Glycosaminoglycans have variable effects on α-synuclein ... · Soumik Ray1, Nitu Singh1, Ashutosh...

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  • S 1

    Supplementary Information

    Glycosaminoglycans have variable effects on α-synuclein aggregation and differentially affect the

    activities of the resulting amyloid fibrils

    Surabhi Mehra1†, Dhiman Ghosh1†, Rakesh Kumar1, Mrityunjoy Mondal1, Laxmikant G. Gadhe1,

    Subhadeep Das1,2, Arunagiri Anoop1, Narendra N. Jha1, Reeba S. Jacob1, Debdeep Chatterjee1,

    Soumik Ray1, Nitu Singh1, Ashutosh Kumar1 and Samir K. Maji1*

    1Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai-

    400076, India 2IITB-Monash Research Academy, Indian Institute of Technology Bombay, Mumbai-400076, India,

    †Equally contributed authors

    Running title: Glycosaminoglycans modulate α-synuclein fibrillation

    *To whom correspondence should be addressed: Dr. Samir K. Maji, Associate Professor, Department of

    Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai 400 076, India, Tel: +(91-22) 2576-7774,

    Fax: +(91-22) 2572 3480. Email: [email protected]

    Keywords: α-synuclein, glycosaminoglycan, amyloid, Parkinson’s disease, neurodegeneration,

    amyloidogenesis

    mailto:[email protected]

  • S 2

    Supplementary Text.

    Secondary structural changes of α-Syn in the presence of various GAGs. α-Syn is an intrinsically

    disordered protein and shows random coil in CD, immediately after dissolution and preparation of LMW

    in physiological buffer. However, after prolonged incubation, the protein converts into β-sheet rich

    structure. Therefore, CD provides valuable information about the conformational transition during

    amyloid formation. The CD spectra of α-Syn in the presence and absence of different GAGs showed

    random coil at 0 h with single minima at ~198-202 nm as expected (Fig. S2). The secondary structural

    content gradually increased during incubation as shown by a decrease in minima at 198 nm and an

    increase of helical character, manifested by two minima, one occurring at 205-208 nm and other at 220-

    222 nm. The conformational transition ultimately leads to β-sheet-rich structure as observed by negative

    ellipticity minima (~218 nm after 108 h of incubation). In agreement with ThT study, in the presence of

    Hep 0.25, α-Syn showed an accelerated conformational transition to β-sheet at 72 h. In the presence of

    Hep 2, α-Syn remained mostly unstructured until 72 h and converted to β-sheet at ~130 h. α-Syn in the

    presence of CSA also showed random coil to β-sheet transformation. CSB stabilized the helical

    intermediate for an extended time before converting to the β-sheet. In the presence of different

    concentrations of Dext, α-Syn showed a similar trend in conformation transition as that of α-Syn alone

    sample.

    In case of PVS, α-Syn underwent rapid conformational transition compared to other GAGs under study.

    All the kinetics experiment with PVS was carried out at a concentration of 10 µM due to its higher

    aggregation rate at a high concentration of protein. In the presence of PVS 0.25, α-Syn remained in

    helical conformation for an extended time and did not convert to β-sheet structure even after 5 days of

    incubation. In contrast, α-Syn instantaneously converted to the β-sheet in the presence of PVS 1 and PVS

    2. Initially, in the presence of PVS 0.5, α-Syn showed the random coil structure and got converted to β-

    sheet after 60 min via a helical intermediate (Fig. S2, PVS). These results suggest that PVS has a stronger

    effect on the conformational transition and amyloid formation of α-Syn.

    Material and Methods

    Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Samples for SDS-

    PAGE were mixed with 5X SDS-loading buffer and boiled at 95oC for 5 mins in a water bath. Equal

    volume of samples was analyzed on 15 % SDS-PAGE followed by Coomassie blue staining. Prestained

    protein ladder (Puregene) was resolved on the same gel as a reference. Densitometric analysis of protein

    bands was done by Image J software and average intensity was calculated.

