NAD+ enhances ribitol and ribose rescue of α-dystroglycan functional glycosylation in human FKRP-mutant myotubes
- Department of Integrative Biology and Physiology, University of Minnesota, United States
- Lillehei Heart Institute, Department of Medicine, University of Minnesota, United States
- Stem Cell Institute, University of Minnesota, United States
- Bioinformatics and Computational Biology Program, University of Minnesota, United States
- Conrad Prebys Center for Chemical Genomics, Sanford Burnham Prebys Medical Discovery Institute, United States
Figures
Figure 1 with 2 supplements
Patient-specific Walker-Warburg syndrome (WWS) induced pluripotent stem (iPS) cell-derived myotubes display reduced functional glycosylation of α-dystroglycan (α-DG).
(A) Representative immunostaining of wild type (WT) and FP4 iPS cell-derived myotubes for myosin heavy chain (MHC) and IIH6 (in red). DAPI stains nuclei (in blue). Scale bar, 200 μm. (B) …
Figure 1—figure supplement 1
Pluripotency characterization of reprogrammed FP4 induced pluripotent stem (iPS) cell line.
(A–C) Representative images show typical (A) pluripotent colony morphology, (B) alkaline phosphatase activity, and (C) immunostaining for OCT3/4, SOX2, and NANOG (red). DAPI stains nuclei (in blue). …
Figure 1—figure supplement 2
Fukutin-related protein (FKRP) overexpression rescues functional glycosylation of α-dystroglycan (α-DG) in Walker-Warburg syndrome (WWS) FP4 induced pluripotent stem (iPS) cell-derived myotubes.
(A) Representative immunofluorescence staining of control (empty vector LV) and FKRP-LV FP4 iPS cell-derived myotubes for myosin heavy chain (MHC) and IIH6 (in red). Wild type (WT) myotubes were …
Figure 2 with 1 supplement
Ribitol and ribose rescue α-dystroglycan (α-DG) functional glycosylation in patient-specific Walker-Warburg syndrome (WWS) myotubes.
(A) Scheme denoting the role of ribose and ribitol in the generation of Rbo5P, which is required by fukutin (FKTN) and fukutin-related protein (FKRP) for glycosylation of α-DG. (B) Representative …
Figure 2—figure supplement 1
Ribitol and ribose dose-response studies in induced pluripotent stem (iPS) cell-derived myotubes.
(A–B) Phase contrast images of FP4 myogenic progenitors that upon induction of differentiation were treated with the indicated concentrations of ribitol for 5 days. (B) Western blot shows enhanced …
Figure 3 with 1 supplement
Ribitol and ribose increase the levels of ribitol-5-P and cytidine diphosphate (CDP)-ribitol in patient-specific Walker-Warburg syndrome (WWS) myotubes.
(A) Detection of ribitol, ribose, ribitol-5-P, and CDP-ribitol in WWS FP4 myotubes that had been treated or not with ribitol or ribose for 5 days (n = 3 for each cohort). Error bars represent …
Figure 3—figure supplement 1
Liquid chromatography with tandem mass spectrometry (LC/MS-MS) standard curves.
(A–D) Standard curves from serial dilution of metabolites (A) ribitol, (B) ribose, (C) ribitol-5-P, and (D) cytidine diphosphate (CDP)-ribitol.
Figure 4 with 2 supplements
NAD+ potentiates the capacity of ribitol and ribose to rescue α-dystroglycan (α-DG) functional glycosylation.
(A–B) Western blots show increased IIH6 staining in Walker-Warburg syndrome (WWS) FP4 myotubes that had been supplemented with ribitol and NAD+ compared to ribitol alone (A) or with ribose and NAD+ …
Figure 4—figure supplement 1
NAD+ rescue functional glycosylation of α-dystroglycan (α-DG) in FP4 induced pluripotent stem (iPS) cell-derived myotubes.
(A) Representative immunostaining of FP4 myotubes treated with NAD+ and respective control wild type (WT) myotubes for myosin heavy chain (MHC) and IIH6 (in red). DAPI stained nuclei (in blue). …
Figure 4—figure supplement 2
NAD+, ribitol/NAD+, and ribose/NAD+ do not increase ribitol-5-P or cytidine diphosphate (CDP)-ribitol in FP4 myotubes.
(A) Liquid chromatography with tandem mass spectrometry (LC/MS-MS) detection of ribitol, ribose, ribitol-5-P, and cytidine diphosphate (CDP)-ribitol from FP4 myotubes untreated or supplemented with …
Figure 5 with 1 supplement
Functional fukutin-related protein (FKRP) is required for NAD+ ribitol/ribose-mediated rescue of α-dystroglycan (α-DG) functional glycosylation.
(A) Representative immunostaining of FKRP knockout (KO) and respective control wild type (WT) isogenic PS cell-derived myotubes for myosin heavy chain (MHC) and IIH6 (in red). DAPI stained nuclei …
Figure 5—figure supplement 1
NAD+ treatment in FKRP C318Y myotubes increases α-dystroglycan (α-DG) levels.
(A–B) Western blot for core α-DG and β-DG in FKRP C318Y myotubes treated with NAD+. (B) Total protein stain was used as loading control. (C) Bar graphs shows quantification of α-DG and β-DG …
Figure 6
Model depicting the Walker-Warburg syndrome (WWS)-associated mutations in the fukutin-related protein (FKRP) catalytic domain.
