Control of Expression, Glycosylation, and Secretion of HIV-1 gp120 by Homologous and Heterologous Signal Sequences
Identifieur interne : 004467 ( Main/Exploration ); précédent : 004466; suivant : 004468Control of Expression, Glycosylation, and Secretion of HIV-1 gp120 by Homologous and Heterologous Signal Sequences
Auteurs : Yan Li [Canada] ; Lizhong Luo [Canada] ; David Y. Thomas [Canada] ; O. Yong Kang [Canada]Source :
- Virology [ 0042-6822 ] ; 1994.
Abstract
Abstract: The HIV-1 gp120 signal sequence of 30 amino acids is longer than most glycoprotein signal sequences and contains an average of 5 positively charged amino acids. The HIV-1 gp120 gene with its natural signal sequence expressed in any prokaryotic or eukaryotic expression systems showed extremely low levels of synthesis and secretion. However, deletion of the HIV-1 gp 120 signal sequence results in production of large quantities of a nonglycosylated form of gp120 in Spodoptera frugiperda cells. Substitution of the gp120 natural signal sequences with the signal sequences from honeybee mellitin or murine interleukin 3 promotes a high level of expression of a glycosylated form of gp120 and efficient secretion. These heterologous signal sequences contain one (mellitin) or no (IL-3) positively charged amino acids and led us to investigate the role of the positively charged amino acids in the signal sequence of HIV-1 gp120. Four charge-altered forms of the gp120 signal sequence of HIV-1 were constructed by site-directed mutagenesis in which the positively charged amino acids were sequentially substituted with neutral amino acids. The results of these experiments showed that the expression and secretion of gp120 was progressively increased by eliminating the positively charged amino acids in a stepwise fashion. However, the substitution of all positively charged amino acids resulted in the accumulation of nonglycosylated gp120 within the cells with decreased amounts of the glycosylated form of gp120. These results demonstrate that the positively charged amino acids in the signal sequence of HIV-1 gp120 are key factors in determining its poor expression and secretion. Analyses of intracellular transport and folding of gp120 further indicate that the presence of a highly charged, uncleaved signal sequence is an important factor limiting transport of gp120 from the rough ER to the Golgi apparatus.
Url:
DOI: 10.1006/viro.1994.1531
Affiliations:
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Control of Expression, Glycosylation, and Secretion of HIV-1 gp120 by Homologous and Heterologous Signal Sequences</title><author><name sortKey="Li, Yan" sort="Li, Yan" uniqKey="Li Y" first="Yan" last="Li">Yan Li</name></author><author><name sortKey="Luo, Lizhong" sort="Luo, Lizhong" uniqKey="Luo L" first="Lizhong" last="Luo">Lizhong Luo</name></author><author><name sortKey="Thomas, David Y" sort="Thomas, David Y" uniqKey="Thomas D" first="David Y." last="Thomas">David Y. Thomas</name></author><author><name sortKey="Kang, O Yong" sort="Kang, O Yong" uniqKey="Kang O" first="O. Yong" last="Kang">O. Yong Kang</name></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:3F963D354A845B627306B4A01709E20AAAC60AE6</idno><date when="1994" year="1994">1994</date><idno type="doi">10.1006/viro.1994.1531</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-C8S34T3N-K/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000E00</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000E00</idno><idno type="wicri:Area/Istex/Curation">000E00</idno><idno type="wicri:Area/Istex/Checkpoint">001B04</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Checkpoint">001B04</idno><idno type="wicri:doubleKey">0042-6822:1994:Li Y:control:of:expression</idno><idno type="wicri:Area/Main/Merge">004530</idno><idno type="wicri:Area/Main/Curation">004467</idno><idno type="wicri:Area/Main/Exploration">004467</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Control of Expression, Glycosylation, and Secretion of HIV-1 gp120 by Homologous and Heterologous Signal Sequences</title><author><name sortKey="Li, Yan" sort="Li, Yan" uniqKey="Li Y" first="Yan" last="Li">Yan Li</name><affiliation wicri:level="1"><country>Canada</country><wicri:regionArea>Department of Zoology, Faculty of Science, and Department of Microbiology and Immunology, Faculty of Medicine, The University of Western Ontario, London, Ontario, Canada N6A 5B7; and Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Avenue Royalmount, Montreal, Quebec</wicri:regionArea><wicri:noRegion>Quebec</wicri:noRegion></affiliation></author><author><name sortKey="Luo, Lizhong" sort="Luo, Lizhong" uniqKey="Luo