Discovery and biological significance of the sialate:O-sulfotransferases in vertebrates

概要

It is well known that sialic acid (Sia), an essential monosaccharide for survival of vertebrates, modifies glycoconjugates on the cell surface and extracellular matrix to mediate and regulate cellular recognition and signaling events. Sia shows an extremely large structural diversity that emerges from modification by acetylation, methylation, sulfation, and so on. However, metabolism and biological functions of those modified Sias have not been clarified, except for a limited knowledge of acetylated Sia. Sulfation of Sia residues is known to occur in various animals including mammals and sea urchin. It has been shown that sulfated Sia (SiaS) is involved in sperm-egg interaction and sperm motility in sea urchin. In contrast, nothing is known about the occurrence, biosynthesis, and function of SiaS in vertebrates. No enzyme responsible for sulfation of Sia has ever been described. Thus, the objective of my thesis is to elucidate biological significance of the SiaS, and three lines of experiments were performed.

　(1) Elucidation of expression profiles of SiaS in vertebrates: First, several mammalian cell lines and tissues from mouse and medaka were analyzed for the existence of SiaS not only by immunochemical methods (Western blotting and flow cytometry) with monoclonal antibody 3G9 recognizing 8-O-sulfated Neu5Ac residue, but also by the chemical method (DMB derivatization-fluorometric high performance liquid chromatography). SiaS were found to occur ubiquitously in vertebrate cells and tissues, although their expression was dynamically changed depending on the cell type and developmental stages. Second, the cell surface expression of SiaS were shown to be dependent on the cell density, as well as on the type of cell lines. Interestingly, SiaS was induced by the antibiotic G418 treatment. Taken together, this is the first observation that the expression level of SiaS is regulated by the intrinsic and extrinsic factors. It is thus suggested that there exists a sulfotransferase responsible for sulfation of Sia, whose gene expression and/or enzyme activation are regulated under various conditions.

　(2) Identification and characterization of the sialate:O-sulfotransferases (SulT-Sias): Of those genes for possible sulfotransferases with unknown acceptor substrates, two sulfotransferase gene candidates, SulT-Sia1 and 2, were cloned from mouse. The transfection of CHO cells with these cDNAs showed an increase of the surface expression of SiaS. On the other hand, when the cDNA for inactive form of SulT-Sia1 with mutations on the donor PAPS-binding domain was transfected, the surface SiaS expression remained unchanged. In addition, cell surface SiaS-positive HEK cells that endogenously expressed SulT-Sia1 gene lost the SiaS expression, when transfected with the corresponding shRNA. Furthermore, in vitro activity was measured using recombinant enzymes obtained by over-expression of the SulT-Sia1 and 2. These two enzymes showed the SulT-Sia activity; however, their acceptor substrate specificity was different between them, suggesting that they have different roles in the sulfation of Sia.

　(3) Biological significance of SiaS at animal level: To understand biological significance of SiaS, we established inherited frameshift mutations on the SulT- Sia1 or the SulT-Sia2 genes in medaka, SulT1-KO and SulT2-KO, respectively, using the gene editing method. Both SulT1- and 2-KO were found to show low survival rates, and dead at young fry before maturation. They also showed different phenotypes than each other. SulT1-KO had defective in cardiac muscle function, while SulT2-KO were poor in innate immunity and showed delayed development especially in brain and eye. These results indicate that sulfation is one of the most critical modifications of Sia that affects the survival of animal.

　Taken together, in this thesis I discovered and identified the sialate:O- sulfotransferases responsible for the biosynthesis of SiaS as the first example in nature, and demonstrated that critical importance of the enzymes in vertebrates.