Molecular Identification of Antigen Recognition Structures in Immune Complexes for Immunotherapeutic Applications by
Proteolytic and Mass Spectrometric Methods
Dissertation
zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften
an der Universität Konstanz
vorgelegt von
Raluca Stefanescu
Konstanz 2007
Tag der műndlichen Prűfung: Donnerstag, den 13. Dezember 2007 Vorsitzender und műndlicher Prűfer: Herr Professor Dr. Fischer Műndliche Prűfer: Herr Professor Dr. Dr. h.c. Przybylski
Herr Professor Dr. Jeschke
Konstanzer Online-Publikations-System (KOPS) URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-89395
URL: http://kops.ub.uni-konstanz.de/volltexte/2009/8939/
It is not the possession of truth, but the success which attends the seeking after it, that enriches the seeker and brings happiness to him.
Max Planck
For my wonderful parents Angela and Radu Stefanescu
The present work has been performed in the time from September 2003 to June 2007 in the Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry of the University of Konstanz, under the supervision of Prof. Dr. Dr. h. c. Michael Przybylski.
Special thanks to:
Prof. Dr. Dr. h. c. Michael Przybylski for giving me the opportunity to work in his group, for the interesting research topic and discussions concerning my work and for his entire support;
Prof. Dr. Gunnar Jeschke for writing the second evaluation of my dissertation;
Prof. Dr. Sylvie Rebuffat and Dr. Severine Zirah for the collaboration at the Zn2+
effect on the antibody interaction to amyloid peptides.
Prof. Dr. Michael Ehrmann for providing the recombinant HtrA and C99.
Prof. Dr. Richard Dodel and Dr. Michael Bacher for providing human Aß-antibody samples (IVIG, AD77, 105, 005, 006) employed in epitope identification.
Prof. Dr. Beat Ernst and Dr. Rita Born for providing the antibody and the recombinant H1CRD employed in this work.
All members of the group for the nice and inspiring atmosphere, but most of all I want to thank to Dr. Xiaodan Tian, for her didactic attitude at the beginning of the work in the research group, Dr. Marilena Manea, Dr. Eugen Damoc, Dr. Roxana Iacob, Dr. Andreas Marquardt, Dr. Catalina Damoc, Iuliana Susnea, Alina Petre, Dr. Suzanne Becker, Madalina Maftei, Adrian Moise, for scientific discussions and interesting advices during my work; Claudia Cozma, Camelia Vlad and Stefan Slamnoiu for the dedicated work during the practicals; Lana Mack, Gabriela Paraschiv and Ute Schad for all the help they gave me, Reinhold Weber for the organization and expert group leading in the Swiss Alps.
Special thanks also to my friends: Marilena, Eduard, Ana-Maria, Lacramioara, Livia and Ioana for their support and the wonderful time spent together.
Last but not least I wish to thank and to express my deep gratitude to my family, for supporting and encouraging me during this time.
This dissertation has been published in part, and presented at the following conferences:
Peer-Reviewed Publications
1. Zirah S., Stefanescu R., Manea M., Tian X., Cecal R., Kozin D.A., Debey P., Rebuffat S., Przybylski M. (2004) “Zinc binding agonist effect on the recognition of the ß-amyloid (4-10) epitope by anti-ß-amyloid antibodies”, Biochem. Biophys. Res.
Commun. 321(2), 324-328.
2. Grau S., Baldi A., Bussani R., Tian X., Stefanescu R., Przybylski M, Richards P., Jones S.A., Shridhar V., Clausen T., Ehrmann M. (2005) “Implications of the serine protease HtrA1 in amyloid precursor protein processing”, Proc. Natl. Acad.
Sci. 102(17),6021-6026.
3. Tian X., Cecal R., McLaurin J., Manea M., Stefanescu R., Grau S., Harnasch M., Amir S., Ehrmann M., St. George-Hyslop P., Kohlmann M., Przybylski M. (2005)
"Identification and structural characterisation of carboxy-terminal polypeptides and antibody epitopes of Alzheimer's amyloid precursor protein using high resolution mass spectrometry", Eur. J. Mass Spectrom. 11(5), 547-556.
4. Bilkova Z., Stefanescu R., Cecal R., Korecka L., Ouzka S., Jezova J., Viovy J.L., Przybylski M. (2005) “Epitope extraction technique using a proteolytic magnetic reactor combined with Fourier-transform ion cyclotron resonance mass spectrometry as a tool for the screening of potential vaccine lead peptides”, Eur. J.
Mass Spectrom. 11(5), 489-495.
5. Stefanescu R., Iacob R., Damoc N., Marquardt A., Amstalden E., Manea M., Perdivara I., Maftei M., Paraschiv G, Przybylski M., (2007) “Mass spectrometric approaches for elucidation of antigen-antibody recognition structures in molecular immunology”, Eur. J. Mass Spectrom, 13(1), 69-75.
Conference presentations
1. Stefanescu R., Cecal R., McLaurin J., Tian X., Manea M., St. George-Hyslop P., Przybylski M., (2003) “Mass spectrometric and immunoanalytical characterisation of an amyloid plaque-specific epitope of a therapeutically active anti-Aß42 antibody from transgenic mice”, DGMS, Münster, Germany.
2. Stefanescu R., Tian X., Grau S., Ehrmann M., Przybylski M., (2004)
“Characterisation of the Proteolytic Reactivity and Specificity of Enzymes on Alzheimer-Target Proteins by High Resolution Mass Spectrometry”, 52nd Conference of Mass Spectrometry and Allied Topics, Nashville, Tennessee, USA,
3. Stefanescu R., Tian X., Grau S., Ehrmann M., Przybylski M., (2005) “High resolution mass spectrometric cleavage site elucidation of the novel serine protease HtrA involved in amyloid precursor protein processing”, Human Proteome Organisation (HUPO) 4th Annual World Congress, München, Germany.
4. Stefanescu R., Born R., Slamnoiu S., Ernst B., Przybylski M., (2006) “Mass spectrometric and immunoanalytical epitope characterisation of a specific antibody to the H1-carbohydrate recognition domain of the asialoglycoprotein receptor”, 17th International Mass Spectrometry Conference, Prague, Czech Republik.
