Mariafrancesca Scalise

Membrane transporters for the special amino acid glutamine: structure/function relationships and relevance to human health

Glutamine together with glucose is essential for bodys homeostasis. It is the most abundant amino acid and is involved in many biosynthetic, regulatory and energy production processes. Several membrane transporters which differ in transport modes, ensure glutamine homeostasis by coordinating its absorption, reabsorption and delivery to tissues. These transporters belong to different protein families, are redundant and ubiquitous. Their classification, originally based on functional properties, has recently been associated with the SLC nomenclature. Function of glutamine transporters is studied in cells over-expressing the transporters or, more recently in proteoliposomes harboring the proteins extracted from animal tissues or over-expressed in microorganisms. The role of the glutamine transporters is linked to their transport modes and coupling with Na+ and H+. Most transporters share specificity for other neutral or cationic amino acids. Na+-dependent co-transporters efficiently accumulate glutamine while antiporters regulate the pools of glutamine and other amino acids. The most acknowledged glutamine transporters belong to the SLC1, 6, 7, and 38 families. The members involved in the homeostasis are the co-transporters B0AT1 and the SNAT members 1, 2, 3, 5, and 7; the antiporters ASCT2, LAT1 and 2. The last two are associated to the ancillary CD98 protein. Some information on regulation of the glutamine transporters exist, which, however, need to be deepened. No information at all is available on structures, besides some homology models obtained using similar bacterial transporters as templates. Some models of rat and human glutamine transporters highlight very similar structures between the orthologs. Moreover the presence of glycosylation and/or phosphorylation sites located at the extracellular or intracellular faces has been predicted. ASCT2 and LAT1 are over-expressed in several cancers, thus representing potential targets for pharmacological intervention.

OCTN Cation Transporters in Health and Disease Role as Drug Targets and Assay Development

The three members of the organic cation transporter novel subfamily are known to be involved in interactions with xenobiotic compounds. These proteins are characterized by 12 transmembrane segments connected by nine short loops and two large hydrophilic loops. It has been recently pointed out that acetylcholine is a physiological substrate of OCTN1. Its transport could be involved in nonneuronal cholinergic functions. OCTN2 maintains the carnitine homeostasis, resulting from intestinal absorption, distribution to tissues, and renal excretion/reabsorption. OCTN3, identified only in mouse, mediates also carnitine transport. OCTN1 and OCTN2 are associated with several pathologies, such as inflammatory bowel disease, primary carnitine deficiency, diabetes, neurological disorders, and cancer, thus representing useful pharmacological targets. The function and interaction with drugs of OCTNs have been studied in intact cell systems and in proteoliposomes. The latter experimental model enables reduced interference from other transporters or enzyme pathways. Using proteoliposomes, the molecular bases of toxicity of some drugs have recently been revealed. Therefore, proteoliposomes represent a promising experimental tool suitable for large-scale molecular screening of interactions of OCTNs with chemicals regarding human health.

Impaired Amino Acid Transport at the Blood Brain Barrier Is a Cause of Autism Spectrum Disorder

Autism spectrum disorders (ASD) are a group of genetic disorders often overlapping with other neurological conditions. We previously described abnormalities in the branched-chain amino acid (BCAA) catabolic pathway as a cause of ASD. Here, we show that the solute carrier transporter 7a5 (SLC7A5), a large neutral amino acid transporter localized at the blood brain barrier (BBB), has an essential role in maintaining normal levels of brain BCAAs. In mice, deletion of Slc7a5 from the endothelial cells of the BBB leads to atypical brain amino acid profile, abnormal mRNA translation, and severe neurological abnormalities. Furthermore, we identified several patients with autistic traits and motor delay carrying deleterious homozygous mutations in the SLC7A5 gene. Finally, we demonstrate that BCAA intracerebroventricular administration ameliorates abnormal behaviors in adult mutant mice. Our data elucidate a neurological syndrome defined by SLC7A5 mutations and support an essential role for the BCAA in human brain function.

The human OCTN1 (SLC22A4) reconstituted in liposomes catalyzes acetylcholine transport which is defective in the mutant L503F associated to the Crohn's disease.

