Evidence for an O ‐glycan sialylation system in brain

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [ 14 C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [ 14 C]sialylated glycans were synthesized: N ‐glycans and monosialylated and disialylated O ‐glycans. The varying effects of N ‐ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N ‐ethylmaleimide, was identified as the Neu5Acα2→3Galβ1→3GalNAc‐R:α2→6 sialyltransferase previously described [Baubichon‐Cortay, H., Serres‐Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149 , 209–223]. This enzyme was responsible for the synthesis of disialylated O ‐glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O ‐glycan. N ‐ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N ‐glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O ‐glycan synthesis, we used another substrate: Galβ1→3GalNAc–protein obtained after galactosylation of desialylated ovine mucin by a GalNAc‐R:β1→3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N ‐glycans and of NeuAc in α2→3 linkages on the galactose residue. When using N ‐ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH 2 columns, we identified this product as Neu5Acα2→3Galβ1→3GalNAc. The enzyme leading to synthesis of this monosialylated O ‐glycan was identified as a Galβ1→3GalNAc‐R:α2→3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Acα2→3Galβ1→3GalNAc‐R:α2→6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the α2→3 sialyltransferase regulates the α2→6 sialyltransferase activity since it synthesizes the α2→6 sialyltransferase substrate.