5. Stefanescu R., Iacob R., Manea M., Tian X., Perdivara I., Maftei M., Paraschiv G., McLaurin J., St. George-Hyslop P., Przybylski M., (2007) “Epitope identification and structure determination of Aß-specific antibodies upon Aß-Immunisation using High-Resolution Mass Spectrometry”, 8th International Conference AD/PD, Salzburg, Austria.
TABLE OF CONTENTS
1. INTRODUCTION ... 1
1.1. Biochemical and immunological basis of antigen-antibody recognition ... 1
1.1.1. Immune response: generation of antibody diversity ... 1
1.1.2. Structure of antibodies and recognition of antigens ... 3
1.2. Analytical methods for epitope identification ... 6
1.3. Identification of epitopes by proteolytic cleavage and mass spectrometry ... 7
1.4. Pathophysiological characteristics and therapeutic perspectives of Alzheimer´s Disease ... 13
1.5. Immunotherapeutic strategies for Alzheimer´s Disease ... 17
1.6. Scientific goals of the dissertation ... 21
2. RESULTS AND DISCUSSION ... 23
2.1. Characterisation of Aß-plaque specific antibodies ... 23
2.1.1. Metodology of mass spectrometric epitope identification ... 23
2.1.2. Mass spectrometric elucidation of the epitope recognized by polyclonal anti-Aß42 and monoclonal anti-Aß(1-17) antibodies ... 25
2.1.3. Identification of functional amino acid residues within the ß-amyloid plaque specific epitope using alanine-scanning mutagenesis ... 28
2.1.4. Effect of zinc ions on the recognition of N-terminal ß-amyloid epitope by plaque specific antibodies ... 34
2.2. Epitope elucidation of Aß-specific human antibodies ... 42
2.2.1. Therapeutic potential of amyloid-specific autoantibodies from human serum ... 42
2.2.2. Purification of Aß-autoantibodies from pooled immunoglobulin gamma preparations ... 44
2.2.3. Identification of the epitope recognized by Aß-autoantibodies isolated from pooled immunoglobulin gamma ... 46
2.2.4. Clonal diversity and sequence analysis of human serum Aß antibodies by 2D-gel electrophoresis and peptide mass fingerprint ... 56
2.2.5. Identification of the epitope recognized by Aß-autoantibodies isolated from the serum of an Alzheimer disease patient ... 63
2.2.6. Synthesis of biotinylated amyloid peptides encompassing the epitope .... 65
2.2.7. Characterisation of the human Aß-autoantibodies binding to amyloid peptides by ELISA ... 67
2.2.8. Concluding discussion of the Aß-autoantibody epitope ... 72
2.3. Investigation of the cleavage specificity of ß-amyloid peptides by HtrA1 protease ... 74
2.3.1. Structure and biological functions of HtrA1 ... 74
2.3.2. Clearance of cerebral Aß by enzymes ... 75
2.3.3. Analytical characterization of C99 and HtrA ... 76
2.3.4. Mass spectrometric identification of cleavage sites in C99 and Aß ... 77
2.4. Epitope identification of a monoclonal antibody to the H1- carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor ... 83
2.4.1. Structure and biological functions of H1CRD ... 83
2.4.2. Primary structure characterisation of H1CRD using mass spectrometric methods ... 85
2.4.3. Epitope identification of a monoclonal antibody to H1CRD ... 96
2.4.4. Affinity of synthetic epitope peptides to the monoclonal antibody ... 100
3. EXPERIMENTAL PART ... 103
3.1. Proteins, Enzymes and Antibodies ... 103
3.2. Materials and reagents ... 103
3.3. Solid phase peptide synthesis ... 104
3.4. Chromatographic and electrophoretic separation methods ... 106
3.4.1. Reversed phase-high performance liquid chromatography ... 106
3.4.2. Sample concentration and desalting using Zip Tip pipette tip ... 108
3.4.3. One-dimensional gel electrophoresis ... 108
3.4.3.1. SDS-PAGE according to Laemmli ... 109
3.4.3.2. SDS-PAGE according to Schägger and Jagow ... 110
3.4.4. Two-dimensional gel electrophoresis ... 111
3.4.5. Colloidal Coomassie staining... 112
3.4.6. Silver staining ... 112
3.5. Immunological methods ... 113
3.5.1. Preparation of immobilised antibodies ... 113
3.5.2. Epitope excision and extraction experiments... 113
3.5.3. Preparation of immobilized antigen column ... 114
3.5.4. Separation of Aß-autoantibodies from pooled IgG preparations ... 114
3.5.5. Enzyme-linked immunosorbent assay ... 115
3.5.5.1. Alanine scanning mutagenesis ... 115
3.5.5.2. Zinc binding effects on the antigen-antibody interaction ... 117
3.6. Chemical modification reactions and enzymatic fragmentation of
proteins ... 119
3.6.1. Reduction and alkylation of disulfide bonds in solution ... 119
3.6.2. Proteolytic digestion of proteins in solution using trypsin ... 119
3.6.3. Proteolytic digestion of proteins in solution using endoproteinase GluC ... 119
3.6.4. Proteolytic digestion of proteins in solution using alpha-chymotrypsin ... 120
3.6.5. Proteolytic digestion of proteins in solution using Pronase ... 121
3.6.6. Proteolytic digestion of proteins in solution using Endoproteinase LysC ... 121
3.6.7. In-gel trypsin digestion procedure of Coomassie Brilliant Blue stained proteins ... 122
3.7. Mass spectrometric methods ... 122
3.7.1. Time of flight mass spectrometry ... 122
3.7.2. Fourier-transform Ion-Cyclotron Resonance mass spectrometry ... 124
3.7.2.1. MALDI-FT-ICR mass spectrometry ... 125
3.7.2.2. ESI-FT-ICR mass spectrometry ... 126
3.7.3. Liquid chromatographic/Ion trap mass spectrometric investigation ... 127
3.8. Bioinformatic tools for mass spectrometry ... 128
3.8.1. GPMAW ... 128
3.8.2. Search engines for identifying proteins ... 129
3.8.3. BALLView 1.1.1 ... 129
3.8.4. PDQuest 2-D gel analysis software ... 130
4. SUMMARY ... 131
5. ZUSAMMENFASSUNG ... 135
6. REFERENCE LIST ... 139
7. APPENDIX ... 156
7.1. Appendix 1 ... 156
7.2. Appendix 2 ... 158
7.3. Appendix 3 ... 159
7.4. Appendix 4 ... 163
1. INTRODUCTION
1.1. Biochemical and immunological basis of antigen-antibody recognition 1.1.1. Immune response: generation of antibody diversity
The immune response is activated by the presence of foreign microorganisms that elude the anatomic and physiologic host barriers, and by genetically modified own cells that express unknown proteins. The two branches of the immune responses, i.e.
