EXPRESSED SEQUENCE TAGS ANALYSIS OF
MAJOR ALLERGENS PRODUCING DUST MITES
AND MOLECULAR CHARACTERIZATION
OF THEIR ALLERGENS








ONG SEOW THENG
(B. Sc. (Hons.), UPM)






A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF PAEDIATRICS
NATIONAL UNIVERSITY OF SINGAPORE
2003
PDF created with pdfFactory Pro trial version
www.pdffactory.com

i

Acknowledgements

My gratitude goes to my supervisor Dr. Chew Fook Tim and co-supervisor Dr.
Lim Saw Hoon for always being encouraging, supportive, and optimistic. I also
appreciate that you always put trust in my abilities and given me a lots of flexibilities
in pursuing the research work. I would also like to specially thank Dr. Chew for the
invaluable recommendations, ideas, guidance and perseverance that you have provided
that shaped this project.

I am greatly indebted to Dr. Shang Huishen, for his tremendous support and
generous sharing of his expertise in molecular biology, for always being optimistic and
calm, especially in times when things are difficult. I owe a big thank-you to Dr. Bi
Xuezhi, for sharing his excellent experience in protein work and his prompt assistance
whenever the computers are not working properly.

I wish to thank everyone in Dr. Chew’s and Dr. Lim’s lab, past and present, for
making my stay in the laboratory a pleasant one. Thanks especially to Aaron, for
always being there whenever we need your help and amazing us by offering better
solutions than we can expect. Xiaoshan, I owe you for your tremendous help in the
statistical analyses. Fei Ling, Tan Ching, Yang Rui, Wun Long, Kuee Theng, Sook
Mei, Teng Nging and Yun Feng, I really appreciate your concern, encouragements and
fruitful discussions. Hai Sim, Winnie, Chye Fong, Madam Toh, Cheng Hui, Wee Kee
and Xiao Le I am grateful your warm support and guidance when I first started my
work. Dr. Adrian Loo, Dr. Li Bin, Puay Ann, Kavita, Bee Leng, Gek Huey, Resma,
PDF created with pdfFactory Pro trial version
www.pdffactory.com

ii


Lubna, Shirley, Qingqing, Hema, Saurabh, Su Yin and Kelly, thanks for your
assistance and moral supports. There are just too many people that I would like to
express my gratitudes for their concern and dedications to the completion of this work.
I may not have listed you here but you know who you are. Thank you!

Last but not least, I cannot thank my family enough for their immeasurable
love, support, patience, encouragement and every persistent effort to guide me to
where I am now. All my friends outside the lab, I thank you for reminding me of the
more important aspects of life, for friendship and for lots of fun.






















PDF created with pdfFactory Pro trial version
www.pdffactory.com

iii

Table of contents

Acknowledgements i

Table of contents iii

Summary ix

List of figures xi

List of tables xv

List of abbreviations xvi



Chapter 1: Introduction


1.1 The immune system as a defense system 1

1.2 Allergy- Type I immediate hypersensitivity 2

1.3 Mechanism of allergy 3


1.4 Immunoglobulin E

9

1.4.1 IgE and allergy 9

1.4.2 Roles of IgE in laboratory diagnosis 10

1.5 Increasing prevalence of allergy 12

1.6 House dust mites as an important cause of allergy 13

1.6.1 General aspects of mites 13

1.6.2 D. farinae as an importance source of house dust mite allergens 15

1.6.3 Cross-reactivity- a common feature especially among taxonomic
related mites 17

1.7 Allergen 19

1.7.1 General features of allergens 19

1.7.2 Mite allergens- important source of indoor allergens 20

1.7.3 Isoallergens- many allergens in nature exist in several isoforms 26
PDF created with pdfFactory Pro trial version
www.pdffactory.com


iv

1.7.4 Cross-reactivity- common feature among proteins derived from
taxonomically related organisms and/or evolutionary conserved
proteins (pan allergen) 28

1.8 Molecular biology for allergen research, diagnosis and treatment 30
1.8.1 Expression systems for recombinant allergens production 30

