Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity.
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 Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----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: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----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 Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.
Baubichon-Cortay
H
H
Department of General and Medical Biochemistry, University of Lyon, France.
Broquet
P
P
George
P
P
Louisot
P
P
D000818:Animals; D001212:Asialoglycoproteins; D001921:Brain / Q000201:enzymology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005033:Ethylmaleimide / Q000494:pharmacology; D060748:Fetuins; D008244:Lysophosphatidylcholines; D008297:Male; D009077:Mucins / Q000302:isolation & purification; D009844:Oligosaccharides / Q000096:biosynthesis; D011134:Polysaccharides / Q000096:biosynthesis*; D051381:Rats; D011919:Rats, Inbred Strains; D012799:Sialyltransferases / Q000032:analysis* / Q000037:antagonists & inhibitors; D013379:Substrate Specificity; D014357:Trypsin; D000509:alpha-Fetoproteins / Q000378:metabolism; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D001212:Asialoglycoproteins; D060748:Fetuins; D008244:Lysophosphatidylcholines; D009077:Mucins; D009844:Oligosaccharides; D011134:Polysaccharides; D000509:alpha-Fetoproteins; C017029:asialofetuin; D012799:Sialyltransferases; D014357:Trypsin; D005033:Ethylmaleimide; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase