Oligomerization, Degradation and Aggregation Reactions and Products of Synuclein Polypeptides Related to Parkinson’s Disease
Oligomerization, Degradation and Aggregation Reactions and Products of Synuclein Polypeptides Related to Parkinson’s Disease
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2011
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Oligomerisierungs-, Abbau- und Aggregationsreaktionen und Produkte von Synuclein Polypeptide bezüglich der Parkinson’schen Krankheit
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Dissertation
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Abstract
In recent years, mass spectrometry (MS) has become a major analytical tool in biochemistry and structural biology. In particular, electrospray mass spectrometry (ESI-MS) has emerged as a powerful technique for analyzing intact gas phase ions from large biomolecules and supramolecular complexes. However, in contrast to the large number of ESI-mass spectrometric studies of protein structures, structure modifications and proteomics, the molecular characterization of protein “misfolding” and aggregation species by mass spectrometry had hitherto little success; possible explanations are (i), the low concentration of intermediate species, and (ii), the slow rates of aggregate formation in vitro. Conventional “soft-ionization” mass spectrometric methods such as ESI-MS and HPLC-MS are not suitable to direct “in-situ” analysis of conformational states and intermediates at different concentrations. Recently, ion-mobility mass spectrometry (IMS-MS) is emerging as a new tool to probe complex biomolecular structures, due to its potential to separate mixtures of protein complexes by conformation state, spatial shape and topology. Thus, IMS-MS implements a new mode of separation that allows the differentiation of protein conformational states.
One of the hallmarks of Parkinson’s disease (PD) and related neurological disorders is the accumulation in human brain of intracellular high molecular weight α-synuclein (αSyn) aggregation products as fibrils. Oligomeric intermediates have been suggested to represent major neurotoxic species; however, chemical structures of αSyn oligomers and possible intermediates have not been hitherto identified. The first parts of this thesis deal with the isolation and structure identification of αSyn oligomerization-aggregation products in vitro and in vivo.
The first part was focused on the molecular characterization of human αSyn (wt-αSyn) and two mutants, αSyn (A53T) and αSyn (A30P) using HPLC, high resolution mass spectrometry, gel electrophoresis and circular dichroism spectroscopy. Additional information about the wt-αSyn structure was obtained by molecular dynamic simulation, which indicated a flexible, random coil structure. Using mass spectrometric methods, proteolytic degradation studies of synucleins were carried out for identifying the cleavage specificity and preferred cleavage sites of different proteases.
The second part of the thesis was focused on the in vitro and in vivo characterization of αSyn oligomerization-aggregation products, employing gel electrophoresis, Dot blot and Western immunoblotting. The in vitro oligomerization of αSyn was carried out by incubation at 37°C in sodium- phosphate (pH 7.5) for up to seven days. The formation of oligomers was monitored by Tris-tricine polyacrylamide gel electrophoresis which revealed bands corresponding to monomeric and oligomer-like αSyn at approximately 37 and 48 kDa. In addition, three bands of minor abundances with molecular weights lower than full-length αSyn indicated the formation of truncation or degradation products. The bands corresponding to αSyn monomer and dimer were excised from the gel, digested with trypsin and analyzed by HPLC-ESI-MS. ESI-MS and tandem-MS of the tryptic peptides revealed the presence of monomeric and dimeric αSyn with full-length sequences, respectively; additional structural characterization was obtained by Edman sequencing. Direct ESI-MS of αSyn upon incubation in an aqueous buffer for 3 hours provided the identification of small amounts of N-terminally truncated αSyn (7-140). In contrast to these results, attempts to identify the truncation and degradation products by direct mass spectrometric analysis and HPLC-MS were unsuccessful, presumably due to their low concentration.
The application of ion-mobility mass spectrometry (IMS-MS) to oligomerization-aggregation mixtures of αSyn in vitro enabled the first identification of specific fragments corresponding to truncation and degradation products that have been previously observed by gel electrophoresis, but not identified. Most important, a highly aggregating fragment was identified, resulting from cleavage at the central aggregation domain of αSyn, between residues Val-71 and Thr-72 (7.2 kDa). These results showed that IMS-MS can be successfully applied to the identification of hitherto undetected truncation products of neurodegenerative target proteins. Aggregation studies of the corresponding carboxy-terminal fragment, αSyn (72-140) prepared by both chemical synthesis and recombinant expression, showed a substantially faster rate of fibrillization compared to the intact full-length αSyn protein. The chemical structure elucidation of in vivo αSyn oligomerization products from human αSyn transgenic mouse brain homogenates and human neuroblastoma cell cultures was performed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and high resolution mass spectrometry, as well as affinity binding studies by online combination of a surface acoustic wave (SAW) biosensor and ESI-MS. Immunoblotting in combination with high resolution- MS provided the identification of αSyn (A30P) from soluble mouse brain homogenate proteins. Spots that were detected as immunoreactive with the anti-αSyn mBD antibody were analyzed by high resolution- MS, leading to the identification of αSyn and of two methionine oxidized products (116Met and 127Met).
