Novel assay for direct fluorescent imaging of sialidase activity
Novel assay for direct fluorescent imaging of sialidase activity
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唾液酸酶活性直接荧光成像的新测定法
DOI：
10.1117/12.889172
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发表时间：
2011
影响因子：
15
通讯作者：
R. Bilyy
中科院分区：
文献类型：
--
作者：
A. Tomin;T. Shkandina;R. Bilyy
在这里，我们描述了一种新的方法来唾液酸酶活性估计。唾液酸酶（EC 3.2.1.18，外切-α-唾液酸酶），也称为神经氨酸酶，是水解糖蛋白和糖脂中末端唾液酸与随后的碳水化合物残基之间结合的糖苷的酶组。唾液酸是一组具有酸性的单糖，因为它们是神经氨酸的乙酰化或乙醇化衍生物。流感病毒和其他一些病毒利用神经氨酸酶的活性来感染宿主细胞。唾液酸化水平与肿瘤细胞的侵袭和转移能力密切相关，唾液酸化水平也决定了衰老或病毒感染细胞的清除。因此，唾液酸酶活性的检测对于临床诊断以及生命科学研究是至关重要的。作者开发了用于活细胞中唾液酸酶活性的可视化和估计的测定。先前已知的用于唾液酸酶活性检测的方法需要破坏细胞材料，或者是低灵敏度的，或者不提供关于在某些细胞内区室中的活性定位的信息。为了克服这些问题，利用荧光神经氨酸酶底物4-MUNA，并提出了使用荧光显微镜检测神经氨酸酶活性的方法，它提供了高信号水平和所研究的酶的细胞定位信息。通过使用这种方法，与活的和原发性坏死细胞相比，证实了凋亡细胞上唾液酸酶活性的增加。
Here we describe a novel approach to sialidase activity estimation. Sialidases (EC 3.2.1.18, exo-α-sialidases), also known as neuraminidases, are the group of enzymes, which hydrolyze the glycoside bound between terminal sialic acid and subsequent carbohydrate residue in glycoproteins and glycolipids. Sialic acids are the group of monosaccharides with acidic properties, since they are acetylated or glycolylated derivates of neuraminic acid. Flu and some other viruses use neuraminidase activity to infect host cells. The level of sialylation was shown to be tightly connected with tumor cell invasiveness and metastatic potential, sialylation level also determines the clearance of aged or virus-infected cells. Thus, detection of sialidase activity is of primary importance for clinical diagnostics as well as life science research. The authors developed the assay for both visualization and estimation of sialidase activity in living cells. Previously known methods for sialidase activity detection required destruction of cellular material, or were low-sensitive, or provided no information on the activity localization in certain intracellular compartment. To overcome these problems, a fluorogenic neuraminidase substrate, 4-MUNA was utilized, and the method for detection of neuraminidase activity using fluorescent microscopy was proposed, it provided a high signal level and information on cellular localization of the studied enzyme. By using this approach the increase of sialidase activity on apoptotic cells was demonstrated in comparison to viable and primary necrotic cells.