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. 44 Table 2.3 - Localisation of allergens within pollen grains by electron microscopic methods 46 Table 2.4- Biological functions of pollen allergens 47 Table 2.5 - Nomenclature of Serine peptidase familles. 51 Table 2.6 - Nomenclature of Cysteine peptidase families 53 Table 2.7 - Nomenclature of Aspartic peptidase families. 55 Table 2.8 - Nomenclature of Metallopeptidase families. 57 Table 2.9 - Nomenclature of naturally occurring peptidase inhibitors 61 Table 2.10 - Commercially available natural class-specific peptidase inhibitors 62 Table 2.11 - Common synthetically manufactured class-specific peptidase inhibitors 63 Table 3.1 – Different types of solid phase assays 70 Table 3.2 - Chromogenic peptide substrates commonly used for the assessment of peptidase activity of different mechanistic classes 73 Table 3.3 - Common substrate coupled, quenched fluorescent groups used for the assessment of peptidase activity of different classes 74 Table 3.4 - Protein fractionation techniques 75 Table 3.5 - Commonly employed ion-exchange groups 78 Table 3.6 - Common group-specific ligands used in affinity chromatography 80 Table 3.7 - Proteomic separation techniques 86 Table 5.1 - MALDI-TOF/MS and LC/MS-MS analysis of proteins from 1D- SDS/PAGE of pollen diffusates 121 Table 5.2 - Summary of proteins of Bermuda grass pollen diffusate identified by micro C18 RP-HPLC and ESI/MS-MS analysis 127 Table 6.1 - Analysis of protein bands from anion-exchange HPLC separated fractions of Bermuda grass pollen diffusate by LC/MS-MS 145 Table 6.2 - Characteristics of Concanavalin A Sepharose separated fractions 149 Table 6.3 - Summary of the proteins identified by LC/MS-MS of Concanavalin A Sepharose separated fractions of Bermuda grass pollen diffusate 158

8 ABBREVIATIONS

2D-PAGE 2-dimensional polyacrylamide gel electrophoresis Arg Arginine AIA Aspirin induced asthma ASA Acetyl salicylic acid AEBSF 4-(2-aminoethyl) benzene sulphonyl fluoride amu atomic mass unit Bcl-6 B-cell lymphoma 6 protein BSA Bovine serum Albumin CC β-chemokines CXC α - chemokines CCR chemokine and chemoattractant receptors Con A Concanavalin A Cys Cysteine CD Clusters of differentiation CTLA-4 C T leukocyte antigen-4 c-maf Transcription factor - musculoaponeurotic fibrosarcoma (maf) Da Dalton DTT Dithiothretiol DC-SIGN Dendritic cell-specific ICAM-3-grabbing nonintegrin DEC-205 Lymphocyte antigen 75 DMP Dimethyl phosphate DC Dendritic cells E-64 ([N-(L-3-trans-transcarboxyoxiran 2carbonyl)-L-Leucyl]- agmatine EC Endothelial cells EDTA Ethylenediaminetetraacetic acid ECRHS European Commision for Research and Health Studies ESI Electrospray Ionisation eV electron volts Fig Figure FT-ICR Fourier transform- Ion cyclotron resonance mass spectrometer FEV1 Fixed expiratory volume/sec FVC Fixed vital capacity FcεRII Fc high affinity receptor 2 GM-CSF Granulocyte/macrophage-colony stimulating factor GATA Erythroid Transcription factor H Hour HPLC High performance Liquid Chromatography HLA Human Leukocyte antigen HLA-DM Human Leukocyte antigen – DM ICAM Intracellular adhesion molecule IEF Iso-electric focusing IFN Interferon Ig Immunoglobulin IL Interleukin ISAAC International study on asthma and allergies in children IMAC Ion- metal affinity chromatography

9 Kda Kilodalton keV Kilo electron volts LT Leukotriene Lys Lysine LAR Late asthma reaction Μltr Microlitre Mab Monoclonal antibody Min Minute mM Millimolar μM Micromolar MHC Major histocompatibility complex MMP Matrix metalloproteases MIP-3β Macrophage inhibitory protein -3β MS Mass Spectrometry MS-MS Tandem mass spectrometry MDC Macrophage derived chemokine NF-κB Nuclear factor-κB NSAID Non-steroidal anti inflammatory drugs OVA Ovalbumin pI Iso-electric point PVDF Polyvinylidene difluoride PMF Peptide mass fingerprinting PMSF Phenyl methane sulphonyl fluoride RP-HPLC Reverse phase-high performance liquid chromatography Rpm Revolutions per mineute RANTES Regulated on activation normal T-cell expressed and secreted RF Radiofrequency SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis Ser Serine SOCS Suppresors of cytokine signalling TNF Tumor necrosis factor TIMP Tissue inhibitor of metalloprotease TRANCE TNF related activation - induced cytokine TARC Thymus and activation related chemokine TPCK Tosyl phenylalanyl chloromethy ketone TLCK Tosyl lysyl chloromethyl ketone TFMK Trifluoro methyl ketone TFA Trifluoroacetic acid

10 ACKNOWLEDGEMENTS

I would like to express my heartfelt gratitude and acknowledge many people for their individual and group contributions:

Firstly, to my supervisors, Professor Rakesh K. Kumar and Dr. Mark J. Raftery for giving me the opportunity to undertake this research project under their guidance and their invaluable insight and friendship. To Professor Denis Wakefield and the School of Medical Sciences for the opportunity to learn in a highly stimulating academic environment. To Dr. Mark Raftery for his guidance and assistance in all aspects of Proteomics and Mass Spectrometry. To Professor Carolyn Geczy for allowing me to conduct my research work and avail all the facilities in her laboratory and her invaluable advice and support. To all the members of the Biomedical Mass Spectrometry Facility, UNSW for their assistance, friendship and encouragement, throughout this research project. To Dr. Anne Poljack and Dr. Valerie Wasinger for their advice on procedures relating to proteomics and their warmth and friendship. To Dr. Nicodemus Tedla and Dr. Zheng Yang for all their friendship and assistance during the initial periods, in the lab and outside. To all the members, past and present of the Inflammatory Diseases Research Unit (IDRU) for their friendship and support. I thank Mr. Farid Rahimi, Dr. Changie Song and Dr. Hong Cai for their constant support in teaching me the ropes around a new lab and their warm disposition. To my family, for their unwavering support and encouragement both financially and emotionally throughout my studies. Finally to my fiancée, Natasha for her love, belief and patient support through the long and arduous times. You’re my pillar of support.

11 PUBLISHED WORK FROM THIS THESIS

Mark J. Raftery, Rohit G. Saldanha*, Carolyn L. Geczy and Rakesh K. Kumar. Mass Spectrometric analysis of electrophoretically separated allergens and proteases in grass pollen diffusates. Respir Res. 2003; 4(1):10. Epub 2003 Sep 20.

(*RGS contributions to the publication included development of the fluorescent substrate assay for the detection of peptidase acitivity, analysis of pollen diffusates by one- and two-dimensional electrophoresis, Western blotting, zymography and editing of the manuscript). ABSTRACTS

Rohit Saldanha, Mark J. Raftery, Carolyn L. Geczy and Rakesh K. Kumar (2003). Characterization of pollen enzymes and their relationship to pollen allergens. – American Society of Mass Spectrometry Conference, Montreal 2003.

Rohit Saldanha, Mark Raftery, Carolyn Geczy and Rakesh Kumar (2003). Characterization of pollen enzymes and their relationship to pollen allergens. – The 24th Protein Structure and Function Conference, Lorne, Victoria, Australia February 2003.

Rohit Saldanha, Mark Raftery, Carolyn Geczy and Rakesh Kumar (2002). The role of pollen proteases and pollen enzymes in allergic diseases. Student Research Induction day Poster presentation sponsored by Merck Sharpe and Dohme, Australia. (Best student poster presentation).

12 ABSTRACT

Pollen grains are ubiquitous triggers of allergic asthma and seasonal rhinitis. Proteolytic enzymes in pollen as well as other sources are capable of disrupting airway epithelial integrity in vivo and in vitro. This provides a plausible mechanism for the initiation of sensitisation of the respiratory immune system to inhaled pollen allergens, comparable to that suggested for Group 1 allergens from house dust mites and cat dander, which are known to possess intrinsic proteolytic activity. This thesis explores the relationship between pollen allergens and proteolytic enzymes. It describes the different strategies used for the characterisation, purification and identification of immunogenic and proteolytic proteins in the complex mixtures of pollen diffusates. The peptidases in the diffusates of Kentucky blue grass, ryegrass and Bermuda grass pollens were characterised by a sensitive fluorescence assay and gelatin zymography. Among these, Bermuda grass pollen demonstrated the presence of a serine peptidase at Mr ~30,000 Da, which corresponded to an intense band by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen, Phl p 1. Purification of this enzyme from Bermuda grass was complicated by the low levels of the enzyme present in the diffusate, as well as by its autohydrolysis. Partial purification of the serine peptidase activity by affinity chromatography using Concanavalin A Sepharose demonstrated that the diffusate contained a trypsin-like peptidase, detected by the fluorescent assay, in addition to the ~30,000 Da serine endopeptidase, detected on gelatin zymography. Proteomic analysis of the ~30,000 Da protein using one- and two-dimensional electrophoresis and mass spectrometry identified it as the major pollen allergen of Bermuda grass, Cyn d 1. The studies reported here provide, for the first time, evidence that a pollen allergen may possess intrinsic proteolytic activity. This activity may play a role in the initiation of airway inflammation and allergic sensitisation.

13 1. ASTHMA AND SENSITISATION IN ALLERGIC DISEASES

1.1 Defining Asthma and its Clinical Presentation

Allergic diseases such as asthma, allergic rhinitis and atopic eczema are the commonest manifestations of a type 1 hypersensitivity reaction. The immune response generated in hypersensitivity mediated diseases results in tissue damage and related patho-physiological changes (Bukantz 2002). Allergic asthma is a complex disease process of the respiratory system. It is a chronic inflammatory disease of the airways, brought on by external antigens, which is primarily characterised by bronchial hyperresponsiveness and airway obstruction that is reversible, spontaneously or with treatment (Toelle, Peat et al. 1997). While most asthma is allergic and characterised by the presence of allergen specific IgE, in some patients, no immunological mechanism can be detected; this is sometimes referred to as intrinsic asthma (Rackemann 1947). In spite of extensive research, the aetiology and pathogenesis of asthma are still poorly understood. A number of genetic and environmental factors contribute to the development of the disease. In many cases the clinical symptoms overlap with other associated atopic diseases such as allergic rhinitis and/or atopic dermatitis, indicating a common mechanism. The clinical presentation in asthma is a spectrum ranging from relatively mild episodes of wheezing to extreme respiratory distress as seen in status asthmaticus. The common symptoms include wheezing, cough, dyspnoea, chest tightness and the production of sputum (Mintz 2004). The symptoms correlate well with the clinical signs in asthmatic patients such as bronchial hyperresponsiveness and airway obstruction. These manifestations are primarily attributed to -: 1. Contraction of the airway smooth muscles 2. Oedema of the walls of the upper and lower airways 3. Increase in the luminal contents as a result of inflammatory exudation and mucus secretion. 4. Remodelling of the airway walls, reducing the internal diameter of the airway lumen (Bousquet, Jeffery et al. 2000) 5. Reduced patency of the smaller airways, possibly due to an increase in the surface tension and surfactant dysfunction (Enhorning, Duffy et al. 1995; Liu, Wang et al. 1996).

14 All these factors could potentially induce the closure of the non-cartilaginous smaller airways that results in a decrease in lung function as assessed by the timed vital capacity (FEV1) and fixed vital capacity (FVC). Manifestations of diminished lung function, in part the result of compensatory mechanisms, include hyperventilation, tachycardia, occasional cyanosis, the use of accessory muscles and intercostal recession (Bousquet 2000).

1.2 Inflammatory Responses in Asthma

Inflammation of the airways is a well established central feature in the pathogenesis of asthma and its clinical manifestations. The presence of airway inflammation has been demonstrated in early stages of the disease, even when the clinical symptoms are minimal or absent. The characteristic pattern of inflammation observed following exposure to a triggering stimulus has led to the description of a biphasic response-: 1. A brief early phase response, followed by 2. A more prolonged late phase response.

1.2.1 The Early Phase Response

The early phase reaction in asthma occurs as a result of the interaction between an allergic stimulus and the immune machinery of the sensitised individual. The interaction between the allergen and the IgE on the surface of pro-inflammatory cells such as mast cells and eosinophils results in their rapid activation, leading to the production of a variety of mediators (Busse and Lemanske 2001). Many of these mediators induce airway smooth muscle contraction, which is a prominent feature in acute asthmatic attacks. Histamine is the archetypal mast cell mediator, characterised by broncho- constrictor effects on the airway smooth muscle as well as the ability to cause vasodilation and to increase vascular permeability. These effects are mediated by H1 receptors whereas increased mucus secretion is mediated by the stimulation of H2 receptors (Fraenkel and Holgate 1996; Tamaoki, Nakata et al. 1997). Tryptase, a proteolytic enzyme secreted by mast cells, can potentiate histamine-induced airway contraction. It is also capable of activating protease activated receptors (PAR) on the epithelial and endothelial cells, resulting in the accentuation of other inflammatory cascades such as the up-regulation of adhesion molecules which selectively attract

15 inflammatory cells like eosinophils and basophils (Holgate 1999; Asokananthan, Graham et al. 2002). Eosinophils also produce and/or release important mediators, including major basic protein and eosinophilic cationic protein (which are stored in granules) as well as the sulphidopeptide leukotrienes, LTC4/D4 and E4 (synthesised) (Wardlaw, Brightling et al. 2000). All of these are capable of injuring the respiratory epithelium.

1.2.2 The Late Phase Reaction

The early phase response is transient and subsides within an hour, usually corresponding with the decline in short-acting mediators. However it also results in the activation of the late phase response, which sets in after about 6-12 hours. This is clinically characterised by the presence of bronchial hyperreactivity, which is not seen during the early phase response (Bochner and Schleimer 2001). The late phase response occurs as a result of the activation of T-lymphocytes by the antigen presenting cells. This specifically results in the release of Th2 type cytokines described later. The release of Th2 cytokines results in the recruitment of additional inflammatory cells such as eosinophils, basophils, neutrophils and macrophages (Jarjour, Calhoun et al. 1997; Peters, Zangrilli et al. 1998). This prolonged inflammatory response is essentially maintained by the Th2 cytokines and can persist for more than 24 hours. Repetitive cycles of inflammation and tissue damage result in the progression of the disease into a chronic phase.

1.3 Effects of Airway Inflammation

1.3.1 Respiratory Epithelium

The bronchial epithelium acts as a protective physical barrier between allergens in the external environment and the immune system. Allergen-induced responses have two distinct detrimental effects on the epithelium: 1. Physical destruction, resulting in the loss of epithelial integrity 2. Activation of the respiratory epithelium, contributing to airway inflammation. The physical destruction of the respiratory epithelium occurs due to the partial shedding of the columnar cells and is characterised by the presence of epithelial cell clumps known as “Creola bodies” in the sputum (Woltmann, Ward et al. 1999).

16 Damage by inflammation results in the disruption of the epithelial tight junctions. This opens the para-cellular channels and allows the subsequent ingress of foreign, antigenic proteins (Wan, Winton et al. 1999). It also results in the decreased production of epithelium derived relaxation factors causing the accentuation of bronchial hyperresponsiveness (Jeffery, Wardlaw et al. 1989; Rabe, Dent et al. 1995). Other inflammation-related changes include goblet cell hyperplasia and mucus hypersecretion (Bousquet, Jeffery et al. 2000). Damage to the respiratory epithelial cells results in their activation, which accentuates the inflammatory cascade. Activated epithelial cells are capable of producing a large number of pro-inflammatory mediators such as 15-HETE (Campbell, Chanez et al. 1993), eotaxin (Lilly, Nakamura et al. 1997), TNF-α, GM- CSF (Pertovaara, Kaipainen et al. 1994) and matrix metalloproteases (Yao, Buhler et al. 1996). These factors promote the differential recruitment of inflammatory cells and amplify the inflammatory process. The airway epithelial cells also secrete an array of growth factors, extra-cellular matrix proteins and fibronectin, which are involved in the process of airway repair and remodelling (Erjefalt and Persson 1997).

1.3.2 Airway Remodelling

Damage to the inflamed epithelium initiates a remodelling response, leading to the restoration of structural integrity. However, in asthma this response is abnormal, altered and uncontrolled. The prominent features of airway remodelling are -: 1. Hypertrophy and hyperplasia of airway smooth muscle 2. Increased numbers of myofibroblasts and fibroblasts in the sub- epithelial region, with collagen deposition. 3. Increased numbers of mucus secreting cells 4. Neo-vascularisation The persistence of chronic inflammation is traditionally accepted as the leading cause of airway remodelling. However, airway biopsy studies in young children demonstrate that tissue restructuring can occur four years before they are even symptomatic, indicating the parallel onset of structural changes and inflammation (Warner, Marguet et al. 1998). One possible explanation for this observation, which has gained popularity, is the effect of the early inflammatory

17 response on the epithelial-mesenchymal interface in the airways, known as the “Epithelial-mesenchymal trophic unit” (EMTU) (Holgate, Davies et al. 2000). The damaged epithelium is a source of pro-inflammatory mediators, growth factors and fibrogenic factors such as EGF, IGFs, etc (Knobil and Jacoby 1998). These factors contribute to the terminal activation and differentiation of myofibroblasts, resulting in the secretion of interstitial collagen into the sub-basement membrane as well as the expression of additional growth factors such as vascular endothelial growth factor, which is mitogenic for epithelial cells and smooth muscle cells (Knobil and Jacoby 1998). Transforming growth factor-β (TGF-β) plays a crucial role in the activation of the EMTU. An increased expression of TGF-β by myofibroblasts is inhibitory for epithelial proliferation, which results in the further activation/differentiation of myofibroblasts by TGF-β in an autocrine fashion. This repetitive cycle leads to the thickening of the lamina reticularis due to collagen deposition, leading to “sub- epithelial fibrosis” of the airways (Moses, Yang et al. 1991; Holgate, Davies et al. 2000). The functional consequences of these remodelling changes include exacerbations in bronchial hyper-responsiveness, and a decrease in the lung capacity of the asthmatic patient brought about by a decrease in airway distensibility.

1.4 Classification of Asthma

The relatively poor understanding of the aetiology of asthma has always complicated its classification. There is no clear consensus on how and what extent variable environmental factors such as antigen exposure, life style changes including diet and vaccination state influence the genetic susceptibility of an individual, skewing the immune status towards an atopic phenotype (Cookson 1999). Therefore it comes as no surprise that asthma is classified in a number of ways, addressing some but not all facets of the aetiology. A traditional system of classification proposed by Rackemann in 1947 (Rackemann 1947) (and still widely used) is on the basis of the demonstration of an IgE dependent mechanism in the disease process. Asthma mediated by an identifiable IgE mediated mechanism is termed “Extrinsic Asthma” whereas cases in which no such mechanism is identifiable are termed non-allergic or “Intrinsic Asthma”.

18 1.4.1 Extrinsic Asthma

Most diagnosed asthma is of the extrinsic variety. The hallmark of extrinsic asthma is the increased production of allergen specific IgE following allergen exposure of the immune system. Interaction between allergens and specific IgE bound to the surface of pro-inflammatory cells results in the initiation of the asthmatic response (Busse and Lemanske 2001). In extrinsic asthma, involvement of the humoral immune system requires prior sensitisation to the environmental allergen. This is a function of at least four factors namely, genetic predisposition, age of exposure, dosage of allergens and duration of exposure to the allergens. Secondary factors such as concomitant respiratory infections, socio-economic factors and pollution contribute to the exacerbation of the process and play an important role in the ultimate prognosis (Warner and Warner 2000).The most common environmental allergens that can cause atopic/extrinsic asthma are aeroallergens, such as those from the house dust mite and allergens from the pollens of grasses, trees and weeds (Chua, Stewart et al. 1988; Suphioglu 2000).

1.4.2 Intrinsic Asthma

In contrast to extrinsic asthma, Rackemann had described “intrinsic asthma” as a sub-set of the disease, where an immune mediated mechanism could not be identified, and hence this was deemed to be a non-allergic variant of asthma (Rackemann 1947). Bronchial constriction in these cases occurs via a number of non- immune pathways, such as exposure to chronic irritants, pharmacological agents, weather conditions (McFadden, Lenner et al. 1986; McFadden and Gilbert 1994), etc. Similarly damage to the respiratory epithelium due to chronic pulmonary infections or inhalation of toxic chemicals can mimic bronchial hyperresponsivness, causing wheezing (Bernstein 1992; Kabalin and Greenberger 1996; Zacharisen 1996). However, a number of studies indicate that despite the lack of allergen specific IgE in intrinsic asthma, the two sub-sets of asthma possess many common immunopathological features (Humbert, Menz et al. 1999). These include the presence of a pro-eosinophilic environment, increased IL-4 and IL-5 levels and a increase in the total serum levels of IgE in both sub-sets of asthma (Romanet-Manent, Charpin et al. 2002).

19 The most well documented type of intrinsic asthma is drug-induced, aspirin sensitive asthma (ASA) (Babu and Salvi 2000). Although the presentation of ASA is similar to a type 1 hypersensitivity reaction, attempts to demonstrate antibodies to acetylsalicylic acid or its derivatives have proved futile. There is significant cross- reactivity in ASA subjects to other non-steroidal anti inflammatory drugs (NSAID) or cycloxygenase inhibitors such as diclofenac, indomethacin etc (Simon 1996). This correlates well with the mounting evidence implicating the cycloxygenase (COX) and lipoxygenase (LO) pathways in the pathogenesis of ASA. The inhibition of the COX pathway by NSAID results in the deviation of the arachidonic acid metabolites towards the LO pathway, resulting in the increased production of cysteinyl leukotrienes, which are potent bronchoconstrictors (Babu and Salvi 2000). Consistent with this finding is the fact that bronchoconstriction in ASA can be inhibited with leukotriene receptor antagonists (Kallos and Schlumberger 1980). However, an immunological input to this disease cannot be ignored. Szczeklik and colleagues reported that approximately one third of the aspirin induced asthma (AIA) patients in their cohort study exhibited both clinical and immunological markers of atopy, which is similar to prevalence data reported in other large community surveys in European adults (Szczeklik, Nizankowska et al. 2000).

1.5 Prevalence of Asthma

It is now a well established fact that the world-wide prevalence of asthma is on the rise with some countries exhibiting greater sustained prevalence rates than others. However, the interpretation of these emerging patterns is hampered by a lack of uniformity in diagnosis, methods for the measurement of prevalence and demographic differences between different cohort studies. Nevertheless, it is clear that in spite of established avoidance and therapeutic protocols in place for the management of asthma, there has been a steady increase in the incidence of asthma admissions over the past three decades (Halken 2003). Genetic predisposition fails to account for such a sharp increase in prevalence rates over such a short period. There is compelling data to indicate that changes in the environment over this period have played a dominant role. The two most well validated, global cohort studies on asthma prevalence are the ISAAC (International Study of Asthma and Allergies in Children) study in children (Strachan, Sibbald et al. 1997) and the ECRHS (The European Community

20 Respiratory Health Survey) study in adults (ECRHS 1996). Both studies have compared international and regional prevalence on a large scale using standardised methods throughout in the assessment and evaluation of the symptoms of asthma and related allergic diseases. In addition, the findings of the two studies have been compared to study any causal relationship between asthma in children and adults. The studies indicate that childhood asthma is a risk factor for the development of adult asthma and that the highest prevalence rates are mostly confined to Western countries such as UK, New Zealand, Australia and Canada. In comparison, prevalence rates of asthma in underdeveloped or developing countries such as China, India and Africa have been reported to be much lower (Lai, Douglass et al. 1996; Pearce, Sunyer et al. 2000). The polarisation of prevalence rates serves to suggest that changes in environmental pollution levels, dietary changes and vaccination can alter the genetic susceptibility of individuals to asthma. These results also seem consistent with the recently proposed theory of the protective role of infections during early childhood (von Mutius, Illi et al. 1999; Ball, Castro-Rodriguez et al. 2000; Matricardi, Rosmini et al. 2000). The variations in the prevalence rates of asthma in populations of similar ethnic backgrounds, but living in different regions further implicates environmental conditions as a decisive factor in modulating asthma susceptibility. Von Mutius et al found a higher prevalence of asthma in children in West Germany compared to East Germany (von Mutius, Martinez et al. 1994). A similar prevalence pattern was noted in some regions of Asia such as Hong Kong, which is more affluent in comparison to others like San Bu, a rural region 200 kilometres away from Hong Kong and Kota Kinabalu, near the island of Borneo (Leung and Ho 1994). Australia currently ranks as the third highest in the world for the prevalence of wheezing in the age group of 6-7 year olds (24.6%) and second highest in the 13-14 year old age group (29.4%) (Robertson, Dalton et al. 1998). According to the ECRHS study, Australian ranks fourth highest for the prevalence of self-reported current wheeze symptoms in the age group of 22-44 (ECRHS 1996). There is a paucity of similar data on the prevalence of asthma in indigenous Australian children, which needs to be addressed. However, recent studies in line with the ISAAC studies demonstrated that there is a high prevalence rate amongst the Aboriginal and Torres- Strait Islander populations (Veale, Peat et al. 1996). This is in contrast to the view that the genetic susceptibility to asthma is greater in Caucasian populations. Additional

21 data may provide a framework for the development and testing of new hypotheses, which will better explain the aetiology of the disease.

1.6 Immunological Sensitisation

The high prevalence rate of asthma in children indicates that the inflammatory process is initiated early in life. Although atopic individuals have a genetic predisposition towards developing allergic immune responses (Blumenthal and Blumenthal 2002), they do not exhibit a classical Mendelian, single gene inheritance pattern. Genetic predisposition in susceptible individuals is manifested in their ability to develop altered T cell responses during childhood, probably unmasked by the exposure of the immune system to different environmental conditions (Halken 2003). Several studies have demonstrated that at birth, the immune response state is predominantly humoral, characterised by the presence of allergen specific T cells in the cord blood (Prescott, Macaubas et al. 1998). This “allergen inclined” state of responsiveness usually gets modified after birth to a non-allergic phenotype by several environmental and lifestyle factors such as vaccination (Wjst, Dold et al. 1994), childhood infection, diet (Black and Sharpe 1997), tobacco smoke (Kulig, Luck et al. 1999) and air pollution (Knox and Suphioglu 1996). Failure in the modulation of this default immune state after birth can result in the progression of a “Th2 type” immune response, which is associated with atopic disease (Holt, Sly et al. 1997; Warner and Warner 2000).

1.7 Antigen Presentation and development of T cell Responses.

The activation of an atopic immune response fundamentally requires cognate interactions between the immunocompetent T cells and the relevant antigens. This cognate interaction is highly specific and is dependent on the appropriate presentation of the antigens by dedicated antigen presenting cells, which are capable of modulating a humoral or cell mediated immune response. In this context, dendritic cells are the most competent antigen presenting cells (APC), located at the interface between the external milieu and the internal immune system. They form an extensive network of cells extending from the mucosa of the mouth, nose and airways down to the gut and genito-urinary tract (Lambrecht 2001). These cells are bone marrow derived and possess heterogenous antigen presenting capabilities (Rissoan, Soumelis et al. 1999; Reid, Penna et al. 2000). Dendritic cell recruitment is initiated and accelerated during

22 periods of acute inflammation, triggered by different antigens such as bacteria (McWilliam, Nelson et al. 1994), viruses (McWilliam, Napoli et al. 1996) and allergens such as pine dust (Schon-Hegrad, Oliver et al. 1991; McWilliam, Napoli et al. 1996) and house dust mites (Jahnsen, Moloney et al. 2001; Upham, Denburg et al. 2002). Allergens that have traversed the airway barriers come into contact with the circulating dendritic cells and subsequently get endocytosed by a number of different mechanisms (Banchereau, Briere et al. 2000; Geijtenbeek, Krooshoop et al. 2000; Geijtenbeek, Torensma et al. 2000; Mahnke, Guo et al. 2000; Noirey, Rougier et al. 2000). These antigenic proteins are cleaved into short immunogenic peptides exposing the T cell epitopes that might otherwise have been sterically hindered. These immunogenic peptides form a complex with the appropriate major histocompatibility complex (MHC) proteins and get expressed on the surface of dendritic cells (Cella, Engering et al. 1997). This allows for naïve, circulating T cells to survey the dendritic cell’s surfaces, recognise the antigens and mount the appropriate immune response. The interactions between the dendritic cells and the CD4+ T cells can occur both locally, at inflammatory sites as well as distantly within the regional lymph nodes (Harris, Watt et al. 2002). Myeloid dendritic cells in mice have demonstrated the capacity to retain allergens for prolonged periods and are capable of T cell activation about 8 weeks post-allergen challenge (Julia, Hessel et al. 2002). This is important since such a mechanism would allow dendritic cells to maintain inflammatory events despite maximal allergen avoidance. Among the lymphocytes, the Antigen-MHC class II complex is exclusively recognised by the CD4+ subset of T-lymphocytes (Seder, 1994). They are also termed “T helper” cells (Th) as they interact with B-lymphocytes, influencing their clonal proliferation, differentiation and production of antigen specific antibodies. The CD4+ T lymphocytes can exhibit two distinct phenotypes known as the Th1 or Th2 cells (Mosmann, Cherwinski et al. 1986). Table 1.1 provides an overview of the functional differences between the two phenotypes. While Th cells exhibiting overlapping phenotypic characteristics certainly exist, the Th1/Th2 paradigm is helpful for understanding the role of T cells in cell-mediated immunity and allergic disease. The Th2 phenotype of CD4+ T cells is largely responsible for the activation and mediation of the allergen induced inflammatory response. Activated cells release Th2 type pro- inflammatory cytokines, which induce the transcription of germ line ε mRNA in B

23 cells and induce the isotype switch from IgG to IgE (Zurawski and de Vries 1994). The activation of B cells results in their differentiation and clonal proliferation into plasma cells, resulting in the secretion of large amounts of allergen specific IgE antibodies. The allergen specific IgE circulates in the blood stream before coming into contact with the high affinity IgE receptors, FcεRI on the surface of pro-inflammatory cells such as mast cells and basophils. Alternatively, it can also bind to low affinity IgE receptors on the surface of lymphocytes, eosinophils and macrophages. Molecular bridging of these IgE receptors with the circulating antigens results in the subsequent activation of the cells and the release of their mediators (Corry and Kheradmand 1999).

Table 1.1 - Factors influencing the polarisation of Th responses Adapted from (Constant and Bottomly 1997).

Favouring Th1 Favouring Th2 Variable factors response response Antigen Dosage High dosage Low dosage Type Candida albicans, yeast Candida albicans hyphae Toxoplasma antigens Allergens ? Adjuvants Gram positive cell wall Toxins Occurs early after LPS Occurs late after LPS stimulation stimulation Histocompatibilty interactions High affinity Low affinity Short interaction Sustained interactions Dendritic cell Maturity Mature Immature interactions Lineage Monocyte derived DC Macrophage derived Lymphoplasmacytoid dendritic DC cells CD1+ a myeloid DC Co-stimulatory influences Low expression CD80/CD86 OX40-L ICAM-1 ICOS-L (B7RP-1 or –2) CD40 Local cytokine profile Low IL-12 High IL-12 High IL-10 Low IL-10 High IL-6

24 1.8 Factors Influencing T cell Activation Responses

The allergen induced inflammatory cascade is tightly regulated at a number of levels. These include -: 1. Co-stimulatory interactions 2. Cognate cellular interactions 3. Soluble pro-inflammatory factors.

1.8.1 Co-Stimulatory Interactions

The simple interaction between the antigen complex on dendritic cells and the T cell receptors normally result in tolerogenic effects and apoptosis of the T lymphocytes rather than their activation (Banchereau and Steinman 1998). This tolerogenic effect is circumvented in allergic reactions by a concomitant co- stimulatory signal, which results in T cell proliferation and the generation of effector and memory cells. The most important co-stimulatory signals molecules are provided by the CD28 family of receptors located on naïve T cells and their corresponding ligands, CD80 (B-7.1) and CD86 (B-7.2), expressed on the surface of dendritic cells (Djukanovic 2000; Turley, Inaba et al. 2000). The interaction between the peptide- MHC II complex and the T cell receptor, on the surfaces of the dendritic cells and T cells respectively, results in the recruitment of the TCR and CD28 molecules towards the site of interaction with dendritic cells (Wulfing and Davis 1998). Similarly, dendritic cells concentrate the CD 86 molecules and the peptide MHC complexes in lipid rafts on their surfaces, towards the sites of T cell contact (Anderson, Hiltbold et al. 2000). These interactions result in the upregulation of CD40 ligand-receptor interaction, which in turn induces T-cells to secrete cytokines, upregulate CD80 and CD86 on dendritic cells and stimulate the migration of T-cells and B-cells to the B cell follicles (Banchereau, Briere et al. 2000). As a regulatory mechanism, expression of the CD28 ligand homologue, CTLA-4 on the T cells results in the inhibition of the T cell response (Corry and Kheradmand 1999). In vivo studies in murine models of asthma have demonstrated that blocking of the CD28/B-7 interactions using CTLA-4 Ig (a fusion protein) resulted in the abrogation of the T cell responses as well as airway hyperresponsiveness and inflammatory cell infiltrate (Krinzman, De Sanctis et al.

25 1996). CTLA-4 has much higher affinity for the B-7 ligand, which possibly helps in the control and inhibition of the inflammatory response.

1.8.2 Cognate Cellular Interactions

The differentiation and clonal proliferation of B cells capable of producing allergen specific IgE are dependent on cognate interactions with effector Th2 lymphocytes and the Th2 cytokine environment. It was initially assumed that recognition signals from the TCR/CD3 complex on the T cells and its corresponding ligands on the B cells were sufficient for the induction of IgE synthesis (Stanciu, Roberts et al. 2001). However more recent studies have demonstrated a requirement for the interaction between CD40 receptors on the surface of B cells and the CD40L on activated but not resting T cells, for the activation of B cells into IgE producing plasma cells (Mehlhop, van de Rijn et al. 2000). The importance of this cognate cellular interaction is highlighted by the fact that soluble CD40 inhibits T cell dependent IgE synthesis and that PBMC from patients with hyper IgM syndrome, whose T cells lack the CD40L molecules, are unable to synthesise IgE in the presence of IL-4 (Fuleihan, Ramesh et al. 1993).