In a third part of the thesis, the epitope structures recognized by anti-αSyn specific antibodies were identified. The mass spectrometric analysis was combined with epitope excision and extraction procedures previously developed in our laboratory. For the epitope excision and extraction experiments, affinity columns were prepared by immobilizing the antibodies on Sepharose. For protein digestion trypsin and Glu-C endoproteases were employed, and for analysis MALDI-TOF-MS, as well as ESI-ion trap-MS used. The epitope excision, followed by mass spectrometric analysis provided direct information that the epitopes recognized by polyclonal antibodies (pASY-1; pC20) were discontinuous and located in the N-terminal (1-23) and central (59-80) regions. Comparative binding studies of anti-αSyn specific antibodies with the epitope peptides αSyn (1-23) and αSyn (59-80), and synthetic mutant model peptides were performed by ELISA, Dot blot and affinity- mass spectrometry. The results showed that αSyn (1-23) and αSyn (59-80) bound to the anti-αSyn pASY-1 antibody in a concentration- dependent manner.
A further part of the dissertation was focused on applications of an online combination of a surface acoustic wave (SAW) biosensor with ESI-MS that enabled the direct detection, identification, and quantification of affinity-bound ligands from synuclein- anti-synuclein antibodies complexes on a biosensor chip. The specific antibodies were covalently immobilized on the gold coated surface of the quartz chip. The interactions with wt-αSyn, mutants αSyn (A53T), αSyn (A30P), αSyn peptides and βSyn were determined. The obtained KD values were in the low nano-molar range and in good agreement with bioaffinities investigated by Dot Blot, ELISA, SAW biosensor and SAW-ESI MS. Moreover, the SAW biosensor has been employed as an affinity detection method in conjunction with MALDI-MS for epitope determination of αSyn.
In the last part of the thesis, chemical stability studies of wt-αSyn, αSyn (A53T), αSyn (A30P) and βSyn proteins were performed. The formation of oligomers was monitored by Tris-tricine polyacrylamide gel electrophoresis, Dot blot, Western blot, and Thioflavin-T assay. It was shown that aggregation rates increased in the order αSyn (A30P) < wt-αSyn < αSyn (A53T). In comparison to the sequence of αSyn, βSyn which lacks 11 central hydrophobic residues did not form any aggregates. First interaction studies between αSyn (59-80 T72A) peptide and αSyn at various molar ratios showed that this peptide comprising the aggregation domain provided a significant increase of the aggregation rate of wt-αSyn. In contrast, chemical modification of αSyn by succinylation stabilized its structure and completely inhibited degradation and oligomerization-aggregation.
One of the hallmarks of Parkinson’s disease (PD) and related neurological disorders is the accumulation in human brain of intracellular high molecular weight α-synuclein (αSyn) aggregation products as fibrils. Oligomeric intermediates have been suggested to represent major neurotoxic species; however, chemical structures of αSyn oligomers and possible intermediates have not been hitherto identified. The first parts of this thesis deal with the isolation and structure identification of αSyn oligomerization-aggregation products in vitro and in vivo.
The first part was focused on the molecular characterization of human αSyn (wt-αSyn) and two mutants, αSyn (A53T) and αSyn (A30P) using HPLC, high resolution mass spectrometry, gel electrophoresis and circular dichroism spectroscopy. Additional information about the wt-αSyn structure was obtained by molecular dynamic simulation, which indicated a flexible, random coil structure. Using mass spectrometric methods, proteolytic degradation studies of synucleins were carried out for identifying the cleavage specificity and preferred cleavage sites of different proteases.
The second part of the thesis was focused on the in vitro and in vivo characterization of αSyn oligomerization-aggregation products, employing gel electrophoresis, Dot blot and Western immunoblotting. The in vitro oligomerization of αSyn was carried out by incubation at 37°C in sodium- phosphate (pH 7.5) for up to seven days. The formation of oligomers was monitored by Tris-tricine polyacrylamide gel electrophoresis which revealed bands corresponding to monomeric and oligomer-like αSyn at approximately 37 and 48 kDa. In addition, three bands of minor abundances with molecular weights lower than full-length αSyn indicated the formation of truncation or degradation products. The bands corresponding to αSyn monomer and dimer were excised from the gel, digested with trypsin and analyzed by HPLC-ESI-MS. ESI-MS and tandem-MS of the tryptic peptides revealed the presence of monomeric and dimeric αSyn with full-length sequences, respectively; additional structural characterization was obtained by Edman sequencing. Direct ESI-MS of αSyn upon incubation in an aqueous buffer for 3 hours provided the identification of small amounts of N-terminally truncated αSyn (7-140). In contrast to these results, attempts to identify the truncation and degradation products by direct mass spectrometric analysis and HPLC-MS were unsuccessful, presumably due to their low concentration.
The application of ion-mobility mass spectrometry (IMS-MS) to oligomerization-aggregation mixtures of αSyn in vitro enabled the first identification of specific fragments corresponding to truncation and degradation products that have been previously observed by gel electrophoresis, but not identified. Most important, a highly aggregating fragment was identified, resulting from cleavage at the central aggregation domain of αSyn, between residues Val-71 and Thr-72 (7.2 kDa). These results showed that IMS-MS can be successfully applied to the identification of hitherto undetected truncation products of neurodegenerative target proteins. Aggregation studies of the corresponding carboxy-terminal fragment, αSyn (72-140) prepared by both chemical synthesis and recombinant expression, showed a substantially faster rate of fibrillization compared to the intact full-length αSyn protein. The chemical structure elucidation of in vivo αSyn oligomerization products from human αSyn transgenic mouse brain homogenates and human neuroblastoma cell cultures was performed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and high resolution mass spectrometry, as well as affinity binding studies by online combination of a surface acoustic wave (SAW) biosensor and ESI-MS. Immunoblotting in combination with high resolution- MS provided the identification of αSyn (A30P) from soluble mouse brain homogenate proteins. Spots that were detected as immunoreactive with the anti-αSyn mBD antibody were analyzed by high resolution- MS, leading to the identification of αSyn and of two methionine oxidized products (116Met and 127Met).
In a third part of the thesis, the epitope structures recognized by anti-αSyn specific antibodies were identified. The mass spectrometric analysis was combined with epitope excision and extraction procedures previously developed in our laboratory. For the epitope excision and extraction experiments, affinity columns were prepared by immobilizing the antibodies on Sepharose. For protein digestion trypsin and Glu-C endoproteases were employed, and for analysis MALDI-TOF-MS, as well as ESI-ion trap-MS used. The epitope excision, followed by mass spectrometric analysis provided direct information that the epitopes recognized by polyclonal antibodies (pASY-1; pC20) were discontinuous and located in the N-terminal (1-23) and central (59-80) regions. Comparative binding studies of anti-αSyn specific antibodies with the epitope peptides αSyn (1-23) and αSyn (59-80), and synthetic mutant model peptides were performed by ELISA, Dot blot and affinity- mass spectrometry. The results showed that αSyn (1-23) and αSyn (59-80) bound to the anti-αSyn pASY-1 antibody in a concentration- dependent manner.
A further part of the dissertation was focused on applications of an online combination of a surface acoustic wave (SAW) biosensor with ESI-MS that enabled the direct detection, identification, and quantification of affinity-bound ligands from synuclein- anti-synuclein antibodies complexes on a biosensor chip. The specific antibodies were covalently immobilized on the gold coated surface of the quartz chip. The interactions with wt-αSyn, mutants αSyn (A53T), αSyn (A30P), αSyn peptides and βSyn were determined. The obtained KD values were in the low nano-molar range and in good agreement with bioaffinities investigated by Dot Blot, ELISA, SAW biosensor and SAW-ESI MS. Moreover, the SAW biosensor has been employed as an affinity detection method in conjunction with MALDI-MS for epitope determination of αSyn.
In the last part of the thesis, chemical stability studies of wt-αSyn, αSyn (A53T), αSyn (A30P) and βSyn proteins were performed. The formation of oligomers was monitored by Tris-tricine polyacrylamide gel electrophoresis, Dot blot, Western blot, and Thioflavin-T assay. It was shown that aggregation rates increased in the order αSyn (A30P) < wt-αSyn < αSyn (A53T). In comparison to the sequence of αSyn, βSyn which lacks 11 central hydrophobic residues did not form any aggregates. First interaction studies between αSyn (59-80 T72A) peptide and αSyn at various molar ratios showed that this peptide comprising the aggregation domain provided a significant increase of the aggregation rate of wt-αSyn. In contrast, chemical modification of αSyn by succinylation stabilized its structure and completely inhibited degradation and oligomerization-aggregation.
Summary in another language
Die Massenspektrometrie hat sich in den letzten Jahren als eine wichtige Methode der Strukturanalyse in der Biochemie und Molekularbiologie erwiesen. Insbesondere die Elektrospray- Massenspektrometrie (ESI-MS) hat sich als leistungsfähige Methode der Analyse von Biopolymer- Ionen und von nicht-kovalenten supramolekularen Komplexen etabliert. Während ESI- massenspektrometrische Techniken in vielen Untersuchungen von Proteinstrukturen, Strukturmodifikationen sowie zur Proteomanalytik eingesetzt wurden, waren Anwendungen zur Analyse von Protein- Fehlfaltung und –Aggregation bisher nur wenig erfolgreich; mögliche Ursachen hierfür sind (i), die geringen Konzentrationen von reaktiven Intermediaten, sowie (ii), die langsamen Bildungsgeschwindigkeiten der enstehenden Aggregate. Konventionelle „soft-ionization“ Methoden wie ESI-MS und HPLC-MS sind für die direkte Analyse von Intermediaten der Proteinaggregation nicht geeignet. Dagegen hat sich die Ionenmobilitäts- Massenspektrometrie (IMS-MS) in jüngster Zeit als neue, leistungsfähige Methode zur Analyse von komplexen Biomolekülstrukturen in geringen Konzentrationen entwickelt. Aufgrund der effizienten Trennung von Protein- Komplexen und –Gemischen mit unterschiedlicher Konformation und Topographie stellt die IMS-MS eine neue Methode der Trennung und Differenzierung von Intermediaten von Protein- Aggregaten dar.
Die Akkumulierung von hochmolekularen Aggregaten und fibrillären Strukturen des Hirnproteins Alpha-Synuclein (αSyn) ist ein charakteristisches Merkmal der Parkinson’schen Krankheit und verwandter neurologischer Erkrankungen. Dabei wird die Bildung von oligomeren Intermediaten hoher Neurotoxizität generell als Schlüsselreaktion angenommen; jedoch konnten die Strukturen der Oligomeren und deren möglicher Zwischenprodukte bisher nicht identifiziert werden. Zielsetzungen der vorliegenden Dissertation waren die Isolierung, elektrophoretische und chromatographische Trennung, Strukturaufklärung, sowie die Darstellung und biochemische Charakterisierung von αSyn- Oligomeren.
In einem ersten Teil der Arbeit wurde die Primär- und Sekundärstruktur- Charakterisierung von rekombinantem αSyn (wt-αSyn), sowie von zwei Mutanten [αSyn (A53T); αSyn (A30P)] durchgeführt, vor allem mit massenspektrometrischen, elektrophoretischen Methoden sowie CD- Spektroskopie. Zusätzliche Strukturinformationen wurden mittels Molekülmodell- Simulation erhalten, die eine flexible ungeordnete Struktur der α-Synucleine zeigten. Durch massenspektrometrische Peptide-mapping- Analyse wurden proteolytische Abbaureaktionen mit verschiedenen Proteasen charakterisiert.
In einem zweiten Teil wurden Oligomerisierung- Aggregationsprodukte von αSyn in vitro mittels Gelelektrophorese sowie immunanalytischer Methoden (Immunoblot; ELISA) charakterisiert. Oligomerisierungsreaktionen wurden in Phosphat- Puffer (pH 7.5) mittels Tricin- Gelelektrophorese über verschiedene Zeiten bis zu 7 Tagen verfolgt. Die gelelektrophoretische Analyse zeigte Monomere und Oligomere (ca. 37 und 48 kDa) sowie mehrere Banden mit niedrigeren Molekulargewichten und geringerer Intensität als das monomere αSyn (15 kDa), die auf Abbauprodukte hinwiesen. Die Gelbanden entsprechend dem monomeren und dimeren αSyn wurden isoliert und nach Abbau mit Trypsin durch HPLC-MS charakterisiert. Die massenspektrometrischen Ergebnisse sowie zusätzliche Edman- Sequenzierung der Banden ergaben jeweils vollständige αSyn- Sequenzen. Ein erstes Abbauprodukt mit trunkierter N-terminaler Sequenz αSyn (7-140) konnte nach 3-stündiger Inkubation durch ESI-Massenspektrometrie nachgewiesen werden; dagegen waren Versuche zur direkten massenspektrometrischen Analyse der αSyn- Abbauprodukte ohne Erfolg.
Die Identifizierung von αSyn- Proteinfragmente gelang erstmals mit Hilfe der Ionenmobilitäts-Massenspektrometrie (IMS-MS), wobei Abbauprodukte (Trunkierung) sowie proteolytische Fragmente entsprechend den elektrophoretischen Proteinbanden nachgewiesen wurden. Von besonderem Interesse war die Identifizierung eines C-terminalen Proteinfragments durch Spaltung in der zentralen Aggregationsdomäne (Val71-Thr72), das hohe Reaktivität der Aggregationsbildung zeigte. Diese Ergebnisse zeigen die erfolgreiche Anwendung der IMS-MS zur direkten Aufklärung der bisher nicht identifizierbaren Abbauprodukte. Erste Aggregations- Untersuchungen des durch chemische Festphasensynthese sowie durch rekombinante Expression in E. Coli dargestellten Fragments αSyn (72-140) zeigten eine erhebliche höhere Aggregationsgeschwindigkeit im Vergleich zum intakten αSyn. Weitere Strukturuntersuchungen von αSyn- Oligomerisierungsprodukten in vivo aus Hirnhomogenat von transgenen Mäusen, sowie aus humanen Neuroblastomzellen wurden mittels Gelektrophorese, hochauflösender Massenspektrometrie, sowie durch Kombination eines Bioaffinitäts (Biosensor)- Systems und ESI-MS durchgeführt. Die elektrophoretischen Banden wurden mit einem αSyn- spezifischen Antikörper nachgewiesen, und nach Isolierung aus dem Gel die Mutante αSyn (A30P) sowie Strukturmodifikationen durch Met- Oxidation identifiziert.