humoral and cell-mediated, assume different, complementary roles in protecting the host [1-3].
The humoral response is responsible for the neutralization of extracellular invaders and soluble foreign molecules and is mediated by B-cells. Each of the mature B-cells expresses about 105 identical membrane bound antibody molecules (IgM and IgD) with a unique amino acid sequence within the population of B-cells. During B-cell maturation in the bone marrow an enormous number (1010) of antigen-specific membrane antibodies can be generated despite the reduced repertoire of immunoglobulin genes [4-7]. This enormous diversity is the result of random gene rearrangements and is later reduced by a selection process that eliminates any B-cell with membrane-bound antibody that recognizes self components. The first encounter with a foreign entity capable of binding to the membrane antibody leads to the stimulation of the B-cell which divides rapidly generating memory B cells and effector B cells (see Figure 1). Memory B cells express the same antibody bound to the membrane and are able to generate new memory cells and effector cells.
Mature B-cell Expressing unique antibody molecules
Antigen stimulation
Activated B-cell
Division and
differentiation IgM
IgG Class
switching
Memory B-cell Effector B-cell
Effector B-cell Mature B-cell
Expressing unique antibody molecules
Antigen stimulation
Antigen stimulation
Activated B-cell
Division and
differentiation IgM
IgG Class
switching
Memory B-cell Effector B-cell
Effector B-cell
Figure 1: Overview of the humoral immune response. After the interaction with an antigen, B cells divide and differentiate into memory B cells and effector B cells. The latter produce soluble antibodies able to interact specifically to the antigen.
Punctual mutations in the recognition site of the antibody which occur in a random manner, followed by the preferential selection of these memory B cells with highest affinity for the antigen increase the binding efficiency of the population of B cells. The effector B cells, called also plasma cells, secrete soluble antibody molecules which bind to the antigen facilitating its clearance from the body.
In contrast to the humoral immunity which provides protection against extracellular invaders, the cell-mediated immunity is able to detect and eliminate cells that harbor intracellular pathogens and cancerous cells that express non-self molecules. In the cell mediated immune response the antigen is recognized by T cells which carry approximately 105 membrane-bound T-cell receptors whose structural diversity resembles those of the B cell receptors. However, the T cell receptor does not exist in soluble form and recognizes the antigen only if is enzymatically degraded and the peptide fragments are presented by the major histocompatibility complex (MHC) molecules (see Figure 2). Therefore, the T cell epitopes have to be considered as part of a molecular complex formed by the antigen presenting molecules and antigen fragments.
Figure 2: Processing and presentation pathway of the exogenous antigens. The foreign agent is degraded by antigen presenting cells and the peptide fragments resulted are associated with class II MHC molecules and exposed at the surface of the cell. The peptide-MHC complex is recognized by a T-helper cell which binds to the complex.
T helper cell
Antigen presenting cell CD4
Antigen
Class II MHC-peptide complex T cell receptor
T helper cell
Antigen presenting cell CD4
Antigen
Class II MHC-peptide complex T cell receptor
An exogenous antigen, internalized by the antigen-presenting cells (macrophages, dendritic cells and B cells) by endocytosis is degraded into peptides while moving through several acidic, enzyme containing compartments, and is finally associated with class II MHC (major histocompatibility complex) molecules transported in vesicles from the Golgi complex. The class II MHC-peptide complex is ultimately transported to the plasma membrane and exposed at the surface of the cell (see Figure 2). The peptides combined with MHC class II are recognized by T helper cells displaying the coreceptor CD4. Virus-infected host cells and cancerous cells express endogenous antigenic proteins which are degraded within the endoplasmic reticulum into peptide fragments. After binding to class I MHC molecules the complex is transported to the cell membrane where it is recognized by T-cytotoxic cells displaying the coreceptor CD8.
1.1.2. Structure of antibodies and recognition of antigens
Antibodies are antigen-binding proteins present on mature B-cell membrane (monomeric IgM and IgD), and are secreted by the effector B cells as IgM, IgG, IgA, IgE. The secreted immunoglobulins each have identical amino acid sequences within the antigen binding site with the membrane bound immunoglobulin of the B-cell clone activated by the interaction with the antigen, but differ in the constant region as result of the class switch process (see Figure 1) [5]. Although IgG is the most abundant class in serum (about 80 % of the total immunoglobulin), each class has specific structural and functional properties.
As depicted in Figure 3a, immunoglobulin G consists of two types of polypeptide chains, a 25-kDa light (L) chain and a 50-kDa heavy (H) chain. Each heavy chain is linked to a light chain by a cystinyl-bond, and the two heavy chains are bound to each other by disulfide bridges and non-covalent interactions. The complete molecule adopts a conformation that resembles the letter Y. The light and heavy chains are folded in 2 and 4 globular domains comprising approximately 110 amino acids.
a) b)
The immunoglobulin domain consists of a pair of ß-sheets each built of antiparallel ß strands linked by a single disulfide bridge. The amino-terminal immunoglobulin domain on each of the light and heavy chain termed variable domain displays a higher variation of the amino acid sequence and contains three connecting loops at one end of the ß-sheet characterized by a hyper variability of the amino acids. The hypervariable loops are referred to as complementarity determining regions (CDRs) [8]. The CDRs of the variable domains from each pair of heavy and light chain, form a single surface located at the end of each arm which interacts with the antigen. The amino acid residues involved in the interaction with the antigen form the paratope [9].