L" first="Lizhong" last="Luo">Lizhong Luo</name><affiliation wicri:level="1"><country>Canada</country><wicri:regionArea>Department of Zoology, Faculty of Science, and Department of Microbiology and Immunology, Faculty of Medicine, The University of Western Ontario, London, Ontario, Canada N6A 5B7; and Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Avenue Royalmount, Montreal, Quebec</wicri:regionArea><wicri:noRegion>Quebec</wicri:noRegion></affiliation></author><author><name sortKey="Thomas, David Y" sort="Thomas, David Y" uniqKey="Thomas D" first="David Y." last="Thomas">David Y. Thomas</name><affiliation wicri:level="1"><country>Canada</country><wicri:regionArea>Department of Zoology, Faculty of Science, and Department of Microbiology and Immunology, Faculty of Medicine, The University of Western Ontario, London, Ontario, Canada N6A 5B7; and Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Avenue Royalmount, Montreal, Quebec</wicri:regionArea><wicri:noRegion>Quebec</wicri:noRegion></affiliation></author><author><name sortKey="Kang, O Yong" sort="Kang, O Yong" uniqKey="Kang O" first="O. Yong" last="Kang">O. Yong Kang</name><affiliation wicri:level="1"><country>Canada</country><wicri:regionArea>Department of Zoology, Faculty of Science, and Department of Microbiology and Immunology, Faculty of Medicine, The University of Western Ontario, London, Ontario, Canada N6A 5B7; and Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Avenue Royalmount, Montreal, Quebec</wicri:regionArea><wicri:noRegion>Quebec</wicri:noRegion></affiliation></author></analytic><monogr></monogr><series><title level="j">Virology</title><title level="j" type="abbrev">YVIRO</title><idno type="ISSN">0042-6822</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1994">1994</date><biblScope unit="volume">204</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="266">266</biblScope><biblScope unit="page" to="278">278</biblScope></imprint><idno type="ISSN">0042-6822</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0042-6822</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: The HIV-1 gp120 signal sequence of 30 amino acids is longer than most glycoprotein signal sequences and contains an average of 5 positively charged amino acids. The HIV-1 gp120 gene with its natural signal sequence expressed in any prokaryotic or eukaryotic expression systems showed extremely low levels of synthesis and secretion. However, deletion of the HIV-1 gp 120 signal sequence results in production of large quantities of a nonglycosylated form of gp120 in Spodoptera frugiperda cells. Substitution of the gp120 natural signal sequences with the signal sequences from honeybee mellitin or murine interleukin 3 promotes a high level of expression of a glycosylated form of gp120 and efficient secretion. These heterologous signal sequences contain one (mellitin) or no (IL-3) positively charged amino acids and led us to investigate the role of the positively charged amino acids in the signal sequence of HIV-1 gp120. Four charge-altered forms of the gp120 signal sequence of HIV-1 were constructed by site-directed mutagenesis in which the positively charged amino acids were sequentially substituted with neutral amino acids. The results of these experiments showed that the expression and secretion of gp120 was progressively increased by eliminating the positively charged amino acids in a stepwise fashion. However, the substitution of all positively charged amino acids resulted in the accumulation of nonglycosylated gp120 within the cells with decreased amounts of the glycosylated form of gp120. These results demonstrate that the positively charged amino acids in the signal sequence of HIV-1 gp120 are key factors in determining its poor expression and secretion. Analyses of intracellular transport and folding of gp120 further indicate that the presence of a highly charged, uncleaved signal sequence is an important factor limiting transport of gp120 from the rough ER to the Golgi apparatus.</div></front></TEI><affiliations><list><country><li>Canada</li></country></list><tree><country name="Canada"><noRegion><name sortKey="Li, Yan" sort="Li, Yan" uniqKey="Li Y" first="Yan" last="Li">Yan Li</name></noRegion><name sortKey="Kang, O Yong" sort="Kang, O Yong" uniqKey="Kang O" first="O. Yong" last="Kang">O. Yong Kang</name><name sortKey="Luo, Lizhong" sort="Luo, Lizhong" uniqKey="Luo L" first="Lizhong" last="Luo">Lizhong Luo</name><name sortKey="Thomas, David Y" sort="Thomas, David Y" uniqKey="Thomas D" first="David Y." last="Thomas">David Y. Thomas</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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