Multiple challenges in protein structure determination by X-ray
crystallography: Four ventures, one structure solved
Dissertation
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften
an der Universität Konstanz Fachbereich Biologie
vorgelegt von
Stefanie Fischer, geb. Diehl
Tag der mündlichen Prüfung: 13.11.2009
Referent/in: Prof. Dr. Wolfram Welte
Referent/in: Prof. Dr. Iwona Adamska
Contents
Zusammenfassung 1
I. A monoclonal antibody against the nAchR of Torpedo californica as crystallization tool 4
I.1. Introduction 5
I.1.1. Function and assembly of the nicotinic acetylcholine receptor . . . 5
I.1.2. Relevance of monoclonal antibodies for crystallization . . . 6
I.1.3. Objective . . . 8
I.2. Materials & Methods 9 I.2.1. Media and buffers . . . 9
I.2.2. Animals . . . 10
I.2.3. Determination of cell density and vitality . . . 10
I.2.4. Storage of adherent eukaryotic cells . . . 10
I.2.5. Production of monoclonal antibodies against the nAchR of Torpedo californica . . . 11
I.2.5.1. Immunization of mice . . . 11
I.2.5.2. Cultivation of myeloma cells . . . 11
I.2.5.3. Preparation of murine peritoneal macrophages as feeder cells . . . 11
I.2.5.4. Harvesting of B cells and preparation . . . 12
I.2.5.5. Cell fusion . . . 12
I.2.6. Monitoring and selection of hybridoma cells . . . 13
I.2.7. Indirect ELISA to detect specific antibodies . . . 14
I.2.8. Sub-cloning of positive hybridomas . . . 16
I.2.9. Isotype definition of monoclonal antibodies . . . 16
I.2.10. SDS polyacrylamide gel electrophoresis (SDS-PAGE) . . . 17
I.2.11. Characterization of epitope binding of monoclonal antibodies by West- ern Blot . . . 19
I.3. Results 20 I.3.1. Immunization . . . 20
I.3.2. Harvesting of splenocytes and preparation . . . 20
I.3.3. Fusion . . . 21
I.3.4. Confirmation of specific antibodies against the nAchR by ELISA and sub-cloning . . . 21
I.3.5. Isotype definition of monoclonal antibodies . . . 21
I.3.6. Characterization of epitope binding of the monoclonal antibody by Western Blot . . . 22
I.4. Discussion 26 I.4.1. Generation and characterization of mAbs against the nAchR of Tor- pedo californica . . . 26
I.5. Perspective 29
II. Structure and function of the β-N-acetylglucosaminidase BsNag3A of Bacillus subtilis 31
II.1. Introduction 33 II.1.1. The cell wall and its recycling in gram-positive bacteria . . . 33II.1.2. Classification of glycohydrolases . . . 37
II.2. Crystallization 39 II.2.1. Growth of protein crystals . . . 39
II.2.2. Sitting drop and hanging drop technique . . . 40
II.2.3. Features of protein crystals . . . 42
II.3. Introduction to x-ray structure analysis 44 II.3.1. Crystal systems . . . 44
II.3.2. Bravais lattices . . . 44
II.3.3. X-radiation . . . 45
II.3.4. Diffraction at protein crystals . . . 45
II.3.5. The Ewald sphere and reciprocal lattice . . . 47
II.3.6. Calculation of the electron density and phase shift . . . 47
II.3.7. Crystallographic and free R factor . . . 49
II.3.8. Computer programs for structure analysis and phase determination . 50 II.3.9. Validation of X-ray protein structures . . . 51
II.3.10. Data recording - X-ray sources . . . 52
II.4. Results 55 II.4.1. Structure of BsNag3A with and without the inhibitor PUGNAc . . . 55
II.4.2. The active site and inhibitor binding . . . 56
II.4.3. Comparison of BsNag3A with the structurally homologue enzymes ExoI and NagZ . . . 59
II.5. Discussion 63
III. Analyses of the Lysin-N
6-hydroxylase from E.coli con- cerning stability and crystallization 67
III.1. Introduction 68
III.1.1. Iron uptake strategies of microorganisms . . . 68
III.1.2. Siderophores . . . 69
III.1.3. Aerobactin . . . 70
III.1.4. Lysine-N6-hydroxylase from E.coli . . . 71
III.1.5. Objective . . . 72
III.2. Material & Methods 74 III.2.1. Bacterial strains and plasmids . . . 74
III.2.2. Media and growth conditions . . . 75
III.2.3. Growth conditions . . . 75
III.2.4. Cell density of bacteria cultures . . . 75
III.2.5. Construction of bacteria strains . . . 76
III.2.6. Preparation, enzymatic digestion and sequencing of DNA . . . 76
III.2.7. Creation of competent bacterial cells . . . 76
III.2.8. Bacterial transformation . . . 77
III.2.9. Transformation rate . . . 77
III.2.10. Polymerase chain reaction (PCR) . . . 77
III.2.11. Oligonucleotides . . . 77
III.2.12. Analysis of expression . . . 78
III.2.13. Western Blot . . . 78
III.2.14. Overexpression and purification of the LH . . . 78
III.2.15. Detection of inclusion bodies . . . 80
III.2.16. Bradford protein assay . . . 80
III.2.17. Activity assay . . . 80
III.2.18. Dynamic light scattering (DLS) . . . 81
III.2.19. Mass spectrometry . . . 82
III.2.20. Crystallization . . . 82
III.3. Results 83 III.3.1. Polymerase chain reaction . . . 83
III.3.2. Sequencing of DNA plasmids after PCR . . . 83
III.3.3. Creation of competent bacterial cells/transformation rate . . . 83
III.3.4. Detection of inclusion bodies . . . 83
III.3.5. Overexpression and purification of the LH . . . 84
III.3.6. Activity assay . . . 85
III.3.7. Crystallization and X-ray diffractometry . . . 87
III.3.8. Mass Spectrometry . . . 87
III.4. Discussion 88
IV. Overexpression and purification of E587-antigen Ig I-
IV from Carassius auratus 91
IV.1. Introduction 92
IV.1.1. Neural cell adhesion molecules (NCAMs) . . . 92
IV.1.2. E587 antigen, a member of the Ig superfamily . . . 92
IV.2. Material & Methods 95 IV.2.1. Eukaryotic cells, bacteria strains and plasmids . . . 95
IV.2.2. Culture media, buffers and additives . . . 95
IV.2.3. Growth conditions and cell culturing . . . 96
IV.2.4. Cell density and vitality of S2 cells . . . 96
IV.2.5. Polymerase chain reaction . . . 97
IV.2.6. Enzymatic engineering and preparation of transfection plasmids . . . 98
IV.2.7. Propagation, maintenance and sequencing of transfection plasmids . . 99
IV.2.8. SDS-PAGE . . . 99
IV.2.9. Western Blot . . . 99
IV.2.10. Transient transfection of S2 cells for test expression . . . 100
IV.2.11. Stable transfection of S2 cells . . . 100
IV.2.12. Tunicamycin assay to inhibit N-glycosylation of E587-antigen-Ig I-IV 100 IV.2.13. Overexpression and purification of the soluble fragment IgI-IV of E587-antigen . . . 101
IV.2.14. Crystallization and X-ray diffraction . . . 102
IV.3. Results 104 IV.3.1. PCR, enzymatic engineering and preparation of transfection plasmids 104 IV.3.2. Transient transfection of S2 cells for test expression . . . 104
IV.3.3. Stable transfection of S2 cells and inhibition of N-glycosylation by tunicamycin . . . 105
IV.3.4. Overexpression and purification . . . 106
IV.4. Discussion and Outlook 109
V. Appendices 113
List of Abbreviations 114
Bibliography 119
Acknowledgements 133
Zusammenfassung
In der Natur stellen die Proteine neben der DNA und RNA die Grundbausteine aller Zellen dar. Sie übernehmen im Organismus vielfältige Aufgaben; so verleihen sie nicht nur den Zellen ihre Struktur und Stabilität, sondern sind vielmehr Maschi- nen auf molkularer Ebene. Sie sind beispielsweise zuständig für Transportprozesse, Abwehr, Katalyse, Pumpvorgänge und die Erkennung von Signalstoffen. Proteine sind somit ein Gerüst des Lebens und schon ihr Name räumt ihnen ihre Bedeutung an diesem ein. Das Wort "Protein" wurde 1838 von Jöns Jakob Berzelius von dem griechischen Wort "proteuo", "ich nehme den ersten Platz ein" abgeleitet.