1.8.3 Soluble Pro-inflammatory Factors

Cytokines play a pivotal role in the maintenance of atopic responses by regulating the expansion of Th2 cells and mediating many of the inflammatory events that underlie asthma. The two CD4+ T cell subsets secrete mutually distinct profiles of cytokines, co-ordinating different classes of immune responses. Th1 lymphocytes secrete IL-12, IFN-γ, TNF-β, and are associated with cell mediated, cytotoxic responses against infections. Th2 cells on the other hand produce IL-4, IL-5, IL-6, IL-9, IL-10, IL-13 and GM-CSF and influence all the major components of the allergic inflammatory response, such as: 1. Isotype switching from IgG to IgE in B-lymphocytes 2. Eosinophil mobilisation and survival 3. Maturation and activation of mast cells 4. Mucus production

26 Table 1.2 describes some of the major roles of the pro-inflammatory cytokines involved in asthma. In addition to cytokines, chemokines provide a chemotactic gradient for the recruitment of inflammatory cells to the airways, where they can mediate their actions. Chemokines are small chemotactic cytokines (8-10 kDa) involved in attracting leukocytes into tissues. They are sub-divided into four families on the basis of the relative position of their conserved cysteine residues, CXC, CC, C and CX3C family of chemokines (Chung and Barnes 1999). The potential role of chemokines in asthma is supported by the observation that many cells in the asthmatic airways are capable of producing chemokines, particularly monocytes/macrophages, activated T-lymphocytes, airway smooth muscle cells and airway epithelium (Chung and Barnes 1999) The main chemokines of interest in asthma are: 1. Eotaxin-1, eotaxin-2 and MCP-1 which are chemotactic for eosinophils, basophils and Th2 lymphocytes. Eotaxin is highly selective for eosinophils and recruits these cells to inflammatory sites by binding to its receptors CCR3. These chemokines can also influence cell degranulation, angiogenesis and the regulation of IgE responses (Uguccioni, Mackay et al. 1997; Bonecchi, Bianchi et al. 1998; Sallusto, Lanzavecchia et al. 1998; Sallusto, Lenig et al. 1998; Zingoni, Soto et al. 1998) 2. RANTES and macrophage inflammatory protein-1α (MIP-1α) are chemotactic for memory CD4+ T cells. RANTES is the most effective chemoattractant for basophils. 3. IL-8 is secreted by bronchial epithelial cells and is a major neutrophil activator and chemoattractant. It is responsible for the exocytosis of enzymes and proteins from neutrophils and activates neutrophil leukotriene expression. 4. The upregulation and/or down regulation of chemokine receptors on lymphocytes facilitate their migration along an established chemokine gradient. Down regulation of the receptor CR7 on Th2 lymphocytes and the concomitant upregulation of CXCR5 and CCR4 directs the activated T cells from the regional lymph nodes to the B cell follicles (Sallusto and Lanzavecchia 2000). 5. Antigen primed B cells are more responsive to CCL10 (MIP-3β), which attracts them to naïve T cells, thus reciprocally activating T lymphocytes.

27 Table 1.2 - Functions of cytokines influencing the Th response Cytokines Pro-Inflammatory functions in Asthma

IL-4 1. Derived from T-lymphocytes, eosinophils, basophils and mast cells (Chung and Barnes 1999). 2. Strongest influence in driving Th2 mediated differentiation response (Swain, Weinberg et al. 1990). 3. Inhibits the maturation of Th1 lymphocytes (Szabo, Dighe et al. 1997). 4. Increases the expression of MHC class II molecules on B cells, CD23 (FcεRII), CD40 (Chung and Barnes 1999). 5. Stimulates expression of co-stimulatory ligands, B7.1 and B7.2. 6. Promotes isotype switching of IgG to IgE in B cells. 7. Suppresses IFN-γ and down regulates IL-12β receptor chain. 8. Promotes growth of myeloid and erythroid progenitors. 9. Promotes goblet cell metaplasia and mucus hypersecretion (Corry and Kheradmand 2002). IL-13 1. Similar to IL-4 in functions. 2. Shares IL-4 receptor α sub-unit for high affinity receptor binding (Corry 1999). 3. Potent modulator of human monocyte and B cell functions. 4. Induces expression of CD23 on B cells, directing the synthesis of IgE (Punnonen, Aversa et al. 1993). 5. Induces inflammation, mucus hypersecretion and sub-epithelial fibrosis. 6. Unlike IL-4, it can induce airway hyperresponsiveness (Wills-Karp 1999). IL-9 1. Pleiotropic effects on many cell types. 2. Enhances proliferation of activated T cells (Hultner, Druez et al. 1990). 3. Enhances production of IgE in B cells (Louahed, Kermouni et al. 1995). 4. Promotes proliferation and differentiation of mast cells and other haemopoietic progenitor cells (Shimbara, Christodoulopoulos et al. 2000). 5. Up regulates the expression of eotaxin and chemokines by lung epithelial cells (Dong, Louahed et al. 1999). 6. Stimulates mucin production in vivo (Louahed, Kermouni et al. 1995). IL-5 1. Vital in the up regulation of eosinophil responses (Coffman, Seymour et al. 1989; Sampson 2001). 2. Regulates eosinophil activation, maturation and survival (Clutterbuck, Hirst et al. 1989).

28 3. Influences eosinophil adhesion to endothelium via β1 and β2 integrins, increasing eosinophil accumulation and cytotoxic mediator release (Gleich 1990; Walsh, Hartnell et al. 1990). TNF-α 1. Expressed in many cells, including macrophages, T lymphocytes, mast cells and epithelial cells (Kips, Tavernier et al. 1993; Kips 2001). 2. Increases airway responsiveness (Kips, Tavernier et al. 1993). 3. Stimulates additional secretion of cytokines from inflammatory cells in the airways. GM-CSF 1. Expressed in macrophages, T lymphocytes, fibroblasts, endothelial cells and airway smooth muscle. 2. Prolongs eosinophil survival and activity in BAL (Park, 1998). 3. Accounts for the increased secretion of leukotrienes and superoxide (Martinez-Moczygemba and Huston 2003). 4. Implicated in airway remodelling.

29 1.9 Intracellular Signalling Mechanisms Regulating T cell Differentiation

The activation of T-lymphocytes occurs via specific, intracellular signalling pathways. Research on the signalling cascades that influence effector T cell functions reveal that the cytokine stimulation signals are primarily transduced by the JAK-STAT transcription factors (Darnell 1997). However, the distinct profile of cytokines secreted by the CD4+ T cell phenotype influences which intracellular pathway gets activated. The binding of cytokines to their respective receptors results in the activation of the JAK family of receptor-associated kinases. These receptors subsequently activate, via tyrosine phosphorylation, pre-existing cytoplasmic factors known as STATs (signal transducers and activators of transcription). Tyrosine phosphorylation allows the STAT proteins to dimerize and translocate to the nucleus, where they mediate changes in gene expression by binding to specific DNA elements (Pernis and Rothman 2002). In naïve CD4+ T cells, although IL-4 and IL-12 both follow this basic signalling framework, the two cytokines differ in the specific JAK and STAT proteins that they activate (Wurster, Tanaka et al. 2000). IL-4 stimulates JAK 1 and JAK 3 to activate STAT 6. In contrast interaction of IL-12 with its receptors leads to the activation of JAK 2 and TYK 2 and the subsequent phosphorylation of STAT 4. In the differentiation of T helper cells, a change in the configuration of chromatin is postulated to cause the accessibility of specific transcription factors leading to the preferential production of cytokines. Agarwal and Rao hypothesised that naïve T cells possess a closed chromatin structure around the genes regulating the expression of inflammatory cytokines. Once the T cells are stimulated to differentiate by antigens and IL-4, STAT 6 is activated to yield specific demethylation around the IL-4, IL-5 and IL-13 gene loci resulting in a characteristic open chromatin structure (Agarwal and Rao 1998). Activation of the gene loci by demethylation is observed in several genes in the immune system and coincides with chromatin remodelling resulting in a more accessible chromatin structure (Lee, Agarwal et al. 2002). This step is accompanied by the concomitant induction of Th2 specific transcription factors such as GATA 3 and c-maf (Glimcher and Murphy 2000; Murphy, Ouyang et al. 2000). After binding to the DNA, these transcription factors

30 synergise with more widely expressed, non-subset specific factors such as activating protein AP-1 and NF-AT to induce the transcription of Th2 genes such as IL-4, IL-13 etc (Rengarajan, Szabo et al. 2000). These open configurations of the gene loci appears to be maintained in already differentiated effector T cells in the absence of active transcription and are also associated with high levels of GATA3 and c-maf expression. Subsequent activation of the effector/memory T cells results in the rapid induction of the transcription factors AP-1, NF-κB, nuclear factor of activated T cells (NF-Atc) and CAAT/enhancer binding protein (C/EBP) (Muro, Minshall et al. 2000). Both AP-1 and NF-κB are important in asthma because they are responsible for the generation of a wide variety of cytokines in the asthmatic process such as IL-1, TNF-α, IL-2, IL-6 and GM-CSF. The JAK-STAT signalling pathway also plays an important role in IgE regulation. After binding of IL-4 and IL-13, the activated JAKs phosphorylate tyrosines within the cytoplasmic domains of IL-4Rα, which acts as a docking site for STAT 6. Phosphorylated STAT 6 homodimerizes, translocates to the nucleus and activates the transcription of genes involved in B cell differentiation including the germ line Iε and Iγ1 genes in mice and the Iε and Iγ4 in humans (Coffman, Lebman et al. 1993). An induction of these germ line “sterile” transcripts in mice has been shown to be essential for Immunoglobulin class switching in mice (Coffman, Lebman et al. 1993). STAT6 contributes to class switching to produce IgE and IgG1 (Kaplan, Schindler et al. 1996; Shimoda, van Deursen et al. 1996; Takeda, Tanaka et al. 1996). In mice, IFN-γ acts via STAT 1 to activate the transcription of many early response genes and to inhibit the transcription of germ line Iε and Iγ1. This blocks the production by B-lymphocytes of IgG1 and IgE (Linehan, Warren et al. 1998). This regulation appears to be mediated by a new family of inhibitory molecules known as suppressors of cytokine signalling (SOCS-1)(Chen, Losman et al. 2000). These proteins are induced by cytokines and appear to function in a negative feed back mechanism. Another inhibitor of JAK-STAT signalling implicated in atopic immune response is Bcl- 6. Normally Bcl-6 functions as a transcription suppressor and has been shown to bind to STAT 6 binding sites such as one found in the Iε promoter. Mice lacking Bcl-6 develop an inflammatory response characterised by increased levels of IgE and Th2 cells and mast cell infiltrates. B cells here produce high levels of IgE (Dent, Shaffer et al. 1997;

31 Ye, Cattoretti et al. 1997). However the role of this suppression in regulation of JAK- STAT signalling in human asthma is still undefined.

32 2 POLLEN ALLERGENS AND THEIR RELATIONSHIP TO PROTEOLYTIC ENZYMES

2.1 The Role of Pollen Allergens in Asthma

Clinically relevant allergens are highly ubiquitous in nature and possess strong antigenic capabilities. In this regard pollen grains are an important source of allergenic proteins, which can initiate allergic responses in over 90% of sensitised individuals (Knox and Suphioglu 1996). Intact pollen grains do not initiate asthma, as their size prevents their entry into the respiratory tract. They have an average diameter of 10-40 μm and studies using dust deposition models have demonstrated that less than 5% of particles this size can penetrate into the lower airways (Bates, Fish et al. 1966). Photo-scintillation imaging of the upper respiratory tract after inhalation of radioactive, technetium-labelled Kentucky blue grass has demonstrated that intact pollen grains are mainly confined to the upper nasal passages, while radioactive pollen extract (sub-micronic particles) can be readily detected within the lower respiratory passages (Wilson, Novey et al. 1973). This is relevant as allergenic sub-micronic particles, released from hygroscopic pollen, can easily bypass the mucociliary barrier of the upper respiratory tract and activate the respiratory immune response.

2.2 Environmental Factors influencing Pollen Exposure

Many plant species rely on the airborne dispersal of pollen grains for their propagation. Pollen grain release or “anthesis” is a relatively passive process, dependent on humidity, air currents and rainfall (Laaidi 2001). These variable weather conditions can influence the airborne concentration of pollen in a particular region to a large extent and are responsible for the seasonal exacerbation of allergic disease. Hence, deciphering environmental influences on pollen exposure is imperative to understanding the patterns of distribution of allergic diseases and the formulation of preventive treatment by allergen avoidance and prophylactic treatment. The recording and compilation of weather data immediately preceding these pollination periods also allows for the development of

33 forecast models to predict the onset of future pollen seasons (Galan, Garcia-Mozo et al. 2001). During the flowering season of an anemophilous (wind pollinated) plant, pollen concentrations vary from 100-3000 grains/cubic meter (Knox and Suphioglu 1996; Bass, Delpech et al. 2000). The day to day variations in weather conditions seem to have a considerable effect on the onset of pollination and its local concentration. Generally speaking, pollination usually occurs on warm days, at around midday (Galan, Garcia- Mozo et al. 2001). The local transport of pollen, post anthesis is controlled by wind speeds, an increase of which reduces their concentration as they are swept to higher altitudes, thereby reducing ground concentrations. Calm, cool weather results in their resettling towards the ground and thus increasing the concentration. Rainfall also tends to influence pollen concentration and allergen release. Under wet and humid conditions or during thunderstorms, hygroscopic pollen grains tend to rupture as a result of osmotic shock, releasing paucimicronic particles such as starch granules and ubisch bodies (Suphioglu 1998). A single pollen grain is capable of releasing 700 allergen rich starch granules (Figure 2.1), which vary from 0.6-2.5 μm in size (Knox and Suphioglu 1996). These particles can easily be inhaled into the lower airways and have been associated with thunderstorm related asthma (Venables, Allitt et al. 1997). The presence of these sub-micronic particles has important practical implications. The released starch granules cannot be quantified by conventional pollen samplers. Hence, traditional pollen samplers can be misleading as an index of outdoor allergen exposure in particular situations. This would potentially explain some studies wherein an inverse association has been demonstrated between pollen counts and prevalence of symptoms of allergic diseases (Burr, Emberlin et al. 2003). Furthermore, the allergens can bind to other small particles in the atmosphere such as diesel exhaust carbon particles (DECP), which can penetrate deeper into the airways, initiating allergic inflammatory responses (Knox, Suphioglu et al. 1997).

34 Figure 2.1 - Scanning electron micrographs of fresh Poaceae pollen grains in the dry state (A&C) and in the hydrated state (B&D) Reproduced from:(Grote, Dolecek et al. 1994)

2.3 Classification of Pollen Sources

2.3.1 Taxonomy of Pollen Sources

There are numerous families of wind pollinated plants that are implicated in pollen associated allergic diseases. Although the taxonomic classification of these plants is relatively young, it is relatively large, with over 20,000 types of plants having been described. The classification of the plant kingdom follows the Linnean binomial system. Plants were initially differentiated on the basis of their floral and morphogenic characteristics. However, this was found to be insufficient and the classification system was subsequently modified and expanded to take into consideration the evolutionary drifts in plant families along convergent and divergent pathways. The accurate identification of a plant species depends upon its allocation under a distinct group of hierarchical sub-divisions in plant taxonomy. These sub-divisions are enumerated in

35 Table 2.1. The kingdom, phylum and classes are extremely broad based and indicate evolutionary origin. The family, genus and species are more specific and descriptive of the specific plant and are more relevant from a scientific point of view.

Table 2.1 - Plant Nomenclature Sub-Divisions

Unit Example Kingdom Plant Division Trache ophyta (Vascular) Sub-division Pteropsida (Fern and seed bearing plants) Class Angiosperms Sub-class Dicotyledoneae, Rosidae Order Rosales Family Rosaceae Genus Rosa Species Rugosa

The classification of a newly identified plant into the appropriate nomenclature sub-divisions is based on a system of rules known as the International Code of Botanical Nomenclature for Plant taxonomy (IAPT) (www.botanik.univie.ac.at/iapt). This classification method attempts to formulate plant groups based on their inherited evolutionary characteristics and incorporates supporting data from genetic and biochemical sources, as well as from palaeobotany, morphology, anatomy and physiology of the plant. Figures 2.2, 2.3 and 2.4 provide a brief overview of the phylogenetic sub-divisions of common allergenic plant sources, indicating their evolutionary patterns.

36 Figure 2.2 - Nomenclature of allergenic tree pollen sources

Class: Gymnosperms Class: Angiosperms

Subclass: Pinacae Subclass: Hamamelididae Subclass: Dillenidae SubclassRosideae Subclass: Asteridae

Order: Pinales Order: Hamamelidales Order: Tiliaceae Order: Sapindales Order: Scrophulariales

Family: Cupressaceae Family: Hamamelidaceae Family: Malvaceae Family: Hippocastanaceae Family: Oleaceae

Arizona cypress, Mountain cedar Sweet gum Linden Buckeye, Horse chestnut Olive Common Juniper, Western cedar Family: Plantanaceae Order: Salicales Family: Aceraceae Family: Taxodiaceae

Sycamore, London planetree Box elder, Silver maple, Norway maple Bald cypress, Japanese red cedar, Family: Salicaceae Coast redwood Order: Fagales Order: Fabales Cottonwood, Aspen, Black willow, white pepper Family: Pinaceae Family: Fagaceae Order: Euphorbiales Family: Mimosaceae Monetery pine, Ponderosa pine Douglas fir, Norway spruce White oak, Chestnut, American Beech mesquite, black locust Family: Euphorbiaceae Family: Betulaceae Family: Fabaceae Annual mercury White birch, Grey Alder, Hazel alfa alfa Order: Capparales Order: Urticales Order: Myrtales

Family: Moringaceae Family: Ulmaceae Family: Myrtaceae Horse Radish tree American helm, Hackberry Eucalyptus

Family: Ceropiaceae Order: Rosales

Pumpwood Family: Rosaceae Family: Moraceae Hawthorn White mulberry Order: Juglandales

Family: Juglandaceae

English walnut, Pecan, Shagbark hickory

37 Figure 2.3 - Nomenclature of allergenic grass pollen sources

Class: Liliopsida

Subclass: Aerecideae Subclass: Commelindieae

Order: Aereceales Order: Cyperales Order: Typales

Family: Aereceae Family: Cyperaceae Family: Poaceae Family: Typaceae

Date Palm, Royal Palm, Nutsledge, Bulrush, Subfamily: Pooideae Common cattail Coconut Sedge

Tribe: Agrostideae Tribe: Poeae

Timothy grass, Redtop Orchard, Fescue, ryegrass Foxtail June

Tribe: Aveneae Tribe: Tritceae

Sweet vernal, Oat, Wheat, Rye Velvet, Canary Barley, Quackgrass

Subfamily: Panicoideae

Tribe: Paniceae Tribe: Andropogoneae

Crab grass, Bahia Johnson grass, Corn Sugarcane

Subfamily: Chloridoideae

Tribe: Eragostideae Tribe: Aeluropideae

Love grass Salt grass

Tribe: Sporoboleae Tribe: Chlorideae

Dropseed Bermuda grass, Grama, Buffalo grass

Subfamily: Arundinoideae

Tribe: Arundineae

Giant reed, common reed

Subfamily: Bambusoideae

Tribe: Oryzeae

Rice, wild rice

38 Figure 2.4 - Nomenclature of allergenic weed pollen sources

Class: Magnoliopsida

Sub class: CaryophyllidaeSub class: Hamamelididae Sub class: Asteridae

Order: CarophyllalesOrder: PolygonalesOrder: Urticales Order: Lamiales Order: Asterales Order: PlantaginalesOrder: Dipsacales Order: Scrophulariales

Family: AmaranthaceaeFamily: Polygonaceae Family: Urticaceae Family: Boraginaceae Family: Asteraceae Family: PlantaginaceaeFamily: Caprifoliaceae Family: Scrophulariaceae

Redroot pigweed Sheep sorrel Nettle Pattersons curse Sub-family: Heliantheae English plantain Elder, honeysuckle Figwort Palmers amaranth Yellow dock Pellitory Western waterhemp Sunflower, short ragweed False ragweed Family: Chenopodiceae Poverty ragweed Cocklebur

Sub-family: Anthemideae Lambs quarter Russian thistle Mugwort Kocia Sagebrush Wingscale Sub-family: Astereae

Goldenrod

Sub-family: Cichorieae

Dandelion

39 2.3.2 Cross-Reactivity between different Pollen Allergens

As the phylogenetic relationship between different families of plants become clearer, it has become apparent that cross-reactivity between allergens reflects phylogeny in a majority of cases. Plants belonging to the same genus are expected to have a great number of shared allergens; those in the same family, perhaps fewer and distantly related plants would be expected to show little or no cross-reactivity. Amongst the tree pollen, allergens from the family Cupressaceae, which consists of species such as cedars, junipers, pftizers and cypresses are highly cross- reactive (Midoro-Horiuti, Goldblum et al. 1999). Grasses are a diverse source of pollen allergens and constitute about 25-30% of the world wide vegetation. The family Poaceae is particularly important as it comprises of over 9000 species which are grown for food, animal forage and as surface vegetation to prevent soil erosion. They represent a major portion of the airborne allergen load during spring and summer months in most parts of the world. The pollen extracts of about 20-30 species in this large family are recognised as major sources of allergic diseases and are used frequently in the diagnosis of grass pollen allergies. The allergens in group 1, 2, 3 and 5 have been found to dominate the immune response to grass pollen (Andersson and Lidholm 2003). This serves to suggest that although phylogenetically diverse, the allergenic proteins probably serve some common biological functions, characterised by the conservation of the allergen epitope. However, there are certain exceptions to the premise of allergen cross- reactivity reflecting phylogeny. Some important exceptions include the birch pollen allergen (Bet v 1), which is highly cross-reactive with major allergens in various fruits such as cherry, hazelnut, peach, carrot, celery and soybean (Vieths, Scheurer et al. 2002). Similarly, cross-reactivity between ragweed allergens with other plant allergens such as the group 4 allergens of Timothy grass (Phl p 1) and other allergens from Japanese cypress, cedar, corn and parthenium has been reported (Mohapatra and Lockey 1999). These findings reinforce the need to clearly understand the phylogenetic relationship between different pollen sources and incorporate this information into the allergen nomenclature. This will allow for a greater understanding of pollen allergies and the prediction of cross-reactivity in different populations and geographic locations.

40 2.4 Classification of Pollen Allergens

The formulation of a systematic nomenclature for allergens is no easy task given the exponential growth in the identification of novel allergens from an already vast number of species. The allocation of allergens to particular groups in the nomenclature is not rigid and are often can be reclassified or regrouped when new aspects of the allergen structure or functional properties are determined. Hence the allergen nomenclature is usually updated on a regular basis. Novel allergens must satisfy certain basic biochemical and immunological criteria set by the WHO/IUIS (World Health Organisation/ International Union of Immunological Studies) allergen nomenclature sub-committee in order to be allocated a designation within the nomenclature (Marsh, Goodfriend et al. 1986). In order to maintain the integrity of the nomenclature system, investigators are expected to screen a reasonable population size, to establish the frequency of the response to the allergen among patients. These criteria are summarised in Figure 2.5 as follows: - Figure 2.5 - IUIS criteria for the inclusion of allergens into allergen nomenclature Reproduced from (Chapman, 1999.)

41 2.4.1 The Revised Allergen Nomenclature

The comprehensive allergen nomenclature was first published by King et al in 1986 (Marsh, Goodfriend et al. 1986). This was appropriately revised in 1994 due to the development of molecular biology techniques in allergen analysis (King, Hoffman et al. 1994). Allergens in the revised system are named according to the accepted taxonomic name of their source as shown in Figure 2.6 below: Figure 2.6 - Schematics of the designation of allergen names according to IUIS Adapted from (King, Hoffman et al. 1994)

Allergens are designated in an abbreviated form with the first three letters referring to their genus, then a space, a single letter for the species, a space and an Arabic numeral. The numbers are assigned to the allergens in order of their identification and the same number is used to designate other homologous allergens or similar allergens from related species. While the original nomenclature system designated allergens in an italicised form, in the revised nomenclature, the use of italics are reserved for the designation of allergen encoding genes in accordance with the general use. Allergens per se are depicted in regular characters. (King, Hoffman et al. 1994). Table 2.2 is an adaptation of the information provided in the revised nomenclature of some commonly known pollen allergens. A practical addition to the revised nomenclature is the optional use of a single letter prefix indicating the origin of the allergen. Allergens prepared by recombinant methods or by chemical synthesis are designated “r” or “s” respectively compared to “n” for the naturally occurring allergen, followed by the designation of the allergen name (e.g.– rCyn d 1) (Larsen and Lowenstein 1996). As most allergens contain some form of post-translational modification such as glycosylation or acetylation, recombinant or synthetic peptides designated by their respective prefixes (r or s) are

42 taken to indicate that they do not contain the same post-translational modification as the natural allergen. If they do, the prefix is changed to “rn” or “sn” respectively (Larsen and Lowenstein 1996). From a clinical point of view, allergens are termed “major” or “minor” depending on whether 50% of a particular population tested reacts positively with the specific allergen.

2.4.2 Ambiguities in the Allergen Nomenclature

The original nomenclature gave rise to certain ambiguities between allergens from different species as a result of the abbreviation of their taxonomic names. For example, allergens of the American cockroach (Periplaneta americana) were commonly confused with those of avocado (Persea americana ). Another example includes differentiating between allergens of the related species of vespids that cause insect allergies (e.g.- Ves v 5 are the designated allergens for Vespula vulgaris and Vespula vidua; Ves c 5 for Vespula consorbina and Vespula curabo). To overcome this problem and circumvent future issues, the revised nomenclature has included an additional single letter for a more clear designation of either the genus or species. Hence, in the revised species, the new designated names are Ves v 1 and Ves vi 1 indicates Vespula vulgaris and Vespula vidua respectively. (King, Hoffman et al. 1995).

2.4.3 Allergen Isoforms

An allergen from a single species can have several similar forms as a result of nucleotide mutations, differential splicing and/or exon exchange in the allergen encoding gene. They are known as isoforms and share common biochemical properties such as:

1. Similar molecular size. 2. Identical biological function. 3. Amino acid sequence similarity of > 67% (King, Hoffman et al. 1995). Each isoallergen may have multiple forms that differ by a limited number of amino acid substitutions and are designated as variants. The addition of suffixes of four numerals to designate isoallergens and their variants allows for their unambiguous identification.

43 IgE binding Grasses Allergen Functions MW pI Cloned Reactivity Cynodon Cyn d 1 β-expansin 31-34 8.56 Yes High dactylon Cyn d 2 - n.d. 4.74 Yes - BG-60 flavoprotein 60 - No Low (Bermuda grass) Cyn d 7 Ca²⁺-binding 12 4.24 Yes Low Cyn d 12 profilin 14 4.89 yes Low Lolium perenne Lol p 1 β-expansin 31-34 5.4, 5.57 Yes High (ryegrass) Lol p 2 - 10-11 5.25 Yes Intermediate Lol p 3 - 10-11 7.86 No Intermediate Lol p 4 - 60-67 - No Low Lol p 5 - 25-34 - - High Lol p 5a - - 10.2 Yes - Lol p 5b - - 6.03 Yes - Lol p 5c - - 5.11 Yes - Lol p 10 Cytochrome c 12 - No - Lol p 11 Soy bean trypsin 18 5.1 Yes Intermediate inhibitor Phleum Phl p 1 β-expansin 33-37 6.16 Yes High pratense Phl p 2 - 11-12 4.64 Yes Intermediate Phl p 3 - 12 - No - (Timothy grass) Phl p 4 - 55-60 - No Intermediate Phl p 5 Ribonuclease 29-38 - - High Phl p 5a - - 6.89 Yes - Phl p 5b - - 5.99 Yes - Phl p 6 P-particle 13 5.56 Yes High associated protein Phl p 7 Ca²⁺-binding 6 4.19 Yes Low Phl p 11 Soy bean trypsin 20 4.96 Yes - inhibitor Phl p 12 profilin 14 5.07 Yes Low Phl p 13 Polygalacturonase 55-60 7.83 Yes Intermediate - like Poa pratensis Poa p1 β-expansin 33-36 6.4, 9.1 Yes High (Kentucky blue Poa p 2 - n.d. n.d. Yes Low grass) Poa p 5 - 28-34 5.2,5.7, - Hig > 9.5 Poa p 5a - - - Yes - Poa p 5b - - - Yes - Poa p 10 Cytochrome c 12 9.9 Yes - Zea mayes Zea m 1 β-expansin 28-31 9.04 Yes Intermediate (maize) Zea m 13 Soy bean trypsin n.d. 4.52 Yes - inhibitor Zea m 12 profilin 14.2 4.94- Yes Intermediate 4.97

Table 2.2: Revised nomenclature of common pollen allergens. Adapted from papers (Johnson and Marsh 1966; Singh, Smith et al. 1990; Matthiesen, Schumacher et al. 1991; Silvanovich, Astwood et al. 1991; Singh, Hough et al. 1991; Dolecek, Vrtala et al. 1993; Ong, Griffith et al. 1993; Laffer, Duchene et al. 1996)

44 For example, cDNA cloning of the ragweed allergen Amb a 1 demonstrated four isoallergens with 12-24% differences in their amino acid sequences. Each isoallergen was further found to have 1-3 variants with more than 90% sequence similarities. Their designations in the nomenclature are as shown in Figure 2.7. Isoallergens and their variants are denoted by the addition of four Arabic numerals suffixes to the allergen name. The first two numerals distinguish between isoallergens and the last two between the variants.

Figure 2.7 - Revised nomenclature of allergen isoforms and variants

2.5 Localisation and Biological Functions of Pollen Allergens

Attempts to localise allergens within the pollen grains were done on the assumption that most of these glycoproteins were concentrated at the periphery of the pollen grain. However, these attempts were hindered by the lack of highly specific antibodies against allergens and inadequate methods to fix intact pollen grains (Grote, Fischer et al. 1998). The use of conventional aqueous fixation and dehydration techniques usually results in the rapid diffusion of the allergens and has largely been replaced by novel, strictly anhydrous techniques (Knox 1973; Knox and Suphioglu 1996). This, together with the production of highly specific monoclonal and polyclonal antibodies against individual pollen allergens and advancements in colloidal gold/antibody techniques in biological electron microscopy has led to the characterisation of a number of allergens

45 at an ultrastructural level (Staff, Taylor et al. 1990; Grote 1992). Table 2.3 enumerates the various allergens that have been localised in intact pollen grains by these methods. Table 2.3 - Localisation of allergens within pollen grains by electron microscopic methods

Allergen Source Putative Localisation Lol p 1 Rye grass Located in the cytoplasmic matrix of pollen and mitochondria. Also located within the exine walls (Staff, Taylor et al. 1990). Phl p 1 Timothy Cytoplasmic matrix of the pollen grain and the exine walls Grass but not the mitochondria (Grote, Dolecek et al. 1994). Lol p 5 Rye grass Phl p 5 Timothy Amyloplasts ( starch granules) in the pollen cytoplasm but none in the pollen walls (Grote, Dolecek et al. 1994). grass Phl p 6 Timothy Polysaccharide p particles within the cytoplasmic matrix grass (Grote, Fischer et al. 1998).

Bet v 1 Birch Pollen cytoplasm matrix and the nuclei (Grote 1992). Plant Birch Cytoplasmic matrix and nuclei (Grote 1992). profilin 3A4 Birch Located within the matrix, nuclei and mitochondria. Also found in similar sites in Timothy grass, mugwort and celery tissues (Heiss, Fischer et al. 1996; Cadot, Kochuyt et al. 2003; Magnusson, Lin et al. 2003). Ole e 1 Olive Endoplasmic reticulum of olive tree pollen. Cross-reactive allergen found in related species such as Ash and Primer (Fernandez, Olmedilla et al. 1996). Cry j 1 Japanese Located in the pollen cell wall layer and Golgi body in the Cedar cytoplasm. Cross-reactive in several cedar species.

The localisation of allergens within their sources has provided valuable clues towards their putative biological functions. With the advent of molecular biology in the study of allergen structure and functions, most of the clinically significant pollen allergens have now been described at a molecular level and their endogenous biochemical activities have been determined. In addition, the functions of some of the previously described allergens have now been established or inferred on the basis of homology studies. The biological functions of some of the known pollen allergens are described in Table 2.4 as follows-:

46

Table 2.4- Biological functions of pollen allergens

Allergen Source Biological Function

Its activity is similar to calmodulin related proteins, which Bet v 3 Birch affect calcium concentrations and result in cell activation.

Demonstrates sequence identity to isoflavone reductase Bet v 5 Birch proteins (60-80%). It is involved in lignin and isoflavone synthesis and has an important role in plant defences against wood rotting fungi (Gang, Kasahara et al. 1999; Karamloo, Schmitz et al. 1999). Has sequence homology to cyclophilin. Possesses peptidyl- Bet v 7 Birch isomerase activity (Cadot, Diaz et al. 2000). Its functions are unknown but these proteins bind to proteins which are targets of transplantation drug cyclosporin A in humans (Ivery 2000). Also known to be pan-allergens (Horner, Reese et al. 1995). Significant homology to pectin degrading enzymes, Phl p 13 Timothy polygalacturonases (Suck, Petersen et al. 2000). They are grass involved in the degradation of pectin in pollen grains (Ohtsuki, Taniguchi et al. 1995).

Related to a group of plant proteins known as pathogenesis Pru av 2 Cherry related proteins (PRP). These proteins are constitutively expressed in response to physical stress of infection in plants. There are currently 14 groups of PR proteins which have Mal d 2 Apple different functions, many of which possess enzymatic activity such as carbohydrases, ribonucleases. PR-5 demonstrates homology to the sweet tasting protein, Jun a 3 Mountain Thaumatin. Can be involved in plant defences, fruit ripening, etc (Vanek-Krebitz, Hoffmann-Sommergruber et al. 1995; cedar Inschlag, Hoffmann-Sommergruber et al. 1998; Soman, Midoro-Horiuti et al. 2000). Phl p 1 Timothy Group 1 allergens have sequence similarities to plant proteins Poa p 1 grass known as “expansins” and induce extension in the cell wall Lol p 1 Kentucky by “cell loosening”. Expansins are important in the blue grass penetration of the pollen tube into the style during Rye grass germination. However, the mechanism is debatable. Grobe et al demonstrated cysteine peptidase activity in rPhl p 1 and hypothesised that cell loosening activity occurs via proteolytic mechanism (Cosgrove 1997; Grobe, Becker et al. 1999; Cosgrove 2000; Grobe, Poppelmann et al. 2002).

