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 direction 154 References 157 Appendix 175 viii Gehlhar, K., K. R. Rajashankar, E. Hofmann, C. Betzel, W. Weber, S. Werner, and A. Bufe. 2006. Lysine as a Critical Amino Acid for IgE Binding in Phl p 5b C Terminus. Int. Arch. Allergy Immunol. 140: 285-294. Grzesiek, S., and A. Bax. 1993. Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins. J. Biomol. NMR 3: 185-204. Grzesiek, S., J. Anglister, and A. Bax. 1992. Correlation of backbone amide and aliphatic side-chain resonances in 13C/15N enriched proteins by isotropic mixing of 13C magnetization. J. Magn. Reson. B101: 114-119. Guntert, P., C. Mumenthaler, and K. Wuthrich. 1997. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273: 283-298. Gutierrez-Gonzalez, L. H., C. Ludwig, C. Hohoff, M. Rademacher, T. Hanhoff, H. Ruterjans, F. Spener, and C. Lucke. 2002. Solution structure and backbone dynamics of human epidermal-type fatty acid-binding protein (E-FABP). Biochem. J. 364: 725-737. Hamid, Q., M. Azzawi, S. Ying, R. Moqbel, A. J. Wardlaw, C. J. Corrigan, B. Bradley, S. R. Durham, J. V. Collins, P. K. Jeffery, and a. et. 1991. Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma. J. Clin. Invest. 87: 1541-1546. Hart, B. J. 1998. Life cycle and reproduction of house-dust mites: environmental factors influencing mite populations. Allergy 53: 13-17. Herrmann, T., P. Guntert, and K. Wuthrich. 2002. Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol. 162 319: 209-227. Higgins D., Thompson J., Gibson T. Thompson J. D., Higgins D. G. and Gibson T. J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989. Sitedirected mutagenesis by overlap extension using the polymerase chain reaction. Gene 77: 51-59. Holm, J., M. Gajhede, M. Ferreras, A. Henriksen, H. Ipsen, J. N. Larsen, L. Lund, H. Jacobi, A. Millner, P. A. Wurtzen, and M. D. Spangfort. 2004. Allergy vaccine engineering: epitope modulation of recombinant Bet v reduces IgE binding but retains protein folding pattern for induction of protective blocking-antibody responses. J. Immunol. 173: 5258-5267. Hui-Yuen, J. S., T. T. Duong, and R. S. Yeung. 2006. TNF-alpha is necessary for induction of coronary artery inflammation and aneurysm formation in an animal model of Kawasaki disease. J. Immunol. 176: 6294-6301. Ichikawa, S., H. Hatanaka, T. Yuuki, N. Iwamoto, S. Kojima, C. Nishiyama, K. Ogura, Y. Okumura, and F. Inagaki. 1998. Solution structure of Der f 2, the major mite allergen for atopic diseases. J. Biol. Chem. 273: 356-360. Ichikawa, S., T. Takai, T. Inoue, T. Yuuki, Y. Okumura, K. Ogura, F. Inagaki, and H. Hatanaka. 2005. NMR study on the major mite allergen Der f 2: its refined tertiary structure, epitopes for monoclonal antibodies and characteristics shared by ML protein group members. J. Biochem. (Tokyo) 137: 255-263. Ikura, M., L. E. Kay, and A. Bax. 1990. A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three- 163 dimensional NMR spectroscopy. Application to calmodulin. Biochemistry 29: 4659-4667. Jeong, K. Y., W. K. Kim, J. S. Lee, J. Lee, I. Y. Lee, K. E. Kim, J. W. Park, C. S. Hong, H. I. Ree, and T. S. Yong. 2005. Immunoglobulin E reactivity of recombinant allergen Tyr p 13 from Tyrophagus putrescentiae homologous to fatty acid binding protein. Clin. Diagn. Lab. Immunol. 