Biological Significance of C-Domain of the Vertebrate CMP-Sialic Acid Synthetases

The CMP-sialic acid synthetase (CSS/CMAS) is essential for the expression of sialic acid (Sia)-containing glycoconjugates on the cell surface and the extracellular space, as it activates free sialic acid (Sia) to CMP-Sia, the sole donor substrate for all kinds of sialytransferases. The vertebrate CSS comprises two domains: the N-terminal domain (N-domain/CSS-N) and the C-terminal domain (C-domain/CSS-C). While CSS-N is highly conserved among most organisms, CSS-C is found only in vertebrates and certain types of bacteria. To investigate the biological significance of CSS-C in vertebrate CSS, its X-ray structure was determined at a resolution of 1.9 Å. CSS-C forms a tetramer and superimposes well with phosphatases of the haloacid dehalogenase superfamily, although it lacks the phosphatase activity due to the blockage of the active-site entrance. Instead, CSS-C functions as a structural platform for the quaternary organization of the CSS. To explore the biological role of CSS-C at the organism level, a medaka mutant strain whose CSS contains a single amino acid substitution, L304Q, was generated. This reverse genetic study revealed that L304 is critical for the CSS stability and that maintaining appropriate levels of soluble CSS expression is essential for animal survival. To obtain further insight into the involvement of CSS-C in regulating enzymatic activity, the effect of CMP-Kdn on the rainbow trout CSS (rtCSS) activity was examined. It was demonstrated that CSS-C regulates Kdn metabolism in rainbow trout, as the activity was inhibited by CMP-Kdn in a C-domain-dependent manner. Taken together, CSS-C not only influences the physicochemical properties but also plays a key role in regulating its enzymatic activity.