1.8.2 Recombinant allergens essential in for research and diagnosis 35

1.8.3 Molecular modification of recombinant proteins- for allergen
specific immunotherapy 38
1.9 Expressed Sequence Tagging - powerful approach for genome
investigations 43

1.10 Objectives of the study 48

Chapter 2: Materials and methods


2.1 Expressed Sequence Tagging (EST) approach 50

2.1.1. Bacterial host stains 50

2.1.2 D. farinae
λ
ZAP II cDNA library 50

2.1.3 Plasmid DNA isolation and sequencing 51


2.1.4 Automated DNA sequence analysis 52

2.1.5 DNA sequence analysis using software package 52

2.1.6 Sequence homology search 53

2.1.7 Cataloging of ESTs into functional categories 53

2.2 Isolation and sequencing of full length putative D. farinae allergens 54

2.2.1 Bacterial host strains for transformation 54

2.2.2 Computer-based characterization and analysis 56

2.2.3 Sequencing of full length putative allergen EST clones 56

2.2.4 Isolation of full length putative allergen clones 56

2.2.4.1 Preparation of mite total RNA 56


PDF created with pdfFactory Pro trial version
www.pdffactory.com

v

2.2.4.2 RT-PCR to isolate full length clones of known mite
allergens not being identified from EST collection 57


2.2.4.3 RACE for truncated EST clones and DNA fragments
generated from RT-PCR 57

2.2.4.4 Full-length cloning and sequence analysis 58

2.3 Sub-cloning of putative allergens into expression vectors 59

2.3.1 Ligation cloning of putative allergens into PET32a (+)
expression vector 60

2.3.2 Ligation Independent Cloning (LIC) of putative allergens into
pET32a (+) expression vector 60
2.4 Expression of putative allergen genes in E. coli 63

2.4.1 Sample induction 63

2.4.2 Affinity purification of recombinant protein with pET 32a (+)
His-Tag system 64

2.5 Immunoassays 64

2.5.1 Patient sera 64

2.5.2 Dot blot analysis 64

2.5.3 Dot blot inhibition assay 65

2.5.4 Measurement and calculation of the dot blot and inhibition results 66

2.5.5 Western blot analysis 67


2.5.5.1 Sodium Dodecyl Sulphate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE) 67

2.5.5.2 Western blot 68

2.5.5.3 Western blot inhibition 69

2.6 Generation of mite FABP homologues deletion mutants 69

2.6.1 PCR amplification of deletion mutants for epitope localizations 69

2.6.2 Expressions of deletion mutants 71

2.6.3 Immuno-dot blots and Western blots on deletion mutants 71
PDF created with pdfFactory Pro trial version
www.pdffactory.com

vi

2.7 Statistical analyses 72

2.8 Comparative molecular modeling 72

2.9 Southern blot analysis 73

2.9.1 Genomic extraction 73

2.9.2 Digestion and electrophoresis of genomic DNA 74


2.9.3 Labeling of DNA probes 74

2.9.5 Hybridization and immunological detection 74

Chapter 3: Results and discussion

3.1 EST database generate information on dust mite genome 76

3.1.1 D. farinae cDNA library construction 76

3.1.2 Allergen avoidance strategies 78

3.1.3 Analysis of EST sequences 78

3.1.4 Contig Assembly and unigenes identification 81

3.1.5 Estimating library redundancy 84

3.1.6 Submission of ESTs to GenBank 86

3.1.7 Homology search for ESTs 86

3.1.8 Biological function assignments of ESTs based on
sequence homology 89

3.1.9 EST as tool for rapid identification and characterization
of dust mite genes 92

3.2 EST databases aid in dust mite allergy research 94


3.2.1 Identification of allergen homologues in D. farinae EST collection 94

3.2.2 ESTs matching to allergen homologues from various sources 98
3.2.3
3.2.3 Assessment of Allergen Isoforms and Gene Families 99