2.6 General Features of Proteolytic enzymes

Proteolytic enzymes are the most ubiquitously occurring enzymes in all living organisms and constitute about 1-5% of the gene content (Neurath 1999). These

47 enzymes play a pivotal role in all biological functions, ranging from protein turnover and digestion to the highly regulated blood coagulation and complement cascades. An abnormal increase in proteolytic activity is a common feature of many pathological states such as emphysema, strokes and cancer metastasis, thus underscoring the functional importance of these enzymes. The existing literature varies widely in the terminology used in reference to these enzymes, the commonest handles used being proteases, peptidases and peptide hydrolases. This resulted in a great deal of ambiguity in our understanding of proteolytic mechanisms and their appropriate classification into nomenclature groups. Hence, in 1983 the International Union of Biochemistry and Molecular Biology (IUB-MB) nomenclature committee recommended using the term “peptidases” to bring about the standardisation of the descriptive terminology of protease enzymes (1983). The term “peptidase” is now synonymous with the widely used term “protease”. Peptidases primarily catalyse the hydrolysis of peptide bonds. Although they demonstrate hydrolytic activity against a wide variety of substrates, these enzymes are highly specific in their ability to cleave peptide bonds within a protein chain (Barrett 1986). For example, trypsin-like peptidases cleave peptide bonds on the carboxy- terminus of the basic amino acids, lysine and arginine (except if preceeded by a proline). Chymotrypsin on the other hand prefers cleaving after large hydrophobic residues such as phenylalanine, tyrosine, tryptophan, leucine and isoleucine. Although they share a common mechanism, the differences in specificity are largely a function of the side chain of the amino acids forming the active sites of these enzymes. The specificity of peptidases for their substrates can be explained by the model system devised by Schechter and Berger (Schechter and Berger 1967), which explains peptidase interaction with its substrate in the context of the active site residues of the enzymes and the amino acids comprising the scissile bond. The binding site for the substrate on a peptidase is envisioned by a series of subsites, each subsite interacting with an amino acid residue on the substrate.

2.7 Proteolytic Enzyme Nomenclature

The last fifty years has witnessed an explosive growth in the identification, isolation and purification of peptidases from diverse sources. There have been significant advances in deducing the primary amino acid sequences and three dimensional crystallographic structures of the peptidases. Current annotated data

48 bases contain over 450 unique peptidases (exopeptidases and endopeptidases) from over 1400 organisms (bacteria, protozoa, fungi, plants, animals and viruses) and are rapidly growing. From a functional point of view, peptidases are sub-divided into two major groups on the basis of their substrate specificity, namely: 1. Endopeptidases: These are peptidases that cleave peptide bonds internally, within a polypeptide chain. 2. Exopeptidases: These peptidases specifically cleave at the terminal ends of the polypeptide chains, cleaving one, two or three terminal amino acids, at the amino- or the carboxy-terminal ends. Peptidases are either endopeptidases or exopeptidases, with some possessing both types of activity (Barrett 1986). Acknowledging that this broad classification is insufficient, proteolytic enzymes have been more specifically classified into evolutionary clans and families by Rawlings and Barrett (Rawlings and Barrett 1993). This classification has become the cornerstone for the development of the accepted nomenclature and has allowed for the development of databases such as MEROPS (http://www.merops.co.uk), which includes a frequently updated listing of all novel peptidase sequences (Barrett, Rawlings et al. 2001). Peptidases are sub-divided into a number of groups based on the demonstration of a distinct catalytic mechanism (Rawlings and Barrett 1993). Each group consists of a number of clans of peptidase families that demonstrate the same catalytic mechanism and evolutionary similarities. A peptidase family comprises of a group of peptidases that demonstrate evidence of their evolutionary relationship by their similar tertiary structures, the order of the catalytic residues in their sequences or by the presence of conserved sequence motifs around their catalytic residues. The allocation of peptidases into their respective groups in the nomenclature is based on the criteria recommended by the nomenclature committee of the International Union of Biochemistry and Molecular Biology (IUB-MB). There are five important groups in the peptidase nomenclature, namely: • Serine and Threonine peptidases • Cysteine peptidases • Aspartic peptidases • Metallo peptidases • Unknown peptidases

49 Each group is identified by a single letter, representing its catalytic mechanism (e.g. S; Serine, C; Cysteine, T; Threonine, M; Metallo, U; Unknown). The clans in each group are identified by two letters (e.g. SA, MA, etc). The first letter signifies the catalytic group, while the second letter differentiates between the clans and is added in the order of identification. A clan that contains families from overlapping catalytic types (C, S M or T) is designated as P. Although the catalytic mechanism exists both for exopeptidases and endopeptidases, the nomenclature classifies endopeptidases only on the basis of their catalytic mechanism, while exopeptidases are differentiated both on the basis of their catalytic mechanism and their substrate specificity.

2.7.1 Classification of Peptidases by Catalytic Mechanism

2.7.1.1 Serine Peptidases

Serine peptidases are the most widespread and well documented group of peptidases. This group consists of over 40 families of serine and threonine peptidases, grouped into seven clans on the basis of their tertiary structures. Although diverse in their overall three-dimensional structures, the catalytic functional group is arranged in a remarkably similar fashion (Barrett and Rawlings 1995) Examples of peptidases in these clans are enumerated in Table 2.5. The hallmark of the active-site of serine peptidases is their catalytic triad comprising of serine, histidine and aspartate residues

(Ser195, His57 and Asp102 in chymotrypsin). These residues interact together, forming a “proton charge relay network” and facilitating peptide bond hydrolysis by a process of reversible acylation (Hedstrom 2002). During peptide bond hydrolysis, the nucleophilic, hydroxyl (OH) group of the serine residue attacks the susceptible carbonyl (C=O) bond of the substrate. A carbonyl-oxygen bond is formed, resulting in the oxygen receiving a “net negative” charge. The proton released is transferred from the serine residue to histidine via the charge relay network, yielding a tetrahedral intermediate complex. The Asp residue is hydrogen bonded to the His-H+ and results in the stabilisation of the imidazole ring. At this point, the acyl component of the substrate is esterified to the serine while the amine portion is hydrogen bonded to the His-Asp complex. The protonated histidine then acts as a general acid, assisting in the peptide bond cleavage by transferring the proton to the amine portion of the peptide bond. This results in the breakdown of the

50 tetrahedral intermediate, resulting in the release of the acyl-enzyme complex and liberation of the protonated leaving group (in this case the amine group) thus causing peptide bond hydrolysis (Hedstrom 2002). Subsequent deacylation as the name implies is the exact opposite of acylation and results in the regeneration of the peptidase. Here, a water molecule acts as the nucleophile. The histidine residue acts as a general base to deprotonate H2O, making it an effective nucleophile. The nucleophile attacks the carbonyl group of the acyl- enzyme complex, bound to serine. This results in the formation of the tetrahedral intermediate. Histidine donates the proton to the oxygen of the serine residue, resulting in the dissociation of the carbonyl portion of the substrate and the regeneration of the reactive hydroxyl group of serine.

Table 2.5 - Nomenclature of Serine peptidase familles. Source : MEROPS (http://www.merops.co.uk)

Clan Families Examples S1 Trypsin S2 Streptogrisin A S3 Togavirin S6 IgA1-specific serine endopeptidase SA S7 Flavivirin S29 Hepatitis C polyprotein peptidase S30 Helper component proteinase S31 Pestivirus NS2-3/NS3 serine peptidase S32 Arterivirus serine endopeptidase SB S8 subtilisin Carlsberg (Bacillus licheniformis) S9 Prolyl oligopeptidase S10 Carboxypeptidase C S15 X-Pro dipeptidyl-peptidase SC S28 Lysosomal Pro-X carboxypeptidase S33 Prolyl amino peptidase S37 Streptomyces PS-10 peptidase S11 Penicillin-binding protein 5 SE S12 Streptomyces R61 D-Ala-D-Ala carboxypeptidase S13 Penicillin-binding protein 4 S24 Lex A repressor S26 Signal peptidase SF S41 TSP protease S44 Tricorn protease SH S21 Cytomegalovirus assembling protein T1 Proteasome TA S42 α-Glutamyl transpeptidase

51

2.7.1.2 Cysteine Peptidases

Peptidases wherein the sulphydryl group of the cysteine residue (in the catalytic active-site) acts as the nucleophile are known as cysteine peptidases. These peptidases bear resemblance in their catalytic mechanism to serine peptidases by their ability to form tetrahedral intermediate complexes during peptide bond hydrolysis. Cysteine peptidases are found in bacteria, plants as well as animals and are sub- divided into five or more families on the basis of their active-site configuration. Table 2.6 enumerates the different clans of cysteine peptidases in the enzyme nomenclature database.

In these peptidases, the sulphydryl group of the active-site cysteine residue serves as the nucleophile and is akin to the hydroxyl group of serine in serine peptidases. The peptide bond hydrolysis occurs around the catalytic triad, comprising of cysteine, histidine and aspartate residues (Cys25, His159 and Asp158 in papain numbering) (Polgar 1973). Like serine peptidases, the nucleophilic, sulphydryl group of cysteine is assisted by histidine, which acts as the proton donor/general base. The sulphydryl group of cysteine attacks the amide or ester carbonyl group of the substrate, to form a thioester intermediate (acyl-enzyme complex) (Rawlings and Barrett 1998). Histidine then protonates the tetrahedral intermediate, resulting in the release of the amine group. In the deacylation step, water acts as the nucleophile, attacking the thio-ester and is catalysed by the imidazole group of histidine. This results in the release of the carboxylic end of the substrate and regeneration of the enzyme (Otto and Schirmeister 1997).

52 Table 2.6 - Nomenclature of Cysteine peptidase families Source: MEROPS (http://www.merops.co.uk)

Clans Family Examples C40 dipeptidyl-peptidase VI (Bacillus sphaericus) C41 cysteine proteinase (hepatitis E virus) C53 pestivirus Npro endopeptidase (classical swine fever virus) C C60 sortase A (Staphylococcus aureus) C61 small protease (Sulfolobus solfataricus) C66 MAC protein (Streptococcus pyogenes) C1 papain (Carica papaya) C2 calpain-2 (Homo sapiens) C6 potyvirus helper component proteinase (potato virus Y) C7 chestnut blight fungus virus p29 proteinase (Cryphonectria hypovirus) C8 chestnut blight fungus virus p48 proteinase (Cryphonectria hypovirus) C9 sindbis virus-type nsP2 proteinase (Sindbis virus) C10 streptopain (Streptococcus pyogenes) C12 ubiquitinyl hydrolase UCH-L1 (Homo sapiens) C16 murine hepatitis coronavirus papain-like endopeptidase 1 (murine hepatitis virus) C19 ubiquitin-specific protease 14 (Homo sapiens) C21 tymovirus endopeptidase (turnip yellow mosaic virus) C23 carlavirus endopeptidase (apple stem pitting virus) C27 rubella virus endopeptidase (Rubella virus) C28 foot-and-mouth disease virus L-proteinase (foot-and-mouth disease virus) CA C31 porcine respiratory and reproductive syndrome arterivirus-type cysteine proteinase alpha (lactate-dehydrogenase-elevating virus) C32 equine arteritis virus-type cysteine proteinase (porcine reproductive and respiratory syndrome virus) C33 equine arterivirus Nsp2-type cysteine proteinase (equine arteritis virus) C36 beet necrotic yellow vein furovirus-type papain-like endopeptidase (beet necrotic yellow vein virus) C39 bacteriocin-processing peptidase (Pediococcus acidilactici) C42 beet yellows virus-type papain-like endopeptidase (beet yellows virus) C47 staphopain A (Staphylococcus aureus) C51 D-alanyl-glycyl endopeptidase (Staphylococcus aureus) C54 Aut2 peptidase (Saccharomyces cerevisiae) C58 YopT peptidase (Yersinia pestis) C64 Cezanne deubiquitinating peptidase (Homo sapiens) C65 otubain 1 (Homo sapiens) C67 CylD protein (Homo sapiens) C11 clostripain (Clostridium histolyticum) C13 legumain (Canavalia ensiformis) CD C14 caspase-1 (Rattus norvegicus) C25 gingipain R (Porphyromonas gingivalis) C50 separase (Saccharomyces cerevisiae) C5 adenain (human adenovirus type 2) C48 Ulp1 endopeptidase (Saccharomyces cerevisiae) CE C55 YopJ protease (Yersinia pseudotuberculosis) C57 vaccinia virus I7 processing peptidase (vaccinia virus) C63 African swine fever virus processing peptidase (African swine fever virus) CF C15 pyroglutamyl-peptidase I (Bacillus amyloliquefaciens) CH C46 hedgehog protein (Drosophila melanogaster)

53 2.7.1.3 Aspartic Peptidases

Aspartic peptidases seem to have evolved from a single precursor evolutionary superfamily, mainly confined to eukaryotic organisms. Their history in enzymology is the longest and the most perplexing. Unlike serine peptidases, aspartic peptidases do not form covalent intermediates on interacting with their substrates (Rawlings and Barrett 1995). Crystallographic studies have demonstrated that these enzymes possess a substrate cleft formed by two distinct but similar domains (Andreeva, Zdanov et al.

1984). The active site of these peptidases consists of two aspartate residues (Asp32 and

Asp215 in pepsin) contributed by each domain. Peptide bond hydrolysis by aspartic peptidases involves the two aspartate residues and an activated water molecule which behaves as the nucleophile. This process involves two simultaneous transfers and is known as the “push-pull mechanism” (Polgar 1987). Here, binding of the substrate to the active-site cleft results in a conformational change and the protonation of Asp32, making it acidic. The proton is subsequently transferred to the carboxyl-oxygen of the scissile peptide bond. This protonated bond is then concomitantly attacked by the activated water molecule which is polarised into a nucleophilic state by the carboxyl group of the charged

Asp215, resulting in hydrolysis of the bond (Davies 1990). The most thoroughly studied aspartic peptidase is pepsin, involved in food digestion in the stomach of higher animals (Tang, Sepulveda et al. 1973; Tang and Wong 1987). In plants, nepenthesin is an aspartic peptidase found in the digestive juice of the carnivorous pitcher plant Nepenthes (Amagase, Nakayama et al. 1969). Most gastric aspartic peptidases in higher animals behave as zymogens that are converted into the active enzymes by proteolytic cleavage at the N-terminal end. This limited proteolysis is mediated by the zymogens themselves at an acidic pH. Pepsin is irreversibly inactivated at above neutral pH, which is probably a mechanism by which its activity is kept localised in the body. Table 2.7 enumerates the different clans, families and examples of aspartic peptidases known:

54 Table 2.7 - Nomenclature of Aspartic peptidase families. Source: MEROPS (http://www.merops.co.uk)

Class Family Examples A4 aspergillopepsin II (Aspergillus niger) A- A5 thermopsin (Sulfolobus acidocaldarius) A1 pepsin A (Homo sapiens) A2 HIV-1 retropepsin (human immunodeficiency virus 1) A3 cauliflower mosaic virus-type endopeptidase (cauliflower mosaic virus) AA A9 spumapepsin (human spumaretrovirus) A11 Copia transposon (Drosophila melanogaster) A12 retrotransposon bs1 endopeptidase (Zea mays) A6 nodavirus endopeptidase (flock house virus) AB A21 tetravirus endopeptidase (Nudaurelia capensis omega virus) AC A8 signal peptidase II (Escherichia coli) A22 presenilin 1 (Homo sapiens) AD A24 type IV prepilin peptidase 1 (Pseudomonas aeruginosa) A29 PibD g.p. (Sulfolobus solfataricus) AE A26 omptin (Escherichia coli)

2.7.1.4 Metallopeptidases

Metallopeptidases are a large and diverse group of enzymes found ubiquitously in most organisms. Like aspartic peptidases, these enzymes employ a water molecule as their nucleophile. However, the activation of the nucleophile is brought about by divalent cations such as Zn²⁺ and Ca2⁺, hence the name. Metallopeptidases are classified into different clans, on the basis of the amino acid ligands that hold the Zn²⁺ or metal cations. These are enumerated in Table 2.8. The active-site component of these enzymes consists of divalent cations (Zn²⁺), which are bound by the imidazole side chains of two histidine residues along with the carboxyl group of a glutamic acid residue (Kester and Matthews 1977). The glutamic acid side chain, along with the Zn²⁺ cation promotes the nucleophilicity of the water molecule, which attacks the susceptible carbonyl (C=O) bond of the substrate, generating a tetrahedral intermediate. The tetrahedral intermediate is stabilised by the electrostatic interactions of the carbonyl oxygen with the Zn²⁺ ion. The proton from the Glu residue is then transferred to the amine portion of the peptide bond cleaving the peptide bond. Metallopeptidases commonly cleave between amino acids with a non-polar side chain and often a hydrophobic residue such as Gly is preferred in the P1 position (McDonald 1985). The most important metallopeptidases of clinical relavence are known as “matrix metalloproteases”, which are a group of Ca²⁺ and Zn²⁺ dependent endopeptidases,

55 active at neutral pH. They are synthesised as secreted or transmembrane pro-enzymes and processed to an active form by the removal of an amino-terminal pro-peptide. MMPs play a crucial role in the remodelling of the extracellular matrix as well as having other important cellular functions. These include physiological functions such as neurite growth, cell migration, bone elongation, wound healing, angiogenesis, etc. The many pathological processes involving MMPs include tumour growth and migration, fibrosis, aortic aneurysms, arthritis, etc.

2.7.2 Classification of Peptidases based on Substrate Specificity

In addition to their catalytic mechanisms, peptidases can be distinguished on the basis of their substrate specificity and their preference for cleavage sites.

2.7.2.1 Endopeptidases

Endopeptidases hydrolyse the peptide chain internally. These enzymes are sub-divided on the basis of their catalytic mechanism as it not possible to classify them on the basis of the specific sites of cleavage, due to wide variability.

2.7.2.2 Exopeptidases

Exopeptidases are enzymes that cleave amino acids at the terminal ends of polypeptide chains. Exopeptidases that cleave amino acids at the amino-terminal end of the polypeptide chain are called “aminopeptidases” and peptidases that cleave at the carboxy-terminal end are called “carboxypeptidases”. These enzymes are sub- divided on the basis of their mechanistic class as well as their substrate specificity.

56

Table 2.8 - Nomenclature of Metallopeptidase families. Source: MEROPS (http://www.merops.co.uk)

Class Family Examples M23 Beta-lytic metalloendopeptidase (Achromobacter lyticus) M27 aminopeptidase T (Thermus aquaticus) M48 Ste24 endopeptidase (Saccharomyces cerevisiae) M M49 dipeptidyl-peptidase III (Rattus norvegicus) M56 BlaR1 peptidase (Bacillus licheniformis) M67 Poh1 peptidase (Saccharomyces cerevisiae) M1 aminopeptidase N (Homo sapiens) M2 Angiotensin-converting enzyme peptidase unit 1 (Homo sapiens) M3 thimet oligopeptidase (Rattus norvegicus) M4 thermolysin (Bacillus thermoproteolyticus) M5 Mycolysin (Streptomyces cacaoi) M9 microbial collagenase (Vibrio alginolyticus) M13 neprilysin (Homo sapiens) M26 IgA1-specific metalloendopeptidase (Streptococcus sanguinis) MA(E) M27 tentoxilysin (Clostridium tetani) M30 hyicolysin (Staphylococcus hyicus) M32 carboxypeptidase Taq (Thermus aquaticus) M34 anthrax lethal factor (Bacillus anthracis) M36 fungalysin (Aspergillus fumigatus) M41 FtsH endopeptidase (Escherichia coli) M60 enhancin (Lymantria dispar nucleopolyhedrovirus) M61 glycyl aminopeptidase (Sphingomonas capsulata) M6 immune inhibitor A (Bacillus thuringiensis) M7 snapalysin (Streptomyces lividans) M8 leishmanolysin (Leishmania major) M10 collagenase 1 (Homo sapiens) M11 gametolysin (Chlamydomonas reinhardtii) M12 astacin (Astacus astacus) MA(M) M35 Deuterolysin (Aspergillus flavus) M43 cytophagalysin (Cytophaga sp.) M57 prtB g.p. (Myxococcus xanthus) M64 IgA protease (Clostridium ramosum) M66 StcE protease (Escherichia coli) M72 peptidyl-Asp metalloendopeptidase (Pseudomonas aeruginosa) MC M14 carboxypeptidase A1 (Homo sapiens) MD M15 zinc D-Ala-D-Ala carboxypeptidase (Streptomyces albus) M16 pitrilysin (Escherichia coli) ME M44 vaccinia virus-type metalloendopeptidase (vaccinia virus) MF M17 leucyl aminopeptidase (Bos taurus) MG M24 methionyl aminopeptidase 1 (Escherichia coli) M18 aminopeptidase I (Saccharomyces cerevisiae) M20 glutamate carboxypeptidase (Pseudomonas sp.) MH M28 aminopeptidase S (Streptomyces griseus) M42 glutamyl aminopeptidase (Lactococcus lactis) M19 membrane dipeptidase (Homo sapiens) MJ M38 beta-aspartyl dipeptidase (Escherichia coli) MK M22 O-sialoglycoprotein endopeptidase (Pasteurella haemolytica) M52 HybD endopeptidase (Escherichia coli) ML M63 gpr protease (Bacillus megaterium) MM M50 S2P protease (Homo sapiens) MN M55 D-aminopeptidase DppA (Bacillus subtilis)

57

2.7.2.2.1 Aminopeptidases

Aminopeptidases are classified on the basis of the amino acids sequentially cleaved at the amino-terminal end. Dipeptidyl and tripeptidyl peptidases liberate dipeptides and tripeptides from the N-terminal end respectively. Examples of aminopeptidases include cathepsin B and H. Dipeptidyl peptidase 4 has recently been purified from fungi and is allergenic in humans. Tripeptidyl peptidases in mammals include Tripeptidyl peptidases 1 and 2 (TPP I and TPP II) (Tomkinson 1999). TPP I is an aspartic peptidase and is implicated in the genetic disease “Late infantile neuronal ceroid lipofuscinosis”. TPP II is a serine peptidase with an active-site of the “subtilisin” type. It is ubiquitous in nature and has been isolated from human erythrocytes, rat brain and in soybean. McDonald and Barrett further classified aminopeptidases after the residues for which they show greatest rates of hydrolysis, such as leucine aminopeptidase, alanyl aminopeptidase (Barrett 1986).

2.7.2.2.2 Carboxypeptidases

Carboxypeptidases hydrolyse peptide bonds from the carboxy-terminal end of the protein substrate. These peptidases are further classified into four groups on the basis of their catalytic mechanism. These are serine, cysteine and metallopeptidase. The fourth group, known as “omega peptidases” includes exopeptidases which remove terminal residues that are substituted, cyclized or linked by isopeptide bonds (linkages other than that of α-carboxyl and α-amino group). Carboxypeptidases that cleave dipeptides from the C- terminal end are called “peptidyl dipeptidases”. An important example of this class of exopeptidase is the “angiotensin-converting enzyme” (ACE), which plays an important role in the regulation of blood pressure.

2.8 Classification of Peptidase Inhibitors

Understanding the mechanism of peptidase inhibitors is as important and fundamental as that of peptidase activity itself. As peptidases and their products play an important role in numerous physiological and pathological processes, peptidase inhibitors serve as crucial regulatory switches of these processes. In the last decade, the focus on peptidase inhibitors has concentrated on their potential as therapeutic targets for the control of various diseases.

58 The effectiveness of inhibition is dependent upon the interaction between the inhibitor and the active site of the peptidase. Reversible inhibitors are simple, competitive and serve as transition-state analogues. They possess substrate like features and their potency depends upon the relative concentrations of the enzyme or the substrate in a solution. Irreversible inhibitors on the contrary are usually low molecular weight and highly site-specific. These inhibitors contain a reactive group, which reacts irreversibly with the amino acid at the active-site of the enzyme. Their mechanism of action usually involves forming an enzyme-inhibitor complex first, followed by the formation of covalent interactions and the permanent modification of the enzyme. Peptidase inhibitors can be classified as being either natural or synthetic. The MEROPS database includes all the known natural inhibitors that have been isolated and purified from different sources (Barrett, Rawlings et al. 2001). They are classified into different families on the basis of similarities in their tertiary structures and functional activity. There is currently no database for synthetically derived peptidase inhibitors. Each clan of peptidase enzymes has a large repertoire of natural or synthetic inhibitors, some of which are highly specific while others have overlapping inhibitory activities.

2.8.1 Natural Peptidase Enzyme Inhibitors

The presence of peptidase inhibitors in all living organisms is as ubiquitous as of peptidases themselves. They are important regulators of numerous cascades which are dependent on peptidase activity, and are often found compartmentalised in the same organelle as the enzyme, or alternatively coupled non-covalently to the enzyme, maintaining them in a zymogen (pro) from. The structures of many inhibitors have now been elucidated by X-ray crystallography, providing new insight into their mechanisms. This has been the basis for the development of novel synthetic inhibitors that are more stable and specific in their site directed activity. The MEROPS database provides the most comprehensive listing of all the known natural inhibitors. As with peptidases, inhibitors are grouped into families on the basis of similarities in their amino acid sequences. Homology between inhibitors in a family is demonstrated by significant similarities in the amino acid sequences to known inhibitors in the family or to other proteins that have already been shown to be homologous to a type inhibitor. Each family has an identifier formed by the letter “I”

59 followed by a unique number. Families thought to be homologous are grouped into clans. The families in a clan show evidence of evolutionary relationships by demonstrating similarities in the tertiary structures. Each clan has a two letter identifier in which the first letter is I and the second letter is accorded in order of identification. Table 2.9 provides an overview of the numerous families of known natural peptidase inhibitors. Although it is more convenient to classify these peptidase inhibitors according to the specific catalytic mechanism that they are capable of inhibiting, a large number of these naturally occurring inhibitors have broad specificities and are capable of inhibiting peptidases from different mechanistic classes such as serine and cysteine peptidases. Moreover these inhibitors are also capable of inhibiting peptidases from other species, demonstrating their evolutionary divergence from a common ancestor. Table 2.10 provides a list of commercially available natural peptidase inhibitors used in the characterisation of peptidase activity in different mechanistic classes.

60

Table 2.9 - Nomenclature of naturally occurring peptidase inhibitors Source: MEROPS (http://www.merops.co.uk)

Clan Family Inhibitor type IA I1 ovomucoid inhibitor unit 3 (Meleagris gallopavo) I5 ascidian trypsin inhibitor (Halocynthia roretzi) I8 chymotrypsin/elastase inhibitor (Ascaris suum) 120 potato peptidase inhibitor II inhibitor unit (Solanum tuberosum) IB I2 aprotinin (Bos taurus) I52 tick anticoagulant peptide (Ornithodoros moubata) IC I3 soybean Kunitz trypsin inhibitor (Glycine max) ID I4 alpha-1-peptidase inhibitor (Homo sapiens) IE I7 trypsin inhibitor MCTI-1 (Momordica charantia) I37 potato carboxypeptidase inhibitor (Solanum tuberosum) IF I12 Bowman-Birk inhibitor (Glycine max) IG I13 eglin C (Hirudo medicinalis) IH I25 cystatin A (Homo sapiens) II I27 calpastatin inhibitor unit 1 (Homo sapiens) IJ I6 ragi seed trypsin/alpha-amylase inhibitor (Eleusine coracana) IK I38 metallopeptidase inhibitor Erwinia (Erwinia chrysanthemi) IL I39 alpha-2-macroglobulin (Homo sapiens) IM I14 hirudin (Hirudo medicinalis) IN I11 ecotin (Escherichia coli) IO I15 antistasin inhibitor unit 1 (Haementeria officinalis) IP I17 mucus peptidase inhibitor inhibitor unit 2 (Homo sapiens) IQ I50 baculovirus p35 caspase inhibitor (Spodoptera litura nucleopolyhedrovirus) IR I33 ascaris pepsin inhibitor PI-3 (Ascaris suum) IS I46 leech carboxypeptidase inhibitor (Hirudo medicinalis) IT I35 timp-1 (Homo sapiens) IU I36 Streptomyces metallopeptidase inhibitor (Streptomyces nigrescens) IV I32 BIRC-5 protein (Homo sapiens) IW I19 peptidase inhibitor LMPI (Locusta migratoria) IX I31 equistatin (Actinia equina) IY I16 streptomyces subtilisin inhibitor (Streptomyces albogriseolus) IZ I59 triabin (Triatoma pallidipennis) JA I34 saccharopepsin inhibitor (Saccharomyces cerevisiae) I 9 Endopeptidase B inhibitor (Saccharomyces cerevisiae) 10 marinostatin (Alteromonas sp.) 18 mustard trypsin inhibitor (Sinapis alba) 21 secretogranin V (Homo sapiens) 24 pinA endopeptidase La inhibitor (bacteriophage T4) 29 cytotoxic T-lymphocyte antigen-2 alpha (Mus musculus) 40 Bombyx subtilisin inhibitor (Bombyx mori) 42 chagasin (Leishmania major) 43 oprin (Didelphis marsupialis) 44 carboxypeptidase A inhibitor (Ascaris suum) 47 latexin (Homo sapiens) 48 clitocypin (Lepista nebularis) 49 proSAAS (Homo sapiens) 51 carboxypeptidase Y inhibitor (Saccharomyces cerevisiae) 57 staphostatin B (Staphylococcus aureus) 58 staphostatin A (Staphylococcus aureus) 63 pro-eosinophil major basic protein (Homo sapiens) L19 -

61

Table 2.10 - Commercially available natural class-specific peptidase inhibitors

Clan Inhibitor Specificity Serine Soybean trypsin Irreversible inhibitors of trypsin like enzymes from bovine, inhibitor (SBTI) human plant sources etc. Peptidases Bowman Birk Trypsin Reversible inhibitor of trypsin and chymotrypsin like Inhibitor (BBI) enzymes by ternary complex formation. Alpha-1 antiplasmin Reversible inhibitor of plasmin, trypsin and chymotrypsin. Antithrombin Reversibly inhibits thrombin, Factor Xa, trypsin and other trypsin like serine peptidases. Aprotinin Reversible inhibitor of serine peptidases, but does not inhibit thrombin and factor Xa. Cysteine Cystatins Inhibitors for cysteine peptidases. Dipeptidyl peptidases III and cathepsin. Very large family and other inhibitors in this Peptidases family have marked differences in their inhibitory properties. Aspartic Pepstatin A Most important inhibitor from Streptomycese species. Statins bind tightly to the Asp residues of these peptidases and have peptidases irreversible inhibitory properties. Metallo TIMP-1 Endogenously produced by MMP secreting cells. A potent reversible inhibitor of MMPs. Inhibits all MMPs. peptidases TIMP-2 Inhibits all MMPs. TIMP-3 Inhibits all MMPs. TIMP-4 Inhibits MMP-1, 2, 3, 7 and 9.

2.8.2 Synthetic Peptidase Inhibitors

Increasing numbers of synthetic peptidase inhibitors are being used for the detection of peptidases with higher sensitivity and specificity. They are generated to circumvent issues of stability commonly associated with natural inhibitors, and improve the existing capability of inhibitors as safe therapeutic products. There are two commonly used strategies used in the development of peptidase inhibitors. The first involves rapidly screening libraries of small molecules or newly synthesised combinatorial libraries for inhibitory activities on different classes of enzymes. The second approach to synthesising peptidase inhibitors is by structure based drug design, using X-ray crystallography. Synthetic, irreversible inhibitors for serine, cysteine and threonine peptidases were first designed by taking a specific substrate and coupling it to a reactive warhead. Some of the earliest inhibitors generated using this methodology include diazo compounds or halo-ketones (Schoellmann and Shaw 1963; Mares-Guia and Shaw 1965). Table 2.11 provides a list of commonly used synthetic peptidase inhibitors used for different peptidase classes with fairly high specificity:

62

Table 2.11 - Common synthetically manufactured class-specific peptidase inhibitors

Synthetic Effective Mechanism Notes Inhibitor Concentration Serine Peptidase Inhibitors Di-isoropylfluorophosphate Irreversible 0.1mM Inhibits all types of serine peptidases, (DFP) Highly neurotoxic 3,4-dichloroisocoumarin Irreversible 0.005-0.1mM Active against most serine peptidase. (3,4-DCI) Not active against β lactamases Tosyl lysyl chloromethyl ketone Irreversible 0.01-0.1 mM Inhibits trypsin like serine peptidases; (TLCK) unstable above pH 6.5 Tosyl phenylalanyl 0.01-0.1 mM Inhibits chymotrypsin like serine chloromethylketone (TPCK) peptidases; soluble in methanol Phenylmethanesulphonyl fluoride Irreversible 0.1-1mM All serine peptidases, Soluble in (PMSF) organic solvents; reversible by reduced thiols 4-(amidinophenyl) Irreversible 0.01-0.1 mM Inhibits trypsin like serine peptidase, methanesulphonyl fluoride more effective than PMSF (APMSF) Aminoethyl-benzene sulfonyl Irreversible 0.004 mM Inhibitor of trypsin like serine fluoride Hydrochloride (AEBSF) peptidase, very stable. Benzamidine Hydrochloride Reversible 0.1- 50 mM Potent inhibitor of thrombin and trypsin Cysteine Peptidase Inhibitors N-[N-[1-Hydroxycarboxyethyl- Irreversible 0.001-0.010 mM Inhibits all cysteine peptidases, does Carbonyl]Leucylamino-Butyl]- not affect cysteine residues in other Guanidine. (E-64) enzymes. Highly specific Iodoacetamide Irreversible 0.010-0.1 mM Non-specific inhibitor of cysteine peptidase and can inhibit other classes of enzymes. Also reacts with low mol. Wt thiols. Leupeptin Irreversible 0.010-0.1 mM Inhibitor of trypsin like serine peptidases and most cysteine peptidases. Aspartic Peptidase Inhibitors Pepstatin Reversible 0.001 mM Transition state analogue inhibitor of some aspartic peptidases. Potent inhibitor of cathepsin D, pepsin and rennin Metallopeptidase inhibitors 1,10 Phenanthroline Irreversible 1-10 mM Inhibits metallopeptidases and metal activated peptidases by chelation of metal ions. Ethylenediaminetetraacetic acid Irreversible 1-10 mM Effective chelator of active site zinc (EDTA) ion in metallopeptidases but can also inhibit metal ion calcium dependent cysteine peptidases. Phosphoramidon Irreversible 0.001-0.010 mM Inhibitor of many bacterial metallo- endopeptidaes but very few mammalian peptidases Diprotin A Irreversible 0.010-0.050 mM Inhibitor of dipeptidyl aminopeptidase IV Diprotin B Irreversible 0.010-0.050 mM Inhibitor of dipeptidyl aminopeptidase IV

63

2.9 Functional Significance of Peptidase activity in plants and pollen

Proteolytic enzymes play an important role in many aspects of plant cell development and physiology. They play an intricate role in cellular housekeeping, metabolism such as the mobilisation of reserve proteins from seeds during germination or seedling growth and also during programmed cell death of specific plant organelles (Vierstra 1996). Some plant sources contain proteolytic enzymes like papain in the latex of the papaya plant, in quantities far exceeding any plausible role in protein turnover (Brocklehurst and Salih 1983). These “Dispensable proteins” are particularly important for their contributory role in plant defences against numerous parasites and pathogens (Whittaker and Feeny 1971). Similarly, many seeds and tubers also contain peptidase inhibitors, which are protective against animal digestive enzymes but not plant peptidases. The presence of peptidases in pollen grains has been well established although their functional role in the process of germination is still somewhat unclear. It is generally assumed that these enzymes assist with the penetration of the pollen tube from the grain onto the stigma for fertilisation. Hydration of the pollen grain occurs when they come into contact with the stigma and results in the release of the peptidases from the exine, allowing for the pollen tube to digest its way into the stigma and subsequently deposit the sperm cells to the ova and form the zygote. The same exine concentrated peptidases could have a detrimental role on the respiratory epithelium and the mediation of allergic diseases. The respiratory epithelial cells are apposed to each other by tight junctions, which act as effective permeability barriers by occluding the apical ends of the para-cellular diffusion channels of the epithelia (Anderson and Van Itallie 1995). These tight junctions prevent the ingress of foreign proteins such as airborne allergens and thus limit their interactions with dendritic cells. That peptidases could contribute to the disruption of the epithelial tight junctions is well established. In vitro studies using enzyme rich extracts from pollen grains as well as the fungus Aspergillus fumigatus have demonstrated that these are capable of detaching the epithelial cells from their basement membrane in a concentration dependent manner (Robinson, Venaille et al. 1990; Hassim, Maronese et al. 1998). While some allergen enriched sources such as pollen grain extracts

64 contain high peptidase activities, in other cases allergens such as Der p 1 and Fel d 1 (from house dust mite and cat) have been shown to possess intrinsic peptidase activities. Both these cases allow for the concomitant localisation of antigenic as well as enzymatically active proteins on the surface of the respiratory epithelium. This potentially provides a mechanism for the simultaneous disruption of epithelial barriers and delivery of allergen to the immune system (Corry, Folkesson et al. 1996; Seymour, Gershwin et al. 1998; Kheradmand, Kiss et al. 2002).

