Structural and epitope characterization of dust mites allergens, der f 13 and blo t 5

STRUCTURAL AND EPITOPE CHARACTERIZATION OF DUST MITES ALLERGENS, DER F 13 AND BLO T CHAN SIEW LEONG NATIONAL UNIVERSITY OF SINGAPORE 2006 STRUCTURAL AND EPITOPE CHARACTERIZATION OF DUST MITES ALLERGENS, DER F 13 AND BLO T CHAN SIEW LEONG (B. Sc., USM) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgement I would like to pay my greatest gratitude to my supervisor, Dr Henry Mok. His guidance and tremendous passion in research have been a motivation to keep me going throughout my PhD. Thank you in believing in me and giving me your trust. It has been a great experience working with you. Most of the work in this thesis would not be possible without close co-operation with great mentor, colleagues and friends in the Functional Genomics Lab 3. A big thank you to Dr Chew Fook Tim, Seow Theng, Tan Ching, Ken, Su Yin and Kavita for their valuable advices. Without them, most of the immunological experiments would not be possible. It’s been great working in the Structural Biology Lab with all the exceptional people: Yvonne, Xingfu, Yonghong, Mingbo, Gary, Olga, Anir, Jana, Deepti, Rika, Michelle, Zheng Yu and Lin Zhi. Thanks for enduring my presence. Many experiments would not be plausible with special advice and technical contribution from these excellent scientists in NUS, namely Prof. Kini, Dr. Yang, Dr. Wang, Dr. Seah, Dr. Fan, Chye Fong and Mr. Ow. To Yvonne, thank you for always being here for me. You’re the greatest love I could ever find. Life in Singapore has been exciting with all my friends here. My special ‘heavy credit’ to the Makan Marathon bunch, can’t wait to eat with you guys again. Dearest Muddies, I have had absolutely spinning good time with you all. Ultimate i Frisbee has been one of the greatest things to happen to me in Singapore. To Dario @ Picio and Oli, stay geeky . Special gratitude to Rick, Jasmine and little Reuben. It’s been a marvelous years being loved by all of you. You all are just wonderful. Dearest Dad, Mom and Kit Yee, thanks for supporting me all these while. I have always wished that all of you could be here with me all these while. Last but not least, a tribute to Hema and Dawny. You girls are absolutely fantastic. Half the thesis goes to both of you. ii Table of contents Acknowledgement i Table of contents iii Summary ix List of figures xi List of tables xiii List of abbreviations xiv Chapter 1: Introduction 1.1 Allergy 1.1.1 Allergy: An Introduction 1.1.2 Mechanism of allergy 1.2 Dust mite, an important source of allergens 1.3 Structural and epitope characterization of allergens 13 1.3.1 Structural biology of allergens 13 1.3.2 IgE epitope mapping of allergens 17 Specific immunotherapy against allergy: Strategies to create 20 1.4 hypoallergen 1.5 Group 13 dust mite allergens 25 1.5.1 Group 13 dust mite allergens and fatty acid binding proteins 25 1.5.2 Der f 13, a newly characterized group 13 allergen from 28 Dermatophagoides farinae. 1.6 Blo t 5, a major allergen with coiled-coil structure from Blomia 31 iii tropicalis 1.7 1.8 Nuclear Magnetic Resonance (NMR) 34 1.7.1 Basics of NMR 34 1.7.2 Multi-dimensional NMR and sequential assignment 36 1.7.3 Solution structure determination using NMR techniques 39 1.7.4 Preparation of NMR sample 41 Objectives of this study 43 Chapter 2: Materials and Methods 2.1 Generation and subcloning of Der f 13 and its mutants into 45 45 expression vector 2.1.1 Bacterial host strains 45 2.1.2 Generation of DNA insert and Polymerase Chain Reaction 45 2.1.3 Generation of DNA mutant insert for site directed 46 mutagenesis 2.2 2.1.4 Preparation of DH5-α competent cells 49 2.1.5 Sub-cloning 49 2.1.6 Transformation of ligation mix into DH5-α competent cells. 