Autoproteolytic Fragments are Intermediates in the Oligomerization- Aggregation of Parkinson’s Disease Protein Alpha-Synuclein as Revealed by Ion Mobility Mass Spectrometry

Several neurodegenerative diseases are characterized by the formation and accumulation of “misfolded” polymeric protein aggregates^[1-3]. The formation of neurotoxic oligomers is generally thought to precede aggregation, as shown for ß-amyloid polypeptide (Aβ), Tauprotein, and alpha-Synuclein (αSyn) the key protein in Parkinson’s disease (PD) ^[4-6]. αSyn, a 140-aa protein, occurring in the presynaptic terminals of neurons is natively unfolded but adopts an α-helical structure when it binds to lipid vesicles, and forms a β-sheet that facilitates the formation of aggregated morphologies^[7,8]. Intracellular accumulation of αSyn aggregates has been recognized at conditions inducing PD^[8,9]. Although oligomeric intermediates may represent major neurotoxic species^[9], detailed chemical structures of αSyn oligomers and their possible intermediates have not been hitherto identified. The slow rates of formation and the low concentrations of aggregating intermediates^[9] may be major reasons for the failure of conventional methods of mass spectrometry (MS) to detect and identify oligomers.

Although MS (especially with electrospray ionization, ESI) ^[10] is now established in biochemistry and structural biology and is proven highly successful for the structure determination of biopolymers from biological samples^[11], low-concentration intermediates and oligomers in αSyn aggregation have not been identified by HPLC-MS. Nevertheless, oligomeric components were observed by gel electrophoresis, atomic force microscopy (AFM), and electron paramagnetic resonance (EPR)^[12-14].

Ion mobility mass spectrometry (IMS-MS) is now emerging as a new tool for the analysis of molecular assemblies of proteins^[15-18]. The ability of IMS-MS to separate ions as they pass through an electric drift field allows components in protein mixtures to be separated and differentiated according to their size, charge and conformation-dependent topography, as determined by differences in their collisional cross-sections^[16-18]. In applications of IMS-MS to oligomerization-aggregation mixtures of αSyn in vitro, we report here the first identification of specific autoproteolytic truncation and degradation products that were previously observed by gel electrophoresis, but not identified. In particular, a highly aggregating fragment was identified by cleavage at the central aggregation domain of αSyn, between residues Val-71 and Thr-72. Aggregation studies of the carboxy-terminal fragment, αSyn(72-140) prepared by both chemical synthesis and recombinant expression, showed a substantially faster fibrillization compared to the intact protein.

The in vitro oligomerization of αSyn was investigated by incubation at 37 °C in sodium-phosphate (pH 7.5) for up to 25 days using a previously established procedure^[19]. Recombinant αSyn expressed in E. Coli^[20] was purified by HPLC to molecular homogeneity, as confirmed by ESI-MS (Figure S1, Supporting Information). The formation of oligomers was monitored by tris-Tricine PAGE (Figure 1) that revealed bands corresponding to monomeric and oligomer-like αSyn having molecular weights of approximately 17, 35, and 48 kDa. In addition, protein bands with molecular weights lower than full length αSyn were observed with slowly increasing abundances within two weeks of incubation, indicating the formation of truncation and/or degradation products. The bands corresponding to αSyn monomer 1a at 17 kDa and dimer 1a’ at 35 kDa were excised from the gel, digested with trypsin, and analyzed by HPLC-ESI-MS (Table S2, Supporting Information). Sequencing of the tryptic peptides confirmed that the monomers and dimers of αSyn contain full-length sequences, respectively; indeed, we found all the expected tryptic peptides and no truncated sequences. Further structural characterization was obtained by N-terminal Edman sequencing after transfer of the proteins onto a PVDF membrane, which yielded the first 20 amino acids of intact αSyn. Direct ESI-MS analysis of the αSyn incubation mixture after ashort time period (3 h), however, revealed a small amount of N-terminally truncated αSyn lacking the first 6 amino acids (αSyn(7-140); spot 1b in Figure 1; Table S1 and Figure S2, Supporting Information). In contrast to these results, attempts to identify the degradation products (bands 1c, 1d, 1e in Figure 1) by direct ESI-MS and HPLC-MS were unsuccessful, presumably owing to their low concentrations.

Figure 1.

