Continuous nonradioactive method for screening trypanosomal trans-sialidase activity and its inhibitors.
Continuous nonradioactive method for screening trypanosomal trans-sialidase activity and its inhibitors.
复制标题
筛选锥虫转唾液酸酶活性及其抑制剂的连续非放射性方法。
DOI：
10.1093/glycob/cwq056
复制
发表时间：
2010
期刊：
影响因子：
4.3
通讯作者：
deLederkremer,RosaM
中科院分区：
文献类型：
--
作者：
Sartor,PaulaA;Agusti,Rosalia;Leguizamon,MariaS;Campetella,Oscar;deLederkremer,RosaM
美国锥虫病病原体克氏锥虫不能合成唾液酸。不是使用相应的核苷酸糖作为单糖的供体，而是从宿主唾液糖偶联物中的α-2，3-连接SA转移到寄生虫粘蛋白的末端β-半乳糖基单位。为此，克氏毛滴虫表达一种glycosylphosphatidylinositol-anchoredtrans-sialidase(Tcts)，在感染的急性期在血液中检测到该Tcts被排放到环境中。TCTS在感染中的重要作用以及哺乳动物中缺乏类似活性，使该酶成为开发替代化疗药物的一个有吸引力的靶点。然而，目前还没有针对该酶的有效抑制剂。在体外，以3‘-唾液酸乳糖(SL)为供体，以放射性乳糖为受体底物是测定TCTS活性的常用方法。然后用阴离子交换层析分离放射性唾液酸化产物并进行测定。本文介绍了一种以SL或胎球蛋白为供体，以β-d-Fuc-(1-→6)-α-d-GlcNAc(1)为受体的新的非放射性分析方法。α-d-GlcNAc与四-O-苯甲酰基-d-岩藻糖发生区域选择性糖基化反应，再经脱苯甲酰化反应，即可合成双糖1。化合物1缺少受体半乳糖的C-6位羟基，因此不是半乳糖氧化酶的底物。我们的方法依赖于通过分光光度法定量从供体到1的6-脱氧半乳糖(D-Fuc)单元反式唾液酸化产生的末端半乳糖。这种方法还可以通过在没有受体1的情况下进行检测来区分唾液酸酶和反唾液酸化活性。
Trypanosoma cruzi, the agent of American trypanosomiasis is unable to synthesize sialic acid (SA). Instead of using the corresponding nucleotide sugar as donor of the monosaccharide, the transfer occurs from α-2,3-linked SA in the host sialoglycoconjugates to terminal β-galactopyranosyl units of the parasite mucins. For that purpose,T. cruziexpresses a glycosylphosphatidylinositol-anchoredtrans-sialidase (TcTS) that is shed into the milieu, being detected in the blood during the acute phase of the infection. The essential role of TcTS in infection and the absence of a similar activity in mammals make this enzyme an attractive target for the development of alternative chemotherapies. However, there is no effective inhibitor toward this enzyme. In vitro, 3′-sialyllactose (SL) as donor and radioactive lactose as acceptor substrate are widely used to measure TcTS activity. The radioactive sialylated product is then isolated by anion exchange chromatography and measured. Here we describe a new nonradioactive assay using SL or fetuin as donor and benzyl β-d-Fuc-(1→6)-α-d-GlcNAc (1) as acceptor. Disaccharide 1 was easily synthesized by regioselective glycosylation of benzyl α-d-GlcNAc with tetra-O-benzoyl-d-fucose followed by debenzoylation. Compound 1 lacks the hydroxyl group at C-6 of the acceptor galactose and therefore is not a substrate for galactose oxidase. Our method relies on the specific quantification of terminal galactose produced bytrans-sialylation from the donor to the 6-deoxy-galactose (D-Fuc) unit of 1 by a spectrophotometric galactose oxidase assay. This method may also discriminate sialidase andtrans-sialylation activities by running the assay in the absence of acceptor1.