12: 581-585. Johnson, B. A., and R. A. Blevins. 1994. NMRView: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4: 603-614. Jutel, M., M. Akdis, F. Budak, C. Aebischer-Casaulta, M. Wrzyszcz, K. Blaser, and C. A. Akdis. 2003. IL-10 and TGF-beta cooperate in the regulatory T cell response to mucosal allergens in normal immunity and specific immunotherapy. Eur. J. Immunol. 33: 1205-1214. Jutel, M., W. J. Pichler, D. Skrbic, A. Urwyler, C. Dahinden, and U. R. Muller. 1995. Bee venom immunotherapy results in decrease of IL-4 and IL-5 and increase of IFN-gamma secretion in specific allergen-stimulated T cell cultures. J. Immunol. 154: 4187-4194. Kane, M. M., and D. M. Mosser. 2001. The role of IL-10 in promoting disease progression in leishmaniasis. J. Immunol. 166: 1141-1147. Karamloo, F., P. Schmid-Grendelmeier, F. Kussebi, M. Akdis, M. Salagianni, B. R. von Beust, A. Reimers, J. Zumkehr, L. Soldatova, Z. Housley-Markovic, U. Muller, T. Kundig, D. M. Kemeny, M. D. Spangfort, K. Blaser, and C. A. Akdis. 2005. Prevention of allergy by a recombinant multi-allergen vaccine with reduced IgE binding and preserved T cell epitopes. Eur. J. Immuno.l 35: 3268-3276. Karisola, P., J. Mikkola, N. Kalkkinen, K. J. Airenne, O. H. Laitinen, S. Repo, O. T. 164 Pentikainen, T. Reunala, K. Turjanmaa, M. S. Johnson, T. Palosuo, M. S. Kulomaa, and H. Alenius. 2004. Construction of hevein (Hev b 6.02) with reduced allergenicity for immunotherapy of latex allergy by comutation of six amino acid residues on the conformational IgE epitopes. J. Immunol. 172: 2621-2628. Kay, A. B. 2001. Allergy and allergic diseases. N. Engl. J. Med. 344: 30-37, 109-113. Kay, L. E., G. Y. Xu, A. U. Singer, D. R. Muhandiram, and J. D. Forman-Kay. 1993. A gradient-enhanced HCCH-TOCSY experiment for recording side-chain 1H and 13C Correlations in H2O samples of proteins. J. Magn. Reson. B101: 333337. King, T. P., Hoffman, H. Lowenstein, D. G. Marsh, T. A. E. Platts-Mills and W. R. Thomas. 1994. Allergen nomenclature. Bull. World Health Organ. 72:797-80. Kon, O. M., and N. C. Barnes. 1997. New treatment drugs for asthma. In Allergy and allergic diseases. A. B. Kay, ed. Blackwell Scientific. 1726-1738. Koradi, R., M. Billeter, and K. Wuthrich. 1996. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14: 51-52. Kuo, I. C., N. Cheong, M. Trakultivakorn, B. W. Lee, and K. Y. Chua. 2003. An extensive study of human IgE cross-reactivity of Blo t and Der p 5. J. Allergy Clin. Immunol. 111: 603-609. Kussebi, F., F. Karamloo, C. Rhyner, P. Schmid-Grendelmeier, M. Salagianni, C. Mannhart, M. Akdis, L. Soldatova, Z. Markovic-Housley, B. R. Von Beust, T. Kundig, D. M. Kemeny, K. Blaser, R. Crameri, and C. A. Akdis. 2005. A major allergen gene-fusion protein for potential usage in allergen-specific immunotherapy. J. Allergy. Clin. Immunol. 115: 323-329. 165 Laemmli U.K. 1970. Cleavage structural proteins during assembly of the head of T4 bacteriophage. Nature 227: 680-681. Lascombe, M. B., C. Gregoire, P. Poncet, G. A. Tavares, I. Rosinski-Chupin, J. Rabillon, H. Goubran-Botros, J. C. Mazie, B. David, and P. M. Alzari. 