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 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, i PDF created with pdfFactory Pro trial version www.pdffactory.com 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. ii PDF created with pdfFactory Pro trial version www.pdffactory.com 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 1.2 Allergy- Type I immediate hypersensitivity 1.3 Mechanism of allergy 1.4 Immunoglobulin E 1.4.1 IgE and allergy 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 iii PDF created with pdfFactory Pro trial version www.pdffactory.com 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 λZAP 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 iv PDF created with pdfFactory Pro trial version www.pdffactory.com 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 2.3.1 Ligation cloning of putative allergens into PET32a (+) expression vector 59 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 v PDF created with pdfFactory Pro trial version www.pdffactory.com 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 3.2.3 3.2.3 Assessment of Allergen Isoforms and Gene Families 98 3.2.4 Allergen not identified from EST collections 99 101 vi PDF created with pdfFactory Pro trial version www.pdffactory.com 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 allergen 124 3.4.3.3 Group and Group 10 allergens 126 3.4.3.4 Group allergens 130 3.4.3.5 Group 14 allergen 133 3.4.3.6 Mag 29 allergen 137 3.4.3.7 Group 3, and allergens 140 3.4.4 Isoallergen homologues for group and group 13 allergen identified in D. farinae EST collection 3.4.4.1 Isoallergens in dust mite allergy 151 151 vii PDF created with pdfFactory Pro trial version www.pdffactory.com 3.4.4.2 Group 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 homologue in D. farinae EST collection 194 3.4.5.2 Gal d 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 3.5 IgE binding reactivity to allergen homologues can be classified into categories based on IgE binding frequency and magnitude 209 209 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 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 viii PDF created with pdfFactory Pro trial version www.pdffactory.com 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 allergens are important allergenic proteins since they either bind IgE at high frequency and/or bind IgE at high magnitude. ix PDF created with pdfFactory Pro trial version www.pdffactory.com full activities of both cysteine protease and IgE binding. Clinical and Experimental Allergy 2001 31:116-24. Yazdanbaksh M., Kremsner P.G., van Ree R. 2002. Allergy, Parasites and the Hygiene Hypothesis. Science 296: 490-494. Yi F.C., Cheong N., Shek P.C., Wang D.Y., Chua K.Y., Lee B.W. 2002. Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus. Clinical and Experimental Allergy 32: 1203-1210. Yssel H., Johnson K.E., Sscneider P.V., Wideman J., Terr A., Kastelein R., van de Vries J.E. 1992. T cell activation-inducing epitopes of the house dust mite allergen Der p 1. Proliferation and lymphokine production patterns by Der p 1specific CD4+ T cell clones. The Journal of Immunology 148:738-745. Yuuki T., Okumura Y., Okudairo H. 1997. Genomic organization and polymorphism of the major house dust mite allergen Der f 2. International Archives of Allergy and Applied Immunology 112:44-48. Zagari A., Schmidt M., Lundberg M., Scheynius A., Whitley P. 1999. Immunologic characteriation of natural and recombinant Mal f yeast allergen. Journal of Allergy and Clinical Immunology 103:877-884. Zetterstrom O. and Johansson S.G.O. 1981. IgE concentrations measured by PRIST in serum of healthy adults and in patients with respiratory allergy. A diagnostic approach. Allergy 36:537-547. Zhang L., Muradia G., De Vouge M.W., Rode H., Vijay H.M. 1996. An allergenic polypeptide representing a variable region of hsp 70 cloned from a cDNA 277 PDF created with pdfFactory Pro trial version www.pdffactory.com library of Cladosporium herbarum. Clinical and Experimental Allergy 26:8895. Zhang S.P., Zubay G., Goldman E. 1991. Low-usage codons in Escherichia coli, yeast, fruit fly and primates. Gene 105: 61–72. 