The Nutraceutical Alliin From Garlic Is a Novel Substrate of the Essential Amino Acid Transporter LAT1 (SLC7A5)

Abstract

The plasma membrane transporter LAT1 (SLC7A5) is a crucial player for cell homeostasis because it is responsible for providing cells with essential amino acids and hormones. LAT1 forms a functional heterodimer with the cell surface antigen heavy chain CD98 (also known as 4F2hc and SLC3A2), a type II membrane glycoprotein, which is essential for LAT1 stability and localization to the plasma membrane. The relevance of LAT1 for human metabolism is also related to its altered expression in human diseases, such as cancer and diabetes. These features boosted research toward molecules that are able to interact with LAT1; in this respect, the recent resolution of the LAT1-CD98 3D structure by Cryo-EM has opened important perspectives in the study of the interaction with different molecules in order to identify new drugs to be used in therapy or new substrates of natural origin to be employed as adjuvants and food supplements. In this work, the interaction of LAT1 with alliin, a garlic derivative, has been investigated by using a combined approach of bioinformatics and in vitro transport assays. Alliin is a nutraceutical that has several beneficial effects on human health, such as antidiabetic, anticarcinogenic, antioxidant, and anti-inflammatory properties. The computational analysis suggested that alliin interacts with the substrate binding site of LAT1, to which alliin was docked. These data were then confirmed by the competitive type inhibition measured in proteoliposomes. Interestingly, in the same experimental model, alliin was also revealed to be a substrate of LAT1.

Keywords: LAT1; SLC; alliin; garlic; liposomes; membrane transport; nutraceutical.

FIGURE 1

Docking analysis of hLAT1. The cryo-EM structure of hLAT1 in inward conformation (PDB ID: 6IRT, chain B) was represented as ribbon (sand) using Chimera v.1.7 software (https://www.cgl.ucsf.edu/chimera). In (A), docking analysis was performed using InducedFit XP docking 5 as described in Materials and Methods. Molecular docking of alliin (sky blue) in the substrate binding site of hLAT1 with the gate residue F252 represented as sticks (sand). In the zoom, the pose of alliin with the docking score of −6.99 kcal/mol and a glide e-model of −72.237. The membrane and intracellular/extracellular environment are indicated. In (B), 2D visualization of hLAT1 interaction with histidine. The arrows indicate the residues involved in the binding. In (C), 2D visualization of hLAT1 interaction with alliin. The arrows indicated the residues involved in the binding.

FIGURE 2

Inhibition analysis of the recombinant hLAT1 in proteoliposomes. The reconstitution was performed as described in Materials and methods. In (A), dose–response analysis for the inhibition by alliin of the hLAT1. Transport was measured by adding 5 µM [³H]-histidine to proteoliposomes containing 10 mM histidine in the presence of indicated concentrations of alliin. Transport was stopped after 20 min as described in Materials and methods. Percent residual activity with respect to the control (without additions) is reported. In (B), kinetic analysis of the inhibition by alliin. Transport was measured by adding indicated concentrations of [³H]-histidine to proteoliposomes containing 10 mM histidine and stopping the reaction after 20 min; 75 µM (●) or 150 µM (□) alliin was added in comparison to samples without inhibitor (○).The data were plotted according to Lineweaver–Burk as reciprocal transport rate vs. reciprocal histidine concentration. Results are the mean ± S.D. from the three independent experiments.

FIGURE 3

Time course of histidine uptake and efflux in proteoliposomes. The reconstitution was performed as described in Materials and methods. (A) Transport was started by adding 5 μM [³H]-histidine to proteoliposomes containing 1 mM histidine (●), or 1 mM alliin (□), or without the internal substrate (○). The transport reaction was stopped at the indicated times according to the stop inhibitor method as described in Materials and Methods. (B) Uptake of 5 μM [³H]-histidine was performed in 60 min. Then, the efflux of [³H]-histidine was measured in the absence of the external substrate (○), or in the presence of 0.1 mM of external histidine (●) or alliin 0.1 mM (□) at the indicated times. The data are calculated as the percent of residual activity with respect to the control sample (efflux time zero). Results are the mean ± SD of three independent experiments. The experimental model is depicted as a sketch.