Aber so sehr Proteine dem Leben zuträglich sind, so großen Schaden können sie auch verursachen, zum Beispiel in Form von Toxinen, pathogenen Proteinstrukturen oder durch Veränderung ihrer genetischen Grundlage. Der Bauplan jeden Proteins findet sich in seinem Gen, genauer der DNA. Kommt es zu Mutationen in einem Gen, kann es zu schwerwiegenden Veränderungen im Aufbau und in der Funktion des Proteins kommen. Die Folge sind oftmals verheerende Störungen, die den gesamten Orga- nismus betreffen können, und die als spontane oder erbliche Krankheiten letztlich ihre Ausprägung finden. Dies ist zum Beispiel der Fall für das neuronale Zelladhä- sionsmolekül L1. Mutationsbedingte Veränderungen führen hier zum sogenannten CRASH-Syndrom (siehe Abschnitt IV.I.2), das die Lebensqualität der Betroffenen stark beeinträchtigt. Ein Projekt dieser Arbeit war, für die Ig Domänen I-IV des L1 Orthologs aus dem Goldfisch, E587-antigen, ein geeignetes Expressionssystem zu finden, durch welches das Proteinfragment sowohl in ausreichender Menge als auch in nativer Faltung zeitnah exprimiert wird für spätere Kristallisationsversuche. Im Zuge einer vorangegangenen Promotion wurde bereits versucht das Proteinfragment in "inclusion bodies" zu exprimieren, um es anschließend in einer speziellen Puffer- kombination nativ rückzufalten. Doch alle Versuche schlugen bis dato fehl. Von der Struktur von E587-antigen Ig I-IV erhofft man sich Rückschlüsse auf den homologen Bereich in L1 und Aussagen über dessen ausgeprägte "mutation sites".
Ein weiteres Beispiel für eine auf Proteinebene gravierende Störung im Organismus ist die Autoimmunkrankheit Myasthenia gravis (siehe Abschnitt I.I.X). Angriffs- punkt dieser Krankheit ist der humane nikotinische Acetylcholinrezeptor, der eine bedeutende Rolle in der Signaltransduktion spielt. Durch die Bindung körpereigener Antikörper an den Rezeptor wird dieser markiert und somit sein Abbau getrig- gert. Die Betroffenen leiden unter neuronalen und motorischen Ausfallerscheinungen sowie fortschreitender Muskeldegeneration. Die dreidimensionale Struktur des Pro- teins könnte ein besseres Verständnis für die in der Krankheit ablaufenden Prozesse bringen, genauso wie die Möglichkeit, gezielt bindende Medikamente zu entwickeln ("rational drug design"), die schützenden oder inhibierenden Einfluss auf bestimmte körpereigene Strukturen ausüben. Bisher konnte die Struktur des Rezeptors nicht in
Zusammenfassung
atomarer Auflösung für derartige Aussagen aufgeklärt werden. Bis heute existiert lediglich eine recht grobe Struktur bei 4 Å, siehe [Unw03]. Um die Kristallisa- tionseigenschaften des transmembranären Rezeptors zu verbessern, sollte innerhalb dieser Arbeit versucht werden, monoklonle Antikörper zu generieren. Diese sollten gezielt an den extrazellulären hydrophilen Bereich des nAchR binden, ihn dadurch vergrößern und somit für bessere Kristallkontakte zwischen den Proteinmolekülen und verbessertes Kristallwachstum sorgen.
Zwei weitere Projekte dieser Dissertation beschäftigen sich mit dem pathogenen Charakter zweier bakterieller Proteine. Zum einen handelt es sich um die Lysin- N6-Hydroxylase des Gram-negativen Organismus E. coli. Sie ist an der Biosynthese des Siderophor-Rezeptors Aerobactin beteiligt. Siderophore dienen vielen Mikroor- ganismen zur Aufnahme von Eisen, das für ihren Stoffwechsel essentiell ist (siehe Abschnitt III.I.). Hemmt man diesen Mechanismus, zum Beispiel durch Inhibition der Lysin-N6-Hydroxylase, kann kein Stoffwechsel, kein Wachstum und keine Ver- mehrung mehr stattfinden. Der pathogene Organismus kann eliminiert werden. In zahlreichen Arbeiten im Vorfeld wurde auf unterschiedlichen Wegen versucht, die LH zu kristallisieren. Doch nichts führte zur Kristallisation. In dieser Arbeit wurde durch Mutation von großen oberflächenexponierten Aminosäure-Seitenketten hin zu kleinen Seitenketten versucht, die Kristallisation zu erreichen. Darüber hinaus wurde die Ursache des Nicht-Kristallisierens untersucht.
Das zweite Protein, die β-N-Acetylglucosaminidase BsNag3A, stammt aus dem Gram-positiven Organismus B.subtilis. Große Aufnahmemengen dieses Organismus führen beim Menschen zu Übelkeit und Erbrechen. BsNag3A ist beim Zellwand- abbau und -recycling beteiligt; es spaltet die β-1,4-glykosidische Bindung zwis- chen GlcNAc und MurNAc des Mureins. Die Inhibition dieses Proteins würde eine Vermehrung von B.subtilis verhindern und somit das Pathogen unschädlich machen. Darüber hinaus kann die Bakterienzelle die Menge an Mureinabbaupro- dukten messen. Letztere akkumulieren unter Stress und gehemmtem Wachstum wie zum Beispiel unter Penicillin-Behandlung. Eine intrazelluläre Signalkaskade triggert daraufhin die Expression der β-Lactamase AmpC. Diese baut viele Cephalosporine der dritten Generation (Antibiotika) ab und sorgt damit für die Resistenz des Or- ganismus gegenüber diesen Wirkstoffen. Die beschriebene Kaskade ließe sich je- doch ausschalten, durch Inhibition des Mureinabbaus, zum Beispiel durch Hem- mung vonBsNag3A. In dieser Arbeit konnte das Protein auf unterschiedliche Arten kristallisiert und dadurch zwei seiner Konformationen gelöst werden. Zum einen das Protein alleine, zum anderen im Komplex mit dem effektiven Inhibitor PUGNAc.
Die beiden Strukturen ermöglichen Aussagen zum aktiven Zentrum und zum Mech- anismus des Proteins.
Aufgrund der genannten Exempel ist die Strukturaufklärung von Proteinen für mehrere Bereiche von Bedeutung: für die Grundlagenforschung, die forschende phar- mazeutische Industrie oder auch für behandelnden Mediziner, die Aufklärungsarbeit betreiben und Wirkweisen von Medikamenten erläutern müssen.
Allerdings ist es alles andere als trivial, die Struktur von Proteinen zu lösen. Auf dem
2
langen Weg von der Idee über die Expression des Proteins, seine Reinigung, Kristalli- sation und letztlich die röntgendiffraktometrischen Untersuchungen gilt es zahlreiche Hürden ("bottlenecks") zu überwinden. So kann es zu Problemen bei der Expres- sion des Proteins kommen (unzureichende Mengen, inclusion bodies, etc.) oder eine ausreichende Stabilität des Proteins ist nicht gegeben genauso wie eine schlechte bis nicht vorhandene Kristallisation des selbigen. Aus diesem Grund wurde immer wieder nach vielfältigen neuen Möglichkeiten und Hilfsmitteln gesucht, die "bottle- necks" zu umgehen oder abzumildern. Die hier vorliegende Arbeit spiegelt die Meth- odenspektren zur Überwindung der "bottlenecks" wider. Sie umfasst neue Wege zur Proteinexpression und -kristallisation, die Untersuchung der Nicht-Kristallisation eines Proteins, aber auch letztlich die erfolgreiche Kristallisation und Lösung zweier Strukturen eines Proteins.
