Howard D R, Fukuda M, Fukuda M N, Stanley P
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
J Biol Chem. 1987 Dec 15;262(35):16830-7.
Previous studies have shown that the GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferase (alpha (1,3) fucosyltransferase (Fuc-T)) activities expressed by the Chinese hamster ovary cell mutants LEC11 (Fuc-TI) and LEC12 (Fuc-TII) are different enzymes and indicated that Fuc-TI might act on sialylated lactosamine sequences (Campbell, C., and Stanley, P. (1984) J. Biol. Chem. 259, 11208-11214). In this paper we show that CSLEX-1, a monoclonal antibody specific for NeuNac alpha (2,3)Gal beta (1,4)(Fuc alpha (1,3))GlcNAc beta 1 sequences, bound to LEC11 cells but not to LEC12 cells. Direct evidence that Fuc-TI could act on sialylated substrates was sought with a series of glycolipid acceptors. Optimal assay conditions in crude cell extracts were determined with nLc4, a glycolipid which accepted fucose with both Fuc-TI and Fuc-TII to generate the Lex antigenic determinant. The two enzymes differed in their detergent sensitivities, pH optima, Mn2+ requirements, and apparent Km values for nLc4. When sialylated glycolipids were examined as substrates, Fuc-TI added fucose to IV3NeuNAcnLc4 but not to IV6NeuNAcnLc4, whereas Fuc-TII was unable to utilize either glycolipid as a substrate. Further studies showed that Fuc-TI and Fuc-TII possess novel specificities for glycolipids containing two lactosamine sequences as potential fucose acceptors. Fuc-TI exhibited good activities with VI3NeuNAcnLc6 and VI6NeuNAcnLc6 whereas Fuc-TII had very low activity with both substrates. Glycosidase digestions of the labeled products showed that Fuc-TI added fucose primarily to the internal N-acetylglucosamine of both glycolipids. The same preference for the internal N-acetylglucosamine was shown by Fuc-TI when nLc6 was the acceptor. In contrast, Fuc-TII preferred to transfer fucose to the external acceptor site of nLc6, consistent with the low activities of Fuc-TII with sialylated nLc6 derivatives. Thus the two enzymes preferentially add fucose to different N-acetylglucosamines in the same substrate, nLc6. This indicates that the biosynthetic pathway for fucosylation of polylactosamine sequences in glycolipids and glycoproteins will vary depending upon the particular alpha (1,3)fucosyltransferase present.
先前的研究表明，中国仓鼠卵巢细胞突变体LEC11（Fuc-TI）和LEC12（Fuc-TII）所表达的GDP-岩藻糖：N-乙酰葡糖胺3-α-L-岩藻糖基转移酶（α(1,3)岩藻糖基转移酶（Fuc-T））活性是不同的酶，并表明Fuc-TI可能作用于唾液酸化乳糖胺序列（坎贝尔，C.，和斯坦利，P.（1984年）《生物化学杂志》259，11208 - 11214）。在本文中，我们表明，CSLEX-1，一种对NeuNacα(2,3)Galβ(1,4)(Fucα(1,3))GlcNAcβ1序列具有特异性的单克隆抗体，与LEC11细胞结合，但不与LEC12细胞结合。我们使用一系列糖脂受体来寻找Fuc-TI可以作用于唾液酸化底物的直接证据。用nLc4（一种糖脂，Fuc-TI和Fuc-TII都能将岩藻糖加到其上以产生Lex抗原决定簇）来确定粗细胞提取物中的最佳测定条件。这两种酶在去污剂敏感性、最适pH、对Mn2+的需求以及对nLc4的表观Km值方面有所不同。当检查唾液酸化糖脂作为底物时，Fuc-TI将岩藻糖加到IV3NeuNAcnLc4上，但不加到IV6NeuNAcnLc4上，而Fuc-TII不能利用这两种糖脂作为底物。进一步的研究表明，Fuc-TI和Fuc-TII对含有两个乳糖胺序列作为潜在岩藻糖受体的糖脂具有新的特异性。Fuc-TI对VI3NeuNAcnLc6和VI6NeuNAcnLc6表现出良好的活性，而Fuc-TII对这两种底物的活性都非常低。对标记产物的糖苷酶消化表明，Fuc-TI主要将岩藻糖加到两种糖脂的内部N-乙酰葡糖胺上。当nLc6作为受体时，Fuc-TI对内部N-乙酰葡糖胺也表现出相同的偏好。相比之下，Fuc-TII更倾向于将岩藻糖转移到nLc6的外部受体位点，这与Fuc-TII对唾液酸化nLc6衍生物的低活性一致。因此，这两种酶优先将岩藻糖加到同一底物nLc6的不同N-乙酰葡糖胺上。这表明糖脂和糖蛋白中多乳糖胺序列的岩藻糖基化生物合成途径将因存在的特定α(1,3)岩藻糖基转移酶而有所不同。
