Research ArticleHEREDITARY AMYLOIDOSIS

Peptide probes detect misfolded transthyretin oligomers in plasma of hereditary amyloidosis patients

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Science Translational Medicine  13 Sep 2017:
Vol. 9, Issue 407, eaam7621
DOI: 10.1126/scitranslmed.aam7621
  • Fig. 1. The B β-strand of TTR labels non-native TTR oligomers.

    (A) Schematic diagrams illustrating our hypothesis that peptide fragments of TTR integrate into defect sites of TTR-derived oligomers to selectively label them. (B) TTR tetramer structure (Protein Data Bank ID, 4N85) and sequence, indicating the β-strands (in different colors, labeled from A to H) and segments between the β-strands (in black) that were each individually synthesized. Each peptide was fluorescently labeled at the N terminus with fluorescein for detection. (C) Native PAGE of TTR strand–derived peptides (A to H) incubated with MTTR oligomers. (D) SEC of probe B-1 incubated with MTTR oligomers. (E) Quantification of incorporation of each candidate TTR-derived peptide probe into MTTR oligomers using SEC. Inset: AFM image of the MTTR oligomers used in this peptide screen. AU, arbitrary units. (F) Effect of probe B-1 alanine mutations on incorporation into MTTR oligomers. The amino acids are numbered according to their position in the protein amino acid sequence. Error bars represent means ± SD of at least three separate experiments.

  • Fig. 2. Probe B-1 is selective for nascently formed soluble non-native oligomeric TTR.

    (A) Native PAGE of probe B-1 incubated with folded tetramers with the indicated mutations (TTRtet) and A25T-derived aggregates. (B) Probe B-1 incorporation into MTTR oligomers as a function of oligomers’ age measured by SEC. Error bars represent means ± SD of at least three separate experiments. Right: Native PAGE showing the molecular mass and labeling intensity differences between 1-week-old MTTR oligomers and 2-month-old MTTR oligomers. (C) Probe B-1-Biotin labeling of salivary gland biopsies of a FAP patient (n = 2; left panel) and a non-FAP control (n = 2; right panel). Arrows indicate regions with amyloid fibrils [around the glandular acini in the patient sample (40)], stained by thioflavin T (ThT; top row) and the anti-TTR antibody (anti-TTR–Alexa594; third row) not labeled by probe B-1-Biotin (B-1–Alexa594; second row). Asterisks indicate regions inside the glandular acini that are labeled by B-1-Biotin in both the patient and the control samples. Scale bars, 1 mm.

  • Fig. 3. Probe B-1 selectively differentiates FAP patient samples from controls.

    (A) Experimental setup and representative SEC chromatograms, where probe B-1 was incubated with the indicated samples for 24 hours before injection onto an SEC column. Probe B-1 selectively labels the high-MW fraction (800 to 1200 μl and 150 to 660 kDa) in the V30M FAP patient sample. (B) Representative native PAGE of the samples in (A). (C) High-MW peak area (150 to 660 kDa) of healthy donors (n = 62, average age = 43 ± 11 SD), asymptomatic V30M carriers (n = 20, average age = 34 ± 11 SD), and V30M FAP patients (n = 45, average age = 41 ± 14 SD). Comparison by analysis of variance (ANOVA) followed by post hoc analysis with Tukey correction for multiple pairwise comparisons. Box plot whiskers represent the 2.5 to 97.5% range.

  • Fig. 4. Probe B-2 labels non-native TTR in patient plasma.

    (A) Chemical structure of probe B-2, containing a photoactivatable cross-linker (diazirine) and reporter (alkyne) functional group, and probe B-2C, lacking the diazirine. (B) Schematic of the photocrosslinking approach used to generate (C) and (D), whereby B-2 is incubated with patient plasma, photocrosslinked via the diazirine, and then visualized by reducing SDS-PAGE analysis after rhodamine conjugation to the alkyne handle. (C) SDS-PAGE demonstrating probe B-2–selective and covalent labeling of recombinant MTTR oligomers that were added to healthy donor plasma. TTR labeling by probe B-2 is indicated by the magenta box. (D) SDS-PAGE demonstrating that probe B-2 differentially covalently labels TTR in patient plasma but not in control plasma. TTR labeling by probe B-2 is indicated by the magenta box. (E) Experimental setup for (F) to (H). After incubation and photocrosslinking, each sample was fractionated by SEC before conjugation of rhodamine or biotin to the alkyne handle. The high [Cu] in this reaction denatures the proteome; thus, nondenaturing SEC is done first. (F) Top: Representative SEC chromatogram of a patient and control plasma sample. Middle: Reducing SDS-PAGE of probe-B2–rhodamine–conjugated proteins (TTR labeling by B-2 is indicated by the magenta box). Bottom: Anti-TTR immunoblot is indicated by the dark green box. (G) SDS-PAGE band densitometry quantification of probe B-2 TTR labeling in the high-MW SEC fractions (fractions 3 and 4; see fig. S11 for experimental design schematic and additional representative data) from healthy donors (n = 30), V30M asymptomatic mutation carriers (n = 13), and V30M FAP patients (n = 43). P values were calculated by ANOVA test followed by post hoc analysis with Tukey correction for multiple pairwise comparisons. Box plot whiskers represent the 2.5 to 97.5% range. (H) Biotin pulldown and anti-TTR immunoblot of probe B-2–conjugated proteins from the high-MW fraction #4 in (F).