    Preparation of FITC-labeled fibrils in the presence of GAGs. Unlabelled α-Syn was dissolved in 20

    mM Gly-NaOH buffer, pH 7.4 containing 0.01% NaN3 and centrifuged at 13,000 rpm for 30 mins to

    remove any higher order aggregates. The LMW protein was prepared by passing through pre-washed and

    activated 100 kDa MW cut-off filters (Centricon YM-100, Millipore, USA). The concentration of the

    flow through was determined by measuring the absorbance at 280 nm and taking 5960 M-1cm-1 as the

    molar absorptivity (ε) for α-Syn. For the preparation of labeled α-Syn stock, 10 mg of FITC-labeled α-

    Syn was dissolved in 350 µl of 20 mM Gly-NaOH buffer (pH 7.4), and its concentration was measured as

    per the manufacturer's recommendation (Invitrogen).

  • S 3

    Hep, Dext, CSA, CSB, and PVS were dissolved in 20 mM Gly-NaOH buffer, pH 7.4, 0.01% sodium

    azide. Chitosan was dissolved in pre-warmed Gly-NaOH buffer (pH 7.4, 0.01% NaN3) by adding few

    microliters of glacial acetic acid. Final pH was then adjusted to 7.4 for all GAGs, except chitosan where

    the pH was set in range 6.5 to 7.0.

    Finally, unlabelled LMW α-Syn was mixed with 10 % labeled wild-type α-Syn with a final concentration

    of the solution adjusted to 300 µM in 20 mM Gly-NaOH buffer, pH 7.4, 0.01% NaN3 in the presence of

    GAGs. Molar ratio of protein:GAGs were kept as Hep 1:2, Hep 1:0.25, Dext 1:1, CSA 1:1, CSB 1:1,

    Chitosan 1:1, PVS 1:1. Two different molar ratios were chosen for Hep as it showed contrasting results at

    1:2 and 1:0.25. Finally, the reaction mixture was incubated at 37oC in rotating shaker (EchoTherm model

    RT11 (Torrey Pines Scientific, USA) at 30 r.p.m for 10 days for the formation of amyloid fibrils. Labeled

    and unlabelled α-Syn controls (without GAGS) were also kept simultaneously. The fibril formation at the

    end of incubation period was confirmed by CD spectroscopy, and fibril morphology was observed by

    TEM.

    Preparation of FITC-labeled fibril seeds. FITC-labeled α-Syn fibrils formed in the presence of

    GAGs/polymers were centrifuged at 14,000 rpm for 1 h and washed thrice by resuspending in the buffer.

    The supernatant of each of the fibril samples was analyzed on 15 % SDS-PAGE and concentration was

    determined by comparing with known α-Syn standard. To determine the fibril concentration, the

    supernatant concentration was subtracted from original solution concentration i.e. 300 µM. Finally, fibril

    pellet was resuspended in the same volume as that of the supernatant. Fibril seeds were prepared by

    sonication at 20 % amplitude, 3 sec ON and 1 sec OFF cycle for 3 mins. Eventually, sonicated fibrils

    were used for internalization assay.

    Concentration and time optimization for internalization assay in SH-SY5Y. For optimization of

    internalization assay, FITC-labeled α-Syn fibrils (formed in the absence of GAGs) were used. Fibril seeds

    were prepared by sonicating α-Syn fibrils at 20 % amplitude, 3 secs ON and 1 sec OFF pulse for 3 mins.

    This was followed by addition of different concentrations (i.e. 0.5 µM, 1 µM and 5 µM) of α-Syn fibrils

    seeds onto SH-SY5Y cells grown at 37oC for 24 h. It was observed that α-Syn fibrils seeds were readily

    internalized by the cells and formed punctate structures inside the cell body. Moreover, there was a

    concentration-dependent increase in FITC signal (Fig. S16). We chose 1 µM as the optimum

    concentration as too high signal was obtained at 5 µM seed concentration. Next, we cultured SH-SY5Y

    cells in the presence of 1 µM fibril seed for the different time period. We fixed the cells at three different

    time points (i.e. 6 h, 12 h and 24 h) with 4 % paraformaldehyde solution and imaged using Leica DMi8

    microscope in DIC and fluorescent mode. Eventually, we chose 24 h as the optimum time for subsequent

    treatments as efficient uptake for fibrils was observed at this time-point (Fig. S16).

  • S 4

    Figure S1. Analysis of purified α-synuclein (A) 15% SDS-PAGE showing a single monomeric band at

    ~17 kDa corresponding to purified α-Syn protein. (B) MALDI-TOF mass spectrometry showing high

    intensity 14.4 kDa [M] and 7.2 kDa [M/2] peaks.

  • S 5

    Figure S2. Secondary structural transition monitored by CD spectroscopy. CD scans of -Syn in the

    presence and absence of different GAGs showing the structural transition from random coil to β-sheet

    conformation during its aggregation process.

  • S 6

    Figure S3. The plots showing θ218 versus incubation time showing β-sheet transition kinetics for α-

    Syn in the absence and presence of GAGs.

  • S 7

    Figure S4. Percentage of secondary structural transition (random coil to β-sheet content) of α-Syn

    in the absence and presence of GAGs during aggregation obtained from deconvolution of CD

    spectra.

  • S 8

    Figure S5. The secondary structural transition of α-Syn in the presence of chitosan by CD. All the

    samples showed β-sheet secondary structure at the end of aggregation kinetics (Day 10). Inset shows

    zoomed in CD spectra of the fibrils formed in the presence of chitosan at Day 10.

  • S 9

    Figure S6. Determination of aggregation efficiency by 1D NMR spectroscopy. (A) The NMR spectra

    for α-Syn control (100 µM) and supernatant of α-Syn obtained after aggregation. (B-F) NMR spectra

    showing the selected regions for supernatant obtained after aggregation of α-Syn in the presence of Hep,

    CSA, CSB, Dext, and Chitosan at all concentrations (GAG 0.25, GAG 0.5, GAG 1 and GAG 2). α-Syn

    and GAGs control of known concentration (100 µM) are also presented. The regions highlighted in green

    show peaks unique for protein (from supernatant) and α-Syn control.

  • S 10

    Figure S7. Oligomer size distribution profile by DLS. The different α-Syn species present in the

    supernatant obtained after centrifugation of α-Syn fibrils formed in the absence and presence of GAGs

    monitored by DLS. Measurements showing a shift in oligomer size distribution towards larger

    hydrodynamic radii in the presence of all GAGs.

  • S 11

    Figure S8. SDS-PAGE analysis of α-Syn in the absence and presence of GAGs. Left: SDS-PAGE

    showing samples at Day 0 (GAG: protein 1:1 molar ratio) just before incubation. Right: SDS-PAGE

    showing fibrils after 10 days incubation. Briefly, after 10 days of incubation, fibrils formed in the

    absence and presence of GAGs were centrifuged at 14000 rpm for 1 h. Fibril pellets were re-dissolved in

    20 mM Gly-NaOH buffer and analyzed on 15% SDS-PAGE.

  • S 12

    Figure S9. Correlation of lag time of α-Syn aggregation with polymer length. At 1:1 and 1:2 protein:

    GAGs ratios, with increasing polymer length, the lag time decreases. However, at low protein: GAG

    ratios (GAG 0.25 and GAG 0.5), this correlation doesn’t exist. Hep 0.25, in spite of being the low

    molecular GAG, accelerates the aggregation kinetics with a lag time 15 ± 5 h (SD), which is the shortest

    lag time among all the other GAGs studied at this concentration.

  • S 13

    Figure S10. Site-specific interaction of α-Syn with different GAGs studied by NMR spectroscopy. 1H-15N HSQC spectra of α-Syn (shown in blue) and overlaid spectra α-Syn in the presence of different

    GAGs (shown in red).

  • S 14

    Figure S11. Morphological analysis of α-Syn fibrils formed in the presence of GAGs. Electron

    micrographs showing morphological differences of α-Syn aggregates formed in presence and absence of

    Hep, CSA, and CSB at different GAGs: protein molar ratios. Inset showing zoomed in images

    highlighting the differences amongst the fibrils in the presence of GAGs marked by colored arrowheads.

