Supporting InformationNoborn et al. 10.1073/pnas.1101194108SI Materials and MethodsAggregation of Transthyretin (TTR). Recombinant wild-type TTRwas expressed in bacteria and purified as described previously(1). The purified protein was dissolved in 20 mM phosphate buf-fer-citric acid (pH 2.7, 3.5, 4.5, or 7.4) to a final concentration of50 μM for Thioflavin T (ThT) fluorescence or 500 μM for nativePAGE. Heparin (3H-labled or unlabeled) and heparan sulfate(HS) were added to the TTR solution to the concentrations asindicated in the respective figure legend. The samples were incu-bated at 37 °C for the time period as specified.

TTR peptides. Synthesized peptides corresponding to differentsegments of TTR were purchased from United Peptide Corpora-tion or produced by G. Engström (Ludwig Institute, Uppsala,Sweden) The purity and sequence of the peptides were validatedby MS/MS analysis.

Heparin and HS. Heparin (Kabivitrum AB) has an average mole-cular mass of 14 kDa as analyzed by gel chromatography on aSuperose 12 column (GE Healthcare Biosciences). The HS sam-ples were isolated from porcine tissues and characterized by re-verse-phase ion-pairing HPLC after enzymatic cleavage with HS/heparin lyases (2). HS I contained 5% the trisulfated disaccharide(IdoA2S—GlcNS6S) species, and HS II contained 45% of thespecies. The 3H-labled heparin was prepared by N-deacetylationof the heparin followed by re-N-acetylation with N-[3H]acetic an-hydride (3). The heparin oligosaccharides were generated by par-tial deaminative cleavage of the heparin followed by reductionwith NaBH4. The fragmented heparin samples were collectedafter separation on a Biogel P-10 column (BioRad) as described(4). The amount of saccharides was quantified by carbazolereagent reaction (5).

Detection of TTR Aggregates. ThT fluorescence assay. After incuba-tion, 5 μL of the sample was mixed with ThT in water to a finalconcentration of 20 μMThT. The mixture was transferred to a flatbottom-black 96-well plate (Grainer), and the fluorescence wasmeasured at excitation and emission wavelength of 440 and480 nm, respectively (Gain ¼ 55, FARcyte instruments). Samplesaggregated at different pHs were adjusted to neutral pH aftercompleted incubation prior to ThT staining. The experimentswere run in duplicate or triplicate.

Native PAGE. After incubation, 10 μL of the samples was mixedwith 2.5 μL of a 5× sample buffer (0.3 M Tris-HCl, pH 6.8,0.05% bromophenol blue, 50% glycerol) and loaded onto a10% polyacrylamide gel without SDS. After electrophoresis at100 V for 2.5 h, the gel was stained with Coomassie blue andscanned. For detection of heparin, the gel was stained with Alcianblue. For quantification of 3H-labled heparin, the correspondinglanes in the gel were cut into six sections including the stacking gel(see Fig. 3A in main text) that were transferred to a scintillationvial in 3 mL of H2O. After 2 h incubation, 2 mL of scintillationcocktail was added, and the radioactivity was measured.

Surface Plasmon Resonance Analysis. Interaction of TTR with he-parin/HS was evaluated using a Biosensor system (Biacore2000).TTR was dissociated in acidic buffer (pH 2.7) and then dilutedinto a 50 mM NaAc (pH 5.0) coupling buffer and immobilizedonto a CM5 sensor chip to a level of 3000 response units usingan amine-coupling kit (GEHealthcare). Thioflavin Twas injectedover the immobilized TTR to verify that the protein did not

aggregate to any substantial degree during the immobilizationprocedure (Fig. S3D).For binding and kinetic analysis, heparinor HS was injected (concentrations indicated in figure legend)over the surface and equilibrated at different pH. The surface wasregenerated after each sample with a 30 s wash step of 0.5 MNaCl. All obtained sensorgrams were double-reference sub-tracted in Biacore evaluation software 2.0, using the referenceflow cell and buffer blank sample.

