Proteolytic Enzymes in Grass Pollen and their Relationship to Allergenic Proteins By Rohit G. Saldanha A thesis submitted in fulfilment of the requirements for the degree of Masters by Research Faculty of Medicine The University of New South Wales March 2005 TABLE OF CONTENTS TABLE OF CONTENTS 1 LIST OF FIGURES 6 LIST OF TABLES 8 LIST OF TABLES 8 ABBREVIATIONS 8 ACKNOWLEDGEMENTS 11 PUBLISHED WORK FROM THIS THESIS 12 ABSTRACT 13 1. ASTHMA AND SENSITISATION IN ALLERGIC DISEASES 14 1.1 Defining Asthma and its Clinical Presentation 14 1.2 Inflammatory Responses in Asthma 15 1.2.1 The Early Phase Response 15 1.2.2 The Late Phase Reaction 16 1.3 Effects of Airway Inflammation 16 1.3.1 Respiratory Epithelium 16 1.3.2 Airway Remodelling 17 1.4 Classification of Asthma 18 1.4.1 Extrinsic Asthma 19 1.4.2 Intrinsic Asthma 19 1.5 Prevalence of Asthma 20 1.6 Immunological Sensitisation 22 1.7 Antigen Presentation and development of T cell Responses. 22 1.8 Factors Influencing T cell Activation Responses 25 1.8.1 Co-Stimulatory Interactions 25 1.8.2 Cognate Cellular Interactions 26 1.8.3 Soluble Pro-inflammatory Factors 26 1.9 Intracellular Signalling Mechanisms Regulating T cell Differentiation 30 2 POLLEN ALLERGENS AND THEIR RELATIONSHIP TO PROTEOLYTIC ENZYMES 33 1 2.1 The Role of Pollen Allergens in Asthma 33 2.2 Environmental Factors influencing Pollen Exposure 33 2.3 Classification of Pollen Sources 35 2.3.1 Taxonomy of Pollen Sources 35 2.3.2 Cross-Reactivity between different Pollen Allergens 40 2.4 Classification of Pollen Allergens 41 2.4.1 The Revised Allergen Nomenclature 42 2.4.2 Ambiguities in the Allergen Nomenclature 43 2.4.3 Allergen Isoforms 43 2.5 Localisation and Biological Functions of Pollen Allergens 45 2.6 General Features of Proteolytic enzymes 47 2.7 Proteolytic Enzyme Nomenclature 48 2.7.1 Classification of Peptidases by Catalytic Mechanism 50 2.7.1.1 Serine Peptidases 50 2.7.1.2 Cysteine Peptidases 52 2.7.1.3 Aspartic Peptidases 54 2.7.1.4 Metallopeptidases 55 2.7.2 Classification of Peptidases based on Substrate Specificity 56 2.7.2.1 Endopeptidases 56 2.7.2.2 Exopeptidases 56 2.7.2.2.1 Aminopeptidases 58 2.7.2.2.2 Carboxypeptidases 58 2.8 Classification of Peptidase Inhibitors 58 2.8.1 Natural Peptidase Enzyme Inhibitors 59 2.8.2 Synthetic Peptidase Inhibitors 62 2.9 Functional Significance of Peptidase activity in plants and pollen 64 3 IDENTIFICATION AND CHARACTERISATION OF PEPTIDASE ENZYMES 66 3.1 Proteolytic Enzyme Extraction Techniques 66 3.2 Measurement of Enzymatic Activity 68 3.2.1 Solid Phase Assays 68 3.2.2 Liquid Phase Assays 71 3.3 Protein Purification Procedures 74 2 3.3.1 General Purification Procedures 74 3.3.2 Size-Exclusion Chromatography 75 3.3.3 Ion Exchange Chromatography 76 3.3.4 Reverse-Phase Chromatography 78 3.3.5 Affinity Chromatography 79 3.4 Principles of Protein Identification by Proteomics 82 3.4.1 Why Proteomics? 