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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (4): 32-37    DOI: 10.13523/j.cb.20190405
研究报告     
在酿酒酵母中研究人类mOGT基质导向序列的功能 *
李凤,高晓冬,中西秀树()
江南大学糖化学与生物技术教育部重点实验室 无锡 214122
Analysis of Martrix Targeting Sequence of Human Mitochondrial OGT in Saccharomyces cerevisiae
Feng LI,Xiao-dong GAO,Hideki NAKANISHI()
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Jiangnan University, Wuxi 214122, China
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摘要：

人类线粒体N-乙酰氨基葡萄糖转移酶(mitochondrial O-GlcNAc transferse, mOGT)通过其N端基质导向序列(matrix targeting sequence,MTS)定位于线粒体内膜上,mOGT的过表达将导致细胞的凋亡。蛋白质的O-GlcNAc修饰存在于绝大多数真核生物中,而酿酒酵母唯独缺乏该修饰途径。对人体mOGT过表达引起酿酒酵母生长缺陷的机制进行分析,期望利用该模型研究mOGT导致的细胞凋亡调控机制。在酿酒酵母细胞中表达人体mOGT及其截短序列发现:mOGT对酿酒酵母的生长抑制及细胞中的定位依赖于N端全长的MTS序列;MTS序列在酿酒酵母中共表达导致线粒体融合。MTS序列过表达的酿酒酵母菌可以作为研究mOGT引起细胞凋亡的模式细胞。
关键词： 人类mOGT;  基质导向序列;  线粒体融合    
Abstract:

Human mitochondrial O-GlcNAc transferase (mOGT) localized in mitochondria inner membrane through its N-terminal martrix targeting sequence(MTS), mOGT overexpression caused cell spoptosis. O-GlcNAc modification existed in most eukaryote cells except Saccharomyces cerevisiae. In order to analyze mOGT induced mammalian cell apoptosis, the mechanism of mOGT caused yeast cell growth defect was investigated. Herein, mOGT was overexpressed in Saccharomyces cerevisiae. Both yeast growth defect and mOGT localization were largely depends on MTS sequence. Furthermore, MTS expression caused yeast mitochondria fussion. Therefore, MTS overexpressed yeast cells might be applied to analyze mOGT caused cell apoptosis.
Key words: Human mOGT    Matrix-targeting sequence    Mitochondria fussion
收稿日期: 2018-11-02 出版日期: 2019-05-08
ZTFLH:  Q819  
基金资助: * 国家自然科学基金资助项目(21576118)
通讯作者: 中西秀树     E-mail: hideki@jiangnan.edu.cn
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李凤
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引用本文:

李凤,高晓冬,中西秀树. 在酿酒酵母中研究人类mOGT基质导向序列的功能 *[J]. 中国生物工程杂志, 2019, 39(4): 32-37.

Feng LI,Xiao-dong GAO,Hideki NAKANISHI. Analysis of Martrix Targeting Sequence of Human Mitochondrial OGT in Saccharomyces cerevisiae. China Biotechnology, 2019, 39(4): 32-37.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190405    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I4/32