Time-dependent autoproteolytic degradation of αSyn and modified αSyn polypeptides visualized by Coomassie (left) and silver staining (right), at the beginning of incubation (A), 4 days (B) and 13 days (C). 1, wt-αSyn; 2, recombinant αSyn(Ala₆(70-75)); 3, recombinant αSyn(Gly₆(70-75)); 4, αSyn(72-140) synthesized by SPPS; 5, recombinant αSyn(72-140); 6, recombinant βSyn. Full length αSyn monomer and dimer are denoted 1a and 1a’, respectively; N-terminally truncated αSyn(7-140) is denoted 1b. αSyn fragments are denoted 1c, 1d, 1e; 1c’ is the dimer of fragment 1c. β-Synuclein 6 lacks the (72-83) sequence. 1 and 1+ denote incubation of wt-αSyn without and with addition of protease inhibitors (serine-, cysteine-proteases; metalloproteases).

The reaction mixtures upon incubation of αSyn for 4 up to 21 days were subjected to ion mobility-MS analysis, which provides inherently three-dimensional data consisting of ion mobility-dependent drift time and separation according to differences in protein collisional cross sections^[15,16], mass/charge, and relative abundances. Broad-band admittance to the drift region afforded an ion-mobility plot of the incubation mixture after 7 days and showed separation into two peaks corresponding to multiply charged ion series with different drift times (Figure 2A; Figure S3, Supporting Information). These proteins were identified by molecular-mass determinations and ESI-tandem-MS sequencing (Figure 2B-D). Deconvolution of the multiply charged ion series to singly charged ions provided identification of full length αSyn monomer (14459.4 Da) and dimer (28919.6 Da) in peak 2, in agreement with the mass spectrometric identification of the gel electrophoretic bands 1a and 1a’ in Figure 1 (Figure 2C and Table S2, Supporting Information). In contrast to the intact αSyn proteins, the IMS-MS analysis of peak 1 at a decreased drift time revealed proteolytic products arising by N- and C-terminal truncation, αSyn(14-133) (12162.5 Da) and αSyn(40-140) (10436.4 Da), corresponding to the proteolytic gel bands 1c, 1e; Figure 1 and Table S1, Supporting Information). A remarkable fragment was identified in the ion series within peak 1; it arose by cleavage at residues Val-71 and Thr-72 in the central amyloidogenic domain αSyn(61-93), corresponding to the gel electrophoretic band 1d (Figure 1). This carboxy-terminal fragment, αSyn(72-140) was identified by (i), accurate mass determination (7274.412 Da) and tandem-MS sequencing in the IMS-MS experiment (Figure 2C, 2D); (ii), moreover, incubation of αSyn for extended time periods (>14 days) afforded formation of this fragment in amounts sufficient for direct elution of the electrophoretic band and identification by MALDI-MS (Table S1, Supporting Information). Additional characterization of the C-terminal fragments αSyn(40-140) and αSyn(72-140) was obtained by affinity-mass spectrometry^[21] and Western-Blot analysis with a monoclonal αSyn antibody recognizing a C-terminal αSyn epitope, as identified by proteolytic-excision mass spectrometry^[22] (data not shown).

Figure 2.

ESI-IMS- MS identification of full length αSyn and fragment αSyn(72-140) in the incubation mixture of wt-αSyn after 7 days. (A), IMS-MS Drift time vs. m/z of αSyn incubation mixture in PBS buffer (pH 7.5) after seven days at 37 °C. (B), Deconvoluted mass spectrum of ions corresponding to peak 1, proteolytic fragment αSyn(72-140); (C), Deconvoluted mass spectrum of ions corresponding to peak 2 showing full length αSyn monomer and dimer; (D), Tandem-MS sequence determination of αSyn(72-140) , peak 1, showing the partial sequence (101-119).

The proteolytic fragment αSyn(72-140) was synthesized by (i), solid-phase peptide synthesis on a semi-automated peptide synthesizer using the Fmoc-strategy with double coupling and capping in the final 30 cycles (band 4), and (ii), recombinant expression in E. Coli^[20] (band 5; s. Figure 1). Polypeptides prepared by both methods were purified to homogeneity by semi-preparative HPLC; their molecular masses and sequences agreed with expected values as determined by ESI-MS and tandem-MS sequencing (Figure S4, Supporting Information). tris-Tricine PAGE of 4 and 5 showed identical major bands corresponding to MWs of approximately 12 kDa, in agreement with the band of the autoproteolytic fragment 1d of intact αSyn; remarkably, in contrast to intact αSyn PAGE the fragment 5 revealed the rapid formation of oligomers (s. Figure 3A). The in vitro aggregation of αSyn(72-140) 5 was analyzed in comparison with full length αSyn 1 for up to 5 days at 7-30 μM concentrations by using Thioflavin T (ThT) fluorescence^[23] as an established fibrillization assay, and the results compared with the oligomerization analysis using tris-Tricine PAGE (Figure 3). Although exact monitoring of fibrillization kinetics was hampered by increasing insolubility of 5 at longer aggregation times, the results showed a substantially faster aggregation of αSyn(72-140). In contrast to full-length αSyn, the fragment 5 showed a typical sigmoidal curve characteristic of reactive aggregating polypeptides.

