Bergh M L, Hooghwinkel G J, van den Eijnden D H
J Biol Chem. 1983 Jun 25;258(12):7430-6.
Fetal calf liver microsomes were found to be capable of sialylating 14C-galactosylated ovine submaxillary asialomucin. The main oligosaccharide product chain could be obtained by beta-elimination under reductive conditions and was identified as NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol (where GalNAcol represents N-acetylgalactosaminitol) by means of high performance liquid chromatography (HPLC) analysis and methylation. The branched trisaccharide Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)-GalNAcol and the disaccharide NeuAc alpha 2 leads to 6GalNAcol were not formed. Very similar results were obtained when asialofetuin and antifreeze glycoprotein were used as an acceptor. When 3H-sialylated antifreeze glycoprotein ([3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc-protein) was incubated with fetal calf liver microsomes and CMP-[14C]NeuAc, a reduced tetrasaccharide could be isolated. The structure of this product chain appeared to be [3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3([14C]NeuAc alpha 2 leads to 6)GalNAcol, as established by means of HPLC analysis, specific enzymatic degradation with Newcastle disease virus neuraminidase, and periodate oxidation. These data indicate that fetal calf liver contains two sialyltransferases involved in the biosynthesis of the O-linked bisialotetrasaccharide chain. The first enzyme is a beta-galactoside alpha 2 leads to 3 sialyltransferase which converts Gal beta 1 leads to 3 GalNAc chains to the substrate for the second enzyme, a (NeuAc alpha 2 leads to 3Gal beta 1 leads to 3)GalNAc-protein alpha 2 leads to 6 sialyltransferase. The latter enzyme does not sialylate GalNAc or Gal beta 1 leads to 3GalNAc units but is capable of transferring sialic acid to C-6 of GalNAc in NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc trisaccharide side chains, thereby dictating a strictly ordered sequence of sialylation of the Gal beta 1 leads to 3 GalNAc units in fetal calf liver.
研究发现，胎牛肝微粒体能够将14C-半乳糖基化的羊颌下脱唾液酸粘蛋白进行唾液酸化。在还原条件下通过β-消除反应可得到主要的寡糖产物链，借助高效液相色谱（HPLC）分析和甲基化鉴定其为NeuAcα2→3Galβ1→3GalNAcol（其中GalNAcol代表N-乙酰半乳糖胺醇）。分支三糖Galβ1→3(NeuAcα2→6)-GalNAcol和二糖NeuAcα2→6GalNAcol未形成。当使用脱唾液酸胎球蛋白和抗冻糖蛋白作为受体时，得到了非常相似的结果。当将3H-唾液酸化抗冻糖蛋白（[3H]NeuAcα2→3Galβ1→3GalNAc-蛋白）与胎牛肝微粒体及CMP-[14C]NeuAc一起温育时，可分离出一种还原型四糖。通过HPLC分析、用新城疫病毒神经氨酸酶进行特异性酶促降解以及高碘酸盐氧化确定该产物链的结构为[3H]NeuAcα2→3Galβ1→3([14C]NeuAcα2→6)GalNAcol。这些数据表明，胎牛肝中含有两种参与O-连接双唾液酸四糖链生物合成的唾液酸转移酶。第一种酶是β-半乳糖苷α2→3唾液酸转移酶，它将Galβ1→3GalNAc链转化为第二种酶即(NeuAcα2→3Galβ1→3)GalNAc-蛋白α2→6唾液酸转移酶的底物。后一种酶不会使GalNAc或Galβ1→3GalNAc单元唾液酸化，但能够将唾液酸转移至NeuAcα2→3Galβ1→3GalNAc三糖侧链中GalNAc的C-6位，从而决定了胎牛肝中Galβ1→3GalNAc单元严格有序的唾液酸化顺序。
