Interaction between methionine synthase isoforms and MMACHC: characterization in cblG-variant, cblG and cblC inherited causes of megaloblastic anaemia

Abstract

The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalamin (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as 'cblG-variant'. In four 'cblG-variant' cases, we observed a decreased conversion of cyanocobalamin to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full-size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in the proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA, respectively, when compared with control cells. In 3D modelling and docking analysis, both truncated and full-size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of Cbls that is required for Cbl cofactor synthesis.

Alternatively Spliced Methionine Synthase in SH‐SY5Y Neuroblastoma Cells: Cobalamin and GSH Dependence and Inhibitory Effects of Neurotoxic Metals and Thimerosal

“…Thus alternative splicing with exon skipping is a normal feature of MS mRNA in human brain, with different patterns occurring at distinct developmental stages. Deletion of exons 16–18 was also recently reported in skin-derived fibroblasts and Caco2 cells [ 9 ]. In the current study we demonstrate alternative splicing of MS mRNA in SH-SY5Y human neuronal cells and we further show that MS activity is dependent upon MeCbl, whose formation from OHCbl is associated with an absolute dependence upon the availability of GSH.…”

“…Deletion of cap domain exons would be expected to increase vulnerability of Cbl to oxidation, potentially altering the relationship between redox and methylation. Alternative splicing of MS mRNA has also been reported in fetal human brain [ 8 ] and, more recently, in several cultured cell types [ 9 ]. However, the functional significance of alternative splicing remains largely unexplored.…”

Cobalamin C Deficiency Shows a Rapidly Progressing Maculopathy With Severe Photoreceptor and Ganglion Cell Loss

“…1,[4][5][6][7][8][9] Mutations in the MMACHC (methylmalonic aciduria cblC type, with homocystinuria) gene, which encodes a protein involved in cobalamin cofactor synthesis, have been associated with cblC. 4,[9][10][11] The ocular phenotype has been described through case reports, a handful of case series, and a single histopathologic report. 8,[12][13][14][15][16][17][18][19][20][21][22][23] Ocular changes range from limited maculopathies to a progressive retina-wide degeneration and severe central vision loss.…”

Inherited disorders of cobalamin metabolism disrupt nucleocytoplasmic transport of mRNA through impaired methylation/phosphorylation of ELAVL1/HuR

“…Steitz, Yale University) as the template for the creation of ELAVL1/HuR mutations using the QuickChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies; see supplementary methods for details). Cell culture of patient and control fibroblasts was performed as described (21). …”

“…The proximity ligation assay (PLA; Duolink in situ PLA reagents; Olink Bioscience; Eurogentec, France) was performed to visualize and quantify interactions, as described previously (11,21). The PLA signals were counted as described in supplementary methods.…”