51 2.1.7 PCR screening of transformant 51 2.1.8 Isolation of DNA plasmid 52 2.1.9 Plasmid DNA sequencing 53 Protein expression and purification of Der f 13 54 2.2.1 Transformation of plasmid into BL21 (DE3) competent cells 54 2.2.2 Protein expression 54 2.2.3 Protein purification using Nickel-affinity chromatography 55 iv 2.2.4 Protein purification using glutathione-sepharose affinity 56 chromatography 2.2.5 Thrombin digestion 56 2.2.6 Gel filtration FPLC 57 2.2.7 Preparation of NMR sample 57 2.2.8 Sodium Dodecyl Sulphate-Polyacrylamide Gel 58 Electrophoresis (SDS-PAGE) 2.3 2.2.9 Circular dichroism spectropolarimetry 58 2.2.10 Sequence alignment 59 Nuclear magnetic resonance and structural determination 60 2.3.1 NMR chemical shift assignments of Der f 13 60 2.3.1.1 2D 1H-15N HSQC spectrum 60 2.3.1.2 HNCACB and CBCA(CO)NH 61 2.3.1.3 C(CO)NH-TOCSY and HC(CO)NH-TOCSY 62 2.3.1.4 HCCH-TOCSY 62 2.3.1.5 63 H-13C HSQC 2.3.2 NOE distance restraints and hydrogen bond restraints of 64 Der f 13 2.4 2.3.2.1 Hydrogen-deuterium exchange measurement 64 2.3.2.2 15 N-edited NOESY 64 2.3.2.3 13 C-edited NOESY 65 2.3.3 NOE assignments and structure calculation 65 Immunoassay 67 2.4.1 Specific IgE binding ELISA experiment 67 2.4.2 Inhibition ELISA experiment 68 v 2.4.3 Skin prick test 68 2.4.4 Isolation of mononuclear cells using Ficoll-Hypaque gradient 69 centrifugation 2.4.5 PBMC proliferation and cytokine expression 70 2.4.6 Cytokines measurement 70 2.4.7 Mouse immunization 71 2.4.8 Mouse orbital bleeding and sera collection 71 2.4.9 Inhibition of human IgE binding to Der f 13 by specific 72 mouse IgG antibodies 2.4.10 Specific mouse IgG binding to Der f 13 ELISA experiment 73 2.4.11 Specific mouse IgE binding to Der f 13 ELISA experiment 74 2.4.12 Specific mouse IgG binding to human FABP ELISA 74 experiment 2.5 Sub-cloning, expression and purification of Blo t 75 2.6 NMR experiments and chemical shifts assignment of Blo t 75 Chapter 3: Structure characterization and IgE epitope mapping of Der f 13 3.1 3.2 77 Protein expression and purification 77 3.1.1 Expression and purification of His-tag Der f 13 77 3.1.2 Protein expression and purification of GST-tag Der f 13 78 3.1.3 Circular dichroism of Der f 13 78 NMR Structure of Der f 13 81 3.2.1 1D 1H-NMR and 2D 1H-15N HSQC spectra of Der f 13 81 3.2.2 Chemical shifts assignment of Der f 13 81 vi 3.3 3.2.2.1 Stereospecific assignment of methyl groups 87 3.2.2.2 Chemical shift index 89 3.2.3 Hydrogen bond restraints and TALOS torsion angle restraints 91 3.2.4 Automated NOE assignment by CYANA 93 3.2.5 Calculations of protein structure by CYANA and DYANA 97 Sequence analysis and putative IgE epitope prediction 103 3.3.1 Human FABPs neither bind IgE and nor react in skin prick 103 test 3.4 3.3.2 Sequence alignment of Der f 13 with human FABP 106 Site directed mutagenesis, IgE binding ELISA and skin prick 109 reactivity 3.4.1 IgE binding ELISA of Der f 13 single mutants 109 3.4.2 Reduced IgE binding of double mutants and triple mutants 110 3.4.3 Triple mutant 3A cannot inhibit binding of IgE to wild type 114 Der f 13 3.4.4 Skin prick reactivities of wild type sand triple mutant 3A of 114 Der f 13 3.4.5 Circular dichroism and gel filtration chromatography of wild 117 type and 3A mutant of Der f 13 3.5 3.4.6 IgE binding epitope site on Der f 13 117 PBMC proliferation and cytokine release 120 3.5.1 Isolation of PBMC from group 13 allergic patients 120 3.5.2 Stimulation of PBMC proliferation by Der f 13 and 3A 120 mutant 3.5.3 Cytokines release by wild type and 3A mutant of Der f 13 122 vii 3.6 Mouse immunization and generation of IgE blocking IgG 127 3.6.1 IgG and IgE production from mice immunized with wild type 127 or 3A mutant of Der f 13 3.6.2 3A mutant raised IgG blocks binding of human IgE to wild 128 type Der f 13 3.6.3 Binding of mouse sera IgG to group 13 isoforms 130 3.6.4 Binding of mouse sera IgG to human FABP 130 3.7 Hypoallergen immunotherapy: Th1 or Treg? 132 3.8 Charged residues: A preferred epitope residues for IgE ? 