Journal: Nature Communications

Article Title: Interplay of SLC33A1-dependent and -independent Golgi sialic acid O -acetylation in CASD1 catalysis

doi: 10.1038/s41467-026-71333-y

Figure Lengend Snippet: a Immuno-TLC of extracted gangliosides from CHO-Δ Slc33a1 (Ex1-5 del) cells transfected with a plasmid encoding human V5-tagged ST8SIA1, alone or in combination with a plasmid encoding the indicated N-terminally Flag-tagged SLC33A1 variants. Mock transfected cells were used as negative control. Total gangliosides were extracted and separated by TLC followed by staining with anti-9- O -Ac-GD3 mAb M-T6004 or anti-GD3 mAb R24. Representative plates from one of three independent experiments are shown. b Flow cytometric analysis of CHO-Δ Slc33a1 (Ex1-5 del) cells transiently transfected as described in ( a ) and stained with anti-9- O -Ac-GD3 mAb M-T6004. Positive cells were gated as shown in Supplementary Fig. . Data are presented as mean ± s.d. of n = 4 independent experiments. One-way ANOVA followed by Bonferroni’s post-hoc test. **** p < 0.0001 indicates a significant difference in comparison to ST8SIA1/SLC33A1-WT expressing cells. Western blot analysis was performed to validate the expression of all Flag-tagged SLC33A1 variants. Protein bands representing full-length SLC33A1 and the C-terminally truncated variant p.Y366* are marked by an arrow. The bands marked by asterisks are already present in the lysate of mock- and ST8SIA1 -transfected cells and represent non-specific cross-reactivity of the anti-Flag antibody with endogenous cellular proteins. Actin was used as loading controls. c Cartoon representation of the cryo-EM structure of SLC33A1 (PDB ID 9M0S) . The N- and C-terminal six-helix bundles of SLC33A1 are colored in green and beige, respectively. Side chains of the residues in position 110 and 113 are shown as sticks. Dotted red lines visualize the course of the helix axes of TMH2, TMH11, and TMH12. Detail enlargements of the SLC33A1 structure highlighting the positioning of A110 ( d ) and G509 ( e ). Helices are shown as semi-transparent ribbons with selected residues shown in stick representation. Hydrogen bonds are indicated as dotted lines. For clarity, only the main chain atoms of L474 and V471 are depicted. f Schematic representation of the membrane topology of SLC33A1. Positions of patient-derived SLC33A1 mutations are indicated. Source data and exact p values are provided as a file.

Article Snippet: After blocking for 30 min with 1 % bovine serum albumin (BSA) in PBS, cells were stained for 1 h at room temperature with the following primary antibodies or virolectins diluted in 1% BSA in PBS: mouse anti-9- O -Ac-GD3 mAb (1 μg mL −1 ; Santa Cruz cat. Sc-32269, clone UM4D4), mouse anti-GD3 mAb R24 (2.5 μg mL −1 ; purified by protein A affinity chromatography from cell culture supernatant of hybridoma cells ATCC HB-8445), mouse anti-Flag mAb (5 μg mL −1 ; Sigma-Aldrich cat. F1804, clone M2), rabbit anti-Flag pAb (DYKDDDDK-Tag antibody; 0.28 μg mL −1 ; Cell Signaling cat. 2368), mouse anti-Myc mAb (10 μg mL −1 ; ThermoFisher, MA1-980, clone 9E10), mouse anti-V5 mAb (1 μg mL −1 ; Acris cat. SM1691PS, clone SV5-PK1), chicken anti-V5 pAb (2 μg mL −1 ; Abcam cat. ab9113), rabbit anti-α-Man II pAb (1:10,000; kindly provided by Kelley Moremen), BCoV-HE 0 -Fc (40 μg mL −1 ), and ICV-HE 0 -Fc (10 μg mL −1 ).

Techniques: Transfection, Plasmid Preparation, Negative Control, Staining, Comparison, Expressing, Western Blot, Variant Assay, Cryo-EM Sample Prep, Membrane, Derivative Assay