图1  OGT结构示意图
质粒/引物 说明/基因型/引物序列(5'-3') 来源
质粒
pRS424GAL1pr TRP1 标记 多拷贝 GAL1 启动子 [16]
pRS424GAL1pr-mOGT TRP1 标记 多拷贝 GAL1 启动子 含mOGT [17]
pRS424GAL1pr-mOGT-RFP TRP1 标记 多拷贝 GAL1 启动子 含C端RFP标记的mOGT基因 本研究
pRS424GAL1pr-mOGT(N△50) TRP1 标记 多拷贝 GAL1 启动子 含mOGT(N△50)基因 本研究
pRS424GAL1pr- MTS15-RFP TRP1 标记 多拷贝 GAL1 启动子 含MTS15-RFP 本研究
pRS424GAL1pr- MTS50-RFP TRP1 标记 多拷贝 GAL1 启动子 含MTS50-RFP 本研究
pRS426TEF2pr URA3 标记 多拷贝 TEF2 启动子 [18]
pRS426TEF2pr-OGA URA3 标记 多拷贝 TEF2 启动子 含OGA基因 [14]
引物
OGA-F(Cla Ⅰ) GCGCATCGATATGGTGCAGAAGGAGAGTCA 本研究
OGA-R (XhoⅠ) GCGCCTCGAGCTACAGGCTCCGACCAAGTA 本研究
MTS15-F(Spe Ⅰ) GCGCACTAGTATGCTGCAGGGTCACTTTTGGCTGGTCAGAGAAGG
AATAATGATAAAAGGAGAAGAACTTTTCAC		 本研究
MTS50-F(XbaⅠ) GCGCTCTAGAATGCTGCAGGGTCACTTTTG 本研究
MTS50-R(Hind Ⅲ) GCGCAAGCTTAGGAGGGGTTAATGAAAGAA 本研究
mOGT-R(Hind Ⅲ) GCGCAAGCTTTGCTGACTCAGTGACTTCAA 本研究
表1  该研究所用的质粒和引物
图2  OGA不能回复mOGT或mOGT(H382A)导致的酵母生长缺陷
图3  MOGT抑制酵母细胞生长依赖于MTS序列
图4  MOGT位于酿酒酵母细胞的线粒体上
图5  MTS序列导致酿酒酵母线粒体融合
图6  MTS序列抑制酿酒酵母生长
[1] Hart G W . Minireview series on the thirtieth anniversary of research on O-GlcNAcylation of nuclear and cytoplasmic proteins: Nutrient regulation of cellular metabolism and physiology by O-GlcNAcylation. Journal of Biological Chemmistry, 2014,289(50):34422-34423.
doi: 10.1074/jbc.R114.609776
[2] Marshall S, Bacote V, Traxinger R R . Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance. Journal of Biological Chemmistry, 1991,266(8):4706-4712.
doi: 10.0000/PMID2002019 pmid: 2002019
[3] Vaidyanathan K, Wells L . Multiple tissue-specific roles for the O-GlcNAc post-translational modification in the induction of and complications arising from type II diabetes. Journal of Biological Chemmistry, 2014,289(50):34466-34471.
doi: 10.1074/jbc.R114.591560
[4] Ma Z, Vosseller K . Cancer metabolism and elevated O-GlcNAc in oncogenic signaling. Journal of Biological Chemmistry, 2014,289(50):34457-34465.
doi: 10.1074/jbc.R114.577718
[5] Marsh S A, Collins H E, Chatham J C . Protein O-GlcNAcylation and cardiovascular (patho)physiology. Journal of Biological Chemmistry, 2014,289(50):34449-34456.
doi: 10.1074/jbc.R114.585984
[6] Zhu Y, Shan X, Yuzwa S A , et al. The emerging link between O-GlcNAc and Alzheimer disease. Journal of Biological Chemmistry, 2014,289(50):34472-34481.
doi: 10.1074/jbc.R114.601351
[7] Hanover J A, Yu S, Lubas W B , et al. Mitochondrial and nucleocytoplasmic isoforms of O-linked GlcNAc transferase encoded by a single mammalian gene. Archives of Biochemistry Biophysics, 2003,409(2):287-297.
doi: 10.1016/S0003-9861(02)00578-7 pmid: 12504895
[8] Burnham-Marusich A R, Berninsone P M . Multiple proteins with essential mitochondrial functions have glycosylated isoforms. Mitochondrion, 2012,12(4):423-427.
doi: 10.1016/j.mito.2012.04.004 pmid: 22564751
[9] Ma J, Liu T, Wei A C , et al. O-GlcNAcomic profiling identifies widespread O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) in oxidative phosphorylation system regulating cardiac mitochondrial function. Journal of Biological Chemmistry, 2015,290(49):29141-29153.
doi: 10.1074/jbc.M115.691741
[10] Hu Y, Suarez J, Fricovsky E , et al. Increased enzymatic O-GlcNAcylation of mitochondrial proteins impairs mitochondrial function in cardiac myocytes exposed to high glucose. Journal of Biological Chemmistry, 2009,284(1):547-555.
doi: 10.1074/jbc.M808518200 pmid: 19004814