3.2.4 Allergen not identified from EST collections 101

PDF created with pdfFactory Pro trial version
www.pdffactory.com

vii

3.2.5 Identification of Non-Allergen Homologues from EST database 101

3.2.6 EST catalogue can be utilized for expression profiling 102

3.2.7 ESTs as a means of facilitating proteomic studies and
characterizations 103

3.2.9 EST approach has successfully identified most if not all of
the dust mite allergenic protein homologues 104

3.3 Escherichia coli as the choice for expression of recombinant
allergen homologues 106

3.3.1 General analysis of cDNA inserts 106

3.3.2 Expression vector pET 32a (+) as choice for recombinant
protein expression 107


3.3.3 Protein profiles of recombinant allergens 107

3.4 Immuno-characterizations of allergenic homologues cloned from
D. farinae EST collections 111

3.4.1 Prevalence of dust mites in Singapore and Italy 111

3.4.2 Overview of IgE binding reactivity of the allergen
homologues cloned 113

3.4.3 IgE profiles of ESTs homologue to mite allergens reported
previously 124

3.4.3.1 Group 2 allergen 124

3.4.3.3 Group 5 and Group 10 allergens 126

3.4.3.4 Group 8 allergens 130

3.4.3.5 Group 14 allergen 133
3.4.3.6 Mag 29 allergen 137
3.4.3.7 Group 3, 6 and 9 allergens 140
3.4.4 Isoallergen homologues for group 7 and group 13 allergen
identified in D. farinae EST collection 151

3.4.4.1 Isoallergens in dust mite allergy 151


PDF created with pdfFactory Pro trial version www.pdffactory.com


viii

3.4.4.2 Group 7 allergen and its isoallergen homologue showed
significant difference in IgE binding reactivity 152

3.4.4.3 Fatty acid binding protein and its isoallergen homologues
showed significant difference in IgE binding reactivity 163

3.4.5 Allergen homologues other than mite origin- potential pan
allergens 194

3.4.5.1 Mal f 6 homologue in D. farinae EST collection 194

3.4.5.2 Gal d 2 homologue in D. farinae EST collection 197

3.4.5.4 Alt a 10 homologue in D. farinae EST collection 206
3.4.6 Allergen homologues not expressed 209

3.5 IgE binding reactivity to allergen homologues can be classified
into 5 categories based on IgE binding frequency and magnitude 209

Chapter 4: Conclusion and future prospects

4.1 EST catalogue generates information on dust mite genome 212

4.2 Different IgE binding patterns detected from various mite allergens 212

4.3 IgE cross-reactivity profiles of Group 7, serine proteases, FABP
and Gal d 2 allergen homologues 213


4.4 Epitope analysis of FABP homologues by deletion mutants indicating
the presence of a conserved immuno-dominant epitope 214

4.5 General discussion and perspectives 215

References
219

Appendix 279





PDF created with pdfFactory Pro trial version
www.pdffactory.com


ix

Summary
The prevalence of asthma and allergy is increasing and negatively impact the quality of
life. More than 90% of the allergic individuals are sensitized to dust mites, especially
the Dermatophagoides pteronyssinus and D. farinae in the temperate regions, and
Blomia tropicalis in the tropics. Expressed Sequence Tagging (EST) is a useful
approach for genome level transcript profiling. EST database of D. farinae was
generated and the database generated was compared to a parallel study of B. tropicalis.
More than 50% of the ESTs matched a known protein in the GenBank, in which most
of them belong to functional groups related to the general house keeping proteins, such

as metabolism, gene expression and protein synthesis.

About 4% (B. tropicalis) and 6% (D. farinae) of the ESTs encode for more
than 20 distinct allergen homologues identified from mites and other sources. A total
of 20 allergen homologues including their isoforms were cloned and expressed in
Escherichia coli. The IgE binding reactivity of the recombinant proteins were tested
using 55 Singaporean and 42 Italian sera which have reacted to D. farinae crude
extracts. Majority of the known dust mite allergens reacted to the similar patterns as
their homologues in various dust mite species reported previously. No significant
difference was detected between the Singaporean and Italian populations for majority
of the recombinant proteins in terms of IgE binding frequency. Group 2, 3, 5, 6, 7, 10,
13 and Mag 1 allergens are important allergenic proteins since they either bind IgE at
high frequency and/or bind IgE at high magnitude.

