Nuclear localization signal of murine CMP-Neu5Ac synthetase includes residues required for both nuclear targeting and enzymatic activity
Nuclear localization signal of murine CMP-Neu5Ac synthetase includes residues required for both nuclear targeting and enzymatic activity
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DOI：
10.1074/jbc.m201093200
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发表时间：
2002-05-31
影响因子：
4.8
通讯作者：
Gerardy-Schahn, R
中科院分区：
文献类型：
--
作者：
Münster, AK;Weinhold, B;Gerardy-Schahn, R
5-N-乙酰神经氨酸（Neu 5Ac）是动物细胞中发现的主要唾液酸衍生物。作为细胞表面糖缀合物的组分，Neu 5Ac对许多细胞识别和通信过程（包括宿主-寄生虫相互作用）是关键的。合成唾液酸化糖缀合物的先决条件是Neu 5Ac活化为胞苷一磷酸N-乙酰神经氨酸（CMP-Neu 5Ac）。该反应由CMP-Neu 5Ac-合成酶（syn）催化，该合成酶由于未知的原因存在于细胞核中。克隆的小鼠CMP-Neu 5Ac合成酶的序列分析鉴定了三个碱性氨基酸簇（BC 1-BC 3），其可能作为核定位信号（NLS）起作用。在本研究中，嵌合蛋白和诱变策略被用来显示，BC 1和BC 2是活性NLS序列时，连接到绿色荧光蛋白（增强的GFP），但只有BC 2是必要的，足以介导CMP-Neu 5Ac合成酶的核输入。定点突变鉴定出残基（KRXR）-R-198是核转运所必需的，Arg（202）是完成转运过程所必需的。通过BC 2中的单位点突变产生的CMP-Neu 5Ac合成酶的胞质形式证明了（i）酶活性不依赖于核定位，以及（ii）Arg（199）和Arg（202）参与核转运和合成酶活性。所有已知和预测的CMP唾液酸合成酶的比较揭示了Arg（202）和Gln（203）在进化中高度保守，并且对于最佳合成酶活性而不是核定位至关重要。结合，数据表明，核运输和酶活性是独立的功能，共享一些共同的氨基酸要求CMP-Neu 5Ac合成酶。
5-N-Acetylneuraminic acid (Neu5Ac) is the major sialic acid derivative found in animal cells. As a component of cell surface glycoconjugates, Neu5Ac is pivotal to numerous cellular recognition and communication processes including host-parasite interactions. A prerequisite for the synthesis of sialylated glycoconjugates is the activation of Neu5Ac to cytidine-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). The reaction is catalyzed by CMP-Neu5Ac-synthetase (syn), which, for unknown reasons, resides in the nucleus. Sequence analysis of the cloned murine CMP-Neu5Ac synthetase identified three clusters of basic amino acids (BC1-BC3) that might function as nuclear localization signals (NLS). In the present study chimeric protein and mutagenesis strategies were used to show that BC1 and BC2 are active NLS sequences when attached to the green fluorescent protein (enhanced GFP), but only BC2 is necessary and sufficient to mediate the nuclear import of CMP-Neu5Ac synthetase. Site-directed mutations identified the residues (KRXR)-R-198 to be essential for nuclear transport and Arg(202) to be necessary to complete the transport process. Cytoplasmic forms of CMP-Neu5Ac synthetase generated by single site mutations in BC2 demonstrated that (i) enzyme activity is independent of nuclear localization, and (ii) Arg(199) and Arg(202) are involved in both nuclear transport and synthetase activity. Comparison of all known and predicted CMPsialic acid synthetases reveals Arg(202) and Gln(203) as highly conserved in evolution and critically important for optimal synthetase activity but not for nuclear localization. Combined, the data demonstrate that nuclear transport and enzyme activity are independent functions that share some common amino acid requirements in CMP-Neu5Ac synthetase.