278 PDF created with pdfFactory Pro trial version www.pdffactory.com Appendix dbEST_Id ======== 16991846 16991847 16991848 16991849 16991850 16991851 16991852 16991853 16991854 16991855 16991856 16991857 16991858 16991859 16991860 16991861 16991862 16991863 16991864 16991865 16991866 16991867 16991868 16991869 16991870 16991871 16991872 16991873 16991874 16991875 16991876 16991877 16991878 16991879 16991880 16991881 16991882 16991883 16991884 16991885 16991886 16991887 16991888 16991889 16991890 16991891 16991892 16991893 16991894 16991895 16991896 16991897 16991898 16991899 16991900 16991901 16991902 16991903 User_Id GenBank_Accn ======= ============ DF0002 CB283468 DF0003 CB283469 DF0004 CB283470 DF0005 CB283471 DF0006 CB283472 DF0007 CB283473 DF0008 CB283474 DF0009 CB283475 DF0010 CB283476 DF0011 CB283477 DF0012 CB283478 DF0016 CB283479 DF0017 CB283480 DF0019 CB283481 DF0020 CB283482 DF0021 CB283483 DF0022 CB283484 DF0023 CB283485 DF0024 CB283486 DF0025 CB283487 DF0026 CB283488 DF0027 CB283489 DF0029 CB283490 DF0030 CB283491 DF0031 CB283492 DF0032 CB283493 DF0033 CB283494 DF0034 CB283495 DF0035 CB283496 DF0036 CB283497 DF0037 CB283498 DF0038 CB283499 DF0039 CB283500 DF0040 CB283501 DF0041 CB283502 DF0042 CB283503 DF0044 CB283504 DF0045 CB283505 DF0046 CB283506 DF0047 CB283507 DF0048 CB283508 DF0049 CB283509 DF0050 CB283510 DF0051 CB283511 DF0052 CB283512 DF0053 CB283513 DF0054 CB283514 DF0056 CB283515 DF0057 CB283516 DF0058 CB283517 DF0059 CB283518 DF0060 CB283519 DF0062 CB283520 DF0066 CB283521 DF0067 CB283522 DF0069 CB283523 DF0070 CB283524 DF0071 CB283525 16991904 16991905 16991906 16991907 16991908 16991909 16991910 16991911 16991912 16991913 16991914 16991915 16991916 16991917 16991918 16991919 16991920 16991921 16991922 16991923 16991924 16991925 16991926 16991927 16991928 16991929 16991930 16991931 16991932 16991933 16991934 16991935 16991936 16991937 16991938 16991939 16991940 16991941 16991942 16991943 16991944 16991945 16991946 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CB285158 CB285159 CB285160 CB285161 CB285162 CB285163 CB285164 CB285165 CB285166 CB285167 CB285168 CB285169 CB285170 CB285171 CB285172 CB285173 CB285174 CB285175 CB285176 CB285177 CB285178 CB285179 CB285180 CB285181 CB285182 CB285183 CB285184 CB285185 CB285186 CB285187 CB285188 CB285189 292 PDF created with pdfFactory Pro trial version www.pdffactory.com [...]... www.pdffactory.com 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 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 IgE binding reactivity of Der f 9, Der p 9 and Blo t 9 tested by 55 Singaporean sera and 42 Italian... www.pdffactory.com List of Tables IgE binding rates of the cloned dust mite allergens and their biological properties 22 Overview of the productions of recombinant mite allergens: from the aspects of the production yield, bioactivities and types of expression systems used 32 Identity and percentage of homology of EST clones corresponding to the known allergen homologues 55 List of primers used in RACE... 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... version www.pdffactory.com 1.6 House dust mites as an important cause of allergy 1.6.1 General aspects of mites Mites are normal inhabitants in our environment and are abundant in house dust as well as barns and grain stores Mites are small organisms, 0.1–1 mm in size with four pairs of legs and belong to the subclass of Acari (mites and ticks), forming an important division of arthropodan class Arachnida... 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. .. for dust mites are a temperature of 23-30o C and 80-90% relative humidity The house dust mites primarily feed on skin scales and dander while the storage mites feed on plants and micro-organisms (Ho and Nadchatram, 1984; Andersen 1988; Hart, 1998; Arlian et al., 1999) Any mite species present in the human environment in more than 100/g of dust could be considered as risk factors for sensitization and. .. List of figures Allergy mechanism: sensitization; immediate reaction; and late reaction 5 TR and NKT cells regulate the differentiation and function of TH1 cells and TH2 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... astigmatid mites (storage mites) can be found in homes and are a potent source of allergens Most notable are species in the families Glycyphagidae (Glycyphagus domesticus and Lepidoglyphus destructor), Acaridae (Tyrophagus putrescentiae and Acarus siro), and Chortoglyphidae (Chortoglyphus ancutatus) Predaceous mites (e.g Cheyletus) and parasitic mites of plants (Tetranychidae, spider mites and Tarsonemidae)... 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 . EXPRESSED SEQUENCE TAGS ANALYSIS OF MAJOR ALLERGENS PRODUCING DUST MITES AND MOLECULAR CHARACTERIZATION OF THEIR ALLERGENS ONG SEOW THENG. 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. 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: