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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Autores principales:	Di Wu, Hiromu Arakawa, Akiko Fujita, Hisashi Hashimoto, Masahiko Hibi, Kiyoshi Naruse, Yasuhiro Kamei, Chihiro Sato, Ken Kitajima
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e0661301453f45079fe8ecf59607b6f8
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spelling	oai:doaj.org-article:e0661301453f45079fe8ecf59607b6f82021-12-05T12:12:21ZA point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility10.1038/s41598-021-01715-32045-2322https://doaj.org/article/e0661301453f45079fe8ecf59607b6f82021-12-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-01715-3https://doaj.org/toc/2045-2322Abstract 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.Di WuHiromu ArakawaAkiko FujitaHisashi HashimotoMasahiko HibiKiyoshi NaruseYasuhiro KameiChihiro SatoKen KitajimaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q 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
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.
format	article
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
title	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_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_sort	point-mutation in the c-domain of cmp-sialic acid synthetase leads to lethality of medaka due to protein insolubility
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/e0661301453f45079fe8ecf59607b6f8
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A point-mutation in the C-domain of CMP-sialic acid synthetase leads to lethality of medaka due to protein insolubility

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