Identification of core alpha 1,3-fucosylated glycans and cloning of the requisite fucosyltransferase cDNA from Drosophila melanogaster. Potential basis of the neural anti-horseadish peroxidase epitope.
Identification of core alpha 1,3-fucosylated glycans and cloning of the requisite fucosyltransferase cDNA from Drosophila melanogaster. Potential basis of the neural anti-horseadish peroxidase epitope.
复制标题
鉴定核心 α1,3-岩藻糖基化聚糖，并克隆黑腹果蝇所需的岩藻糖基转移酶 cDNA。
DOI：
--
复制
发表时间：
2001
影响因子：
4.8
通讯作者：
I. Wilson
中科院分区：
文献类型：
--
作者：
G. Fabini;A. Freilinger;F. Altmann;I. Wilson
多年来，针对植物糖蛋白辣根过氧化物酶的多克隆抗体已被用于特异性染色果蝇的神经和雄性生殖组织。该表位被认为是碳水化合物来源的，但尚未从果蝇中分离出可解释该交叉反应性的聚糖结构。在这里，我们报告，N-聚糖核心α 1，3-连接岩藻糖，作为判断由预吸收实验，识别果蝇胚胎神经系统的抗辣根过氧化物酶抗体是必不可少的。此外，我们描述了识别基质辅助激光解吸电离飞行时间质谱和高效液相色谱法的两个果蝇N-聚糖，已经在其他昆虫中检测到，携带alpha 1，3-和alpha 1，6-连接岩藻糖残基的近端核心GlcNAc。此外，我们已经从果蝇中分离出三个编码α 1，3-岩藻糖基转移酶同源物的cDNA。其中一个cDNA，当转化到毕赤酵母中时，被发现指导核心α 1，3-岩藻糖基转移酶活性的表达。该重组酶优先选择携带两个非还原性N-乙酰葡糖胺残基的双触角核心α 1，6-岩藻糖基化N-聚糖（GnGnF 6; Km 11 μ m）作为底物，而不是缺乏核心岩藻糖残基的相同结构（GnGn; Km 46 μ m）。果蝇核心α 1，3-岩藻糖基转移酶也被证明能够在体外岩藻糖基化人转铁蛋白的N-聚糖结构，这种修饰与获得与抗辣根过氧化物酶抗体的结合相关。
For many years, polyclonal antibodies raised against the plant glycoprotein horseradish peroxidase have been used to specifically stain the neural and male reproductive tissue of Drosophila melanogaster. This epitope is considered to be of carbohydrate origin, but no glycan structure from Drosophila has yet been isolated that could account for this cross-reactivity. Here we report that N-glycan core alpha1,3-linked fucose is, as judged by preabsorption experiments, indispensable for recognition of Drosophila embryonic nervous system by anti-horseradish peroxidase antibody. Further, we describe the identification by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and high performance liquid chromatography of two Drosophila N-glycans that, as already detected in other insects, carry both alpha1,3- and alpha1,6-linked fucose residues on the proximal core GlcNAc. Moreover, we have isolated three cDNAs encoding alpha1,3-fucosyltransferase homologues from Drosophila. One of the cDNAs, when transformed into Pichia pastoris, was found to direct expression of core alpha1,3-fucosyltransferase activity. This recombinant enzyme preferred as substrate a biantennary core alpha1,6-fucosylated N-glycan carrying two non-reducing N-acetylglucosamine residues (GnGnF6; Km 11 microm) over the same structure lacking a core fucose residue (GnGn; Km 46 microm). The Drosophila core alpha1,3-fucosyltransferase enzyme was also shown to be able to fucosylate N-glycan structures of human transferrin in vitro, this modification correlating with the acquisition of binding to anti-horseradish peroxidase antibody.