2000. Crystal structure of the allergen Equ c 1. A dimeric lipocalin with restricted IgE-reactive epitopes. J. Biol. Chem. 275: 21572-21577. Laskowski, R. A., J. A. Rullmannn, M. W. MacArthur, R. Kaptein, and J. M. Thornton. 1996. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8: 477-486. Liaw, S. H., H. Z. Chen, G. G. Liu, and K. Y. Chua. 2001. Acid-induced polymerization of the group mite allergen from Dermatophagoides pteronyssinus. Biochem. Biophys. Res. Commun. 285: 308-312. Liaw, S. H., H. Z. Chen, I. C. Kuo, and K. Y. Chua. 1998. Crystallization and preliminary X-ray diffraction analysis of group mite allergen from Dermatophagoides pteronyssinus. J. Struct. Biol. 123: 265-268. Liew, F. Y. 2002. T(H)1 and T(H)2 cells: a historical perspective. Nat. Rev. Immunol. 2: 55-60. Lin, K. L., K. H. Hsieh, W. R. Thomas, B. L. Chiang, and K. Y. Chua. 1994. Characterization of Der p V allergen, cDNA analysis, and IgE-mediated reactivity to the recombinant protein. J. Allergy Clin. Immunol. 94: 989-996. Lucke, C., M. Rademacher, A. W. Zimmerman, H. T. van Moerkerk, J. H. Veerkamp, and H. Ruterjans. 2001. Spin-system heterogeneities indicate a selected-fit mechanism in fatty acid binding to heart-type fatty acid-binding protein (HFABP). Biochem. J. 354: 259-266. 166 Lukacs, N. W., R. M. Strieter, S. W. Chensue, M. Widmer, and S. L. Kunkel. 1995. TNF-alpha mediates recruitment of neutrophils and eosinophils during airway inflammation. J. Immunol. 154: 5411-5417. Marcotte, G. V., C. M. Braun, P. S. Norman, C. F. Nicodemus, A. Kagey-Sobotka, L. M. Lichtenstein, and D. M. Essayan. 1998. Effects of peptide therapy on ex vivo T-cell responses. J. Allergy Clin. Immunol. 101: 506-513. Marsh, D. G., L. Goodfriend , T. P. King, H. Lowenstein, T. A. E. Platts-Mills. Allergen nomenclature. Bull. World Health Organ. 64:767-70. Meno, K., P. B. Thorsted, H. Ipsen, O. Kristensen, J. N. Larsen, M. D. Spangfort, M. Gajhede, and K. Lund. 2005. The crystal structure of recombinant proDer p 1, a major house dust mite proteolytic allergen. J .Immunol. 175: 3835-3845. Midoro-Horiuti, T., V. Mathura, C. H. Schein, W. Braun, S. Yu, M. Watanabe, J. C. Lee, E. G. Brooks, and R. M. Goldblum. 2003. Major linear IgE epitopes of mountain cedar pollen allergen Jun a map to the pectate lyase catalytic site. Mol. Immunol. 40: 555-562. Mirza, O., A. Henriksen, H. Ipsen, J. N. Larsen, M. Wissenbach, M. D. Spangfort, and M. Gajhede. 2000. Dominant epitopes and allergic cross-reactivity: complex formation between a Fab fragment of a monoclonal murine IgG antibody and the major allergen from birch pollen Bet v 1. J. Immunol. 165: 331-338. Montelione, G. T., B. A. Lyons, S. D. Emerson, and M. Tashiro. 1992. An efficient triple resonance experiment using carbon-13 isotropic mixing for determining sequence-specific resonance assignments of isotopically-enriched proteins. J. Am. Chem. Soc. 114: 10974-10975. 167 Mori, A., M. Suko, N. Tsuruoka, O. Kaminuma, T. Ohmura, Y. Nishizaki, K. Ito, and H. Okudaira. 1995. Allergen-specific human T cell clones produce interleukin-5 upon stimulation with the Th1 cytokine interleukin-2. Int. Arch. Allergy Immunol. 107: 220-222. Mori, A., M. Suko, O. Kaminuma, Y. Nishizaki, T. Mikami, T. Ohmura, A. Hoshino, S. Inoue, N. Tsuruoka, Y. Okumura, G. Sato, K. Ito, and H. Okudaira. 