The organic cation transporter (OCTN1) plays key roles in transport of selected organic cations, but understanding of its biological functions remains limited by restricted knowledge of its substrate targets. Here we show capacity of human OCTN1-reconstituted proteoliposomes to mediate uptake and efflux of [(3)H]acetylcholine, the Km of transport being 1.0mM with V(max) of 160nmol⋅mg(-1)protein⋅min(-1). OCTN1-mediated transport of this neurotransmitter was time-dependent and was stimulated by intraliposomal ATP. The transporter operates as uniporter but translocates acetylcholine in both directions. [(3)H]acetylcholine uptake was competitively inhibited by tetraethylammonium, γ-butyrobetaine and acetylcarnitine, and was also inhibited by various polyamines. Decreasing intraliposomal ATP concentrations increased OCTN Km for acetylcholine, but V(max) was unaffected. Evaluation of the acetylcholine transporter properties of a variant form of OCTN1, the Crohns disease-associated 503F variant, revealed time course, Km and V(max) for acetylcholine uptake to be comparable to that of wild-type OCTN1. Km for acetylcholine efflux was also comparable for both OCTN1 species, but V(max) of OCTN1 503F-mediated acetylcholine efflux (1.9nmol⋅mg(-1)protein⋅min(-1)) was significantly lower than that of wild-type OCTN1 (14nmol⋅mg(-1)protein⋅min(-1)). These data identify a new transport role for OCTN1 and raise the possibility that its involvement in the non-neuronal acetylcholine system may be relevant to the pathogenesis of Crohns disease.

LAT1 is the transport competent unit of the LAT1/CD98 heterodimeric amino acid transporter.

LAT1 (SLC7A5) and CD98 (SLC3A2) constitute a heterodimeric transmembrane protein complex that catalyzes amino acid transport. Whether one or both subunits are competent for transport is still unclear. The present work aims to solve this question using different experimental strategies. Firstly, LAT1 and CD98 were immuno-detected in protein extracts from SiHa cells. Under oxidizing conditions, i.e., without addition of SH (thiol) reducing agent DTE, both proteins were revealed as a 120kDa major band. Upon DTE treatment separated bands, corresponding to LAT1(35kDa) or CD98(80kDa), were detected. LAT1 function was evaluated in intact cells as BCH sensitive [(3)H]His transport inhibited by hydrophobic amino acids. Antiport of [(3)H]His was measured in proteoliposomes reconstituted with SiHa cell extract in presence of internal His. Transport was increased by DTE. Hydrophobic amino acids were best inhibitors in addition to hydrophilic Tyr, Gln, Asn and Lys. Cys, Tyr and Gln, included in the intraliposomal space, were transported in antiport with external [(3)H]His. Similar experiments were performed in proteoliposomes reconstituted with the recombinant purified hLAT1. Results overlapping those obtained with native protein were achieved. Lower transport of [(3)H]Leu and [(3)H]Gln with respect to [(3)H]His was detected. Kinetic asymmetry was found with external Km for His lower than internal one. No transport was detected in proteoliposomes reconstituted with recombinant hCD98. The experimental data demonstrate that LAT1 is the sole transport competent subunit of the heterodimer. This conclusion has important outcome for following studies on functional characterization and identification of specific inhibitors with potential application in human therapy.

Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris--functional and kinetic asymmetry revealed in proteoliposomes.

The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni(2+)-chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [(3)H]glutamine/glutamine antiport was measured in proteoliposomes which was active only in the presence of external Na(+). Internal Na(+) slightly stimulated the antiport. Optimal activity was found at pH7.0. A substantial inhibition of the transport was observed by Cys, Thr, Ser, Ala, Asn and Met (≥70%) and by mercurials and methanethiosulfonates (≥80%). Heterologous antiport of [(3)H]glutamine with other neutral amino acids was also studied. The transporter showed asymmetric specificity for amino acids: Ala, Cys, Val, Met were only inwardly transported, while Gln, Ser, Asn, and Thr were transported bi-directionally. From kinetic analysis of [(3)H]glutamine/glutamine antiport Km values of 0.097 and 1.8mM were measured on the external and internal sides of proteoliposomes, respectively. The Km for Na(+) on the external side was 32mM. The homology structural model of the hASCT2 protein was built using the GltPh of Pyrococcus horikoshii as template. Cys395 was the only Cys residue externally exposed, thus being the potential target of SH reagents inhibition and, hence, potentially involved in the transport mechanism.

N-linked Glycosylation of human SLC1A5 (ASCT2) transporter is critical for trafficking to membrane

The human amino acid transporter SLC1A5 (ASCT2) contains two N-glycosylation sites (N163 and N212) located in the large extracellular loop. In the homology structural model of ASCT2 these Asn residues are extracellularly exposed. Mutants of the two Asn exhibited altered electrophoretic mobility. N163Q and N212Q displayed multiple bands with apparent molecular masses from 80kDa to 50kDa. N163/212Q displayed a single band of 50kDa corresponding to the unglycosylated protein. The presence in membrane of WT and mutants was evaluated by protein biotinylation assay followed by immunoblotting. The double mutation significantly impaired the presence of the protein in membrane, without impairment in protein synthesis. [(3)H]glutamine transport was measured in cells transiently transfected with the WT or mutants. N163/212Q exhibited a strongly reduced transport activity correlating with reduced surface expression. The same proteins extracted from cells and reconstituted in liposomes showed comparable transport activities demonstrating that the intrinsic transport function of the mutants was not affected. The rate of endocytosis of ASCT2 was assayed by a reversible biotinylation strategy. N212Q and N163/212Q showed strongly increased rates of endocytosis respect to WT. ASCT2 stability was determined using cycloheximide. N163Q or N163/212Q showed a slightly or significantly lower stability with respect to WT. To assess trafficking to the membrane, a brefeldin-based assay, which caused retention of proteins in ER, was performed. One hour after brefeldin removal WT protein was localized to the plasma membrane while the double mutant was localized in the cytosol. The results demonstrate that N-glycosylation is critical for trafficking.

