GROUP ALLERGENS FROM DUST MITE: EPITOPE MAPPING AND FUNCTIONAL CHARACTERIZATION OF DER P 2, AND IDENTIFICATION OF A PARALOGUE OF DER F 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 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 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 was done together with Kway Kwee Theng Quantification of allergen concentration in dust samples was done with Kelly Goh Extraction of native Blo t 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 Allergens In: The 60th American Academy of Allergy and Immunology Annual Meeting, - 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, - 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 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 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 Literature Review 1.1.1 Allergy 1.1.2 Dust mites 1.1.3 Dust mite allergens 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 using degenerate primers 16 2.1.3 DNA sequencing 17 v 2.1.4 Isolation of Blo t isoforms 18 2.1.5 Isolation of the genomic DNA encoding for Der f 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 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 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 31 2.6 Lipid assays 2.6.1 Liposome preparation 33 33 2.6.2 Detection of liposome binding to Der p by liposome sedimentation and SDS-PAGE 33 2.6.3 Lipid ELISA 34 2.6.4 Extraction of lipid fraction from Der p 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 allergens 37 3.1 Introduction 37 3.2 Cloning and sequence analysis of Ale o 2, Sui m and Blo t 39 3.3 IgE reactivity to group allergens 50 3.3.1 IgE reactivity to group allergens in the Singaporean population 50 3.3.2 IgE reactivity to group allergens in the Italian population 56 3.4 Further characterization of Blo t 61 3.4.1 Isolation of native Blo t 61 3.4.2 Histamine release of Blo t 61 3.4.3 Blo t is present in the environmental dust samples 64 3.4.4 Isoforms of Blo t 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 83 4.4 Southern blot analysis 86 4.5 IgE binding capacities of Der f 22 and Der f 88 4.6 Localization of Der f 22 and Der f on sectioned D farinae 92 4.7 Concentration of Der f 22 and Der f in the indoor environment 94 4.8 Der f and Der f 22 binds to cholesterol 96 4.9 Presence of paralogues of group allergens 98 4.10 Discussion 101 Chapter 5: Der p is a cholesterol binding protein 105 5.1 Introduction 105 5.2 Der p binding to liposomes 107 5.3 Binding of Der p to purified lipids 109 5.4 Analysis of lipid extracts from nDer p and rDer p 112 5.5 Characterization of potential cholesterol binding sites in Der p via site directed mutagenesis 114 5.6 Native Der p and two other allergens of the ML domain family bind to cholesterol 117 5.7 Docking of cholesterol on Der p 119 viii 5.8 Discussion 121 Chapter 6: IgE epitope mapping of Der p 125 6.1 Introduction 125 6.2 Design and production of Der p 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 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 151 6.6 Discussion 155 Chapter 7: Evaluation of hypoallergen vaccine candidates for Der p 158 7.1 Introduction 158 7.2 Specific IgE binding to site directed mutants of Der p