83 3.4.2 Protein Separation Techniques 84 3.4.3 Proteomic Analysis by Mass Spectrometry 87 3.5 Types of Analytical Mass Spectrometers 88 3.5.1 MALDI-TOF Mass Spectrometer 88 3.5.2 Tandem Mass Spectrometers 91 3.6 Protein Identification by Mass Spectrometry 93 3.6.1 Peptide Mass Fingerprinting 93 3.6.2 Tandem Mass Spectrometry 94 3.7 Interpretation of Peptide Sequence from Tandem Mass Spectra 95 3.8 Proteomic Analysis of Allergic Diseases 97 4 MATERIALS AND METHODS 100 4.1 General Methods 100 4.1.1 Preparation of Pollen Diffusates 100 4.1.2 Development of Fluorescent Substrate Assay 100 4.1.3 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS- PAGE) and Western Immunoblotting 101 4.1.4 Development of Zymography 101 4.1.5 Two-Dimensional Gel Electrophoresis 103 4.2 Specific Purification Procedures 104 4.2.1 Size-Exclusion Chromatography 104 4.2.2 Ion-Exchange Chromatography 105 4.2.3 Affinity Chromatography using Benzamidine Sepharose 105 4.2.4 Affinity Chromatography using Concanavalin A Sepharose 106 4.3 Gel Destaining Protocols for the Proteomic Analysis of Proteins 107 4.3.1 Destaining of Silver Nitrate Stained gels 107 3 4.3.2 Destaining of Coomassie Stained Gels 107 4.3.3 Peptide Extraction from SDS/PAGE for Mass Spectrometry 108 4.4 Mass Spectrometric Analysis of Proteins 108 4.4.1 Peptide Mass Fingerprinting by MALDI-TOF Mass Spectrometry 108 4.4.2 Microcapillary Liquid Chromatography/Tandem Mass Spectrometric Analysis of Proteins 109 5 SERINE PEPTIDASE ACTIVITY IN POLLEN DIFFUSATES 111 5.1 Peptidase Activity in Pollen diffusates 111 5.1.1 Fluorometric Assay 111 5.1.2 Characterisation of Peptidase Activity based on the Inhibitory Profile 111 5.2 One-Dimensional Electrophoretic Analysis of Pollen Diffusates 112 5.2.1 SDS/PAGE and Western Blotting of Pollen Diffusates 112 5.2.2 Gelatin Zymography 112 5.2.3 Proteomic Analysis of Samples from One-Dimensional SDS/PAGE 118 5.3 Two-Dimensional Electrophoretic Analysis of Bermuda grass Pollen Diffusate 122 5.3.1 Two-Dimensional SDS/PAGE and Western Blotting 122 5.3.2 Two-Dimensional Gelatin Zymography 122 5.3.3 Proteomic Analysis of samples from Two-Dimensional SDS/PAGE of Bermuda grass Pollen 125 5.4 Discussion 128 6 PURIFICATION OF PROTEOLYTIC ACTIVITY IN POLLEN DIFFUSATES 137 6.1 General Chromatographic Separation Techniques 137 6.1.1 Size-Exclusion Chromatography 137 6.1.2 Ion-Exchange Chromatography 141 6.1.3 Discussion 145 6.2 Affinity Chromatography Purification Techniques 147 6.2.1 Benzamidine Sepharose Chromatography 147 6.2.2 Concanavalin A Sepharose Chromatography 149 6.2.2.1 Affinity Purification using Concanavalin A Sepharose 149 6.2.2.2 Analysis of Peptidase activity by Fluorometric Assay 149 4 6.2.2.3 Analysis by One-Dimensional SDS/PAGE and Gelatin Zymography 150 6.2.2.4 Proteomic Analysis of Con A Sepharose Purified Fractions. 154 6.2.2.5 Analysis of the proteolytically active fractions by Anion Exchange HPLC 154 6.2.3 Discussion 158 7 CONCLUDING DISCUSSION 164 BIBLIOGRAPHY 169 APPENDIX: GENERAL REAGENTS 191 5 LIST OF FIGURES Figure 2.1 - Scanning electron micrographs of fresh Poaceae pollen grains in the dry state (A&C) and in the hydrated state (B&D) 35 Figure 2.2 - Nomenclature of allergenic tree pollen sources 37 Figure 2.3 - Nomenclature of allergenic grass pollen sources 38 Figure 2.4 - Nomenclature of allergenic weed pollen sources 39 Figure 2.5 - IUIS criteria for the inclusion of allergens into allergen nomenclature 41 Figure 2.6 - Schematics of the designation of allergen names according to IUIS 42 Figure 2.7 - Revised nomenclature of allergen isoforms and variants 45 Figure 3.1 - Schematic representation of size-exclusion chromatography 76 Figure 3.2 - Schematic representation of ion-exchange chromatography 77 Figure 3.3 - Schematic of the principle of affinity chromatography. 81 Figure 3.4 - General Strategy for proteome characterisation 85 Figure 3.5 - Schematics for an orthogonal Time of Flight mass analyser 90 Figure 3.6 - Schematics for Q-Tof hybrid Tandem Mass Spectrometer 92 Figure 3.7 - The ion series produced by the fragmentation pattern of amino acid residues 96 Figure 3.8 - Typical tandem mass spectrum (MS2) of GluFibrino peptide (+2) 98 Figure 4.1 - Running parameters for isoelectric focussing of pollen diffusates 104 Figure 5.1 - Hydrolysis of fluorescent substrate by crude pollen diffusates 113 Figure 5.2 - Measurement of the hydrolysis of the fluorescent substrate by the pollen diffusates 114 Figure 5.3 - SDS/PAGE analysis of pollen diffusates 115 Figure 5.4 - SDS/PAGE and Western blot analysis of pollen diffusates 115 Figure 5.5 - Gelatin zymography of Pollen Diffusates and sensitivity to inhibitors 117 Figure 5.6 - SDS/PAGE and MALDI reflectron TOF of tryptic peptides derived from band 4 (Lolium perenne) and band 12 (Cynodon dactylon) 119 Figure 5.7 - Two-Dimensional SDS/PAGE and gelatin zymography of the pollen diffusate of Bermuda grass 123 Figure 5.8 - Two-Dimensional Western Blotting of Bermuda grass pollen diffusate 124 Figure 5.9 - Tandem mass spectrometric analysis of tryptic peptides derived from spot 4 (Cynodon dactylon) 126 Figure 5.10 - Multiple sequence alignments of selected allergens and trypsin 135 6 Figure 6.1 - Size-exclusion chromatogram of gel filtration standards 138 Figure 6.2 - Size-exclusion chromatogram of Bermuda grass pollen diffusate 139 Figure 6.3 - SDS/PAGE and Gelatin Zymography of Size-Exclusion HPLC fractions of Bermuda grass pollen diffusate 140 Figure 6.4 - Anion-exchange chromatogram of Bermuda grass pollen diffusate 142 Figure 6.5 - SDS/PAGE and Gelatin zymography of Anion-Exchange chromatography fractions of Bermuda grass pollen diffusate 143 Figure 6.6 - Analysis of the protein bands separated by SDS/PAGE of fractions of Anion exchange HPLC 144 Figure 6.7 - Fluorescent substrate assay on Benzamidine Sepharose purified Fractions of Kentucky blue and Bermuda grass 148 Figure 6.8 - Protein concentrations of the Concanavalin A Sepharose separated fractions of Bermuda grass pollen diffusates by the BCA assay 151 Figure 6.9 - Hydrolysis of the fluorescent substrate (NBAMC) by the Con A Sepharose separated fractions of Bermuda grass pollen diffusate 152 Figure 6.10 - Hydrolysis of fluorescent substrate by the Con A Sepharose separated fractions of Bermuda grass pollen diffusate 153 Figure 6.11 - SDS/PAGE and gelatin Zymography of the Con A Sepharose separated fractions of Bermuda grass pollen diffusate 155 Figure 6.12 - Proteomic analysis of Con A Sepharose separated fractions of Bermuda grass 156 Figure 6.13 - Anion Exchange chromatography of the Bound fractions of Con A Sepharose affinity chromatography of Bermuda grass pollen diffusate 157 7 LIST OF TABLES Table 1.1 - Factors influencing the polarisation of Th responses 24 Table 1.2 - Functions of cytokines influencing the Th response 28 Table 2.1 - Plant Nomenclature Sub-Divisions 36 Table 2.2: Revised nomenclature of common pollen allergens.