In einem dritten Abschnitt der Arbeit wurden Untersuchungen zur Aufklärung der Epitopstrukturen von αSyn- spezifischen Antikörpern unter Anwendung der in unserem Laboratorium entwickelten proteolytischen Excisions- und Extraktions- Methoden in Kombination mit Massenspektrometrie durchgeführt. Hierzu wurden Sepharose-immobiliserte Antikörper zum in situ Abbau mit Trypsin und Glu-C-Endoprotease hergestellt, und MALDI-TOF-MS sowie ESI-MS angewendet. Die massenspektrometrischen Ergebnisse ergaben für zwei polyklonale αSyn- Antikörper (ASY-1; pC20) diskontinuierliche Epitopstrukturen im N-terminalen Bereich (1-23) sowie der zentralen Aggregationsdomäne (59-80). Die Epitope- Peptide sowie weitere αSyn- Partialsequenzen wurden durch Festphasensynthese dargestellt und mit verschiedenen Antikörpern mittels ELISA, Immunoblot und Affinitäts- Massenspektrometrie charakterisiert.
In einem weiteren Abschnitt wurden Epitopstrukturen von αSyn- spezifischen Antikörpern, sowie Antikörper- Interaktionen verschiedener Synucleine und αSyn- Partialpeptide durch direkte (online) Kombination eines SAW (Surface-acoustic-wave)- Biosensors und ESI-MS charakterisiert. Interaktionen wurden für wt-αSyn, αSyn- Mutanten, βSyn sowie verschiedene αSyn- Partialsequenzen bestimmt und ergaben Affinitätskonstanten bzw. KD- Werte im Bereich von 12- ca. 400 nmol, in Übereinstimmung mit den Affinitätsuntersuchungen der Antikörperspezifitäten mit anderen Methoden (ELISA; Affinitäts-Massenspektrometrie).
Im letzten Teil der Arbeit wurden vergleichende Untersuchungen zur chemischen Stabilität und in vitro- Aggregation von αSyn, Mutanten des humanen αSyn, sowie von βSyn mittels Gelelektrophorese, sowie mit dem Thioflavin T-Assay durchgeführt. Mit der Thioflavinmethode konnte gezeigt werden, dass die Aggregationsgeschwindigkeit der αSyn- Mutante (A30P) geringer ist im Vergleich zum wt-αSyn, dagegen zeigte die Mutante αSyn (A53T) eine erhöhte Aggregationsgeschwindigkeit. Im Gegensatz zu den αSyn zeigte das βSyn, bei dem die zentrale hydrophobe Domäne (73-83) fehlt, weder Oligomerisierung noch nachweisbare Aggregation. Erste Untersuchungen zur Interaktion und Aggregation von αSyn- Peptiden mit dem wt-αSyn ergaben eine erhöhte Aggregationsgeschwindigkeit durch die Peptidmutante αSyn (59-80 T72A). Im Gegensatz dazu zeigte ein durch Succinylierung aller Aminogruppen modifiziertes αSyn hohe Stabilität und keine Oligomerisierung und Aggregation.
Die Akkumulierung von hochmolekularen Aggregaten und fibrillären Strukturen des Hirnproteins Alpha-Synuclein (αSyn) ist ein charakteristisches Merkmal der Parkinson’schen Krankheit und verwandter neurologischer Erkrankungen. Dabei wird die Bildung von oligomeren Intermediaten hoher Neurotoxizität generell als Schlüsselreaktion angenommen; jedoch konnten die Strukturen der Oligomeren und deren möglicher Zwischenprodukte bisher nicht identifiziert werden. Zielsetzungen der vorliegenden Dissertation waren die Isolierung, elektrophoretische und chromatographische Trennung, Strukturaufklärung, sowie die Darstellung und biochemische Charakterisierung von αSyn- Oligomeren.
In einem ersten Teil der Arbeit wurde die Primär- und Sekundärstruktur- Charakterisierung von rekombinantem αSyn (wt-αSyn), sowie von zwei Mutanten [αSyn (A53T); αSyn (A30P)] durchgeführt, vor allem mit massenspektrometrischen, elektrophoretischen Methoden sowie CD- Spektroskopie. Zusätzliche Strukturinformationen wurden mittels Molekülmodell- Simulation erhalten, die eine flexible ungeordnete Struktur der α-Synucleine zeigten. Durch massenspektrometrische Peptide-mapping- Analyse wurden proteolytische Abbaureaktionen mit verschiedenen Proteasen charakterisiert.
