Institute of Glyco-core Research, Nagoya University, Nagoya, Chikusa, 464-8601, Japan; Bioscience and Biotechnology Center, and Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Chikusa, 464-8601, Japan.
Univ. Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France; Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada.
Biochem Biophys Res Commun. 2022 Aug 20;617(Pt 1):16-21. doi: 10.1016/j.bbrc.2022.05.031. Epub 2022 May 13.
The CMP-sialic acid synthetase (CSS) activates free sialic acid (Sia) to CMP-Sia using CTP, and is prerequisite for the sialylation of cell surface glycoconjugates. The vertebrate CSS consists of two domains, a catalytic N-domain and a non-catalytic C-domain. Although the C-domain is not required for the CSS enzyme to synthesize CMP-Sia, its involvement in the catalytic activity remains unknown. First, the real-time monitoring of CSS-catalyzed reaction was performed by P NMR using the rainbow trout CSS (rtCSS). While a rtCSS lacking the C-domain (rtCSS-N) similarly activated both deaminoneuraminic acid (Kdn) and N-acetylneuraminic acid (Neu5Ac), the full-length rtCSS (rtCSS-FL) did not activate Kdn as efficiently as Neu5Ac. These results suggest that the C-domain of rtCSS affects the enzymatic activity, when Kdn was used as a substrate. Second, the enzymatic activity of rtCSS-FL and rtCSS-N was measured under various concentrations of CMP-Kdn. Inhibition by CMP-Kdn was observed only for rtCSS-FL, but not for rtCSS-N, suggesting that the inhibition was C-domain-dependent. Third, the inhibitory effect of CMP-Kdn was also investigated using the mouse CSS (mCSS). However, no inhibition was observed with mCSS even at high concentrations of CMP-Kdn. Taken together, the data demonstrated that the C-domain is involved in the CMP-Kdn-dependent inhibition of rtCSS, which is a novel regulation of the Sia metabolism in rainbow trout.
CMP-唾液酸合酶 (CSS) 使用 CTP 将游离唾液酸 (Sia) 激活为 CMP-Sia，是细胞表面糖缀合物发生唾液酸化的前提。脊椎动物 CSS 由两个结构域组成，一个是催化 N 结构域，另一个是非催化 C 结构域。尽管 C 结构域对于 CSS 酶合成 CMP-Sia 不是必需的，但它在催化活性中的参与仍然未知。首先，使用彩虹鳟鱼 CSS（rtCSS）通过 P NMR 实时监测 CSS 催化反应。虽然缺乏 C 结构域的 rtCSS（rtCSS-N）同样可以激活去氨神经氨酸（Kdn）和 N-乙酰神经氨酸（Neu5Ac），但全长 rtCSS（rtCSS-FL）对 Kdn 的激活效率不如 Neu5Ac。这些结果表明，当 Kdn 作为底物时，C 结构域影响 rtCSS 的酶活性。其次，在各种 CMP-Kdn 浓度下测量 rtCSS-FL 和 rtCSS-N 的酶活性。仅在 rtCSS-FL 中观察到 CMP-Kdn 的抑制，但 rtCSS-N 没有，表明抑制是 C 结构域依赖性的。第三，还使用小鼠 CSS（mCSS）研究了 CMP-Kdn 的抑制作用。然而，即使在高浓度的 CMP-Kdn 下，mCSS 也没有观察到抑制作用。总之，数据表明 C 结构域参与了 rtCSS 对 CMP-Kdn 的依赖性抑制，这是彩虹鳟 Sia 代谢的一种新调控。
