Article Figures & Data
Figures
- Figure 1 GLT8D1 p.I290M Variation Identified in This Study
(A) Sanger sequencing traces demonstrating the heterozygous GLT8D1 c.870C>G (p.I290M) variation. (B) The domain diagram of human GLT8D1, the location of the variation identified in this study, and the alignment of multiple GLT8D1 orthologs showing conservation of the I290 residue from human to zebrafish. (C) Pedigree of the patient carrying the GLT8D1 c.870C>G (p.I290M) variation. GLT8D1 = glycosyltransferase 8 domain-containing protein 1.
- Figure 2 In Vitro Expression of the GLT8D1 Variants in HEK293T Cells
(A) Representative Western Blot analysis of steady-state expression of GLT8D1 proteins in HEK293T cells transfected with GLT8D1 constructs. Actin was used as a loading control. Densitometric quantification is shown below. The error bars indicate standard error of the mean (SEM) from 4 independent experiments. The asterisk indicates a statistically significant difference (**p < 0.01). (B) Immunofluorescence analyses of HEK293T cells expressing GLT8D1 mutants. Confocal fluorescence images of transfected cells labeled with an Alexa 488-conjugated anti-FLAG antibody (GLT8D1; green). Cell nuclei were stained with DAPI (blue). (C and D) Subcellular localization of the GLT8D1 mutants. Confocal fluorescence images of HEK293T cells cotransfected with construct encoding GLT8D1 (labeled with FLAG antibody, green) and markers of the Golgi (DsRed-Monomer-Golgi, red) or the ER (DsRed-ER, red). Cell nuclei were stained with DAPI (blue). Scale bar, 10 μm. ER = endoplasmic reticulum; GLT8D1 = glycosyltransferase 8 domain-containing protein 1; ns = means no statistically significant difference; SEM = standard error of the mean; WT = wild-type.
- Figure 3 In Vitro Functional Analysis of the GLT8D1 Variants
(A) The activation of ER stress was evaluated by RT-qPCR, measuring the expression levels of CHOP, BiP, and sXBP1 mRNA. Cells transfected with R98C MPZ expression plasmid represented the positive controls. All values (mean ± SEM, n = 4) were normalized to the GAPDH mRNA levels. (B) Analyses of glycosyltransferase activities of the purified GLT8D1 proteins. FLAG-tagged WT and mutant GLT8D1 proteins were overexpressed in HEK293T cells and purified by immunoprecipitation. The inorganic phosphate generated through glycosyltransferase reactions were quantified to estimate the glycosyltransferase activities (n = 8). (C and D) Expression of I290M GLT8D1 and R92C GLT8D1 impaired cell viability (C) and induced cytotoxicity (D) in HEK293T cells compared with the WT proteins. Values are shown as means ± SEM of 8 independent transfections (*p < 0.05; **p < 0.01). BiP = binding immunoglobulin protein; CHOP = C/EBP homologous protein; ER = endoplasmic reticulum; GLT8D1 = glycosyltransferase 8 domain-containing protein 1; MPZ = myelin protein zero; RT-qPCR = real-time quantitative PCR; SEM = standard error of the mean; sXBP1 = spliced X-box–binding protein 1; WT = wild-type.
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