biotinylated galanthus nivalis lectin  (Vector Laboratories)

Journal: Frontiers in Immunology

Article Title: Immunopathology in PMM2-CDG: Defective glycosylation impact in the TNFα -TNFR1 signalling pathway

doi: 10.3389/fimmu.2025.1655354

Figure Lengend Snippet: N-glycoprofiling in stimulated and non-stimulated PMM2-CDG and control fibroblasts. PMM2-CDG and control fibroblasts were stimulated (S) or non-stimulated (NS) with TNF-α (10 ng/ml) for 24h and analysed using different techniques. (A) Lectin staining was performed resorting to the ConA and GNL lectins and analysed by flow cytometry. Data are represented as the mean ± SD of normalized mean fluorescence intensity (MFI) values to the WT non-stimulated group (n = 3); (B) N-glycosylation biosynthetic pathways with emphasis on the N-glycan structures identified in this study. N-glycan synthesis begins in the cytosolic side of the endoplasmic reticulum (ER) with the attachment of N-acetylglucosamine (GlcNAc) to dolichol monophosphate. This is followed by the addition of one GlcNAc and five mannose residues, using a nucleotide-activated sugar, guanosine diphosphate-mannose (GDP-Man), as the donor. The PMM2 enzyme plays a critical role in the in GDP-Man biosynthesis, by converting mannose-1-phosphate in mannose-6-phosphate. The lipid precursor is then translocated by a flippase to the lumen of the ER, where it undergoes further elongation. Once the oligosaccharide (Glc3Man9GlcNAc2) is completed, it is transferred to an asparagine residue of the nascent protein. The processing of N-glycans is initiated in the ER and continues in the Golgi apparatus, resulting in the formation of various N-glycans forms, such as high mannose, galactosylated, early terminated, fucosylated agalactosylated bi-antennary and hybrid N-glycans. Image created using BioRender ( www.biorender.com ); (C) Semi-quantitative representation of four main N-glycan groups based on their type (high mannose; galactosylated and early terminated; hybrid; and other glycans) determined by the sum of relative intensities of individual structures by MALDI-TOF mass spectrometry; (D) relative distribution of individual high mannose N-glycans (Man3GlcNAc2-Man10GlcNAc2) determined by MALDI-TOF mass spectrometry; and (E) relative distribution of individual galactosylated and early terminated N-glycans determined by MALDI-TOF mass spectrometry. Data from mass spectrometry analysis is based on four technical replicates, except stimulated PMM2-CDG fibroblasts that were measured in triplicate due to limited sample amount. p < 0.05 (*), p < 0.01 (**). green circle – mannose, yellow circle – galactose, blue square – N-acetylglucosamine, red triangle – fucose, purple diamond – sialic acid.

Article Snippet: To analyse the overall cell N-glycosylation, 1x10 5 cells were stained with Concanavalin A (ConA)-biotin (1:100, #B-1005, VectorLabs) and Galanthus nivalis Lectin (GNL)-biotin (1:100, #B-1245, VectorLabs) at 4°C for 20 min. Streptavidin-PE (1:100, #554061, BD Biosciences) was used for secondary detection.

Techniques: Control, Staining, Flow Cytometry, Fluorescence, Glycoproteomics, Residue, Mass Spectrometry