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. HsRhCGR202C 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.

Dog erythrocyte antigen 1: mode of inheritance and initial characterization

“…[23][24][25][26] Disruption of function can have severe implications on cellular or organ function, which can manifest in tissue damage and disease. 27 In conclusion, we demonstrated the inheritance pattern of DEA 1À and weakly to strongly DEA 1+ dogs in a multiallelic autosomal dominant blood system. Like many of the human blood groups, including Rh, we hypothesize that the DEA 1 system may be more complicated than initially thought.…”

“…In addition, the human Rh RBC antigen is an ammonia transporter and despite its popularity in the RBC field, the Rh factor is also found in the cells of the kidney, liver, gastrointestinal tract, testes, and other organs . Disruption of function can have severe implications on cellular or organ function, which can manifest in tissue damage and disease …”

The TORC1 effector kinase Npr1 fine tunes the inherent activity of the Mep2 ammonium transport protein

“…A similar mechanism of activity fine tuning involving the C terminus might also apply to mammalian Rh factors, despite the divergence of this domain. We recently characterized a human polymorphism of RhCG showing a reduced activity and likely affected in the interaction between IL3 and the C terminus 54 . The clinical outcome potentially associated to this variant is currently unknown.…”

De la découverte des transporteurs d’ammonium Mep-Amt microbiens aux facteurs Rhésus humains

“…Ainsi un polymorphisme de RhCG correspondant à la mutation R202C conduit à une altération de la capacité de la protéine à transporter l'ammonium dans les levures [16]. Une réduction de la fonctionnalité de RhCG chez les individus portant cette mutation pourrait être à l'origine de troubles menant à une acidose métabolique, comme cela est observé chez les souris invalidées pour RHcg.…”

“…Des mutations ponctuelles situées dans le gène RHAG ont été par ailleurs trouvées chez des patients atteints de stomatocytose avec hématies hyperhydratées (OHSt), une maladie dominante héréditaire caractérisée par une fuite abondante de cations monovalents à travers la membrane érythrocytaire [17]. L'expression hétérologue de ces versions mutées de HsRHAG dans les levures indique que les protéines correspondantes présentent effectivement une altération de leur fonction de transport de l'ammonium [16] (Figure 1). …”