IgE antibodies which are bound to mast cells upon allergen

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exposure, resulting in the release of inflammatory mediators, such as histamine and
leukotriene (Beaven and Metzger, 1993; Turner and Kinet, 1999).
In atopic individuals, the first step in IgE mediated allergic disease is
sensitization (Figure 1.1). After initial allergen exposure, the antigen presenting cells
(APC) process and present the fragmented allergen peptides on their MHC (major
histocompatibility complex) II molecules. The allergen fragments are then recognized
by the T- cell receptor (TCR), in the context of MHC II. T-helper cells (Th) can be
classified into two populations, based on the cytokines that they produce (Romagnani,
1991). Th1 cells produce interleukin 2 (IL-2), tumor necrosis factor-β (TNF-β) and
interferon-γ (IFN-γ), while Th2 cells produce IL-4, IL-5 and IL-13 cytokines. IL-4
and IL-13 promote Ig isotype switching from IgG to IgE in B-cells (Pene et al., 1988;
Punnonen et al., 1993) and IL-5 plays an important role in the growth, differentiation
and recruitment of eosinophils to the site of allergic reaction (Romagnani, 1991). B
cells secrete IgE which then binds to the high affinity FсεRI receptor on mast cells.
Re-exposure to the allergen leads to binding to and cross-linking of allergen specific
IgE on the surface of the mast cell. This results in the degranulation of mast cells,
releasing pre-formed inflammatory mediators such as histamine, leukotrine and
cytokines (IL-4, IL-5 and IL-13) (Kinet, 1999) which initiates the early phase allergic
reaction that appears within minutes of allergen exposure. Examples of the resulting
symptoms of allergies are asthma, rhinitis and conjunctivitis (Valenta, 2002). The late
phase reaction occurs 2-24 hours after allergen exposure, and is caused by the
proliferation of allergen-activated Th2 cells, releasing pro-inflammatory cytokines
that enhance eosinophil recruitment. Mediators produced by eosinophils such as

major basic protein, eosinophil cationic protein and leukotrienes promote tissue
damage (Dombrowicz and Capron, 2001).

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Figure 1.1 A simplified overview of the allergic reaction. Antigen presenting cell
(APC) recognizes processes and presents the allergen as on its MHC class II receptor.
The allergen fragment is then recognized by the T cell receptor (TCR) in the context
of MHC II. This drives the T cell to mature to Th2 cells, producing cytokines that
stimulate IgE production, or eosinophil recruitment. Cross linking of IgE on mast
cells causes degranulation leading to immediate hypersensitivity.

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