I. A monoclonal antibody against the nAchR of Torpedo californica as
crystallization tool
4
I.1. Introduction
I.1.1. Function and assembly of the nicotinic acetylcholine receptor
During the signal transduction between nerve cells and their target cells, e.g. the neuromuscular end-plate, neurotransmitter molecules are released in the region of the chemical synapse. The transmitter diffuses to the target cell membrane and binds to transmitter-gated ion channels. The nAchR is up to now the best studied ligand-gated ion channel among them [Hil08]. During quiescent state its confor- mation is closed. Upon binding of the neurotransmitter acetylcholine the recep- tor opens transiently for the passage of selected monovalent cations, mainly Na+ and K+. Due to the concentration gradient the cations diffuse into the cells of the motor end plate. Within these cells the electric potential is decreased by the cation influx. Voltage-gated ion channels can open if the decreasing electric po- tential reaches a certain threshold, thus causing a depolarisation and forwarding of a postsynaptic excitation signal. The latter can result in muscular contraction or further saltatoric depolarisation at myelinated nerve fibres. The nAchR is a highly effective channel and can precisely transmit electric signals within neuronal assem- blies. The acetylcholine-driven signal persists until either transmitter degeneration by the acetylcholine esterase sets in, or a temporary receptor desensitisation occurs within∼20ms [Unw03] upon long-lasting presynaptic signalling [Mat92]. Moreover already 20µs after acetylcholine binding several thousands of selected ions can pas- sage the receptor within one millisecond. On the other hand during resting state, the channel represents an effective barrier against concentration dependent ion influx into the cell. The hitherto knowledge of function and composition of the nAchR has been mainly gained by receptors stemming from the electric cytes of torpedo fish.
Their electric organs consist of reorganized muscle tissue, containing vast amounts of the receptor. For molecular investigations of these organs/receptors tissue of the species Torpedo californica and Torpedo marmorata has been established.
The receptor is a pentameric glycoprotein of 290 kDa, whereas ∼ 20 kD refer to its glycosylations comprising different sugar chains. It is assembled from a ring of heterologous subunits: α,γ, α,β and δ [Kar02] [Cor00] and belongs to the pLGICs superfamily, which includes neuronal Ach receptors, GABAA receptors, 5-HT3 re- ceptors and glycine receptors. Its subunit assembly, however, can vary according to tissue, species and developmental stadium of an organism. Furthermore, the nAchR can be divided into three domains: a large N-terminal extracellular ligand-binding domain, a cation-selective membrane-spanning pore and a smaller intracellular do- main [Unw05]. The total length of this assembly is∼160Å normal to the membrane plane. The ligand-binding domain forms a long central vestibule with a diameter of
I.1. Introduction
∼ 20 Å and possesses two binding pockets for Ach. The latter are located in the α subunits on opposite sides of the pore about 40 Å from the membrane surface.
The pore displays a narrow path across the membrane and contains the gate, which opens when Ach occupies both receptor binding sites [Unw03] [Unw05]. The intra- cellular domain contains a smaller vestibule with narrow lateral openings [Miy99]
which act as electrostatic filters for the ions [Miy99] [Kel03]. Concerning the nAchR of mammalian muscle it is reported that the receptor appears to be the target of an antibody-mediated autoimmune response, which causes the disease myasthenia gravis [Kon96]. The autoantibodies decrease the number of nAchR in the postsy- naptic membrane of the muscle end plate. This blockage of the receptor function results in failure of the neuromuscular transmission [Lin88] and leads to muscular weakness and fatigue [Dra94]. This disease led to further research interest in this ligand-gated ion channel.
Figure I.1.1.: Structure of the nicotinic acetylcholine receptor
Ribbon diagrams of the whole receptor, as viewed (a) from the synaptic cleft and (b) parallel with the membrane plane [Unw03]. Only the ligand-binding domain is highlighted in (a) and only the front two subunits are highlighted in (b) for clarity (α, red;β, green;γ, blue;δ, light blue).
I.1.2. Relevance of monoclonal antibodies for crystallization
More than 55.000 structures (as of January 2009) are currently deposited in the Protein Data Bank (PDB), but not just yet 100 of them are membrane protein structures. This displays a significant increase in knowledge compared to eight years ago, when only a handful of membrane protein structures were available [Rea07].
Many advances in protein crystallography and structure analysis have been made over the past few years, but nevertheless the understanding of structure-function relationships still has to be enlightened for many membrane proteins. This is of
6
I.1.2. Relevance of monoclonal antibodies for crystallization
Figure I.1.2.: Fab attachment and crystal packing. On the left figure KcsA (yellow) was crystallized as a complex with an antibody Fab fragment (blue). View down the four-fold crys- tallographic axis of the /4 cell, which corresponds to the molecular four-fold axis of the K+chan- nel [Hun02a]. On the right hand side is the overall structure of the complex between the redox partners cytochrome c (yellow) and cytochrome bc1 complex with an antibody fragment (orange) bound to the catalytic domain. [Zho01]
particular importance for clinically relevant proteins.
Concerning the structure analysis of membrane proteins two main bottlenecks have to be fought. First, the purification of sufficient amounts of protein in detergent is crucible and secondly, the arrangement of protein into a well-ordered three- dimensional crystal. Crystallization of membrane proteins is difficult because of their amphipathic surface, the hydrophobic part of the protein resides in the mem- brane or is covered in a belt-like manner by the detergent micelle after purification.
The hydrophilic surfaces of membrane proteins are in general exposed to the aqueous phases. But these domains are relatively small compared to the hydrophobic region of many membrane proteins. Nevertheless, exactly these small polar domains are essential for the development of a protein crystal as they form crystal contacts with neighbouring protein molecules. For this reason one strategy to obtain valuable crystals for high-resolution studies is the enlargement of the hydrophilic regions.
This can be achieved by connecting polar domains with specific monoclonal anti- body fragments. Native antibodies have flexible linker regions and a bivalent bind- ing behaviour. Both features are undesired for crystallization. Thus, proteolytic cleavage is used to obtain two monovalent Fab fragments. Up to now, all anti- body fragments successfully applied for co-crystallization with membrane proteins were derived from hybridoma cell lines [Hun02b]. Membrane protein structures that could be solved by the aid of Fabs are for example: the cytochrome c oxidase (COX) fromParacoccus denitrificans [Har99], [Hun02a], the yeast cytochrome bc1 complex (QCR) [Hun02c], the KcsA K+ channel fromStreptomyces lividans[Zho01] and the voltage-gated ClC-type chloride channel eriC from E.coli [Dut03] or the human β2 adrenergic G-protein-coupled receptor [Ras07].
I.1. Introduction
I.1.3. Objective
Due to the receptor’s medical relevance with regard to the muscle disease myasthenia gravis, its structure determination for the possible development of pharmaceuticals would be highly interesting. The current state of research provides a 4 Å structure of the nAchR of Torpedo californica [Unw05], lacking any detail information e.g.
discrete residues involved in ligand-binding. Moreover, there do not exist structures of this receptor co-crystallized with auto-antibodies found in sera of myasthenia gravis patients. Such structures could give a hint of the antibody binding to the receptor’s extracellular domain, hence becoming a main target for the receptor’s loss of function and degradation during disease.
Within this work monoclonal antibodies against the nAchR of Torpedo californica should be generated. Those antibodies should later serve for co-crystallization attempts of the receptor to eventually obtain a more detailed structure and to learn more about the antibody binding to receptor’s extracellular domain. As co- crystallization with mAbs already proved to be a powerful means for the crystalliza- tion of membrane proteins [Hun02b] [Hun02a] [Hun02c]this procedure was chosen for the present case.
Figure I.1.3.: Generation of monoclonal antibodies, a scheme
8
I.2. Materials & Methods
I.2.1. Media and buffers
Media
Complete DMEM-10 10 % FCS
10 mM HEPES
1 mM sodium pyruvate
Purpose: 1 day before fusion myeloma cells grow in this medium Complete DMEM-20
20 % FCS 10 mM HEPES
1 mM sodium pyruvate
Purpose: For plating hybridomas after fusion Complete DMEM, serum-free
10 mM HEPES
1 mM sodium pyruvate Purpose: For fusion
Complete DMEM-20-HAT/HT 20 % FCS
10 mM HEPES 1 mM sodium pyruvate 1x HAT or 1x HT
Purpose: Medium for feeding hybridomas after fusion, supplement with HAT or HT depends on selection stadium
Complete means:
1 % non-essential amino acids 50µM 2-Mercapto-ethanol
Penicillin (10.000 U/ml)/Streptomycin (10.000 µg/ml) IMDM
10 % FCS
Penicillin (10.000 U/ml)/Streptomycin (10.000 µg/ml)
I.2. Materials & Methods
Buffers
Ammonium chloride solution, sterile 0.02 M Tris-HCl, pH 7.2
0.14 M NH4Cl
50 % (w/v) PEG 1500, sterile PBS (GIBCO)
I.2.2. Animals
For the production of antibodies against the nicotinic acetylcholine receptor four female BALB/c mice of the same litter were chosen. At the beginning of the im- munizations the mice were nine weeks old. Further six mice of the same inbreeding strain were needed for the preparation of peritoneal macrophages. All mice stemmed from the own breed of the animal research facility of the University of Konstanz.