65 3 IDENTIFICATION AND CHARACTERISATION OF PEPTIDASE ENZYMES

Understanding the role of proteolytic enzymes in physiological and pathological states requires their preliminary characterisation in a relatively pure form. However, unlike most proteins, the characterisation of proteolytic enzymes is relatively difficult because they are unstable (prone to autolysis) and are present in low concentrations (peptidases exert their biological functions at high specificity). The main objectives in the characterisation of proteolytic enzymes are: 1. Defining their substrate specificity: Peptidases are highly specific in their ability to cleave their target substrate for the activation or inhibition of a given reaction (Auld 1999). 2. Identifying catalytic mechanism: All peptidases possess highly conserved, active-site motifs, which in turn are dependent on the three-dimensional folding of the enzyme. 3. Determining optimal environmental conditions: The hydrolysis of a specific substrate is highly dependent on factors such as pH, presence or absence of co- factors, cations, etc all of which determine the rate of reactivity of the enzyme. There is no single method that can universally be employed for the purification of proteolytic enzymes. The basic premise in enzyme purification is to use the minimum number of purification techniques while maintaining optimal enzyme conditions. This chapter describes strategies that are generally used in the purification and characterisation of proteolytic enzymes.

3.1 Proteolytic Enzyme Extraction Techniques

The extraction of a functionally active enzyme from its cellular source is the first step towards its purification. The importance of enzyme extraction is underscored by the observation that most peptidases are highly compartmentalised for the maintenance of their catalytic activity and can be unstable outside their confined environment, due to the presence of specific inhibitors or related contaminating compounds. Thus, the extraction of peptidases from their cellular confines always involves a compromise between recovery of enzyme activity versus relative purity.

66 There are a number of methods/techniques that can be employed in the homogenisation of cells or tissues for the extraction of these enzymes. The application of the right technique depends on whether the peptidase of interest is localised within a specific organelle (which can be isolated prior to extraction) or if the enzyme can be obtained from whole cell homogenisation. Most plant peptidases are contained in vacuoles within the cytoplasm and comprise no more than 1-2% of the total cell protein. In addition, plants contain cellulose, chloroplasts, starch granules and other organelles. The extraction of proteolytic enzymes from these compartments requires a certain degree of cell disruption. Possible approaches include: 1. Moderate to vigorous extraction involving osmotic shock, with chilled neutral buffers of high molar concentration (usually a high percentage of sucrose). More vigorous techniques may involve homogenising the plant tissues in mixers or blenders, or agitation using abrasives such as silica. The latter is usually performed on smaller volumes of tissues, wherein the plant material is frozen and then ground using a mortar and pestle with a mixture of alumina or sand to release intracellular components. 2. Liquid extrusion, which relies on the principle of forcing a cell suspension at high pressure through a narrow orifice. By varying the pressure applied, cells may be partially or completely disrupted. 3. Ultrasonication, involving the application of high-pressure sound waves. This causes cell disintegration by generating shearing forces. Compared to animal cells, plant cells require stronger disruption methods because of the cellulose component of their cell walls. However, disruptive extraction processes are non-specific and can result in the alteration of pH conditions or the oxidation of susceptible proteins during homogenisation. Many plants contain high concentrations of phenolic compounds, which oxidise to form dark pigments. These pigments can interact covalently and inactivate many enzymes. This can be prevented by including thiol compounds such as β-mercaptoethanol or polyvinylpyrolidone as they preferentially react with phenolic compounds. Many intracellular peptidases also occur in precursor forms, where they are prevented from exerting their effects. Releasing these enzymes from the internal milieu can result in their activation, leading to autohydrolysis and destruction. Hence, it is advisable to incubate the solution with a broad spectrum of reversible inhibitors to preserve their functional activity. The extraction of peptidases into solutions also requires strict attention to the

67 maintenance of the appropriate pH, temperature and ionic concentrations. Since most mechanical cell disruption methods result in overheating and consequent denaturation of the proteins, the extracts and equipment need to be cooled to a low temperature with several short phases of mechanical disruption being applied instead of a single, long application.

3.2 Measurement of Enzymatic Activity

The ability to accurately monitor the hydrolytic activity of peptidases is an important prerequisite for the design of experimental methods for enzyme purification. A specific and sensitive assay allows for the detection of activity in crude extracts, monitoring the progress of purification, assessment of factors influencing optimal catalytic activity, the elucidation of its catalytic mechanism and studying enzyme kinetics (Johnson 1992). Such an assay can also be applied to the characterisation of active-site chemistry using site-specific, time dependent inhibitors as reactivity probes (Boghosian and McGuinness 1981). The principle of a peptidase assay is to stoichiometrically measure the loss of substrate or the formation of a product as a result of peptide bond hydrolysis. Assays may be performed in the solid or liquid phase. Although both assay formats employ the same substrates for the detection of peptidase activity, the information generated is qualitatively different.

3.2.1 Solid Phase Assays

As the name implies, these assays involve the use of immobilised substrates for the detection and measurement of peptidase activity. The substrates used can either be general proteins such as gelatin or haemoglobin (when the specificity of the enzyme is unknown) or highly specific peptide substrates. High sensitivity in these assays can be achieved by labelling the substrate with a chromogenic or fluorogenic tag. Undoubtedly, the detection and characterisation of proteolytic activity using electrophoretic techniques has received the widest application because it is relatively inexpensive and is capable of separating enzymes from contaminating proteins in a crude extract (Laemmli 1970). Polyacrylamide gel electrophoresis (PAGE) is the most common technique used in laboratories although other non-convection media such as papers or membranes have also been used (Gabriel and Gersten 1993). The

68 advantages of PAGE over other electrophoretic techniques is the fact that these gels behave as sieve with variably sized pores (that can be controlled by varying the percentage of the acrylamide in the gels) which effectively separate peptidases from other proteins based on differences in their molecular weight. Table 3.1 provides a brief description of different electrophoretic based assays that are routinely applied in enzyme detection and quantitation. A potential disadvantage of electrophoresis in enzyme detection is that it is time consuming compared to liquid phase assays. In addition, enzyme denaturation can occur due to high temperatures during electrophoresis or the presence of denaturing compounds such as SDS or urea. This has largely been overcome by using native gels, which are non-denaturing and spare the activity of the enzyme. However, native gels are suitable for soluble proteins that will not precipitate or aggregate during electrophoresis. Due to the mild running conditions, native gels cannot effectively reduce disulphide bonds, thus affecting the ability of proteins to accurately migrate to their specific molecular weight regions.

69 Table 3.1 – Different types of solid phase assays

Assay Description

SDS-PAGE This type of assay generally involves measuring the ratio between a macromolecular substrate and its digested, lower molecular weight product as a result of peptidase activity. The detection of the product is performed by fractionation of the substrate by SDS/PAGE. The intensity of the substrate and product reflects hydrolysis rate. A calibration curve can then be generated to convert intensity into rate of reaction.

Zymography In these assays, the polyacrylamide gel is co-polymerised with a general or specific protein substrate. Electrophoresis is performed under non-reducing conditions. The separated peptidases subsequently digest the protein in the region they are separated into, resulting in the formation of a substrate free zone, which can be detected and quantitated by staining the gel with Coomassie brilliant blue. Is often used in crude samples where multiple peptidases may be present.

Blotting Electrophoretically separated peptidases are transferred to hydrophobic blots and can be probed for enzymatic activity using techniques substrates or known enzyme inhibitors that are fluorescently or radio labelled. The measurement of the fluorescence or radioactivity reflects the concentration allowing for its quantitation.

Isoelectric Like SDS/PAGE, this method is applicable for general protein analysis and allows for the two-dimensional separation of the Focusing enzyme of interest by isolating it on the basis of its isoelectric point and its molecular weight. The detection is performed the same way in the second phase as in zymography. This method however is not very sensitive as it often results in the denaturation of enzymatic activity.

70 3.2.2 Liquid Phase Assays

Liquid phase assay formats are preferred to solid phase assays because they are relatively easy to set up and are highly versatile in application. Advances in the specificity and sensitivity of these assays allow for high-throughput detection and analysis of peptidase activity, a feature that makes them preferable to solid phase assays. Liquid phase assays are often used to study the functional properties of peptidase activity and the kinetics of the catalysed reaction such as: 1. Monitoring the reaction progression curves. This allows for the detection of lag or burst phases in enzyme kinetics and the substrate utilisation rates.

2. Comparing kinetic parameters such as Vmax and Km of the enzymatic reaction on multiple substrates simultaneously. This is useful in evaluating different substrate specificities and susceptibility to inhibitors (Gul, Sreedharan et al. 1998). 3. Investigating the mechanism of catalysis such as transition state, formation of enzyme-substrate covalent intermediates and steady state kinetics (Johnson 1992). Liquid phase assays are sub-divided into discontinuous assays and continuous, direct or indirect assays. In discontinuous assays, the rate of reaction cannot be measured in real time due to an inability to detect the product formed or differentiate the product from the substrate in a mixture. Commonly used discontinuous assays include radioactive or HPLC based assays (Stewart and Young 1984). In these methods, a progressive curve of the reaction is constructed by quenching the enzymatic reaction at different time points and analysing the reaction mixture (Kuwada and Katayama 1984). However, these assays are prone to errors if the reactions are improperly quenched and/or the linearity of the reaction is not well determined thereby affecting the assessment of the initial reaction rates and enzyme kinetics. Continuous assays are more sensitive, informative and provide a much more reliable estimation of the rate of reaction in real time. Continuous assays can either be measured directly, where the catalysed reaction itself produces a measurable signal or in an indirect format, wherein the catalysed reaction results in the formation of an end product which is not detectable unless it reacts with a highly specific probe present in the assay solution. Most continuous assays are based on a measurable difference between the physical properties of the substrate and the products. Changes in

71 absorbance, fluorescence, pH, conductivity and viscosity have all been used in the accurate measurement of enzymatic activity (Gul, Sreedharan et al. 1998). Photometric assays are the most frequently used continuous assays. These assays rely on chromophoric differences between the substrate and digested products. Some assays use natural substrates, which on cleavage produce a detectable decrease in absorbance (John 1993). However, in most cases, synthetic analogue substrates have been able to achieve better results due to the improved sensitivity and specificity. These chemically generated substrates usually possess one cleavable bond and are coupled to chromogenic substituents either on their amino-terminal or the carboxy- terminal end. Common chromogenic substituents used in photometric assays include p- nitrophenol, 4-nitrophenylalanine, dithio-bis(2 nitrobenzoic acid) (DTNB) (Sarath, Motte et al. 1989). Table 3.2 enumerates the commonly used natural and synthetic chromogenic substituents used in peptidase enzyme analysis. The introduction of a fluorescent tag into a protein/peptide substrate is more sensitive as a method to assay enzymatic activity. The attachment of a fluorophore to the C-terminal end through an ester or amide bond allows for the direct measurement of the activity of some endopeptidases. Furthermore, the incorporation of a fluorophore and its quencher into a single substrate allows for the development of intramolecular quenching assays for both endo- and exopeptidases. The most widely used fluorescent substituents are either 7-amino-4-methylcoumarin (AMC) or 6,1 aminonapthalenesulphonamide (ANSN) for the examination of endopeptidase and aminopeptidase catalysis respectively. Most of the recently designed synthetic fluorogenic substrates are intramolecularly quenched. These substrates contain a fluorophore (donor) quenched by another group (acceptor) present in the substrate, but are separated from each other by a scissile bond. Hydrolysis of the scissile bond by the peptidase results in the release of the fluorescent activity moiety, which is measured by a spectrofluorometer on the basis of its excitation and emission wavelength. Table 3.3 provides examples of commonly used fluorophore/quencher pairs used in the generation of specific substrates for peptidase assays:

72

Table 3.2 - Chromogenic peptide substrates commonly used for the assessment of peptidase activity of different mechanistic classes Adapted from: (Kirschke and Weideranders 1999)

Optimal Enzymes Substrates pH Endopeptidases Z-Lys-SBzl 8.0 Trypsin Z-Arg-SBzl 7.5

Tos-Gly-Pro-Arg-NHPhNO2 7.5 Chymotrypsin Boc-Ala-Ala-Phe-SBzl 7.5 Z-Lys-SBzl 8.0 Thrombin Z-Arg-SBzl 7.5 Boc-Ala-Ala-Phe-SBzl 6.8

Papain Z-Phe-Cit-NHPhNO2 6.2

Bz-Arg-NHPhNO2 7.2

Chymopapain Boc-Ala-Ala-Gly-NHPhNO2 6.8

Tryptase Suc-Val-Pro-Phe-NHPhNO2 8.0

Thermolysin Ac-Pro-Leu-Ala-Nva-Trp-NH2 6.0

Gelatinase A Ac-Pro-Leu-Ala-Nva-Trp-NH2 6.0 Exopeptidases Carboxypeptidase N FA-Ala-Lys-OH 7.5 Carboxypeptidase C FA-Phe-Leu-OH 6.5 H-Leu-NHNap 8.0 Leucine aminopeptidase H-Leu-NHNap 8.0 H-Leu-hydrazide 8.0

Peptidyl dipeptidase A FA-Phe-Gly-Gly-OH 7.5 (ACE) FA-Phe-Ala-Phe-OH 7.5

Cathepsin E H-Pro-Pro-Thr-Ile-Phe-Phe(NO2)-Arg-Leu-OH 3.5

H-Phe-Gly-His-Phe(NO2)-Phe-Val-Leu-Ome 4.0 Cathepsin D H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-leu-OH 3.1

Boc-Ala-Ala-Gly-NHPhNO2 6.8

Glycyl endopeptidase Z-Phe-Cit-NHPhNO2 6.2

Bz-Arg-NHPhNO2 7.2

73

Table 3.3 - Common substrate coupled, quenched fluorescent groups used for the assessment of peptidase activity of different classes

Ex/Em Donor/Acceptor pairs Enzyme wavelengths Abz/ONBzl Aminopeptidase 310/410

Abz/Phe(NO2) HIV protease 337/410

Abz/Try(NO2) Subtilisin/pepsin 320/420

Abz/EDDnp Kallikreins 320/420

EDANS/DABCYL HIV protease 340/475

Mca/Dnp MMP 328/393

Rp/Dns Endopeptidase 285/360

Trp/Dnp MMP 280/360

Assays using these quenched substrates have also been adapted in several novel enzyme assays such as fluorescence resonance energy transfer (FRET), which can generally be used when the fluorescence based assay requires more than a single scissile bond for cleavage (Knight, Willenbrock et al. 1992).

3.3 Protein Purification Procedures

3.3.1 General Purification Procedures

The successful separation of a protein from a complex mixture usually involves exploiting some of its inherent properties; be it size, charge, solubility or the presence of specific binding sites. The initial stage of purification requires the removal of cell debris and contaminating proteins co-localising during the extraction phase. Peptidase enzymes are present in low concentrations and can easily be inactivated by endogenous inhibitors present in the cell extract. Hence the initial fractionation of the cell extract to obtain an enzyme rich fraction is an important prerequisite towards purification of a peptidase. Table 3.4 provides a brief overview of the different types of pre-fractionation methodologies used in the purification of enzymes.

74 Table 3.4 - Protein fractionation techniques

Fractionation Procedure Application Step Precipitation This procedure involves changing the solubility of This technique results in the enzyme either by changing the pH or the ionic selective denaturation and can strength. Common precipitation agents include result either in the inactivation high salt concentrations such as ammonium or reduction of enzymatic sulphate. Alternatively, decreasing the dielectric activity. However, numerous constant using water miscible solvents also cause enzymes on resuspension and the precipitation of large, charged molecules. renaturation demonstrate retained activity. Centrifugation Large organelles can be sedimented by high Depends on a variety of centrifugal speeds (30,000 g), generated by factors such as size and shape ultracentrifugation. More often it is used to of the protein and viscosity of separate insoluble material generated during the the sample. This method is not homogenisation of the sample. usually used to separate samples containing multiple enzymes in a single mixture. Dialysis or Ultra Protein separation method involving size based Cannot separate between filtration fractionation through a semi-permeable complex mixtures of enzyme. membrane, which selectively retains large Dialysis membranes usually globular proteins of a specific molecular range. It can get clogged due to is used more often in the removal of salts, organic precipitating proteins. solvents and inhibitors of low molecular weight. Ultrafiltration involves a similar process, under pressure. It is useful in reducing the volume of the sample and concentrating the proteins. Lyophilisation Used to effectively reduce the solvent component Re-solubilisation of proteins of the enzyme solution. Used when the enzyme can sometimes be an issue. sample is relatively pure and is used for storage purposes.

3.3.2 Size-Exclusion Chromatography

Size-exclusion chromatography is a convenient and highly reproducible method of separating simple protein mixtures whose components differ sufficiently in their molecular weight (Andrews 1965). Smaller proteins require at least 10% difference in their molecular weights for efficient resolution by this method whereas larger molecules such as enzymes require at least two fold differences to resolve efficiently (Porath 1997; Paulsen, Olafsdottir et al. 2002). This chromatography uses an aqueous mobile phase and hydrophilic matrix to separate the proteins in a mixture. The solid phase consists of a matrix made up beads, with pores of defined size, packed in a column through which the mobile phase flows. In principle, large molecular weight proteins that cannot permeate the beads flow through the column faster and elute earlier while smaller proteins within the pore size range are retained within the beads and hence take longer to flow, eluting later. Figure 3.1 provides a brief schematic on protein separation by this method.

75

Figure 3.1 - Schematic representation of size-exclusion chromatography

Size Exclusion column comprising of porous bead (inner column) and dead space between the packed column

Chromatogram displaying the elution of proteins on the basis of their size and retention in the column.

Retention Time (mins)

The primary factors affecting the resolution of protein separation in size- exclusion chromatography are column volume, particle size, pore size distribution, flow rate of the mobile phase, protein conformation, temperature and solvent viscosity. The resolution of separation is directly proportional to the size of the column. However, larger columns result in increased separation/analysis time since effective separation of the proteins requires slow flow rates (Scopes 1995). These shortcomings have been circumvented with the development of more effective particulate solid phase materials that can withstand the high back pressures associated with faster flow rates. These include cross-linked dextrans such as Sephadex, polystyrene packing materials and polyacrylamide based matrices (Bio-gel), etc. Size-exclusion chromatography is well suited for enzyme purification due to its gentle binding and elution techniques and its ability to retain biological activity. However the limitations on its efficiency make it more suitable for purification stages where the mixture is not highly complex.

3.3.3 Ion Exchange Chromatography

Ion exchange chromatography is widely used in protein purification due to its dynamic range and high resolution capabilities. Protein separation by this method is

76 based on differences in electric charge (Roe 1993). Since proteins fundamentally differ in their amino acid sequence, they vary in their net charge at any given pH other than their isoelectric point. The solid phase in ion-exchange chromatography consists of modified derivatives of support materials such as cellulose, sephadex, etc. When a protein sample consisting of different ionic species passes through the column, the proteins are distributed between the mobile phase and the solid phase. The strength of their interaction is dependent upon the degree of counter ion exchange. These interactions are reversible (Glod 1997) by progressively increasing the ionic strength of the mobile phase, so that the counter ions compete with the solutes for the interaction sites on the solid phase (Walsh and Headon 1994). Figure 3.2 explains the ionic interactions. Figure 3.2 - Schematic representation of ion-exchange chromatography

1. Equilibrium 2. Adsorption 3.Desorption 4. Regeneration

C+ C+ C+ S++ S++ C+ C+ C+ S++ C+C+ C+ C+ S+ C+ C+ S+ S+ S+ C+ C+ C+ F- F- F- C+ F- C+ S+ C+ C+ F- C+ F- F- C+ F- C+ S+ C+ C+ C+ F- F- S+ F- C+ F- C+ C+ C+ C+ F- C+ F- S++ F- S++ F- C+ C+ S++ S++ C+ F- F- F- F- C+ S++ S++ C+ F- C+ F- S++ F- S++ F- C+ C+ S++ S++ C+

S++ C+ C+ C+ S++ S+ S+ S++ C+ C+ S++ S+ S+ S++

F-: Cation exchange resin C+: Counter ion in Buffer S+: Analyte S++: Higher affinity analyte

Ion exchange chromatography wherein the stationary phase carries positive charges is known as “anion-exchange chromatography” whereas a negatively charged solid phase is known as “cation-exchange chromatography”. These exchanges allow for the separation of a wide range of differentially charged proteins. Enzymes are usually applied to an ion exchange column in a solution of low ionic strength and pH. The elution of the protein is usually performed by one of two mechanisms, namely: 1. Changing the pH of the mobile phase, resulting in the alteration of the charges of the ionic species bound; i.e. lower pH for anionic exchange and increasing pH for cationic exchange.

77 2. Increasing the ionic strength, thereby weakening the electrostatic interactions between the proteins and the adsorbent material. Commonly used ion exchange solid phases and their optimal pH range are enumerated in Table 3.5. Table 3.5 - Commonly employed ion-exchange groups Adapted from (Karlsson, Ryden et al. 1998)

Group pH range

Anion Exchangers

Quaternary ammonium exchangers 2-12

Diethylaminoethyl groups 2-9

Quaternary aminoethyl groups 2-12

Cation Exchangers

Sulphopropyl groups 2-12

Methyl sulphonate groups 2-12

Carboxymethyl groups 6-11

3.3.4 Reverse-Phase Chromatography

Reversed-Phase chromatography (RPC) is the most widely used chromatographic technique used for the separation of complex protein mixtures. As its name implies, RPC essentially is the reverse of normal-phase chromatography in the sense that it involves the use of a non-polar (hydrophobic) stationary phase and a polar (hydrophilic) mobile phase. The chromatographic separation comprises a stationary phase; usually silica derivatives to which a functional, hydrocarbon based group is chemically bonded. Some of the common functional groups include the alkyl-, cyano- or amino groups. The retention of proteins usually is found to increase with the chain length of the functional group. The retention of proteins or peptides on the stationary phase is assumed to occur because of the hydrophobic behaviour of proteins (Doonan 1996). Retention is highly dependent on the quality of the stationary phase, which includes the type of functional group, the pore distribution of the matrix and the length of the hydrophobic

78 chain. RPC using small chain hydrocarbon groups retains proteins on the basis of adsorption whereas longer chain hydrocarbons (C8-C18) retain proteins by forming a partition between the solid and mobile phase (Syed 1993). Elution of the proteins is brought about by polar solvents, typically water mixed with a gradient of less polar solvent such as methanol or acetonitrile. The addition of 0.1% trifluoroacetic acid (TFA) in the buffers improves the resolution of separation by ion pairing with the proteins present in the mixture. As the polarity of the mobile phase is gradually decreased; the solvophobic behaviour of the proteins to the mobile phase decreases, resulting in their elution. The main consideration in RPC is the instability of the silica support at extremes of pH. The general range of the mobile phase is 2-8, beyond which hydrolysis of the functional group or the disassociation of the matrix may occur. The application of RPC to enzyme purification is limited, because of the denaturing conditions (low pH and organic solvents). However, some biologically active serine peptidases and growth factors have been recovered intact (Chlenov, Kandyba et al. 1993).

3.3.5 Affinity Chromatography

The binding of a ligand to its receptor is usually stereoselective and often of high affinity and specificity. This property of “selective attraction” as depicted in Figure 3.3 is well exploited for the purpose of protein purification and this technique is called “affinity chromatography”. Affinity chromatography is a powerful technique, since it allows for the purification from complex mixtures of target proteins that exist in very low concentrations, using specific functional properties of proteins (Winzor 2000). Affinity matrices are often custom made for the separation of a single target protein. These can be formed from ligands that are either mono-specific or group-specific. Table 3.6 provides a list of the commonly used affinity based matrices and their target proteins.

79

Table 3.6 - Common group-specific ligands used in affinity chromatography

Ligand Target protein

5’AMP, ATP Dehydrogenases NAD, NADP Dehydrogenases Protein A Antibodies Lectin Glycoproteins, Polysaccharides Heparin Lipoproteins, DNA, RNA Gelatin Fibronectin Arginine peptidases Benzamidine Serine peptidase Polymyxin Endotoxin Calmodulin Kinases Cibacron Blue Kinases, phosphatases, albumin

The main requirements for the design and synthesis of a successful affinity adsorbent are: 1. The immobilisation of the ligand and the matrix should not interfere with the ligand’s ability to bind to its protein. 2. A “spacer arm” setting the ligand away from the matrix should be used to make it more accessible to the protein. 3. Non-specific interactions should be minimal, allowing for high-grade purification. 4. The linkages should be sturdy and stable enough to withstand harsh “clean up” procedures, prior to re-use. Most of the early work on affinity chromatography involved the purification of proteins or enzymes using naturally occurring receptors, substrates or specific inhibitors. Subsequent applications employed interactions that do not involve protein ligands such as Cibacron blue (Clonis 1988) and immobilised metal activated cation (IMAC) chromatography (Sulkowski 1989). The advent of recombinant protein production technology and the generation of mono- and polyclonal antibodies has allowed for the affinity purification of proteins on the basis of their biological functions.

80 Group-specific matrices also play an important role in the purification of certain classes of proteins from crude mixtures. These include Protein A and Protein G for the general purification of antibodies (Desai 1990), lectin chromatography for the purification of glycoproteins from diverse sources (Kennedy and Rosevear 1973; Sharma and Mahendroo 1980; Hage 1999), as well as lysine, arginine and benzamidine Sepharose chromatography for the purification of trypsin-like serine peptidases (Ehle and Horn 1990; Labrou 2002). In practice, natural biological ligands for specific protein purification are very difficult to obtain or isolate. The selection of suitable substrates or specific inhibitors in enzyme purification requires an in-depth understanding of the enzyme-substrate and enzyme-inhibitor interaction as well as its kinetics before they can be employed as adsorbents (Wagner 1986). Figure 3.3 - Schematic of the principle of affinity chromatography. The immobilized affinity matrix selectively binds to its stereo-specific ligand, while the other non-specific proteins elute away in the mobile phase. Adapted from (Cutler, 1996)

The ultimate adsorbent for any enzyme or protein is an immobilised antibody, raised against the particular protein and one that specifically interacts with a single surface feature of the protein. Antibodies are not only specific for the exact amino acid sequence and three-dimensional organization, but also have high binding constants (Angal and Dean 1993). A number of allergens and isoallergens have been isolated and

81 purified using these methods (Kahn and Marsh 1986; Huecas, Villalba et al. 1999; Lehmann, Hoffmann et al. 2003). On a similar front, there have been reports of the isolation of cross-reactive allergens from a particular group of proteins from different sources of grass pollen, using monoclonal antibodies raised against the group 1 allergen (Han, Chang et al. 1993; Chang, Liu et al. 1995; Stumvoll, Lidholm et al. 2002).

3.4 Principles of Protein Identification by Proteomics

The “Proteome” is defined as the total protein complement of the genome expressed by an organelle, cell, tissues or an entire organism (Wasinger, Cordwell et al. 1995). The field of proteome analysis, referred to as proteomics, is the comprehensive, multi-faceted analysis of the various aspects of protein expression, post-translational modification, interactions, organisation and function at a global level. Although the rudiments of proteins separation technology have existed since the early 1970’s, a number of advancements have fuelled its application at the forefront of biological sciences. They are as follows: 1. The most beneficial contribution to the field of proteomics has been the rapid advances in genomics, the successful completion of the human genome and the sequencing of other important genomes (available on http://www.ncbi.nlm.nih.gov/genomes). These complete genome databases provide for a sequence based framework for the mining of the proteome. 2. Improvements in the sensitivity and resolution of protein separation technology have significantly lowered the threshold of protein detection. This has allowed for the identification of novel proteins of very low abundance. The developments include advances in the field of gel based protein separation techniques, micro and nano-liquid chromatography. 3. Improved ionisation techniques, compatible with protein/peptide analysis and signicifcant developments in mass spectrometric instrumentation have enhanced the applications of biological mass spectrometry. 4. The introduction of bioinformatics has greatly facilitated the analysis of vast volumes of sequence data. The completion of entire genome sequences of different organisms has led to the development of the Genbank sequence databases, comprising of publicly available nucleotide sequences and their

82 protein translation. This has also led to the development of mining software that allows for the rapid screening and comparison of proteins identified from a species to any known related protein in the database. This is no easy task, considering that these databases contain over 29.3 billion nucleotide bases in over 23 million sequences, a figure that is exponentially increasing with time. The analysis and characterisation of the proteome of an organism can essentially be sub-divided into four active area of research, namely: 1. Protein Mining to determine all the proteins and their phenotypes present in any particular proteome. 2. Protein Expression Profiling to accurately quantify expression levels for individual proteins during different cell states. 3. Post-translational Modification: Identifying protein modification and their functional roles 4. Protein-Protein Interaction: Identifying the interactions between different classes of proteins (ligand-receptors, antigen-antibody and enzyme-inhibitors), which mediate cell functions.

3.4.1 Why Proteomics?

The post-genomic era has resulted in a compelling need to interpret the data from sequenced genomes at a functional level in cells and tissues. The sequencing of the human genome exposed several inherent limitations in the application of the data, the most significant being the inability to correlate gene regulation to the transcription, translation and expression of proteins. In this regard, protein analysis has several advantages compared to gene expression profiling. Proteins are strategically placed to provide the missing link between specific gene(s) and the cellular function with which they are associated. Moreover, while disease aberrations are caused at a genetic level, the functional consequences are expressed at a protein level. Some of the qualitative differences between the analysis of the genome versus the proteome are: 1. The genome is relatively static and finite and hence largely uninformative of the constant changes that occur at a cellular level. The proteome, on the contrary is multi-dimensional and dynamic, stemming from the large number of cell lineages and sub-types, each with its own unique proteome (Godovac- Zimmermann and Brown 2001).

83 2. Although the genome of an organism is amenable to environmental stimuli, the most prominent and visible effects of exogenous or environmental stimulation on cellular functions occurs at a protein level. 3. The paradigm of 1 gene = 1 protein is a wrong assumption. The complete sequence of the 3.3 billion nucleotides comprising the human genome has recently been completed (http://www.ncbi.nih.gov/genome/guide/human). These include the location and nearly complete sequences of the 26,000 to 30,000 protein encoding genes (Eichler and Frazer 2004). However, there are an estimated 1.5 million proteins, which translates to roughly 50 proteins for every gene (Meri and Baumann 2001) 4. Although proteins are fundamentally the end products of gene expression, they are often modified post-translationally for the mediation of their functions. The type or location of these post-translation modifications cannot be derived from the gene sequence data, hence limiting its application in understanding cellular function at a molecular level. In fact, even though there are only 20 amino acids, Uy and Wold (Uy and Wold 1977) listed a total of 140 possible amino acid forms that result from chemical modifications of the parent 20 (eg. Glycosylation, acetylation, glucuronidation, phosphorylation, methylation, etc.). 5. The analysis of the complete mRNA complement of the genome (transcriptomics) is also inadequate. If most of the regulatory events controlling the levels of expression of proteins are dictated during the production of the corresponding mRNA, it would be sufficient to study the functional aspects of genomics (Gygi, Rochon et al. 1999) However, comparisons between the mRNA and corresponding protein levels in experiments that have been carried out using liver and yeast cells have yielded very low correlation levels (< 0.4). This means that protein levels cannot simply be deduced from levels of mRNA expression or vice versa. Furthermore, a variation at the level of a given mRNA species cannot be equated with a corresponding variation in the levels of the proteins (Gygi, Rochon et al. 1999).

3.4.2 Protein Separation Techniques

Analysing the proteome of an organism involves two principal processes; effective separation of the cellular constituents of the proteome and their subsequent

84 characterisation. Figure 3.4 provides a schematic representation of the analytical approach towards protein identification. Figure 3.4 - General Strategy for proteome characterisation

Protein Sample

Protein Extraction

Protein Concentration

One-dimensional Two-dimensional Chromatographic Electrophoresis Electrophoresis Separation RP-HPLC IEC-HPLC

Solubilised protein

Peptide digest solution

Mass Spectrometric Analysis

MALDI-TOF/MS LC-ESI-MS/MS

Database searches

Identification Characterisation Quantitation Post translation modification Protein-protein interaction

Most proteomic based experimental studies are based on this approach, incorporating rapidly evolving technologies and improving on the sensitivity and resolution of separation and detection techniques. Effective protein separation is a complicated task as cellular proteins have to be isolated from other interfering biological molecules such as carbohydrates, lipids and nucleic acids. Moreover, proteins isoforms are highly homologous, making it difficult to separate them effectively. Unlike gene expression analysis, which is relatively straightforward, there is no standard biochemical technique that can be uniformly applied to protein separation. In addition, the complexity of the proteome requires these separation techniques to be interfaced with analytical instruments that can identify the individual components in a high-throughput fashion. Two approaches that fulfil most of these criteria are the gel based, electrophoretic separation techniques and HPLC separation. Table 3.7 provides an overview of the different protein separation techniques that are currently employed in proteomic based studies along with their specific strengths and weaknesses.