  • Fig. 5. Targeted MS confirms non-native TTR as a target of the B-peptide.

    (A) Experimental scheme where either probe B-2 or a probe B-2 mutant (V28A or B-2-Mut) is incubated with either patient or control plasma. After photocrosslinking, biotin is conjugated to the alkyne handle and the probe B-2–biotin conjugates were enriched with streptavidin. After trypsin digestion, the trypsin fragments are labeled with one of six TMT tags and then combined and subjected to MudPIT LC-MS/MS analysis. (B) Rhodamine fluorescence labeling of non-native TTR oligomers from a V30M FAP patient incubated with probe B-2 compared to probe B-2-Mut (magenta box). (C) Venn diagram showing the overlap of proteins identified from healthy blood donors, V30M asymptomatic mutation carriers, and V30M FAP patients. (D) Intensity of TTR peptide signal from plasma of three FAP patients treated with either probe B-2 or probe B-2-Mut. (E) Volcano plot of identified proteins quantified by TMT isobaric mass tags shown as a ratio of the intensity in the probe B-2 TMT channel to that of the corresponding probe B-2-Mut TMT channel. Enrichment ratios are shown as an average (n = 3), and P values were calculated from the log2-transformed values using a two-tailed t test assuming equal SDs followed by a correction for multiple testing using a false discovery rate (FDR) of 5% (table S1), as described in Materials and Methods. (F) Probe B-2 to probe B-2-Mut ratios for a select number of proteins previously identified in TTR amyloid deposits. P values were calculated by an ANOVA test followed by post hoc analysis with Tukey correction for multiple pairwise comparisons. P values were calculated using a two-tailed t test.

  • Fig. 6. Labeling of high-MW non-native TTR oligomers decreases in V30M FAP patients treated with tafamidis or liver transplantation.

    (A) Top: Schematic representing the experimental workflow. Bottom: Representative probe B-2–rhodamine gel images from the high-MW SEC fraction from 15 Portuguese FAP patients whose blood was taken before treatment and after 12 months of treatment with the kinetic stabilizer tafamidis. The NIS-LL score for each patient and the change after 12 months are listed below each gel. (B) Quantification of probe B-2 non-native TTR labeling by band densitometry (left) and fold change after 12 months of treatment (right). P value was calculated by a two-tailed unpaired t test comparing the pre-tafamidis and 12-month time points. Box plot whiskers represent the minimum-maximum. (C) Non-native TTR labeling by B-2 of seven V30M FAP patients (Japan) who underwent liver transplantation (0.8 ± 0.3; means ± SD).

  • Fig. 7. Non-native TTR is detected in predominantly neuropathic hereditary TTR amyloidosis and not detected in cardiomyopathy-associated genotypes.

    (Top) Experimental schematic. (Bottom) Non-native TTR labeling in TTR amyloidosis genotypes other than V30M (individual mutations as indicated) as detected by B-2 photocrosslinking and SDS-PAGE. The y axis refers to values calculated by gel densitometry. P values were calculated using an ANOVA test followed by post hoc analysis with Tukey correction for multiple pairwise comparisons. V30M FAP data from Fig. 4G are presented for comparison. Box plot whiskers represent the 2.5 to 97.5% range.

  • Table 1. Sample age and demographics for the non-native TTR detection by the B-2 SDS-PAGE assay presented in Figs. 4G, 6, and 7.
    Genotype (n)Female/maleAge (means ± SD)Origin
    Healthy donors (30)18/1251 ± 16United States (28), Japan (2)
    V30M asymptomatic (13)6/736 ± 13Portugal (12), United States (1)
    V30M FAP (43)21/2243 ± 14Portugal (34), Japan (7), United States (2)
    WT cardiomyopathy (15)1/1476 ± 8United States (15)
    V122I (6)4/276 ± 8United States (6)
    Other: T60A (4), F44S (2), T79K (1), T49P (1), I84N (1), S50I (1), and S50R (1)5/656 ± 11United States (6), Japan (5)

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/407/eaam7621/DC1

    Materials and Methods

    Fig. S1. The B β-strand of TTR labels TTR50–127 oligomers.

