Mutations in DPAGT1 in CMS and CDG
DPAGT1-CMS and CDG-Ij are recessive disorders, caused by loss of DPAGT1 function (Table S1), through loss of enzymatic activity, protein truncation or RNA splicing defects. Structures, catalytic activity (Figure 4A), and thermostability measurements (Figure 4B) for purified mutant proteins allowed us to examine how DPAGT1 variants cause disease (Figures 4 and S3, Table S1). Surprisingly, in most cases mutated DPAGT1 protein had close to WT thermostability (Figure 4B), suggesting correct folding. Only two (Ile29Phe and Arg218Trp, Basiri et al., 2013, Iqbal et al., 2013) gave protein that was too unstable to purify, due to insertion of large hydrophobic sidechains either at the ER surface or in the core of the protein (Figure 4C).
Figure 4 DPAGT1 CMS and CDG-Ij Missense Variants Analyses
(A) Relative activity and (B) thermostability for DPAGT1 missense variants found in CMS (dark grey) or CDG-Ij (mid grey). WT activity/stability is indicated by an orange line. Mutations are colored in panels A, D, E-G as follows: 25% or less activity: red; 50%–100% WT activity: pink; >WT activity: green; instability on purification: cyan; confirmed splicing variants indicated with a ∗; Spl: unknown splicing variant. For all measurements n=9.
(C) Location of variants mapped onto tunicamycin (purple) complex, colored as above.
(D) Changes in thermostabilization of selected variants by substrates UDP-GlcNAc and Dol-P (n=9).
(E) Location of variants near UDP-GlcNAc binding site.
(F) Variants near predicted Dol-P binding site. Dol-P (pale green) has been modelled based on the tunicamycin lipid tail.
(G) Environment of Val264. Structures of WT (grey) and V264G variant (orange) are shown illustrating the subtle conformational difference in TM4b and EL4.
For all panels data presented are means ± SD.
Figure S3 The c.478G>A; Gly160Ser and c.791T>G; Val264Gly Mutations Are Associated with Exon Splicing Errors, Resulting in the Loss of Exons 2 and 3 from the Transcript Harbouring c.478G>A; Gly160Ser and the Loss of Exons 6 and 7 from the Transcript Harbouring c.791T>G; Val264Gly, Related to Figure 4
(A) Schematic of pET01 exon trap vector with DPAGT1 exons 2-4 inserted showing the location within the genomic sequence of c.478G>A; Gly160Ser.
(B) Sequencing data and schematic diagrams showing aberrant splicing that results from genomic sequence harbouring the c.478G>A; Gly160Ser variant. RT-PCR on RNA produced in TE671 muscle cell line following transfection with the “exon trap” vector gave wild type RNA sequence, but also some transcripts that excluded exons 2 and 3. When the genomic sequence contained the c.478G>A; Gly160Ser variant was transfected only RNA missing exons 2 and 3 was detected.
(C) Schematic of pET01 exon trap vector with DPAGT1 exons 6-8 inserted showing the location within the genomic sequence of the c.791T>G; Val264Gly variant.
(D) Sequencing data and schematic diagrams showing sequences obtained following RT-PCR on TE671 cells transfected with the “exon trap” vector containing human genomic DNA that is either wild type or has the c.791T>G; Val264Gly variant. Wild type sequence generated RNA harbouring only exons 6, 7 and 8, as shown. The c.791T>G; Val264Gly variant generated some RNA transcripts containing exons 6, 7 and 8 (as for wild type) but for the majority of transcripts exons 6 and 7 were excluded and only exon 8 was present.
Many CMS patients have compound heterozygous mutations, and in general one allele had a much greater impact on DPAGT1 function, with either loss of at least 75% of the WT catalytic activity (Leu120Met, Gly192Ser, Met108Ile, Figure 4A, Table S1) or protein quantity (e.g., truncation: Thr234Hisfs∗116, (Belaya et al., 2012); low protein yield: Ile29Phe (Klein et al., 2015); exon skipping: Leu120Leu (Selcen et al., 2014). However, expressed protein from the second allele had 50% or more WT catalytic activity (Val117Ile, Pro30Ser, Val264Met) or even increased activity (Val264Gly and Gly160Ser, Belaya et al., 2012) (Figure 4B, Table S1). The only homozygous CMS mutation, Arg218Trp (Basiri et al., 2013), gave protein that was too unstable to purify. Patients with the more severe CDG-Ij disease often had significant loss of function for both alleles, with less than 25% activity (Leu168Pro, Tyr170Cys, Arg301His, Arg301Cys), low protein yield/stability (Ile29Phe), or disrupted splicing (with WT activity and thermostability for the purified protein, e.g., Ala114Gly, Würde et al., 2012). The Leu385Arg (Carrera et al., 2012) and Ile69Asn (Timal et al., 2012) mutations are unusual in that they did not show loss of stability or activity, suggesting that splicing changes should be investigated.
As expected, mutations with less than 25% activity generally lie in or near the substrate-binding regions. In two variants found in patients with CDG-Ij (Arg301His and Arg301Cys, Carrera et al., 2012, Imtiaz et al., 2012), the Arg301 that “pincers” the UDP-GlcNAc pyrophosphates is mutated (Figures 2D and 2F) and these mutations gave reduced thermostabilization by UDP-GlcNAc (Figure 4D), confirming a role in UDP-GlcNAc binding. Similarly, the CMS variant Leu120Met lies close to the Dol-P/UDP-GlcNAc interface on the critical CL3 loop that forms one side of the active site (Figure 4E) and the variant Gly192Ser (Belaya et al., 2012) lies within the highly conserved CL5 loop in the uridyl recognition pocket (Figure 4E) where it would disrupt substrate binding. Two CDG-Ij mutations (Leu168Pro and Tyr170Cys, Iqbal et al., 2013, Wu et al., 2003) are of particular interest as they lie on the ER side of the membrane, at the top of the predicted Dol-P binding site, an extensive, conserved intramembrane groove between TMH4, TMH5, and TMH9, below the EL4 loop (Figure 4F). Both mutations cause a reduction in thermostabilization by Dol-P, confirming their involvement in Dol-P binding. The Met108Leu mutation lies within a hydrophobic cluster in the core of DPAGT1 where a mutation would disrupt the positions of TMH4 and TMH5, which form the back of the Dol-P binding site (Figure 4F). Conversely, many of the mutations that maintain at least 50% of WT activity lie on the protein surface (e.g., Pro30Ser, Ala114Gly, Val117Ile) and involve small sidechain changes (Figure 4C).
Given that DPAGT1-CMS is a recessive disorder, we were surprised to find an increased enzymatic activity with the Val264Gly and Gly160Ser variants. Val264 is mutated to either Gly or Met (Selcen et al., 2014), giving either a 2.5-fold increase or a slight (18%) decrease in catalytic activity. Val264 is located on TMH8 in the core of the protein adjacent to TMH3/4 (Figure 4G). Comparing the WT and Val264Gly structures showed a small (1–1.5 Å) movement at the C-terminal end of TMH4b towards TMH8 (Figure 4G), which would affect both the dimer interface and the exact position of EL4, which forms the top of the Dol-P lipid-binding site. Conversely, the Val264Met variant would be poorly accommodated at this buried site. As the Gly160Ser mutation lies in the disordered EL4 luminal loop (Figure 4F), it is unclear why the activity increases. Since these missense variants cause an unexpected increase in enzyme activity, we explored other causes of pathogenicity. Tissue from muscle biopsies were unavailable, so we used the “exon trap” system to detect abnormal RNA splicing. Both mutations, c.478G>A, p.Gly160Ser, and c.791T>G, p.Val264Gly, gave rise to abnormal RNA splicing of their respective RNA transcripts (Figure S3), thus explaining the pathogenicity of these variants.