Rabbit Anti-Human SLC52A1 (internal) Polyclonal antibody

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.

Subramanian VS, Ghosal A, Subramanya SB, Lytle C, Said HM. Differentiation-dependent regulation of intestinal vitamin B2 uptake: studies utilizing human-derived intestinal epithelial Caco-2 cells and native rat intestine. Am J Physiol Gastrointest Liver Physiol 304: G741-G748, 2013. First published February 14, 2013; doi: 10.1152/ajpgi.00018.2013.-Intestinal epithelial cells undergo differentiation as they move from the crypt to the villi, a process that is associated with up-and downregulation in expression of a variety of genes, including those involved in nutrient absorption. Whether the intestinal uptake process of vitamin B2 [riboflavin (RF)] also undergoes differentiation-dependent regulation and the mechanism through which this occurs are not known. We used humanderived intestinal epithelial Caco-2 cells and native rat intestine as models to address these issues. Caco-2 cells showed a significantly higher carrier-mediated RF uptake in post-than preconfluent cells. This upregulation was associated with a significantly higher level of protein and mRNA expression of the RF transporters hRFVT-1 and hRFVT-3 in the post-than preconfluent cells; it was also accompanied with a significantly higher rate of transcription of the respective genes (SLC52A1 and SLC52A3), as indicated by the higher level of expression of heterogeneous nuclear RNA and higher promoter activity in post-than preconfluent cells. Studies with native rat intestine also showed a significantly higher RF uptake by epithelial cells of the villus tip than epithelial cells of the crypt; this again was accompanied by a significantly higher level of expression of the rat RFVT-1 and RFVT-3 at the protein, mRNA, and heterogeneous nuclear RNA levels. These findings show, for the first time, that the intestinal RF uptake process undergoes differentiation-dependent upregulation and suggest that this is mediated (at least in part) via transcriptional mechanisms.