TTR Aggregation in Cell Culture. Wild-type Chinese hamster ovary(CHO-WT) and HS-deficient CHO-pgsD-677 (6) cells werecultured in F12 medium (Gibco) supplemented with 10% fetalbovine serum (FBS), penicillin/streptomycin (60 and 50 μg∕mL,respectively). The cells were seeded at a density of 6000 cells∕well in an 8-well LabTec chamber (Nunc) and cultured for48 h. TTR was dissociated in acidic buffer (pH 2.7) and neutra-lized with NaOH to neutral pH prior to adding to the cells (finalconcentration 5 μM), which were cultured in starvation media(0.1% FBS). After 48 h, medium was removed and cells werewashed extensively in PBS. The cells were thereafter lysed with1% NaOH. The lysate was centrifuged (10 min at 8;100 × g), andthe supernatant was mixed with ThT in water to a finalconcentration of 20 μM ThT. The fluorescence was measured asdescribed above. Cells cultured without the addition of TTR wereused as control to subtract background fluorescence.

For immunocytostaining, the cells were fixed in ice-cold 95%ethanol and 5% acetic acid and washed with PBS. Cells were per-meabilized with 0.4% Triton-X-100 and incubated with antiTTRantibody (1 μg∕mL, HPA002550, Atlas Antibodies) followed byincubation with Alexa flour 488 antirabbit IgG (8 μg∕mL, Mole-cular Probes) for 1 h. The slides were mounted with Vectashield(Vector Labs) containing DAPI counterstain. The TTR immuno-signal was captured with an AxioCam instrument (Carl Zeiss).

Detection of TTR Deposition in Cardiac Tissues. Formalin-fixed, par-affin-embedded sections (15 μm) of cardiomyopathic and controlcardiac tissue were deparaffinized in xylene (30 min) and rehy-drated at 10-min intervals through ethanol baths (99.9–70%).For Congo red staining, sections were first incubated in saturatedNaCl solution (80% ethanol/0.1% NaOH; 30 min) and thentransferred to a filtered 0.4% Congo red (Sigma Aldrich) satu-rated NaCl solution (30 min). For staining of the sulfated glyco-saminoglycans, sections were first incubated in 95% ethanol/10%AcOH (1–2 min) and then stained with sulfated Alcian blue(0.45% Alcian Blue 8GX Sigma Aldrich) solution (0.45%Na2SO4∕45% ethanol∕10% AcOH; 45 min). For immumostain-ing of TTR and HS, the sections were heated in microwave forantigen retrieval and then permeablized in 0.4% Triton-X-100(15 min). Primary antibody incubations were carried out over-night at 4 °C (antiTTR: as described above; antiHS, 1∶250 10E4,gift from Dr. Guido David, Center for Human Genetics, Univer-sity of Leuven, Belgium). For fluorescent detection, sections wereincubated with 4 μg∕mL of the relevant secondary Alexa fluor488 or 594 (Molecular Probes) antibodies for 30 min at roomtemperature; nuclei were counterstained with DAPI. For chro-mogenic detection, the relevant Mach 3 Alkaline PhosphatasePolymer detection kit (Biocare Medical) was developed withVulcan Fast Red (Biocare Medical). Fluorescent and bright fieldimages were captured with a Nikon DXM1200F instrument.

Analysis of Transgenic Drosophila TTR Model. The transgenic Droso-phila melanogaster strain (Dhet TTR-A) overexpressing a mutant

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TTR was generated as described (7). TTR-transgenic (w; GMR-Gal4/+; UAS-TTR-A/+) and non-TTR-transgenic control flies(w; GMR-Gal4/+; +/+) were used in the study. The TTR expres-sion is under the control of GMR-Gal4 driver, directing TTR ex-pression to the photoreceptor cells during the eye-developingstage. The animals were cultivated at room temperature on stan-dard mashed-potato/yeast/agar media. Eggs were reared up withthe standard food supplemented with either heparin (KabivitrumAB) or low molecular weight heparin (Klexane®) 10 mg∕mLthat was mixed with the media. After 9–17 d, the animals werecollected for analysis. For ThT staining, 15 heads of each groupwere homogenized in 75 μL 0.15 M NaCl. After homogenization,the samples were centrifuged (10 min at 8100 × g), and resultingsupernatants were mixed with ThT (final concentration of 20 μMThT). The fluorescence was measured as described above. Non-TTR-transgenic flies were used as control to subtract background