85 Table 3.7 - Proteomic separation techniques

Method Principle Strengths Weakness One- One of the most widely used analytical separation Very simple and inexpensive methodology. Resolution and sensitivity have always been Dimensional techniques for complex protein mixtures Proteins are Easily reproducible. Proteins can be extracted issues. Cannot resolve between proteins with Electrophoresis separated on the basis of their molecular weight from the gel for proteomic based analysis. similar molecular weights. Sensitivity of the through an acrylamide gel which acts as a sieve. method is highly dependent on the detection method used (e.g. Silver stain vs. Coomassie). Preparative Iso- Another commonly used protein separation The method is ideal for the application of large This method is suitable for proteins that can be Electric technique. Complex protein mixtures are separated in quantities of proteins, which is a shortcoming solubilised. Hydrophobic proteins tend to Focusing the liquid phase rather than the solid phase. Proteins of gel based methods. Typical IEF runs can precipitate when they reach their isoelectric are separated on the basis of their isoelectric pH, accommodate milligram to gram amount of point. which is achieved along a pH gradient generated proteins. High resolution based on protein using a mixture of ampholytes. quantities. Two- Advancement over the above mentioned techniques The method possesses high resolution and Protein solubilisation is a major factor in the Dimensional and one of the core techniques in proteome research. sensitivity. Capable of separating over 1000 improvement in the resolution of the separating Electrophoresis Complex proteins are separated in two dimensions; proteins in a single run. Is capable of proteins. Hydrophobic proteins are particularly the first on the basis of their isoelectric point and then differentiating between numerous isoforms of a hard to solubilize. Detection limits are usually a subsequently in the second dimension on the basis of single protein. Can also be used in protein function of the technique Separation of highly their molecular weight. The reproducibility is expression comparisons. basic proteins (≥ pH 10) is difficult. achieved by the use of immobilised pH gradients that are incorporated into the gels. High This method involves interfacing HPLC techniques Much higher resolution than any other method Expensive method. Column can often get Performance like reverse phase or ion-exchange chromatography of protein separation technique. Requires no blocked due to protein overload. The buffers Liquid separately or in tandem (2D-LC or MUDPIT) for sample handling avoiding contamination from used to run one type of chromatography are Chromatography complex protein mixtures separation. The HPLC human manipulations. The method circumvents incompatible with the second chromatographic systems have been miniaturized to micro or nano-LC, the problems associated with the solubilisation run and often, buffer exchange is necessary. allowing for their effective interfacing with high- and separation of hydrophobic and hydrophilic Effective separation results in the generation of throughput detection systems. Separation of the proteins. Requires less sample material due to large volumes of data which needs to be sifted protein mixtures occurs on the basis of protein its high sensitivity. through. hydrophobicity (RP) or ionic charges (IEC).

86 3.4.3 Proteomic Analysis by Mass Spectrometry

In spite of the advances in the electrophoretic and chromatographic separation of complex protein mixtures, proteomics suffered a lag phase due to the lack of appropriate biochemical methods capable of identifying proteins in a high-throughput fashion, and of bioinformatics tools capable of correlating oligonucleotide sequence information from genomic databases to protein sequence tags. These bottlenecks have largely been addressed in the last decade or so, with rapid development in the field of biological mass spectrometric detection and analysis of proteins as well as bioinformatics software to effectively correlate expressed sequence tags with identified proteins and validate the results of high-throughput proteomic analysis (Dass 2001). Mass spectrometry is an analytical technique that is used to measure the masses of molecules with great accuracy. The scientific principle behind this technique is simple and involves the conversion of analyte molecules into a gaseous phase and their subsequent ionisation. Once ionised, the masses may be determined on the basis of the relationship with net charge (mass/charge ratio) using a variety of mass spectrometers (e.g. time of flight mass spectrometers, Tof-MS). Heavier ions traverse the flight-path more slowly than lighter ions and are detected later. The mass resolved ion current is displayed in the form of a spectrum, consisting of the relative abundance of the detected ions. Typical instruments are capable of measuring masses to within 0.01% error of the total molecular weight of the molecule i.e., within 4 Da error for a protein of MW 40,000 Da (Leibler 2002). This is accurate enough to allow the measurement of minor changes such as amino acid substitution or post- translational modifications. The application of mass spectrometry in protein identification was initially limited by the fact that biological samples could not withstand the harsh ionisation techniques and were frequently degraded on analysis coupled with the poor sensitivity and detection capabilities for small sample volumes (Dass 2001). This changed in the last 15 years with the advent of gentle ionisation techniques such as “Electrospray ionisation” (ESI) and “Matrix assisted laser desorption/ionisation” (MALDI) (Karas and Hillenkamp 1988). These ionisation techniques coupled with the development of sensitive analysers have revolutionised the application of mass spectrometry in protein chemistry. Proteomic analysis by mass spectrometry is accomplished by two

87 distinct methods using two different aspects of mass spectrometry derived data. They are: 1. Peptide mass fingerprinting 2. Tandem mass spectrometry Instruments used for these purposes are described below.

3.5 Types of Analytical Mass Spectrometers

There have been great advancements in the development of analytical mass spectrometers over the past 100 years since J.J. Thomson first built his “parabola mass spectrograph”. Although mass spectrometric analysis of biological and non- biological compounds is in principle the same, the method by which this is achieved varies in different instruments. The three main functional components of an analytical mass spectrometer are: 1. An ion source, used to convert the liquid analyte sample into gaseous, charged ion species. 2. Mass analysers capable of resolving these ions on the basis of their m/z ratio with great sensitivity 3. Detectors that can measure the abundance of the mass resolved ions. All these components are operated in an automated fashion in order to obtain maximum accuracy and sensitivity of the instrument. Most commercially available mass spectrometers vary in the ionisation source used and the type of mass analyser that is coupled to it. The mass analyser is the heart of the instrument and each type has its own characteristics, applications, benefits and limitations. This chapter will not attempt to discuss the principles of all the different types of ionisation sources and mass analysers that are currently available. The most common analytical mass spectrometers used in biological research are the MALDI-TOF mass spectrometers capable of peptide mass fingerprinting and the hybrid, tandem mass spectrometers used for peptide amino acid sequence analysis. The following sections will focus on the MALDI-TOF MS instrument and briefly on the different types of tandem mass spectrometers.

3.5.1 MALDI-TOF Mass Spectrometer

Matrix assisted laser desorption/ionisation (MALDI) based mass spectrometry was first introduced by Karas and Hillenkamp in 1988 (Karas and Hillenkamp 1988)

88 It is based on the use of organic matrices to assist in the ionisation of large biomolecules such as proteins and peptides. Under normal circumstances, proteins, peptides and DNA are highly resistant to ionisation and can get destroyed due to high thermal instability. However, the addition of suitable organic compounds that can absorb the wavelength of the laser allows for the excitation of the matrix coupled proteins resulting in their subsequent ionisation (Spengler 2000). The crystallization of the proteins and the matrix allows the uniform transfer of energy to the peptides resulting in their non-destructive ionisation. UV lasers are most commonly used due to their high photon energies. Most of the matrices are chosen due to their strong absorption in the UV range. These include dihydroxybenzoic acid, sinnapinic acid and nicotinic acid. Although there are a number of sensitive and high resolution mass analysers currently available, the time-of flight mass analyser is ideally suited for MALDI ion sources due to its ability to measure and resolve ions in a discontinuous fashion and its rapid response time. A time of flight (Tof) analyser behaves as a velocity spectrometer by measuring the time taken for ions to fly from one end of the analyser to the other, before they strike the detector. The principle of mass analysis is based on the fact that after acceleration to a constant kinetic energy, ions travel at a velocity that is inversely proportional to their m/z ratio. To this effect, Tof analysers are used in two modes: linear and reflectron (Cotter 1992; Guilhaus, Selby et al. 2000). Linear Tof instruments were the first analysers of this type. These analysers detected ions on measuring their time of flight, after they were continuously extracted from the ionisation source. However, inherent difficulties were encountered in detecting and distinguishing between ions having very minor differences in their m/z values. This was attributed to minor differences in the kinetic energies and velocities of ions of the same mass due to inherent differences in the time of their ionisation. This problem was resolved with the introduction of the “reflectron” mode of the analysers. A “reflectron” is basically an electrostatic mirror, which consists of a number of electrical lenses, each with a progressively increasing repelling potential. Ions entering the flight tube have variable velocities. As they travel through the first field free region in the flight tube, their velocities are slowed down when they strike the reflectron, until they come to a rest and their direction is then reversed and they are finally reaccelerated into the second field free region, onto the detector (Kovtoun, English et al. 2002). Ions with greater velocities spend less time in the field free

89 regions, but penetrate deeper into the reflectron fields, consequently resulting in the correction of the velocity differences between ions of the same m/z values (Doroshenko and Cotter 1999). This results in dramatic improvement in the resolution of the Tof instrument. A schematic representation of a Tof analyser is shown in Figure 3.5. Figure 3.5 - Schematics for an orthogonal Time of Flight mass analyser Reproduced from http://www.ivv.fhg.de/ms/ms-analyzers.html

The evolution of the MALDI-TOF mass spectrometer has made it the most versatile tool for the measurement of large mass biomolecules. Modern instruments are capable of measuring analytes in the range of 400 to >100,000 Da. The ability of MALDI spectrometers to analyse different classes of biomolecules (peptides, proteins, polysaccharides, polynucleotide, etc) has been thoroughly demonstrated. There have been numerous additional developments in the field of MALDI based mass spectrometry. In 1995, Brown and Lennon introduced a time lag focusing (or delayed extraction), wherein an extraction voltage is applied over the ion source resulting in the compensation of the initial kinetic energies, so that ions of identical m/z ratios will arrive at the detector at the same time (Brown and Lennon 1995). This has allowed for a dramatic gain in resolution and sensitivity of MALDI-Tof instruments in comparison to other mass analysers. In addition, the application of an orthogonal Tof flight tube (right angled to the sample ion beam) improves the resolution in terms of adjusting the velocity of the extracted ions (Guilhaus, Selby et al. 2000). These instruments, in theory are also capable of performing tandem mass spectrometric analysis by in-flight fragmentation of the parent ion in the Tof analyser

90 (called post-source decay or PSD) (Spengler 2000). However, it is more complicated and suffers in its resolution compared to general tandem mass spectrometers. This shortcoming has also been resolved with the development of MALDI source coupled to a Tof/Tof tandem mass analyser. The development of superior optics, capable of low and high collision induced fragmentation have allowed for the analysis of peptide sequences and structure based studies.

3.5.2 Tandem Mass Spectrometers

Tandem mass spectrometry has gained wide application in biological sciences due to its obvious advantages over peptide mass fingerprinting. Methods for protein sequencing using tandem mass spectrometry began in the 1970’s and with the development of ionisation techniques suitable for proteins, namely “electrospray ionisation” or ESI. In this method, the analyte is forced through a fine needle with a high voltage applied at its end. The thin needle disperses the analyte into a fine mist of charged droplets, which get ionised when they are exposed to this high voltage, forming precursor ions (Whitehouse, Dreyer et al. 1985). There are a number of instrument designs that are capable of the MS/MS analysis of biological compounds. Some instruments perform this analysis in different regions of the instruments (magnetic sector and quadrupole instruments) while others are capable of MS/MS analysis in the same instruments, but in a sequential mode (Quadrupole Ion trap and FT-ICR instruments). Perhaps the best instruments in use today are the “Hybrid tandem mass spectrometers” using two different mass analysers for the measurement of precursor and product ions (Dass 2001). The advantage of using such systems is that different mass analysers can be chosen on the basis of their performance capabilities and interfacing properties. Popular in this category is the combination of a quadrupole and an orthogonal acceleration (oa) time of flight (Tof) instrument (which is right angled to the beam of ions generated from the quadrupole mass analyser) (Morris, Paxton et al. 1997). Figure 3.6 shows the schematic components of this type of an instrument.

91 Figure 3.6 - Schematics for Q-Tof hybrid Tandem Mass Spectrometer Reproduced from (Chernushevich, Loboda et al. 2001)

The quadrupole part of the instrument comprises of a normal quadrupole, which operates under the influence of direct current (DC) and radio frequency (RF) voltage and an (RF) only quadrupole respectively. The normal quadrupole serves as the first analyser, separating the mass selected precursor ion from the other ions. The selected ion then passes into the RF only quadrupole, which acts as a wide band mass filter/collision chamber. Fragmentation of the precursor ion into its product ions occurs here. The m/z ratios of the product ions are analysed at high resolution by the Tof instrument to provide their accurate masses. The Tof can also be used to obtain a normal MS spectrum of all the precursor ions. Here, both quadrupoles behave as wide band mass filters, allowing the transmission of ions of a broad mass range. The advantage of using an orthogonal acceleration Tof instrument is that it allows for the accurate measurement of ions formed continuously from the ion source, increasing its sensitivity and resolution (Loboda, Krutchinsky et al. 2000).These instruments have the advantage of good resolution, high transmission, ultra high sensitivity (low femtomole range for peptide sequencing) and good mass accuracy. Other hybrid tandem mass spectrometers worth mentioning are the magnetic sector tandem mass spectrometer which consists of different configurations of electrostatic and magnetic

92 field analysers, capable of performing high resolution MS/MS analysis and MSn analysis. Another frequently used tandem mass spectrometer is the triple quadrupole tandem mass spectrometer. These instruments consist of three quadrupole devices connected sequentially to each other. The first and third quadrupole function as ion analysers while the middle quadrupole serves as the ion collision chamber. The advantages of these instruments are their low cost and operational simplicity (Dass 2001). Recent developments in the field of tandem mass spectrometry have been well reviewed (Reid and McLuckey 2002; Hopfgartner, Varesio et al. 2004; Sleno and Volmer 2004).

3.6 Protein Identification by Mass Spectrometry

Protein identification by mass spectrometry is accomplished either by peptide mass fingerprinting (PMF) or amino acid sequence determination by tandem mass spectrometry. Peptide mass fingerprinting is usually but not necessarily performed on MALDI-Tof mass spectrometers while amino acid sequence based identification of proteins is performed using tandem mass spectrometers. The principles, advantages and disadvantages of both methods are desribed in the section below.

3.6.1 Peptide Mass Fingerprinting

Protein identification by peptide mass fingerprinting (PMF) is a rapid method used to identify proteins that are usually but not necessarily separated by one-or two- dimensional electrophoresis. The principle behind PMF is simple and is based on the fact that proteins are primarily composed of amino acids and differ from each other in their amino acid composition. The cleavage of individual proteins by sequence specific enzymes (e.g. trypsin) or chemicals such as cyanogen bromide (cleaves the amide bond on the C-terminal end of methionine) results in the formation of a unique peptide pattern of the protein, akin to a unique fingerprint. These fragment peptides are then converted into ions of specific m/z ratios by ionisation techniques and are accurately detected and measured by mass spectrometers. Protein identification is achieved by matching the experimentally determined peptide masses with the theoretical peptide masses of proteins in annotated databases. Commonly used software matching algorithms in peptide mass fingerprinting include PeptIdent/MultIdent and Profound (Wilkins, Gasteiger et al. 1998; Zhang and Chait 2000). The proteins with the highest number of experimental and theoretical peptide

93 matches are ranked highest in probability of identification. Judging from these criteria, it is clear that the successful identification of proteins by PMF depends on: 1. The number of peptide ion masses that can be detected by the mass spectrometer. 2. The ability to accurately measure the peptide masses (resolution). 3. A comprehensive and accurate database of protein sequences for referencing (Berndt, Hobohm et al. 1999). Among the numerous databases used in protein identification, “NCBInr” is the most comprehensive. “SwissProt” is a smaller but accurately annotated repository to remove duplicated proteins whose amino acid sequences have been determined. In addition, the entries in the non redundant (nr) database of NCBI have been compiled from the known oligonucleotide sequence databases such as Genbank and SWISSPROT. Peptide mass fingerprinting is not suitable for the analysis of complex mixtures, as it is impossible to determine from which protein a series of peptides originated (Fenyo, Qin et al. 1998).

3.6.2 Tandem Mass Spectrometry

The determination of the amino acid sequence of proteins has two useful functions in proteomics, namely: 1. Identification of highly homologous proteins, possessing similar peptide mass profiles, which cannot be differentiated by peptide mass fingerprinting. 2. As proteomic applications proceed from simple protein identification to more complicated, structural and functional based studies, the peptide sequence data become much more informative than peptide masses alone. The determination of the amino acid sequence of a protein not only allows for its unambiguous identification but provides crucial clues about how protein structure and composition are altered under different developmental and pathological states. Peptide sequencing represents the additional information that can be derived from the analysis of peptide ion masses and thus forms the core of proteomic data. Elucidation of the amino acid sequence of a peptide by mass spectrometry is achieved by a two mass analysis steps being performed in tandem and hence the name “tandem mass spectrometry” (Boyd, Bott et al. 1987). Initial mass analysis of the fragmented peptide proceeds in a similar fashion to peptide mass fingerprinting. Tandem mass analysis refers to the ability of the analyser

94 to selectively isolate a specific precursor ion from the ion population by imposing limits on the m/z range. The selected ion is then directed into a collision cell where it undergoes multiple collisions with neutral gas atoms (e.g. argon). This results in the ion absorbing the collision energy and subsequently fragmenting. The resultant fragment ions (product ions) are then mass analysed, resulting in a tandem mass spectrum (MS/MS scan) which may be representative of the amino acid sequence of the specific, ionised peptide. Peptide bond fragmentation is usually facilitated by the presence of positive charges on the ion. Obtaining tandem mass spectra also depends upon the effective collision of the precursor ion. Low collision energy (10 eV-30 eV) may result in the effective fragmentation of the peptide bond and the formation of product ions whereas high collision energy (1000 eV-3000 eV) results in the fragmentation of the side chains of the individual amino acids, providing additional data (Papayannopoulos 1995). Other interacting factors that influence effective fragmentation include the charge on the ion species and energy deposition on the precursor ions (the higher the atomic cross-sectional area of a gas molecule, the larger the energy transmission e.g.; Xe ≥ Ar ≥ He) (Staudenmann and James 2000).

3.7 Interpretation of Peptide Sequence from Tandem Mass Spectra

The interpretation of the sequence data from tandem mass spectra depends on our understanding of the fragmentation pattern of the peptide as it undergoes dissociation. Peptide chains are formed by the end to end condensation of amino acids with the loss of a water molecule. In addition to the peptide backbone, each amino acid possesses side chains attached to its α-carbon which are responsible for its unique characteristics. The fragmentation pattern of the peptide ion induced by neutral gas collisions predictably occurs along the peptide back bone. However, as mentioned earlier, peptide ion fragmentation depends on the collision energy and hence fragmentation can also occur along the amino acid side chains, in addition to the peptide back bone. This can potentially complicate the interpretation of the mass spectrum. Roepstorff proposed a systematic nomenclature, classifying the different types of fragment ions that can be generated by the cleavage of the peptide bond (Roepstorff and Fohlman 1984). This was further modified by Biemann in 1988 (Biemann 1988). Figure 3.7 provides a schematic representation of the plausible fragmentation pattern of the amino acid residues describing the ion series nomenclature.

95 Figure 3.7 - The ion series produced by the fragmentation pattern of amino acid residues Reproduced from (Roepstorff and Fohlman 1984)

From the figure it is clear that the peptide bond can be fragmented at three specific locations, namely:

1. The alkyl-carbonyl bond (CH-CO) – a and x cleavage sites 2. The peptide-amide bond (CO-NH) – b and y cleavage sites 3. The amino-alkyl bond (NH-CH) – c and z cleavage sites Peptide bond cleavage results in the formation of a neutral, uncharged species and a charged species. Only the charged species are detected by the mass spectrometric detector, while the neutral fragment is lost. Hence in principle there are six plausible sequence specific ions that can be generated based on whether the charge is retained on the carboxyl-terminus or the amino-terminus. Ions generated from the

N-terminal end are represented by the symbols an, bn and cn where n corresponds to the amino acid number in the peptide chain while ions from the carboxyl-terminus are

designated as xn, yn and zn respectively (Biemann 1988). An increase in the collision energy results in the internal energy being redirected into the amino acid side chains. Cleavage of the side chains results in the formation of secondary ions designated as

wn, vn and dn based on the location where the side chain cleavage occurs. The internal fragmentation is particularly useful in differentiating between isobaric amino acids such as leucine and isoleucine (Dass 2001). The translation of the product ion spectrum into a specific string of amino acids is performed by de novo sequencing and is quite predictable since the atomic

96 mass and structural chemistry of each amino acid is well defined. The product ion spectrum theoretically corresponds to the amino acid occurrence in the particular peptide. Although peptide ions are capable of generating a complete N-terminal and C-terminal ion series, in most cases only one series predominates. In a typical tandem mass analysis run, a precursor ion is selected, fragmented in the collision cell and the product ion m/z ratio determined. An illustration of the how this information is derived is provided in Figure 3.8 for the peptide EGVNDNEEGFFSAR. Examination of MS-MS profile of this peptide demonstrates that cleavages from the N-terminal end yield an ascending series of ion peaks (y- series), which is complementary to the descending series of ion peaks (b -series). The series of peaks in the MS/MS spectra are indicative of the amino acid sequence that

makes up the peptide. If we consider the y series of ions, the gap between y10 and y9 is 114.1 amu, which corresponds to the mass of the residue of asparagine. Similarly, the gap between the ions y9 and y8 ions is 129, which corresponds to glutamic acid, between y8 and y7 is close to 129 or glutamic acid again. The complete y ions series

(y11-y2) indicates the NDNEEGFFSA motif. In summary, the complementary b and y sets of ions describe the same amino acid sequence in two different directions, hence providing definitive confirmation of the sequence, but these complete series are rarely observed in most fragmentation spectra.

3.8 Proteomic Analysis of Allergic Diseases

Proteomics is potentially useful in the study of allergic diseases. The application of proteomics to the study of the molecular and pathological changes that occur in allergic inflammatory responses remains largely unexplored (Toda and Ono 2002). Moreover, proteomics has the potential to be valuable for the analysis of environmental allergens. Understanding how they selectively initiate an atopic response may be possible by identifying the functional components of the allergen molecules. Such information may subsequently allow for the development of attenuated, synthetic recombinant allergens, which could go a long way towards making the testing of allergen-sensitive individuals safer and may have some use in immunotherapy. The application of proteomics to the study of allergens is a major focus of this thesis.

97 Figure 3.8 - Typical tandem mass spectrum (MS2) of GluFibrino peptide (+2) (A): Product ion spectrum of the precursor (785.91) ion of GluFibrino peptide (+2). (B): The precursor ion peak of m/z 785.91 was selected from the MS1 spectrum and subjected to low energy CID, resulting in the MS-MS spectrum. The ascending y ion series are labelled y1-y10 and the amino acids they correspond to are labelled in red.

+TOF Product (785.9): 4.816 to 6.415 min from Sample 2 of 18.wiff Max. 142.1 counts. a=3.56385499999999980e-004, t0=7.50669213025000060e+001

480.2946 140

130

333.2112 120

110

100 187.0845

90 684.3957 80 246.1734 70 813.4609 175.1275 60 240.1534 497.2402 50

40 382.1999 785.9161 942.5100 230.0953 30 286.1651 214.1370 400.2165 515.2605 740.3457 1056.5948 20 1285.6090 169.0711 354.2006 473.1998 612.2649 723.3138 924.5008 1171.5561 10 199.0855

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z, amu

98 +T+TOFOF Product (785.9): 4.816 to 6.415 min from Sample 2 of 18.wiff Max. 142.1 countcounts.s. a=3.a=3.56385499999999980e-004,56385499999999980e-004, t0=7.50669213025000060e+001 y5 480.2946 140 De novo 130 y4 333.2112 sequencing 120

110

100 187.0845 y 90 7 684.3957 80 y F 3 y8 246.1734 813.4609 70 y2 GF 175.1275 S 60 240.1534 A 497.2402 50 E E y9 40 382.1999 785.9161 942.5100 230.0953 30 N y 286.1651 10 214.1370 400.2165 515.2605 740.3457 1056.5948 y12 20 D y 1285.6090 169.0711 354.2006 473.1998 11 612.2649 723.3138 924.5008 1171.5561 10 199.0855 N

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z, amu

99 4 MATERIALS AND METHODS

4.1 General Methods

4.1.1 Preparation of Pollen Diffusates

Dry, unprocessed pollen of Kentucky blue grass (Poa pratensis) and rye grass (Lolium perenne) was purchased from Bayer (USA). Bermuda grass pollen (Cynodon dactylon) was purchased from three different sources (on the basis of availability at different times) namely, Bayer, Sigma and Greer Laboratories (USA). All pollens were certified as ≥ 97% pure with very low quantities of spore contamination. All other reagents used and their sources are listed in the appendix (page 191). These three pollens were chosen on the basis of their importance in triggering allergic diseases (Bass 1984; Knox and Suphioglu 1996; Bass, Delpech et al. 2000).The pollen diffusates from the grasses were prepared by thoroughly mixing dry pollen (200 mgs) with 2 ml of sterile saline (pH 7.5, Baxter, USA) for the fluorescent substrate assay and one-dimensional gel electrophoresis, with deionised water for proteomic analysis and the appropriate equilibration buffer for liquid chromatography analysis. The mixtures were incubated without agitation at 37°C for 60 minutes to allow for the leaching out of soluble proteins (Widmer, Hayes et al. 2000). The mixtures were then centrifuged at 20,800 g for 10 minutes and the supernatant removed. The pellets were washed (1× 500 μl) with sterile saline/deionised water. The supernatants were pooled, filtered using a 0.22 μm filter (Millex GV, Millipore, USA) to remove any trace sediments and were stored at −20°C.

4.1.2 Development of Fluorescent Substrate Assay

The pollen diffusates were analysed for trypsin-like peptidase activity using a fluorogenic mono-peptide substrate in a micro-titre plate assay. Pollen diffusates of Kentucky blue, rye and Bermuda grass (4 μl, 1 mg/ml) were incubated with the arginine mono-peptide substrate, Nα-benzoyl–L–Arg 7–amido–4–methylcoumarin (NBAMC, 4 μl, 1 mg/ml, Sigma) and PBS (25 mM sodium phosphate, 250 mM NaCl, 100 μl) in a 96 well plate (Nunc, Roskilde, Denmark) for 1 minute. Fluorescence

(Ex360 nm, Em460 nm) was measured at one-minute intervals for 60 minutes using a Cytofluor plate reader (Perseptive Biosystems, Framingham CT). Titrations

100 established that optimal results were achieved with the incubation of 4 μl of both the pollen diffusates and the fluorescent substrate. The assay was assessed under a range of conditions including measuring peptidase activity over a buffer pH range of 3-10 and the presence/absence of divalent cations such as Ca2+ or Mg2+. Optimal peptidase activity was determined to be at pH 7.5 and the presence of divalent cations such as Ca 2+ or Mg 2+ did not influence the rate of the enzymatic reaction.

4.1.3 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS- PAGE) and Western Immunoblotting

SDS/PAGE was performed using a Mini-Protean II apparatus (BioRad, Hercules, USA) with 10 or 12.5 % gels and the Tris/Tricine buffer system (Schagger and von Jagow 1987). Following SDS/PAGE, proteins were detected by silver staining methods or Coomassie brilliant blue R-250 according to published procedures (Neuhoff, Arold et al. 1988) (Shevchenko, Wilm et al. 1996). The molecular weights of the protein bands were determined by including ten kilodalton (kDa) benchmark protein ladder standards (Invitrogen, USA) in one lane. For western blotting, the proteins were transferred to polyvinylidene (PVDF) membranes (Immobilon–P, Millipore, Bedford, Ma) at 60V for 60 minutes. The membranes were then probed with a monoclonal antibody to the group 1 allergen of timothy grass, Phl p 1 (a generous gift from Dr. Kay Grobe, UCSD, La Jolla California, USA and Dr. Arnd Petersen, Research Centre, Borstel, Germany) (Grobe, Becker et al. 1999), detected with horseradish peroxidase conjugated to goat anti mouse IgG (1:10,000 Bio-Rad) and visualised by enhanced chemiluminescence (ECL detection system, Perkin Elmer, USA).

4.1.4 Development of Zymography

Proteolytic activity in pollen diffusates was also assessed by zymography according to published procedures (Sarath, Motte et al. 1989). The procedure was optimised by analysing the diffusates against a panel of substrates such as gelatin (1%, Sigma), casein (1%), bovine serum albumin (1%, Sigma) and haemoglobin (1%, Sigma). The best results were obtained with gelatin, which was used as the substrate for all further detection of proteolytic activity by zymography. Bovine type B gelatin (3 mg/ml) was co-polymerised with 10% or 12.5% SDS-polyacrylamide resolving gels. The pollen diffusates of Kentucky blue, rye and

101 Bermuda grass (10 μl, 1 mg/ml) were diluted in a ratio of 1:3 with non–reducing zymogram sample buffer (BioRad) and loaded directly into the wells of the 4% acrylamide stacking gels. Heating the samples was avoided to minimise denaturation and subsequent loss of activity. In the case of two–dimensional zymograms, the immobilised pH gradient strips (IPG strips, Immobiline, 7 cm, Amersham) were loaded onto the 4% acrylamide stacking gels and overlaid with agarose (0.5% agarose, 0.01% bromophenol blue). The gels were run using the Mini–Protean II apparatus (BioRad, Hercules, USA) at 40 V for 30 minutes through the stacking gels, followed by 100 V for 120 minutes through the resolving gel. Despite the precautions taken to minimise denaturation, some loss of activity was expected due to the presence of the detergent SDS in the polyacrylamide gel. The use of native polyacrylamide gels was considered but was rejected because of the need to correlate the proteolytic activity with the appropriate protein band in the SDS- PAGE/Coomassie stained gel. After electrophoresis, the gels were washed in zymogram renaturation buffer (25 mM Tris, 250 mM NaCl, 0.5% Triton-X100, 20 ml, 15 minutes × 2) to remove excessive SDS, followed by incubation in tris-buffered saline for 14 hours. The gels were then stained with 0.4% Coomassie brilliant blue (CBB, R–250, Sigma) in water/methanol/acetic acid (6:3:1 v/v) for 1 hour at room temperature. After staining, the gels were destained with water:methanol:acetic acid (6:3:1, v/v) mixture for three hours (4× 30 ml). Negative staining on a blue-stained background indicated zones of enzymatic activity. An image was then obtained using the G-700 Gel Doc imaging system (BioRad, USA). The characterisation of peptidase activity in the zymograms was performed using irreversible inhibitors, because non-covalent interactions between reversible inhibitors and endopeptidases can easily be broken as the complex migrates through the gel during electrophoresis. Two potential methods of characterising the proteolytic activity in zymograms were explored. The first involved performing the initial electrophoresis for zymography and subsequently incubating the gel overnight in substrate buffer containing the appropriate class-specific inhibitor at the specified concentration. This method did not result in any inhibition of proteolytic activity, possibly because the inhibitors diffused into the polyacrylamide gel too slowly to affect the digestion of the protein substrate. The second method involved incubating the pollen diffusate with the class specific inhibitor for a period of 1 hour, followed by

102 the addition of the sample buffer and subsequent analysis of the mixture by gelatin zymography and was found to be more effective.

4.1.5 Two-Dimensional Gel Electrophoresis

Two–dimensional electrophoresis was performed in accordance with published procedures (Gorg, Boguth et al. 1995; Gorg and Weiss 2000). To obtain satisfactory results, Bermuda grass pollen diffusate (250 μl, 1 mg/ml) was concentrated and desalted to 50 μl, using a Centricon 10 kDa cut-off centrifugal filtration device (Millipore, USA). The concentrated diffusate was dissolved in 75 μl of iso-electric focusing rehydration buffer containing 8 M urea, 2% w/v CHAPS, 2% carrier ampholytes (100X, Bio-lyte, pH range 3-10, Bio-Rad, USA) and 0.1 % bromophenol blue. Reducing agents such as DTT or tri-butyl phosphine were excluded from the rehydration solution as they destroyed the proteolytic activity in the diffusate. The sample/rehydration solution mixture was then pipetted into the ceramic IPG isoelectric focusing strip holder (Amersham). The IPG strip was gently overlaid onto the sample mixture, taking care to avoid air bubbles. The strip was covered with IPG dry-strip cover fluid (Amersham Biosciences, Uppsala, Sweden) and covered. The running conditions for the first dimension are descrined in Figure 4.1. Following isoelectric focussing, the IPG gel strips were equilibrated for 30 minutes in 20 ml of SDS-equilibration buffer (50 mM Tris-HCl, pH 8.8, 8 M Urea and 30% glycerol, 2% SDS) to diminish endosmotic effects (Gorg, Boguth et al. 1995). After the equilibration step, the proteins were then analysed in the second dimension by 12.5% SDS-PAGE and/or gelatin zymography as described above.

103 Figure 4.1 - Running parameters for isoelectric focussing of pollen diffusates

4.2 Specific Purification Procedures

4.2.1 Size-Exclusion Chromatography

All high performance liquid chromatographic (HPLC) separation of pollen diffusates was performed on a non metallic LC 625 HPLC system (Waters, Bedford, MA, USA). Protein fractionation was monitored by UV absorbance on a Waters 484 tunable absorbance detector (Waters, Bedford, USA) at 280 nm. Size-exclusion HPLC was performed using a Biosil SEC 125-5 steel column (300 mm x 7.8 mm, BioRad) with a flow rate of 0.5 ml/min and phosphate buffer (25 mM sodium phosphate, 500 mM NaCl, pH 7.5) that was filtered using a 0.45 μm filter (Whatman, USA) to remove contaminating particulate matter. The column was initially calibrated with 5 μl of gel filtration standards (BioRad) followed by a wash and then an injection of 25 μl of the crude pollen diffusate. Fractions were collected at

104 90 seconds intervals, until the absorbance reached baseline levels. The protein content in all the fractions was too low to be detected by the BCA protein assay. Fractions corresponding to major A280 nm readings were concentrated using Centricon 10 kDa cut-off centrifugal filtration devices (Millipore, USA) and analysed by 12.5% SDS/PAGE and gelatin zymography.

4.2.2 Ion-Exchange Chromatography

Crude Bermuda grass pollen diffusate was also subjected to anion-exchange HPLC, using a “Mono Q HR” 5/5 column (Amersham). The column was initially equilibrated with buffer A (25 mM Tris-HCl, 10 mM NaCl, pH 7.5), then 25μl of the crude pollen diffusate was injected and subsequently fractionated over a linear gradient of NaCl from 10 mM (buffer A) to 1 M (buffer B) over a period of 30 minutes at 0.5 ml/min. Following elution the column was re-equilibrated in buffer A for 10 minutes. Protein fractions were collected at 1 minute intervals until the absorbance returned to base line levels. The fractions were then concentrated and desalted using the Centricon devices and subsequently stored at -20°C until further analysis. Analysis of the fractions was performed by 10 or 12.5% SDS/PAGE and gelatin zymography.

4.2.3 Affinity Chromatography using Benzamidine Sepharose

Benzamidine Sepharose 6B resin was purchased from Pharmacia (Uppsala, Sweden) and the column prepared according to the manufacturer’s instructions. The resin was prepared by pouring 1 ml of the pre-swollen Sepharose beads in a polystyrene column (5 ml capacity, Pharmacia) and equilibrating the column with a binding buffer (100 mM NaCl, 50 mM Tris-HCl, pH 8.0 10 minutes × 2 ml). After washing the gel resin twice to remove the ethanol solution, the binding buffer was eluted. Affinity chromatography was performed by incubating crude pollen diffusate of Kentucky blue or Bermuda grass (2 ml, 100 mg/ml) with the pre-washed resin for two hours at 4° C. Incubation of the diffusates for 24 hours at 4°C did not increase the binding capacity of the resin. The unbound components of the diffusate were collected in the flow through and eluted washes with binding buffer (Wash 1 and 2). Serine peptidases that were potentially bound to the column were eluted with a buffer

105 containing a high concentration of benzamidine-HCl (500 mM benzamidine-HCl, 50 mM Tris-HCl, pH 8.0, 2 ml × 30 min, Elute 1) (Runge, Bode et al. 1987). Elution of high affinity proteins bound to the column was also performed using a high salt concentration, low pH buffer (200 mM Glycine, 50 mM Tris-HCl, pH 2.0, 2 ml×10 min, Elute 2). All the fractions were concentrated and desalted with Centricon 10 kDa cut-off devices prior to analysis by the fluorescent substrate assay, SDS/PAGE and/or gelatin zymography.