    Scale bar of the image and inset is 500 and 200 nm, respectively. The TEM images of α-Syn fibrils in the

    presence of GAG 1 is again reintroduced here (as of Fig. 4A) for comparative analysis.

  • S 15

    Figure S12. Morphological analysis of α-Syn fibrils formed in the presence of GAGs. Electron

    micrographs showing morphological differences of α-Syn aggregates formed in the presence and absence

    of Dext, chitosan and PVS at different GAGs: protein molar ratios. Inset showing zoomed in images

    highlighting the differences amongst fibrils formed in the presence of GAGs marked by colored

    arrowheads. Scale bar of the image and inset is 500 and 200 nm, respectively. The TEM images of α-Syn

    fibrils in the presence of GAG 1 is again reintroduced here (as of Fig. 4A) for comparative analysis.

  • S 16

    Figure S13. Effect of chitosan on pre-formed fibrils. AFM images showing the dissociation of pre-

    formed α-Syn fibrils upon incubation with chitosan. Three different fields of α-Syn pre-incubated with

    chitosan are shown. Corresponding scale bar and heights are shown.

  • S 17

    Figure S14. ThT binding assay of freshly dissolved fibrils immediately after addition of equimolar

    concentration of CSA/B. ThT binding remains unaltered in the pre-formed α-Syn fibrils alone and upon

    addition of CSA/B.

  • S 18

    Figure S15. Morphology of FITC-labeled -Syn fibrils formed in the presence and absence of

    GAGs. (A) TEM images showing FITC-labeled fibrils formed by α-Syn as well as those formed in the

    presence of GAGs/polymers. α-Syn formed amorphous structures in the presence of PVS. (B) TEM

    showing FITC-labeled fibril seeds after sonication. Scale bar represents 200 nm. (C) Box plot showing

    the fibril length (nm) of the sonicated FITC-labeled fibrils. At least 80 different sonicated fibrils were

    analyzed from 10 different fields for generating the boxplot.

  • S 19

    Figure S16. Optimization of cellular internalization assay for α-Syn fibrils. Top panel: Internalization

    of α-Syn fibril seeds in SH-SY5Y cells at three different concentrations i.e. 0.5 µM, 1 µM, and 5 µM.

    Bottom panel: Internalization of α-Syn fibril seeds (1 µM) in SH-SY5Y treated for 6 h, 12 h, and 24 h.

    The inset represents corresponding images taken at DIC and fluorescent modes.

  • S 20

    Figure S17. The standard concentration curve for each GAG analyzed by Alcian Blue assay. The

    response curve showing linear equation and coefficient of correlation (R2) for each GAG. In the equation,

    Y is equal to the absorbance at 600 nm and x is the concentration of GAG in mg/ml.

  • S 21

    Table S1. Deconvolution of CD spectra using CD Pro Software showing the percentage of the

    secondary structures of α-Syn during aggregation in the presence and absence of GAGs.

    Percentage of all secondary structures (α-helix, β-sheet, turn and random coil) are shown. “h”

    indicates hours and “m” indicates minutes.

    α-Syn

    Time α-helix (%) (±S.D) β-sheet (%) (±S.D) Turn (%) (±S.D) Random Coil

    (%)

    (±S.D)

    0h 2 0.1 3 1.5 11.5 2.7 84 2

    16h 5 2.8 16.7 14.8 13.35 6.9 61.5 15.1

    24h 4.15 0.5 22 0.7 14.15 6.15 58.9 7.5

    48h 6.4 1.1 29.45 1.8 16.2 1.7 47.95 1.3

    72h 4.55 1.1 24.65 6.4 14.8 5.8 55.4 12.2

    80h 16.35 4.3 26.85 5 17.7 5.2 37 9

    100h 19.8 1.75 27.5 3.3 20.7 0.6 33 2.3

    108h 18.2 2.5 30.35 2.3 23.75 2.5 27.95 4

  • S 22

    Hep 0.25

    Time α-helix

    (%)

    (±S.D) β-sheet

    (%)

