Fig. 7
Loss of GORAB causes defective terminal N-glycosylation of proteins. a N-glycome analysis of WT and GO fibroblasts. Quantification of relative intensities of MALDI-TOF-MS signals for N-glycan species detected in lysates from wild-type (N = 3 cell lines) and GO fibroblasts (N = 4 cell lines). Error bars represent the mean ± SEM from 4 independent experiments, *p < 0.05, unpaired t-test. GlcNAc N-acetylglucosamine, NeuAc N-acetylneuraminic acid, NeuGc N-glycolylneuraminic acid. Yellow shading indicates differences between WT and GO fibroblasts. b Analysis of sialylated plasma membrane proteins in WT and GO fibroblasts using MAL and SNA lectins. Top, glycan chains recognized by the lectins. Bottom, non-permeabilized fibroblasts stained with FITC-conjugated lectins. Scale bar, 10 µm. c Quantification of fluorescence intensities from b (150 cells analyzed per cell line in each of 3 independent experiments, min to max box and whisker plot, **p < 0.01, Mann–Whitney U test. d Representative flow cytometry histogram of WT and GO fibroblasts (N = 3 cell lines) stained with FITC-conjugated MAL and SNA lectins. e Analysis of metabolic labeling of WT and GO fibroblasts with alkynyl-tagged sialic acid precursor ManNAl. Co-cultured WT and GO cells were incubated with ManNAI for 10 h, fixed and labeled with antibodies to GORAB and TGN46. Scale bar, 10 µm. f Quantification of ManNAI labeling assessed as fluorescence intensity against that of a Golgi marker, with 300 cells analyzed per cell line in 3 independent experiments, min to max box and whisker plot, ***p < 0.001, Mann–Whitney U test. g N-glycome analysis of control and GorabNull mouse skin tissue. Symbols representing monosaccharide residues are as in a. Yellow shading indicates N-glycans different between control and GorabNull samples. h, i Left, lectin blot analysis of skin lysates of control and GorabNull E18.5 embryos with E-PHA (h) or SNA lectin (i). Right, quantification of E-PHA (h) or SNA (i) levels. Error bars represent the mean + SD, n = 4 independent experiments, *p < 0.05, **p < 0.01, unpaired t-test. In a, g, the glycan colored symbols are drawn according to the Symbol Nomenclature For Glycans convention. The structures shown are those most probable for compositions determined from accurate m/z measurements on the basis of the well-accepted biosynthetic route for N-glycans. Glycan assignments and accompanying masses in g are shown in Supplementary Table 1
