scholarly journals Cryo-EM structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc

2019 ◽  
Author(s):  
Yongchan Lee ◽  
Pattama Wiriyasermkul ◽  
Chunhuan Jin ◽  
Lili Quan ◽  
Ryuichi Ohgaki ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transmembrane Helix ◽  
Amino Acid Transporter ◽  
Molecular Assembly ◽  
Surface Glycoprotein ◽  
Binding Modes ◽  
Cell Surface Glycoprotein ◽  
Solute Carrier ◽  
Acid Transporter

SummaryThe L-type amino acid transporter 1 (LAT1) transports large neutral amino acids and drugs across the plasma membrane and is crucial for nutrient uptake, brain drug delivery and tumor growth. LAT1 is a unique solute carrier that forms a disulfide-linked heterodimer with the cell-surface glycoprotein CD98 heavy chain (CD98hc), but the mechanisms of its molecular assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.4 Å resolution, revealing the hitherto unprecedented architecture of a solute carrier-glycoprotein heterocomplex. LAT1 features a canonical LeuT-fold while exhibiting an unusual loop structure on transmembrane helix 6, creating an extended cavity to accommodate bulky hydrophobic amino acids and drugs. CD98hc engages with LAT1 through multiple interactions, not only in the extracellular and transmembrane domains but also in the interdomain linker. The heterodimer interface features multiple sterol molecules, corroborating previous biochemical data on the role of cholesterols in heterodimer stabilization. We also visualized the binding modes of two anti-CD98 antibodies and show that they recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities despite the glycan heterogeneity. Furthermore, we mapped disease-causing mutations onto the structure and homology models, which rationalized some of the phenotypes of SLC3- and SLC7-related congenital disorders. Together, these results shed light on the principles of the structural assembly between a glycoprotein and a solute carrier, and provide a template for improving preclinical drugs and therapeutic antibodies targeting LAT1 and CD98.

scholarly journals Amino Acid Carriers of the Solute Carrier Families 7 (SLC7) and 38 (SLC38) Are Involved in Leucine Sensing in the Brain of Atlantic Salmon (Salmo salar)

2021 ◽  
Vol 8 ◽  
Author(s):  
Sara Comesaña ◽  
Floriana Lai ◽  
Ann-Elise Olderbakk Jordal ◽  
Tiziano Verri ◽  
Marit Espe ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Atlantic Salmon ◽  
Feed Intake ◽  
Amino Acid Transporter ◽  
Limited Information ◽  
Brain Areas ◽  
Solute Carrier ◽  
Acid Transporter ◽  
Amino Acid Sensing

Sensing of amino acids in fish brain, especially branched-chain amino acids (BCAA) like leucine, is involved in regulation of feed intake through different mechanisms. However, there is limited information regarding the possible involvement of mechanisms dependent on amino acid carriers of the solute carrier families (SLC) known to be key regulators of intracellular leucine concentration, namely L-type amino acid transporter 1 (LAT1), and sodium-dependent neutral amino acid transporter 2 (SNAT2) and 9,(SNAT9), for which evidence of their participation is available in mammals. Comparative analysis amongst sequences revealed a complex pattern of paralogues in Atlantic salmon, for LAT1 (slc7a5aa, slc7a5ab, slc7a5ba, slc7a5bb, slc7a5ca, and slc7a5cb), SNAT2 (slc38a2a and slc38a2b) and SNAT9 (slc38a9). After establishing phylogenetic relationships of the different paralogues evaluated, samples of the selected brain areas were taken from Atlantic salmon to assess tissue distribution of transcripts. In an additional experiment, fish were fed two diets with different levels of leucine (high leucine: 35 g/kg vs. control leucine: 27.3 g/kg). The high leucine diet resulted in lower feed intake and increased mRNA abundance of specific paralogues of LAT1 (slc7a5aa, slc7a5ab, and slc7a5bb) and SNAT2 (slc38a2a and slc38a2b) though apparently not for SNAT9 in brain areas like hypothalamus and telencephalon involved in food intake regulation. The results obtained suggest a role for members of the SLC family in the anorectic effect of leucine and thus their involvement as additional amino acid sensing mechanism not characterised so far in fish regulation of feed intake.

scholarly journals Neutral amino acid transporter ASCT2 displays substrate-induced Na+ exchange and a substrate-gated anion conductance