    Fig. S2. The minimal binding motif is VAVHVF.

    Fig. S3. Determination of probe stoichiometry in MTTR oligomers.

    Fig. S4. Additional microscopy images of salivary gland biopsies stained with the B-peptide.

    Fig. S5. Probe B-1 linearly labels MTTR oligomers in human healthy plasma.

    Fig. S6. A similar probe B-1 structure-activity relationship is found when MTTR oligomers are incubated in healthy plasma.

    Fig. S7. Labeling of the high-MW SEC fraction in FAP patients by the B-peptide is fluorophore-independent.

    Fig. S8. Alanine substitutions on probe B-1 have identical effects on incorporation of the peptide into the high-MW fraction of FAP V30M patient plasma.

    Fig. S9. B-1 is the only peptide of the TTR β-strands that incorporates into the high-MW fraction of patient plasma.

    Fig. S10. Diazirine-containing probe B-2 selectively labels oligomeric TTR.

    Fig. S11. Schematic of probe B-2 non-native TTR gel quantification method and representative data.

    Fig. S12. Probe B-1 does not cross-react with the anti-TTR antibody (DAKO, catalog no. A0002).

    Fig. S13. Correlation of spectral counts in the MS1 spectra of the diazirine-containing B-2 targets from V30M FAP patients (average of three patients) with plasma concentration.

    Fig. S14. Validation of N-terminally cleaved non-native TTR as a target of the B-peptide in V30M FAP patient plasma.

    Table S1. Full summary of MudPIT LC-MS/MS data presented in Fig. 5 (Excel format).

    Table S2. All raw data for experiments where n < 20 (Excel format).

  • Supplementary Material for:

    Peptide probes detect misfolded transthyretin oligomers in plasma of hereditary amyloidosis patients

    Joseph D. Schonhoft, Cecilia Monteiro, Lars Plate, Yvonne S. Eisele, John M. Kelly, Daniel Boland, Christopher G. Parker, Benjamin F. Cravatt, Sergio Teruya, Stephen Helmke, Mathew Maurer, John Berk, Yoshiki Sekijima, Marta Novais, Teresa Coelho, Evan T. Powers, Jeffery W. Kelly*

    *Corresponding author. Email: jkelly{at}scripps.edu

    Published 13 September 2017, Sci. Transl. Med. 9, eaam7621 (2017)
    DOI: 10.1126/scitranslmed.aam7621

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. The B β-strand of TTR labels TTR50–127 oligomers.
    • Fig. S2. The minimal binding motif is VAVHVF.
    • Fig. S3. Determination of probe stoichiometry in MTTR oligomers.
    • Fig. S4. Additional microscopy images of salivary gland biopsies stained with the B-peptide.
    • Fig. S5. Probe B-1 linearly labels MTTR oligomers in human healthy plasma.
    • Fig. S6. A similar probe B-1 structure-activity relationship is found when MTTR oligomers are incubated in healthy plasma.
    • Fig. S7. Labeling of the high-MW SEC fraction in FAP patients by the B-peptide is fluorophore-independent.
    • Fig. S8. Alanine substitutions on probe B-1 have identical effects on incorporation of the peptide into the high-MW fraction of FAP V30M patient plasma.
    • Fig. S9. B-1 is the only peptide of the TTR β-strands that incorporates into the high-MW fraction of patient plasma.
    • Fig. S10. Diazirine-containing probe B-2 selectively labels oligomeric TTR.
    • Fig. S11. Schematic of probe B-2 non-native TTR gel quantification method and representative data.
    • Fig. S12. Probe B-1 does not cross-react with the anti-TTR antibody (DAKO, catalog no. A0002).
    • Fig. S13. Correlation of spectral counts in the MS1 spectra of the diazirine-containing B-2 targets from V30M FAP patients (average of three patients) with plasma concentration.
    • Fig. S14. Validation of N-terminally cleaved non-native TTR as a target of the B-peptide in V30M FAP patient plasma.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Full summary of MudPIT LC-MS/MS data presented in Fig. 5 (Excel format).
    • Table S2. All raw data for experiments where n < 20 (Excel format).

    [Download Tables S1 and S2]

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