Figure 2.
GALE variants associated with reduced GALE protein levels, impaired enzymatic activity, platelet hypoglycosylation, and increased apoptosis. (A) Western blotting from platelet lysates of patients from both pedigrees, unaffected relatives, and controls. Membranes were blotted with anti-GALE and anti-β-actin as an internal control. A.II.2 presented total levels of GALE protein similar to controls, but 2 GALE bands with different electrophoretic mobility were observed (top, GALE-WT; bottom, GALE p.Thr150Met), whereas A.I.2 displayed a 50% reduction of total GALE protein. Probands from pedigree A (II.1 and II.4) showed reduced protein levels (50%) and a tiny band corresponding to GALE p.Thr150Met. Individuals from pedigree B: I.1, I.2, and II.2 showed reduced total GALE protein levels (51%, 65%, and 35%, respectively) vs control. (B) Enzymatic activity of UDP-galactose-4-epimerase assessed in members of pedigrees A and B. The activity of a healthy control was evaluated in parallel. Probands of both families showed a sharp reduction in the GALE enzymatic activity (A.II.1, 15%; A.II.4, 15%; and B.II.2, 7.5%), whereas heterozygous carriers in both families displayed a moderate reduction (A.II.2, 42%; A.I.1, 45%; B.I.1, 31%; and B.I.2, 24%). (C) To assess the glycosylation profile, platelet lysates were probed with lectin ECL, which specifically binds to LacNAc, a dimer of N-acetyl-glucosamine and galactose molecules. β-actin staining, shown in panel A, was used as an internal control. A sharp decrease in ECL binding was found in all probands, less pronounced in heterozygous carriers of a single GALE variant. (D) Platelet lysates were probed with antibodies against procaspase 8 (56 kDa) (top band) or the active-cleaved form of caspase 8 (bottom bands). β-actin (shown in panel A) was the internal protein control. Proband from pedigree A displayed increased procaspase 8 and active caspase 8 level, whereas in the proband from the pedigree B only the active caspase 8 was increased. Band intensities were quantified by densitometric analysis using the ‘Image J’ software. ECL, Erythrina crista-galli lectin; LacNAc, N-acetyl-lactosamine; UDP-Glc, UDP-glucose; UDP-Gal, UDP-galactose.

GALE variants associated with reduced GALE protein levels, impaired enzymatic activity, platelet hypoglycosylation, and increased apoptosis. (A) Western blotting from platelet lysates of patients from both pedigrees, unaffected relatives, and controls. Membranes were blotted with anti-GALE and anti-β-actin as an internal control. A.II.2 presented total levels of GALE protein similar to controls, but 2 GALE bands with different electrophoretic mobility were observed (top, GALE-WT; bottom, GALE p.Thr150Met), whereas A.I.2 displayed a 50% reduction of total GALE protein. Probands from pedigree A (II.1 and II.4) showed reduced protein levels (50%) and a tiny band corresponding to GALE p.Thr150Met. Individuals from pedigree B: I.1, I.2, and II.2 showed reduced total GALE protein levels (51%, 65%, and 35%, respectively) vs control. (B) Enzymatic activity of UDP-galactose-4-epimerase assessed in members of pedigrees A and B. The activity of a healthy control was evaluated in parallel. Probands of both families showed a sharp reduction in the GALE enzymatic activity (A.II.1, 15%; A.II.4, 15%; and B.II.2, 7.5%), whereas heterozygous carriers in both families displayed a moderate reduction (A.II.2, 42%; A.I.1, 45%; B.I.1, 31%; and B.I.2, 24%). (C) To assess the glycosylation profile, platelet lysates were probed with lectin ECL, which specifically binds to LacNAc, a dimer of N-acetyl-glucosamine and galactose molecules. β-actin staining, shown in panel A, was used as an internal control. A sharp decrease in ECL binding was found in all probands, less pronounced in heterozygous carriers of a single GALE variant. (D) Platelet lysates were probed with antibodies against procaspase 8 (56 kDa) (top band) or the active-cleaved form of caspase 8 (bottom bands). β-actin (shown in panel A) was the internal protein control. Proband from pedigree A displayed increased procaspase 8 and active caspase 8 level, whereas in the proband from the pedigree B only the active caspase 8 was increased. Band intensities were quantified by densitometric analysis using the ‘Image J’ software. ECL, Erythrina crista-galli lectin; LacNAc, N-acetyl-lactosamine; UDP-Glc, UDP-glucose; UDP-Gal, UDP-galactose.

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