4.2.4 Affinity Chromatography using Concanavalin A Sepharose

The Concanavalin A Sepharose 4B matrix was prepared by transferring 2 ml of the pre-swollen Sepharose beads (Pharmacia) into a 50 ml Falcon tube and equilibrating the resin with binding buffer BA, (20 mM Tris-HCl, 0.5 M NaCl, 1 mM

MnCl2 and 1 mM CaCl2, pH 7.4, 20 ml, 10 minutes× 2) on a rotating wheel at room temperature. The gel resin was then centrifuged at 1,000 rpm for 10 minutes and the binding buffer decanted. Affinity chromatography was performed by incubating 10 ml of freshly prepared Bermuda grass pollen diffusate with the pre-equilibrated resin (Kennedy and Rosevear 1973). The sample was allowed to interact with the resin for 3 hours at 4° C, before collecting the supernatant containing the unbound components (F1). The resin was washed with binding buffer BA, (10 ml, 10 minutes × 2) and the supernatants collected (F2 and F3). Specifically bound glycoproteins were then eluted by washing the resin with an elution buffer BB, (20 mM Tris-HCl, 500 mM methyl αD- glucopyranoside, 1 mM CaCl2 and 1 mM MnCl2, 0.5 NaCl, pH 7.4, 5 ml, 30 minutes × 2). The glycoprotein rich fractions were centrifuged at 1000 rpm for 5 minutes and the supernatant collected for analysis (F4 and F5). For the elution of strongly bound glycoproteins, the resin was incubated with a borate buffer BC, (100 mM boric acid, 50 mM Tris-HCl, pH 6.5, 5 ml, 10 minutes × 2) and the supernatant collected after centrifugation at 1000 rpm for 5 minutes (F6 and F7). All the fractions collected were stored at -70°C, prior to analysis for peptidase activity by the fluorescent peptide substrate assay, followed by one- and two-dimensional SDS-PAGE/gelatin zymography. In the fluorescent substrate assay, the optimal rate of hydrolysis (over a period of 60 minutes) was observed when 5 μl of the fractions (total protein concentrations adjusted to 500 μg/ml) was incubated with 4 μl of the fluorescent

106 substrate (1 mg/ml) in a total volume of 125 μl of PBS at pH 7.5. The measurement of the fluorescence was calculated after subtraction of background fluorescence from the plate as well as fluorescence from the unhydrolysed substrate (controls). For tandem affinity purification methods, fractions containing the specific, bound glycoproteins that were eluted from the concanavalin A Sepharose column (F4 and F5) were pooled together, desalted and concentrated using Centricon 10 kDa cut-off centrifugal filtration devices. 25 μl of the pooled, concentrated fraction was subjected to anion exchange HPLC using the “Mono Q HR” column under the same running conditions as the crude pollen diffusate of Bermuda grass (section 4.2.2). Fractions were collected at one minute intervals for the entire duration of the chromatographic separation (30 minutes), concentrated and then subjected to analysis by fluorescent substrate assay, 12.5% SDS/PAGE and/or gelatin zymography.

4.3 Gel Destaining Protocols for the Proteomic Analysis of Proteins

4.3.1 Destaining of Silver Nitrate Stained gels

Protein bands from SDS-PAGE detected by silver staining were excised and destained prior to peptide digestion according to published procedures (Shevchenko, Wilm et al. 1996). Briefly, the gels pieces were incubated in 50 μl of a working solution comprising of KFe(CN)3 (Potassium ferricyanide, 30 mM, BDH) and

Na2S2O3 (Sodium thiosulphate, 100 mM, BDH) in a ratio of 1:1 v/v. Incubation periods varied from 1 minute to 5 minutes, depending upon the intensity of the silver stain. After incubation, the gel pieces were washed in dH2O (100 μl, 10 minutes ×3).

The transparent bands were incubated in NH4HCO3 (100 mM, 100 μl × 15minutes).

After incubation, the gel pieces were washed in CH3CN (100 μl, 10 minutes × 3). The gel pieces, which appeared white in colour were dried using a speedvac (Savant,

Farmingdale, NY) for 10 minutes. The dried gel pieces were rehydrated in NH4HCO3 (10 mM, 25 μl) containing 50 ng of trypsin and incubated for 14 hours at 37°C.

4.3.2 Destaining of Coomassie Stained Gels

Stained bands from the one- and two-dimensional electrophoresis, stained with

Coomassie brilliant blue were excised and washed with NH4HCO3 (100 μl, 100 mM,

15 minutes) followed by 200 μl of a 1:1 solution of NH4HCO3 (20 mM) and CH3CN (100 μl, 10 minutes) (Neuhoff, Arold et al. 1988). This was repeated until the bands

107 were clear, followed by a final incubation in CH3CN (150 μl, 20 minutes). The bands were then dried using a speedvac for 10 minutes. The pieces were rehydrated as described above. Following digestion with trypsin, the gel pieces were subjected to a peptide extraction procedure.

4.3.3 Peptide Extraction from SDS/PAGE for Mass Spectrometry

In order to improve the sensitivity of the detection of the peptides by mass spectrometry, the excised gel pieces were subjected to an additional peptide extraction step as per established procedures. Briefly, the digest buffer containing the tryptic peptides was removed and the gel pieces were incubated in an extraction buffer comprised of 1% formic acid (25 μl, 15 minutes × 2). The extract buffers were then pooled with the digest buffer. The gel pieces were subsequently washed in a solution of water/acetonitrile/formic acid made up in the ratio of 49.5:49.5:1 (25 μl, 15 minutes ×2). The wash buffers were also pooled with the digest buffer (total volume of 125 μl) and then dried using a speedvac for 2 hours or to dryness. The dried peptide samples were stored at -20°C. Prior to mass spectrometric analysis; the samples were reconstituted with 1% formic acid (5-20 μl).

4.4 Mass Spectrometric Analysis of Proteins

4.4.1 Peptide Mass Fingerprinting by MALDI-TOF Mass Spectrometry

The reconstituted buffer containing the peptides (0.5 μl) were desalted using C18 micro Ziptips (according to the manufacturers instructions, Millipore, MA), which would otherwise interfere with the ionisation procedure. The peptides were mixed with 2,5-dihydroxy benzoic acid matrix (2,5-DHB, 1 μl, 10 mg/ml) and spotted onto the MALDI plate. The samples were allowed to air dry and analysed by reflectron-TOF mass spectrometry over a m/z range of 550 to 5000. Approximately

250 spectra were acquired and analysed. Positive ions were generated using a N2 laser (337 nm, 3 nsec pulse width) and accelerated to 25 keV after an extraction delay of 175 nsec (Voyager STR, Perspective Biosystems, Framingham, MA). The spectra were calibrated externally, using the monoisotopic masses of angiotensin I and oxidized insulin B chain. The peptide masses were manually entered into the peptide mass fingerprinting search program, MASCOT (www.matrixscience.com). Non- redundant protein databases (NCBInr) were searched, with the taxonomic selection set

108 on the green plant database (Viridaeplantae), the search results tabulated and scores assigned allowing assignment of likely proteins.

4.4.2 Microcapillary Liquid Chromatography/Tandem Mass Spectrometric Analysis of Proteins

Fused silica capillaries (200 μm × 15 μm) were packed with an acetonitrile slurry of C18 resin (C18 Widepore, Bakerbond, Phillipsburg, NJ, USA) with a 1 cm piece of capillary (50 μm × 190 μm) preventing leaching of the resin at the outlet. The column was coupled to a low volume stainless steel connector where high voltage (~ 2 kV) was applied and the outlet was connected to a piece of fused silica (~2 cm) that was pulled to a tip diameter of ~ 25 μm. The tip was positioned ~ 2-3 mm from the heated capillary (175°C) of a TSQ 7000 mass spectrometer (Finnigan, San Jose, Ca).

A HPLC 1090 LC system (Hewlett Packard) forming a gradient of 100% H20O (1% formic acid) to 60% acetonitrile (1% formic acid) over 40 minutes was applied at a flow rate of 100 μl/min, which was split 1:100 such that the flow from the column was ~ 1 μl/minute. Peptide solutions from the gel extracts (up to 5 μl) in 1% formic acid were injected manually into the liquid chromatography system using a Rheodyne 815 injector (5 μl loop, Rheodyne, CA, USA). Electrospray ionisation mass spectra were acquired from m/z 400 to 1600 in 1 second. The most intense ion for each spectrum that exceeded a preset threshold was automatically selected for low energy collision induced dissociation (CID) for MS-MS analysis with a collision energy of 23V and collision gas (Argon) at a manifold pressure ~ 1.2 Torr. The identities of the peptides were confirmed by searching amino acid sequence databases SwissProt or

NCBInr with tandem mass spectra, using the SEQUEST algorithm (ThermoFinnigan, San Jose, Ca) or the MS/MS search program from matrix science (www.matrixscience.com). The results from both searches were compared and found to be similar. Alternatively, the digest peptides were also separated by nano-LC using an Ultimate HPLC and Famos autosampler system (LC-Packings, Amsterdam, Netherlands). The samples (5 µl) were concentrated and desalted onto a micro C18 precolumn (500 µm x 2 mm, Micron Bioresources, Auburn, CA) with water: acetonitrile buffer (H2O: CH3CN, 98:2, 0.1 % formic acid) at 20 μl/min. After a four- minute wash, the pre-column was switched (Switchos, LC Packings) into line with the

109 analytical column containing C18 RP silica (PEPMAP, 75 µm x 15 mm, LC- Packings) or a fritless nano column manufactured according to Gatlin (Gatlin,

Kleemann et al. 1998). Peptides were eluted using a linear gradient of H2O:CH3CN

(95:5, 0.1 % formic acid) to H2O:CH3CN (40:60, 0.1 % formic acid) at 200 nl/min over 30 min. The column was connected via a fused silica capillary to a low volume tee (Upchurch Scientific) where high voltage (2300 V) was applied and a nano electrospray needle (New Objective) was positioned ~ 1 cm from the orifice of an API QStar Pulsar I hybrid tandem mass spectrometer (Applied Biosystems, Foster City CA). Positive ions were generated by electrospray while the instrument operated in an information dependent acquisition mode (IDA). A Tof MS survey scan was acquired from m/z 350-1700 in 0.75 sec. The two most intense, multiply charged ions (counts > 15) were sequentially selected by Q1 for MS-MS analysis. Nitrogen was used as the collision gas and an optimum collision energy chosen (based on charge state and mass). Tandem mass spectra were accumulated for 5 seconds (m/z 50-2000). A processing script generated data suitable for submission to the database search program (Mascot, Matrix Science). High scores indicated a likely match.

110 5 SERINE PEPTIDASE ACTIVITY IN POLLEN DIFFUSATES

5.1 Peptidase Activity in Pollen diffusates

5.1.1 Fluorometric Assay

Serine peptidase activity in the pollen diffusates of Kentucky blue grass (Poa pratensis), rye grass (Lolium perenne) and Bermuda grass (Cynodon dactylon) was quantitated using a fluorescent arginine mono-peptide ester substrate (NBAMC), which has trypsin-like specificity. As a positive control, trypsin-EDTA (4μl, 100μg/ml, Sigma, Australia) was used. All the pollen diffusates digested the fluorescent substrate immediately. However, at similar protein concentrations, the rate of hydrolysis of the three diffusates differed markedly. The relative rate of hydrolysis of rye grass was comparable to that of trypsin and was over four fold greater than that of Bermuda and Kentucky blue grass (Figure 5.1). Titration of the crude pollen diffusate and the fluorescent substrate established that optimal rates of hydrolysis over a period of 60 minutes were obtained using 4 μl of the pollen diffusates (1 mg/ml) and 4 μl of the fluorescent substrate (1 mg/ml).

5.1.2 Characterisation of Peptidase Activity based on the Inhibitory Profile

The characterisation of the catalytic mechanism of the peptidase activity in the pollen diffusates was performed by analysing the effects of a panel of commercially available, class specific enzyme inhibitors on the rate of hydrolysis. The inhibitors employed broadly covered all the known groups of proteolytic enzymes on the basis of their catalytic mechanism of action. The peptidase activity in all three pollen diffusates was effectively inhibited by the complete cocktail inhibitorTM (1 μl, stock solution as per manufacturers instruction, Roche) as well as by AEBSF (1 μl, 4 μM), benzamidine-HCl (1 μl, 100 μM) and TLCK (4 μl, 100 μM) as recommended by the manufacturers instruction. Surprisingly, only a 30% decrease in substrate hydrolysis was observed with PMSF (1 μl, 200 mM) on all three diffusates (Figure 5.2). An increase in the concentration of PMSF to 300 mM did not result in further inhibition of the peptidase activity. Incubation of the diffusate with iodoacetamide (1 μl. 10 mM), an irreversible inhibitor

111 of cysteine peptidases resulted in ~ 20-30% inhibition of the enzymatic activity in Kentucky blue and rye grass, relative to the controls. No significant inhibition of peptidase activity was observed with TPCK (a chymotrypsin specific inhibitor at 4 μl, 100 μM) or EDTA (a metallopeptidase inhibitor at 1μl, 500 mM). The activity in all three pollen diffusates analysed was therefore inferred to be predominantly due to one or more trypsin-like serine peptidase enzymes.

5.2 One-Dimensional Electrophoretic Analysis of Pollen Diffusates

5.2.1 SDS/PAGE and Western Blotting of Pollen Diffusates

SDS/PAGE of crude pollen diffusates of Kentucky blue grass, rye grass and Bermuda grass revealed numerous protein components in the Mr ~10,000 to 100,000 (Fig 5.3). Western blot analysis, using a monoclonal antibody against the group 1 allergen of timothy grass (Phl p 1) revealed an intense, immuno-reactive band at Mr~ 35,000 in diffusates of Kentucky blue grass and Bermuda grass, indicating the presence of a cross-reactive determinant in the two pollen extracts (Figure 5.4).

5.2.2 Gelatin Zymography

Zymography of the three pollen diffusates demonstrated intense proteolytic activity in different molecular weight regions. All three pollen diffusates demonstrated proteolytic activity at Mr ~95,000 Da. In addition, rye grass demonstrated an intense negative band in the Mr ~60,000 Da region. Interestingly, Bermuda grass pollen demonstrated an intense proteolytic band at Mr ~35,000 Da which coincided with a strong Coomassie stained band on SDS/PAGE and the immunoreactive band on Western blotting, potentially suggesting the presence of an allergenic peptidase in Bermuda grass pollen. Characterisation of the mechanistic classes of the peptidases in the zymograms was performed by assessing the effects of inhibitors on the activity. The peptidase activity in all three pollen diffusates was completely inhibited by the complete cocktail inhibitor (1μl, stock solution), partially inhibited by AEBSF (1 μl, 4μM) and moderately inhibited by PMSF (1 μl, 200 mM) (Figure 5.5). No inhibition was observed with iodoacetamide at the recommended concentration. These results suggested that the enzymatic activities demonstrated on gelatin zymograms were of serine family peptidases, complementing the fluorescent substrate assay data.

112 100000

75000 RYE GRASS TRYPSIN BERMUDA GRASS 50000 KENTUCKY BLUE FLUORESCENCE FLUORESCENCE 25000

0 0 10 20 30 40 50 60 TIME (MINUTES)

Figure 5.1 - Hydrolysis of fluorescent substrate by crude pollen diffusates An increase in fluoresence indicates the hydrolysis of peptide substrate. Typical fluorescence vs time profle of trypsin (…●…) (4μl, 100μg/ml), Kentucky blue grass ( ♦ ), Rye grass (─── ───) and Bermuda (----+----) (4μl, 1 mg/ml) after incubation with Nα-benzoyl-L- Arg 7- amido-4-methylcoumarin (4μl, 1 mg/ml) for 1 minute. Fluorescence was then measured every minute, for 60 minutes. Results are the averages of three experiments performed at different times ± 1SEM.

113 Figure 5.2 - Measurement of the hydrolysis of the fluorescent substrate by the pollen diffusates The maximum rate of hydrolysis has been adjusted to the maximum relative rate of trypsin. Maximum fluorescence was reached at 60 minutes and inhibition was expressed as the reduction in fluorescence compared to the maximum. Pollen diffusates of Kentucky blue grass ( ) rye grass ( ) and Bermuda grass ( ) (4μl,1mg/ml) were incubated with fluorescent substrate, NBAMC (4μl, 1 mg/ml) and characterisation was performed using four serine peptidase inhibitors (AEBSF, 4 μM; Benzamidine HCl, 100 μM; TLCK, 100 μM and TPCK, 100 μM), a cysteine peptidase inhibitor (IAC, 100 mM), a metallopeptidase inhibitor (EDTA, 500 mM) and a broad spectrum cocktail inhibitor (1 μl, stock solutionl). Experiments were repeated three times and the values are the means of three readings.Error bars are included for the mean of the 3 experiments ± SEM. Kentucky blue grass Rye grass Bermuda grass 120

100 80

60

40

20 Relative Fluorescence 0

rol F C A K K dine A C nt cktail i I LC o PMS EDT T TP C Co AEBSF Benzam Pollen Diffusate/Enzyme Inhibitors

114

Figure 5.3 - SDS/PAGE analysis of pollen diffusates Approximately 10 µg of diffusate was loaded in each lane of a 12.5% SDS/PAGE and silver-stained (lanes 1-3). Lane K, Kentucky blue grass (Poa pratensis); Lane R, rye grass (Lolium perenne) and Lane B, Bermuda grass (Cynodon dactylon). 5 μl of 10 kDa ladder markers was loaded in lane 1 (Mr). Protein detection was performed by silver staining according to published procedures.

Figure 5.4 - SDS/PAGE and Western blot analysis of pollen diffusates Approximately 10 µg of diffusate was loaded in each lane of a 12.5% SDS/PAGE and silver-stained. K, Kentucky blue grass (Poa pratensis); R, rye grass (Lolium perenne) and B, Bermuda grass (Cynodon dactylon). Kw, Rw, Bw refer to the same diffusates after Western blotting with a monoclonal antibody (IG 12) to allergen Phl p 1. Molecular weight markers are shown (Mr). Comparison between SDS/PAGE and Western blotting demonstrates the presence of an intensely stained protein band in Bermuda grass diffusate at Mr ~35,000 which corresponds with a strong immunoreactive band at the same molecular weight region in the Western blot. Although a strong immunoreactive band in Kentucky blue grass and a faint band in rye grass can also be seen by Western blotting, they do not coincide with any major protein band in SDS/PAGE.

115 Mr K R B

Mr KRK B w Rw Bw

116 1 2 34 56 7 8 9101112131415 16 Kentucky blue grass Rye grass Bermuda grass

Figure 5.5 - Gelatin zymography of Pollen Diffusates and sensitivity to inhibitors Approximately 20 µg of Kentucky blue grass (lanes 2-6) rye grass (lanes 7-11) and Bermuda grass (lanes 12-16) diffusates were separated on 12.5% SDS/PAGE. Trypsin (~500 ng) was loaded in lane 1. Crude diffusates (lanes 2, 7 and 12) contain several high MW bands (~95,000) with proteolytic activity and Bermuda grass diffusate also contains intense proteolytic activity at ~35,000 (lane 12). PMSF partially inhibited proteolytic activity in rye grass (lane 8) but not in Kentucky or Bermuda grass (lanes 3 and 13); activity was completely inhibited in all three pollen diffusates by the CompleteTM cocktail inhibitor (lanes 4, 9 and 14) and moderately inhibited by AEBSF (lanes 5, 10 and 15). Activity was unaffected by iodoacetamide (lanes 6, 11 and 16).

117 5.2.3 Proteomic Analysis of Samples from One-Dimensional SDS/PAGE

A proteomic approach was employed to enable the rapid identification and analysis of the protein bands at Mr ~35,000 Da in Bermuda grass. Other intense bands in the SDS/PAGE gel were also analysed to validate the suitability of this approach for the analysis of proteins from pollen grains. The bands were excised, destained and digested with trypsin overnight (Figure 5.6A). The peptide digests were then extracted from the gel pieces using an established procedure and analysed using the MALDI (reflectron)- Tof/ peptide mass fingerprinting method and micro C18 LC/MS-MS method. In both cases, the most intense protein band from SDS/PAGE, corresponding to the immunoreactive band in the Western blot and the proteolytically active band in the gelatin zymogram of Bermuda grass pollen diffusate (band 12) was identified as the major group 1 allergen of Bermuda grass (Cyn d 1, Cynodon dactylon). The intense band in rye grass (band 4) was identified as the major group 1 allergen of rye grass (Lol p 1, Lolium perenne). The peptide mass fingerprints from MALDI-Tof/MS analysis of bands 4 and 12 are shown in Figures 5.6 B and C respectively. From the 16 bands that were analysed, 5 proteins corresponding to known allergens were readily identified, validating the usefulness of the proteomic approach. The results of mass spectrometric analysis of these bands are summarised in Table 5.1.

118

Figure 5.6 - SDS/PAGE and MALDI reflectron TOF of tryptic peptides derived from band 4 (Lolium perenne) and band 12 (Cynodon dactylon)

A. Pollen diffusates (lane 1: Kentucky blue grass, lane: 2 rye grass and lane 3: Bermuda grass) were separated using SDS/PAGE, stained with Coomassie brilliant blue and labelled bands excised, destained and digested with trypsin. B. Peptides from band 4 (rye grass) were analysed using MALDI reflectron Tof; digest peptides with masses corresponding to allergen Lol p 1 are labelled (*). C. Peptides from band 12 (Bermuda grass) were analysed using MALDI relfectron Tof, digest peptides with masses corresponding to allergen Cyn d 1 are labelled (*).

119

120 Table 5.1 - MALDI-TOF/MS and LC/MS-MS analysis of proteins from 1D- SDS/PAGE of pollen diffusates

Pollen Gel (Mr) MALDI µLC-ESI Mass Allergen (calc) Kentucky Band 1 (14) ? Dac g 3 (Dactylis glomerata) 10926 Y

Kentucky Band 2 (32) Hol l1 Hol l 1 (Holcus lanatus) 28329 Y

Rye Band 3 (14) Lol p2A Lol p 2, Lol p 3 (Lolium perenne) 10873, Y grass + p3 10901 Rye Band 4 (32) Lol p1 Lol p 1 (Lolium perenne) 28439 Y grass Bermuda Band 5 (10) ? ? ? ?

Bermuda Band 6 (12) ? ? ? ?

Bermuda Band 7 (14) ? profilin [Cyn d 12] (Cynodon 14135 Y dactylon) Bermuda Band 8 (18) ? calmodulin (wheat) 16077 ?

Bermuda Band 9 (25) ? ? ? ?

Bermuda Band 10 (27) ? ? ? ?

Bermuda Band 11 (30) Cyn d 1 Cyn d 1 (Cynodon dactylon) 26872 Y

Bermuda Band 12(32) Cyn d 1 Cyn d 1 (Cynodon dactylon) 28391 Y

Bermuda Band 13 (37) Cyn d 1 Malate dehydogenase 36968 ? (Arabidopsis thaliana) Bermuda Band 14 (40) ? Putative legumin protein (Oryza 40238 ? sativa) Bermuda Band 15 (45) ? phosphoglycerate kinase 42096 ? (cytosolic) Bermuda Band 16 (70) ? phosphoglucomutase (Oryza 62910 ? sativa)

121 5.3 Two-Dimensional Electrophoretic Analysis of Bermuda grass Pollen Diffusate

5.3.1 Two-Dimensional SDS/PAGE and Western Blotting

Analysis of the concentrated diffusates of Bermuda grass pollen by the 2D- SDS/PAGE method resulted in a much more effective separation of the individual protein components of the mixture (Figure 5.7 A). Optimal detection was achieved with 250 μg of diffusate concentrated and solubilised in 125 μl of rehydration solution. A number of spots migrated at Mr ~30,000 to 32,000 Da and predominantly focussed in the pH range of 5-8, suggesting the presence of numerous isoforms of the same protein. The procedure was repeated three times and was generally found to be reproducible. Western blotting of the two-dimensional SDS/PAGE of Bermuda grass pollen diffusate, run under identical conditions and using the monoclonal antibody IG12, demonstrated immunoreactive spots in a similar pattern as the resolved proteins in the Mr ~30,000–32,000 Da and pI range of 5-9 (Figure 5.8). A few cross-reactive spots were also detected at the Mr ~80,000 Da, indicating either the presence of dimers or other cross-reactive proteins.

5.3.2 Two-Dimensional Gelatin Zymography

Isoelectric focusing/gelatin zymography of the concentrated Bermuda grass pollen diffusate, run under identical conditions as the two-dimensional SDS/PAGE, yielded diffuse proteolytically active streaks around the molecular weight region of 30,000- 32,000 Da (Figure 5.7 B). These observed activity approximately corresponded to the pattern of intense spots on the two-dimensional SDS/PAGE and Western blotting of the diffusate. However, the relatively weaker activities compared to the one-dimensional gelatin zymogram, coupled with the trailing effect of substrate hydrolysis made it particularly difficult to precisely align and correlate the activity to any particular set of spots. The experiments were repeated, the gels aligned as accurately as possible and all the proteins that potentially correlated with the peptidase activity were excised, digested and analysed by the micro C18 LC/MS-MS technique.

122

Figure 5.7 - Two-Dimensional SDS/PAGE and gelatin zymography of the pollen diffusate of Bermuda grass (A). Two-dimensional SDS/PAGE stained with Coomassie brilliant blue. Labelled spots were excised, destained and digested with trypsin for LC/MS-MS analysis. (B). Two- dimensional gelatin zymograms. Negatively stained streaks in the Mr ~30,000-32,000 correspond on alignment to spots 1-7. 2D-SDS/PAGE and 2D-zymography were repeated three times and generally found to be reproducible.

123 Figure 5.8 - Two-Dimensional Western Blotting of Bermuda grass pollen diffusate Approximately 250 μg of diffusate was loaded onto IPG strip (pH 3-10). Following 2D- SDS/PAGE, western blotting was performed using monoclonal antibody (IG12) to allergen Phl p 1. The immunoreactive streak at ~32,000 (pI 5-8) corresponded with spots 2-7 on 2D-SDS/PAGE (Fig 5.7A). The immunoreactive streak at ~29,000 (pI 7-10) and ~80,000 (pI 5-6) did not correlate with any clear protein spot in the 2D-SDS/PAGE.

124

5.3.3 Proteomic Analysis of samples from Two-Dimensional SDS/PAGE of Bermuda grass Pollen

The proteins spots resolved by two-dimensional SDS/PAGE of the Bermuda grass

pollen diffusate were excised and digested with trypsin (50 ng/25 μl of NH4HCO3). Tryptic peptide digests were then separated by micro C18 RP-HPLC followed by automated, data dependent, low energy CID/MS-MS analysis of the most intense, multiply charged peptide ions. Peptide sequence tags generated from the MS-MS spectra of the precursor ions from each spot were used to determine the protein identity by database searches. A summary of the proteins identified by this technique is presented in Table 5.2. A number of spots that corresponded with the diffuse peptidase activity in the two- dimensional zymogram (Figure 5.7B) and the immunoreactive streak in the two- dimensional Western blot of Bermuda grass pollen diffusate (Figure 5.8) were identified with high confidence as the group 1 allergen, Cyn d 1 and its isoforms. A typical base peak chromatogram of the tryptic peptides (of spot 4 in Figure 5.7A) is shown in figure 5.9A. The MS-MS spectrum of the most intense, multiply charged precursor ion (741.72) is shown in Figure 5.9 B. The database search of all the MS-MS spectra of the particular spot identified the protein as the acidic Cyn d 1 isoallergen isoform 2 precursor. Some sequence ions in the tandem mass spectrum of the [M+3H]3+ precursor are labelled

(Figure 5.9B) and correspond to the peptide sequence Cyn d 1111-129 (isoform 2). Although the multiple peptides from the protein allowed for high confidence matching of protein identity, it was insufficient to effectively differentiate between the isoforms 2, 3 and 4 of the allergen Cyn d 1 due to their extremely high sequence similarities (≥ 99%). The database searches also identified proteins from other plant species. This is because the current annotated plant database is incomplete and/or the fact that the derived peptides may be common to one or more species.

125 Figure 5.9 - Tandem mass spectrometric analysis of tryptic peptides derived from spot 4 (Cynodon dactylon) (A) Peptides from tryptic digests of spot 4 (Figure 5.7A) were separated by micro C18 RP-HPLC and the eluate analysed by ESI-MS/MS. Base peak chromatogram with each peak labelled with their retention time, mass and charge state. (B) MS/MS spectrum of a triple charged precursor ion at m/z 741.7 corresponding to the sequence ITDKNYEHIAAYHFDLSGK from the allergen Cyn d 1 (identified by MASCOT database searches).

741.72, 17.26 (+3) C1 A 3.4e5 711.42, 16.62 111-129 (+2) C1 737.14 3.0e5 185-198 403.57, 16.14 729.66 (+3) C1 2.5e5 145-154 737.74

2.0e5 675.38, 21.44 534.29, 14.49 (+3) C1201-217 (+3) C196-110 1.5e5 507.24, 9.22 631.38, 22.05 (+2) C1 (+2) C141-49 422.25, 10.74 155-166 In ten sity, cp s 1.0e5 (+2) C1166-172 540.33 5.0e4 430.95

0.0 2 4 6 8 10121416182022242628303234 Time, min

100 B Y’’10 1108.5661

75 501.2416 Cyn d 1111-129 86.1025 ITDKNYEHIAAYHFDLSGK 129.1012 50 3+ [M+3H] Y’’9 110.0665 Y’’8 741.7213 966.4639 1037.5170 Y’’ Y’’ 5 187.1571 3 519.2824 Y’’6 291.1795 487.2458 666.3700 Y’’7 25 803.4229 Y’’11

Relative Abundance % Abundance Relative 1221.6588

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z, amu

126 Table 5.2 - Summary of proteins of Bermuda grass pollen diffusate identified by micro C18 RP-HPLC and ESI/MS-MS analysis

Spot Gel micro LC-ESI Score Peptide Mass (calc) (Mr) Match 1 30,000 Acidic Cyn d 1 isoallergen isoform 3 precursor 231 7 28,378 2 30,000 Acidic Cyn d 1 isoallergen isoform 4 precursor 690 25 28,407 3 30,000 Acidic Cyn d 1 isoallergen isoform 2/4 precursor 743 25 28,391 4 30,000 Acidic Cyn d 1 isoallergen isoform 2/4 precursor 723 21 28,407 5 25,000 Acidic allergen Cyn d 1 precursor 643 18 28,391 6 25,000 Acidic allergen Cyn d 1 precursor 486 14 26,645 7 25,000 Acidic allergen Cyn d 1 precursor 562 15 26,645 8 35,000 Acidic allergen Cyn d 1 precursor 526 15 26,645 9 50,000 Acidic Cyn d 1 isoallergen isoform 1 precursor 144 5 26,663 10 50,000 Acidic Cyn d 1 isoallergen isoform 1 precursor 206 5 26,663 11 50,000 Phosphoglucomutase, cytoplasmic (PGM2) 366 7 63,002 12 35,000 Acidic Cyn d 1 isoallergen isoform 1 precursor 65 2 26,663 13 35,000 L-Ascorbate peroxidase, cytosolic isozyme, maize 218 4 27,295 14 35,000 L-Ascorbate peroxidase, cytosolic isozyme, maize 106 3 26,663 15 35,000 Phosphoglucomutase, cytoplasmic 2 345 7 63,002 16 40,000 Acidic Cyn d 1 isoallergen isoform 1 precursor 140 3 26,663 17 40,000 Triosephosphate isomerase, cytosolic (TIM) 183 4 27,008 18 40,000 Enolase 2 102 3 48,132 19 40,000 Profilin 3 (ZmPRO3) 85 2 14,228 20 50,000 Pistilata homolog ScPI (Sanguinaria Canadensis) 42 1 24,075 21 60,000 Acidic Cyn d 1 isoallergen isoform 1 precursor 195 6 26,663 22 70,000 Acidic allergen Cyn d 1 precursor 308 9 26,645 23 60,000 Acidic allergen Cyn d 1 precursor 433 11 26,645 24 60,000 Acidic Cyn d 1 isoallergen isoform 2 precursor 561 12 28,391 25 90,000 Acidic allergen Cyn d 1 459 12 26,645 26 12,000 Acidic allergen Cyn d 1 402 10 26,645 27 18,000 Acidic allergen Cyn d 1 precursor 311 10 26,645

127 5.4 Discussion

The aim of this study was to characterise the serine peptidase activity in allergenic pollens. This was based on the results of a previous study by Widmer and colleagues who examined the substrate specificity profiles of peptidases in the crude diffusates of allergenic pollen grains. Widmer et al found that the proteolytic activity in Kentucky blue grass exhibited a marked preference for the cleavage of substrates with arginine or lysine in the P1 position, which is similar to the activity profile of trypsin (Widmer, Hayes et al. 2000). The profiles of rye grass and Bermuda grass had an increased preference for cysteine, leucine and methionine and relatively low affinity for cleaving arginine and lysine, indicating a mixture of enzymes, none of which seemed to possess trypsin like specificity. A fluorescence based assay approach, using the mono-peptide substrate (NBAMC) was used in these experiments because it was more sensitive compared to photometric based assays. The free 7-amino-4-methylcoumarin group in the substrate is approximately 500-700 times more fluorescent in its free form than when conjugated to an amino acid (Neurath 1989). Surprisingly and in contrast to the findings of Widmer et al, the fluorescent assay demonstrated efficient cleavage of the arginine substrate by all three pollen diffusates. In fact ryegrass, which apparently possessed no specificity for arginine in the earlier assay, demonstrated at least four- fold greater cleavage of the substrate than Kentucky blue or Bermuda grass. These observations could partly be attributed to the differences in the assay conditions and the substrates employed. In the assay used by Widmer et al, the initial pH of all the mixtures was markedly alkaline and occurred in the presence of DMSO. This was done to facilitate the solubility of the substrate and activity was revealed by a progressive fall in the pH (Widmer, Hayes et al. 2000). The current assay employed a more physiological approach, using phosphate buffered saline at pH 7.5. The characterisation of peptidase activity by continuous solution based assays is clearly useful when a single protein is present, which is not the case in the crude pollen diffusate. It is not possible to clearly differentiate between multiple serine peptidase activities using these assays. Moreover, not all the serine and cysteine peptidases present in the mixture might be detected as the assay provides for the cleavage of only a single amino acid target. To complement the results of the

