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Functional and Molecular Aspects of Biotin Uptake via SMVT in Human Corneal Epithelial (HCEC) and Retinal Pigment Epithelial (D407) Cells

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ABSTRACT

Sodium-dependent multivitamin transporter (SMVT) is a vital transmembrane protein responsible for translocating biotin and other essential cofactors such as pantothenate and lipoate. Unlike primary cultures of corneal and retinal pigment epithelial (RPE) cells, immortalized cells can be subcultured many times, yet maintain their physiological properties. Hence, the purpose of this study was to delineate the functional and molecular aspects of biotin uptake via SMVT on immortalized human corneal epithelial (HCEC) and RPE (D407) cells. Functional aspects of [3H] biotin uptake were studied in the presence of different concentrations of unlabeled biotin, pH, temperature, metabolic inhibitors, ions, substrates, structural analogs and biotinylated prodrug (Biotin-Acyclovir (B-ACV)). Molecular identity of SMVT was examined with reverse transcription–polymerase chain reaction. Biotin uptake was found to be saturable in HCEC and D407 cells with K m of 296.2 ± 25.9 and 863.8 ± 66.9 μM and V max of 77.2 ± 2.2 and 308.3 ± 10.7 pmol/mg protein/min, respectively. Uptake was found to be pH, temperature, energy, and sodium-dependent. Inhibition of biotin uptake was observed in the presence of structural analogs and specific substrates. Further, uptake was lowered in the presence of B-ACV indicating the translocation of biotinylated prodrug by SMVT. A distinct band at 774 bp confirmed the molecular existence of SMVT in both the cells. This study shows for the first time the functional and molecular presence of SMVT in HCEC and D407 cells. Therefore, these cell lines may be utilized as in vitro models to study the cellular translocation of biotin-conjugated prodrugs.

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ACKNOWLEDGMENTS

This study has been supported by NIH grants R01EY09171-16 and R01EY010659-14. The authors would like to acknowledge Dr. Vadivel Ganapathy and Dr. Pamela Martin from the Department of Biochemistry and Molecular Biology at Georgia Health Sciences University for their generous help in providing human corneal RNA used in our studies. Also, the authors would like to thank Matthew Scrivner at the UMKC Writing Center for his assistance during the preparation of this manuscript.

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  1. Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri—Kansas City, 2464 Charlotte Street, Kansas City, Missouri, 64108-2718, USA

    Aswani Dutt Vadlapudi, Ramya Krishna Vadlapatla, Dhananjay Pal & Ashim K. Mitra

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  1. Aswani Dutt Vadlapudi
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  2. Ramya Krishna Vadlapatla
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  4. Ashim K. Mitra
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Correspondence to Ashim K. Mitra.

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Vadlapudi, A.D., Vadlapatla, R.K., Pal, D. et al. Functional and Molecular Aspects of Biotin Uptake via SMVT in Human Corneal Epithelial (HCEC) and Retinal Pigment Epithelial (D407) Cells. AAPS J 14, 832–842 (2012). https://doi.org/10.1208/s12248-012-9399-5

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