PDF created with pdfFactory Pro trial version
www.pdffactory.com

x

The rest of the proteins could also be unique and important to a specific
individual. Allergenic proteins with house keeping functions such as tropomyosin,
proteases, heat shock proteins, arginine kinase are normally abundant and can be easily
identified from various organisms crossing taxonomic kingdoms, justifying its
potential to be important pan allergens. Moreover, the newly identified allergen
homologues from fungus, yeast and food also show 30-42% of IgE binding reactivity
in dust mite allergic sera.

IgE binding and cross-reactivity of group 7 and 13 isoallergens have been
studied. IgE binding reactivity of group 7 isoform, DF167 is postulated to be largely
attributed to the shared epitopes that most probably derived from sequence and/or

structural similarity to Der f 7 (DF47) and other group 7 allergens. Southern analyses
indicate that the isoforms of group 13 allergen could be members of the multigenic
family of fatty acid binding protein (FABP). Different IgE binding rates were observed
for all the 5 FABP isoallergens, ranging from 6%-55% and these isoallergens also
demonstrate some degree of cross-reactivity. Deletion studies and molecular modeling
of the group 13 isoallergens suggest the presence of immuno-dominant epitope(s)
within the FABP N-terminal region and/or this region is important for maintaining the
proper protein folding.

Understanding the allergenic components in mites is essential for effective
diagnosis and immunotherapy. EST approach is indeed a rapid and efficient way of
isolating allergenic components for which the sequence and polymorphic variants of
the proteins can be characterized and cloned for further studies.
PDF created with pdfFactory Pro trial version
www.pdffactory.com

xi

List of figures


Figure 1: Allergy mechanism: sensitization; immediate reaction; and late
reaction. 5

Figure 2: T
R
and NKT cells regulate the differentiation and function of T
H
1
cells and T

H
2 cells. 8

Figure 3: Taxonomic distribution of common dust mites. 16

Figure 4: An outline of the generation of EST collection. 44

Figure 5: Redundancy rate of ESTs generated for D. farinae and B. tropicalis. 85

Figure 6: Determination of E-value as cut-off point using first 1000 ESTs
generated. 88

Figure 7: Classification of 1722 and 1432 ESTs from the D. farinae and B.
tropicalis cDNA libraries, respectively. 91

Figure 8:
Comparison of the highly redundant genes between the two dust
mites cDNA libraries. 93

Figure 9: Coomassie brilliant blue-stained 15% SDS-PAGE of purified
recombinant allergens incorporated with fusion tags. 110

Figure 10: Representative IgE dot blots of 43 positive sera and 7 negative sera
of 2 geographical distinct populations on the recombinant allergens. 116

Figure 11: Percentage of IgE binding frequency and IgE binding strength
to D. farinae, D. pteronyssinus and B. tropicalis recombinant
allergens of Singaporean patients allergic to D. farinae. 117

Figure 12: Percentage of IgE binding frequency and IgE binding strength

to D. farinae, D. pteronyssinus and B. tropicalis recombinant
allergens of Italian patients allergic to D. farinae. 118

Figure 13: Comparison of the sensitization to D. farinae, D. pteronyssinus
and B. tropicalis recombinant allergens between the Singaporean
and Italian populations allergic to D. farinae. 119

Figure 14: Western blots of 4 individual sera on D. farinae recombinant
allergens. 120

Figure 15: Multiple sequences alignment of group 2 mite allergens. 125

PDF created with pdfFactory Pro trial version
www.pdffactory.com

xii

Figure 16:
Multiple sequences alignment of Der f 5, Der p 5, Blo t 5 and
Lep d 5. 129

Figure 17: Multiple sequences alignment of Der f 8 and Der p 8. 131

Figure 18: Nucleotide and deduced amino acid sequence of the DF1894. 132

Figure 19: Multiple sequences alignment of the mite group 14 and Mag 1
allergens. 135

