A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility
Abstract Vertebrate CMP-sialic acid synthetase (CSS), which catalyzes the synthesis of CMP-sialic acid (CMP-Sia), consists of a 28 kDa-N-domain and a 20 kDa-C-domain. The N-domain is known to be a catalytic domain; however, the significance of the C-domain still remains unknown. To elucidate the fun...
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Nature Portfolio
2021-12-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-021-01715-3 |
| _version_ | 1818742528026869760 |
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| author | Di Wu Hiromu Arakawa Akiko Fujita Hisashi Hashimoto Masahiko Hibi Kiyoshi Naruse Yasuhiro Kamei Chihiro Sato Ken Kitajima |
| author_facet | Di Wu Hiromu Arakawa Akiko Fujita Hisashi Hashimoto Masahiko Hibi Kiyoshi Naruse Yasuhiro Kamei Chihiro Sato Ken Kitajima |
| author_sort | Di Wu |
| collection | DOAJ |
| description | Abstract Vertebrate CMP-sialic acid synthetase (CSS), which catalyzes the synthesis of CMP-sialic acid (CMP-Sia), consists of a 28 kDa-N-domain and a 20 kDa-C-domain. The N-domain is known to be a catalytic domain; however, the significance of the C-domain still remains unknown. To elucidate the function of the C-domain at the organism level, we screened the medaka TILLING library and obtained medaka with non-synonymous mutations (t911a), or single amino acid substitutions of CSS, L304Q, in the C-domain. Prominently, most L304Q medaka was lethal within 19 days post-fertilization (dpf). L304Q young fry displayed free Sia accumulation, and impairment of sialylation, up to 8 dpf. At 8 dpf, a marked abnormality in ventricular contraction and skeletal myogenesis was observed. To gain insight into the mechanism of L304Q-induced abnormalities, L304Q was biochemically characterized. Although bacterially expressed soluble L304Q and WT showed the similar V max/K m values, very few soluble L304Q was detected when expressed in CHO cells in sharp contrast to the WT. Additionally, the thermostability of various mutations of L304 greatly decreased, except for WT and L304I. These results suggest that L304 is important for the stability of CSS, and that an appropriate level of expression of soluble CSS is significant for animal survival. |
| first_indexed | 2024-12-18T02:13:57Z |
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| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-18T02:13:57Z |
| publishDate | 2021-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-e0661301453f45079fe8ecf59607b6f82022-12-21T21:24:25ZengNature PortfolioScientific Reports2045-23222021-12-0111111510.1038/s41598-021-01715-3A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubilityDi Wu0Hiromu Arakawa1Akiko Fujita2Hisashi Hashimoto3Masahiko Hibi4Kiyoshi Naruse5Yasuhiro Kamei6Chihiro Sato7Ken Kitajima8Institute of Glyco-Core Research, Nagoya UniversityBioscience and Biotechnology Center, and Graduate School of Bioagricultural Sciences, Nagoya UniversityBioscience and Biotechnology Center, and Graduate School of Bioagricultural Sciences, Nagoya UniversityGraduate School of Science, Nagoya UniversityGraduate School of Science, Nagoya UniversityNational Institute of Basic BiologyNational Institute of Basic BiologyInstitute of Glyco-Core Research, Nagoya UniversityInstitute of Glyco-Core Research, Nagoya UniversityAbstract Vertebrate CMP-sialic acid synthetase (CSS), which catalyzes the synthesis of CMP-sialic acid (CMP-Sia), consists of a 28 kDa-N-domain and a 20 kDa-C-domain. The N-domain is known to be a catalytic domain; however, the significance of the C-domain still remains unknown. To elucidate the function of the C-domain at the organism level, we screened the medaka TILLING library and obtained medaka with non-synonymous mutations (t911a), or single amino acid substitutions of CSS, L304Q, in the C-domain. Prominently, most L304Q medaka was lethal within 19 days post-fertilization (dpf). L304Q young fry displayed free Sia accumulation, and impairment of sialylation, up to 8 dpf. At 8 dpf, a marked abnormality in ventricular contraction and skeletal myogenesis was observed. To gain insight into the mechanism of L304Q-induced abnormalities, L304Q was biochemically characterized. Although bacterially expressed soluble L304Q and WT showed the similar V max/K m values, very few soluble L304Q was detected when expressed in CHO cells in sharp contrast to the WT. Additionally, the thermostability of various mutations of L304 greatly decreased, except for WT and L304I. These results suggest that L304 is important for the stability of CSS, and that an appropriate level of expression of soluble CSS is significant for animal survival.https://doi.org/10.1038/s41598-021-01715-3 |
| spellingShingle | Di Wu Hiromu Arakawa Akiko Fujita Hisashi Hashimoto Masahiko Hibi Kiyoshi Naruse Yasuhiro Kamei Chihiro Sato Ken Kitajima A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility Scientific Reports |
| title | A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility |
| title_full | A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility |
| title_fullStr | A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility |
| title_full_unstemmed | A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility |
| title_short | A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility |
| title_sort | point mutation in the c domain of cmp sialic acid synthetase leads to lethality of medaka due to protein insolubility |
| url | https://doi.org/10.1038/s41598-021-01715-3 |
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