Exploring the Biosynthesis of O‐acetylated Sialic Acids using CASD1‐null Mice

Sialic acids (Sias) are a family of nine‐carbon backbone monosaccharides naturally occurring at the outer ends of vertebrate cell surface glycoconjugates. O‐acetyl esters at C4, C7, C8 or C9 of Sias are very common modification of Sias, and can play key roles in a number of biological processes. Cell‐, tissue‐ and individual‐specific variations in Sia O‐acetylation have been reported. Several studies also showed that different cell types incorporated 9(7)‐O‐acetylated Sias differentially into different classes of glycoconjugates. Sialate:O‐acetyltransferase activities were also studied in various systems. But the genetic basis and the regulation mechanisms involved remain largely unknown until the discovery of CASD1 as the first candidate gene responsible for sialate:O‐acetylation in eukaryotic systems, and in vitro studies confirmed the enzymatic activity on CMP‐Neu5Ac. Systemic CASD1 knockout (KO) mice, (Casd1 tm1b/tm1b ) showed a marked reduction of 9‐O‐acetylation on blood cells. These mice now provide a tool for us to explore the role of CASD1 in sialate:O‐acetylation of Sias in other tissues, and existence of potential O‐acetyltransferase other than CASD1. A combination of different analytical approaches were used to detect residual O‐acetylation in various tissues and cell types of the CASD1‐deficient mice. 9‐O‐acetyl‐Sia distribution was analyzed by histochemistry staining of wildtype (WT) and CASD1 KO mouse tissues with the recombinant 9‐O‐acetyl‐Sia recognizing probe originated from porcine torovirus (PToV) and its non‐binding mutant (PToV‐mut). Significant loss of PToV staining was seen in multiple tissues from CASD1 KO mice, compared to those from WT mice. This is further confirmed by western blot analysis on tissue samples from the WT and CASD1 homozygous KO mouse probing with PToV. O‐acetyl‐Sia levels in various tissue homogenates from CASD1 KO mice in comparison to WT mice was analyzed by mild acid or sialidase release, 1,2‐diamino‐4,5‐methylenedioxybenzene‐2HCl (DMB) derivatization and HPLC and compared to CASD1 heterozygous mice. Base treatment was also applied to Sias released with acetic acid or sialidase to provide a more accurate quantification of O‐acetyl‐Sias. Marked reduction of 9‐O‐acetyl‐Sias was observed in homozygous CASD1 KO tissue (particularly colon, brain and testis and blood) with a small amount of residual 9‐O‐acetyl‐Sias still found in certain tissue like adrenals and bladders. This work suggested that CASD1 plays a major role in the biosynthesis of 9‐O‐acetyl Sias in different mouse tissue‐ and cell‐types. Whether CASD1 participates in the 4‐O‐acetylation of sialic acids and whether different O‐acetyltransferases work on glycolipid and glycoprotein‐bound sialic acids needs further study. These mice also provide a model system to study the in vivo functions of sialic acid 9(7)‐O‐acetylation.