Figure 20: A magnified version of multiple sequences alignment of the mite
group 14 and Mag 1 allergens. 136


Figure 21: Multiple sequences alignment of the D. farinae Mag 29 allergens. 139

Figure 22: Multiple sequences alignment of 18 N-terminal amino acid residues
of the mature protein of Der p 9, Der f 9 and Blo t 9 allergens. 142

Figure 23: Multiple sequences alignment of Der f 9, Der p 9 and Blo t 9
allergens. 143

Figure 24: IgE binding reactivity of Der f 9, Der p 9 and Blo t 9 tested by
55 Singaporean sera and 42 Italian sera that are sensitized to
D. farinaecrude extract. 145

Figure 25: Multiple sequences alignment of Der f 3 and the newly cloned
isoform of Der f 3 (Der f 3-RTPCR). 147

Figure 26: Multiple sequences alignment of Der f 3, Der f 6 and Der f 9. 149

Figure 27:
Venn diagram showing IgE binding of a total of 97 D. farinae
sensitized sera to Der f 3, Der f 6 and Der f 9 allergens. 150

Figure 28: Amino acid sequence alignment between DF47 and Der f 7. 154

Figure 29: Amino acid sequence alignment between DF47 and DF167. 155

Figure 30: Multiple sequences alignment of the group 7 allergens, DF47,
DF167, Der f 7, Der p 7, Blo t 7 and Lep d 7. 156

Figure 31:

Phylogenetic tree was created based on distance matrix of DF47,
DF167, Der f 7, Der p 7, Blo t 7 and Lep d 7. 157

Figure 32: IgE binding reactivity of DF47 and DF167 tested by 55
Singaporean sera and 42 Italian sera that are sensitized to
D. farinae crude extract. 159

PDF created with pdfFactory Pro trial version
www.pdffactory.com

xiii

Figure 33:
Dose dependent inhibition curve of IgE immunoblot for group 7
allergens isolated from EST collection. 160

Figure 34: Inhibition of IgE binding to group 7 allergens. 162

Figure 35: Multiple sequences alignment of mites FABP homologues,
DF414, DF1096, BT796, BT1313 and BT1694. 166

Figure 36: Phylogenetic tree of all the group 13 allergens (Aca s 13, Blo t 13
and Lep d 13), mites FABP homologues (DF414, DF1096, BT796,
BT1313 and BT1694) and FABPs identified from various sources
including the nematode Sm14, Fh15, adipose FABP, brain FABP,
heart FABP, retinoic FABP (crustacean) and liver FABP. 168

Figure 37: Genomic DNA of D. farinae and B. tropicalis analyzed by
electrophoresis on a 0.8% agarose gel. 171


Figure 38: Restriction enzyme analysis of the cDNA sequence of DF414;
DF1096; BT796; BT1313 and BT1694. 172
Figure 39:
Southern blot analysis of mites FABP homologues. 173

Figure 40: IgE binding frequency of the Singaporean and Italian sera towards
DF414, DF1096, BT796, BT1313 and BT1694. 175
Figure 41: Venn diagram showing IgE binding to DF414 and DF1096;
DF414 and BT796; and BT796, BT1313 and BT1694. 176

Figure 42: IgE binding reactivity of mites FABP homologues tested by 55
Singaporean sera and 42 Italian sera that are sensitized to
D. farinae crude extract. 179

Figure 43:
Dose dependent inhibition curve of IgE immunoblots for mites
FABP homologues. 182

Figure 44: Inhibition of IgE binding to mites FABP homologues and group
13 allergens. 184

Figure 45: Diagrammatic presentation of the mutants generated for DF414,
DF1096, BT796, BT1313 and BT1694. 186

Figure 46:
IgE binding to FABP homologues and their deletion mutants. 187

Figure 47: Secondary structure of DF414 based on Chemical Shift Index
(CSI) calculation. 189


Figure 48: Homology models of DF414, DF1096, BT796, BT1313 and
BT1694 as predicted by SWISS MODEL. 190

