2. University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0436, USA.
Background: The equilibrative nucleoside transporter 1 (ENT1), a protein encoded by the SLC29A1 gene, is able to carry nucleosides across cell membranes. The high affinity for adenosine makes it relevant to anti-retroviral therapy (ART), where the drugs are adenosine analogs. The ENT1 protein might transport adenosine drug analogs such as tenofovir (TFV) and its prodrugs into and out of cells, removing them from their site of action.
Methods: A commercial polyclonal antibody to the ENT1 protein was applied to a vaginal cultured cell line and on vaginal epithelial cells (VEC) donated by female volunteers. Antibody labeling was tested by western blotting, by light and electron microscopy, and by quantitative analysis of anti-ENT1 label on V19 multilayer cultures. Antibody specificity was tested by affinity adsorption.
Results: The ENT1 protein was present on VEC from human donors and on V19 cultured cells. The ENT1 antibody revealed protein bands at 50 kDa and 30 kDa on a western blot of V19 proteins. Light microscopy revealed only limited information on the sub-cellular location of the ENT1 protein, but immuno-electron microscopy showed the ENT1 protein predominantly located on the plasma membrane and lysosome-like structures. Label was also present over the endoplasmic reticulum (ER), Golgi complex, and cytoplasmic vesicles.
Conclusions: The results suggest a synthesis and degradation pathway for the ENT1 protein. However, the presence of ENT1 in junctional complexes and mitochondria suggest a more complex role for the protein. The ENT1 protein may potentially transport adenosine drug analogs such as tenofovir (TFV) and its prodrugs into and out of cells. The presence of ENT1 on VEC, with a rapid turnover, may have wideranging implications on anti-retroviral treatment (ART) therapies by removing them from their site of action. However, the bidirectional nature of ENT1 could mean that drugs such as TFV are also removed from cells. Transporter proteins such as ENT1 could shuttle the TFV through the vaginal epithelium to immune cells located in lower cell layers. Future studies will focus on determining if the ENT1 protein is active on vaginal cell plasma membranes, and if it has a role in the transport of nucleoside analogs.
Keywords: Tenofovir, Electron Microscopy, Retrovirus, Drug Delivery, Protein Expression, Transporter, Immunolabeling