    (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 4.15 0.35 16.3 0.5 12.6 1.55 61.5 4.5

    24h 15.5 1.9 25.4 3 12.9 3.7 40.6 5.8

    36h 16.1 1.9 25.7 8.8 13.6 4.15 38.4 8.0

    48h 8.5 0.45 23.0 1.4 14. 2.2 51.0 3.1

    54h 17.1 1.15 26.3 5.8 18.9 2.5 38.6 6.9

    66h 8.4 7.3 31.3 12.6 19.8 2.8 46.2 11.0

    72h 13.3 7.7 37.7 9.05 24.7 2.5 26.6 2.4

    Hep 0.5

    Time α-helix

    (%)

    (±S.D) β-sheet

    (%)

    (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 3.4 0.28 8.7 1.1 16.8 10.3 52.2 22.6

    16h 3.25 0.1 28.95 1.2 13.5 2.3 53.9 1.55

    24h 10.1 5.9 20.45 6.4 11.65 3.91 57.45 4.45

    36h 5.75 0.5 23.5 2.1 15.15 4.2 54.75 6.6

    48h 13.8 5.5 30.7 5.5 11.3 2.9 40.3 15.6

    66h 16.0 1.9 23.2 3.2 17.0 2.2 43 4.9

    80h 18.2 13.5 19.6 1.4 16.2 2.8 38.2 6.0

    90h 28 3.7 27.3 2.8 19.9 0.6 24.4 2.05

    96h 19.3 1.1 33.3 1.95 22.2 0.5 25.8 2.5

  • S 23

    Hep 1

    Time α-helix

    (%)

    (±S.D) β-sheet

    (%)

    (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 3.05 0.2 22.65 1.5 8.25 1.3 60.45 12.4

    16h 2.7 0.7 24.3 8.6 5.65 4.0 57 3

    24h 1.9 0.1 21.9 7.2 13.65 6.6 61.65 15.2

    48h 2.5 0.3 26.45 11.8 15.55 8.8 55 21.2

    66h 2.75 1.5 24.05 1.6 12.9 3.4 58.5 2.8

    72h 3.3 1.8 27.0 7.8 12.6 10.9 44.7 13.8

    80h 5.5 1.4 20.1 9.7 15.1 6.4 56.1 13.9

    90h 9.5 1.6 23.8 2.4 13.7 7.45 48.7 4.6

    100h 29.6 0.95 22.75 0.2 19.3 1.4 27.9 0.75

    108h 30.3 1.3 23.6 0.5 21.5 2.6 25.7 1.5

    120h 15.85 2.5 37.5 1.4 23.75 1.1 22.5 2

    Hep 2

    Time α-helix

    (%)

    (±S.D) β-sheet

    (%)

    (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 2.7 0.67 17.3 8.9 9.7 7.1 65.4 11.7

    24h 1.9 0.6 17.7 4.9 10.25 4.9 69.8 11.8

    36h 2.6 0.7 15.5 6.7 8.8 4.6 74 13.3

    54h 5.1 4.4 17.8 2.1 10.9 1.9 68.4 4.2

    72h 4.9 0. 8 19.5 5.75 13.5 3.2 59.5 7.2

    90h 10.7 3.1 28.2 3 18.6 0.6 40.2 9.25

    108h 16.7 0.15 24.4 5.3 22.3 0.1 31. 0.9

  • S 24

    120h 33.3 1.8 30.2 0.9 18.8 2.7 23.3 4.4

    130h 11.6 7.9 44.1 13.0 21.75 2.05 22.45 2.6

    CSA 0.25

    Time α-helix

    (%)

    (±S.D) β-sheet (%) (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 2.15 0.9 15.25 4.8 8.5 3.7 73.35 7.8

    16h 2.6 0.3 13.2 6.9 8 5.6 75.2 12.3

    24h 7.1 5.9 20.1 5.15 12.6 4.6 59.9 10.45

    36h 8.1 1.5 18.6 3.9 15.9 5.0 57 8.3

    66h 13 5.8 20.7 2.3 15.1 0.65 51 2.4

    80h 11.9 6.2 19.25 1.6 15.05 0.1 53.8 4.8

    90h 24.4 1.25 27.1 0.7 20.8 0.75 28.3 1.7

    108h 23.3 1.0 37.3 4.4 21.8 5.7 18.8 2.5

    CSA 0.5

    Time α-helix

    (%)