In einem zweiten Teil wurden Oligomerisierung- Aggregationsprodukte von αSyn in vitro mittels Gelelektrophorese sowie immunanalytischer Methoden (Immunoblot; ELISA) charakterisiert. Oligomerisierungsreaktionen wurden in Phosphat- Puffer (pH 7.5) mittels Tricin- Gelelektrophorese über verschiedene Zeiten bis zu 7 Tagen verfolgt. Die gelelektrophoretische Analyse zeigte Monomere und Oligomere (ca. 37 und 48 kDa) sowie mehrere Banden mit niedrigeren Molekulargewichten und geringerer Intensität als das monomere αSyn (15 kDa), die auf Abbauprodukte hinwiesen. Die Gelbanden entsprechend dem monomeren und dimeren αSyn wurden isoliert und nach Abbau mit Trypsin durch HPLC-MS charakterisiert. Die massenspektrometrischen Ergebnisse sowie zusätzliche Edman- Sequenzierung der Banden ergaben jeweils vollständige αSyn- Sequenzen. Ein erstes Abbauprodukt mit trunkierter N-terminaler Sequenz αSyn (7-140) konnte nach 3-stündiger Inkubation durch ESI-Massenspektrometrie nachgewiesen werden; dagegen waren Versuche zur direkten massenspektrometrischen Analyse der αSyn- Abbauprodukte ohne Erfolg.
Die Identifizierung von αSyn- Proteinfragmente gelang erstmals mit Hilfe der Ionenmobilitäts-Massenspektrometrie (IMS-MS), wobei Abbauprodukte (Trunkierung) sowie proteolytische Fragmente entsprechend den elektrophoretischen Proteinbanden nachgewiesen wurden. Von besonderem Interesse war die Identifizierung eines C-terminalen Proteinfragments durch Spaltung in der zentralen Aggregationsdomäne (Val71-Thr72), das hohe Reaktivität der Aggregationsbildung zeigte. Diese Ergebnisse zeigen die erfolgreiche Anwendung der IMS-MS zur direkten Aufklärung der bisher nicht identifizierbaren Abbauprodukte. Erste Aggregations- Untersuchungen des durch chemische Festphasensynthese sowie durch rekombinante Expression in E. Coli dargestellten Fragments αSyn (72-140) zeigten eine erhebliche höhere Aggregationsgeschwindigkeit im Vergleich zum intakten αSyn. Weitere Strukturuntersuchungen von αSyn- Oligomerisierungsprodukten in vivo aus Hirnhomogenat von transgenen Mäusen, sowie aus humanen Neuroblastomzellen wurden mittels Gelektrophorese, hochauflösender Massenspektrometrie, sowie durch Kombination eines Bioaffinitäts (Biosensor)- Systems und ESI-MS durchgeführt. Die elektrophoretischen Banden wurden mit einem αSyn- spezifischen Antikörper nachgewiesen, und nach Isolierung aus dem Gel die Mutante αSyn (A30P) sowie Strukturmodifikationen durch Met- Oxidation identifiziert.
In einem dritten Abschnitt der Arbeit wurden Untersuchungen zur Aufklärung der Epitopstrukturen von αSyn- spezifischen Antikörpern unter Anwendung der in unserem Laboratorium entwickelten proteolytischen Excisions- und Extraktions- Methoden in Kombination mit Massenspektrometrie durchgeführt. Hierzu wurden Sepharose-immobiliserte Antikörper zum in situ Abbau mit Trypsin und Glu-C-Endoprotease hergestellt, und MALDI-TOF-MS sowie ESI-MS angewendet. Die massenspektrometrischen Ergebnisse ergaben für zwei polyklonale αSyn- Antikörper (ASY-1; pC20) diskontinuierliche Epitopstrukturen im N-terminalen Bereich (1-23) sowie der zentralen Aggregationsdomäne (59-80). Die Epitope- Peptide sowie weitere αSyn- Partialsequenzen wurden durch Festphasensynthese dargestellt und mit verschiedenen Antikörpern mittels ELISA, Immunoblot und Affinitäts- Massenspektrometrie charakterisiert.
In einem weiteren Abschnitt wurden Epitopstrukturen von αSyn- spezifischen Antikörpern, sowie Antikörper- Interaktionen verschiedener Synucleine und αSyn- Partialpeptide durch direkte (online) Kombination eines SAW (Surface-acoustic-wave)- Biosensors und ESI-MS charakterisiert. Interaktionen wurden für wt-αSyn, αSyn- Mutanten, βSyn sowie verschiedene αSyn- Partialsequenzen bestimmt und ergaben Affinitätskonstanten bzw. KD- Werte im Bereich von 12- ca. 400 nmol, in Übereinstimmung mit den Affinitätsuntersuchungen der Antikörperspezifitäten mit anderen Methoden (ELISA; Affinitäts-Massenspektrometrie).