in five allergic individuals 159 7.3 Skin Prick Test 167 7.4 Mouse IgG antibodies raised against mutant E102A and unfolded Der p are able to block allergic individuals’ IgE binding to WT Der p 169 7.5 T cell reactivity and cytokine profile 172 7.6 Discussion 175 ix Ko DC, Binkley J, Sidow A and Scott MP 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allergens in Singapore Clin Exp Allergy 27(8): 876-885 209 Appendix I List of primers A Primers for the amplification of group allergens Lepd2_BamH1(f) : 5’-CGGGATCCGGCAAGATGACCTTCAAG-3’ Lepd2_EcoR1(r): 5’-CGGAATTCTTATTCGACTTGTCCGTG-3’ Glyd2_BamH1(f): 5’-CGGGATCCGGCAAGATGAAGTTTAAG-3’ Glyd2_EcoR1(r): 5’-CGGAATTCTTATTCGACTTGCCGGCC-3’ Derf2_BamH1(f): 5’-CGGGATCCGATCAAGTCGATGTTAAAG-3’ Derf2_EcoR1(r): 5’-CGGAATTCTTAATCACGGATTTACC-3’ Derp2_BamH1(f): 5’-CGGGATCCCGTGATCAAGTCGATGTC-3’ Derp2_EcoR1(r): 5’-CGGAATTCTTAATCGCGGATTTCGC-3’ Tyrp2_BamH1(f): 5’-CGGGATCCGGCAAGGTCAAGTTCACC-3’ Tyrp2_EcoR1(r): 5’-CGGAATTCTTAGGGCTTGACGTCGGT-3’ Aleo2_BamH1(f): 5’-CGGGATCCGGTCAAGTTCGGTTCGAG-3’ Aleo2_EcoR1(r): 5’-CGGAATTCTTAGTTGGCAATGTCGCC-3’ B Primers for alanine mutants of Der p R128A_F: 5'-TGCTACTCATGCTAAAATCGCCGATTAAGAATTCGAGCTCC-3' 78.2°C R128A_R: 5'-GGAGCTCGAATTCTTAATCGGCGATTTTAGCATGAGTAGCA-3' 78.2°C H11A_F: 5'-TCGATGTCAAAGATTGTGCCAATGCCGAAATCAAAAAAGTTTTGGTACCAG-3' 78.5°C H11A_R: 5'-CTGGTACCAAAACTTTTTTGATTTCGGCATTGGCACAATCTTTGACATCGA-3' 78.5°C W92A_F: 5'-AGGACAACAATATGATATTAAATATACAGCGAATGTTCCAAAAATTGCACCAAAATC3' 78.4°C W92A_R: 5'-GATTTTGGTGCAATTTTTGGAACATTCGCTGTATATTTAATATCATATTGTTGTCCT3' 78.4°C H22A_F: 5'-AAAGTTTTGGTACCAGGATGCGCCGGTTCAGAACCATGTATCATT-3' 78.1°C H22A_R: 5'-AATGATACATGGTTCTGAACCGGCGCATCCTGGTACCAAAACTTT-3' 78.1°C K55A_F: 5'-CTCAAAAACAGCTAAAATTGAAATCGCAGCTTCAATCGATGGTTTAGAAGT3'78.8°C 210 K55A_R: 5'-ACTTCTAAACCATCGATTGAAGCTGCGATTTCAATTTTAGCTGTTTTTGAG3'78.8°C K89A_F: 5'-TGGTTAAAGGACAACAATATGATATTGCATATACATGGAATGTTCCAAAAATTGC-3' 78.3°C K89A_R: 5'-GCAATTTTTGGAACATTCCATGTATATGCAATATCATATTGTTGTCCTTTAACCA-3' 78.3°C K77A_F: 5'-TCCAAATGCATGCCATTATATGGCCTGTCCATTGGTTAAAGGACAAC-3' 78.2°C K77A_R: 5'-GTTGTCCTTTAACCAATGGACAGGCCATATAATGGCATGCATTTGGA-3' 78.2°C L61A_F: 5'-AATCAAAGCTTCAATCGATGGTGCCGAAGTTGATGTTCCCGGTAT-3' 78.1°C L61A_R: 5'-ATACCGGGAACATCAACTTCGGCACCATCGATTGAAGCTTTGATT-3' 78.1°C E25A_F: 5'-CAGGATGCCATGGTTCAGCCCCATGTATCATTCATCGTG-3' 80.1°C E25A_R: 5'-CACGATGAATGATACATGGGGCTGAACCATGGCATCCTG-3' 80.1°C Q85A_F: 5'ATGAAATGTCCATTGGTTAAAGGACAAGCCTATGATATTAAATATACATGGAATGTTCC-3' 78.2°C Q85A_R: 5'GGAACATTCCATGTATATTTAATATCATAGGCTTGTCCTTTAACCAATGGACATTTCAT-3' 78.2°C N10A_F: 5'-GTCGATGTCAAAGATTGTGCCGCCCATGAAATCAAAAAAGTTTTGGTAC-3' 78.3°C N10A_R: 5'-GTACCAAAACTTTTTTGATTTCATGGGCGGCACAATCTTTGACATCGAC-3' 78.3°C K6A_F: 5'-CGCCACCACCACTGAGAGCCGATTGTGCCAATCATGAAATC-3' 79.7°C K6A_R: 5'-GATTTCATGATTGGCACAATCGGCTCTCAGTGGTGGTGGCG-3' 79.7°C H30A_F: 5'-GGTTCAGAACCATGTATCATTGCCCGTGGTAAACCATTCCAATTG-3' 78.1°C H30A_R: 5'-CAATTGGAATGGTTTACCACGGGCAATGATACATGGTTCTGAACC-3' 78.1°C K96A_F: 5'-GATATTAAATATACATGGAATGTTCCAGCAATTGCACCAAAATCTGAAAATGTTG-3' 78.3°C K96A_R: 5'-CAACATTTTCAGATTTTGGTGCAATTGCTGGAACATTCCATGTATATTTAATATC-3' 78.3°C I97A_F: 5'-ATATTAAATATACATGGAATGTTCCAAAAGCCGCACCAAAATCTGAAAATGTTGTCG3' 78.1°C I97A_R: 5'-CGACAACATTTTCAGATTTTGGTGCGGCTTTTGGAACATTCCATGTATATTTAATAT3' 