    (±S.D) β-sheet (%) (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 4.1 2.1 12.9 9.9 9.9 3.8 69.5 6.5

    24h 5.55 1.45 13.4 0.05 8.6 0.9 72.4 1.9

    36h 8 1.5 13.3 3.1 9.8 1.5 68.6 5.9

    48h 8.2 2.7 15.7 4.6 11.6 4.95 63.5 8.8

    66h 12.6 2.9 22.7 3.27 16.6 0.9 47.5 4.3

    80h 22.7 1.4 23.5 2 20.6 1.6 33.0 1.5

    90h 20.05 1.2 39.85 3.9 23.15 1.5 16.8 3.7

  • S 25

    CSA 1

    Time α-helix

    (%)

    (±S.D) β-sheet (%) (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 2.7 0.87 13.07 6.7 8.1 6.5 75.1 13.1

    16h 4.6 1.55 14.1 0.85 8.7 1.15 72.5 2.2

    24h 11.1 4.2 20.3 4.7 15.3 4.2 52 10.2

    36h 20.8 3.1 18.7 6.55 13.9 4.7 45.1 10

    48h 18.0 0.6 17.5 1.5 16.1 0.9 48.6 2.05

    66h 19 1 23.8 1.9 20.4 1.3 36.9 2.7

    80h 17.45 2.5 37.2 5.1 23.75 0.9 21.15 3.6

    CSA 2

    Time α-helix

    (%)

    (±S.D) β-sheet (%) (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 8.9 7.2 12 6.2 15.3 8.3 64.75 2.9

    16h 4.6 2.3 16.6 3.4 10.7 4.2 65.7 6.25

    24h 7 1.9 18.5 2.3 12.8 3.05 59.8 4.4

    36h 17.5 1.25 18.0 2.4 15.3 1.6 48.8 4.95

    48h 22.3 1.8 19.2 0.1 18.1 1.7 38.9 4.25

    66h 12.75 7.0 42.55 2.6 22 0.4 17.9 3.4

  • S 26

    CSB0.25

    Time α-helix

    (%)

    (±S.D) β-sheet (%) (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 4.7 2.2 12.8 2.4 8.1 2.7 75.3 5.9

    24h 5.1 2.2 6.3 7.5 10 2.3 69.9 3.6

    36h 8.5 3.7 12.9 2.3 10.8 4.7 68.5 8.3

    48h 17.2 0.3 11.5 4 9.2 6.4 58.7 6.1

    54h 12.1 4.1 14.5 6.3 15.8 4.6 57.9 7.3

    80h 13.25 7.0 31.05 5.6 21.15 2.5 26.55 1.3

    96h 15.1 6.9 36.15 3.9 26.3 5.7 22.7 4.1

    CSB 0.5

    Time α-helix

    (%)

    (±S.D) β-sheet (%) (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 3.6 1.5 10.7 4.4 6.1 2.8 80.2 8.4

    16h 7.1 4.8 13.6 7.5 8.1 5.2 74.5 14

    24h 12.8 5.4 25 12.6 19.7 8.3 42.1 18

    36h 9.4 3.4 13.9 3.8 12.2 1.1 64.5 4.5

    48h 17.1 1.9 14.6 2.9 14.4 1.9 55.7 2.6

    54h 17.6 1.3 18.6 2.3 16.9 1.1 47.3 3.0

    66h 18.8 1.7 20.7 2.2 18.4 0.3 43.2 1.5

    72h 18.5 1 21.5 2.1 21.3 0.97 39.4 1.3

    80h 19.35 1.3 37.25 5.0 22 0.3 21.6 4.1

  • S 27

    CSB 1

    Time α-helix

    (%)

    (±S.D) β-sheet (%) (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 1.9 0.7 11.6 6.3 5.9 4.4 76.6 5.75

