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

• Published in: Orphanet journal of rare diseases Vol. 14; no. 1; p. 63
• Main Authors: Rotoli, Bianca Maria, Barilli, Amelia, Ingoglia, Filippo, Visigalli, Rossana, Bianchi, Massimiliano G, Ferrari, Francesca, Martinelli, Diego, Dionisi-Vici, Carlo, Dall'Asta, Valeria
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 04.03.2019; BioMed Central; BMC
• Subjects: Adult; Amino Acid Metabolism, Inborn Errors - genetics; Amino acids; Animals; Arginine; Arginine - metabolism; Arginine transport; Cells, Cultured; Child; Child, Preschool; CHO Cells; Confocal microscopy; Cricetulus; Cytosol; Cytosol - metabolism; Defects; Diagnosis; eGFP fusion proteins; Female; Food intolerance; Fusion proteins; Fusion Regulatory Protein 1, Light Chains - genetics; Fusion Regulatory Protein 1, Light Chains - metabolism; Gene expression; Genetic aspects; Genotypes; Humans; Immunology; Intestine; Localization; Lymphoblasts; Lysine; Male; Medical research; Messenger RNA; Metabolic diseases; Microscopy; Monocytes; mRNA; Mutagenesis; Mutation; Ornithine; Phenotypes; Protein Transport - genetics; Proteins; Rare diseases; RNA; Site-directed mutagenesis; Subcellular protein localization; Urea; y+L transport system; Italy; Subcellular protein localization; y+L transport system; eGFP fusion proteins; Site-directed mutagenesis; Arginine transport
• ISSN: 1750-1172; 1750-1172
• DOI: 10.1186/s13023-019-1028-2

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, LPI ), 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. 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. 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.