PDF created with pdfFactory Pro trial version
www.pdffactory.com

xiv

Figure 49:
Box plots drawn based on densitometric index scored in

IgE
binding assay on deletion mutants of DF414, DF1096, BT796,
BT1313 and BT1694. 192

Figure 50: Multiple sequences alignment of DF Mal f 6 homologues and
Mal f 6 allergen. 296

Figure 51: Multiple sequences alignment of DF Gal d 2 homologue and
Gal d 2 allergen. 200

Figure 52: Venn diagram showing IgE binding of a total of 120 allergic
sera to DF Gal d 2 homologue and egg white; and
DF Gal d 2 homologue and ovalbumin. 201

Figure 53: Evaluation of cross-reactivity between DF Gal d 2 homologue,
ovalbumin and egg white extract. 202

Figure 54: A space-filled model of 10VA with IgE binding fragments 41-172
and 301-305 colored in green, with the rest of the atoms in grey. 204


Figure 55: Predictive homology model of DF Gal d 2 homologue was created
using the crystal structure of ovalbumin (10VA) as template. 205

Figure 56: Multiple sequences alignment of DF Alt a 10 homologue,
Alt a 10 and Cla h 3 allergens. 208





















PDF created with pdfFactory Pro trial version
www.pdffactory.com


xv

List of Tables


Table 1: IgE binding rates of the cloned dust mite allergens and their
biological properties. 22

Table 2: Overview of the productions of recombinant mite allergens:
from the aspects of the production yield, bioactivities and types
of expression systems used. 32

Table 3: Identity and percentage of homology of EST clones corresponding
to the known allergen homologues. 55

Table 4: List of primers used in RACE protocol as well as obtaining full
length clones. 58

Table 5: Synthetic oligonucleotides used in PCR to amplify the putative
allergens for pET32a (+) vector cloning. 61

Table 6: Concentrations of inhibitors used in dot blot inhibition assay. 65

Table 7:
Synthetic oligonucleotides used as PCR primers to amplify the
deletion mutants of the mites FABP homologues. 70

Table 8: Overview of the characteristics and statistical data of the
dust mites EST collections. 80


Table 9: Summary and comparisons of gene expression levels in D. farinae
and B. tropicalis cDNA libraries. 83

Table 10: Number of D. farinae and B. tropicalis EST copy number matching
to allergen homologues from dust mites and other sources. 96

Table 11: Estimated molecular weight of the expressed allergen with the
fusion protein. 109

Table 12: Profile of allergen collections of D. farinae and B. tropicalis. 114

Table 13: Percentage of amino acid sequence homology between the
mites FABP homologues. 167

Table 14: IgE reactivity profiles of the sera selected for inhibition studies. 181

Table 15: Classification of allergen homologues based on their IgE binding
rate and IgE binding strength according to the populations tested. 211
PDF created with pdfFactory Pro trial version
www.pdffactory.com

xvi

List of abbreviations

Chemical and reagents

BCIP 5-bromo-4-chloro-3-indolyl phosphate
BSA bovine serum albumin
dH

2
0 distilled water
EDTA ethylenediaminetetraacetic acid
IPTG isopropyl-β-thiogalactopyranoside
NBT nitroblue tetrazolium
PBS phosphate-buffered saline
PVDF polyvinyldiflouride
SDS Sodium dodecylsulphate
SDS-PAGE Sodium dodecylsulphate polyacrylamide gel electrophoresis
SSC saline-sodium citrate
TEMED N,N,N,N,-tetramethylethylenediamine
TE tris-EDTA
Tris Tris (hydroxymethyl)-aminomenthane

Units and Measurements

bp base pair
kb kilo base pair
kDa kilo Dalton
PDF created with pdfFactory Pro trial version
www.pdffactory.com

xvii

IU international unit
M Molar
min minute
pH abbreviation of "potential of hydrogen"
rpm round per minute
sec second

