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 tissuespecific, 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 GD3, which accumulates in mitochondria during apoptosis and is the best substrate for the sialidase.
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Research Article|
August 09 2005
Evidence for mitochondrial localization of a novel human sialidase (NEU4)
Kazunori Yamaguchi;
Kazunori Yamaguchi
*Division of Biochemistry, Miyagi Cancer Center Research Institute, Natori, Miyagi, 981-1293, Japan
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Keiko Hata;
Keiko Hata
*Division of Biochemistry, Miyagi Cancer Center Research Institute, Natori, Miyagi, 981-1293, Japan
†CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan
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Koichi Koseki;
Koichi Koseki
*Division of Biochemistry, Miyagi Cancer Center Research Institute, Natori, Miyagi, 981-1293, Japan
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Kazuhiro Shiozaki;
Kazuhiro Shiozaki
*Division of Biochemistry, Miyagi Cancer Center Research Institute, Natori, Miyagi, 981-1293, Japan
†CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan
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Hirotoshi Akita;
Hirotoshi Akita
‡Second Department of Oral Anatomy, School of Dentistry, Tohoku University, Sendai, 980-8575, Japan
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Tadashi Wada;
Tadashi Wada
*Division of Biochemistry, Miyagi Cancer Center Research Institute, Natori, Miyagi, 981-1293, Japan
†CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan
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Setsuko Moriya;
Setsuko Moriya
*Division of Biochemistry, Miyagi Cancer Center Research Institute, Natori, Miyagi, 981-1293, Japan
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Taeko Miyagi
Taeko Miyagi
1
*Division of Biochemistry, Miyagi Cancer Center Research Institute, Natori, Miyagi, 981-1293, Japan
†CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan
1To whom correspondence should be addressed (email tmiyagi@mcc.pref.miyagi.jp).
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Publisher: Portland Press Ltd
Received:
January 04 2005
Revision Received:
April 14 2005
Accepted:
April 22 2005
Accepted Manuscript online:
April 22 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 390 (1): 85–93.
Article history
Received:
January 04 2005
Revision Received:
April 14 2005
Accepted:
April 22 2005
Accepted Manuscript online:
April 22 2005
Citation
Kazunori Yamaguchi, Keiko Hata, Koichi Koseki, Kazuhiro Shiozaki, Hirotoshi Akita, Tadashi Wada, Setsuko Moriya, Taeko Miyagi; Evidence for mitochondrial localization of a novel human sialidase (NEU4). Biochem J 15 August 2005; 390 (1): 85–93. doi: https://doi.org/10.1042/BJ20050017
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