Figure 2.

Functional assays confirm a lack of B4GALNT1 function in primary fibroblasts derived from the proband and in cell models.

A) Overview of the ganglioside biosynthesis pathway. B4GALNT1 is responsible for the synthesis of GA2, GM2, GD2 and GT2 and downstream complex gangliosides. B) Thin-layer chromatography of acidic glycosphingolipids from fibroblasts of a healthy control and the proband. Glycosphingolipids were detected using orcinol-sulfuric acid reagent. The proband’s fibroblasts lack GM2 and downstream gangliosides. C) Immuno-thin-layer chromatography of acidic glycosphingolipids from control and proband fibroblasts, detected using anti-GM2 antibody. This confirms the lack of GM2 in the proband’s fibroblasts. D) Flow cytometry analysis of cell-surface staining of GM1 using CTXb-FITC (red line). The black line indicates an unstained control sample. The proband’s fibroblasts show no detectable levels of GM1. E) Western blotting for B4GALNT1 in whole cell lysates of B78 cells transfected with full length, wild-type B4GALNT1 or plasmids carrying the p.R519P mutation or a deletion of exon 6. Untransfected B78 cells do not express detectable levels of endogenous B4GALNT1. B78 cells transfected with wild-type cDNA show robust levels of B4GALNT1, whereas the p.R519P mutant protein is expressed at a lower level and the plasmid carrying an exon 6 deletion shows no expression above background. F) Immunocytochemistry for B4GALNT1 in B78 cells transfected with full length, wild-type B4GALNT1 or plasmids carrying the p.R519P mutation or a deletion of exon 6. Wild-type B4GALNT1 localizes predominantly to the Golgi network, marked by GM130. p.R519P mutant B4GALNT1 shows a more diffuse, cytoplasmic distribution and exon 6 deleted B4GALNT1 shows no expression. Scale bar = 50μm. G) In vitro B4GALNT1 enzyme activity is diminished below the detection level in B78 cells expressing either B4GALNT1 variant. H) Flow cytometry analysis of GM2 and GA2 (also known as asialo-GM2) in B78 cells and L cells, respectively, transfected with full length, wild-type B4GALNT1 or plasmids carrying the p.R519P mutation or a deletion of exon 6. Expression of either B4GALNT1 variant does not lead to any detectable levels of GM2 and GA2, confirming loss of B4GALNT1 enzyme activity.