Figure 3.

Thioflavin T fibrillization of the αSyn(72-140) fragment 5 (A) compared to intact wt- αSyn 1 (B). Proteins were incubated at 30 μM concentrations for 5 days in 20 mM Na₂HPO₄ pH 7.5 at 37°C, and ThT fluorescence determined 450 nm excitation and 486 nm emission (F_450/486). The final assay volume (100 μL) contained 25 μM ThT and 7 μM αSyn sample. The inserts show tris-Tricine-PAGE at the begin of incubation (left) and after 4 days (right) using silver nitrate staining.

The autoproteolytic degradation of αSyn and the significance of the fragment αSyn(72-140) were ascertained by a number of additional mass spectrometric and gel electrophoretic data, and by control experiments with several synuclein polypeptides. Comparative in vitro studies of the non-aggregating brain protein, β-synuclein (βSyn) that lacks the central amyloidogenic domain (72-83), showed neither oligomerization-aggregation, nor any autoproteolytic cleavage within 21 days of incubation (Figure 1, 6); identical expression systems and HPLC purification procedures were employed for ßSyn and αSyn, thus excluding αSyn degradation by an external protease. Further results provided evidence to exclude a proteolytic cleavage of αSyn by contaminating proteases; : (i), incubation of αSyn with and without addition of a broad spectrum of protease inhibitors to yield identical autoproteolytic degradation (Figure 1; 1+ and 1, right lane); (ii), boiling of samples prior to incubation and use of sterile buffers to show no change of αSyn autoproteolysis; and (iii), observation to show autoproteolytic degradation for αSyn mutants 2 and 3 in which the six residues (70-75) were exchanged against Ala and Gly residues (s. Figure 1). Moreover, a recent study on the interaction of αSyn with ß-glucocerebrosidase (GCase), the target enzyme of Gaucher’s disease, in cortical neurons lends additional support to the validity of our results, showing that the neurotoxicity-enhancing effect of αSyn upon depletion of GCase is abolished in a mutant αSyn lacking the sequence (71-82)^[24].

Further support for the importance of the central domain for the autoproteolysis of αSyn was obtained from initial hydrogen-deuterium exchange mass spectrometry (HDX-MS)^[25] studies using high resolution ESI-MS. HDX-MS of full length αSyn 1 showed rapid exchange (<3 hrs) for 115 of 134 backbone hydrogens while 19 aa remained resistant to exchange for >14 days (data not shown). Remarkably, an identical number of 19 aa in the αSyn(72-140) fragment 5 was found resistant to HDX (exact determination of the shielded domain is currently performed). These results suggest shielding of a C-terminal part of the hydrophobic amyloidogenic domain within residues 73/74 and 93, and are consistent with autoproteolytic accessibility at residues 71/72.

In conclusion, ion-mobility MS, owing to its capability to separate αSyn and its autoproteolytic products even though the concentration of the latter is small, enabled the characterization of intermediate fragments in the in vitro oligomerization-aggregation; particularly, IMS-MS revealed a possible key fragment, αSyn(72-140). The yet unknown mechanism of autoproteolysis, possibly proceeding via initial N-terminal truncation products observed, may be amenable to examination of specific functional αSyn residues. Thus, modification of full length αSyn 1 by amino-succinylation^[26] provided a stable derivative succinylated at all Lys residues and at His50 as confirmed by ESI-MS; this succinylated αSyn did not show any aggregation or fragmentation, but did form a dimer (Figure S5, Supporting Information). The detailed biochemical evaluation of autoproteolytic products, presently carried out in our laboratories, may provide a key to the elucidation of the oligomerization-aggregation mechanism of αSyn underlying its neurotoxicity. Moreover, the application of IMS-MS is expected to be highly valuable for identifying proteolytic products in the in vivo aggregation of αSyn.

