DmSAS Is Required for Sialic Acid Biosynthesis in Cultured Drosophila Third Instar Larvae CNS neurons
DmSAS Is Required for Sialic Acid Biosynthesis in Cultured Drosophila Third Instar Larvae CNS neurons
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DOI：
10.1021/cb200238k
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发表时间：
2011-11-01
影响因子：
4
通讯作者：
Recio-Pinto, Esperanza
中科院分区：
文献类型：
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作者：
Granell, Annelise E. von Bergen;Palter, Karen B.;Recio-Pinto, Esperanza
唾液酸化是糖复合物的一种重要的碳水化合物修饰，已被证明可以调节脊椎动物中的许多细胞/分子相互作用。在果蝇 (Dm) 中，利用序列同源性，克隆了唾液酸化途径的几种酶，并在表达系统中测试了它们的功能。在这里，我们研究了培养的 Dm 中枢神经系统 (CNS) 神经元中唾液酸的掺入是否需要内源表达的 Dm 唾液酸合酶 (DmSAS)。我们将源自野生型 Dm 幼虫的神经元与含有 DmSAS 突变（148 bp 缺失）的神经元进行了比较。这些细胞从 Ac(4)ManNAz（叠氮化物衍生的 N-乙酰甘露糖胺）产生 Sia5NAz（唾液酸形式）并将其掺入其糖缀合物的能力是通过 Click-iT 化学用荧光剂标记 Sia5NAz 的叠氮基团来测量的。我们发现大多数野生型 Dm CNS 神经元将 Sia5NAz 掺入其糖缀合物中。胞体处的唾液酸掺入量高于神经突处的唾液酸掺入量，并且也可以在核周区域和质膜处检测到。相反，来自 DmSAS 突变体的神经元不会整合 Sia5NAz，除非重新引入 DmSAS（救援突变体）。大多数神经元表达α2,6-唾液酸转移酶。这些结果证实该突变是无效突变，并且Dm细胞中不存在多余的唾液酸生物合成活性，即只有一个DmSAS。他们还提供了迄今为止最有力的证据，证明 DmSAS 是 Dm CNS 神经元唾液酸生物合成的关键酶，观察到的新合成唾液酸的亚细胞分布为了解其生物学功能提供了见解。
Sialylation is an important carbohydrate modification of glycoconjugates that has been shown to modulate many cellular/molecular interactions in vertebrates. In Drosophila melanogaster (Dm), using sequence homology, several enzymes of the sialylation pathway have been cloned and their function tested in expression systems Here we investigated whether sialic acid incorporation in cultured Dm central nervous system (CNS) neurons required endogenously expressed Dm sialic acid synthase (DmSAS). We compared neurons derived from wild type Dm larvae with those containing a DmSAS mutation (148 bp deletion). The ability of these cells to produce Sia5NAz (sialic acid form) from Ac(4)ManNAz (azide-derivatized N-acetylmannosamine) and incorporate it into their glycoconjugates was measured by tagging the azide group of Sia5NAz with fluorescent agents via Click-iT chemistry. We found that most of the wild type Dm CNS neurons incorporated Sia5NAz into their glycoconjugates. Sialic acid incorporation was higher at the soma than at the neurite and could also be detected at perinuclear regions and the plasma membrane. In contrast, neurons from the DmSAS mutant did not incorporate Sia5NAz unless DmSAS was reintroduced (rescue mutant). Most of the neurons expressed alpha 2,6-sialyltransferase. These results, confirm that the mutation was a null mutation and that no redundant sialic acid biosynthetic activity exists in Dm cells, i.e., there is only one DmSAS. They also provide the strongest proof to date that DmSAS is a key enzyme in the biosynthesis of sialic acids in Dm CNS neurons and the observed sub-cellular distribution of the newly synthesized sialic acids offers insights into their biological function.