(A) Schematic representation of FKRP protein highlighting the N-terminal stem domain and the localization of C318Y and F473C mutations in the C-terminal catalytic domain. (B) Crystal structure of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens, male) | FP4 | This study | Available from the Anne Bang lab | |
| Genetic reagent (Homo sapiens, male) | FKRP C318Y | This study | TC1133 FKRP C318Y | Available from the Rita Perlingeiro lab |
| Genetic reagent (Homo sapiens, male) | FKRP KO | This study | H9 FKRP KO | Available from the Rita Perlingeiro lab |
| Cell line (Homo sapiens, male) | WT | PMID:22560081 | PLZ | Control line, available from the Rita Perlingeiro lab |
| Cell line (Homo sapiens, male) | Parental WT FKRP KO | WiCell | H9 | ESC control line (WA09) |
| Cell line (Homo sapiens, male) | Parental WT FKRP C318Y | PMID:26411904 | TC-1133 | Control line, available with RUCDR Infinite Biologics |
| Chemical compound, drug | CHIR99021 | Tocris | Cat# 4423 | 10 µM |
| Chemical compound, drug | LDN193189 | Cayman chemical | Cat# 19396 | 200 nM |
| Chemical compound, drug | SB431542 | Cayman chemical | Cat# 13031 | 10 µM |
| Chemical compound, drug | DAPT | Cayman chemical | Cat# 13197 | 10 µM |
| Chemical compound, drug | Dexamethasone | Cayman chemical | Cat# 11015 | 10 µM |
| Chemical compound, drug | Forskolin | Cayman chemical | Cat# 11018 | 10 µM |
| Chemical compound, drug | Ribitol | Sigma-Aldrich | Cat# A5502 | 50 mM |
| Chemical compound, drug | D-(−)-ribose | Sigma-Aldrich | Cat# R9629 | 10 mM |
| Chemical compound, drug | NAD+ | Sigma-Aldrich | Cat# N0632 | 100 µM |
| Chemical compound, drug | Sorbinil | Sigma-Aldrich | Cat# S7701 | 100 μM |
| Chemical compound, drug | Doxycycline | Sigma-Aldrich | Cat# D9891 | 1 µg/ml |
| Recombinant protein | Recombinant human FGF-basic | Peprotech | Cat# 100-18B | 5 ng/ml |
| Antibody | Anti-alpha dystroglycan (mouse monoclonal) | Millipore | Cat# 05–593, RRID:AB_309828 | Dilution 1:1000 (WB) |
| Antibody | Anti-alpha dystroglycan (mouse monoclonal) | DSHB | Cat# IIH6 C4, RRID:AB_2617216 | Dilution 1:50 (IF) |
| Antibody | Anti-human dystroglycan (sheep polyclonal) | R and D Systems | Cat# AF6868, RRID:AB_10891298 | Dilution 1:1000 (WB) |
| Antibody | Anti-MHC (mouse monoclonal) | DSHB | Cat# MF20, RRID: AB_2147781 | Dilution 1:50 (IF) 1:200 (WB) |
| Antibody | Anti-laminin (rabbit polyclonal) | Sigma-Aldrich | Cat# L9393, RRID:AB_477163 | Dilution 1:1000 (WB) |
| Antibody | Anti-beta dystroglycan, concentrated (mouse monoclonal) | DSHB | Cat# MANDAG2 clone 7D11, RRID:AB_2211772 | Dilution 1:1500 (WB) |
| Antibody | Anti-OCT3/4 (mouse monoclonal) | SCBT | Cat# C-10, RRID: AB_628051 | Dilution 1:50 (IF) |
| Antibody | Anti-SOX2 (goat polyclonal) | SCBT | Cat# Y-17, RRID: AB_2286684 | Dilution 1:50 (IF) |
| Antibody | Anti-NANOG (mouse monoclonal) | SCBT | Cat# H-2, RRID: AB_10918255 | Dilution 1:50 (IF) |
| Antibody | Anti-SSEA4 (mouse monoclonal) | SCBT | Cat# sc-21704, RRID: AB_628289 | Dilution 1:50 (IF) |
| Antibody | Alexa fluor 555 goat anti-mouse IgG and IgM (goat polyclonal) | Thermo Fisher Scientific | Cat# A-21424, RRID: AB_141780 Cat# A-21042, RRID:AB_2535711 | Dilution 1:500 (IF) |
| Antibody | DyLight 680 anti-rabbit IgG (goat polyclonal) | Thermo Fisher Scientific | Cat# 35568, RRID:AB_614946 | Dilution 1:10000 (WB) |
| Antibody | DyLight 680 anti-mouse IgM (goat polyclonal) | Thermo Fisher Scientific | Cat# SA5-10154, RRID:AB_2556734 | Dilution 1:10000 (WB) |
| Antibody | DyLight 680 anti-sheep IgG (rabbit polyclonal) | Thermo Fisher Scientific | Cat# SA5-10058, RRID:AB_2556638 | Dilution 1:10000 (WB) |
| Antibody | DyLight 800 anti-mouse IgG (goat polyclonal) | Thermo Fisher Scientific | Cat# SA5-10176, RRID:AB_2556756 | Dilution 1:10000 (WB) |
| Other | DAPI stain | SCBT | sc-3598 | (1.5 µg/ml) |
| Other | Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane | Millipore Sigma | Cat# L2020 | 1 µg/µl |