2000 ◽  
Vol 346 (3) ◽  
pp. 705-710 ◽  
Author(s):  
Angelika BRÖER ◽  
Carsten WAGNER ◽  
Florian LANG ◽  
Stefan BRÖER
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Anion Channel ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Xenopus Laevis Oocytes ◽  
Anion Conductance ◽  
Neutral Amino Acid Transporter ◽  
Inward Currents ◽  
Acid Transporter

The neutral amino acid transporter ASCT2 mediates electroneutral obligatory antiport but at the same time requires Na+ for its function. To elucidate the mechanism, ASCT2 was expressed in Xenopus laevis oocytes and transport was analysed by flux studies and two-electrode voltage clamp recordings. Flux studies with 22NaCl indicated that the uptake of one molecule of glutamine or alanine is accompanied by the uptake of four to seven Na+ ions. Similarly to the transport of amino acids, the Na+ uptake was mediated by an obligatory Na+ exchange mechanism that depended on the presence of amino acids but was not stoichiometrically coupled to the amino acid transport. Other cations could not replace Na+ in this transport mechanism. When NaCl was replaced by NaSCN in the transport buffer, the superfusion of oocytes with amino acid substrates resulted in large inward currents, indicating the presence of a substrate-gated anion channel in the ASCT2 transporter. The Km for glutamine derived from these experiments is in good agreement with the Km derived from flux studies; it varied between 40 and 90 μM at holding potentials of -60 and -20 mV respectively. The permeability of the substrate-gated anion conductance decreased in the order SCN- NO3- > I- > Cl- and also required the presence of Na+.

scholarly journals Intestinal peptidases form functional complexes with the neutral amino acid transporter B0AT1

2012 ◽  
Vol 446 (1) ◽  
pp. 135-148 ◽  
Author(s):  
Stephen J. Fairweather ◽  
Angelika Bröer ◽  
Megan L. O'Mara ◽  
Stefan Bröer
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Amino Acid Transporter ◽  
The Body ◽  
Site Directed Mutagenesis ◽  
Substrate Affinity ◽  
Xenopus Laevis Oocytes ◽  
Acid Transporter

The brush-border membrane of the small intestine and kidney proximal tubule are the major sites for the absorption and re-absorption of nutrients in the body respectively. Transport of amino acids is mediated through the action of numerous secondary active transporters. In the mouse, neutral amino acids are transported by B0AT1 [broad neutral (0) amino acid transporter 1; SLC6A19 (solute carrier family 6 member 19)] in the intestine and by B0AT1 and B0AT3 (SLC6A18) in the kidney. Immunoprecipitation and Blue native electrophoresis of intestinal brush-border membrane proteins revealed that B0AT1 forms complexes with two peptidases, APN (aminopeptidase N/CD13) and ACE2 (angiotensin-converting enzyme 2). Physiological characterization of B0AT1 expressed together with these peptidases in Xenopus laevis oocytes revealed that APN increased the substrate affinity of the transporter up to 2.5-fold and also increased its surface expression (Vmax). Peptide competition experiments, in silico modelling and site-directed mutagenesis of APN suggest that the catalytic site of the peptidase is involved in the observed changes of B0AT1 apparent substrate affinity, possibly by increasing the local substrate concentration. These results provide evidence for the existence of B0AT1-containing digestive complexes in the brush-border membrane, interacting differentially with various peptidases, and responding to the dynamic needs of nutrient absorption in the intestine and kidney.

scholarly journals Size does matter: 18 amino acids at the N-terminal tip of an amino acid transporter in Leishmania determine substrate specificity

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Doreen Schlisselberg ◽  
Eldar Mazarib ◽  
Ehud Inbar ◽  
Doris Rentsch ◽  
Peter J. Myler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Transporter ◽  
Acid Transporter

scholarly journals Microarray analysis reveals the inhibition of intestinal expression of nutrient transporters in piglets infected with porcine epidemic diarrhea virus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Junmei Zhang ◽  
Di Zhao ◽  
Dan Yi ◽  
Mengjun Wu ◽  
Hongbo Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Transporter ◽  
Nutrient Transporters ◽  
Solute Carrier Family ◽  
Diarrhea Virus ◽  
Expression Of Genes ◽  
Sodium Dependent ◽  
Solute Carrier ◽  
Porcine Epidemic Diarrhea ◽  
Acid Transporter