Im letzten Teil der Arbeit wurden vergleichende Untersuchungen zur chemischen Stabilität und in vitro- Aggregation von αSyn, Mutanten des humanen αSyn, sowie von βSyn mittels Gelelektrophorese, sowie mit dem Thioflavin T-Assay durchgeführt. Mit der Thioflavinmethode konnte gezeigt werden, dass die Aggregationsgeschwindigkeit der αSyn- Mutante (A30P) geringer ist im Vergleich zum wt-αSyn, dagegen zeigte die Mutante αSyn (A53T) eine erhöhte Aggregationsgeschwindigkeit. Im Gegensatz zu den αSyn zeigte das βSyn, bei dem die zentrale hydrophobe Domäne (73-83) fehlt, weder Oligomerisierung noch nachweisbare Aggregation. Erste Untersuchungen zur Interaktion und Aggregation von αSyn- Peptiden mit dem wt-αSyn ergaben eine erhöhte Aggregationsgeschwindigkeit durch die Peptidmutante αSyn (59-80 T72A). Im Gegensatz dazu zeigte ein durch Succinylierung aller Aminogruppen modifiziertes αSyn hohe Stabilität und keine Oligomerisierung und Aggregation.
Subject (DDC)
540 Chemistry
Keywords
Parkinson’s disease,analytical chemistry,ion-mobility mass spectrometry,alpha-synuclein
Conference
Review
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Cite This
ISO 690
VLAD, Camelia, 2011. Oligomerization, Degradation and Aggregation Reactions and Products of Synuclein Polypeptides Related to Parkinson’s Disease [Dissertation]. Konstanz: University of KonstanzBibTex
@phdthesis{Vlad2011Oligo-14788,
year={2011},
title={Oligomerization, Degradation and Aggregation Reactions and Products of Synuclein Polypeptides Related to Parkinson’s Disease},
author={Vlad, Camelia},
address={Konstanz},
school={Universität Konstanz}
}
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<dc:contributor>Vlad, Camelia</dc:contributor>
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<dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-08-31T10:58:37Z</dc:date>
<dcterms:alternative>Oligomerisierungs-, Abbau- und Aggregationsreaktionen und Produkte von Synuclein Polypeptide bezüglich der Parkinson’schen Krankheit</dcterms:alternative>
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<dcterms:abstract xml:lang="eng">In recent years, mass spectrometry (MS) has become a major analytical tool in biochemistry and structural biology. In particular, electrospray mass spectrometry (ESI-MS) has emerged as a powerful technique for analyzing intact gas phase ions from large biomolecules and supramolecular complexes. However, in contrast to the large number of ESI-mass spectrometric studies of protein structures, structure modifications and proteomics, the molecular characterization of protein “misfolding” and aggregation species by mass spectrometry had hitherto little success; possible explanations are (i), the low concentration of intermediate species, and (ii), the slow rates of aggregate formation in vitro. Conventional “soft-ionization” mass spectrometric methods such as ESI-MS and HPLC-MS are not suitable to direct “in-situ” analysis of conformational states and intermediates at different concentrations. Recently, ion-mobility mass spectrometry (IMS-MS) is emerging as a new tool to probe complex biomolecular structures, due to its potential to separate mixtures of protein complexes by conformation state, spatial shape and topology. Thus, IMS-MS implements a new mode of separation that allows the differentiation of protein conformational states.<br /><br /><br />One of the hallmarks of Parkinson’s disease (PD) and related neurological disorders is the accumulation in human brain of intracellular high molecular weight α-synuclein (αSyn) aggregation products as fibrils. Oligomeric intermediates have been suggested to represent major neurotoxic species; however, chemical structures of αSyn oligomers and possible intermediates have not been hitherto identified. The first parts of this thesis deal with the isolation and structure identification of αSyn oligomerization-aggregation products in vitro and in vivo.<br /><br /><br />The first part was focused on the molecular characterization of human αSyn (wt-αSyn) and two mutants, αSyn (A53T) and αSyn (A30P) using HPLC, high resolution mass spectrometry, gel electrophoresis and circular dichroism spectroscopy. Additional information about the wt-αSyn structure was obtained by molecular dynamic simulation, which indicated a flexible, random coil structure. Using mass spectrometric methods, proteolytic degradation studies of synucleins were carried out for identifying the cleavage specificity and preferred cleavage sites of different proteases.<br /><br /><br />The second part of the thesis was focused on the in vitro and in vivo characterization of αSyn oligomerization-aggregation products, employing gel electrophoresis, Dot blot and Western immunoblotting. The in vitro oligomerization of αSyn was carried out by incubation at 37°C in sodium- phosphate (pH 7.5) for up to seven days. The formation of oligomers was monitored by Tris-tricine polyacrylamide gel electrophoresis which revealed bands corresponding to monomeric and oligomer-like αSyn at approximately 37 and 48 kDa. In addition, three bands of minor abundances with molecular weights lower than full-length αSyn indicated the formation of truncation or degradation products. The bands corresponding to αSyn monomer and dimer were excised from the gel, digested with trypsin and analyzed by HPLC-ESI-MS. ESI-MS and tandem-MS of the tryptic peptides revealed the presence of monomeric and dimeric αSyn with full-length sequences, respectively; additional structural characterization was obtained by Edman sequencing. Direct ESI-MS of αSyn upon incubation in an aqueous buffer for 3 hours provided the identification of small amounts of N-terminally truncated αSyn (7-140). In contrast to these results, attempts to identify the truncation and degradation products by direct mass spectrometric analysis and HPLC-MS were unsuccessful, presumably due to their low concentration.