I.2.3. Determination of cell density and vitality
For counting, the used eucaryotic cells were mixed with trypan blue/crystal violet in the rate 1:10. The cell count was determined by the use of the Neubauer cham- ber. Healthy living cells are able to exclude the trypan blue dye actively, dead cells appear bluish colored in the microscope. Thus, only living cells were included in cell counting. Crystal violet was used as dye exclusively for the counting of freshly harvested splenocytes. This dye lysed remaining red blood cells and killed spleen cells. The latter became blue and could easily be counted without disturbance of erythrocytes.
Counting formula
Total number of cells (quadrants 1-4)/0.4µl (volume quadrants 1-4) = x cells/original cell volume after resuspension · dilution factor
I.2.4. Storage of adherent eukaryotic cells
Freezing
For freezing the confluent hybridoma cells were harvested from a small 10 ml culture flask, spinned at 1200 rpm for 5 min in a 15 ml vial and resuspended in 2 ml freezing medium at 4◦C. The cryo vial was kept in a styropor box in a -20◦C freezer for two days, so cells cooled down slowly this way. During this process DMSO diffused into the cells, preventing ice crystal formation and the destruction of cell membranes.
For long term storage the vial was transferred into liquid nitrogen at -196◦C.
10
I.2.5. Production of monoclonal antibodies against the nAchR of Torpedo californica
Freezing medium 40 % Medium 40 % FCS 20 % DMSO Thawing
Cells were obtained from the liquid nitrogen dewar and shortly incubated in a water bath at 37◦C. Afterwards cells were immediately incubated with some fresh medium to dilute the DMSO containing freezing medium to avoid further damage. Cells were resuspended by slowly pipetting, then centrifuged at 1200 rpm for 5 min and resuspended in 10 ml fresh medium. The cell suspension was transferred into a small culturing flask for further growth.
I.2.5. Production of monoclonal antibodies against the nAchR of Torpedo californica
I.2.5.1. Immunization of mice
Before the first immunization eye bleeds were taken from the four mice and tested by ELISA as pre-immune control sera. A few days later mice were immunized for the first time with 100µg purified nAchR in complete Freund’s adjuvans, mixed 1:1 in a total injection volume of 100µl. For the following injections the protein was applied in incomplete Freund’s adjuvans. Mice were re-immunized two times in the interval of two weeks with the same amount of protein to increase the immune response that should mainly consist of IgG. After the third immunization again eye bleeds were taken to control the antigen titer of the mice and to select one for the final two immunizations. The last immunization took place three days before the fusion. All immunizations were performed by a member of the animal research facility of the University of Konstanz.
I.2.5.2. Cultivation of myeloma cells
Four weeks prior to cell fusion the myeloma cell line SP 2/0-Ag14 was thawn. Cells were cultivated in IMDM medium. When cells grew stable after thawing they were splitted every second day at a ratio of 1:4. The day before the fusion cells were not splitted, but only brought into new medium to avoid a too strong thinning of cells, which would cause stress.
I.2.5.3. Preparation of murine peritoneal macrophages as feeder cells
Two days before the fusion six BALB/c mice were sacrificed by neck break for the extraction of feeder cells. These cells were gained by rinsing the abdominal cavity with 10 ml ice cold PBS. Afterwards the cells were counted and adjusted to 1·104
I.2. Materials & Methods
cells/ml. In this concentration they were plated in twelve 96-well plates, whereas only the inner 60 wells were filled with 100 µl, due to the danger of contamination.
I.2.5.4. Harvesting of B cells and preparation
The immunized mouse was sacrificed and the spleen was harvested under sterile conditions by a staff member of the animal research facility of the University of Konstanz. The spleen was immediately placed in 10 ml pre-warmed DMEM medium in a 15 ml tube. The next steps were performed in a sterile hood. The spleen was put into a sterile petri dish and remaining fatty tissue around the organ was removed, without impairing the spleen’s capsule. Now, the spleen was transferred into a new sterile petri dish that contained 10 ml serum-free DMEM medium, being dispersed into a single-cell suspension by chopping with fine-tipped dissection scissors. The capsule was removed and the spleen cells were further solubilized by passage through a fine-mesh metal grid. The B cell solution was transferred to a sterile 50 ml tube and incubated with sterile complete serum-free DMEM. Cells were centrifuged for 5 min at 1300 rpm and RT, the supernatant was discarded. Remaining red blood cells were lysed by resuspension of the pellet in 5 ml ammonium chloride solution for 5 min at RT. Thereafter 45 ml complete serum-free DMEM medium was adjoined and centrifuged for 5 min at 1500 rpm, RT. The pellet was then washed twice by resuspension in 50 ml of complete serum-free DMEM and following this centrifuged at 1300 rpm for 5 min at RT. During the wash steps of the lymphocytes the myeloma SP 2/0 cells were harvested and transferred in 50 ml tubes. The myeloma cells were centrifuged at 1200 rpm for 5 min at RT and resuspended in serum-free DMEM medium. All cells were pooled in one 50 ml tube and washed three times with serum-free DMEM medium, followed by centrifugation at 1300 rpm for 5 min at RT. Before cell fusion, the lymphocytes were resuspended in 20 ml and the myeloma cells in 50 ml serum-free DMEM. For each cell suspension the cell count, as well as the cell viability, was defined by trypan blue incubation for the myelomas and by crystal violet incubation for the lymphocytes in the Neubauer chamber. Based on the cell counts, the amount of complete DMEM-20 for the plating of the cells was calculated.
I.2.5.5. Cell fusion
In order to generate a stable cell line that expresses monoclonal antibodies in a great amount, it is possible to fuse B cells with myeloma cells and further clone the resulting hybridoma cells. Concerning these hybrid cells, the B cells provide for the capability of antibody production, while the myeloma cells account for unrestricted growth and continuous secretion of antibodies [Köh75]. As fusion partner for the B cells in this experiment the murine HAT-sensitive SP 2/0-Ag14 myeloma cell line was chosen. These cells exhibit a failure in their "salvage pathway" of nucleic acid biosythesis. The essential enzyme "hypoxanthine-guanine ribosyltransferase"
(HGPRT) lacks. It catalyses the synthesis of a corporate precursor of AMP and
12
I.2.6. Monitoring and selection of hybridoma cells
GMP, evolved from hypoxanthine. Using aminopterine in HAT medium for the se- lection of myeloma cells, the main metabolic pathway in the purine- and pyrimidine- metabolism is blocked. Cells that are capable to synthesize purines and pyrimidines from thymidine and hypoxanthine via the "salvage pathway" can survive the HAT treatment. Hybridoma cells can get this capability by the fusion with B lympho- cytes, whereas myeloma cells decay due to the collapse of both metabolic pathways.
Lymphocytes and myeloma cells were compounded at a ratio of 1:1 (each 5.2·107 cells) in a 50 ml falcon and filled up with complete serum-free DMEM medium.
During centrifugation of the cell mixture at 1300 rpm for 5 min at RT, three 37 ◦C double-beaker water baths were prepared under the hood. In one of the water baths a 400 ml beaker filled with 100 ml of 37 ◦C water was placed into another 600 ml beaker filled with 80 ml of 37◦C water. Another water bath contained the tubes of 50 % PEG 1500 solution and complete serum-free DMEM medium. The supernatant of the centrifuged cell pellet was aspirated and discarded. The cell fusion was then initiated by placing the falcon with the mixed cell pellet in a double-beaker water bath at 37 ◦C. With a 2 ml plastic pipet 1 ml pre-warmed 50 % PEG solution was added drop-by-drop to the cell pellet over 1 min. Thereby cells were agitated with the pipet tip after each drop and afterwards for an additional minute. Subsequent to the PEG solution 1 ml pre-warmed complete serum-free DMEM medium was added drop-by-drop to the cell mixture over 1 min accompanied by constant stirring. This step was repeated over again. Then 7 ml pre-warmed complete serum-free DMEM was adjusted drop-by-drop over 2 to 3 min. During this process macroscopic clumps of fused cells became visible and were centrifuged later at 1300 rpm for 5 min at RT.