V Volt
(v/v) volume: volume ratio
(w/v) weight: volume ratio

Others
AAAAI American Academy of Allergy, Asthma & Immunology, Inc
ACR Ancient Conserved Regions
APC Antigen Presenting Cell
BLAST Basic Local Alignment Search Tool
CD spectra circular dichroism spectra
cDNA complementary deoxyribonucleic acid
cLTP cytosolic lipid transfer transport proteins
COI Cytochrome Oxidase subunit I
CSI Chemical Shift Index
DNA deoxyribonucleic acid
dbEST GenBank database of Expressed Sequence Tags
dsDNA double stranded deoxyribonucleic acid
EMBL European Molecular Biology Laboratory
PDF created with pdfFactory Pro trial version
www.pdffactory.com

xviii

EST expressed sequence tag
FABP fatty acid binding protein
Fc R Fc receptors
GM-CSF granulocyte-macrophage colony-stimulating factor
GST glutathione-S-transferase
HPLC High Performance Liquid Chromatography
IFN interferon

IgE immunoglobulin E
IgG
1
immunoglobulin G, class 1

IgG
4
immunoglobulin G, class 4
IgM immunoglobulin M
IL interleukin
ISAAC International Study of Asthma and Allergies in Childhood
IUIS/WHO International Union of Immunologic Societies Subcommittee/
World Health Organization
LIC ligation independent cloning
MALDI-TOF Matrix-Assisted Laser Desorption/Ionization- Time of Flight
mRNA messenger ribonucleic acid
MHC major histocompatibility complex
MW molecular weight
NCBI National Center for Biotechnology Information
NIH National Institution of Health
NLM National Library of Medicine
NMR Nuclear Magnetic Resonance Spectroscopy
ORF open reading frame
PDF created with pdfFactory Pro trial version
www.pdffactory.com

xix

PCR polymerase chain reaction
PDB Protein Data Bank

pET expression vector (Novagen)
pI isoelectric point
r recombinant
RACE Rapid amplification cDNA ends
RAST radioallergosorbent Test
RNA ribodeoxyribonucleic acid
RT-PCR reverse transcription- polymerase chain reaction
spp. species
TCR T cell receptor

T
C
cytotoxic T cell

T
H
T-helper cell
TNF tumor necrosis factor
WHO World Health Organization









PDF created with pdfFactory Pro trial version
www.pdffactory.com



1
Chapter 1: Introduction

1.1 The immune system as a defense system

One of the most easily understood functions of the immune system is its role in
protecting against foreign antigens. The immune responses fall into 2 broad categories,
the innate and adaptive immunities. The innate immune responses act as first line of
defense and are mediated by the phagocytic cells such as the monocytes, macrophages
and neutrophils. They engulf, present and initiate inflammatory responses using non-
specific recognition systems. The important difference between innate and adaptive
immunity is that the latter is highly specific for a particular antigen. The adaptive
immune system also “remembers” the antigen and will respond more vigorously on
second exposure.

Lymphocytes are central to all adaptive immune responses and these cells fall
into 2 basic categories, the T cells and B cells. The antigen specificity of the immune
system is due to both the B cell receptor and the T cell receptor (TCR) for antigens.
The antigen receptor for B cell is a membrane bound IgM molecule. Having
recognized its specific antigen, B cells proliferate and differentiate into plasma cells,
which produce large amounts of antibodies that activate other parts of the immune
system. T cells come in several different types and they have a variety of functions. T
helper (T
H
) cells carry the CD4 marker and recognize their specific antigens in
association with major histocompatibility complex (MHC) class II molecules. T
H
cells

are subdivided into T
H
1 cells that mediate cytotoxicity and local inflammatory
reactions by secreting interleukin (IL)-2, tumour necrosis factor (TNF)-
β
and
PDF created with pdfFactory Pro trial version
www.pdffactory.com


2
interferon (IFN)-
γ
; while T
H
2 cells on the other hand produce cytokines such as IL-4,
IL-5, IL-13 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and are
more effective at stimulating B cells to proliferate and produce antibodies (Romagnani,
1991). Another type of T cells, the cytotoxic T (T
C
) cells carry CD8 marker and
recognize antigens in association with MHC class I molecules, and are responsible for
destruction of host cells which have been infected.

