2. Academic Validation
  3. A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility

A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility

  • Sci Rep. 2021 Dec 1;11(1):23211. doi: 10.1038/s41598-021-01715-3.
Di Wu 1 2,  Hiromu Arakawa 2,  Akiko Fujita 2,  Hisashi Hashimoto 3,  Masahiko Hibi 3,  Kiyoshi Naruse 4,  Yasuhiro Kamei 4 5,  Chihiro Sato 1 2,  Ken Kitajima 6 7
Affiliations

Affiliations

  • 1 Institute of Glyco-Core Research, Nagoya University, Chikusa, Nagoya, 464-8601, Japan.
  • 2 Bioscience and Biotechnology Center, and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601, Japan.
  • 3 Graduate School of Science, Nagoya University, Chikusa, Nagoya, 464-8601, Japan.
  • 4 National Institute of Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, 444-8585, Japan.
  • 5 Department of Basic Biology, School of Life Science, The Graduate University for Advanced Studies, Shonan Village, Hayama, Kanagawa, 240-0193, Japan.
  • 6 Institute of Glyco-Core Research, Nagoya University, Chikusa, Nagoya, 464-8601, Japan. kitajima@agr.nagoya-u.ac.jp.
  • 7 Bioscience and Biotechnology Center, and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601, Japan. kitajima@agr.nagoya-u.ac.jp.
PMID: 34853329 DOI: 10.1038/s41598-021-01715-3
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 Vmax/Km 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.

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