fluorescence. For immunostaining, cryostat sections (15 μm) pre-pared from heads of Drosophila flies (10–14 d of age) were air-dried and fixated in PFA (4%). After washing in PBS, the sectionswere dehydrated at 10-min intervals through methanol baths(25–100%). H2O2 treatment (2%) was followed by rehydrationthrough methanol baths, and the sections were blocked with5% BSA in PBS with 0.2% Triton-X 100. Primary antibodies(antiTTR and antiHS/heparin, as described above) were incu-bated over night in blocking solution. For fluorescent detection,sections were incubated with 2 μg∕mL of the relevant secondaryAlexa fluor 555 and 633 antibodies for 1 h in blocking solution.After washing in PBS, the sections were stained with DAPI inmounting media. Images were captured with an inverted confocalmicroscope (LSM 700, Carl Zeiss), using a PlanApochromat 20x/0.16 or a C-Apochromat 63x/1.2 objective.

1. Furuya H, et al. (1991) Production of recombinant human transthyretin with biologicalactivities toward the understanding of the molecular basis of familiar amyloidoticpolyneuropathy (FAP). Biochemistry 30:2415–2421.

2. Staatz WD, Toyoda H, Kinoshita-Toyoda A, Chhor K, Selleck SB (2001) Analysis of pro-teoglycans and glycosaminoglycans from Drosophila. Methods Mol Biol 171:41–52.

3. Hook GE, Gilmore LB (1982) Hydrolases of pulmonary lysosomes and lamellar bodies.J Biol Chem 257:9211–9220.

4. Maccarana M, Lindahl U (1993) Mode of interaction between platelet factor 4 andheparin. Glycobiology 3:271–277.

5. Bitter T, Muir HM (1962) A modified uronic acid carbazole reaction. Anal Biochem4:330–334.

6. Esko JD, Stewart TE, Taylor WH (1985) Animal cell mutants defective in glycosamino-glycan biosynthesis. Proc Natl Acad Sci USA 82:3197–3201.

7. Pokrzywa M, Dacklin I, Hultmark D, Lundgren E (2007) Misfolded transthyretin causesbehavioral changes in a Drosophila model for transthyretin-associated amyloidosis.Eur J Neurosci 26:913–924.

8. Klabunde T, et al. (2000) Rational design of potent human transthyretin amyloid dis-ease inhibitors. Nat Struct Biol 7: 312–321.

A

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Fig. S1. pH-dependent fibrillization of wild-type TTR. TTR (50 μM) was dissolved in phosphate-citric acid buffer at the pH-value as indicated and incubated at37 °C for 48 h. After incubation, the samples were analyzed for their fibril content using ThT fluorescence (A). Fibril formation was only detected in the samplesincubated at pH 2.7, in a much smaller amount [200 arbitrary units (AU)] in comparison with the samples incubated in the presence of heparin (8000 AU; seeFig. 2A in main text) at the same pH. TTR (500 μM) was incubated under the same conditions as above and analyzed by native PAGE (B). The strong bandsmigrated to the bottom of the gel represent mono- and dimers.

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Fig. S2. Analysis of heparin incubated with preaggregated TTR. TTR dissolved in phosphate buffer (pH 2.7) was incubated without heparin at 37 °C for 48 h.Heparin (mixture of 3H-labeled and unlabeled) was then added, and the TTR–heparin sample was incubated for an additional hour at 37 °C. The sample wasthereafter separated by native PAGE, and the gel was cut into six sections as illustrated in Fig. 3A. Radioactivity in each section was measured by scintillationcounting. Data represents triplicate experiments (mean� SEM).