[11] Lazarus M B, Nam Y, Jiang J , et al. Structure of human O-GlcNAc transferase and its complex with a peptide substrate. Nature, 2011,469(7331):564-567.
doi: 10.1038/nature09638 pmid: 21240259
[12] Sacoman J L, Dagda R Y , Burnham-Marusich A R , et al. Mitochondrial O-GlcNAc transferase (mOGT) regulates mitochondrial structure, function and survival in HeLa cells. Journal of Biological Chemmistry, 2017,292(11):4499-4518.
doi: 10.1074/jbc.M116.726752
[13] Halim A, Larsen I S, Neubert P , et al. Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast. Proceedings of the National Academy of Science of the United States of America, 2015,112(51):15648-15653.
[14] Nakanishi H, Li F, Han B X , et al. Yeast cells as an assay system for in vivo O-GlcNAc modification. Biochimica et Biophysica acta -General Subjects , 2017,1861(5):1159-1167.
doi: 10.1016/j.bbagen.2017.03.002
[15] 韩宝仙, 高晓冬, 中西秀树 . 人体线粒体N-乙酰氨基葡萄糖转移酶在酿酒酵母中的表达. 食品与生物技术学报, 2016,35(9):987-992.
Han B X, Gao X D, Nakanishi H . Expression of a human mitochondrial N-acetylglucosamine transferase in Saccharomyces cerevisiae. Journal of Food Science and Biotechnology, 2016,35(9):987-992.
[16] Longtine M S, Mckenzie Iii A, Demarini D J , et al. Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae. Yeast, 2010,14(10):953-961.
[17] Lubas W A, Frank D W, Krause M , et al. O-linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats. Journal of Biological Chemmistry, 1997,272(14):9316-9324.
doi: 10.1074/jbc.272.14.9316
[18] Mumberg D, Müller R, Funk M . Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene, 1995,156(1):119-122.
doi: 10.1016/0378-1119(95)00037-7 pmid: 7737504
[19] Banerjee P S, Ma J, Hart G W . Diabetes-associated dysregulation of O-GlcNAcylation in rat cardiac mitochondria. Proceedings of the National Academy of Science of the United States of America, 2015,112(19):6050-6055.
doi: 10.1073/pnas.1424017112
[20] Love D C, Kochan J, Cathey R L , et al. Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase. Journal of Cell Science, 2003,116(4):647-654.
doi: 10.1242/jcs.00246 pmid: 12538765
[21] Shin S H, Love D C, Hanover J A . Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis. Amino Acids, 2011,40(3):885-893.
doi: 10.1007/s00726-010-0719-8 pmid: 20824293
[22] Bocharova N, Chave-Cox R, Sokolov S , et al. Protein aggregation and neurodegeneration: clues from a yeast model of Huntington’s disease. Biochemistry, 2009,74(2):231-234.
doi: 10.1134/S0006297909020163 pmid: 19267681
[23] Zha H, Fisk H A, Yaffe M P , et al. Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells. Molecular and Cellular Biology, 1996,16(11):6494-6508.
doi: 10.1128/MCB.16.11.6494 pmid: 231651
[24] Bossywetzel E, Barsoum M J, Godzik A , et al. Mitochondrial fission in apoptosis, neurodegeneration and aging. Current Opinion in Cell Biology, 2003,15(6):706-716.
doi: 10.1016/j.ceb.2003.10.015 pmid: 14644195
[25] Mignotte B, Vayssiere J L . Mitochondria and apoptosis. European Journal of Biochemistry, 1998,252(1):1-15.
doi: 10.1046/j.1432-1327.1998.2520001.x
[26] 安志远, 丁文一 . 鲍曼不动杆菌Omp34经线粒体途径诱导HeLa细胞凋亡. 生物技术, 2018,28(4):323-328.
An Z Y, Ding W Y . The outer membrane protein of Acinetobacter baumannii induces apoptosis in HeLa cells through mitochondria signal pathway. Biotechnology, 2018,28(4):323-328.
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