ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitol phosphate onto α-dystroglycan

Mutations in genes required for the glycosylation of α-dystroglycan lead to muscle and brain diseases known as dystroglycanopathies. However, the precise structure and biogenesis of the assembled glycan are not completely understood. Here we report that three enzymes mutated in dystroglycanopathies can collaborate to attach ribitol phosphate onto α-dystroglycan. Specifically, we demonstrate that isoprenoid synthase domain-containing protein (ISPD) synthesizes CDP-ribitol, present in muscle, and that both recombinant fukutin (FKTN) and fukutin-related protein (FKRP) can transfer a ribitol phosphate group from CDP-ribitol to α-dystroglycan. We also show that ISPD and FKTN are essential for the incorporation of ribitol into α-dystroglycan in HEK293 cells. Glycosylation of α-dystroglycan in fibroblasts from patients with hypomorphic ISPD mutations is reduced. We observe that in some cases glycosylation can be partially restored by addition of ribitol to the culture medium, suggesting that dietary supplementation with ribitol should be evaluated as a therapy for patients with ISPD mutations.


Supplementary Figure 3. ISPD mutations in HAP1 cells induced by CRISPR/Cas9 double-nickase approach.
The three selected clones contain deletions in exon 1 of ISPD that lead to frameshifts. Locations of the guide RNAs are underlined.

Supplementary Figure 4. ISPD inactivation in HAP1 cells leads to reduced dystroglycan glycosylation.
Glycosylation status of HAP1 parental cells and three independent knockout clones was assessed by flow cytometry using the antibody IIH6. Supplementary Figure 6. Purification of FKTN, FKRP and rabbit -dystroglycan by affinity purification. a, Fusion proteins of N-terminal fragments of rabbit -dystroglycan with a C-terminal SFBtag were purified in a single step with Sepharose beads covalently coupled to streptavidin (lanes 2-4), were resolved by SDS-PAGE and are visualized by silver staining. b, Fusion proteins comprising full-length mouse Fktn and Fkrp with a C-terminal SFB-tag were partially purified with Sepharose beads covalently coupled to streptavidin, resolved by SDS-PAGE and stained with Coomassie Blue. c, Affinity-purified FKTN and FKRP containing a C-terminal SFB-tag were analyzed by western blot using an antibody directed against FKTN and the FLAG epitope tag.

Supplementary Figure 7. SDS-PAGE analysis and silver-staining/Coomassie Blue staining of samples used in ribitol-phosphorylation experiments.
a, Coomassie Blue staining of the gel used to obtain the 32 P-signal shown in Figure 5. The position of the alkaline phosphatase (AP) protein is indicated. b, Silver staining of a parallel gel analysing the samples used to obtain the 32 P signal shown in Figure 5d (left two panels). The samples shown in the two right panels of Figure 5d contain 10 µg BSA, which largely obscures other proteins of interest. Hence the corresponding gel is not shown.

Supplementary Figure 8. Identification of ribitol bound to -dystroglycan.
a&b, HEK293-cells engineered to express an -dystroglycan fragment (comprising amino acids 1-485 and a C-terminal SFB tag) and ISPD under the control of a doxycycline-regulated promoter were incubated with doxycycline for four days. -dystroglycan was purified by affinity chromatography from the cell supernatant. N-and O-glycans were released from dystroglycan and depolymerized by treatment with methanolic HCl before re-N-acetylation and derivatisation with TMS. The EI spectrum obtained for the peak at 12.084 min (a) shows a fragment pattern of a pentitol (b) and its retention time is consistent with ribitol. The fragments with an m/z of 512, 409, 307, 205 and 103 represent fragments that have lost one, two, three or four carbon units as indicated in the schematic (b). c-e, Arabitol (c) and ribitol (d) were added to hydrolyzed dystroglycan glycans (e) before derivatization in order to show that the observed pentitol coelutes with ribitol. The three selected clones contain deletions that lead to frameshifts. Locations of the guide RNAs are highlighted in color. Clones C12, C2 and D4 correspond to clones #1, #2 and #3 in Figure 6, respectively.

Supplementary Figure 11. FKTN mutations in HEK293 cells induced by CRISPR/Cas9 double-nickase.
The three selected clones contain deletions that lead to frameshifts. Locations of the guide RNAs are highlighted in color. Clone A3 and A5 correspond to clones #1 and #2 in Figure 6, respectively.

Supplementary Figure 12. FKRP mutations in HEK293 cells induced by CRISPR/Cas9 double-nickase.
The clones contain deletions that lead to a frameshift. Locations of the guide RNAs are highlighted in color.

Supplementary Figure 13. POMT1 mutations in HEK293 cells induced by CRISPR/Cas9 double-nickase.
The three selected clones contain deletions that lead to frameshifts. Locations of the guide RNAs are highlighted in color. Clones A8, A9 and B10 correspond to clones #1, #2 and #3, respectively.