1996. A critical role of IL-2 for the production and gene transcription of IL-5 in allergen-specific human T cell clones. Int. Immunol. 8: 1889-1895. Moverare, R., L. Elfman, E. Vesterinen, T. Metso, and T. Haahtela. 2002. Development of new IgE specificities to allergenic components in birch pollen extract during specific immunotherapy studied with immunoblotting and Pharmacia CAP System. Allergy 57: 423-430. Mueller, G. A., A. M. Smith, M. D. Chapman, G. S. Rule, and D. C. Benjamin. 2001. Hydrogen exchange nuclear magnetic resonance spectroscopy mapping of antibody epitopes on the house dust mite allergen Der p 2. J. Biol. Chem. 276: 9359-9365. Mueller, G. A., D. C. Benjamin, and G. S. Rule. 1998. Tertiary structure of the major house dust mite allergen Der p 2: sequential and structural homologies. Biochemistry 37: 12707-12714. Muller, U., C. A. Akdis, M. Fricker, M. Akdis, T. Blesken, F. Bettens, and K. Blaser. 1998. Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. J. Allergy Clin. Immunol. 101: 747-754. 168 Nadler, M. J., S. A. Matthews, H. Turner, and J. P. Kinet. 2000. Signal transduction by the high-affinity immunoglobulin E receptor Fc epsilon RI: coupling form to function. Adv. Immunol. 76: 325-355. Neudecker, P., K. Lehmann, J. Nerkamp, T. Haase, A. Wangorsch, K. Fotisch, S. Hoffmann, P. Rosch, S. Vieths, and S. Scheurer. 2003. Mutational epitope analysis of Pru av and Api g 1, the major allergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgE reactivity with threedimensional structure. Biochem. J. 376: 97-107. Niederberger, V., F. Horak, S. Vrtala, S. Spitzauer, M. T. Krauth, P. Valent, J. Reisinger, M. Pelzmann, B. Hayek, M. Kronqvist, G. Gafvelin, H. Gronlund, A. Purohit, R. Suck, H. Fiebig, O. Cromwell, G. Pauli, M. van Hage-Hamsten, and R. Valenta. 2004. Vaccination with genetically engineered allergens prevents progression of allergic disease. Proc. Natl. Acad. Sci. U. S. A. 101 Suppl 2: 14677-14682. Nutku, E., Q. Zhuang, A. Soussi-Gounni, F. Aris, B. D. Mazer, and Q. Hamid. 2001. Functional expression of IL-12 receptor by human eosinophils: IL-12 promotes eosinophil apoptosis. J. Immunol. 167: 1039-1046. Olsson, S., M. van Hage-Hamsten, and P. Whitley. 1998. Contribution of disulphide bonds to antigenicity of Lep d 2, the major allergen of the dust mite Lepidoglyphus destructor. Mol. Immunol. 35: 1017-1023. Ong, S. T. 2003. Expressed sequence tags analysis of major allergens producing dust mites and molecular characterization of their allergens. PhD Thesis, National University of Singapore. Pearlman, D. A., D. A. Case, J. W. Caldwell, W. S. Ross, T. E. Cheatham, S. DeBolt, D. Ferguson, G. Seibel, and P. Kollman. 1995. AMBER, a package of 169 computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules. Comp. Phys. Commun. 91: 1-41. Plebanski, M., K. L. Flanagan, E. A. Lee, W. H. Reece, K. Hart, C. Gelder, G. Gillespie, M. Pinder, and A. V. Hill. 1999. Interleukin 10-mediated immunosuppression by a variant CD4 T cell epitope of Plasmodium falciparum. Immunity 10: 651-660. Puerta, L., M. W. Kennedy, S. Jimnez, and L. Caraballo. 