    16h 3 1 13.25 1.2 8.05 1. 8 75 2.8

    24h 7. 7 3.8 21.1 2.7 14.4 3.4 55.4 9.5

    48h 10.5 4.5 27.4 7.6 15.7 3.4 43.8 18.5

    54h 10 2.8 32.3 2.02 20.5 0.6 36.7 1.6

    66h 2.5 1.6 42 1.7 24 1.15 31.5 0.8

    CSB 2

    Time α-helix

    (%)

    (±S.D) β-sheet (%) (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 3.8 1.3 18.35 2.3 10.45 3.6 66.85 0.9

    16h 5 2.3 16.5 2.8 10.1 2.7 65.5 6.85

    24h 12.7 9.0 23.7 0.7 14.3 5.4 51.65 2.8

    36h 10.9 5.5 21.45 2.2 12.1 0.3 55.2 6.9

    48h 11.6 4.7 28.65 4.7 18.4 1.4 42.95 1.1

    54h 5.4 1.8 29.5 1.2 16.1 5.9 44.7 1.15

  • S 28

    Dext 0.25

    Time α-helix (%) (±S.D) β-sheet

    (%)

    (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 7 4.8 8.9 2.7 12.8 4.4 72.6 11.2

    16h 6 2.1 14.45 2.5 8.1 1.3 73 1.3

    24h 4.7 2.2 14.2 4.9 8 3.0 74 8.5

    36h 3.3 0.5 13.9 5.8 8.5 5.2 72.6 10.1

    48h 6.6 2.3 17.3 7.4 10.6 8.8 60.8 7.5

    66h 9.7 1.9 19.1 1.5 14.7 0.8 55.2 5.7

    72h 12.2 2.75 26.1 2.1 19.1 1.4 39.3 9.1

    96h 16.65 0.8 39.9 2.7 22.55 1.5 20.95 3.6

    Dext 0.5

    Time α-helix

    (%)

    (±S.D) β-sheet

    (%)

    (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 5.8 2.2 8.4 6.1 11.6 3 70.8 4.3

    16h 4.9 2.3 11.2 5.9 8.5 2.7 72.8 5.6

    24h 7.1 6.7 21 5.4 13.6 5.9 60.6 11.6

    36h 6.2 1.3 16.7 6.1 12.3 4.3 63.7 9.1

    48h 10.9 3.6 24 3.6 16.8 0.9 48.1 3

    72h 25 1.1 27.0 0.65 20.6 0.85 26.7 1

    90h 2.8 0.6 60 10.7 23.85 4.3 13 14.0

  • S 29

    Dext 1

    Time α-helix

    (%)

    (±S.D) β-sheet

    (%)

    (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 7.4 2.65 7 4.0 9.3 2.2 72 7.8

    16h 4.9 2.95 15.5 2.0 10.1 3.4 70.5 5.7

    24h 4.1 1.7 18.75 2.2 10.25 4.7 66 5.7

    36h 3.9 1.0 11.6 4.1 7.5 2.45 75.3 4.95

    48h 5.6 1.0 9.8 5.0 8.6 1.8 72.3 3

    54h 13 2.9 21.5 1.25 16.7 0.3 47.5 4.5

    72h 19.4 1.2 22.5 0.15 18.5 0.9 39 2.6

    80h 11.9 3.4 28.1 20.6 21.7 1.7 19.2 11.3

    Dext 2

    Time α-helix

    (%)

    (±S.D) β-sheet

    (%)

    (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0h 4.9 1 9 4.4 9.2 3.8 70.4 9.1

    16h 5.8 3.1 16.3 5.3 10.1 3.8 67.5 7.7

    24h 7.3 2.4 20.4 1.25 13.9 2.6 57.1 7.3

    36h 7.6 1.6 13.6 6.6 13.2 0.8 61.7 3.6

    48h 16.6 1.4 18.7 3.1 8.7 6.0 48.9 5.1

    54h 15.4 4.9 21.8 0.1 18.1 0.5 39.7 2.9

    72h 32.9 9.3 31.25 4.8 18.4 3.3 23.8 5.6

  • S 30

    PVS 0.25

    Time α-helix

    (%)