135 Chapter 4: Structure characterization of Blo t 140 4.1 Protein expression and purification of Blo t 140 4.2 Circular dichroism spectrum of Blo t 142 4.3 1D 1H-NMR and 2D 1H-15N HSQC spectra of Blo t 143 4.4 Backbone chemical shifts assignment and chemical shift index of Blo 146 t5 Chapter 5: Conclusion 5.1 Structure and IgE epitope mapping of Der f 13 leading to 149 149 development of hypoallergenic mutant 5.2 Structure characterization of Blo t 153 5.3 Future 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Biol. 33: 865-876. 174 Appendix I Recipe for M9 medium To 773 ml of sterile water, add the following: 200 ml 5X M9 ml 1M MgSO4 ml 20% Glucose 0.1 ml 1M CaCl2 ml 0.25 g/ml NH4Cl ml 0.1 g/ml Ampicillin ml Kanamycin ml Tetracycline Composition: 1. 5X M9 30 g Na2HPO4 15 g KH2PO4 2.5 g NaCl Add water to 1L (Autoclave) 2. 0.25 g/ml NH4Cl 50 g NH4Cl, add water to 200 ml (Autoclave, or filter-sterilize for 15N) 3. 20% Glucose 40 g Glucose, add water to 200 ml (Autoclave or filter-sterilize for 13C) 4. 1M MgSO4 19.72 g MgSO4, add water to 80 ml (Autoclave) 1M CaCl2 11.76 g CaCl2, add water to 80 ml (Autoclave) 175 Appendix II Buffers for Ni-NTA Affinity Chromatography • Nickel binding buffer mM Immidazole 0.5 M NaCl 20 mM Tris pH 7.9 • Elution Buffer 0.5 M 0.5 M 20 mM Immidazole NaCl Tris pH 7.9 • Strip Buffer 100 mM 0.5 M 20 mM EDTA pH 8.0 NaCl Tris pH 7.9 • Ni-Charge buffer 50 mM NiSO4 Buffers for Glutathione-Sepharose Chromatography • Phosphate Buffer Saline (PBS) 140 mM NaCl 2.7 mM KCl 10 mM Na2HPO4 1.8 mM KH2PO4 • GST Elution Buffer 50 mM 10 mM • Column Regeneration Buffer 0.1 M Tris-Cl pH 8.5 0.5 M NaCl • Column Regeneration Buffer 0.1 M Sodium acetate pH 4.5 0.5 M NaCl Tris-Cl pH 7.9 L-Gluthathione (reduced) 176 Appendix III Recipe for SDS-PAGE 30% Acrylamide/0.8% Bis Resolving Buffer Stacking Buffer 10% SDS Water 10% Ammonium Persulphate TEMED 15% Separating Gel 3.5 ml 1.75 ml 70 µl 1.68 ml 42 µl 4.2 µl 4% Stacking Gel 0.4 ml 0.75 ml 30 µl 1.8 ml 30 µl µl 1. Acrylamide solution (30%, 0.8 % Bis) 2. Resolving gel buffer: 1.5 M Tris, pH 8.8 For 200 ml, 36.3 g of Tris adjust to pH 8.8 with HCl 3. Stacking gel buffer: 0.5 M Tris, pH 6.8 For 200 ml, 12.1 g Tris, adjust to pH 6.8 with HCl 4. 10% Ammonium persulphate: g Ammonium persulphate in 10 ml water 5. 10X Tank buffer: for liters 60 g Tris base 288g Glycine 200 ml 10% SDS solution water to liters 8. 2X SDS gel sample buffer: for 50 ml ml Glycerol 6.25 ml 0.5 M Tris pH 6.8 12.5 ml 10% SDS 2.5 ml β-mercaptoethanol 0.01 g Bromophenol blue water to 50 ml 9. Stain stock: 1% Coomassie Brilliant Blue G (Stir and filter) 10. Destaining solution: for liters 140 ml Acetic acid 100 ml Methanol water to liters 177 [...]... Expression and purification of His-tag Blo t 5 140 Figure 4.2 Gel filtration profile of Blo t 5 141 Figure 4.3 Circular dichroism spectrum of Blo t 5 142 Figure 4.4 One dimensional 1H-NMR spectrum of Blo t 5 144 Figure 4 .5 1 1 45 Figure 4.6 Sequential assignment of backbone chemical shifts of Blo t 5 148 Figure 4.7 Chemical shift index of Blo t 5 148 H-15N HSQC spectrum of Blo t 5 xii List of Tables Table... mutants 112 Figure 3.21 IgE binding assay for double mutants and triple mutant 3A of Der f 13 113 Figure 3.22 Inhibition of IgE binding by wild type and 3A mutant of Der f 13 116 Figure 3.23 Circular dichroism and gel filtration profiles of the wild type and 3A mutant of Der f 13 118 Figure 3.24 IgE binding epitope residues of Der f 13 119 Figure 3. 25 PBMC proliferation by wild type and 3A mutant of. .. topology of Der f 13 100 Figure 3. 