Translational and clinical immunologyAlanine-scanning mutagenesis of human signal transducer and activator of transcription 1 to estimate loss- or gain-of-function variants
Section snippets
Functional assay based on systematic alanine-scanning mutagenesis
A vector from which to express HaloTag STAT1 was obtained from the Kazusa cDNA/ORF clone collection (FHC013013). The codons of the vector-encoding residues from L136 to F487 of STAT1, except the 10 alanines (A188, A230, A246, A254, A267, A401, A402, A415, A469, and A479), were individually substituted with GCC, the codon most frequently encoding alanine in human subjects, by using site-directed mutagenesis. The activities of the mutants were measured with a luciferase reporter assay with the
Functional assay based on systematic alanine-scanning mutagenesis
Alanine-scanning mutagenesis is a widely used technique in the determination of the catalytic or functional role of protein residues. We systematically investigated the effects of alanine substitutions in the CCD/DBD of STAT1 with a GAS reporter assay after IFN-γ stimulation. We generated 176 alanine mutants in the CCD and 166 alanine mutants in the DBD. We defined the individual alanine mutants as LOF or GOF if they showed less than 0.3 times or greater than 1.6 times the GAS transcriptional
Discussion
We systematically evaluated the effect of amino acid substitutions (342 alanine mutants) in the CCD/DBD of STAT1 using a GAS reporter assay. The GAS reporter assay is an accurate and practical method with which to assess LOF and GOF STAT1 mutations.11, 12, 15, 19, 20, 21 This assay allowed us to explain 100% of known LOF mutations and 78.1% (86.7% in the CCD and 64.3% in the DBD) of known GOF mutations in the CCD/DBD of STAT1 by using alanine substituents of the same residues. It showed that
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Supported in part by Grants in Aid for Scientific Research from the Japan Society for the Promotion of Science (22591161 to M.K. and 25713039, 16H05355, and 16K15528 to S.O.) and was supported in part by the Practical Research Project for Rare/Intractable Diseases from Japan Agency for Medical Research and development (AMED). This study was also supported in part by Research on Measures for Intractable Diseases funding from the Japanese Ministry of Health, Labour and Welfare (H22-Nanchi-ippan-078 to M.K.), GSK Japan Research Grant 2014, and the Kurozumi Medical Foundation. The Laboratory of Human Genetics of Infectious Diseases is supported by institutional grants from INSERM, University Paris Descartes, the Rockefeller University, the St Giles Foundation, the US National Institute of Allergy and Infectious Diseases (grant no. R37AI095983 and U01AI109697), and grants from the French National Research Agency (ANR) under the “Investments for the future” program (grant no. ANR-10-IAHU-01). The sequence analysis was supported by the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University.
Disclosure of potential conflict of interest: J.-L. Casanova serves on the ADMA Scientific Advisory Board and serves as a consultant for ADMA, Nimbus, and Vitae Pharmaceuticals. O. Ohara received grant support from the Japan Agency for Medical Research and Development. S. Okada receives grant support from the Japan Agency for Medical Research and Development, Grants in Aid for Scientific Research from the Japan Society, GlaxoSmithKline, and Kurozumi Medical Foundation. The rest of the authors declare that they have no relevant conflicts of interest.
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These authors contributed equally to this work.