Analysis of LPI-causing mutations on y+LAT1 function and localization

Bianca Maria Rotoli, Amelia Barilli, Filippo Ingoglia, Rossana Visigalli, Massimiliano G Bianchi, Francesca Ferrari, Diego Martinelli, Carlo Dionisi-Vici, Valeria Dall'Asta

Research output: Contribution to journalArticle

Abstract

BACKGROUND: y+LAT1, encoded by SCL7A7, is the protein mutated in Lysinuric Protein Intolerance (LPI), a rare metabolic disease caused by a defective cationic amino acid (CAA, arginine, lysine, ornithine) transport at the basolateral membrane of intestinal and renal tubular cells. The disease is characterized by protein-rich food intolerance with secondary urea cycle disorder, but symptoms are heterogeneous with lung and immunological complications that are not explainable by the CAA transport defect. With the exception of the Finnish founder mutation (c.895-2A > T, LPIFin), LPI-causative mutations are heterogeneous and genotype-phenotype correlations have not been found. Here we addressed system y+L-mediated arginine uptake in monocytes from three LPI Italian patients and in lymphoblasts carrying the same mutations; in parallel, the genetic defects carried by the patients were reproduced as eGFP-tagged y+LAT1 mutants in transfected CHO cells to define the function and localization protein.

RESULTS: System y+L activity is impaired in monocytes isolated from all LPI patients, and in CHO cells transfected with the three eGFP-y+LAT1 mutants, but not in lymphoblasts bearing the same mutations. The analysis of protein localization with confocal microscopy revealed that the eGFP-tagged mutants were retained inside the cytosol, with a pattern of expression quite heterogeneous among the mutants.

CONCLUSIONS: The three mutations studied of y+LAT1 transporter result in a defective arginine transport both in ex vivo (monocytes) and in vitro (CHO transfected cells) models, likely caused by the retention of the mutated proteins in the cytosol. The different effect of y+LAT1 mutation on arginine transport in monocytes and lymphoblasts is supposed to be due to the different expression of SLC7A7 mRNA in the two models, supporting the hypothesis that the impact of LPI defect largely depends on the relative abundance of LPI target gene in each cell type.

Original languageEnglish
Pages (from-to)63
JournalOrphanet Journal of Rare Diseases
Volume14
Issue number1
DOIs
Publication statusPublished - Mar 4 2019

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Mutation
Arginine
CHO Cells
Monocytes
Proteins
Cytosol
Inborn Urea Cycle Disorder
Ornithine
Metabolic Diseases
Genetic Association Studies
Rare Diseases
Confocal Microscopy
Lysine
Lysinuric Protein Intolerance
Kidney
Amino Acids
Food
Lung
Messenger RNA
Membranes

Cite this

Rotoli, B. M., Barilli, A., Ingoglia, F., Visigalli, R., Bianchi, M. G., Ferrari, F., ... Dall'Asta, V. (2019). Analysis of LPI-causing mutations on y+LAT1 function and localization. Orphanet Journal of Rare Diseases, 14(1), 63. https://doi.org/10.1186/s13023-019-1028-2

Analysis of LPI-causing mutations on y+LAT1 function and localization. / Rotoli, Bianca Maria; Barilli, Amelia; Ingoglia, Filippo; Visigalli, Rossana; Bianchi, Massimiliano G; Ferrari, Francesca; Martinelli, Diego; Dionisi-Vici, Carlo; Dall'Asta, Valeria.

In: Orphanet Journal of Rare Diseases, Vol. 14, No. 1, 04.03.2019, p. 63.

Research output: Contribution to journalArticle

Rotoli, BM, Barilli, A, Ingoglia, F, Visigalli, R, Bianchi, MG, Ferrari, F, Martinelli, D, Dionisi-Vici, C & Dall'Asta, V 2019, 'Analysis of LPI-causing mutations on y+LAT1 function and localization', Orphanet Journal of Rare Diseases, vol. 14, no. 1, pp. 63. https://doi.org/10.1186/s13023-019-1028-2
Rotoli BM, Barilli A, Ingoglia F, Visigalli R, Bianchi MG, Ferrari F et al. Analysis of LPI-causing mutations on y+LAT1 function and localization. Orphanet Journal of Rare Diseases. 2019 Mar 4;14(1):63. https://doi.org/10.1186/s13023-019-1028-2
Rotoli, Bianca Maria ; Barilli, Amelia ; Ingoglia, Filippo ; Visigalli, Rossana ; Bianchi, Massimiliano G ; Ferrari, Francesca ; Martinelli, Diego ; Dionisi-Vici, Carlo ; Dall'Asta, Valeria. / Analysis of LPI-causing mutations on y+LAT1 function and localization. In: Orphanet Journal of Rare Diseases. 2019 ; Vol. 14, No. 1. pp. 63.
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abstract = "BACKGROUND: y+LAT1, encoded by SCL7A7, is the protein mutated in Lysinuric Protein Intolerance (LPI), a rare metabolic disease caused by a defective cationic amino acid (CAA, arginine, lysine, ornithine) transport at the basolateral membrane of intestinal and renal tubular cells. The disease is characterized by protein-rich food intolerance with secondary urea cycle disorder, but symptoms are heterogeneous with lung and immunological complications that are not explainable by the CAA transport defect. With the exception of the Finnish founder mutation (c.895-2A > T, LPIFin), LPI-causative mutations are heterogeneous and genotype-phenotype correlations have not been found. Here we addressed system y+L-mediated arginine uptake in monocytes from three LPI Italian patients and in lymphoblasts carrying the same mutations; in parallel, the genetic defects carried by the patients were reproduced as eGFP-tagged y+LAT1 mutants in transfected CHO cells to define the function and localization protein.RESULTS: System y+L activity is impaired in monocytes isolated from all LPI patients, and in CHO cells transfected with the three eGFP-y+LAT1 mutants, but not in lymphoblasts bearing the same mutations. The analysis of protein localization with confocal microscopy revealed that the eGFP-tagged mutants were retained inside the cytosol, with a pattern of expression quite heterogeneous among the mutants.CONCLUSIONS: The three mutations studied of y+LAT1 transporter result in a defective arginine transport both in ex vivo (monocytes) and in vitro (CHO transfected cells) models, likely caused by the retention of the mutated proteins in the cytosol. The different effect of y+LAT1 mutation on arginine transport in monocytes and lymphoblasts is supposed to be due to the different expression of SLC7A7 mRNA in the two models, supporting the hypothesis that the impact of LPI defect largely depends on the relative abundance of LPI target gene in each cell type.",
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T1 - Analysis of LPI-causing mutations on y+LAT1 function and localization