The remaining immunoglobulin domains on each heavy and light chain are similar in all antibodies and are referred to as constant domains. A ribbon representation of the crystal structure of the non-covalent complex formed by the hen eggwhite lysozyme (HEL) and the Fab of the HyHEL5 antibody is shown in Figure 3b [10, 11].
Figure 3: Schematic representation of the immunoglobulin G and the antigen binding site. a) The heavy chain consists of 3 constant domains (CH) and a variable domain (CV) and is linked through a disulfide bridge to a light chain composed of a constant (CL) and a variable (VL) domain. Two heterodimers made of a heavy and a light chain are linked to form the IgG molecule. The CDRs are depicted according to the Kabat numbering [10];
b) Ribbon diagram from the crystal structure of the anti-HEL Fab shown in complex with the lysozyme. The hypervariable regions (CDRs) (yellow) located on the variable domains of the heavy (red) and light (blue) chain form the binding cleft which interacts with the antigen (Protein Data Bank [9] accession number 1YQV).
Antigen NH3+
-S-S- -S-S-
-S-S- -S-S- NH3+
COO-COO- COO-
Fc
VL
CL VH
CH1
CH2
CH3 COO-
Antigen binding domain
Fab 31-35
50-65 95-102 24-34
50-56 89-97
VH
CH1
VL
CL
CDRs
Antigen NH3+
-S-S- -S-S-
-S-S- -S-S- NH3+
COO-COO- COO-
Fc
VL
CL VH
CH1
CH2
CH3 COO-
Antigen binding domain
Fab 31-35
50-65 95-102 24-34
50-56 89-97
VH
CH1
VL
CL
CDRs
Antibodies recognize discrete sequences of amino acids located at the surface of the antigen called epitopes or antigenic determinants. To form an immune complex, non- covalent interactions (hydrogen bonds, ionic bonds, hydrophobic interactions, Van der Waals interactions) are established between the surfaces of the 2 molecules, complementary in their topology [12]. Approximately 90 % of the antibodies raised against intact proteins recognize a discontinuous epitope (see Figure 4) consisting of amino acid residues localized on different regions of the linear amino acid sequence of the antigen, that are folded together in the native molecule. Denaturation of the protein leads in most cases to the loss of antibody affinity. Only 10 % of the antibodies are estimated to interact with continuous epitopes that are composed of contiguous amino acid residues along the polypeptide chain [13].
Proteins with more complex structures have usually multiple antigenic determinants.
The x-ray crystallographic analyses of the immune complexes of 3 antibodies HyHEL-10, HyHEL-5 and D1.3 raised against the globular antigen hen egg-white lyzozyme revealed that all recognize discontinuous epitopes. While the HyHEL-5 and D1.3 interact with distinct epitopes consisting of 2 streches of polypeptide chains, the sites that interact with HyHEL-10 are more dispersed within the primary structure of the lyzozyme [14, 15].
The ability to induce a humoral immune response is called immunogenicity and depends on the capacity of the immune system to distinguish between self and nonself agents, molecular size of a foreign agent, its chemical composition and heterogeneity. In contrast to the immunogens, antigens are molecules that interact specifically with the immunoglobulins without having the ability to elicit an immune
H2N
COOH
continous epitope discontinous
epitope
H2N
COOH
H2N
COOH
continous epitope discontinous
epitope
D isco ntin u o us e pitop e
C ontinu ou s e pitop e
H2N
COOH
continous epitope discontinous
epitope
H2N
COOH
H2N
COOH
continous epitope discontinous
epitope
D isco ntin u o us e pitop e
C ontinu ou s e pitop e
Figure 4: Schematic representation of a continuous and a discontinuous epitope.
response. Upon immune challenge by a foreign agent, different B-cell clones, each with unique amino acid sequence and conformation of the combining site, are able to interact with the immunogen. Each clone interacts with a different sequence of amino acids on the surface of the immunogen and will secrete a specific antibody for this sequence. The affinity separation of the IgG molecules interacting with the immunogen produces a purified polyclonal antibody. In contrast, by fusion of an activated, antibody-producing B cell with a myeloma cell (a cancerous plasma cell) a hybrid cell is formed, called a hybridoma, that posseses the immortal-growth properties of the myeloma cell and secretes only the antibody produced by the activated B cell. The affinity separation of the IgG molecules using the immobilized immunogen will lead to a purified monoclonal antibody.
1.2. Analytical methods for epitope identification
In the past decade a variety of methods, including mass spectrometry-based approaches have been developed to provide structural information about antigen- antibody complexes. In particular, the elucidation of the fine structure of the epitopes provides a basis for the design of diagnostic tools and improved immunogens as lead structures in the development of vaccines [16-18]
One approach to the identification of the epitope is termed Pepscan and consists of the synthesis of overlapping peptide sequences that will be further scanned for the binding to the specific antibody by enzyme-linked immunosorbent assay (ELISA) [19- 22]. There are different strategies used for the synthesis of the peptides. The peptides can be synthesized in larger amounts on a resin, cleaved from the support and purified for the binding assay, or they can be synthesized directly on the surface on which the binding assay takes place (multi-pin synthesis) [23]. The first approach allows the use of fresh peptides for each test while the second implies the use of regenerated peptides after dissociation of the immune complex. The use of synthetic peptides for testing the reactivity to the antibody allows also the replacement of specific amino acids present in the antigen sequence with alanine providing the identification of critical amino acids (alanine scanning mutagenesis) [24, 25].
Alternatively, proteins containing alanine mutated amino acids are produced by expression [26, 27].
Phage display has been also successfully used for the identification of linear epitopes [28, 29]. The method is based on the incorporation of a nucleotide sequence encoding a foreign peptide into a phage genome as a fusion to a gene encoding a phage coat protein. This fusion ensures the display of the foreign peptide. Selection of the phage displaying the interacting peptide is achieved by exposing the phage particles to an immobilized antibody, removal of non-binding and non-specifically bound phages by several washes and recovery of the bound phages by acid elution.
