Butor C, Diaz S, Varki A
University of California, San Diego, Cancer Center, La Jolla 92093.
J Biol Chem. 1993 May 15;268(14):10197-206.
O-Acetylation of sialic acids has previously been considered an uncommon modification found on certain salivary mucins and neural gangliosides. We show here that glycosidically bound sialic acids from total membranes of rat liver have surprisingly high levels (approximately 20%) of O-acetylation at the 7- or 9-position. This O-acetylation is further enriched in N-linked oligosaccharides but is barely detectable in ganglioside fractions from the same tissue. The position of O-acetylation on the sialic acid side chain varies between different subcellular fractions. In particular, 7-O-acetylation was enriched in lysosomal membranes and 9-O-acetylation in plasma membranes, whereas Golgi membranes contained both types. This distribution fits with the ability of the rat liver sialate: O-acetyltransferase(s) to synthesize both 7- and 9-O-acetyl esters (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426) and the fact that 7-O-acetyl esters can migrate to the 9-position at physiological temperature but only under neutral or mildly alkaline conditions. Subcellular fractionation shows that sialate:O-acetyltransferase activity directed toward endogenous acceptors is enriched in Golgi fractions, whereas an intralumenal sialic acid-specific O-acetylesterase activity is not. The O-acetyltransferase is labile and difficult to solubilize in the intact state and cannot be assayed with exogenous acceptors. However, a prelabeled [3H]acetyl intermediate can be solubilized from Golgi membranes with Triton X-100 and is stable for a prolonged time in the cold. In contrast to the transferase, the lumenal esterase is easily released in a stable and water-soluble form from membrane fractions by saponin permeabilization or repeated freeze-thaw. In keeping with this finding, differential subcellular fractionation and continuous sucrose gradients indicate that this enzyme is enriched in lysosomal fractions (see also the accompanying paper (Butor, C., Higa, H. H., and Varki, A. (1993) J. Biol. Chem. 268, 10207-10213). Based upon findings reported in this and previous studies, a model is proposed for the biosynthesis, maturation, and turnover of 7- and 9-O-acetyl esters on the sialic acids of N-linked oligosaccharides that are attached to membrane-bound proteins in the rat liver.
唾液酸的O-乙酰化以前被认为是在某些唾液粘蛋白和神经节苷脂上发现的一种不常见的修饰。我们在此表明，大鼠肝脏总膜中糖苷键结合的唾液酸在7或9位具有惊人的高O-乙酰化水平（约20%）。这种O-乙酰化在N-连接寡糖中进一步富集，但在同一组织的神经节苷脂部分中几乎检测不到。唾液酸侧链上O-乙酰化的位置在不同亚细胞部分之间有所不同。特别是，7-O-乙酰化在溶酶体膜中富集，9-O-乙酰化在质膜中富集，而高尔基体膜中则同时含有这两种类型。这种分布与大鼠肝脏唾液酸：O-乙酰转移酶合成7-和9-O-乙酰酯的能力相符合（迪亚兹，S.，希加，H. H.，海斯，B. K.，和瓦尔基，A.（1989年）《生物化学杂志》264，19416 - 19426），以及7-O-乙酰酯在生理温度下但仅在中性或轻度碱性条件下可迁移至9位这一事实相符。亚细胞分级分离表明，针对内源性受体的唾液酸：O-乙酰转移酶活性在高尔基体部分中富集，而腔内唾液酸特异性O-乙酰酯酶活性则不然。O-乙酰转移酶不稳定，难以完整状态下溶解，且不能用外源性受体进行测定。然而，预标记的[3H]乙酰中间体可用Triton X-100从高尔基体膜中溶解出来，并且在低温下可长时间稳定。与转移酶不同，腔内酯酶通过皂苷通透或反复冻融很容易以稳定且水溶性的形式从膜部分释放出来。与这一发现一致，差异亚细胞分级分离和连续蔗糖梯度表明该酶在溶酶体部分中富集（另见随附论文（布托尔，C.，希加，H. H.，和瓦尔基，A.（1993年）《生物化学杂志》268，10207 - 10213））。基于本研究和先前研究报告的结果，提出了一个关于大鼠肝脏中与膜结合蛋白相连的N-连接寡糖唾液酸上7-和9-O-乙酰酯的生物合成、成熟和周转的模型。
