The human SLC52A1 gene encodes the riboflavin transporter-1 (RFVT-1), a plasma membrane protein that transports vitamin B2 (riboflavin, RF) into cells, and thus, plays a role in controlling cellular homeostasis of RF in those tissues that express the carrier protein (e.g. placenta and intestine). Currently, there is nothing known about transcriptional regulation of the SLC52A1 gene, therefore, we aimed to clone and characterize its 5'-flanking region. Using rapid amplification of the cDNA ends (5'-RACE), we identified one transcription start site (TSS). A 579 bp segment of the 5'-flanking region of this gene was cloned which exhibited robust promoter activity upon transfection in human intestinal epithelial cells. Deletion analysis revealed that the core promoter activity to be embedded in a region between -234 and -23 that lacked TATA element, was GC-rich, and harbored several putative cis-regulatory sites including KLFs, AP-2, EGRF and Sp-1. Mutating each of these sites led to a significant decrease in promoter activity (which was highest for the Sp-1 site), suggesting their possible involvement in regulating SLC52A1 transcription. Focusing on the Sp-1 site, EMSA, super-shift and ChIP analysis was performed that established the interaction of the Sp-1 transcription factor with the SLC52A1 promoter; also, co-transfection of the minimal SLC52A1 promoter with an Sp-1 containing vector in Drosophila SL-2 cells led to significant promoter activation. These results are the first to reveal the identity of the minimal SLC52A1 promoter and to establish an important role for Sp-1 in its activity.

Background

Riboflavin (RF) is essential for normal cellular metabolic activities. Human cells obtain RF from their surroundings via a carrier-mediated process that involves RF transporters -1, -2 & -3 (hRFVT -1, -2 & -3; products of SLC52A1, -A2 and -A3 genes, respectively). Little is known about the structural features of these transporters that are important for their function/cell biology. Our aim in this study was to address these issues for the hRFVT-2, a transporter linked to the neurodegenerative disorder Brown-Vialetto-Van Laere Syndrome (BVVLS).

Methods

We used comparative protein-structure modelling to predict residues that interact with two amino acids known to be critical for hRFVT-2 function (the clinical mutants L123 and L339), site-directed mutagenesis, and truncation approach in the human-derived brain U87 cell model.

Results

First we showed that the defect in the function of the L123 and L339 hRFVT-2 clinical mutants is related to a reduction in protein stability/translation efficiency and to retention of the protein in the ER. Mutating V120 and L121 (residues predicted to interact with L123) and L342 (a residue predicted to interact with L339) also led to a significant inhibition in hRFVT-2 function (with no change in membrane expression); this inhibition was associated with changes in protein stability/translation efficiency (in the case of V120A and L342A) and an impairment in transport function (in the case of L121). Truncating the N- and C- terminals of hRFVT-2 led to significant inhibition in RF uptake, which was associated with changes in protein stability/translation efficiency (it was also associated with a partial impairment in membrane targeting in the case of the N-terminal truncation).

Conclusion

These investigations report on identification of residues/sequences in the hRFVT-2 protein that is important for its physiological function and cell biology.

The intestinal absorption process of vitamin B2 (riboflavin, RF) is carrier-mediated, and all three known human RF transporters, i.e., hRFVT-1, -2, and -3 (products of the SLC52A1, 2 & 3 genes, respectively) are expressed in the gut. We have previously shown that the intestinal RF uptake process is adaptively regulated by substrate level, but little is known about the molecular mechanism(s) involved. Using human intestinal epithelial NCM460 cells maintained under RF deficient and over-supplemented (OS) conditions, we now show that the induction in RF uptake in RF deficiency is associated with an increase in expression of the hRFVT-2 & -3 (but not hRFVT-1) at the protein and mRNA levels. Focusing on hRFVT-3, the predominant transporter in the intestine, we also observed an increase in the level of expression of its hnRNA and activity of its promoter in the RF deficiency state. An increase in the level of expression of the nuclear factor Sp1 (which is important for activity of the SLC52A3 promoter) was observed in RF deficiency, while mutating the Sp1/GC site in the SLC52A3 promoter drastically decreased the level of induction in SLC52A3 promoter activity in RF deficiency. We also observed specific epigenetic changes in the SLC52A3 promoter in RF deficiency. Finally, an increase in hRFVT-3 protein expression at the cell surface was observed in RF deficiency. Results of these investigations show, for the first time, that transcriptional and post-transcriptional mechanisms are involved in the adaptive regulation of intestinal RF uptake by the prevailing substrate level.