128 fluorescent substrate assay, further analysis was performed, using gelatin zymography. The presence of a number of proteolytically active bands on zymography of all three pollen diffusates confirmed the presence of multiple peptidases in the pollen grains. To characterise the catalytic activity of the enzymes detected by both these assays, their susceptibility to different class-specific peptidase inhibitors was assessed. Preliminary results suggested serine peptidase activity by both methods, although not necessarily the same enzyme (Figures 5.2 & 5.5). PMSF resulted in the partial inhibition (30%) of peptidase activity in all three pollen diffusate tested. This partial effect was probably because PMSF is highly unstable in aqueous solutions and rapidly decays at a physiological pH of 7.5. Moreover its specificity is poor as it can reversibly react with cysteine peptidases. In order to further confirm the mechanistic class of peptidase activity in the pollen diffusate, a wider range of serine peptidase inhibitors was used. 4-(2-Aminoethyl benzene sulphonyl fluoride hydrochloride) (AEBSF) is an alternative sulphonyl-fluoride based serine peptidase inhibitor. It is much more effective than PMSF and reacts about 100 fold faster than PMSF with chymotrypsin-like serine peptidases (Markwardt, Drawert et al. 1974; Walsmann, Landmann et al. 1974). Its mechanism of action is similar to other sulphonyl fluorides, occurring via the acylation of the active site serine in serine peptidases, and it is not subject to the shortcomings of PMSF (Sarath, Motte et al. 1989). AEBSF was effective in its inhibition of the peptidase activity in all three pollen diffusates, both in the solution and solid phase assay. Comparable inhibition was also observed in the liquid phase assay using benzamidine hydrochloride, which is a reversible competitive inhibitor of serine peptidases. To differentiate between enzymes with trypsin and chymotrypsin like specificity, the inhibitory effects of two different chloromethyl ketones on the serine peptidase activity were examined. Tosyl-Phe-CH2Cl2 (TPCK) and Tosyl-Lys-CH2Cl2 (TLCK) are irreversible inhibitors, specific for chymotrypsin and trypsin like serine peptidases respectively. These chloromethyl ketones irreversibly alkylate the histidine residue in the active site of serine peptidases (Petra, Cohen et al. 1965; Tsilikounas, Rao et al. 1996; Powers, Asgian et al. 2002). TPCK is also known to inhibit some cysteine peptidases. The peptidase activity of all three diffusates in the solution phase assay was found to be trypsin–like, based on its sensitivity to TLCK in comparison to TPCK (Figure 5.2). The application of these inhibitors could not be effectively tested

129 in gelatin zymography because the proteolytic activity in the pollen diffusate was inactivated by the harsh buffer conditions used to solubilise the inhibitors. TLCK is soluble at pH 3.0 and is very unstable above 6.0 while TPCK requires 100 mM methanol for its effective solubilisation. TPCK marginally inhibited peptidase activity in the diffusate of Kentucky blue grass in the solution phase assay, with no inhibition being observed in the diffusates of rye or Bermuda grass, suggesting the presence of a minor chymotrypsin (or cysteine peptidase) component in Kentucky blue grass. The partial inhibition was further confirmed by the partial inhibition of peptidase activity in Kentucky blue grass using iodoacetamide (IAC), which is a cysteine peptidase inhibitor. Although IAC inhibited peptidase activity by about 20% in rye grass, the activity was completely inactivated by TLCK, suggesting that the activity was more likely to be that of a serine peptidase. No metallopeptidase activity was demonstrated based on the inhibition using the metal chelator, EDTA, in either the solid or liquid phase assays. The presence of proteolytic activity in pollen grains has been well known for over 50 years and various roles for peptidases have been well documented (Vierstra 1996). However, there has been little focus on whether any relationship exists between these proteolytically active proteins and allergenicity. Exactly how important pollen enzymes may be in the induction of allergic sensitisation and whether any of the pollen allergens possess intrinsic proteolytic activity thus remains unknown. These association between enzymatic activity and allergenicity is clearly important in the case of allergens of the house dust mite (Der p 1, 2, 9, etc) and cat (Fel d 1) (Chua, Stewart et al. 1988; Ring, Wan et al. 2000; Wan, Winton et al. 2001), which are known to possess intrinsic peptidase activity in addition to their ability to initiate a strong IgE mediated immune response in sensitised individuals (Lind and Lowenstein 1983). Although exo- and endo-peptidases have been identified in the pollen grains of Mesquite (Prosopis velutinia) (Matheson, Schmidt et al. 1995; Matheson and Travis 1998), maize pollen (Kalinowski, Radlowski et al. 2002; Wu, Suen et al. 2002) and ragweed (Ambrosia artemisiifolia), their roles in allergic sensitisation have not been addressed. Analysis of the crude pollen diffusates of Kentucky blue, rye and Bermuda grass by SDS/PAGE and gelatin zymography demonstrated that all three extracts consisted of a complex mixture of proteins, some of which were proteolytically active. The activity in the diffusates appeared to be due to the serine family

130 peptidases, based on their inhibitory profiles. Furthermore, there appeared to be a correlation between one band exhibiting proteolytic activity and the 35 kDa, immunoreactive band in Bermuda grass. To unequivocally determine the identity of the protein(s) exhibiting peptidase activity, a proteomic approach was employed, which also permitted assessment of any relationship to known allergenic proteins. These experiments permitted identification of several well known allergens in the various diffusates. More importantly, in the extract of Bermuda grass pollen, the intense band in SDS/PAGE at ~35,000 (which aligned accurately with the proteolytically active band in the zymograms) was identified as the major allergen Cyn d 1, both by peptide mass fingerprinting and micro LC/MS-MS methods. This result suggested that the allergen might possess serine peptidase activity. Group 1 allergens are the major sensitising allergens in grasses. They are glycoproteins with an estimated Mr ~30,000 – 35,000 Da and a carbohydrate content of about 5% (Andersson and Lidholm 2003). More than 95% of individuals allergic to grass pollen possess specific IgE antibodies to group 1 allergens, which are usually cross-reactive with homologous allergens from other plant species. The biological function of the group 1 allergens in plants has recently been correlated with a group of cell wall loosening proteins in plants known as “expansins” on the basis of sequence similarities (Cosgrove 1997; Cosgrove 2000). However, the underlying biochemical mechanism of expansin activity is controversial. Cosgrove and colleagues suggested that expansins from a number of plant sources mediated cell wall loosening by a novel mechanism resulting in the weakening of the non-covalent interactions between the cell wall polysaccharides and the cellulose microfibrils (Cosgrove 1997; Cosgrove 2000). In contrast, work from Grobe et al suggested that the expansin activity in the group 1 allergen from timothy grass, Phl p 1, was due to proteolytic activity. They argued that this protein was related to cathepsins, a family of C1 cysteine peptidases, and that Phl p 1 exhibited a conserved cathepsin catalytic sequence. (Grobe, Becker et al. 1999; Grobe, Poppelmann et al. 2002). Grobe and colleagues had raised a monoclonal antibody designated IG12 against Phl p 1 and this was employed in the present study to investigate whether intrinsic proteolytic activity was present in proteins that might be immunologically cross-reactive with Phl p 1. The allergen Phl p 1 possesses 70%, 90% and 93% sequence similarities to its homologous allergens in Kentucky blue grass (Poa p 1),

131 rye grass (Lol p 1), and Bermuda grass (Cyn d 1) respectively (Suphioglu 2000), indicating the presence of shared antigenic determinants and possibly common biological functions (Petersen, Schramm et al. 1995; Andersson and Lidholm 2003). Western blotting of the diffusates with IG12 demonstrated cross-reactive bands in Kentucky blue and Bermuda grass diffusates. The immunoreactive band in Bermuda grass pollen migrated in the same region as the peptidase activity in the gelatin zymography (Mr ~35,000) and the intense, Coomassie-stained band that was identified as Cyn d 1. However, the resolution of one-dimensional electrophoresis is insufficient to confidently assign enzymatic and antigenic properties to a single protein entity. To achieve better resolution of the mixture, 2D-SDS/PAGE was performed (Gorg and Weiss 2000; Andersson and Lidholm 2003). Peptidase activity in the diffusate was analysed using two-dimensional zymography and immunoreactivity by two- dimensional Western blotting. The proteins detected by 2D-electrophoresis were identified using tandem mass spectrometry and database searches. Separation of complex protein mixtures by two dimensional electrophoretic techniques yields much higher resolution than one dimensional separation techniques (Gorg, Weiss et al. 2004). Application of this method has gained popularity with rapid advances in high-throughput mass spectrometric analysis of proteins (Hunter, Andon et al. 2002; Sellers and Yates 2003). The analysis of the Bermuda grass pollen diffusate by two-dimensional SDS/PAGE resulted in the separation of a number of proteins in the molecular weight range of 30,000 – 32,000 Da over a wide isoelectric range from 5-8. This pattern correlated with the previously reported two-dimensional patterns of Cyn d 1 (Chang, Liu et al. 1995). Moreover, the spots correlated well with the pattern of spots obtained by Western blotting of the diffusate with the monoclonal antibody IG12 against Phl p 1. Although a number of comparative studies have demonstrated that allergens from Bermuda grass have limited cross-reactivity with other grasses, due to their unique immunochemical characteristics (Schumacher, Grabowski et al. 1985; Sridhara, Singh et al. 1995), there appeared to be strong antigenic cross-reactivity between Phl p 1 and a number of isoforms of Cyn d 1, indicating the presence of common antigenic determinants. The correlation of these protein spots with the proteolytic activity observed by two-dimensional gelatin zymography is consistent with the interpretation that the

132 allergen (or at least some of its isoforms) possesses intrinsic proteolytic activity. Enzymatic activity by two-dimensional electrophoretic techniques has rarely been demonstrated by other investigators (Tyagi, Kumar et al. 1996; Park, Kho et al. 2002). In this study, isoelectric focussing/gelatin zymography of the Bermuda grass pollen diffusate was successfully performed, with the demonstration of diffuse proteolytic streaks in the Mr~ 30,000-32,000 Da. The activity was predominantly located in the pI range of 5-8. Retaining enzymatic activity necessitated less than optimal preparative procedures for iso electric focussing. The diffusates were initially concentrated and desalted using Centricon, 10 kDa cut-off centrifugal filtration devices, since salt accumulation can result in the formation of high conductivity zones and subsequent streaking (Rabilloud and Chevallet 2000) Potentially interfering plant compounds such as lignins, polyphenols, tannins, alkaloids, pigments etc (Gegenheimer 1990) could not be removed prior to IEF by precipitation because this would result in the inactivation of proteolytic activity (Cremer and Van de Walle 1985; Thiellement, Bahrman et al. 1999). In addition, the rehydration buffer used to solubilise the proteins in the diffusate was modified and comprised of a low urea- detergent-ampholyte mixture to unfold the proteins and prevent their non-covalent interactions. Reducing agents such as DTT and tributyl phosphine were excluded because they cleave disulphide bonds resulting in the total loss of serine peptidase activity in the diffusate. Despite the optimisation steps, the proteolytic activity was still weaker than that observed in the one-dimensional SDS/PAGE, probably as a consequence of denaturation and less efficient, subsequent renaturation. Together with the relative streaking of the proteins in the two-dimensional SDS/PAGE and proteolytic activity in the two-dimension/gelatin zymograms, this made it particularly difficult to determine precisely which isoform(s) possessed enzymatic activity. The streaking of proteolytic activity in zymography has commonly been attributed to the interaction between the enzyme that is being resolved and the substrate incorporated into the polyacrylamide gel (Hummel, Penheiter et al. 1996). The inclusion of gelatin in the gel has been known to slow the relative migration rate of proteins to a small extent, which can affect the accurate alignment of the regions of enzymatic activity with particular protein spots (Hummel, Penheiter et al. 1996). Moreover, gelatin has been shown to bind differentially to proteolytic enzymes from complex extracts during electrophoretic migration (Hummel, Penheiter et al. 1996), resulting in some

133 instances, in the formation of hydrolytic trails, thus causing an overestimation of the peptidase in the mixture at a particular molecular range and isoelectric point. To address these concerns, the experiments were repeated a number of times to examine the reproducibility. Since all the gels were similar in their migratory patterns, all the protein spots that potentially corresponded to the proteolytic activity in the two dimensional gelatin zymogram were subjected to tandem mass spectrometric analysis in order to elucidate the identity of the enzymatically active protein or proteins. As indicated in Table 5.2, the identity of the majority of the protein spots which corresponded to the serine peptidase activity was determined to be Cyn d 1 and/or its isoforms. Chang et al classified the two-dimensional, resolved isoforms of Cyn d 1 into acidic and basic groups, which also vary slightly in their molecular weights (Chang, Liu et al. 1995; Levy, Davies et al. 2001) There are currently about 15 known isoallergens of Cyn d 1, spanning over a pH range of 4-11. Although the different acidic isoforms and basic isoforms possess about 97% and 99% sequence similarities respectively, there is a significant difference in the sequence identity (14%) between the two categories of isoallergens (Chang, Liu et al. 1995). It is possible that not all isoforms exhibit proteolytic activity because of variations in the amino acid sequences. Despite this, attributing the proteolytic activity to Cyn d 1 by this method is not unambiguous. It is not possible to exclude the presence of a co-migrating, low abundance protein, responsible for the proteolytic activity. A comparison of the amino acid sequences between trypsin, the major pollen allergen Cyn d 1 and the four major isoforms identified by tandem mass spectrometry did not result in the identification of any conserved sequence motifs that might link the proteolytic activity to the allergens (Figure 5.10). To exclude the possibility of the abundant Cyn d 1 overshadowing the detection of a low level protein, additional experiments needed to be performed with pre-fractionation of the crude pollen diffusates by chromatography.

134 Figure 5.10 - Multiple sequence alignments of selected allergens and trypsin Sequence of the allergens Cyn d 1 and its isoforms with the major pollen allergen of timothy grass, Phl p 1. The aligned sequences were compared with the sequence of human trypsin. Sequences were aligned using the CLUSTAL W sequence alignment program. All sequences were obtained from the SWISS-PROT protein sequence database. Common amino acid sequences are marked in bold and marked with * sign. The amino acids comprising of the catalytic triad of trypsin are high lighted in yellow. Alignment demonstrates the absence of any common motif that is capable of catalytic activity in the aligned allergens.

Cyn d I isoallergens 4 1 -----MLAVVAVVLASMVGGALCAMGDKPGPNITATYGDKWLDAKATFYGSDPRGAAPDD Cyn d I isoallergens 3 1 -----MLAVVAVVLASMVGGALCAMGDKPGPNITATYGDKWLDAKATFYGSDPRGAAPDD Cyn d I isoallergens 2 1 -----MLAAVAVVLASMVGGAWCAMGDKPGPNITATYGDKWLDAKATFYGSDPRGAAPDD Pollen allergen Cyn d 1 1 ------AIGDKPGPNITATYGSKWLEARATFYGSNPRGAAPDD Pollen allergen Phl p 1 1 MASSSSVLLVVVLFAVFLGSAYGIPKVPPGPNITATYGDKWLDAKSTWYG-KPTGAGPKD Trypsin (human) 1 ------MNPLLILTFVAAALAAPFDDDDK-IVGGYN---CEENSVPY---QVSLNSGY . *.. *. : .:. * . .

Cyn d I isoallergens 4 61 HGGACGYKDVDKAPFDSMTGCGNEPIFKDGLGCGSCYEIKCKEPAECSGEPVLIK-ITDK Cyn d I isoallergens 3 61 HGGACGYKDVDKAPFDGMTGCGNEPIFKDGLACGSCYEIKCKEPAECSGEPVLIK-ITDK Cyn d I isoallergens 2 61 HGGACGYKDVDKAPFDGMTGCGNEPIFKDGLGCGSCYEIKCKEPAECSGEPVLIK-ITDK Pollen allergen Cyn d 1 61 HGGACGYKDVDKPPFDGMTACGNEPIFKDGLGCRACYEIKCKEPVECSGEPVLVK-ITDK Pollen allergen Phl p 1 61 NGGACGYKDVDKPPFSGMTGCGNTPIFKSGRGCGSCFEIKCTKPEACSGEPVVVH-ITDD Trypsin (human) 61 H--FCGGSLINEQWVVSAGHCYKSRIQVR-LGEHN-IEVLEGNEQFINAAKIIRHPQYDR : ** . ::: . . * : * . *: : .. :: : *

Cyn d I isoallergens 4 121 NYEHIAAYHFDLSGKAFG-AMAKKGEEDKLRKAGELMLQFRRVKCEYPSDTKIAFHVEKG Cyn d I isoallergens 3 121 NYEHIAAYHFDLSGKAFG-AMAKKGEEDKLRKAGELMLQFRRVKCEYPSDTKIAFHVEKG Cyn d I isoallergens 2 121 NYEHIAAYHFDLSGKAFG-AMAKKGEEDKLRKAGELMLQFRRVKCEYPSDTKITFHVEKG Pollen allergen Cyn d 1 121 NYEHIAAYHFDLSGKAFG-AMAKKGQEDKLRKAGELTLQFRRVKCKYPSGTKITFHIEKG Pollen allergen Phl p 1 121 NEEPIAPYHFDLSGHAFG-AMAKKGDEQKLRSAGELELQFRRVKCKYPEGTKVTFHVEKG Trypsin (human) 121 KTLNNDIMLIKLSSRAVINARVSTISLPTAPPATGTKCLISGWGNTASSGADYPDELQCL : :.**.:*. * ... . . * : ...:. . .::

135

Cyn d I isoallergens 4 181 SNPNY--LALLVKY-AAGDGNIVSVDIKSKGSDEFLPMKQSWGAIWRIDPPKPLK--GPF Cyn d I isoallergens 3 181 SNPNY--LALLVKY-AAGDGNIVSVDIKSKGSDEFLPMKQSWGAIWRIDPPKPLK--GPF Cyn d I isoallergens 2 181 SSPNY--LALLVKY-AAGDGNIVGVDIKPKGSDVFLPMKLSWGAIWRMDPPKPLK--GPF Pollen allergen Cyn d 1 181 SNDHY--LALLVKY-AAGDGNIVAVDIKPRDSDEFIPMKSSWGAIWRIDPKKPLK--GPF Pollen allergen Phl p 1 181 SNPNY--LALLVKY-VNGDGDVVAVDIKEKGKDKWIELKESWGAIWRIDTPDKLT--GPF Trypsin (human) 181 DAPVLSQAKCEASYPGKITSNMFCVGFLEGGKD--SCQGDSGGPVVCNGQLQGVVSWGDG . ..* .::. *.: ..* * *.: . . : *

Cyn d I isoallergens 4 241 TIRLTSESGGHVEQEDVIPEDWKPDTVYKSKIQF-- Cyn d I isoallergens 3 241 TIRLTSESGGHVEQEDVIPEDWKPDTVYKSKIQF-- Cyn d I isoallergens 2 241 TIRLTSESGGHVEQEDVIPEDWKPDTVYKSKIQF-- Pollen allergen Cyn d 1 241 SIRLTSEGGAHLVQDDVIPANWKPDTVYTSKLQFGA Pollen allergen Phl p 1 241 TVRYTTEGGTKTEAEDVIPEGWKADTSYESK----- Trypsin (human) 241 CAQKN-KPGVYTKVYNYVK--WIKNTIAANS----- * * *

136 6 Purification of Proteolytic Activity in Pollen Diffusates

6.1 General Chromatographic Separation Techniques

6.1.1 Size-Exclusion Chromatography

Size exclusion HPLC of the crude pollen diffusate of Bermuda grass was performed on a Bio-Sil SEC 125-5 column with a wide exclusion range of 5-100 kDa. Initial calibration of the column, using gel filtration standards, demonstrated good resolution in the separation of the individual protein standards (Figure 6.1). In an experiment using 25 μl of the crude pollen diffusate of Bermuda grass (120 μg of total protein) which was close to the upper limit of capacity of the column, nine peaks were separated on the basis of absorbance at 280 nm, with retention times varying between 15.5 and 29.71 minutes (Figure 6.2). However, the resolution was poor, probably due to lateral diffusion of proteins as they passed through the column, resulting in peak broadening and incomplete separation. This was further evident from the failure of individual peaks to reach baseline absorbance before the elution of the next peak. In an attempt to differentiate between the resolved proteins as much as possible, fractions were collected at 1.5 minute intervals, rather than retention times corresponding to the individual peaks. SDS/PAGE of the concentrated fractions demonstrated that the proteins in the M r~32,000 range were resolved in fractions 4 to 6 (retention time of 14.5 minutes to 19 minutes, Figure 6.3A). However, no proteolytic activity could be demonstrated in any of the fractions by gelatin zymography (Figure 6.3B). Although repeating the experiments demonstrated good reproducibility, proteolytic activity could not be demonstrated even after attempting to optimise the conditions by running the experiments at 4°C.

137 0.07

Figure 6.1 - Size-exclusion chromatogram of gel filtration standards 5μl of0.06 protein standards of known MW were injected into the Biosil 125-5 column and separated over a period of 30 minutes in the isocratic mode. Chromatogram indicated good resolution of the individual protein components.

0.05

670kDa

0.04

1.35kDa 0.03

17kDa 0.02 44kDa Absorbance 280 nm nm 280 280 Absorbance Absorbance 158kDa 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Time (minutes)

0.01

0.00

138

Figure 6.2 - Size-exclusion chromatogram of Bermuda grass pollen diffusate 25μl (120μg) of clarified, crude pollen diffusate was injected into the Biosil 125-5 column and separation performed in the isocratic mode over 30 minutes. Size-exclusion separation of the diffusate resulted in the separation of 9 peaks (P1-P9) with the retention times denoted in the chromatogram. Fractions were collected at 1.5 minute intervals from 10-30 minutes (fractions 1-13) to differentiate between the separated proteins as much as possible and analysed by SDS/PAGE and gelatin zymography.

P521.500

0.02 P621.983

P827.100 0.02

P723.700

P420.850 0.01 P319.783

AbsorbanceAbsorbance (280nm (280nm0.01 ) )

P929.717 0.00

P217.083 P115.550

-0.01

(minutes)

0.00 10.00 139 20.00 30.00

Time Figure 6.3 - SDS/PAGE and Gelatin Zymography of Size-Exclusion HPLC fractions of Bermuda grass pollen diffusate (A) 1.5 ml fractions (Lanes 1-13) were collected at 1.5 min intervals from 10-30 minutes of Size-exclusion chromatography of Bermuda grass pollen. 500 μl of each fraction was concentrated 10 fold to 50 μl. 10 μl of the concentrated fraction was mixed with non-reducing sample buffer in a ratio of 1:3 and analysed by 12.5% SDS/PAGE. Molecular weight markers and 10 μg of the crude pollen diffusate (BGP) were loaded in the lanes labelled Markers and BGP respectively. (B): Gelatin zymography of the concentrated fractions from size-exclusion HPLC of Bermuda grass pollen diffusate.10μl of the same concentrated fraction was mixed with sample buffer and simultaneously analysed by 12.5 % gelatin Zymography. Lane markings are the same as SDS/PAGE.

Markers BGP 13245 6 7 8910111213

220 97.4 66 46

30 21.6

14.3

Markers BGP 13245 6 7 8910111213

220 97.4 66 46

30 21.6

14.3

140 6.1.2 Ion-Exchange Chromatography

The analysis of 25 μl (120 μgm of total protein extract) of the crude pollen diffusate of Bermuda grass pollen by anion exchange HPLC resulted in the separation of 4 distinct peaks over a linear gradient of NaCl from 10 mM (buffer A) to 1 M (buffer B), (Figure 6.4). As the absorbance readings reached baseline at 20 minutes in the 30 minute chromatographic run, fractions were collected at 1 minute intervals (0.5 ml/min) from 1-20 minutes, corresponding to the detection of peaks. Analysis of fractions 1-16 by 12.5% SDS/PAGE and gelatin zymography demonstrated that the proteolytically active protein of Mr~ 32,000 Da eluted in fractions 2, 3 and 4 and corresponded with a single dominant band in the same molecular weight region on SDS/PAGE (Figure 6.5). All three fractions corresponded with a minor peak that eluted immediately after the first peak, with a retention period of 2 to 5 minutes in the chromatogram. Concentration of the three fractions containing peptidase activity at Mr~ 32,000 Da, followed by SDS/PAGE, revealed that a large number of proteins eluted early in fractions 2 and 3, which were not apparent in the dilute fractions (Figure. 6.6). However, the most prominent bands observed in the fractions, that closely corresponded with the peptidase activity, were found to be a major band at 32,000 Da and a minor band at 30,000 Da. The major proteins in the SDS/PAGE - Coomassie staining were excised, digested and analysed by micro LC/MS-MS methods to determine the identity of the proteolytically active bands (Figure. 6.6). Database searches using the MASCOT search program identified the protein at ~ 32,000 Da (band 3 or 7) as acidic Cyn d 1 isoallergen isoform 1. The minor component band at Mr~ 30,000 Da (band 4) was also identified with high confidence as the allergen Cyn d 1 and might correspond to either a deglycosylated or fragmented form of the allergen. These bands corresponded to the negatively-stained peptidase activity at Mr~ 32,000 on gelatin zymography. It was not possible to effectively differentiate between Cyn d 1 and the isoforms identified in database searches because of the high levels of sequence similarity between the proteins (band 3 and 7).

141 Figure 6.4 - Anion-exchange chromatogram of Bermuda grass pollen diffusate 25 μl (120 μg) of the clarified, crude pollen diffusate was injected into the Mono Q HR column and separated in a linear gradient mode from 100 mM, NaCl to 1 M, NaCl buffers. Ion-exchange chromatography resulted in the fractionation of 4 major peaks (P1-P4). Fractions were collected at 1 minute intervals from 0-20 minutes to separate the protein fractions and subsequently analysed by SDS/PAGE and gelatin zymography. 1.20

P11.967

1.00

0.80

0.60 AbsorbanceAbsorbance 214 214nmnm

0.40 P28.717 P312.917

P415.033

0.20

0.00 10.00 20.00 30.00 40.00 0.00

Time (Minutes)

142 Figure 6.5 - SDS/PAGE and Gelatin zymography of Anion-Exchange chromatography fractions of Bermuda grass pollen diffusate Fractions were collected from 1 to 16 minutes at a flow rate of 0.5 ml/min, until absorbance read at baseline. Fractions were concentrated 10 fold from 500 μl to 50 μl with Centricon centrifugal filtration devices. 10 μl of the fraction was solubilised with 20 μl of non-reducing sample buffer and analysed. Lane markings in SDS/PAGE and zymography (1-16) correspond to 1 minute fractions collected from the HPLC, trypsin (TRY) and Bermuda grass pollen diffusate (BGP) are labelled respectively. A&B: 12.5% SDS/PAGE. C&D: Gelatin zymograms.

ABTRY BGP 1234567 8 9 10 11 12 13 14 15 16

94 63 43 30 20

14

CDTRY BGP 12 34 5 67 8 9 10 11 12 13 14 15 16

94 63 43 30 20

14

143

Figure 6.6 - Analysis of the protein bands separated by SDS/PAGE of fractions of Anion exchange HPLC 100 μl of fractions 1-7 from the Anion-Exchange HPLC of Bermuda grass were concentrated further to 10 μl using Centricon devices. Samples were mixed with non-reducing sample buffer in a ratio of 1:3 and analysed by 12.5% SDS/PAGE. Labelled protein bands were excised, destained and digested overnight using trypsin (5 μl, 15 ng/μl). Peptide digests were analysed by LC/MS-MS analysis and identity determined by searching MASCOT protein database, using peptide sequence tags.

BGP12345 67

94 1 67 2 6 7 10 43 3 4 30 8 5 20 9

14

144 Table 6.1 - Analysis of protein bands from anion-exchange HPLC separated fractions of Bermuda grass pollen diffusate by LC/MS-MS

Spot Gel μLC/ESI Score Peptide Mass (Mr) Match (calc) 1 80,000 Phosphoglucomutase, cytoplasmic 2 123 4 63,002 2 60,000 UDP-glucose pyrophosphorylase 209 8 51,330 3 35,000 Acidic Cyn d 1 isoallergen isoform 1 436 15 26,663 (fragment) 4 30,000 Major pollen allergen Cyn d 1 440 18 26,872 5 20,000 Enolase 1(2-phosphoglycerate 249 11 48,033 dehydratase) 6 60,000 Phosphoglucomutase (Oryza sativa) 122 4 62,910 7 35,000 Major pollen allergen Cyn d 1 417 11 26,872 8 25,000 Acidic Cyn d 1 isoallergen isoform 3 137 6 28,738 precursor 9 20,000 Ascorbate peroxidase 99 3 19,254 10 35,000 Acidic allergen Cyn d 1 precursor 432 19 26,645 (Cynodon dactylon)

6.1.3 Discussion

The rationale for using size-exclusion HPLC in initial attempts to purify the peptidase activity was that proteins of Mr~ 32,000 Da might be separated from the complex mixture without inactivating the enzyme. While this technique has been successfully applied to the purification of a number of allergens and/or enzymes from plant sources, including Bermuda grass pollen diffusate (Chang, Liu et al. 1995; Su, Peng et al. 2003), in the current experiments the resolution was found to be inadequate. The successful purification of Cyn d 1 from pollen diffusate necessitated pre-concentration of the proteins (e.g., ammonium sulphate precipitation) in order to remove potentially interfering compounds that are usually present in plant material (lignins, polyphenols, etc.) (Matthiesen, Schumacher et al. 1989). This pre-concentration step could not be used in the current study because precipitation of the proteins resulted in the total inactivation of peptidase activity in the pollen diffusate.

145 Disappointingly, although size-exclusion HPLC of the crude diffusate resulted in the partial purification of the proteins of Mr~ 32,000 Da, it caused complete loss of peptidase activity on gelatin zymography. Proteolytic activity could not be retained even if the experiment was performed at 4°C. This suggested that the peptidase might be autolysed during the separation procedure, possibly due to the removal of an endogenous inhibitor or a surrogate substrate during purification. Optimising the resolution of the separation by using larger columns (larger number of theoretical plates) was not attempted as it would require longer separation times, which would inevitably increase the likelihood of loss of proteolytic activity. Hence size- exclusion HPLC was determined to be unsuitable for the partial purification of the peptidase activity in Bermuda grass. The application of anion exchange HPLC for the purification of the peptidase was attempted because two-dimensional gelatin zymography of the crude pollen diffusate of Bermuda grass demonstrated that the activity of Mr~ 30,000-32,000 Da focussed in a pI range of 5.5 to 6.5. Moreover, the allergen Cyn d 1 is known to possess a number of isoforms in the acidic range (Chang, Liu et al. 1995). It was hoped that application of a buffer at pH 7.5 would allow for the development of a net negative charge on these acidic proteins, permitting separation without compromising the enzymatic activity in the sample. This procedure yielded much more encouraging results, as the peptidase activity was recovered 2-5 minutes after commencement of elution with a salt gradient. While the technique was relatively successful in purifying the peptidase activity from the crude pollen mixture, the elution of a number of protein components in the early fractions implied that the differences in the affinity of different proteins for the ion exchanger were minor. The retention of charged molecules in an ion exchange column is also dependent on the gradient established by the eluting buffers, which is influenced by the molar concentration of the counter ions present in the column (Neue 1997). Identification of the protein bands that corresponded with the proteolytic activity in the purified fractions as Cyn d 1 further strengthened the proposition that Cyn d 1 might possess intrinsic peptidase activity. However, these results were still open to the criticism mentioned earlier viz. that co-migration of an unrelated protein could be responsible for the peptidase activity. Analytical separation by varying the pH of the eluting buffer provides an alternative approach for improving the resolution of separation between closely eluting proteins. Proteins

146 co-eluting at a particular pH are not likely to co-elute at another pH due to variations in their affinity for the ion exchanger. However, this was not attempted because of the high likelihood of loss of proteolytic activity. Nevertheless, the chromatographic technique was found to be potentially useful as a partial purification step to study the biochemical properties of the peptidase. It was considered that coupling this technique with an affinity purification approach might be useful to establish whether the pollen allergen did indeed exhibit enzymatic activity.

6.2 Affinity Chromatography Purification Techniques

6.2.1 Benzamidine Sepharose Chromatography

The purification of the serine peptidase activity in Kentucky blue and Bermuda grass was attempted by affinity chromatography using the immobilised serine peptidase inhibitor, Benzamidine Sepharose (Amersham). All the fractions that were eluted from the column were desalted, depleted of any residual benzamidine and concentrated using Centricon 10 kDa cut-off centrifugal filtration devices at 4°C. Analysis by the fluorescent substrate assay demonstrated that the peptidase activity in the diffusate of Kentucky blue grass pollen did not bind to immobilised benzamidine but was present in the flow-though fractions. Disappointingly, all of the fractions from Bermuda grass pollen diffusate were completely devoid of peptidase activity (Figure 6.7 A&B). Further analysis of the fractions of Kentucky blue grass by 12.5% SDS/PAGE demonstrated that no proteins from the crude pollen diffusate had bound to the immobilised inhibitor. Analysis by gelatin zymography did not detect proteolytic activity in any of the eluted fractions, including the flow-through fractions (not shown). Similarly, no activity was recovered from either the flow-through or the eluted fractions of Bermuda grass pollen diffusate. These results indicated that the peptidase activity was lost on interaction with benzamidine, with no obvious binding occurring between any of the proteins in the pollen diffusates and the inhibitor. Attempts to optimise the binding (by increasing the incubation time) or the elution (by minimising the number of washes with PBS) did not result in any improvement.

147 Figure 6.7 - Fluorescent substrate assay on Benzamidine Sepharose purified Fractions of Kentucky blue and Bermuda grass Fractions eluted from the benzamidine column (2 ml) were desalted and concentrated to 200μl. Protein concentrations were adjusted to 100 μg/ml. 5 μl of each fraction was analysed with 4μl of fluorescent substrate (NBAMC). Typical fluorescence vs time profile was monitored over a period of 60 minutes. Assay was repeated thrice and found to be reproducible. Trypsin (1μl, 40 ng/μl) was used as a positive control and found to be highly effective in substrate hydrolysis (not shown).

