Evidence for mitochondrial localization of a novel human sialidase (NEU4)
Evidence for mitochondrial localization of a novel human sialidase (NEU4)
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DOI：
10.1042/bj20050017
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发表时间：
2005-08-15
影响因子：
4.1
通讯作者：
Miyagi, T
中科院分区：
文献类型：
--
作者：
Yamaguchi, K;Hata, K;Miyagi, T
基于公共数据库中预测代表NEU4唾液酸酶基因的人类cDNA序列，从人脑中分离出涵盖整个编码序列的cDNA，并在哺乳动物细胞中表达。该cDNA编码两种异构体：一种具有N末端12个氨基酸序列，预测为线粒体靶向序列，另一种缺少这些氨基酸。通过逆转录 - PCR评估，异构体的表达具有组织特异性。脑、肌肉和肾脏含有两种异构体；肝脏表达量最高，且在该器官中短异构体占主导。在瞬时转染的COS - 1细胞中，以神经节苷脂以及糖蛋白和寡糖为底物时，酶活性与对照水平相比显著增加。这与其他人类唾液酸酶的研究结果不同。尽管两种异构体在底物特异性方面无法区分，但它们表现出不同的亚细胞定位。免疫荧光显微镜和生化分级分离表明，在几种人类培养细胞类型中，外源表达的带有血凝素标签的NEU4长异构体集中在线粒体中，而短异构体存在于细胞内膜中，这表明包含N末端12个氨基酸残基的序列作为线粒体的靶向信号。将N末端区域与增强型绿色荧光蛋白融合后能有效靶向线粒体，以及该区域一个氨基酸发生替换的突变体靶向失败，进一步支持了长异构体定位于线粒体。NEU4可能通过调节神经节苷脂G(D3)参与细胞凋亡的调控，神经节苷脂G(D3)在细胞凋亡过程中在线粒体中积累，并且是唾液酸酶的最佳底物。
Based on the human cDNA sequence predicted to represent the NEU4 sialidase gene in public databases, a cDNA covering the entire coding sequence was isolated from human brain and expressed in mammalian cells. The cDNA encodes two isoforms: one possessing an N-terminal 12-amino-acid sequence that is predicted to be a mitochondrial targeting sequence, and the other lacking these amino acids. Expression of the isoforms is tissue-specific, as assessed by reverse transcription-PCR. Brain, muscle and kidney contained both isoforms; liver showed the highest expression, and the short form was predominant in this organ. In transiently transfected COS-1 cells, enzyme activity was markedly increased with gangliosides as well as with glycoproteins and oligosaccharides as substrates compared with the control levels. This differs from findings with other human sialidases. Although the isoforms were not distinguishable with regard to substrate specificity, they exhibited differential subcellular localizations. Immunofluorescence microscopy and biochemical fractionation demonstrated that an exogenously expressed haemagglutinin-tagged long form of NEU4 was concentrated in mitochondria in several human culture cell types, whereas the short form was present in intracellular membranes, indicating that the sequence comprising the N-terminal 12 amino acid residues acts as a targeting signal for mitochondria. Co-localization of the long form to mitochondria was further supported by efficient targeting of the N-terminal region fused to enhanced green fluorescent protein, and by the targeting failure of a mutant with an amino acid substitution in this region. NEU4 is possibly involved in regulation of apoptosis by modulation of ganglioside G(D3), which accumulates in mitochondria during apoptosis and is the best substrate for the sialidase.