In the course of centrifugation the beaker water baths were rewarmed and complete DMEM-20 (the amount of final plating medium as calculated before, see above) was placed in one of them at 37 ◦C. After centrifugation the supernatant was discarded and the falcon with pelleted cells placed in a beaker water bath. 10 ml pre-warmed complete DMEM-20 medium were effectually attached with a 10 ml pipet to the cell pellet. This was repeated until the amount of plating medium (42 ml) was de- pleted. During pipetting, cell clumps were allowed to settle and if necessary they were disrupted with the pipet tip. At this point the fusion process was terminated and further warming of the cell suspension was no longer essential. For plating, 2 ml of the cell suspension was gently aspirated with a 2 ml pipet. One drop (100-125 µl) of suspension was adjusted per well of a 96-well flat-bottom plate, charged with feeder cells two days before. The plating was continued until the entire cell suspen- sion was depleted. The charged 96-well plates were then incubated in a humidified 37◦C incubator with 5 % CO2 overnight.
I.2.6. Monitoring and selection of hybridoma cells
The first five days after fusion cells were checked daily for the control of growth and the removal of toxic products due to cell death. Especially at day two and three after fusion drastic cell death was apparent by reason of the donation of HAT and a
I.2. Materials & Methods
resulting viable hybridoma formation<10−5 [Har88]. Therefore, half the volume of each well is extracted and replaced by two drops (2 ml pipet) of complete DMEM- 20-HAT. During the following days cells were fed as necessary, depending on the actual number of cells remaining in the wells and their growth status. At day 14 after fusion the feeding medium was changed to complete DMEM-20-HT. Cells were only fed once with this medium, since the aminopterin was diluted out sufficiently this way. Afterwards cells are capable to survive without another donation of HT.
The subsequent days cells were fed with complete DMEM-20. The supernatant in the wells was considered to be ready for screening, when it changed its colour from neutral red to acidic yellow. At this visible change the metabolism rate of the cells was regarded to be high enough for measureable antibody secretion into the supernatant and screening by ELISA.
I.2.7. Indirect ELISA to detect specific antibodies
The ELISA test is an immunological verification procedure, based on an enzymatic color reaction. For the detection of a specific substance (antigen), the capability of certain antibodies to bind to this substance is used. The antibody or the antigen is previously labeled with an enzyme. The latter catalyzes a reaction that proves the presence of the antigen. The product of the reaction can be detected as shift in colour, fluorescence or chemoluminescence. In general, the signal strength is a function of the antigen concentration, hence the ELISA can also serve as quantita- tive confirmation method. In the current work this assay was at first performed to screen the antibody titer in the blood of the BALB/c mice before and during im- munization. Moreover, it served to analyze the hybridoma supernatants for specific antibodies.
ELISA buffers
Buffer A/Wash solution 50 mM Tris, pH 7.4 200 mm NaCl
0.15 % decylmaltoside Coating solution Buffer A
20µg/ml nAchR Blocking solution Buffer A
5 % BSA
14
I.2.7. Indirect ELISA to detect specific antibodies
Primary antibody solution
Either: Control/test sera diluted 1:100 in Buffer A + 2 % BSA, or: Hybridoma supernatant diluted 1:1 in 2x Buffer A + 2 % BSA
Secondary antibody solution
1:2000 goat α mouse horseradish peroxidase conjugate (Invitrogen) in Buffer A + 2 % BSA for hybridoma supernatant, test sera and negative controls
1:4000 mouse α rat horseradish peroxidase conjugate (Invitrogen) in Buffer A + 2 % BSA for positive controls
Substrate solution
ABTS (2,2’-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid)
Stop solution 1 % SDS
The day before the assay 50 µl of coating solution were added to each well of a 96-well ELISA plate and stored at 4 ◦C overnight. The following day the coating solution was removed by emptying the plate face down over the sink. The wells were washed 3x with 200 µl wash solution/well, shaking for one minute. The wash solution was removed as the coating buffer before. Afterwards 300 µl of blocking solution were added to each well, plates were incubated at RT for two hours. Then the blocking solution was removed and replaced by 3x 200 µl washing solution.
Subsequently 50 µl 2x Buffer A were augmented to each well, with the exception of the positive and negative controls. This was followed by the addition of 50 µl hybridoma supernatant in each well. For the testing of antisera only 100 µl sera diluted 1:100 in Buffer A + 2 % BSA is added to the well after the wash step. The positive control (monoclonal antibody 124, donated by Institute Pasteur Athens, but without impact on crystallization) as well as the negative control (eye bleeds before immunization) were each added to a coated and uncoated well. The plates were incubated for one hour at RT. Thereafter the primary antibody solution was exhausted and wells were washed threefold with 200 µl washing solution. Accor- dingly 100 µl secondary antibody solution were filled into the wells and incubated at RT for one hour. Finally wells were washed five times with washing solution and 100 µl substrate solution were added. The reaction was allowed to proceed until a bright blue colouration had developed. At this point the reaction was stopped with 1 % SDS solution. In the end the plate was read in the SPECTRAFluor Plus Reader (TECAN) at 405 nm. Whereas the clearest coloring was considered to display the strongest antibody binding/production.
I.2. Materials & Methods
I.2.8. Sub-cloning of positive hybridomas
Considering hybridoma cell lines, they are accounted to be monoclonal and stable, if they could have been sub-cloned for at least two times. Hence it was necessary to isolate a positive clone (ELISA) from a mixture of different cell populations in a well. For this purpose cells in a well were resuspended and diluted in a way that only one cell per well would be statistically found.
After cell counting the hybridoma solution was adjusted to ∼60cells in 6 ml. This dilution was plated in a 96-well plate with 100 µl/well. Finally 100 µl complete DMEM-20 medium were added to each well and the plate was stored at 37 ◦C in the incubator. Cells were allowed to grow for one week without being disturbed.
Thereafter, wells were inspected and single colonies were marked. In the end cells were again tested by ELISA and positive clones were further sub-cloned into 48- and 24-well plates and later frozen as resource.
I.2.9. Isotype definition of monoclonal antibodies
Blocking buffer
0.5 % Tween-20 in PBS Antibody solution G/M
1:5000 goatαmouse IgG/IgM horseradish peroxidase conjugate in 0.05 % Tween-20 10 % FCS
0.02 % Thimorosal Antibody solution M
1:5000 goat α mouse IgM horseradish peroxidase conjugate in 0.05 % Tween-20 10 % FCS
0.02 % Thimorosal Washing buffer
0.05 % Tween-20 in PBS
For the determination of the isotype of monoclonal antibodies the supernatant of the cultured cells was used. The elucidation of the immunoglobuline class (IgG, IgM) of the mouse monoclonal antibodies for characterization was determined by the Dot Blot procedure. This method also belongs to the group of immuno assays.
Proteins are conveyed onto a nitrocellulose membrane, e.g. by pipetting, and adhere due to hydrophobic interactions. Moreover, they contain their structure, thus, they are accessible for other methods like immuno-labelling or -staining.
First of all two drops of 1 µl of hybridoma culture supernatant were placed on a nitrocellulose membrane. They were allowed to dry for 2 min and the drop contours were marked. Afterwards the membrane was cut into two pieces to separate the
16
I.2.10. SDS polyacrylamide gel electrophoresis (SDS-PAGE)
drops. Subsequently the membrane pieces were blocked with blocking buffer for one hour at 37 ◦C on a tumbler. One drop was then incubated with antibody solution G/M, the other drop was incubated with the solution M, shaking for two hours at 37
◦C. Thereafter the membranes were washed 3x with washing buffer. The Dot Blots were developed with the Duo ECL kit (PIERCE). The two components "Luminol Enhancer Solution" and "Stable Peroxide Solution" were mixed 1:1 and equally dispensed on the membranes. Those were incubated for 3 min at RT. Afterwards the membranes were transferred into a film cassette and fixed. The cassette was brought into a dark room and a light sensitive film was placed onto the membranes for 5 min. Subsequently the two films were inserted into the developer. The dots that emitted luminescence due to the enzymatic reaction of horseradish peroxidase with the peroxide solution appeared as black labelled points on the film. Those points were the criterion for a positive result.