The interacting peptide sequence is identified by sequencing the phage genome which contains the fusion gene coding the peptide.
To date X-ray crystallography is the most powerful technique for structure determination and analysis of proteins because it is capable of providing atomic coordinates of an entire assembly [30]. However, a critical step is the crystallization of the sample which requires large amounts of protein with high purity for screening a wide range of conditions (pH, temperature, salt, protein concentration) to find the appropriate conditions. In contrast NMR spectroscopy experiments require solubilisation of the protein in aqueous buffer under conditions similar to the physiological conditions. The major drawbacks of the method are the time consuming data collection and data analysis, the size constraints of the proteins and amount of sample required for analysis.
1.3. Identification of epitopes by proteolytic cleavage and mass spectrometry
Mass spectrometry has emerged as a widespread technique for the study of protein structure, function, quantity and interaction with other biomolecules. Important features of the mass spectrometric protein analysis are the high sensitivity, high mass accuracy, short analysis time and low sample consumption. To identify molecules within complex protein mixtures and to dissect the structure of the molecular recognition domains diverse applications have been developed in
conjunction with mass spectrometry. These methods include chromatographic and electrophoretic separations, proteolytic assays, and differential chemical modification of specific amino acid functions, sample preparation and bioinformatic tools for data analysis.
One of the most important applications of MS is that it provides structural identification of epitopes, unlike any of the other methods. The first attempts concerning the investigation of the antigenic determinant resulted by limited proteolytic cleavage of immune complexes were carried out by PAGE [31] and HPLC [32]. However both methods are unsuitable for unambiguous epitope identification. A general, molecular approach for identification of epitopes from peptide and protein antigens using mass spectrometry was developed by our laboratory [33, 34]. This method combines the advantage of the proteolytic stability of antibodies, and the shielding of the epitope with the unambiguous molecular identification provided by MS [16, 35, 36].
For epitope excision, the antigen is digested by proteolytic enzymes following the formation of the immune complex (see Figure 5). The epitope will be resistant to the fragmentation while the peptide sequences exposed to the enzymes will be cleaved.
Cleavage sites of the protease located inside the antigenic determinant that are not fragmented provide information on the epitope. The peptide fragments resulting after cleavage by the proteolytic enzyme as well as the epitope fragments collected after acidic dissociation of the complex are analyzed by mass spectrometry which is capable to provide unambiguous identification of the peptide sequences. In epitope extraction, the proteolytic fragmentation of the antigen in solution yields peptide sequences that might contain the intact epitope if the enzyme has no specificity for the amino acids contained by the antigenic determinant. The resulting peptide fragments are allowed to react with the antibody. Due to the high specificity of the antigen-antibody interaction only the peptides that contain the antigenic determinant in a similar conformation as in the intact protein will interact. The characterization of discontinuous epitopes is usually more difficult involving a combination of proteolytic epitope excision, chemical modification and mass spectrometry [37-41].
a) b)
The methodology for epitope excision using proteolytic cleavage of the unbound peptide sequences followed by mass spectrometric identification of the remaining affinity bound peptides can be employed for the identification of the binding partners of any isolated protein, and therefore, emerged as an important tool in the characterization of protein function [42]. The identification of the specific antigen from a complex mixture of proteins is achieved based on the data base search using the peptide masses determined from the elution fraction. An immobilized anti-troponin antibody and bovine heart cell lysate were used as model system.
A new, recent development, as an analogous method to the determination of epitopes has been the identification of antibody paratope structures using antigen columns containing the immobilized epitope. The antibody is exposed to the antigen column either after digestion in solution (proteolytic paratope extraction), or as an intact molecule that will be digested by a protease after the immune complex is formed (proteolytic paratope excision). The characterization of antibody-paratopes is
Complex formation
Proteolytic digestion
Intact
antigen Intact
antigen
Complex formation Proteolytic digestion Epitope excision Epitope extraction
Washing Dissociation
MS MS
Complex formation
Proteolytic digestion
Intact
antigen Intact
antigen
Complex formation Proteolytic digestion Epitope excision Epitope extraction
Washing Dissociation
MS MS
Intact
antigen Intact
antigen
Complex formation Proteolytic digestion Epitope excision Epitope extraction
Washing Dissociation
MS MS
Figure 5: The principle of mass spectrometric epitope identification. (a) For epitope excision the antigen is bound to the immobilized antibody and digested with proteases. Unbound peptides are washed off and the affinity bound peptides are dissociated from the antibody.
Both fractions are analysed by mass spectrometry. (b) In epitope extraction, the antigen is first digested in solution, and the proteolytic fragments are allowed to bind to the antibody.
considerably more difficult due to i) higher stability to proteolytic digestion of the antibody compared to the antigen, which makes difficult the application of the proteolytic excision of the paratope; ii) the fact that the antigen binding sites might be scattered within the 6 complementarity determining regions (CDRs) of the heavy and light chains and the individual sequences resulted by digestion in solution during epitope extraction might not display affinity to the antigen and iii) the lack of genomic data for most of the antibodies to provide fast identification of the paratope sequences by comparing the set of peptide masses obtained in the mass spectrum of the paratope elution with the theoretical masses from the sequence database. A first example of paratope identification by mass spectrometry was described for the camel anti-lysozyme antibody cAbLys3 (see Figure 6) [43]. Camel antibodies lack the light chains, therefore containing only three CDRs [44]. The CDR3 is significantly larger than those in human and mouse immunoglobulin and forms an exposed loop of 24 amino acids that fits into the active site cleft of lysozyme [45]. Mass spectrometric paratope identification was applied in this case for a 26 amino acid synthetic peptide containing the CDR3 sequence.
Although mass spectrometry has been an established technique in organic chemistry, the involatility of the macromolecules has limited the applications in the biological and medical field in the past. This difficulty has been overcome by the introduction of “soft”
ionization techniques for effectively dispersing proteins and other molecules into the gas phase with no or little fragmentation. The predominant methods are today i) matrix-assisted laser desorption and ionization (MALDI), and ii) electrospray ionization (ESI).
Lysozyme
cAbLys3
CDR3 Lysozyme
cAbLys3
CDR3
Figure 6: Ribbon representation of the non-covalent complex formed by cAbLys3 camel antibody and hen eggwhite lysozyme (HEL). The CDR3 region of the cAbLys3 is marked in yellow. The structure was prepared with BAllView 1.1 based on the crystal structure with the PDB accession number 1MEL.
In MALDI mass spectrometry applications, the analyte is co-crystallized with a large excess of a matrix. The matrix molecules are typically organic acids which have an absorption in the wavelength at which the laser is used (UV, visible or infrared) [46, 47]. Pulses of laser light (1-10 ns) are applied to the surface of the sample, causing desorption and ionization of the analyte-matrix mixture. The thermal-spike model proposes that the energy absorbed by the matrix molecules causes rapid heating of the irradiated layers of sample followed by evaporation of matrix together with the analyte molecules. The ionization of the analyte occurs probably subsequent to the ejection of the molecules from the support [48]. A schematic representation of the MALDI process is illustrated in Figure 7a. Typically, the mass spectrum of a sample ionized by MALDI contains singly charged molecular ions and ions of low charge states.
In contrast to MALDI-MS, electrospray ionization (ESI) sources operate at atmospheric pressure and provide the transfer of the ions present in liquid samples in the gas phase as isolated entities [49]. Analyte ions are generated by solubilisation in suitable solvents such as acidic aqueous solutions containing methanol or acetonitrile. The analyte solution is pushed through a very small metal capillary. The high electric field applied between the needle and the counter electrode forces the solution to emerge from the tip of the needle giving rise to the so called Taylor cone.
If the electric field is high enough small charged droplets form. The formation of fine droplets from the solution emerging from the needle is facilitated by a sheet flow of nitrogen gas. According to the “ion-evaporation” model, the solvent from each droplet evaporates yielding a higher charge density. When the Coulomb repulsion becomes of the same order as the surface tension, the droplet undergoes fissions producing smaller droplets that also evaporate. The process leads eventually to the formation of droplets containing a single ion. Ultimately fully desolvated ions result from complete evaporation of the solvent.
a)
b)
Figure 7: (a) Schematic representation of the ion formation in MALDI mass spectrometry; (b) Schematic representation of the electrospray process.
The advances in the development of ionization techniques led to an increasing interest in the technological improvements of time-of-flight, ion trap and Fourier transform mass spectrometers for applications in peptide and protein analysis. While FTMS combines all the high performance characteristics (accuracy, resolution, sensitivity), the high cost of magnets and maintenance and the complexity of operation have limited their widespread use to industrial laboratories; however several mass spectrometry research laboratories recently focused on extending the applications and performance of the FTICR-MS methods. Triple quadrupole, TOF and quadrupole ion trap mass spectrometers are three other types of mass analysers with widespread use primarily owing to their cost and ease to use.
nebulising gas
Solution with analyte
Atmospheric pressure
Taylor cone
Counter electrode
+ -
Skimmer
Vacuum stages
To the mass analyser nebulising gas
Solution with analyte
Atmospheric pressure
Taylor cone
Counter electrode
+ -
Skimmer
Vacuum stages
To the mass analyser To the mass
analyser A2
A3
A1
Analyt co-crystallized with matrix on sample
target
Laser shot
Desorption plume A1 A2
A3
H+
H+ H+ H+ H+
H+
H+
A2
A1
A3 H+ H+ H+
H+ H+ H+
H+
Matrix-analyt clusters
A2
A1
AH3+ H+ H+
H+ H+
H+ H+
Analyt ions Extraction grid
+ 20 kV
To the mass analyser A2
A2
A3
A3
A1 A1
Analyt co-crystallized with matrix on sample
target
Laser shot
Desorption plume A1 A1 A2
A2
A3 A3
H+
H+ H+ H+ H+
H+
H+
A2 A2
A1
A1
A3 A3 H+
H+ H+
H+ H+ H+
H+
Matrix-analyt clusters
A2 A2
A1
A1
A3 AH3+
H+ H+
H+ H+
H+ H+
Analyt ions Extraction grid
+ 20 kV
1.4. Pathophysiological characteristics and therapeutic perspectives of Alzheimer´s Disease
“She sits on the bed with a helpless expression. What is your name? Auguste. Last name? Auguste. What is your husband’s name? Auguste, I think.” [50]
It has been already a century ago since the German psychiatrist Alois Alzheimer presented the case of Auguste D., a 51-year-old lady who had shown progressive loss of cognitive functions and psychosocial competence. A. Alzheimer described for the first time the clinical picture of presenile dementia as well as the histological findings of amyloid plaques, neurofibrillary tangles and arteriosclerotic changes.
Alzheimer´s disease is clinically characterized by a progressive decline of cognitive functions from mild forgetfulness and cognitive impairment, to widespread loss of memory, language and logical thinking having impact on the ability to perform everyday activities and changing the patient’s behavior. Death occurs, on average, 10 years after the diagnosis. In addition to its direct effects on patients, advanced AD loads a tremendous burden on family caregivers and causes substantial nursing costs for the society [51]. Due to the increase of life expectancy of the population, the absolute number of people afflicted by AD is expected to grow substantially. It is estimated that there are currently 26 million people worldwide suffering of Alzheimer´s disease, and the global prevalence is expected to increase to more than 100 million by 2050.
Current medications approved for the treatment of Alzheimer´s disease are based on the modulation of neurotransmission. Acethylcholinesterase (AchE) inhibitors attempt to address the cholinergic deficits seen in AD and are used for mild to moderate cases. Memantine an (N-methyl-D-aspartate)-receptor antagonist that has been used for the treatment of moderate to severe Alzheimer dementia aims to prevent the neuronal excitotoxic effect exerted by high levels of glutamate. Although producing moderate symptomatic improvements of the cognitive function, none of these drugs appears to be able to cure Alzheimer´s disease [52].
