title: SNPs altering ammonium transport activity of human Rhesus factors characterized by a yeast-based functional assay.
abstract: Proteins of the conserved Mep-Amt-Rh family, including mammalian Rhesus factors, mediate transmembrane ammonium transport. Ammonium is an important nitrogen source for the biosynthesis of amino acids but is also a metabolic waste product. Its disposal in urine plays a critical role in the regulation of the acid/base homeostasis, especially with an acid diet, a trait of Western countries. Ammonium accumulation above a certain concentration is however pathologic, the cytotoxicity causing fatal cerebral paralysis in acute cases. Alteration in ammonium transport via human Rh proteins could have clinical outcomes. We used a yeast-based expression assay to characterize human Rh variants resulting from non synonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinical phenotypes and assessed their ammonium transport efficiency, protein level, localization and potential trans-dominant impact. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditary stomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsic bidirectional ammonium transport. Moreover, this study reveals that the R202C variant of HsRhCG, the orthologue of mouse MmRhcg required for optimal urinary ammonium excretion and blood pH control, shows an impaired inherent ammonium transport activity. Urinary ammonium excretion was RHcg gene-dose dependent in mouse, highlighting MmRhcg as a limiting factor. HsRhCG(R202C) may confer susceptibility to disorders leading to metabolic acidosis for instance. Finally, the analogous R211C mutation in the yeast ScMep2 homologue also impaired intrinsic activity consistent with a conserved functional role of the preserved arginine residue. The yeast expression assay used here constitutes an inexpensive, fast and easy tool to screen nsSNPs reported by high throughput sequencing or individual cases for functional alterations in Rh factors revealing potential causal variants.
authors: Biologie du Transport Membranaire, Université Libre de Bruxelles, Gosselies, Belgium.
authors: Aude
authors: A
authors: Deschuyteneer
authors: Mélanie
authors: M
authors: Boeckstaens
authors: Christelle
authors: C
authors: De Mees
authors: Pascale
authors: P
authors: Van Vooren
authors: René
authors: R
authors: Wintjens
authors: Anna Maria
authors: AM
authors: Marini
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D064751:Ammonium Compounds / Q000378:metabolism* / Q000652:urine; D000818:Animals; D001692:Biological Transport; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D005799:Genes, Dominant; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D012204:Rh-Hr Blood-Group System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D064751:Ammonium Compounds; D001798:Blood Proteins; D027682:Cation Transport Proteins; D008562:Membrane Glycoproteins; D021122:Protein Subunits; C498523:RHAG protein, human; C413213:RHCG protein, human; D012204:Rh-Hr Blood-Group System
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