I.2.10. SDS polyacrylamide gel electrophoresis (SDS-PAGE)
The SDS-PAGE is a frequently used method in molecular biology to separate pro- teins due to their molecular weight. It was performed according to [Lae70] [Lae73].
The composition of a 12.5 % SDS gel is depicted in table I.2.1.
SDS-PAGE upper buffer (pH 6.8) 0.4 M Tris-HCl
0.4 % w/v SDS
SDS-PAGE lower buffer (pH 8.8) 1.5 M Tris-HCl
0.4 % w/v SDS
SDS-PAGE running buffer (10x) 3 % w/v Tris-HCl
14.4 % w/v Glycine 10 % w/v SDS
30 % Acrylamide stock solution
10 % w/v Ammonium persulfate (APS) TEMED
Isopropanol
I.2. Materials & Methods
SDS-PAGE sample buffer 60 mM Tris-HCl, pH 6.8 2 % SDS
10 % Glycerine
0.1 % Bromphenole blue
25 % 2-mercapto ethanol, only in reducing sample buffer Mark12TM molecular weight marker (Invitrogen) or
SeeBluerPlus2 Prestained Standard (Invitrogen) for Western Blot gels
Composition Stacking gel Resolving gel 30 % acrylamid 0.33 ml 2.15 ml
Tris-HCl (pH 6.8) 0.63 ml -
Tris-HCl (pH 8.8) - 1.25 ml
H2O bidest. 1.55 ml 1.65 ml
APS 40 µl 75 µl
TEMED 15 µl 40 µl
Table I.2.1.:Composition of a 12.5 % SDS gel
In the case of monoclonal antibody binding characterization, 2µg of nicotinic acetyl- choline receptor were mixed with 25 µl reducing and non-reducing sample buffer.
The reduced sample was boiled at 98 ◦C for 5 min. Samples and markers were filled into the gel pockets with a Hamilton pipet and the gel was run at 160 V for 1:10 h.
Coomassie staining of SDS gels
The SDS gels were stained with Coomassie according to [Sil84]
Staining solution 30 % Ethanol 10 % Acetic acid
1.4 % Coomassie Brilliant Blue R-250 De-staining solution
30 % Ethanol 10 % Acetic acid
18
I.2.11. Characterization of epitope binding of monoclonal antibodies by Western Blot
I.2.11. Characterization of epitope binding of monoclonal antibodies by Western Blot
To investigate the binding characteristics of monoclonal antibodies based on their antigen, it is useful to check the epitope binding under native and reduced conditions.
For this reason the antibody binding behaviour was controlled by Western blotting according to [Tow79]. Therefore, an unstained SDS gel loaded with nAchR (native and reduced) was transferred to a PVDF membrane (Pall). Afterwards the blot was developed by using the antibody solution G/M and the DuoECL kit [see section I.2.9].
I.3. Results
I.3.1. Immunization
The antigen was applied subcutaneously in complete Freund’s adjuvans, and later on subcutaneously in incomplete Freund’s adjuvans. Expertise of the collaborating group of Prof. Dr. Markus Groettrup proved that immunogenicity of membrane proteins was enforced, when immunizations were performed the latter way.
The immune response to an applied antigen can vary even in a group of genetically homogeneous species, therefore, four animals were immunized. In order to increase specific B cell development and to get the maximum exploitation of fusion, the mouse (No. 0) with highest immune response got a last boost immunization three days prior to cell fusion.
Figure I.3.1.: Immune response of mice after last immunization
I.3.2. Harvesting of splenocytes and preparation
After harvest of the spleen, splenocytes and myeloma cells were counted. Thereby
∼2.5·106 total cells/ml of mixture were scheduled. The total number of splenocytes
20
I.3.3. Fusion
was 5.2·107, for the later fusion the same amount of myeloma cells was prepared.
The amount of medium to plate cells at the above mentioned concentration was determined as follows: Total amount of cells =2·5.2·107 cells, divided by∼2.5·106 cells/ml = 42 ml. The amount of medium was pre-warmed in a 37 ◦C water bath.
I.3.3. Fusion
As antibody secreting B cells of the spleen are unable to survive in cell culture, they were fused with the immortal myeloma cell line SP 2/0-Ag14 at the ratio 1:1. During plating, right after the fusion of cells, vigorous pipetting was implicitly avoided, since the newly generated hybrid cells tended to be very unstable. The modus operandi at plating was to leave free the outermost ranks of wells, as the introduction of contaminants tended to happen preferentially at the brim of the plates.
I.3.4. Confirmation of specific antibodies against the nAchR by ELISA and sub-cloning
About 14 days after fusion the inspection of the culture supernatants of the emerged hybridoma cells began. To detect the binding of a newly evolved antibody, the bot- toms of the ELISA wells were coated with nAchR, the antigen. Afterwards the specific/investigated antibody was allowed to bind to the antigen. This binding was detected by the binding of a secondary goat α mouse antibody with conjugated horseradish peroxidase (HRP). After addition of ABTS as a substrate, it was hy- drolysed by the HRP and a considerable blue coloring appeared in the well in the case a positive binding had appeared. After halting the color reaction, the plate was examined in the SPECTRAFluor Plus Reader to measure the optical density at 405 nm. An antibody was selected for further cloning and testing, when the numeric value of the measurement exceeded the other measured values by more than double.
The positive and negative controls served solely to prove the functionality of the assay and not as scale type to identify a well as positive or negative.
In total 720 different supernatants were investigated. After the first round of ELISA screening, 27 hybridoma wells were selected for further sub-cloning and testing by ELISA.
I.3.5. Isotype definition of monoclonal antibodies
The isotype definition of monoclonal antibodies was done via Dot Blot. An early characterization of an antibody’s isotype is fundamental for a number of purposes, such as purification, structural assembly and generation of Fabs. It had to be tested to find out whether the monoclonal antibodies belong to the class IgG or IgM.
The latter is the first class of antibody that is built upon the first contact with an
I.3. Results
Figure I.3.2.: Example of an successful ELISA within the first round of screening In the wells A7, B10 and C11 examples of positively tested hybridoma supernatants are displayed.
The positive and negative controls are labeled with + and -.
antigen and marks a new infection. IgM is a pentamer, consisting of five subunits that are linked by the "J peptide". IgM antibodies tend to be unstable and to easily agglutinate during purification, what makes them undesirable for purposes like crys- tallization. IgG in contrast is first built in a period of delayed immune response (∼ 3 weeks post antigen contact) but remains long in the immune system. During the Dot Blot two different antibodies were used for detection: goat α mouse IgG/IgM- HRP and goat α mouse IgM-HRP, specific for the µ-chain of IgM antibodies. As positive control for an IgG/IgM antibody served an already confirmed mouse IgG antibody (AG Stürmer, University of Konstanz). For the IgM antibody was no positive control available, but the antibody gave a positive result in the above men- tioned group only a few days before. Hence it was not supposed to degrade in the meantime. There were no hits for monoclonal IgM antibodies, but after the second round of sub-cloning six positive hits for IgG were found by Dot Blot. However, all these tested antibodies/hybridoma cells stemmed from the same colony (b’2) and were merely splitted into several sub-clones. Thus, one monoclonal antibody was obtained at the end of the experiment.
I.3.6. Characterization of epitope binding of the monoclonal antibody by Western Blot
For the purpose of protein crystallography it was determined to which kind of epi- tope the monoclonal antibody binds. In the case of binding to a discontinuous epitope, which consists of disjointed sections of the amino acid sequence, the anti- body exclusively recognizes the native conformation of its antigen. It is dependent on the structural assembly of the antigen. A continuous epitope is characterized by a coherent amino acid sequence, displaying a rather linear fragment of the antigen.