AbstractPorcine epidemic diarrhea virus (PEDV) infection can induce intestinal dysfunction, resulting in severe diarrhea and even death, but the mode of action underlying these viral effects remains unclear. This study determined the effects of PEDV infection on intestinal absorption and the expression of genes for nutrient transporters via biochemical tests and microarray analysis. Sixteen 7-day-old healthy piglets fed a milk replacer were randomly allocated to one of two groups. After 5-day adaption, piglets (n = 8/group) were orally administrated with either sterile saline or PEDV (the strain from Yunnan province) at 104.5 TCID50 (50% tissue culture infectious dose) per pig. All pigs were orally infused D-xylose (0.1 g/kg BW) on day 5 post PEDV or saline administration. One hour later, jugular vein blood samples as well as intestinal samples were collected for further analysis. In comparison with the control group, PEDV infection increased diarrhea incidence, blood diamine oxidase activity, and iFABP level, while reducing growth and plasma D-xylose concentration in piglets. Moreover, PEDV infection altered plasma and jejunal amino acid profiles, and decreased the expression of aquaporins and amino acid transporters (L-type amino acid transporter 1, sodium-independent amino acid transporter, B(°,+)-type amino acid transport protein, sodium-dependent neutral amino acid transporter 1, sodium-dependent glutamate/aspartate transporter 3, and peptide transporter (1), lipid transport and metabolism-related genes (lipoprotein lipase, apolipoprotein A1, apolipoprotein A4, apolipoprotein C2, solute carrier family 27 member 2, solute carrier family 27 member 4, fatty acid synthase, and long-chain acyl-CoA synthetase (3), and glucose transport genes (glucose transporter-2 and insulin receptor) in the jejunum. However, PEDV administration increased mRNA levels for phosphoenolpyruvate carboxykinase 1, argininosuccinate synthase 1, sodium/glucose co-transporter-1, and cystic fibrosis transmembrane conductance regulator in the jejunum. Collectively, these comprehensive results indicate that PEDV infection induces intestinal injury and inhibits the expression of genes encoding for nutrient transporters.

scholarly journals The zebrafish cationic amino acid transporter/glycoprotein-associated family: sequence and spatiotemporal distribution during development of the transport system b0,+ (slc3a1/slc7a9)

2021 ◽  
Author(s):  
Ståle Ellingsen ◽  
Shailesh Narawane ◽  
Anders Fjose ◽  
Tiziano Verri ◽  
Ivar Rønnestad
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transport System ◽  
Expression Patterns ◽  
Amino Acid Transporter ◽  
Rapid Screening ◽  
Cationic Amino Acid ◽  
Large Neutral Amino Acids ◽  
Neutral Amino Acids ◽  
Acid Transporter

AbstractSystem b0,+ absorbs lysine, arginine, ornithine, and cystine, as well as some (large) neutral amino acids in the mammalian kidney and intestine. It is a heteromeric amino acid transporter made of the heavy subunit SLC3A1/rBAT and the light subunit SLC7A9/b0,+AT. Mutations in these two genes can cause cystinuria in mammals. To extend information on this transport system to teleost fish, we focused on the slc3a1 and slc7a9 genes by performing comparative and phylogenetic sequence analysis, investigating gene conservation during evolution (synteny), and defining early expression patterns during zebrafish (Danio rerio) development. Notably, we found that slc3a1 and slc7a9 are non-duplicated in the zebrafish genome. Whole-mount in situ hybridization detected co-localized expression of slc3a1 and slc7a9 in pronephric ducts at 24 h post-fertilization and in the proximal convoluted tubule at 3 days post-fertilization (dpf). Notably, both the genes showed co-localized expression in epithelial cells in the gut primordium at 3 dpf and in the intestine at 5 dpf (onset of exogenous feeding). Taken together, these results highlight the value of slc3a1 and slc7a9 as markers of zebrafish kidney and intestine development and show promise for establishing new zebrafish tools that can aid in the rapid screening(s) of substrates. Importantly, such studies will help clarify the complex interplay between the absorption of dibasic amino acids, cystine, and (large) neutral amino acids and the effect(s) of such nutrients on organismal growth.

