Biallelic Mutations of Methionyl-tRNA Synthetase Cause a Specific Type of Pulmonary Alveolar Proteinosis Prevalent on Réunion Island

Abstract

Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to tRNA and is critical for protein biosynthesis. We identified biallelic missense mutations in MARS in a specific form of pediatric pulmonary alveolar proteinosis (PAP), a severe lung disorder that is prevalent on the island of Réunion and the molecular basis of which is unresolved. Mutations were found in 26 individuals from Réunion and nearby islands and in two families from other countries. Functional consequences of the mutated alleles were assessed by growth of wild-type and mutant strains and methionine-incorporation assays in yeast. Enzyme activity was attenuated in a liquid medium without methionine but could be restored by methionine supplementation. In summary, identification of a founder mutation in MARS led to the molecular definition of a specific type of PAP and will enable carrier screening in the affected community and possibly open new treatment opportunities.

Figure 1

MARS Variants in PAP (A) Sequencing reads showing the different biallelic variants identified in individuals from Réunion, Tunisia, and France. (B) Amino acid conservation across MARS orthologs. (C) Scheme of the domain structure of MARS with the location of the variants. (D) Predicted tertiary structure. MARS contains a nucleotide-binding (Rossmann) fold (green); a region called the connective polypeptide, which contains the zinc-binding sites (orange); the stem-contact fold domain (red); and the α-helix bundle domain that forms the anticodon-binding site (violet). The positions of the variants are indicated relative to the reference sequence (GenBank: NM_004990.3). The structure of human MARS was predicted by homology modeling based on the Aquifex aeolicus structure of MARS complexed with methionyl sulfamoyl adenosine (MSA) and the elongator tRNA^Met (PDB: 2CT8) as templates. The model was constructed with the SWISS-MODEL automated protein-structure homology-modeling server. The predicted structure was superimposed with MSA and tRNA^Met with SPDBviewer and was visualized with Rasmol.

Figure 2

Growth of MES1 Wild-Type and mes1 Mutant Strains Growth (A) without methionine or (B) with 20 μg/ml methionine. Cells were inoculated at the concentration of 0.1 OD₆₀₀/ml and grown until the stationary phase was reached after 28 hr. At regular intervals, aliquots were used for measurement of cell density by UV-visible spectrophotometry at 600 nm. Sampling times are indicated by x-axis ticks. Tables show division times (minutes) calculated during the exponential phase of growth. Division times are the mean of three independent growth curves. The S. cerevisiae strain used in this work was W303-1B (Matα ade2-1 leu2-3,112 ura3-1 trp1-1 his3-11,15 can1-100). The MES1 wild-type allele was cloned in the centromeric vector pFL38. Genomic MES1 was disrupted in the pFL38MES1-transformed W303-1B strain by one-step gene disruption with a KanMX expression cassette.mes1 mutant and double-mutant alleles were constructed via site-directed mutagenesis through the PCR overlap extension technique with the oligonucleotides listed in Table S4, cloned into vector pFL39, and introduced into W303-1B mes1Δ pFL38MES1. In a second step, strains devoid of pFL38MES1^WT and containing the pFL39-borne MES1^WT or mes1 mutant alleles were selected through plasmid shuffling. NS, not significant in a two-tailed, unpaired t test; ^∗∗p < 0.01.

Figure 3

³⁵S Incorporation of MES1 Wild-Type and mes1 Mutant Strains Incorporation (A) without methionine or (B) with 20 μg/ml methionine. Values are normalized to the wild-type strain, which was set as 100%. Four replicates were performed for the experiments without methionine and three replicates for the experiments with methionine. The error bars indicate SDs. Cells were inoculated at a final concentration of 0.1 OD₆₀₀/ml in synthetic-complete-dextrose medium (0.69% yeast nitrogen base, 0.1% yeast amino acid and nucleobase mixture, 2% glucose) with or without 20 μg/ml methionine and grown at 37°C. After 16 hr, cells were diluted to a final concentration of 1.2 OD₆₀₀/ml. After 5 min, cells were supplemented with 1 μl, if grown without methionine, or 10 μl, if grown with methionine, of EasyTag [³⁵S]-protein labeling mix having a specific activity of 1,000 Ci/mmol (Perkin Elmer). Once we verified that the incorporation signal was linear between 2 and 10 min, we blocked protein synthesis after 6 min by adding a mix containing 200 μg cycloheximide, 1 mg erythromycin, 100 μg cold L-methionine, and 100 μg cold L-cysteine and chilling the mixture on ice. We used the trichloroacetic (TCA) method to precipitate total proteins by chilling the cells supplemented with 25% TCA on ice, then resuspended the proteins in 30 μl of 60-mM Tris-HCl (pH 6.8). For each sample, counts per minute/OD₆₀₀ were measured on 10 μl aliquots and normalized to values for the wild-type strain, which was set as 100%. NS, not significant in a two-tailed, paired t test; ^∗∗p < 0.01; ^∗∗∗p < 0.001.