This kind of epitope is recognized by the antibody in the native as well as in dena-
22
I.3.6. Characterization of epitope binding of the monoclonal antibody by Western Blot
Figure I.3.3.: Isotype definition of a monoclonal IgG antibody by Dot Blot
After the second round of sub-cloning several positive hits for IgG were found by Dot Blot, among them the hybridoma colonies B’3, B’4 and E’3. But the according cells all stemmed from the same original colony b’2 and were merely splitted into several pure sub-colonies. The positive control is labeled with +.
tured conformation.
At first a gel electrophoresis with a 12.5 % SDS gel was run. Three different sam- ples of nAchR as antigen were prepared: 2 µg nAchR in non-reducing buffer, 2 µg nAchR in reducing buffer and as positive control for staining the serum from the immunized mouse No. 0. The antigen was subsequently blotted onto a nitrocellu- lose membrane and developed with the DuoECL kit. The protocol was the same as for the development of the Dot Blot, except for two antibody solutions that were applied successively. At first the culture supernatant that contained the monoclonal antibody, secondly, the antibody solution G/M known from Dot Blot procedure [see section I.2.9].
On the left hand side of the blot one can see the bands of the nAchR in non-reducing buffer. There exists extensive black labeling in the area of the 250 and 148 kD band.
At 250 kD the whole receptor can be found in the gel as hetero pentamer (2x α = 76 kD, β = 50 kD, γ = 57 kD and δ = 64 kD). At ∼148 kD another thick band is visible, a multimeric part of the receptor and also at 64 kD, which represents the δ subunit of the receptor. As positive control serves the serum of the selected mouse 0, which contains a high antibody titer. The serum is also applied in non-reducing buffer, thus most of the immunoglobulins are labeled extensively at∼148 kD (IgG = 150 kD) and above (aggregates). On the right hand side the bands of the antigen in reducing buffer are visible. The bands are in general stronger labeled, but the same pattern as on the left side of the blot is recognizable. The δ subunit is recognized
I.3. Results
Figure I.3.4.: Binding character of the monoclonal antibody towards its antigen I The positive control for staining is labeled with +.
Figure I.3.5.: Binding character of the monoclonal antibody towards its antigen II
separately as well as in multimeric assemblies.
On the second blot the same pattern of labeling is apparent. On the left hand side again the antigen is applied in non-reducing buffer twice, on the right hand side the reducing buffer was used for application. The only alterations to the first blot are the absence of the serum positive control and the additional lane occupied by the reduced and boiled sample of the nAchR at the right side of the Blot. The latter sample shows compared to the reduced but not boiled sample of the nAchR strong labeling in the area above the actual separation zone. According to this, a certain amount of receptor got stuck in the pockets of the upper gel of the gel electrophoresis after heating. This observance is congruent with former SDS-PAGES of the nAchR, which were done and assigned by the lab member Alexander Brosig. Furthermore, it is obvious that the bands of the two lanes on the reducing side of the second gel are variably strong coloured compared with each other. The observation of differing strength in colouration has already been made for the first Blot, where the reducing
24
I.3.6. Characterization of epitope binding of the monoclonal antibody by Western Blot
and non-reducing sides of the blot have considerably differed in labeling.
I.4. Discussion
I.4.1. Generation and characterization of mAbs against the nAchR of Torpedo californica
By immunization of BALB/c mice with the purified nAchR in native conformation it was possible to generate a monoclonal antibody against this receptor.
The screening via ELISA was a suitable method to ensure a precocious selection of the hybridoma cells. Moreover, during screening, all tested cells were subjected to the identical metabolic conditions (culture supernatant turned acidic), which should provide for the comparability of the data. After sub-cloning the hitherto positive hybridoma colonies were tested by Dot Blot for their antibody isotype. It was dif- ferentiated between IgG and IgM. In mice and humans the class of IgG antibodies encompasses around 75 % of the immune system’s antibodies, whereas the IgM anti- bodies represent∼8 % thereof [Jan01]. Thus, it is rather likely to gain an antibody that belongs to the dominant IgG class. In addition the IgG isotype can easily be purified (protein A or G affinity chromatography) as well as Fabs can be easily produced by papain cleavage [Har88]. For protein crystallization Fabs are a desired tool for getting well ordered crystals of membrane proteins [Hun02b], thus, an IgG antibody, as gained in this work, is virtually the means of choice.
Beside the isotype class also the binding characteristics of the monoclonal antibody were investigated. For this reason the antigen was applied to a SDS gel at reducing and non-reducing/non-denaturating conditions. Concerning the latter conditions the antigen could be considered as "in native conformation". One percent SDS in the running buffer hardly exerts such an influence on the detergent-solubilized re- ceptor that it would loose its native conformation without previous treatment of reducing agents. Moreover, it was discovered in our lab that reducing and non- reducing sample buffers do not interfere with each other, when they are separated on the gel by two or more lanes (personal communication K. Lohner, technical assis- tant). After Western blotting and development of the luminescence films it has been concluded that the δ subunit (64 kD) is isolated on the blot and labeled by black colouring. Thus, the monoclonal antibody has recognized this part of the receptor as its epitope. This would also explain the band at 250 kD, where the δ subunit is also present. Probably at the band slightly below 148 kD one could find the two α and the δ subunits in assembly (140 kD) and hence the black labeling. For the intense and varying intensity of colouring on the Western Blots several reasons could exist. On the one hand differing amounts of antigen could have been loaded onto the gel or the efficiency of blotting varied within the gels/among gels. On the other hand the washing steps performed after incubation with the antibody solution G/M were not sufficient, thus, the obtained signal was very strong.
26
I.4.1. Generation and characterization of mAbs against the nAchR of Torpedo californica
In general, the binding patterns are the same for the non-reduced/non-denatured and the reduced receptor on the blots. This means the monoclonal antibody binds its antigen in native as well as in linearized conformation. Thus, the binding region is supposed to be a continuous epitope rather than a discontinuous epitope, which would not be recognized by the antibody in denatured conformation [Hun02b]. The explanation for binding to a continuous epitope could be as follows: The nAchR is a membrane protein, which has to be solubilized in detergent (n-decyl-β-D- maltopyranoside) for purification. This detergent forms charactristic micelles (∼ 48.3 kD) around the receptor. Although the nAchR is of respectable size, each subunit comprises around 30 Å x 40 Å x 160 Å [Unw05], the micelles only leave a small amount of hydrophilic residues/loops accessible at the extracellular region of the receptor. Moreover the outmost part of the EC region mainly consists of several flexible loops that are essential for ligand binding and receptor function [Unw05].
Such loops often exhibit areas which are rather outstretched or linear. Thus, it is not unlikely that an antibody binds such a "stable" extended region. The latter is easier accessible as epitope instead of an epitope that is flexible and composed of different structural regions (discontinuous residues).
For crystallization antibodies are preferred which bind to the native epitope [Hun02b], probably to provide for the elucidation of the "truly native" structure of a protein.
But concerning this really native or "in vivo" structure, one should be cautious. If a membrane protein is isolated of its natural environment and taken up in detergent micelles, can guarantees be made that this purification procedure has not changed its natural conformation? Nevertheless, also an antibody was investigated that binds to a continuous epitope of NhaA (Na+/H+ antiporter) of E.coli. The results of these investigations implied that the epitope recognized by the mAb is important for antiporter activity and is exposed only during certain binding or reconstitution conditions. Hence, this mAb together with other monoclonal antibodies recognizing an essential epitope provide a new tool to study the mechanism of the antiporter and the relationship between structure and function [Pad98]. Moreover, for crys- tallization of the human β2 adrenergic receptor for example a monoclonal antibody was chosen that did not recognize the native epitope exclusively [Day07]. In fact, it also bound to the denatured epitope, even though this binding was weaker. In the case of the present monoclonal antibody against the nAchR one has to state that the binding characteristics determined by Western blot combined with DuoECL stain- ing are not definite. The extent to which this antibody recognizes the denatured epitope differs. Certainly the amount of antibody differs within diverse expression cultures, likewise the extent of binding and staining. For this reason another method for characterization should be applied, which is more insensitive to antibody con- centration or staining artefacts, for example mass spectrometry.
