Evidence for an O‐glycan sialylation system in brain

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Abstract
We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]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 SiNH2 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.

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