Hence, an enormous need exists for the development of new medications for AD with strong disease-modifying properties, and research is focused on the development of new therapeutic strategies that target the underlying pathogenic mechanisms of Alzheimer´s disease.
A comparative examination of the brains from AD patients and normal elderly individuals reveals a dramatic loss of brain tissue [53]. Shrinkage of the brain is extremely severe in the hippocampus, temporal and parietal lobes and is mainly observed in the widened cortical sulci and ventricular dilatation as depicted in Figure 8b. The histopathological hallmarks of Alzheimer´s disease are loss of cholinergic and glutamatergic neurons, intracellular and extracellular deposits of proteins and microvascular angiopathy. Many neurons in the brain regions typically affected in AD contain abnormal protein deposits called neurofibrillary tangles that occupy much of the perinuclear cytoplasm (see Figure 8a). The neurofibrillary tangles consist of microtubule-associated protein Tau in abnormally phosphorylated form [54]. The in vitro phosphorylation of tau has been reported to inhibit the polymerization of tubulin [55] into the microtubules. Microtubules are crucially important structures which run through the cell and are involved in axonal transport, synaptic transmission, cell support and shape.
a) b)
Normal Alzheimer´s Disease Normal Alzheimer´s Disease
Normal Alzheimer´s Disease Normal Alzheimer´s Disease
Figure 8: Pathophysiological characteristics of Alzheimer´s Disease compared with a healthy individual: a) neurofibrillary tangles and amyloid plaques; b) brain cross section showing atrophy of the brain tissue affecting predominantly the language and memory lobes. Copyright © 2000-2009 American Health Foundation. All rights reserved.
The extracellular deposits are referred to as neuritic or senile plaques and consist of aggregated amyloid-ß protein [56] surrounded by astrocytes and neurites emanating from local neurons. Microvascular angiopathy caused by the deposition of amyloid-ß protein on the walls of the arterioles and venules was found outside the brain as well as within the cerebral cortex of the brains from patients with Alzheimer´s disease [57, 58]. Cerebral amyloid angiopathy can lead to hemorrhages which may contribute to the cognitive decline [59]. The hypothesis that states the fundamental role of the overproduction and accumulation of Aß in senile plaques in the pathology of AD has been extesively studied in the last two decades. An overview concerning the origin of amyloid-ß protein and the accumulation in senile plaques as well as the main therapeutic strategies that are currently pursued will be discussed in the following sections of the introduction. In Alzheimer´s disease, excessive activation of NMDA receptors by L-Glutamate (L-Glu) is believed to cause elevated cytosolic Ca2+ which then initiates pathological events that ultimately lead to neurodegeneration [52, 60].
Amyloid-ß (Aß) was first sequenced from the meningeal blood vessels of AD patients and individuals with Down syndrome by George G. Glenner [58, 61] and then identified in the senile plaques [62]. Aß is proteolytically cleaved from the amyloid precursor protein (APP) that contains a single transmembrane domain, with a longer N-terminal amino acid sequence emanating out of the cell and a shorter C-terminal domain jutting into the cytosol. APP is encoded by a gene located on the chromosome 21 [63-65] and although is produced by many cells and tissues its precise biological role has remained unknown. Several forms of APP that differ mainly at the amino-terminal end of the sequence have been described to arise by alternative splicing: APP-695, APP-751 and APP-770 [66-68]. The enzymes that play a central role in the proteolytic processing of APP are α-, β- and γ-secretases (see Figure 9). The proteolytic cleavage by α-secretase occurs 12 amino acids NH2- terminal to the transmembrane domain and releases a large soluble fragment (α- APPs) into the extracellular space. The 83-amino acid residue COOH-terminal fragment is retained in the membrane and is further cleaved by γ-secretase, generating the p3 peptide fragment and a 57/59 amino acid residue carboxy-terminal fragment (CT57/59). Alternatively, APP is cleaved 16 amino acids N-terminal to the α-secretase cleavage site by ß-secretase releasing ß-APPs into the extracellular space and retaining a 99-amino-acid residue in the membrane. The cleavage by γ-
secretase produces a 40/42 peptide fragment referred to as Aß and the CT57/59.
The α-secretase activity was described to be exerted by three related metalloproteases of the ADAM (a disintegrin and metalloprotease) family, ADAM-9 [69], ADAM-10 [70] and ADAM-17 [71]. Two aspartyl enzymes responsible for the ß- secretase cleavage have been identified in 1999 referred to as BACE (ßAPP cleaving enzyme) [72-75] and BACE-2 [76, 77]. BACE activity can also generate fragments of APP cleaved at secondary sites such as Glu11 within the Aß sequence [78]. The second enzymatic activity required for Aß generation is exerted heterogeneously by γ-secretase. Most of the full-length Aß species produced is a 40- residue peptide (Aß40), whereas a smaller proportion is a 42-residue carboxy- terminal form.
Figure 9: Proteolytic processing of APP. The conjoint cleavage of APP by α- and γ-secretase produces the harmless fragment p3, the carboxy-terminal C57/59 and the longer soluble α-APP; Alternatively the cleavage by ß- and γ-secretase releases the 40/42 residues long fragment called Aß that is prone to aggregation.
Under normal circumstances, Aß generated in the CNS is cleared with a half-life of 1- 2 h [79]. Initially, Aß42 which is more prone to aggregation than Aß40 is deposited in diffuse (nonfibrillar) plaques with little or no detectable neuritic dystrophy. The mechanism through which the aggregated Aß exerts its toxic effects is still controversial. It was shown that aggregated forms of synthetic Aß peptides can
H2N COOH APP770
Kunitz protease inhibitor domain
Ox-2 antigen domain
289 345 364 672 713
α
β γ
sAPPα C83
p3
sAPPβ C99
αααα-secretase
672DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA713
β α γ γ
γγγγ-secretase γγγγ-secretase
ββββ-secretase
H2N COOH APP770
Kunitz protease inhibitor domain
Ox-2 antigen domain
289 345 364 672 713
α
β γ
H2N COOH APP770
Kunitz protease inhibitor domain
Ox-2 antigen domain
289 345 364 672 713
α
β γ
sAPPα C83
p3
sAPPβ C99
αααα-secretase
672DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA713
β α γ γ
γγγγ-secretase γγγγ-secretase
ββββ-secretase
cause damage to cultured neuronal cells [80, 81]. However, more recent findings suggest that soluble oligomeric prefibrillar forms of Aß may represent the neurotoxic species that causes neurotoxicity and synaptic dysfunction [82, 83].