For crystallization people focus on the good binding capabilities of the antibodies and, thus, on the enlargement of the hydrophilic part of the membrane protein to augment the area for possible crystal contacts. Why should antibodies binding to a continuous epitope not serve for these purposes? Antibodies preferring the one or the other epitope should be tested likewise in crystallization trials. Protein crys-
I.4. Discussion
tallization is a field in biology, where no one can predict in advance if a protein crystallizes or not and there exist no rules to unerringly obtain protein crystals.
Especially membrane proteins are difficult to purify and crystallize. Less than one percent of all published protein structures make membrane proteins and not re- ally numerous are co-crystallized with Fabs of monoclonal antibodies. Hence, the knowledge concerning this subject is not sufficiently substantial to create dogmas.
28
I.5. Perspective
The monoclonal antibody was successfully generated for the co-crystallization of its Fabs with the nAchR. The next queuing steps are to do a large scale production of the monoclonal antibody and to purify it by either protein A or G affinity chromatog- raphy. Afterwards Fabs should be generated (e.g. ImmunoPurer Fab Preparation Kit, PIERCE) and again purified by affinity chromatography. Subsequently a co- crystallization approach could be set up. Additionally, it would be interesting to learn more about localization and character of the binding epitope. For this reason the antigen could be digested by trypsin and the fragments could be separated by gel electrophoresis. The gel would thereafter be blotted on a membrane and incu- bated at first with the monoclonal antibody and second with a labeling antibody [see section I.2.11]. A specific fragment to which the monoclonal antibody binds could be identified by the according staining/labelling. Furthermore, the antigen could also be first digested by trypsin and second the monoclonal antibody could be applied. Afterwards this mixture could be examined by mass spectrometry, that would yield thus the exact amino acid sequence and the localization of the Fab’s binding epitope.
II. Structure and function of the
β -N-acetylglucosaminidase Bs Nag3A of
Bacillus subtilis
I.5. Perspective
This work is to be published in:
LITZINGER, S.*,FISCHER, S.*, WITWORTH, G., VOCADLO, D., DIEDERICHS, K., WELTE, W. and MAYER, C. (2009) An Asp...His catalytic dyad is the general acid/base catalyst of family 3 β-N-acetylglucosaminidase.
*Contributed equally to this work.
32
II.1. Introduction
II.1.1. The cell wall and its recycling in gram-positive bacteria
In nature different bacterial species can be found and with them a wide variety of habitats, occupied by them. But common to all these diverse environments is the persistent competition among the microbes for the finite nutrition resources. There- fore, the assimilation of substances for metabolism has to work efficiently and the nutrients should be kept available in the cytoplasm. Thus, high concentrations of so- lutes arise within a bacterial cell. In order to control the cellular import and export of substances a permeability barrier formed by a lipid bilayer was developed during evolution. On the contrary, a certain disadvantage of this structure is its susceptibil- ity. As bacterial cells often populate hypotonic habitats and are frequently subjected to fluctuating conditions they have to cope with an immense turgor pressure that impacts on the cell’s structure. For this reason an additional coat reinforces the cell with its membrane like an exoskeleton. This coat consists of a highly complex and essential macromolecule called peptidoglycan or murein [Bon05]. It is present in most eubacteria and maintains the cellular viability and shape [Atr99] as well as counteracting the high intracellular turgor [May05]. Contrary to gram-negative bac- teria that possess one to three peptidoglycan layers in the periplasm, gram-positive bacteria possess no outer membrane but they are opulently encompassed by 10-20 layers of peptidoglycan, associated with teichoic acid. These layers show a thickness of up to 100 nm and represent 20-70 % of the cells dry weight [Sel02].
Figure II.1.1.: Isolated murein sacculus fromE.coli visualized by electron microscopy The sacculus reflects the cell’s shape from that it was prepared. It resembles an exoskeleton that protects the bacterial cell from barren environments. Picture from [Hei02].
The peptidoglycan is a very dynamic structure and provides for unhampered growth and cell division. Moreover, its chemical assembly is quite similar among eubacte- ria. For this reason the following implementations concerning PG and the according
II.1. Introduction
pathways will mainly refer to the model organism E.coli instead of B.subtilis as the first is best examined.
Peptidoglycan - a biopolymer
In bacterial cells, the peptidoglycan or murein is formed into a three-dimensional structure. It displays a hollow body that completely surrounds the bacterial cell [Höl85] [Par96] [Rog80] [Wei64]. It has the shape and size of the bacterium and is a gigantic sacculus-shaped molecule. Furthermore, the peptidoglycan, also called murein, consists of linear glycan strands that are made of the aminosugars N- acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). These sugars are linked together byβ-1,4 glycosidic bonds. Unlike chitin, murein is held together by covalent bonds in two or even three dimensions, resulting in a mono- or multi- layered structure [Höl98]. The presence of the D-lactyl group of the muramic acid allows the covalent attachment of peptides, which are cross-linked to form the char- acteristic net structure of the peptidoglycan. Whereas the chemistry of the glycan strands shows only few variations among different bacteria, such as O or N acetyla- tion, the peptides vary considerably [Rog80] [Sch72a]. In E.coli and B.subtilis their composition is most often: L-alanine-γ-D-glutamate-meso-diaminopimelic acid-D- alanine-D-alanine. Importantly, the dibasic amino acid, meso-diaminopimelic acid, has to be present to enable the cross-linking peptide bond between the amino group and the carboxyl group of the D-alanine in the neighbouring peptide chain (see figure II.1.2.). Meso-diaminopimelic acid is an intermediate in the biosynthetic pathway and leads to lysine [Höl98] [Atr99]. A peculiar structure of the peptido- glycan of E.coli and B.subtilis is a 1,6-anhydromuramic acid present at one end of the peptidoglycan strands [Höl75]. All of the normally reducing ends seem to be blocked by this intramolecular glycosidic bond, since free reducing ends have not been found [Hen72]. The stem peptides are arranged helically along the gly- can strand, protruding in all directions and forming angles to one another of about 90◦ [Lab85]. Therefore, in monolayered peptidoglycan, only every second peptide can be cross-linked by turns to the left and right [Höl98]. The peptidoglycan struc- ture is composed of rather stiff glycan strands with restricted flexibility but associ- ated with highly flexible peptides [Lab79] [Lab85] that can be reversibly stretched four-fold in length [May05]. This special arrangement provides for a certain elas- ticity, hence the peptidoglycan envelope can expand as well as shrink, depending on cell turgor. Concerning the three-dimensional architecture of the peptidoglycan, several hypotheses exist. On the one hand, the glycan strands of the peptidoglycan are supposed to be arranged parallel to the membrane [Vol04]. On the other hand, the recently introduced scaffold model suggests the glycan strands to be stretched perpendicular to the membrane and to grow in a linear manner [Dmi99] [Dmi03].
However, up to now neither hypothesis could be verified.
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II.1.1. The cell wall and its recycling in gram-positive bacteria
Figure II.1.2.: Peptidoglycan assembly in E.coli
Scheme of two glycan strands, associated with a processed tetra-peptide and a non-processed penta-peptide. The formation of a new D,D-peptide cross-linkage, energised by release of the last D-alanine of the adjacent peptide chain is depicted (see arrow). GlcNAc = grey circle, MurNAc = grey square, 1,6-anhydroMurNAc with intramolecular 1,6-glycosidic bond, represents the reducing end of a glycan strand = grey square with a half-circle above. From [Jäg07]
Turnover and recycling of the cell wall peptidoglycan
The phenomenon of peptidoglycan turnover in E.coli was detected rather late as turnover is coupled to peptidoglycan recycling [Par93] [Goo85]. The components that are released during turnover at a rate of up to 50 % per generation are effi- ciently recycled, see fig. II.1.3. About 90 % of the components are reinserted into the peptidoglycan sacculus. 1,6-anhydro-MurNAc-β-1,4-GlcNAc-tetra- and tripeptides represent the main turnover products, indicating the involvement of lytic transgly- cosylases in their release. The lack of dimers and trimers indicates that turnover is subjected to the concerted actions of transglycosylases and endopeptidases. The turnover products accumulate in the periplasm, from where they are reimported into the cytoplasm [Höl98].
The major pathway for the uptake of turnover products is via the transporter protein AmpG [Lin93] [Par93], a transmembrane protein that acts as a specific permease for intact muropeptides [Jac94]. In the cytoplasm, the muropeptides have to be reduced