1999. Structural and ligand binding analysis of recombinant Blo t 13 allergen from Blomia tropicalis mite, a fatty acid binding protein. Int. Arch. Allergy Immunol. 119: 181-184. Rademacher, M., A. W. Zimmerman, H. Ruterjans, J. H. Veerkamp, and C. Lucke. 2002. Solution structure of fatty acid-binding protein from human brain. Mol. Cell. Biochem. 239: 61-68. Reid, D. G. 1997. Protein NMR Techniques. Humana Press. Ringom, R., E. Axen, J. Uppenberg, T. Lundback, L. Rondahl, and T. Barf. 2004. Substituted benzylamino-6-(trifluoromethyl)pyrimidin-4(1H)-ones: a novel class of selective human A-FABP inhibitors. Bioorg. Med. Chem. Lett. 14: 4449-4452. Roos, T. C., S. Geuer, S. Roos, and H. Brost. 2004. Recent advances in treatment strategies for atopic dermatitis. Drugs 64: 2639-2666. Schulz, O., H. F. Sewell, and F. Shakib. 1998. Proteolytic Cleavage of CD25, the Subunit of the Human T Cell Interleukin Receptor, by Der p 1, a Major Mite Allergen with Cysteine Protease Activity. J. Exp. Med. 187: 271-275 Schulz, O., P. Laing, H.F. Sewell, and F. Shakib. 1995. Der p I, a major allergen of the house dust mite, proteolytically cleaves the low-affinity receptor for 170 human IgE (CD23). Eur. J. Immunol. 25: 3191–3194. Spangfort, M. D., O. Mirza, H. Ipsen, R. J. Van Neerven, M. Gajhede, and J. N. Larsen. 2003. Dominating IgE-binding epitope of Bet v 1, the major allergen of birch pollen, characterized by X-ray crystallography and site-directed mutagenesis. J. Immunol. 171: 3084-3090. Spera, S., and A. Bax. 1991. Empirical correlation between protein backbone conformation and Cα and Cβ 13C nuclear magnetic resonance chemical shifts. J. Am. Chem. Soc. 113: 5490-5492. Stingl, G., and D. Maurer. 1997. IgE-mediated allergen presentation via Fc epsilon RI on antigen-presenting cells. Int. Arch. Allergy Immunol. 113: 24-29. Suzuki, K., J. J. Lareyre, D. Sanchez, G. Gutierrez, Y. Araki, R. J. Matusik, and M. C. Orgebin-Crist. 2004. Molecular evolution of epididymal lipocalin genes localized on mouse chromosome 2. Gene 339: 49-59. Szyperski, T., D. Neri, B. Leiting, G. Otting, and K. Wuthrich. 1992. Support of 1H NMR assignments in proteins by biosynthetically directed fractional 13Clabeling. J. Biomol. NMR 2: 323-334. Takai, T., S. Ichikawa, H. Hatanaka, F. Inagaki, and Y. Okumura. 2000. Effects of proline mutations in the major house dust mite allergen Der f on IgE-binding and histamine-releasing activity. Eur. J. Biochem. 267: 6650-6656. Takai, T., T. Yokota, M. Yasue, C. Nishiyama, T. Yuuki, A. Mori, H. Okudaira, and Y. Okumura. 1997. Engineering of the major house dust mite allergen Der f for allergen-specific immunotherapy. Nat. Biotechnol. 15: 754-758. Tan, T. N., D. L. Lim, B. W. Lee, and H. P. Van Bever. 2005. Prevalence of allergyrelated symptoms in Singaporean children in the second year of life. Pediatr. Allergy Immunol. 16: 151-156. 171 Thomas, W. R., W. A. Smith, B. J. Hales, K. L. Mills, and R. M. O'Brien. 2002. Characterization and immunobiology of house dust mite allergens. Int. Arch. Allergy Immunol. 129: 1-18. Tovey, E. R., M. C. Johnson, A. L. Roche, G. S. Cobon, and B. A. Baldo. 1989. Cloning and sequencing of a cDNA expressing a recombinant house dust mite protein that binds human IgE and corresponds to an important low molecular weight allergen. J. Exp. Med. 170: 1457-1462. Tovey, E. R., M. D. Chapman, and T. A. Platts-Mills. 