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Fig. S3. Binding affinity of HS and heparin for TTR. Dissociated TTR was immobilized onto a Biacore sensor chip. Heparin (A), HS II (B), or HS I (C) was injected ina concentration series (ranging from 0.001–70 μM) over the surface at pH 5.0. The response for each concentration was measured at the steady-state region ofthe curve. The apparent equilibrium dissociation constant (KD) was calculated by plotting the response at steady-state for each concentration versus heparin/HSconcentration. The data were fitted to the equation Response ¼ ðRmax � CÞ∕ðKD þ CÞ, where C is the concentration of heparin/HS. Two analyte flow rates(20 and 30 μL∕min) were tested to assess possible effects of mass transport limitations in the system. Altering the flow rate had no significant effect onthe obtained KD value, indicating that mass transport effects were not a limiting factor in the assay. The presented values are average value of three inde-pendent experiments. (D) Thioflavin T (50 μM) injected over the surface immobilized with dissociated TTR resulted in no binding (blue). In contrast, ThioflavinT injection over the surface immobilized with aggregated TTR revealed significant binding (red).

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Fig. S4. SPR analysis of TTR peptides binding to heparin. Heparin was immobilized onto a Biacore sensor chip (CM5) using the surface thiol coupling kit(GE Healthcare). Peptides derived from regions on the TTR sequence (Fig. 5A in main text) containing a histidine residue (24-35, 51-61, and 84-94), wereallowed to interact with the heparin surface at pH 5.0 (peptide conc. 25 μg∕mL; flow rate 20 μL∕min). In consistence with the result analyzed by the nitro-cellulose filter-binding assay (Fig. 5B in main text), only the peptide of aa 24-35 interacted with heparin (red).

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Fig. S5. Reduced HS binding activity to native TTR. (A) Equal amount of dissociated or native protein was immobilized onto a Biacore sensor chip, and HS II wasallowed to interact with the immobilized TTR at pH 5.0. (B) The values of the HS II–TTR interaction signal were obtained by repeated experiments using threechips with different ligand densities (250–3,000 response units). The signals were normalized to a ligand density of 1,000 response units, and average value isshown. Statistical analysis was done by t test.

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Fig. S6. Effect of HS on TTR fibril formation in cell culture. Dissociated TTR was added toWT (CHO-WT) and HS-deficient (CHO-pgsD-677) cells and cultured for48 h. (A) After extensive washing with PBS, the cells were lysed in 1%NaOH to extract cell-associated TTR fibrils. The lysed material was mixed with ThT, and thefluorescence was measured (seeMaterials and Methods in main text). Values represent triplicate experiments (means� SEM). Statistical analysis was done by ttest. (B) Cells were fixed and immunostained with antiTTR antibody (HPA002550; green). DAPI counterstaining is shown in blue. Original magnification, 20X.Scale bar, 20 μM.

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Fig. S7. Effect of heparin on TTR fibril formation in vivo. (A) Eggs of TTR transgenic flies were cultured on regular medium (control) or medium supplementedwith low molecular weight heparin (Klexane®) or heparin. Heads were collected 9–17 d posteclosion and lysed in saline. The extract was analyzed by ThTstaining. Values represent the average of duplicate experiments. (B) The presence of heparin in the Drosophila head lysate was analyzed with dot-blot stainedAlcian blue. Heparin (25 ng and 50 ng) was used as positive control. Twenty-five microliters of the lysate (same samples as used for ThTanalysis) was applied to anylon membrane (Hybond Nþ, GE Healthcare) and stained with Alcian blue (0.5% Alcian Blue dissolved in 25% isopropanol and 3% acetic acid).

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31-HVFRK-35

31-HVFRK-35

Fig. S8. Heparin/HS binding domain on native TTR. A model of a TTR dimer, generated by the PyMol program [Protein Data Bank ID code 1DVQ (8)], showingthe heparin/HS binding site on native TTR. The cluster of basic amino acids (31-HVFRK-35) in this domain, which are expected to be critical for TTR–heparin/HSbinding, is marked in magenta. The binding site is partly embedded in the structure of native TTR tetramer form, providing a rational for the low HS bindingactivity of native TTR.

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