Peptidase activity in Benzamidine Sepharose Peptidase activity in Benzamidine Sepharose 30000 Purified fractions of Kentucky blue grass Purified fractions of Bermuda grass Blank 35000 Blank Poa P diffusate 25000 Cyn D diffusate Flow through 30000 Flow through Wash 1 Wash 1 Wash 2 Wash 2

)) 20000 25000 Elute 1 Elute 1 Elute 2 Elute 2 20000 15000

15000 Fluoresence (360/460) (360/460) Fluoresence Fluoresence Fluoresence (360/460 (360/460 Fluoresence Fluoresence 10000 10000

5000 5000

0 0 0 102030405060 0 Time (Minutes) 002 103040506 Time (Minutes)

148 6.2.2 Concanavalin A Sepharose Chromatography

6.2.2.1 Affinity Purification using Concanavalin A Sepharose

As an alternative to separation of the peptidase based on physical properties, partial purification of native Cyn d 1, which is a glycoprotein, was attempted using Concanavalin A Sepharose beads (Pharmacia). A large volume (10 ml) of the crude diffusate of Bermuda grass was incubated with the lectin coupled beads at 4°C for 3 hours. Prolonging the incubation period overnight (24 hr) did not result in an appreciable increase in proteins binding to the matrix, indicating that binding was relatively rapid. Hence, incubation periods were maintained at 3 hours. Fractions were then eluted and collected sequentially after short incubation periods (10 minutes). The characteristics of the fractions are summarised in Table 6.2. The fractions collected were desalted and concentrated to remove the excessive sugars or salts from the buffers, using a Centricon 10 kDa cut-off centrifugal ultrafiltration device, and were stored at -70°C until used for assessment of peptidase activity or for further purification. A BCA assay revealed that most of the proteins in the mixture did not bind to the lectin-Sepharose matrix. The bound fraction was estimated to be approximately 1.5% of the total protein content of the crude diffusate (Figure 6.8).

Table 6.2 - Characteristics of Concanavalin A Sepharose separated fractions

Volume Fraction no. Elution buffer Protein content collected F1 Con A binding buffer 10mls Proteins not bound to the column F2 Con A binding buffer 8mls Proteins not bound to the column F3 Con A binding buffer 8mls Proteins not bound to the column F4 α -Methyl glucopyranoside 5mls Bound glycoproteins F5 α -Methyl glucopyranoside 5mls Bound glycoproteins F6 Sodium Borate buffer pH 6.8 5mls Strongly bound glycoproteins F7 Sodium Borate buffer pH 6.8 5mls Strongly bound glycoproteins

6.2.2.2 Analysis of Peptidase activity by Fluorometric Assay

Peptidase activity in the Concanavalin A Sepharose fractions of Bermuda grass pollen diffusate was initially quantitated using the fluorescent peptide ester substrate (NBAMC), which

149 has trypsin-like specificity. The protein concentrations in all the fractions were adjusted to 500 μg/ml prior to analysis. The assay demonstrated that peptidase activity was only present in fractions F1 and F2 which were of the unbound, flow-through fraction and its wash (Figure 6.9). No peptidase activity was detected in any of the bound fractions (F4 - F7). At the concentrations tested (5 μl, 500 μg/ml), the relative rate of hydrolysis by fraction F1 was approximately four- fold greater than that of Bermuda grass diffusate (Figure 6.10). Peptidase activity in fraction F2 was equivalent to that of the crude diffusate.

6.2.2.3 Analysis by One-Dimensional SDS/PAGE and Gelatin Zymography

Analysis of the concentrated fractions of Bermuda grass pollen diffusate separated by Concanavalin A Sepharose by SDS/PAGE confirmed the results of the protein concentration assay i.e. that over 90% of the proteins in the crude diffusate flowed through the column. Fractions F4 and F5, comprised of glycoproteins that bound to the Concanavalin A matrix, contained proteins of the Mr ~10,000 to 65,000 (Figure 6.11 A). Gelatin zymography of the fractions revealed peptidase activity at Mr ~30,000 Da which was detected only in fractions F4 and F5, in contrast to the results of the fluorescent peptide substrate assay (Figure. 6.11 B). The higher molecular weight peptidase of Mr ~100,000 Da was present in all fractions, but progressively decreased with each wash, indicating non-specific binding. There was no peptidase activity demonstrated in fractions F6 and F7, either by gelatin zymography or by the fluorescent substrate assay, presumably either due to denaturation by the eluting buffer or to the very low levels of protein in the fractions. The protein bands in fractions F4 and F5, which corresponded to the peptidase activity at ~30,000 Da were subjected to mass spectrometric analysis (Figure 6.12).

150 160

140

120

100

80

60 Total protein (mg)

40

20

0 Bermuda F1 F2 F3 F4 F5 F6 F7 Fractions from Con A Sepharose

Figure 6.8 - Protein concentrations of the Concanavalin A Sepharose separated fractions of Bermuda grass pollen diffusates by the BCA assay Dry pollen grains (1 gm) were incubated in 10 ml of dH2O for 1 hour at 37° C. The diffusate was then centrifuged at 20,800 g for 15 minutes and the pellet was washed. The supernatants were pooled and filtered (0.22 μm, Millex-GV, Millipore, Bedford MA). The crude pollen diffusate was then subjected to affinity chromatography, using Concanavalin A Sepharose. The protein content of the glycoprotein-rich fractions that bound to Concanavalin A Sepharose was estimated to be approximately 1.5% of the total protein.

151 100000

Trypsin 75000 Bermuda grass F1 F2 50000 F3 F4 F5 Fluorescent activity F6 25000 F7

0 0 10 20 30 40 50 60 Time (minutes)

Figure 6.9 - Hydrolysis of the fluorescent substrate (NBAMC) by the Con A Sepharose separated fractions of Bermuda grass pollen diffusate Typical fluorescence vs. time profile over a period of 60 minutes. The legends for the individual fractions are depicted in the graph. 5 μl (500 μg/ml) of the affinity separated fractions of Bermuda grass pollen diffusate was incubated with 4 μl (1 mg/ml)of the fluorescent substrate, NBAMC. An increase in fluorescence indicates substrate hydrolysis. Peptidase activity was confined to the unbound fractions of Con A Sepharose (F1 & F2). The hydrolytic activity of fraction F1 was equivalent to trypsin and was four-fold greater than that of the Bermuda grass diffusate. Fraction F2 possessed hydrolytic activity equivalent to that of the crude pollen diffusate. No peptidase activity was detected in the bound, glycoprotein rich fractions (F4 & F5).

152

Figure 6.10 - Hydrolysis of fluorescent substrate by the Con A Sepharose separated fractions of Bermuda grass pollen diffusate The maximum rate of hydrolysis has been adjusted to hydrolysis by trypsin. The peptidase activity in the fractions is expressed relative to the trypsin control. The protein concentration of all fractions has been adjusted to 500 μg/ml. 5μl (2.5 μg total protein) of the affinity separated fractions of Bermuda grass pollen diffusate was incubated with 4 μl (1 mg/ml) of fluorescent substrate NBAMC. Highest enzymatic activity was observed in the unbound fractions of Concanavalin A Sepharose (F1 & F2). No activity was observed in any of the bound fractions (F4, F5, F6 & F7).

100

Trypsin Bermuda 75 F1 F2 F3 F4 50 max) F5 F6 F7 25 Relative Fluoresence activity (% relative to relative (% activity Fluoresence Relative

0 1 2 sin da F F F3 F4 F5 F6 F7 p u Try Berm

153 6.2.2.4 Proteomic Analysis of Con A Sepharose Purified Fractions.

The protein bands selected for analysis by tandem mass spectrometry were excised, digested and subjected to LC/MS-MS analysis. The peptide sequence tags from tandem mass spectra were used for database searches. LC/MS-MS analysis of bands 8 and 9 (Figure 6.12), which corresponded to the peptidase activity at Mr ~30,000 Da in the gelatin zymograms, identified the proteins as Cyn d 1 and its isoform Cyn d 1 isoallergen 1 respectively (Table 6.3). Due to the high degree of homology between the different isoallergens, it was not possible to ascertain if the proteolytic activity was limited to this isoform, as other isoallergens of Cyn d 1 were also identified with high confidence in the database search.

6.2.2.5 Analysis of the proteolytically active fractions by Anion Exchange HPLC

The bound protein fractions of the Concanavalin A Sepharose column (F4 and F5) were pooled together (250 μl) and further concentrated down to 25 μl using Centricon 10 kDa cut-off centrifugal filtration devices to remove the α-methyl D glucopyranoside present in the elution buffer. Analysis of the pooled fraction sample by anion-exchange HPLC as described earlier, using the same buffer gradient (10 mM to 1 M, NaCl), resulted in weak, non resolving peaks, which could not be differentiated from the base line (Figure 6.13). Nevertheless, one minute fractions were collected for the entire chromatographic run, concentrated and analysed by gelatin zymography to determine whether peptidase activity was still retained. The absence of peptidase activity in the gelatin zymography of the fractions indicated that the enzymatic activity was not resilient enough to withstand an additional ion-exchange HPLC step after purification by Concanavalin A Sepharose chromatography. Due to the loss of peptidase activity and lack of time, optimisation of anion-exchange HPLC to improve the resolution of separation of the Concanavalin A Sepharose purified fractions was not pursued further.

154 Figure 6.11 - SDS/PAGE and gelatin zymography of the Con A Sepharose separated fractions of Bermuda grass pollen diffusate (A). Fractions (10μl) were separated by 12.5% SDS/PAGE and stained by Coomassie brilliant blue. (B). Gelatin zymography of the fractions. Peptidase activity at Mr ~30,000 Da was detected in the bound fractions of Concanavalin A Sepharose affinity chromatography (F4&F5). No peptidase activity was detected in the unbound flow-through fractions (F1, F2 & F3) or the fractions eluted with sodium borate buffer (F6 & F7). Lanes with trypsin (10 μg) and Bermuda grass pollen diffusates are labelled respectively.

155

Figure 6.12 - Proteomic analysis of Con A Sepharose separated fractions of Bermuda grass The fractions were separated by 12.5% SDS/PAGE and stained with Coomassie brilliant blue. The bands labelled for analysis were excised, destained and digested with trypsin. Digest peptides were subjected to LC/MS-MS analysis and their identities determined by searching the plant database (Viridaeplantae) using the MASCOT Daemon program. Proteins identified by this method are listed in Table 6.3.

TRYF1F2F3F4F5F6F7BGP

156 0.35

0.30

0.25

0.20

0.15 Absorbance nm 214

0.10

0.05

0.00

0.00 10.00 20.00 30.00 40.00 Time (Minutes)

Figure 6.13 - Anion Exchange chromatography of the Bound fractions of Con A Sepharose affinity chromatography of Bermuda grass pollen diffusate 25 μμl (75 g) of pooled fractions that bound to Con A Sepharose were desalted and then injected into the Mono Q HR column and separated over a gradient of 100 mM, NaCl to 1M, NaCl over 30 minutes. However, analysis resulted in very poor separation of the fractions. 1 minute fractions were collected for the entire run of the chromatogram and subjected to analysis by SDS/PAGE and gelatin zymography. No peptidase activity was detected in any of the fractions.

157 Table 6.3 - Summary of the proteins identified by LC/MS-MS of Concanavalin A Sepharose separated fractions of Bermuda grass pollen diffusate

Spot Gel μLC/ESI Score Peptide Mass (Mr) Match (calc) 1 60,000 Acidic Cyn d 1 isoallergen isoform 1 185 65 26,663 (fragment) 2 50,000 Calcium-binding protein precursor 138 6 47,983 3 45,000 Calcium binding protein 486 17 47,983 4 30,000 Acidic Cyn d 1 isoallergen isoform 1 440 18 26,663 (fragment) 5 25,000 Lectin 592 11 31,389 6 30,000 Lectin 320 6 31,389 7 30,000 Lectin (fragment) 317 3 31,389 8 30,000 Major pollen allergen Cyn d 1 157 5 26,872 9 30,000 Acidic Cyn d 1 isoallergen isoform 1 70 1 26,872 (fragment) 10 25,000 Major pollen allergen Cyn d 1 217 9 26,872 11 15,000 Lectin 76 4 31,389

6.2.3 Discussion

The allergen Cyn d 1 is a glycoprotein, abundantly present in Bermuda grass pollen. Like other group 1 allergens, it is recognised as one of the most prominent allergenic determinants in the extracts of grass pollen (Trinca 1962; Orren and Dowdle 1977; Matthiesen, Schumacher et al. 1989; Matthiesen, Schumacher et al. 1991). About 5% of the allergen is made up of oligosaccharides (Andersson and Lidholm 2003). Although group 1 allergens from all pollen sources in general are glycoproteins, only allergens from Cynodon and Phragmites pollen have any affinity to bind to lectins (Karlstam and Nilsson 1982). Watson et al found that Concanavalin A was useful in differentiating Cynodon pollen from most other grass pollen, with specific binding of cell wall components in the Cynodon pollen to the Concanavalin lectin (Watson, Knox et al. 1974). Karlstram and Nilsson further demonstrated that out of nine grass

158 pollens of importance in allergy, only Cynodon and Phragmites allergens bound to Concanavalin A Sepharose, while binding of Lolium and Phleum pollen allergens was not observed (Karlstam and Nilsson 19 82). Matthiesen et al concluded that the affinity of Cyn d 1 f or Concan avalin A was due to the presence of carbohydrates with free terminal α- D- mannop yranoside or α- D- gluc opyranoside residues, which are either not present or acce ssible i n other majo r pollen allergens (Matthiesen, Schumacher et al. 1989). The carbohydrate components of the allergen Cyn d 1 and some of its isoallergens have been comprehensively analysed by Su and co lleagues (Su, Shu et al. 1996; Su, Peng et al. 2003). The purification p rotocol f or Cyn d 1 allergens curr ently involv es combining Concanavalin A Sepharose chromato graphy with a wide variety of other HPLC techniques, but there is no “gold standard method” and different combinations have been used with variable success (Matthiesen, Schumacher et al. 1989; Su, Shu et al. 1996; Su, Peng et al. 2003). For these studies, Concanavalin A Sepharose chromatog raphy was used as a partial puri fication meth od because: • The fractionating procedure and buffers were not denaturing to the proteolytic activity in the diffusate. • Lectin chromatography would allow for the relative purification of Cyn d 1 and its corresponding basic and acidic isoallergen by binding to the gly can componen ts of the allergen. • If Cyn d 1 did indeed possess intrinsic proteolytic activity, the activity would be demonstrable in the eluted, bound fractions by the fluorescent assay and/or gelatin zymography. • If Cyn d 1 did not possess intrinsic enzyme activity, the proteolytic activity would be demonstrable only in the unbound fractions, which would allow for its purification by the removal of excessive Cyn d 1, enabling detection of the low abundance protein by mass spectrometric techniques. One cannot exclude the possibility that the peptidase could also bind to Concanavalin A Sepharose, resulting in its co-purification with Cyn d 1. Nevertheless, this would result in the partial purification of the enzyme since ~ 90% of the proteins in the crude diffusate do not bind the lectin. Hence, affinity chromatography would make the proteomic analysis of the mixture easier.

159 All the chromatography procedures and incubations were performed at 4°C to preserve proteolytic activity in the fractions. The fractions were desalted to remove excessive salts and oligosaccharides in the different elution buffers, which would otherwise interfere with the analysis of the proteolytic activity. In initial experiments, dialysis tubing (3000 Da cut-off) was used for buffer exchange with PBS (pH 7.5) at 4°C, overnight but this prolonged incubation led to the loss of the proteolytic activity and hence was replaced by centrifugal ultra-filtration. Interestingly, analysis of the eluted fractions using the fluorescent peptide substrate assay demonstrated that the peptidase activity was located in the unbound, flow-through fraction (F1) of the Concanavalin A Sepharose column. Moreover, the rate of hydrolysis was four-fold greater than that of the crude pollen diffusate of Bermuda grass pollen at similar protein concentrations (Table 6.10). The increase in peptidase activity could probably be attributed to the removal of some inhibitor on passing through the column. Fraction F2, which comprised the second wash from the column, demonstrated peptidase activity equivalent to that of the crude pollen diffusate of Bermuda grass. The two fractions combined accounted for 80% of the total protein and a two- fold i ncr ease in the hydrolytic activity, compared to the crude diffusate. The absence of peptidase activity in the bound fractions of Concanavalin A Sepharose (F4 to F7) as assessed by the soluti on phase assay was in contrast to the analysis of the same fractions by gelatin zymography. Gelatin zymography of the fractions demonstrated that endopeptidase activity of Mr ~30,000 Da was detected in fractions F4 and F5, which comprised of bound glycoproteins that had been eluted fr om the column. This enzyme was of slightly lower Mr than the 32,000-35,000 Da activity observed in crude Bermuda grass diffusate, but was the only peptidase activity recovered from the column. There was no residual activity in the fractions eluted using the sodium borate buffe r, w hich indicated that little if any proteolytic activity was strongly bound to the matrix. The results from the Concanavalin A Sepharose experiments confirmed the suggestion (based on enzyme inhibitory profiles) that the proteolytic activity in the pollen diffusate might consist of at least two distinct serine peptidase profiles. The peptidase present in the unbound fractions of concanavalin A Sepharose possessed trypsin-like specificity based on the inhibitory profile but was incapable of digesting whole protein substrates like gelatin on zymography, suggesting that it was probably an exopeptidase. The peptidase in the bound fractions of Concanavalin A Sepharose was not found to be arginine specific, as it did not hydrolyse the fluorescent mono-peptide substrate, but was capable of digesting gelatin on zymography and was

160 inhibited by serine peptidase inhibitors. This could explain why benzamidine Sepharose did not selectively bind the peptidase activity at 30,000-35,000 Da from the crude diffusate of Bermuda grass were unsuccessful. In light of these findings, the quantitation of the serine peptidase in the bound fractions of Concanavalin A Sepharose chromatography was attempted in a liquid assay format, using a fluorescently quenched gelatin substrate (DQ gelatin, Molecular probes). However, the assay sensitivity was found to be poor; there was high background fluorescence from the substrate itself, and prolonged incubation was required. Attempts to optimise the assay by adjusting the gain on the instrument and varying the substrate concentrations did not result in any improvement in the sensitivity of the assay. Further analysis and optimisation of this assay was not pursued. Two-dimensional SDS/PAGE was also attempted on the affinity purified, proteolytically active fractions, to enhance the resolution of separation. Although this method demonstrated a number of spots of Mr 32,000-35,000 Da, it was not helpful as no proteolytic activity could be demonstrated in the two-dimensional gelatin zymograms of the pooled fractions. This was attributed to the denaturing conditions associated with the rehydration buffer. Varying the concentration of urea in the rehydration solution was attempted, compromising on the resolution of separation in order to retain peptidase activity, but was unsuccessful and hence not pursued. The proteomic analysis of the SDS/PAGE separated bands corresponding with the active peptidase in the Concanavalin A purified fractions identified the protein as Cyn d 1 and/or its isoallergens with high confidence, strongly suggesting that the activity was associated with Cyn d 1. However, since Concanavalin A binds to glycoproteins which possess α-D-Mannopyranosyl sugars, with free hydroxyl groups at the C3, C4 and C6 position, it is not specific for Cyn d 1. Hence one still cannot completely exclude the possibility that another co-localising protein with the same specificity could be responsible for the proteolytic activity in the diffusate. In an attempt to address the possibility that the proteolytic activity could be a minor component, co-migrating with Cyn d 1, which was missed by proteomic methods due to the abundance of Cyn d 1, the fractions that bound to Concanavalin A Sepharose were subjected to tandem chromatography. The purification of native Cyn d 1 by Matthiesen and colleagues involved the concentration of the proteins in the diffusate by ammonium sulphate precipitation, followed by Concanavalin A Sepharose and cation-exchange chromatography, which was

161 performed using an acetate based buffer at pH 5.0 (Matthiesen, Schumacher et al. 1991). Although these tandem purification steps were successful in purifying Cyn d 1 (as demonstrated by the ability of the purified protein to bind to IgE), the approach could not be used in the current study, because the peptidase activity in the pollen diffusate was rapidly inactivated below pH 6.0 as well as by concentration by precipitation. The current study attempted anion-exchange HPLC in conjunction with Concanavalin A Sepharose as it was the only other technique that was successful in retaining peptidase activity on purification. However, analysis of the pooled fractions that retained proteolytic activity at Mr ~32,000 by anion exchange HPLC demonstrated very weak, non-resolving peaks, which could not be differentiated from the base line. The lack of peptidase activity in any of the fractions collected demonstrated that the peptidase activity could not withstand tandem purification. This is not surprising considering that during purification of enzymes; inactivation can often result from minor changes in their environment. Moreover, peptidases in crude mixtures usually exist in precursor forms and/or are non-covalently associated with inhibitors, the removal of which results in their rapid self-degradation. Another possible approach to purification that might permit peptidase activity to be retained is immunoaffinity chromatography. As the IG 12 antibody to Phl p 1 appears to cross-react with Cyn d 1, purification of Cyn d 1 by immobilising IG12 on an immunoaffinity column might be feasible. Unfortunately it was not possible to perform this experiment because of the lack of availability of sufficient antibody. The recent development of activity-based probes offers another potential alternative for isolating and further characterising the serine peptidase activity in Bermuda grass pollen. The principle underlying the synthesis of these probes involves designing specific chemical reagents that are able to recognise and bind to conserved catalytic/functional/structural motifs in active enzymes in a covalent manner (Kozarich 2003). The probes can be detected or quantified by coupling them either to a fluorogenic or affinity tag like biotin. This allows the covalently bound protein to be captured, quantified and identified by chromatographic, electrophoretic and mass spectrometric techniques. Proof of concept of specificity and sensitivity of these probes has been demonstrated recently by Cravatt and colleagues using a fluorophosphonate probe (FP) to detect serine hydrolases (Liu, Patricelli et al. 1999). The FP group is a selective inhibitor of active serine peptidases, which was coupled to a biotin molecule via a linker. The biotin is useful for the visualization and capture of the covalently labelled proteins by standard avidin-based

162 methods. The biotin could also be substituted with a fluorescent group (trifunctional probe), allowing for more sensitive detection and quantitation (Kidd, Liu et al. 2001). Using this technique, Cravatt and colleagues have demonstrated tissue expression levels of serine peptidases in several proteomes (Kidd, Liu et al. 2001; Jessani, Liu et al. 2002), confirming the success of the technique in complex samples. Bogyo and co-workers have employed a similar approach, using epoxide inhibitor-coupled probes for the profiling of cysteine peptidases (Bogyo, Verhelst et al. 2000; Greenbaum, Medzihradszky et al. 2000). Employing this method with the Concanavalin A separated fractions of Bermuda grass pollen diffusate would allow for definitive confirmation of the enzymatic property of the Cyn d 1 allergen. Unfortunately, these probes are currently not commercially available, but they will undoubtedly be useful for the identification and purification of serine peptidases in the future.

163 7 Concluding Discussion

The focus of the studies described in this thesis was to explore the relationship between pollen allergens and proteolytic enzymes present within pollen grains. Although pollens are implicated as major triggers of allergic diseases, little is understood about how this process is initiated at a biochemical or molecular level. Pollen grains have long been recognised to contain a number of peptidases, but the putative functions of these enzymes have largely been ignored. In fact, very little is known about the biological significance of the various enzymes present in pollens, which include lyases (Turcich, Hamilton et al. 1993), esterases (Albani, Altosaar et al. 1991) and peptidases (Knox and Heslop-Harrison 1970; Travis, Whitworth et al. 1996). A large proportion of proteolytic enzymes are localised within the exine and intine layers of the pollen grain wall, allowing them to leach out rapidly on hydration (Knox and Heslop- Harrison 1970). It has been assumed that the localisation of these enzymes in the walls of the pollen grain contributes to the process of fertilisation. When the pollen grain comes into contact with the stigma of the flower, the proteolytic enzymes released presumably allow the pollen tube to break through the cellular layers of the stigma, allowing fertilisation of the plant (Knox and Heslop-Harrison 1970; Knox 1973; Knox and Suphioglu 1996). Studies have now begun to focus on the effects of peptidases on respiratory epithelium and their contribution to allergic sensitisation (Chua, Stewart et al. 1988; Hassim, Maronese et al. 1998; Wan, Winton et al. 1999; Wan, Winton et al. 2000). An important step in characterising the biological functions of pollen peptidases is being able to accurately determine substrate specificity in order to detect and quantitate the enzymatic activity. Previous studies from this laboratory, together with the experiments undertaken in this thesis, highlight the importance of standardising the analytical conditions under which the assay is performed. For example, in the study by Widmer and colleagues, peptidases in several pollen grain diffusates were determined to have tryptic-like specificity (Widmer, Hayes et al. 2000). In contrast, the diffusates of the same pollens tested by two different assays methods (fluorescent monopeptide substrate assay with trypsin-like specificity, and gelatin zymography) under more “physiological” assay conditions yielded quite different results. The fluorescent assay demonstrated that all three pollen diffusates tested in the current study possessed “trypsin-like” serine peptidase activity whereas two of the pollens had not been known to exhibit trypsin-like activity in the earlier study.

164 Analysis of the same diffusates by gelatin zymography revealed the presence of multiple peptidases of different molecular weig hts, some of which were found to possess serine peptidase activity. The partial purification of the peptidases in Bermuda grass pollen using Concanavalin A Sepharose affinity chromatography demonstrated that the assay methods used in the present study actually detected and quantitated two different serine peptidase enzymes. While the fluorescent peptidase substrate assay readily detected a trypsin-like exopeptidase, this enzyme could not digest gelatin on zymography. The serine peptidase at Mr ~32,000 Da readily hydrolysed gelatin, but exhibited no specificity for arginine in the fluorescent substrate assay. These results emphasise that although peptide substrate assays can generally be used to screen for proteolytic activity, they do not necessarily reflect the activities of all of the enzymes in the sample. The studies on Bermuda grass pollen diffusate provided strong evidence of concordance between one of the major proteolytically active molecular species (Mr ~32,000) in Bermuda grass and the major pollen allergen Cyn d 1. To confirm the identity of the serine peptidase, two different proteomic techniques were used, namely peptide mass fingerprinting using MALDI- TOF and tandem mass spectrometry using micro or nano LC/MS-MS. The only protein that was consistently identified in the protein spots that corresponded to the proteolytic activity in the gelatin zymograms was the allergen Cyn d 1 and/or its isoallergens. An unresolved issue is whether all the isoallergens of Cyn d 1 exhibit proteolytic activity. Although a number of spots of Mr ~30,000–35,000 Da that were separated by 2D-gel electrophoresis were identified as Cyn d 1 or its isoallergens, not all of them demonstrated enzymatic activity by zymography. It is plausible that isoallergen species might vary in catalytic activity, with some being more sensitive than others to the harsh denaturing/renaturing conditions of isoelectric focusing/gelatin zymography. Alternatively, minor variations in the amino acid sequences of the different isoallergens could influence which isoform is proteolytically active. The key question posed by the results of this study is “Does Cyn d 1 possess biologically relevant peptidase activity?” It is difficult to answer this unambiguously. While the results from these experiments have demonstrated a strong association between immunoreactivity or molecular sequence and enzymatic activity, they do not irrefutably prove that Cyn d 1 is a

165 peptidase. Moreover, proteolytic activity has so far only been demonstrated in vitro by gelatin zymography. Testing the in vivo effects of this protein would require assessment in an appropriate in vivo model of allergic sensitisation or of asthma, which would require large amounts of protein. The standard approach to this problem would be to generate the protein via a recombinant expression system. Unfortunately, there are numerous caveats associated with this. Perhaps the most important is the problem of contaminating enzymes derived from the expression system when preparing recombinant proteins (Poppelmann, Becker et al. 2002). In studies by Grobe et al, the recombinant major pollen allergen of Timothy grass, rPhl p 1, apparently possessed intrinsic cysteine peptidase activity (Grobe, Becker et al. 1999). However, Poppelmann and colleagues later demonstrated that the recombinant allergen did not possess any inherent peptidase activity and that the observed activity was an artefact due to a contaminating peptidase synthesised and secreted by the yeast expression system (Poppelmann, Becker et al. 2002). Because it is impossible to completely exclude the presence of contaminating enzymes in recombinant protein preparations, this severely limits the application of such an approach in an in vivo model. To circumvent these issues, native allergen purified from pollen diffusates would be required. In the present experiments, attempts to further purify the active fractions obtained after Concanavalin A Sepharose affinity chromatography of Bermuda grass pollen diffusate always led to loss of enzymatic activity. It seems likely that progressive purification of the enzyme of interest from its crude mixture resulted in autohydrolysis, leading to the total loss of activity. This would make the purification of large amounts of proteolytically active Cyn d 1 particularly difficult. A possible solution to this problem would be to add a carrier protein of known molecular weight to the partially purified fractions. If this was able to behave as a surrogate substrate, it would prevent autohydrolysis. Because the goal of purification is to obtain an intact, enzymatically active protein for the analysis of antigenicity in vivo in animal models, it would be important to choose a carrier protein that did not impact upon the immunological response. Numerous studies, both in vitro and in vivo have established that proteolytic enzymes have a role in triggering the inflammatory response in allergic diseases. In vitro studies using peptidases from diverse sources have consistently demonstrated airway epithelial cell damage and/or detachment (Robinson, Venaille et al. 1990; Hassim, Maronese et al. 1998; Wan, Winton et al. 2001). Perhaps the most comprehensive studies of the effects of peptidases in allergic

166 asthma are those of the house dust mite allergen Der p 1, which is known to possess intrinsic cysteine peptidase activity (Chua, Stewart et al. 1988). This enzyme (as with other enzymes, mostly serine peptidases, in house dust mite faecal pellets) has been shown to play a potentially important role in airway epithelial damage by disrupting the tight junctions at the apices of the epithelial cells. Degradation of the transmembrane protein occludin and the intracellular protein zona occludan-1 (ZO-1) (Wan, Winton et al. 2001) in the tight junctions could open up the normally sealed paracellular channels and allow the ingress of foreign allergens. These allergens might then traverse the epithelial barrier and thus gain access to the sub-epithelial antigen presenting dendritic cells (Robinson, Kalsheker et al. 1997). Other allergens that have been shown to have peptidase activity include Fel d 1 (Ring, Wan et al. 2000), the major cat allergen and Pen ch 1 from Penicillium chrysogenum (Chou, Lai et al. 2002). However, the role of these enzymatic activities in eliciting airway epithelial injury in vivo is unknown. In addition to the degradative activity, the peptidases in pollens may contribute to the amplification of the airway inflammatory response by promoting the recruitment of inflammatory cells such as T-lymphocytes, neutrophils, eosinophils and mast cells, and activating them to release potent pro-inflammatory mediators (Chung and Barnes 1999; Bousquet, Jeffery et al. 2000; Boyce 2003). Peptidases are capable of exerting these effects via protease-activated receptors (PAR). Miike et al demonstrated that serine proteolytic activity could induce the release of pro-inflammatory mediators from eosinophils via PAR-2 (Miike, McWilliam et al. 2001), which might be a pathway for the peptidase activity associated with allergens to potentiate the inflammatory response (Asokananthan, Graham et al. 2002). In vitro studies have shown that the proteolytically active Der p 1 can also play an important role in driving an IgE response. Der p 1 is capable of cleaving several cell surface molecules which are involved in the synthesis of IgE. The cleavage of CD40 on the surface of dendritic cells results in the production of significantly less IFN-γ and more IL-4 by the CD4+ T cells, skewing the immune response towards a Th2 phenotype (Ghaemmaghami, Gough et al. 2002). Cleavage of the low affinity IgE Fc receptor (CD23) on the surface of B lymphocytes by Der p 1 can result in increased IgE production via inhibition of the negative feedback mechanism in B-cells (Hewitt, Brown et al. 1995; Schulz, Sewell et al. 1998). Der p 1 is also capable of cleaving CD25 on T lymphocytes and biasing the immune response towards allergy by

167 decreasing the production of IFN-γ (Schulz, Sewell et al. 1998; Ghaemmaghami, Robins et al. 2001). The results of the in vitro studies correlate well with those of in vivo studies in animal models of allergic sensitisation. Gough et al demonstrated that intraperitoneal immunization of mice with proteolytically active Der p 1 resulted in significant and selective enhancement of total IgE and Der p 1 specific IgE, compared with mice immunized with Der p 1 which had been irreversibly blocked by a potent cysteine peptidase inhibitor, E-64 (Gough, Schulz et al. 1999). They also demonstrated that in the presence of an active enzyme, the IgE response to proteolytically inactive bystander allergens such as ovalbumin was enhanced (Gough, Sewell et al. 2001). In this initial study, the allergenic peptidases were administered systemically. To be relevant to asthma and allergic rhinitis, the enzymatically active allergens need to be evaluated following delivery by inhalation. Acknowledging this, Gough and colleagues further tested a model of this type using intranasal exposure and demonstrated that proteolytically active Der p 1 elicited significantly higher levels of cellular infiltration in the lungs and an increase in the circulating total and Der p 1 specific IgE, compared to proteolytically inactivated Der p 1 (Gough, Campbell et al. 2003). This result mirrored earlier findings with respect to both IgG and IgE in mouse and guinea pig models (Robinson, Babcock et al. 1996; Robinson, Horn et al. 1998). The biological relevance of the proteolytic activity of Cyn d 1 will ultimately need to be determined using a comparable appropriate in vivo model. From a pathophysiological point of view, this might best be achieved by intranasal exposure to the highly purified, proteolytically active Cyn d 1, compared to Cyn d 1 in which the peptidase activity has been irreversibly inactivated using a highly specific inhibitor.

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190 APPENDIX: GENERAL REAGENTS

α-1 Hydroxy cinaminic acid Agilent Technologies Acetic acid, glacial Ajax Fine Chemicals Acetonitrile, HPLC grade Ajax Fine Chemicals Acrylamide, electrophoresis grade Bio-Rad APS Bio-Rad AEBSF (Pefabloc) Roche, USA Benzamidine HCl BDH chemicals, Poole, England Benzamidine 6B Sepharose Pharmacia, Upsalla, Sweden Bermuda grass pollen Bayer, Sigma and Greer Labs, USA Bicinchoninic acid dye protein assay Sigma Biolyte 3-10 ampholyte Bio-Rad Bromophenol blue Bio-Rad BSA, low endotoxin Sigma Benchmark protein ladder marker Invitrogen, USA Complete protease cocktail inhibitors Roche, Mannhein, Germany Coomassie Blue R-250 Sigma Collagenase Sigma Concanavalin A Sepahrose Pharmacia, Upsalla, Sweden Dithithretiol (DTT) Bio-Rad EDTA (disodium salt) Sigma Ethanol, absolute Ajax Fine Chemicals Formaldehyde Riedel-de Haen, Germany Formic acid Sigma Gelatin, bovine type B Sigma Glycerol BDH Hydrochloric acid BDH IPG dry strips pH 3-10 Amersham Pharmacia, Sweden IPG buffer Amersham Pharmacia, Sweden

191 Iodoacetamide BDH Kentucky blue grass pollen Bayer, USA Methanol, HPLC grade Ajax Fine Chemicals Nunc 100 well plates Nunc, Roskilde, Denmark PMSF Sigma Polypropylene columns Pierce, USA PVDF membranes Millipore, Australia Potassium Ferricyanide Ajax chemicals Saline Baxter, Australia Sodium Azide Sigma Sodium Borate BDH Sodium Carbonate APS fine chemicals Sodium Chloride Merck Sodium dodecyl sulphate (SDS) Bio-Rad Sodium hydroxide BDH Sodium thiosulphate Merck Silver nitrate BDH TEMED Bio-Rad Tricine Sigma Trifluoracetic acid (TFA) Aldrich Triton X-100 Sigma Trizma Sigma Tween-20 Sigma Zymogram buffer Bio-Rad

192