Éva Margittai1, Csilla Németh2, Angelo Benedetti3, Gábor Bánhegyi2
1 Semmelweis University, Institute of Clinical Experimental Research
2 Semmelweis University, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry
3 University of Siena, Department of Molecular and Developmental Medicine
Introduction: GLUT10 belongs to a family of transporters that catalyze the uptake of sugars/polyols by facilitated diffusion. Loss-of-function mutations in the gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS), a rare connective tissue disorder mainly characterized by tortuosity, stenosis and aneurysm of main arteries.
Aims: Eventhough the transporter was described long ago, neither the subcellular localization of GLUT10, nor its transported ligand(s) have been clearly identified. We aimed to characterize the transporter and in light of this, to propose a novel hypothesis for the pathomechanism of ATS.
Methods: As a model system, fibroblasts isolated from control and ATS cells were used. The localization of the transporter was verified by immunocytochemistry and Western blot analysis. The transport activity of the protein was examined by rapid filtration technique.
Results: The results showed a perinuclear distribution of GLUT10 as demonstrated by in fibroblasts from healthy controls. Immunoblotting revealed that GLUT10 protein was present in the microsomal fraction of the cells. Dehydroascorbic acid transport and accumulation was markedly reduced in fibroblasts from ATS patients and in GLUT10 shRNAi-silenced fibroblasts whose plasma membrane was selectively permeabilized. Re-expression of GLUT10 in patients‘ fibroblasts restored dehydroascorbic acid transport activity. Measurement of dehydroascorbic acid uptake in subcellular fractions of fibroblasts showed that endoplasmic reticulum transport was reduced in patients. GLUT10 protein produced by in vitro translation and incorporated into liposomes efficiently transported dehydroascorbic acid. Lower intracellular ascorbate content was measured in patients’ fibroblasts incubated in the presence of physiological concentration of ascorbate.
Conclusion: The results demonstrate that GLUT10 is a dehydroascorbic acid transporter in the endoplasmic reticulum and nuclear envelope. Furthermore, they suggest that a reduced transport of dehydroascorbic acid into the endoplasmic reticulum and nucleoplasm – where ascorbate functions as a cofactor for Fe2+/2-oxoglutarate dependent dioxygenases – can be a causal factor of the pathomechanism in ATS.
ÚNKP postdoctoral fellowship winner