The dominating hypothesis concerning the mechanisms leading to Alzheimer’s disease assigns a central role of the accumulation of Aß in brain to the initiation of a cascade of pathological events that ultimately lead to neurodegeneration and dementia [52, 84]. A first argument supporting a causal role of Aß in Alzheimer´s disease came from the identification of the APP gene locus on chromosome 21 and the earlier finding that individuals affected by Down syndrome posses 3 copies of the chromosome 21 and develop invariably AD pathology. Additionally, several mutations that are responsible for early-onset forms of familial Alzheimer’s disease (FAD) have been identified in the APP gene. These mutations are located directly adjacent close to the ß- and γ-secretase cleavage sites favoring the proteolytic processing of APP and leading to increased Aß production. Mutations in the genes encoding PS1 and PS2 are also responsible for elevated production of Aß. Increased Aß plaque deposition in brain has been associated with the presence of apolipoprotein E (apoE)-protein although the mechanism remains unknown [85]. Recent data showed a reduction of Aß deposition in the offspring by crossing APP overexpressing transgenic mice with apoE-deficient mice [86].
1.5. Immunotherapeutic strategies for Alzheimer´s Disease
A primary goal of research on Alzheimer´s disease is to develop therapeutics able to prevent or reverse the cognitive decline of AD patients. As discussed in the previous section the most studied hypothesis emphasizes the neurodegenerative effect exerted by amyloid deposits. Based on the current knowledge provided by these studies, the development of anti-Aß therapeutics appears as a rational approach for treatment.
Present approaches are focused on (i) the modulation of Aß production, (ii) preventing Aß aggregation and (iii) clearance of soluble Aß and amyloid deposits from brain (see Figure 10). The attempt to reduce the production of Aß led to the
development of potent inhibitors that block the activity of ß- and γ-secretase.
Although the inhibiting compounds have been tested in mice and were effective in reducing Aß, testing in humans has been barely attempted due to the concern regarding potential side effects. Preventing the formation of Aß aggregates has been attempted by chelating the metal ions (Zn2+, Cu2+) reported to enhance Aß deposition [87-89].
The idea of using the ability of the immune system to produce specific antibodies that recognise soluble Aß or amyloid deposits and lead to their clearance from brain has gained increasing interest in recent years. Several immunotherapeutic strategies have been under investigation including active immunization with synthetic Aß, protofibrillar Aß assemblies or Aß peptide fragments conjugated to a carrier protein, and passive immunization with monoclonal Aß-specific antibodies [90, 91]. The studies carried out by B. Solomon and collaborators provided first evidence that antibodies recognizing Aß were effective in blocking the formation of amyloid fibrils in vitro [92], dissolving pre-existing amyloid fibrils [93] and preventing neurotoxicity of Aß fibrils. These observations performed in vitro were followed by the immunization of transgenic mice overexpressing APP with Aß. The first results of immunization with Aß42 showed a dramatic reduction of fibrillar Aß deposition in young PDAPP
APP
Aß ß γ
Aß
Aß Amyloid plaque Production
Clearance
Aggregation
Clearance ß, γ-secretase
inhibitors
Aggregation inhibitors
Immunotherapy Immunotherapy
Reduced levels of Aß in brain APP
Aß ß γ
Aß
Aß Amyloid plaque Production
Clearance
Aggregation
Clearance ß, γ-secretase
inhibitors
Aggregation inhibitors
Immunotherapy Immunotherapy
Reduced levels of Aß in brain
Figure 10: Schematic presentation of the therapeutic strategies aiming to prevent and reverse the pathological events leading to amyloid plaque formation.
transgenic mice, and reduction or even reversal of amyloid deposition if the immunization was performed in older animals [94]. A correlation between plaque reduction and the ability to perform memory tasks was not possible due to the observation that in PDAPP mice cognitive impairment precedes amyloid deposition [95]. However, improvements in cognitive function associated with plaque reduction were described in two parallel studies in which TgCRND8 transgenic mice were immunized with protofibrillar Aß42 [95] and APP/PS1 mice with Aß42 [96]. Both studies did not observe any adverse effect of the vaccine. On the basis of the promising preclinical findings, a first clinical trial was initiated in which 300 patients received active immunisation with AN1792 (Aß42 and adjuvants). Unfortunately, 6%
of the patients developed symptoms of meningoencephalitis and the trial was suspended [97]. At present, the inflammatory response encountered is attributed to the infiltration of the brain with activated T cells [98, 99]. However, the patients with high anti-Aß titers had significantly less deterioration of cognitive performance in the year following the clinical trial, than patients with little or no anti-Aß antibodies [100, 101].
In recent years, the immunotherapeutic studies have been concentrated on the development of passive immunization strategies. The intravenous administration of amyloid specific antibodies might offer potential advantages over the active immunization approach: (i) avoid the variability of the immune response across the individuals receiving the immunogen by providing known amounts of antibody of known epitope specificity; (ii) do not trigger T cell activation; and (iii) can be withdrawn if adverse reaction are encountered [102]. A number of controversies have arisen regarding the antibody specificity for passive immunization studies. While Bard et al. showed that only N-terminal domain antibodies are able to clear amyloid plaques [103], DeMattos et al. argue that an antibody to the Aß mid-domain with little reactivity to brain amyloid might be effective [104]. Alternatively, Morgan et al.
reported that passive administration of antibodies specific to a carboxy terminal domain of Aß was able to reverse cognitive deficits in transgenic mice.
Several mechanisms have been proposed to explain the therapeutic action of anti- amyloid antibodies (see Figure 11). The plaque breakdown hypothesis relies on the ability of a small amount of Aß-antibodies (0.1%) to cross the blood-brain barrier into