Transport mechanism and regulatory properties of the human amino acid transporter ASCT2 (SLC1A5)

The kinetic mechanism of the transport catalyzed by the human glutamine/neutral amino acid transporter hASCT2 over-expressed in P. pastoris was determined in proteoliposomes by pseudo-bi-substrate kinetic analysis of the Na+-glutamineex/glutaminein transport reaction. A random simultaneous mechanism resulted from the experimental analysis. Purified functional hASCT2 was chemically cross-linked to a stable dimeric form. The oligomeric structure correlated well with the kinetic mechanism of transport. Half-saturation constants (Km) of the transporter for the other substrates Ala, Ser, Asn and Thr were measured both on the external and internal side. External Km were much lower than the internal ones confirming the asymmetry of the transporter. The electric nature of the transport reaction was determined imposing a negative inside membrane potential generated by K+ gradients in the presence of valinomycin. The transport reaction resulted to be electrogenic and the electrogenicity originated from external Na+. Internal Na+ exerted a stimulatory effect on the transport activity which could be explained by a regulatory, not a counter-transport, effect. Native and deglycosylated hASCT2 extracted from HeLa showed the same transport features demonstrating that the glycosyl moiety has no role in transport function. Both in vitro and in vivo interactions of hASCT2 with the scaffold protein PDZK1 were revealed.

Regulation by physiological cations of acetylcholine transport mediated by human OCTN1 (SLC22A4). Implications in the non-neuronal cholinergic system.

AIMS This study aimed to investigate the influence of physiological ions on the transport of acetylcholine which is catalyzed by the recombinant human Organic Cation Transporter Novel 1 (hOCTN1), thus being involved in the function of the non neuronal cholinergic system. MAIN METHODS The experimental model of proteoliposomes reconstituted with the hOCTN1 transporter obtained by over-expression in E. coli has been used. Uptake and efflux of [(3)H]acetylcholine in the proteoliposome system have been followed in the presence of different cations, mimicking the cell environment. KEY FINDINGS Internal K(+) stimulated, while external Na(+) strongly inhibited the uptake of [(3)H]acetylcholine in proteoliposomes. Strong inhibition was exerted also by external K(+) while Mg(2+) or sucrose had no effect. Differently, the efflux of [(3)H]acetylcholine from proteoliposomes was not influenced by external or internal Na(+) and was only marginally stimulated by internal K(+). By dose response analysis of the Na(+) inhibition, an IC(50) of 1.3 mM was derived. The kinetic analysis of the Na(+) effect revealed a competitive type of inhibition on acetylcholine uptake, i.e., Na(+) interacts with the same external binding site of acetylcholine with a Ki of 1.2 mM. SIGNIFICANCE Acetylcholine transport catalyzed by hOCTN1 revealed an asymmetric regulation by Na(+). Since the orientation of the transporter in the liposomal membrane is the same as in native membranes, and on the basis of sidedness of inhibition, physiological acetylcholine is principally exported by the transporter. This implies a role in autocrine and paracrine effects in non neuronal tissues.

Reconstitution in liposomes of the functionally active human OCTN1 (SLC22A4) transporter overexpressed in Escherichia coli

The hOCTN1 (human organic cation transporter 1) overexpressed in Escherichia coli and purified by Ni-chelating chromatography has been reconstituted in liposomes by detergent removal with a batch-wise procedure. The reconstitution was optimized with respect to the protein concentration, the detergent/phospholipid ratio and the time of incubation with Amberlite XAD-4 resin. Time-dependent [(14)C]tetraethylammonium, [(3)H]carnitine or [(3)H]ergothioneine uptake was measured in proteoliposomes with activities ratios of 8:1.3:1 respectively. Optimal activity was found at pH 8.0. The transport depended on intraliposomal ATP. [(14)C]tetraethylammonium transport was inhibited by several compounds. The most effective were acetyl-choline and γ-butyrobetaine, followed by acetylcarnitine and tetramethylammonium. Reagents such as pyridoxal 5-phosphate, MTSES [sodium (2-sulfonatoethyl) methanethiosulfonate] and mercurials strongly inhibited the transport. From kinetic analysis of tetraethylammonium transport a K(m) of 0.77 mM was calculated. Acetylcholine and γ-butyrobetaine behaved as competitive inhibitors of TEA (tetraethylammonium) transport with K(i) values of 0.44 and 0.63 mM respectively.