Molecular Identification and Functional Characterization of the Human Colonic Thiamine Pyrophosphate Transporter [Membrane Biology]

December 30th, 2013 by Nabokina, S. M., Inoue, K., Subramanian, V. S., Valle, J. E., Yuasa, H., Said, H. M.

Colonic microbiota synthesizes considerable amount of thiamine in the form of thiamine pyrophosphate (TPP). Recent functional studies from our laboratory have shown the existence of a specific, high-affinity, and regulated carrier-mediated uptake system for TPP in human colonocytes. Nothing, however, is known about the molecular identity of this system. Here we report on molecular identification of the colonic TPP uptake system as the product of the SLC44A4 gene. We cloned the cDNA of SLC44A4 from human colonic epithelial NCM460 cells, which upon expression in ARPE19 cells led to a significant (P < 0.01; > 5 folds) induction in 3H-TPP uptake. Uptake by the induced system was also found to be: 1) temperature- and energy-dependent, 2) Na+-; slightly higher at acidic buffer pH and highly sensitive to protonophores, 3) saturable as a function of TPP concentration with an apparent Km of 0.17 ± 0.064 μM, and 4) highly specific for TPP and is not affected by free thiamine, thiamine monophosphate, or choline. Expression of the human TPP transporter (hTPPT) was found to be high in the colon and negligible in the small intestine. Cell surface biotinylation assay and live cell confocal imaging studies showed the hTPPT protein to be expressed at the apical membrane domain of polarized epithelia. These results report, for the first time, the molecular identification and characterization of a specific and high affinity TPP uptake system in human colonocytes. The findings lend further support that the microbiota-generated TPP is absorbable and could contribute toward host thiamine homeostasis, and especially toward cellular nutrition of colonocytes.