Experimental Section

Gel electrophoresis of αSyn oligomerization in vitro

αSyn oligomers were prepared by incubation of several batches in triplicate using 5 - 30 μM solutions for up to 25 days in sodium phosphate buffer (pH 7.5), as previously described^[19]. Proteins were solubilized and denatured by using stock solutions of sample buffer containing 4 % SDS, 25 % glycerol, 50 mM tris-buffer and 6 M urea, pH 6.8; gels were run at 100 V until the tracking dye reached the bottom. Separations were performed by tris-Tricine PAGE on a Mini-Protean-3 cell (BioRad, München, Germany) using 12-15 % PAGE, and protein bands visualized by Coomassie Blue and silver staining (90 × 60 × 1 mm gels).

For the protease inhibitor mixture (Complete-Mini; Roche Applied Science, Mannheim, Germany) ca. 50 μM was dissolved in 1.5 mL 20 mM phosphate buffer pH 7.5.

Ion mobility mass spectrometry

Ion mobility mass spectrometry was performed on a Synapt-G1 QTOF-mass spectrometer (Waters, Manchester, UK) equipped with an electrospray ionization source. Ions were passed through a quadrupole, either set to transmit a substantial mass range or to select a particular m/z ion before entering the Triwave ion mobility unit comprising a T-Wave trap unit for ion accumulation; stored ions were gated (500 μsec) into the IMS T-Wave unit for separation according to their mobilities and passed to the T-Wave unit for transfer into the orthogonal-TOF analyzer^[15a] (Figure S3; Supporting Information). Pressures in the T-Wave trap and transfer regions were 7e-2 mbar (Ar) and 0.5 mbar (nitrogen), respectively; sample injection volume was 5 μL. IMS acquisition was performed at 350 - 4000 m/z range, 25 V cone voltage, 0.45 bar IMS pressure, and 5-15 V wave height. Sample injection was performed by using an Advion Triversa Nanomate, as previously described^[18a,b].

Synthesis of αSyn polypeptides

Solid-phase synthesis of αSyn(72-140) 4 was performed on a semi-automated peptide synthesizer EPS-221 (Intavis, Köln, Germany) using a NovaSyn TGR resin; Fmoc protection was employed with double coupling and capping within residues (73-98), followed by mass spectrometric monitoring of intermediate crude products^{[21a, 27]}. Fmoc amino acids, NovaSyn TGR resin, and other reagents for activation and protection were obtained from Novabiochem (Laufelfingen, Switzerland). The general protocol applied was: DMF washing followed by deprotection with 20% piperidine in DMF; 8 min deprotection, 30 min coupling. Coupling was carried out in a mixture solution containing PyBOP and N-methylmorpholine (NMM) in DMF; the resin was then washed with DMF, ethanol and dried under vacuum^[27]. Final deprotection of side chains and cleavage from the resin was performed with trifluoroacetic acid / triethylsilane / water (95: 2.5: 2.5, v/v/v) for 2 h. Resin and crude peptide were separated by filtration, and the crude peptide submitted to HPLC purification.

Recombinant expression of full length αSyn 1 and αSyn(72-140) 4 was performed by using the E. Coli expression system BL 21 (DE3) [pLys] strain and the T7 RNA polymerase system. Harvested cells were centrifuged, resuspended in PBS and heated for 2 min to 100 °C; the cell suspension was centrifuged for 15 min at 4300 rcf and the protein redissolved in PBS buffer. HPLC purification of synthetic and recombinant αSyn polypeptides was performed with a BioRad-3000 semipreparative system (BioRad, Richmond, CA) on a Vydac-C4 column (250 × 4.6 mm) with a linear gradient (0.1 % TFA/0.1 % TFA/80% acetonitrile) at a flow rate of 1 mL/min (Figure S4; Supporting Information).

Thioflavin-T (ThT) aggregation

Purified αSyn 1 and αSyn(72-140) 5 were dissolved in 20 mM Na₂HPO₄ containing 0.03% NaN₃ (pH 7.5) at 7 up to 30 μM in microcentrifuge tubes, and triplicates incubated at 37 °C with agitation. For ThT assay a 23 μL sample was withdrawn at regular time intervals. The final assay volume (100 μL) contained 25 μM ThT/7 μM protein; a blank of 25 μM ThT was used as a control. Determinations were performed in 96-well MTP with a Victor-2 fluorescence plate reader (Perkin-Elmer, Überlingen, Germany) at 450 nm excitation and 486 nm emission. Data were analyzed with Graph Pad Prism wherein the sigmoidal increase of the ThT fluorescence was analyzed.