Journal: bioRxiv
Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy
doi: 10.64898/2026.03.02.708854
Figure Lengend Snippet: A) SLC35A2 encodes a transporter for UDP-galactose, which is required for the synthesis of O-GalNAc glycans. B) The lectin VVA binds terminal alpha-GalNAc (Tn-antigen), while PNA binds terminal Gal of Core 1 O-GalNAc glycans (T-antigen), which can be removed by O-glycosidase (OGA). C) Breeding strategy of Emx1-Cre mice with floxed- Slc35a2 mice, with blue regions highlighting the forebrain expression Emx1 . D) VVA binding is minimal in protein blots of wild-type mouse cortex; female (+/+) and male (+/y) with floxed- Slc35a2 . In contrast, mice lacking Slc35a2 in cells expressing the forebrain-specific promoter Emx1 ( Emx1-Cre ) display robust VVA binding in both females (+/-) and males (-/y), consistent with inhibition of O-GalNAc extension. Binding of PNA after Neuraminidase A (NeuA) treatment was significantly reduced in both +/- and -/y lines, and specificity of PNA binding was confirmed via OGA treatment of a +/+ sample. ConA, which binds all N-glycans, was unaffected in all mouse lines, and specificity was confirmed using peptide N-glycosidase F (PNGase F) treatment of a +/+ sample. Each lane contains 15 µg cortical protein lysate from an individual mouse of the corresponding genotype. Experiment was replicated three times with similar results. E) Quantification of lectin binding from D normalized to total protein (TP) staining within the same lane of each blot. Data presented as the mean binding intensity +/- SEM, with data points for each individual mouse included, N = 3/genotype. One-way ANOVA confirmed significant group differences ( p < 0.05) for VVA and PNA but not ConA, followed by post hoc Student’s t-tests to confirm genotype effects in each sex: ** p -value = 0.01, **** p -value = 0.0001. F) Schematic of coronal mouse brain section highlighting the cortex (CTX), corpus callosum (CC), and basal ganglia (BG). G) VVA binding is minimal across all regions in wildtype mouse cortex (+/y), while PNA binding localizes to neuronal tracts of the CC and axon bundles in the BG. In contrast, mice lacking forebrain expression of Slc35a2 (-/y) display a build-up of truncated O-GalNAc glycans in cortex that fail to properly localize. Mature O-GalNAc glycans are entirely absent from the CTX and CC, but preserved in the BG, which does not express Emx1 -Cre. H) Relative quantification of lectin binding across brain regions (CTX, CC, BG) from G. Data presented as the mean binding intensity +/- SEM, with individual points representing the average of 6 measures across independent mice. Colored bars correspond with the specific brain region indicated in (F): CTX, purple; CC, orange; BG, blue. N = 2 for +/y VVA; N = 4 for +/- PNA, -/y VVA, and -/y PNA. Fluorescence was quantified as arbitrary intensity (a.i.) after subtracting background signal from each sample.
Article Snippet: Biotinylated lectins ConA (VectorLabs,#B-1005-5, 25 μg/mL) and VVA (VectorLabs, #B-1235-2, 25 μg/mL) were diluted in TBS-T and incubated for 1 hour at room temperature.
Techniques: Expressing, Binding Assay, Inhibition, Staining, Quantitative Proteomics, Fluorescence
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