    (±S.D) β-sheet

    (%)

    (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0 m 5.5 1.4 14.9 2.4 14.3 1.1 59.0 5.1

    10m 6.5 1 17.15 1.5 11.4 4.1 54.35 0.9

    90m 7.7 2.3 17.1 2.8 17.5 9.4 49.1 5.1

    150m 8.5 2.8 18 3.9 16.1 0.9 51.9 3.2

    d5 10.6 4.9 18.35 2.3 8.9 7.4 47.35 7.7

    PVS 0.5

    Time α-helix

    (%)

    (±S.D) β-sheet (%) (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0m 4.4 1.6 22.0 2.2 9.1 4.7 57.3 3.2

    5m 17.5 0.9 23.7 3.5 9.8 6.5 41.0 6.0

    10m 10.2 2.5 27.5 6.2 12.4 11.8 39.8 7.4

    15m 9.8 2.0 29.6 0.8 16.5 1.6 43.7 1.3

    20m 7.2 2.2 21.6 5.4 12.3 6.3 55.5 7.8

    25m 10.6 1.9 28.3 2.9 17.9 2.8 41.5 5.25

    30m 9 5. 18.6 10.1 19.3 0.7 34.7 5.5

    40m 9.2 1.55 28.6 0.8 20.2 1.15 38.6 5.2

    50m 6.7 2.0 27.8 1.5 16 5.6 45.6 1.2

    60m 6.7 4.1 29.6 3 23.3 1.2 36.9 2.4

  • S 31

    PVS 1

    Time α-helix

    (%)

    (±S.D) β-sheet

    (%)

    (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0m 9.7 3.3 25.2 1.2 15.4 3.6 47.6 1.1

    5m 9.0 3.1 19.6 12 22.6 7.5 47.1 6.9

    10m 9.4 4.2 22.0 10.3 22 2.7 44 10.9

    15m 7.5 2.3 28.5 0.5 21.5 0.6 28.6 20.8

    20m 7.5 2.5 30.2 0.7 20.5 0.5 34.3 4.7

    PVS 2

    Time α-helix

    (%)

    (±S.D) β-sheet

    (%)

    (±S.D) Turn (%) (±S.D) Random

    coil (%)

    (±S.D)

    0m 16.5 1.6 29.6 1.2 16.3 6.4 33.4 1.5

    3m 8.7 2.45 33 2.2 22.1 1 35.5 1.5

    6m 8.25 1.3 36.15 0.6 24.05 0.6 30.8 1

  • S 32

    Table S2. Table showing the degree of fibrillation of α-Syn in the absence and presence of

    GAGs analyzed by 1D NMR. The concentration of supernatant and fibrils at the end of the

    aggregation formed in the presence of GAGs at different protein: GAG molar ratio is shown.

    NMR measurements were recorded with two independent sets. The error associated with NMR

    measurements is ~10-15%.

    Sample Protein: GAGs ratio

    Supernatant Concentration

    (µM)

    Fibril Concentration

    (µM)

    % Fibrillation

    α-Syn - 105 195 65

    Hep 0.25 25 275 91

    0.5 21 279 93

    1 48 252 84

    2 61 239 80

    CSA 0.25 75 225 75

    0.5 81 219 73

    1 109 191 64

    2 66 234 78

    CSB 0.25 52 248 83

    0.5 32 268 89

    1 42 258 86

    2 41 259 86

    Dext 0.25 92 208 69

    0.5 76 224 75

    1 90 210 70

    2 97 203 68

    Chitosan 0.25 17 283 94

    0.5 3 297 99

    1 5 295 98

    2 15 285 95

  • S 33

    Table S3: GAGs estimation by Alcian blue assay. For each GAG, two independent sets were

    performed. The error was found to be in the range of 5-12%.

    GAGs GAGs Concentration in supernatant (µM)

    GAGs Concentration in Fibrils (µM)

    % Incorporation in fibrils

    Hep 210 90 30

    CSA 107 193 64

    CSB 97 203 68

    Dext 186 114 38