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. 2020 Dec 15;9:e63997. doi: 10.7554/eLife.63997

Figure 2. Identification of UDP-glucose:glycoprotein glucosyltransferase (UGGT)1- and UGGT2-specific substrates.

(A) Reglucosylation substrates in ALG6/UGGT2-/- cells were identified and quantified as previously described in Figure 1. Localizations as annotated by UniprotKB are depicted. Data are representative of two independent experiments. Error bars represent SEM. (B) Reglucosylated substrates in ALG6/UGGT1-/- cells were identified and quantified as previously above. (C) The distribution of localizations as annotated by UniprotKB for reglucosylation substrates identified in both ALG6/UGGT2-/- and ALG6/UGGT1-/- cells is depicted. (D) The overlap of reglucosylated substrates identified in both ALG6/UGGT2-/- cells (purple) and ALG6/UGGT1-/- cells (gray) is visualized by a Venn diagram. (E) Reglucosylated substrate enrichment in either ALG6/UGGT1-/- or ALG6/UGGT2-/- cells is depicted by dividing the tandem mass tag quantification for each protein in ALG6/UGGT1-/- cells by the associated value in ALG6/UGGT2-/- cells on a log10 scale. Positive and negative values represent enrichment in ALG6/UGGT1-/- and ALG6/UGGT2-/- cells, respectively. Localization (coloring) and topology (soluble [circles] or transmembrane [squares]) are depicted based on UniprotKB annotation.

Figure 2—source data 1. TMT quantification results for Figure 2A and Figure 2B.
elife-63997-fig2-data1.xlsx (161.7KB, xlsx)

Figure 2.

Figure 2—figure supplement 1. UDP-glucose:glycoprotein glucosyltransferase (UGGT)1 and UGGT2 expression.

Figure 2—figure supplement 1.

(A) The indicated cells were lysed and whole cell lysates were resolved by SDS-PAGE and imaged by immunoblotting against UGGT1 and GAPDH. Asterisk denotes background band. Data are representative of three independent experiments with quantification shown in (B). UGGT1 expression was normalized to that of ALG6-/- cells. Error bars represent standard deviation. Asterisk denotes a p-value of less than 0.05. (C) Counts per million of UGGT2 mRNA generated by RNAseq from Supplementary file 4 was analyzed for the level of UGGT2 mRNA expression in the indicated cell lines. Counts per million of all genes were averaged and the standard deviation from the average for UGGT2 mRNA was determined. Error bars represent the standard deviation. Data are representative of three independent experiments.
Figure 2—figure supplement 2. mRNA expression analysis of UDP-glucose:glycoprotein glucosyltransferase (UGGT)1 and UGGT2 substrates.

Figure 2—figure supplement 2.

(A) Reglucosylated substrates identified in ALG6-/- (A), ALG6/UGGT1-/- (B), and ALG6/UGGT2-/- (C) cells were compared to the average expression for the N-glycoproteome in counts per million. The standard deviation from the average is plotted, with the error bars representing the standard deviation. Blue dots above each gene represent the level of fold increase (GST-CRT/GST-CRT-Y109A) found by tandem mass tag (TMT) mass spectrometry. The Pearson’s correlation coefficient (R) between the mRNA expression and TMT mass spectrometry fold increase is shown. Data is representative of three independent experiments.
Figure 2—figure supplement 3. β-hexosaminidase subunit β trafficking and hypoglycosylation and CI-M6PR hypoglycosylation.

Figure 2—figure supplement 3.

(A) Cells treated without or with deoxynojirimycin for 12 hr, lysed and whole cell lysate samples were resolved and imaged by immunoblotting against β-hexosaminidase subunit β. Data is representative of three independent experiments and quantification is shown in (B). (C) The indicated cell lines were lysed and samples were split evenly between non-treated and PNGaseF treated. Asterisks denote deglycosylated protein (D). As described for panel C, except immunoblotting was against CI M6PR.