78.1°C K100A_F: 5'-GAATGTTCCAAAAATTGCACCAGCCTCTGAAAATGTTGTCGTCACTG-3' 78.2°C K100A_R: 5'-CAGTGACGACAACATTTTCAGAGGCTGGTGCAATTTTTGGAACATTC-3' 78.2°C H74A_F: 5'-GGTATCGATCCAAATGCATGCGCCTATATGAAATGTCCATTGGTTAAAG-3'78.3°C H74A_R: 5'-CTTTAACCAATGGACATTTCATATAGGCGCATGCATTTGGATCGATACC-3'78.3°C K15A_F: 5'-GTGCCAATCATGAAATCAAAGCAGTTTTGGTACCAGGATGC-3' 78.2°C K15A_R: 5'-GCATCCTGGTACCAAAACTGCTTTGATTTCATGATTGGCAC-3' 78.2°C N114A_F: 5'-GTCACTGTTAAAGTTATGGGTGATGCAGGTGTTTTGGCTTGTGCTATTG-3' 79.2°C N114A_R: 5'-CAATAGCACAAGCCAAAACACCTGCATCACCCATAACTTTAACAGTGAC-3' 79.2°C N93A_F: 5'GACAACAATATGATATTAAATATACATGGGCCGTTCCAAAAATTGCACCAAAATCTGAA-3' 78.2°C N93A_R: 5'TTCAGATTTTGGTGCAATTTTTGGAACGGCCCATGTATATTTAATATCATATTGTTGTC-3' 78.2°C 211 E102A_F: 5'-TCCAAAAATTGCACCAAAATCTGCCAATGTTGTCGTCACTGTTAAAG-3' 78.6°C E102A_R: 5'-CTTTAACAGTGACGACAACATTGGCAGATTTTGGTGCAATTTTTGGA-3' 78.6°C E62A_F: 5'-AAGCTTCAATCGATGGTTTAGCCGTTGATGTTCCCGGTATC-3' 79.2°C E62A_R: 5'-GATACCGGGAACATCAACGGCTAAACCATCGATTGAAGCTT-3' 79.2°C V110A_F: 5'-AAATGTTGTCGTCACTGTTAAAGCAATGGGTGATAATGGTGTTTTGG-3' 78.6°C V110A_R: 5'-CCAAAACACCATTATCACCCATTGCTTTAACAGTGACGACAACATTT-3' 78.6°C V108A_F: 5'-ATCTGAAAATGTTGTCGTCACTGCAAAAGTTATGGGTGATAATGGTG-3' 78.6°C V108A_R: 5'-CACCATTATCACCCATAACTTTTGCAGTGACGACAACATTTTCAGAT-3' 78.6°C V106A_F: 5'-GCACCAAAATCTGAAAATGTTGTCGCAACTGTTAAAGTTATGGGTGATAAT-3' 78.8°C V106A_R: 5'-ATTATCACCCATAACTTTAACAGTTGCGACAACATTTTCAGATTTTGGTGC-3' 78.8°C V104A_F: 5'-AAAAATTGCACCAAAATCTGAAAATGCAGTCGTCACTGTTAAAGTTATGGG-3' 78.8°C V104A_R: 5'-CCCATAACTTTAACAGTGACGACTGCATTTTCAGATTTTGGTGCAATTTTT-3' 78.8°C F41A_F: 5'-GTAAACCATTCCAATTGGAAGCTTTAGCAGAAGCCAATCAAAACTCAAAAACAGC-3' 79.4°C F41A_R: 5'-GCTGTTTTTGAGTTTTGATTGGCTTCTGCTAAAGCTTCCAATTGGAATGGTTTAC-3' 79.4°C V16A_F: 5'-GTGCCAATCATGAAATCAAAAAAGCATTGGTACCAGGATGCCATG-3' 79.4°C V16A_R: 5'-CATGGCATCCTGGTACCAATGCTTTTTTGATTTCATGATTGGCAC-3' 79.4°C Y90A_F: 5'-GGTTAAAGGACAACAATATGATATTAAAGCAACATGGAATGTTCCAAAAATTGCACC3' 78.1°C Y90A_R: 5'-GGTGCAATTTTTGGAACATTCCATGTTGCTTTAATATCATATTGTTGTCCTTTAACC3' 78.1°C L37A_F: 5'-TCATCGTGGTAAACCATTCCAAGCAGAAGCTTTATTCGAAGCCAATC-3' 78.2°C L37A_R: 5'-GATTGGCTTCGAATAAAGCTTCTGCTTGGAATGGTTTACCACGATGA-3' 78.2°C V18A_F: 5'-CATGAAATCAAAAAAGTTTTGGCACCAGGATGCCATGGTTC-3' 79.6°C V18A_R: 5'-GAACCATGGCATCCTGGTGCCAAAACTTTTTTGATTTCATG-3' 79.6°C W92A_F: 5'-AGGACAACAATATGATATTAAATATACAGCGAATGTTCCAAAAATTGCACCAAAATC3' 78.4°C W92A_R: 5'-GATTTTGGTGCAATTTTTGGAACATTCGCTGTATATTTAATATCATATTGTTGTCCT3' 78.4°C F35A_F: 5'-GTATCATTCATCGTGGTAAACCAGCACAATTGGAAGCTTTATTCGAAGC-3' 78.3°C F35A_R: 5'-GCTTCGAATAAAGCTTCCAATTGTGCTGGTTTACCACGATGAATGATAC-3' 78.3°C 212 Appendix II A B Supplementary Figure Microtome-cut D farinae sections probed with preimmune sera of rabbits which were later immunized with (A) Der f and (B) Der f 22 Sections were viewed using light microscope with 200X magnification 213 ... 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. .. 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 in dust samples 95 Figure 4.13 Binding of Der f 22 and Der. .. Cystein pairing of Der f 22 and Der f 80 Figure 4.4 Ribbon structures of Der f 22 and Der f 80 Figure 4.5 CD spectra of Der f 22 (solid line) and Der f (dashed line) 82 Figure 4.6 Location of intron