ACE2 and gut amino acid transport

2020 ◽  
Vol 134 (21) ◽  
pp. 2823-2833 ◽  
Author(s):  
Simone M.R. Camargo ◽  
Raphael N. Vuille-dit-Bille ◽  
Chantal F. Meier ◽  
François Verrey
Keyword(s):  
Amino Acids ◽  
Small Intestine ◽  
Amino Acid ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Amino Acid Transporters ◽  
Type I ◽  
Small Intestinal Mucosa ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter

Abstract ACE2 is a type I membrane protein with extracellular carboxypeptidase activity displaying a broad tissue distribution with highest expression levels at the brush border membrane (BBM) of small intestine enterocytes and a lower expression in stomach and colon. In small intestinal mucosa, ACE2 mRNA expression appears to increase with age and to display higher levels in patients taking ACE-inhibitors (ACE-I). There, ACE2 protein heterodimerizes with the neutral amino acid transporter Broad neutral Amino acid Transporter 1 (B0AT1) (SLC6A19) or the imino acid transporter Sodium-dependent Imino Transporter 1 (SIT1) (SLC6A20), associations that are required for the surface expression of these transport proteins. These heterodimers can form quaternary structures able to function as binding sites for SARS-CoV-2 spike glycoproteins. The heterodimerization of the carboxypeptidase ACE2 with B0AT1 is suggested to favor the direct supply of substrate amino acids to the transporter, but whether this association impacts the ability of ACE2 to mediate viral infection is not known. B0AT1 mutations cause Hartnup disorder, a condition characterized by neutral aminoaciduria and, in some cases, pellagra-like symptoms, such as photosensitive rash, diarrhea, and cerebellar ataxia. Correspondingly, the lack of ACE2 and the concurrent absence of B0AT1 expression in small intestine causes a decrease in l-tryptophan absorption, niacin deficiency, decreased intestinal antimicrobial peptide production, and increased susceptibility to inflammatory bowel disease (IBD) in mice. Thus, the abundant expression of ACE2 in small intestine and its association with amino acid transporters appears to play a crucial role for the digestion of peptides and the absorption of amino acids and, thereby, for the maintenance of structural and functional gut integrity.

scholarly journals Amino acid sensing by enteroendocrine STC-1 cells: role of the Na+-coupled neutral amino acid transporter 2

2010 ◽  
Vol 298 (6) ◽  
pp. C1401-C1413 ◽  
Author(s):  
Steven H. Young ◽  
Osvaldo Rey ◽  
Catia Sternini ◽  
Enrique Rozengurt
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Isobutyric Acid ◽  
Amino Acid Transporter ◽  
Membrane Depolarization ◽  
Neutral Amino Acid ◽  
Acid Transport ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter

The results presented here show that STC-1 cells, a model of intestinal endocrine cells, respond to a broad range of amino acids, including l-proline, l-serine, l-alanine, l-methionine, l-glycine, l-histidine, and α-methyl-amino-isobutyric acid (MeAIB) with a rapid increase in the intracellular Ca2+ concentration ([Ca2+]i). We sought to identify the mechanism by which amino acids induce Ca2+ signaling in these cells. Several lines of evidence suggest that amino acid transport through the Na+-coupled neutral amino acid transporter 2 (SNAT2) is a major mechanism by which amino acids induced Ca2+ signaling in STC-1 cells: 1) the amino acid efficacy profile for inducing Ca2+ signaling in STC-1 cells closely matches the amino acid specificity of SNAT2; 2) amino acid-induced Ca2+ signaling in STC-1 cells was suppressed by removing Na+ from the medium; 3) the nonmetabolized synthetic substrate of amino acid transport MeAIB produced a marked increase in [Ca2+]i; 4) transfection of small interfering RNA targeting SNAT2 produced a marked decrease in Ca2+ signaling in response to l-proline in STC-1 cells; 5) amino acid-induced increase in [Ca2+]i was associated with membrane depolarization and mediated by Ca2+ influx, since it depended on extracellular Ca2+; 6) the increase in [Ca2+]i in response to l-proline, l-alanine, or MeAIB was abrogated by either nifedipine (1–10 μM) or nitrendipine (1 μM), which block L-type voltage-sensitive Ca2+ channels. We hypothesize that the inward current of Na+ associated with the function of SNAT2 leads to membrane depolarization and activation of voltage-sensitive Ca2+ channels that mediate Ca2+ influx, thereby leading to an increase in the [Ca2+]i in enteroendocrine STC-1 cells.

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