15 Solution structure of Der f 13 101 Figure 3.16 Surface diagram and hydrophobic core of Der f 13 102 Figure 3.17 Skin prick test on patient H3 1 05 H-15N HSQC of Der f 13 H-13C HSQC spectrum of Der f 13 xi Figure 3.18 IgE binding for Der f 13 and human FABP 1 05 Figure 3.19 Sequence alignment of Der f 13 with human FABPs 107 Figure 3.20 IgE binding assay of Der f 13 single... Classification of dust mite allergens 12 Table 1.2 List of allergen structures 16 Table 3.1 Structural statistics of Der f 13 solution structure 99 Table 3.2 Skin prick test for Der f 13, Der p 2 and human FABP 104 Table 3.3 Solvent accessibility of residues selected for mutagenesis in Der f 13 108 Table 3.4 Skin prick test of 3A mutant 1 15 Table 3 .5 Analysis of charged amino acids of major allergens and. .. growth factors to promote the proliferation of these cells, and at the same time act as inhibitor for the growth of the cells of the opposite type (Liew, 2002; Gajewski and Fitch, 1988; Fernandez-Botran et al., 1988) IL-4 would induce the growth of Th2 cells, but at the same time would inhibit the proliferation of Th1 cells In contrast, IFN-γ, a cytokine released by Th1 subset of cells, is able to promote... site-directed mutants by PCR-based overlap extension 47 Figure 2.2 List of primers used for PCR and mutagenesis studies 48 Figure 3.1 Expression and purification of His-tag Der f 13 79 Figure 3.2 Expression and purification of GST-tag Der f 13 79 Figure 3.3 Gel filtration profile and CD spectrum of Der f 13 80 Figure 3.4 One dimensional 1H-NMR of Der f 13 83 Figure 3 .5 1 84 Figure 3.6 Sequential assignment of backbone... reactivity The 3A mutant also showed similar PBMC proliferation induction as wild type Der f 13 and is able to stimulate release of Th1 cytokines while at the same time reducing the secretion of Th2 cytokines Although the IgE epitopes of 3A mutant have been removed, it is still able to stimulate production of mouse blocking IgG antibodies that are able to inhibit the binding of patients’ sera IgE to... 2002) The allergens are grouped based on their function and sequence similarity, and are numbered according to the order of isolation 12 1.3 Structural and epitope characterization of allergens 1.3.1 Structural biology of allergens Structural studies of allergenic proteins are essential in identification of structural attributes that would give rise to allergenic properties of a protein Both x-ray crystallography... beds, carpets, blankets, and clothing It has become clear now that bodies and feces of mites are the 9 most important sources of allergens (Arlian et al., 1987) They contain mainly enzymes and other proteins from the mites that react potently as allergens To date, more than 20 groups of proteins have been characterized from dust mites showing the wide diversity of different proteins that are involved... region of Blo t 5 may be disordered and contain no secondary structures x List of Figures Figure 1.1 Mechanisms of allergic reaction 8 Figure 1.2 Structures of human FABP 27 Figure 1.3 Sequence alignment of Der f 13 with other homologous group 13 allergens 29 Figure 1.4 DNA sequence and translated amino acid sequence of Der f 13 30 Figure 1 .5 Sequence of Blo t 5 33 Figure 2.1 Generation of site-directed . Chapter 5: Conclusion 5. 1 Structure and IgE epitope mapping of Der f 13 leading to development of hypoallergenic mutant 5. 2 Structure characterization of Blo t 5 5. 3 Future direction 149. mutants and triple mutants 3.4.3 Triple mutant 3A cannot inhibit binding of IgE to wild type Der f 13 3.4.4 Skin prick reactivities of wild type sand triple mutant 3A of Der f 13 3.4 .5 Circular. spectrum of Blo t 5 144 Figure 4 .5 1 H- 15 N HSQC spectrum of Blo t 5 1 45 Figure 4.6 Sequential assignment of backbone chemical shifts of Blo t 5 148 Figure 4.7 Chemical shift index of