1981. Mite faeces are a major source of house dust allergens. Nature 289: 592-593. Turner, H., and J. P. Kinet. 1999. Signalling through the high-affinity IgE receptor Fc epsilonRI. Nature 402: B24-B30. Valenta, R. 2002. The future of antigen-specific immunotherapy of allergy. Nat. Rev. Immunol. 2: 446-453. van Neerven, R. J., T. Wikborg, G. Lund, B. Jacobsen, A. Brinch-Nielsen, J. Arnved, and H. Ipsen. 1999. Blocking antibodies induced by specific allergy vaccination prevent the activation of CD4+ T cells by inhibiting serum-IgEfacilitated allergen presentation. J. Immunol. 163: 2944-2952. Veldman, C., A. Nagel, and M. Hertl. 2006. Type I regulatory T cells in autoimmunity and inflammatory diseases. Int. Arch. Allergy Immunol. 140: 174-183. Vrtala, S., C. A. Akdis, F. Budak, M. Akdis, K. Blaser, D. Kraft, and R. Valenta. 2000. T cell epitope-containing hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1, induce blocking antibodies. J. Immunol. 165: 6653-6659. 172 Vrtala, S., K. Hirtenlehner, M. Susani, P. Hufnagl, B. R. Binder, L. Vangelista, A. Pastore, W. R. Sperr, P. Valent, C. Ebner, D. Kraft, and R. Valenta. 1999. Genetic engineering of recombinant hypoallergenic oligomers of the major birch pollen allergen, Bet v 1: candidates for specific immunotherapy. Int. Arch. Allergy Immunol. 118: 218-219. Vrtala, S., T. Ball, S. Spitzauer, B. Pandjaitan, C. Suphioglu, B. Knox, W. R. Sperr, P. Valent, D. Kraft, and R. Valenta. 1998. Immunization with purified natural and recombinant allergens induces mouse IgG1 antibodies that recognize similar epitopes as human IgE and inhibit the human IgE-allergen interaction and allergen-induced basophil degranulation. J. Immunol. 160: 6137-6144. Wachholz, P. A., K. T. Nouri-Aria, D. R. Wilson, S. M. Walker, A. Verhoef, S. J. Till, and S. R. Durham. 2002. Grass pollen immunotherapy for hayfever is associated with increases in local nasal but not peripheral Th1:Th2 cytokine ratios. Immunology 105: 56-62. Wills-Karp, M., J. Luyimbazi, X. Xu, B. Schofield, T. Y. Neben, C. L. Karp, and D. D. Donaldson. 1998. Interleukin-13: central mediator of allergic asthma. Science 282: 2258-2261. Wishart, D. S., B. D. Sykes, and F. M. Richards. 1992. The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry 31: 1647-1651. Wishart, D. S., C. G. Bigam, A. Holm, R. S. Hodges, and B. D. Sykes. 1995. 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J. Biomol. NMR 5: 67-81. Wittekind, M., and L. Mueller. 1993. HNCACB, a high-sensitivity 3D NMR experiment to correlate amide-proton and nitrogen resonances with the alpha- 173 and beta-carbon resonances in proteins. J. Magn. Reson. B 101: 201-205. Wutrich, K. 1986. NMR of proteins and nucleic acids. Wiley, New York. Xu, Y., Z. Lin, C. Ho, and D. Yang. 2005. A general strategy for the assignment of aliphatic side-chain resonances of uniformly 13C,15N-labeled large proteins. J. Am. Chem. Soc. 127: 11920-11921. Zimmerman, A. W., H. T. van Moerkerk, and J. H. Veerkamp. 2001. Ligand specificity and conformational stability of human fatty acid-binding proteins. Int. J. Biochem. Cell. 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