AU - Rotoli, Bianca Maria

AU - Barilli, Amelia

AU - Ingoglia, Filippo

AU - Visigalli, Rossana

AU - Bianchi, Massimiliano G

AU - Ferrari, Francesca

AU - Martinelli, Diego

AU - Dionisi-Vici, Carlo

AU - Dall'Asta, Valeria

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N2 - BACKGROUND: y+LAT1, encoded by SCL7A7, is the protein mutated in Lysinuric Protein Intolerance (LPI), a rare metabolic disease caused by a defective cationic amino acid (CAA, arginine, lysine, ornithine) transport at the basolateral membrane of intestinal and renal tubular cells. The disease is characterized by protein-rich food intolerance with secondary urea cycle disorder, but symptoms are heterogeneous with lung and immunological complications that are not explainable by the CAA transport defect. With the exception of the Finnish founder mutation (c.895-2A > T, LPIFin), LPI-causative mutations are heterogeneous and genotype-phenotype correlations have not been found. Here we addressed system y+L-mediated arginine uptake in monocytes from three LPI Italian patients and in lymphoblasts carrying the same mutations; in parallel, the genetic defects carried by the patients were reproduced as eGFP-tagged y+LAT1 mutants in transfected CHO cells to define the function and localization protein.RESULTS: System y+L activity is impaired in monocytes isolated from all LPI patients, and in CHO cells transfected with the three eGFP-y+LAT1 mutants, but not in lymphoblasts bearing the same mutations. The analysis of protein localization with confocal microscopy revealed that the eGFP-tagged mutants were retained inside the cytosol, with a pattern of expression quite heterogeneous among the mutants.CONCLUSIONS: The three mutations studied of y+LAT1 transporter result in a defective arginine transport both in ex vivo (monocytes) and in vitro (CHO transfected cells) models, likely caused by the retention of the mutated proteins in the cytosol. The different effect of y+LAT1 mutation on arginine transport in monocytes and lymphoblasts is supposed to be due to the different expression of SLC7A7 mRNA in the two models, supporting the hypothesis that the impact of LPI defect largely depends on the relative abundance of LPI target gene in each cell type.

AB - BACKGROUND: y+LAT1, encoded by SCL7A7, is the protein mutated in Lysinuric Protein Intolerance (LPI), a rare metabolic disease caused by a defective cationic amino acid (CAA, arginine, lysine, ornithine) transport at the basolateral membrane of intestinal and renal tubular cells. The disease is characterized by protein-rich food intolerance with secondary urea cycle disorder, but symptoms are heterogeneous with lung and immunological complications that are not explainable by the CAA transport defect. With the exception of the Finnish founder mutation (c.895-2A > T, LPIFin), LPI-causative mutations are heterogeneous and genotype-phenotype correlations have not been found. Here we addressed system y+L-mediated arginine uptake in monocytes from three LPI Italian patients and in lymphoblasts carrying the same mutations; in parallel, the genetic defects carried by the patients were reproduced as eGFP-tagged y+LAT1 mutants in transfected CHO cells to define the function and localization protein.RESULTS: System y+L activity is impaired in monocytes isolated from all LPI patients, and in CHO cells transfected with the three eGFP-y+LAT1 mutants, but not in lymphoblasts bearing the same mutations. The analysis of protein localization with confocal microscopy revealed that the eGFP-tagged mutants were retained inside the cytosol, with a pattern of expression quite heterogeneous among the mutants.CONCLUSIONS: The three mutations studied of y+LAT1 transporter result in a defective arginine transport both in ex vivo (monocytes) and in vitro (CHO transfected cells) models, likely caused by the retention of the mutated proteins in the cytosol. The different effect of y+LAT1 mutation on arginine transport in monocytes and lymphoblasts is supposed to be due to the different expression of SLC7A7 mRNA in the two models, supporting the hypothesis that the impact of LPI defect largely depends on the relative abundance of LPI target gene in each cell type.

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DO - 10.1186/s13023-019-1028-2

M3 - Article

C2 - 30832686

VL - 14

SP - 63

JO - Orphanet Journal of Rare Diseases

JF - Orphanet Journal of Rare Diseases

SN - 1750-1172

IS - 1

ER -