A spectrum of mutations in the C20orf54
gene (which encodes hRFT-2) has been identified in patients with BVVLS [2
]. The hRFT-2 is expressed in a variety of tissues with high expression in the small intestine [7
]. In the gut, the protein is localized exclusively at the apical membrane domain of the polarized enterocytes [7
], and is believed to play an important role in intestinal RF absorption [6
]. Patients with BVVLS/Fazio Londe syndrome are riboflavin deficient despite normal dietary intake of the vitamin suggesting impairment in normal riboflavin assimilation [4
]; RF supplementation improves the clinical symptoms and the biochemical abnormalities in the affected patients [4
]. Based on this knowledge, we designed a study to examine the effect of hRFT-2 mutations identified in patients with BVVLS on functionality of the carrier protein in transporting riboflavin. The human intestinal epithelial Caco-2 cells were chosen for the study because of their suitability to study riboflavin transport [16
] and because intestinal epithelial cells express high levels of hRFT-2 [9
]. We generated six clinically relevant missense hRFT-2 mutants carrying single amino acid substitutions and examined the effect of the individual mutation on riboflavin uptake and expression profile of the mutated proteins in living cells.
Results of our study showed that five hRFT-2 mutants (W17R, P28T, E36K, E71K, and R132W) to cause a significant (P < 0.01) inhibition in RF uptake by Caco-2 cells. It is interesting that three of the mutated sites (W17, P28 and E36) are highly conserved in RFT-2 of different species, whereas the other two residues (E71 and R132) are not highly conserved (E71 can also be a D and T, and R132 can also be a Q). The mutated residues important for RF uptake are predicted to be located in the transmembrane domain 1 (W17), extracellular loop (P28 and E36K), and intracellular loops (E71K and R132W) of the hRFT-2 polypeptide (). The decrease in riboflavin uptake found in the five hRFT-2 missense mutants were not due to decrease in their transcriptional or translational efficiencies as judged by the observation that expression level of their mRNA and protein were similar to that of wild-type. The finding that the L350M mutation in hRFT-2 does not affect riboflavin uptake when expressed in Caco-2 cells is consistent with the previous suggestion that this mutation does not have pathogenic phenotype [2
]. This mutation, which is found in some BVVLS patients with compound homozygous mutations, is tolerated by SIFT algorithm and the amino acid involved is not conserved in mammalian RFT-2 [2
The hRFT-2 mutants P28T, E36K, E71K, and R132W were found by confocal imaging to be retained intracellular, most probably in the endoplasmic reticulum. This retention was uniformly associated with impairment in riboflavin uptake. This finding suggests an important role for these amino acid residues in the movement of the hRFT-2 protein to the cell membrane. Similar observations of impaired delivery to cell surface of clinically relevant mutants have been seen with other membrane transporters including the D-glucose transporter SGLT-1 in patients with glucose-galactose malabsorption and the thiamine transporter hTHTR-1 in patients with thiamine-responsive megaloblastic anaemia (TRMA) [21
]. A different situation was found for the W17R mutant where confocal imaging showed the protein to be expressed at the cell surface at the same level as that of the wild-type hRFT-2; nevertheless the mutated protein is functionality impaired. Confirmation of cell surface expression the W17R hRFT-2 mutant came from the results of the biotinylation assay, which again showed a similar level of membrane expression of the mutant and wild-type hRFT. These findings suggest that the tryptophan affected at position 17 of the hRFT-2 polypeptide is important for the transport function of hRFT-2, but not for its membrane targeting. Further studies at the cellular/molecular levels are needed to determine how this single mutation affects the functionality of hRFT-2.
In summary, our data demonstrate for the first time that missense mutations in hRFT-2 found in patients with BVVLS affect the functionality of the transporter in human intestinal epithelial cells and that this effect is mediated via alteration in membrane targeting and/or transporter activity.
- This study provides evidence that some of the Brown-Vialetto-Van Laere Syndrome (BVVLS) causing mutations in human riboflavin transporter -2 (hRFT-2) affect the transporter functionality in intestinal epithelial cells.
- This study also provides evidence that the BVVLS associated impairment in hRFT-2 functionality is mediated via alterations in membrane targeting and/or activity of the transporter.
- This study is important for better understanding of molecular mechanism of the BVVLS disorder and in order to gain insight into the structure-function/biology of the hRFT-2 protein.