<br /><br />The application of ion-mobility mass spectrometry (IMS-MS) to oligomerization-aggregation mixtures of αSyn in vitro enabled the first identification of specific fragments corresponding to truncation and degradation products that have been previously observed by gel electrophoresis, but not identified. Most important, a highly aggregating fragment was identified, resulting from cleavage at the central aggregation domain of αSyn, between residues Val-71 and Thr-72 (7.2 kDa). These results showed that IMS-MS can be successfully applied to the identification of hitherto undetected truncation products of neurodegenerative target proteins. Aggregation studies of the corresponding carboxy-terminal fragment, αSyn (72-140) prepared by both chemical synthesis and recombinant expression, showed a substantially faster rate of fibrillization compared to the intact full-length αSyn protein. The chemical structure elucidation of in vivo αSyn oligomerization products from human αSyn transgenic mouse brain homogenates and human neuroblastoma cell cultures was performed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and high resolution mass spectrometry, as well as affinity binding studies by online combination of a surface acoustic wave (SAW) biosensor and ESI-MS. Immunoblotting in combination with high resolution- MS provided the identification of αSyn (A30P) from soluble mouse brain homogenate proteins. Spots that were detected as immunoreactive with the anti-αSyn mBD antibody were analyzed by high resolution- MS, leading to the identification of αSyn and of two methionine oxidized products (<sup>116</sup>Met and <sup>127</sup>Met).<br /><br /><br />In a third part of the thesis, the epitope structures recognized by anti-αSyn specific antibodies were identified. The mass spectrometric analysis was combined with epitope excision and extraction procedures previously developed in our laboratory. For the epitope excision and extraction experiments, affinity columns were prepared by immobilizing the antibodies on Sepharose. For protein digestion trypsin and Glu-C endoproteases were employed, and for analysis MALDI-TOF-MS, as well as ESI-ion trap-MS used. The epitope excision, followed by mass spectrometric analysis provided direct information that the epitopes recognized by polyclonal antibodies (pASY-1; pC20) were discontinuous and located in the N-terminal (1-23) and central (59-80) regions. Comparative binding studies of anti-αSyn specific antibodies with the epitope peptides αSyn (1-23) and αSyn (59-80), and synthetic mutant model peptides were performed by ELISA, Dot blot and affinity- mass spectrometry. The results showed that αSyn (1-23) and αSyn (59-80) bound to the anti-αSyn pASY-1 antibody in a concentration- dependent manner.<br /><br /><br />A further part of the dissertation was focused on applications of an online combination of a surface acoustic wave (SAW) biosensor with ESI-MS that enabled the direct detection, identification, and quantification of affinity-bound ligands from synuclein- anti-synuclein antibodies complexes on a biosensor chip. The specific antibodies were covalently immobilized on the gold coated surface of the quartz chip. The interactions with wt-αSyn, mutants αSyn (A53T), αSyn (A30P), αSyn peptides and βSyn were determined. The obtained KD values were in the low nano-molar range and in good agreement with bioaffinities investigated by Dot Blot, ELISA, SAW biosensor and SAW-ESI MS. Moreover, the SAW biosensor has been employed as an affinity detection method in conjunction with MALDI-MS for epitope determination of αSyn.<br /><br /><br />In the last part of the thesis, chemical stability studies of wt-αSyn, αSyn (A53T), αSyn (A30P) and βSyn proteins were performed. The formation of oligomers was monitored by Tris-tricine polyacrylamide gel electrophoresis, Dot blot, Western blot, and Thioflavin-T assay. It was shown that aggregation rates increased in the order αSyn (A30P) < wt-αSyn < αSyn (A53T). In comparison to the sequence of αSyn, βSyn which lacks 11 central hydrophobic residues did not form any aggregates. First interaction studies between αSyn (59-80 T72A) peptide and αSyn at various molar ratios showed that this peptide comprising the aggregation domain provided a significant increase of the aggregation rate of wt-αSyn. In contrast, chemical modification of αSyn by succinylation stabilized its structure and completely inhibited degradation and oligomerization-aggregation.</dcterms:abstract>
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<dc:language>eng</dc:language>
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<dc:creator>Vlad, Camelia</dc:creator>
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<dcterms:title>Oligomerization, Degradation and Aggregation Reactions and Products of Synuclein Polypeptides Related to Parkinson’s Disease</dcterms:title>
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<dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2012-07-30T22:25:07Z</dcterms:available>
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Examination date of dissertation
July 27, 2011