The B cell receptor binds antigen directly whereas the TCR recognizes antigen-
derived processed peptides in the context of MHC molecules that are present on
antigen presenting cells (APC). The professional APC includes B cells, dendritic cells
and monocyte/macrophages.

1.2 Allergy- Type I immediate hypersensitivity


When an individual has been immunologically primed, further contact with antigen
will lead to secondary boosting of the immune response. However, the reaction may be
excessive and lead to gross tissue changes (hypersensitivity) if the antigen is present in
relatively large amounts or if the humoral and cellular immune state is at a heightened
level. It should be emphasized that the mechanisms underlying these inappropriate
reactions are those normally employed by the body in combating infections (Roitt,
1997). Coombs and Gell (1975) described 4 types of hypersensitivity reaction, to
which a fifth, viz. “stimulatory” was added later. Types I, II, III and V hypersensitivity
are antibody-mediated; the fourth is primarily mediated by T-cells and macrophages.
PDF created with pdfFactory Pro trial version
www.pdffactory.com


3
Allergy is commonly regarded as type I or immediate hypersensitivity, in which IgE
antibody responses to the allergen.

1.3 Mechanism of allergy

As illustrated in figure 1, when an innocuous environmental antigen is taken up by a
local APC, the antigen is processed and presented as antigenic peptides to a T helper
cell. Non-atopic populations will mount a low grade immunological response and
produce allergen specific IgG
1
and IgG
4
antibodies (Kemeny et al., 1989). Their T
cells respond to the antigen with a modest degree of proliferation and production of
IFN-

γ
which is typical of T
H
1 cells (Romagnani, 1991; Ebner et al., 1995; Till et al.,
1997). It is well known that IFN-
γ
inhibits the effects of IL-4 (Pène et al., 1988) thus
inhibits the development of T
H
2 cells. Atopic individuals, by contrast, build up
exaggerated allergen specific IgE response, a typical type I hypersensitivity response
to an antigen. T
H
2 cells seem to be preferentially stimulated by the APCs, resulting in
the release of IL-4 and IL-13 which promote the isotype switch of B cells to IgE
production (Pène et al., 1988; Finkelman et al., 1988; Punnonen et al., 1993; Emson et
al., 1998; Till et al., 1997) and IL-5 which is required for eosinophil growth and
enhances basophil histamine release (Weller, 1992; Hogan et al., 1998).

Secreted IgE is then bound to the high-affinity receptors, FcεRI on mast cells
and/or basophils. Upon re-exposure to allergen, cross-linking of allergen specific IgE
occurs. This causes mast cell (and basophil) degranulation and the release of
preformed mediators (histamine, tryptase, and heparin), along with newly synthesized
PDF created with pdfFactory Pro trial version
www.pdffactory.com


4
mediators (leukotrienes, and cytokines IL-4, IL-5 and IL-13) (Kinet, 1999). Immediate
hypersensitivity reactions are then developed and the symptoms include weal and flare

eruptions on the skin, itching, coughing, wheezing, sneezing, blocked nose, watery
eyes, and more serious conditions such as asthma and anaphylaxis (The Allergy
Report, 2000).

Late reactions begin 2 to 4 hours after exposure to allergen and can last for 24
hours before subsiding. This is mainly caused by the presentation of allergens to T
H
2
cells, which become activated, proliferate and release proinflammatory cytokines that
promote IgE production, eosinophil chemoattraction and increased numbers of
mucosal mast cells. Inflammatory leukocytes (e.g., neutrophils, basophils, eosinophils)
are involved but the late response is primarily mediated by eosinophils and mast cells
in atopic individuals. Eosinophils produce mediators (e.g. major basic protein,
eosinophil cationic protein and leukotrienes) that promote tissue damage associated
with chronic allergic reactions (Dombrowicz and Capron, 2001; The Allergy Report,
2000).








PDF created with pdfFactory Pro trial version
www.pdffactory.com


5
Figure 1:

Allergy mechanism: (a) sensitization; (b) immediate reaction; and (c) late
reaction (adapted from Valenta, 1999).








PDF created with pdfFactory Pro trial version www.pdffactory.com