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The Effect of a Novel rBAT Mutation on the Expression and Function of System b 0,+
Author(s) -
Rice Sarah,
Edwards Noel,
Sayer John,
Thwaites David
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.666.28
Subject(s) - cystinuria , microbiology and biotechnology , biology , missense mutation , mutation , arginine , genetics , cystine , chemistry , amino acid , biochemistry , gene , cysteine , enzyme
To identify the causative mutations within a cohort of cystinuria patients, the two genes ( SLC3A1 and SLC7A9 ) encoding the subunits (rBAT and b 0,+ AT) of the amino acid carrier System b 0,+ were analysed. A novel homozygous missense mutation (M465K) in the heavy chain chaperone protein rBAT was identified in one patient and investigation of obligate heterozygotes showed an autosomal recessive penetrance of phenotype. Homology modelling was used to create a model of the extracellular domain of rBAT based on the known crystal structure of oligo‐1,6‐glucosidase from B. cereus (PDB code 1UOK). Using this model, M465K is predicted to be located on the 7 th alpha helix of the triosephosphate isomerase (TIM)‐like structure. Protein expression and function were characterised following cRNA injection in Xenopus laevis oocytes by [ 3 H]arginine uptake, western blotting, and immunocytochemistry. Reduced protein expression and arginine uptake were observed in M465K‐injected oocytes versus wild‐type rBAT suggesting a decrease in transporter trafficking to the oocyte membrane. M465K expression was dependent upon time (post‐injection) and quantity of cRNA injected and followed a pattern similar to that observed with the neighbouring mutation M467T, the most common cystinuria‐causing mutation in several European populations. Cystinuria in this patient likely results from reduced cystine reabsorption in the renal proximal tubule due to defective trafficking of M465K and decreased System b 0,+ activity in vivo. Supported by the Medical Research Council and Northern Counties Kidney Research Fund.