Anchoring secreted proteins in endoplasmic reticulum by plant oleosin: the example of vitamin B12 cellular sequestration by transcobalamin
Oleosin is a plant protein localized to lipid droplets and endoplasmic reticulum of plant cells. Our idea was to use it to target functional secretory proteins of interest to the cytosolic side of the endoplasmic reticulum of mammalian cells, through expressing oleosin-containing chimeras. We have d...
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| Veröffentlicht in: | PloS one 2009-07, Vol.4 (7), p.e6325-e6325 |
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| creator | Pons, Laurent Battaglia-Hsu, Shyue-Fang Orozco-Barrios, Carlos Enrique Ortiou, Sandrine Chery, Celine Alberto, Jean-Marc Arango-Rodriguez, Martha Ligia Dumas, Dominique Martinez-Fong, Daniel Freund, Jean-Noel Gueant, Jean-Louis |
| description | Oleosin is a plant protein localized to lipid droplets and endoplasmic reticulum of plant cells. Our idea was to use it to target functional secretory proteins of interest to the cytosolic side of the endoplasmic reticulum of mammalian cells, through expressing oleosin-containing chimeras. We have designed this approach to create cellular models deficient in vitamin B12 (cobalamin) because of the known problematics associated to the obtainment of effective vitamin B12 deficient cell models. This was achieved by the overexpression of transcobalamin inside cells through anchoring to oleosin.
chimera gene constructs including transcobalamin-oleosin (TC-O), green fluorescent protein-transcobalamin-oleosin (GFP-TC-O) and oleosin-transcobalamin (O-TC) were inserted into pAcSG2 and pCDNA3 vectors for expression in sf9 insect cells, Caco2 (colon carcinoma), NIE-115 (mouse neuroblastoma), HEK (human embryonic kidney), COS-7 (Green Monkey SV40-transfected kidney fibroblasts) and CHO (Chinese hamster ovary cells). The subcellular localization, the changes in vitamin B12 binding activity and the metabolic consequences were investigated in both Caco2 and NIE-115 cells.
vitamin B12 binding was dramatically higher in TC-O than that in O-TC and wild type (WT). The expression of GFP-TC-O was observed in all cell lines and found to be co-localized with an ER-targeted red fluorescent protein and calreticulin of the endoplasmic reticulum in Caco2 and COS-7 cells. The overexpression of TC-O led to B12 deficiency, evidenced by impaired conversion of cyano-cobalamin to ado-cobalamin and methyl-cobalamin, decreased methionine synthase activity and reduced S-adenosyl methionine to S-adenosyl homocysteine ratio, as well as increases in homocysteine and methylmalonic acid concentration.
the heterologous expression of TC-O in mammalian cells can be used as an effective strategy for investigating the cellular consequences of vitamin B12 deficiency. More generally, expression of oleosin-anchored proteins could be an interesting tool in cell engineering for studying proteins of pharmacological interest. |
| doi_str_mv | 10.1371/journal.pone.0006325 |
| format | Article |
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chimera gene constructs including transcobalamin-oleosin (TC-O), green fluorescent protein-transcobalamin-oleosin (GFP-TC-O) and oleosin-transcobalamin (O-TC) were inserted into pAcSG2 and pCDNA3 vectors for expression in sf9 insect cells, Caco2 (colon carcinoma), NIE-115 (mouse neuroblastoma), HEK (human embryonic kidney), COS-7 (Green Monkey SV40-transfected kidney fibroblasts) and CHO (Chinese hamster ovary cells). The subcellular localization, the changes in vitamin B12 binding activity and the metabolic consequences were investigated in both Caco2 and NIE-115 cells.
vitamin B12 binding was dramatically higher in TC-O than that in O-TC and wild type (WT). The expression of GFP-TC-O was observed in all cell lines and found to be co-localized with an ER-targeted red fluorescent protein and calreticulin of the endoplasmic reticulum in Caco2 and COS-7 cells. The overexpression of TC-O led to B12 deficiency, evidenced by impaired conversion of cyano-cobalamin to ado-cobalamin and methyl-cobalamin, decreased methionine synthase activity and reduced S-adenosyl methionine to S-adenosyl homocysteine ratio, as well as increases in homocysteine and methylmalonic acid concentration.
the heterologous expression of TC-O in mammalian cells can be used as an effective strategy for investigating the cellular consequences of vitamin B12 deficiency. More generally, expression of oleosin-anchored proteins could be an interesting tool in cell engineering for studying proteins of pharmacological interest.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0006325</identifier><identifier>PMID: 19623264</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>5-Methyltetrahydrofolate-homocysteine S-methyltransferase ; Analysis ; Anchoring ; Animal models ; Animals ; Apoptosis ; Base Sequence ; Binding ; Biochemistry ; Biochemistry, Molecular Biology ; Biophysics ; Biosynthesis ; Blotting, Western ; Calreticulin ; Cell Biology/Gene Expression ; Cell culture ; Cell Line ; Cells (Biology) ; Chimeras ; Cloning ; Colon ; Colon cancer ; Cyanocobalamin ; Deoxyribonucleic acid ; DNA ; DNA methylation ; DNA Primers ; DNA, Complementary ; Electrophoresis, Polyacrylamide Gel ; Endoplasmic Reticulum ; Endoplasmic Reticulum - metabolism ; Expression vectors ; Fibroblasts ; Fluorescence ; Food engineering ; Green fluorescent protein ; Homocysteine ; Humans ; Insect cells ; Insects ; Life Sciences ; Localization ; Mammalian cells ; Mammals ; Metabolism ; Methionine ; Microscopy, Confocal ; Nervous system ; Neuroblastoma ; Neurosciences ; Nutrition/Deficiencies ; Oleosin ; Pharmacology ; Physiology ; Plant cells ; Plant Proteins ; Plant Proteins - metabolism ; Plasmids ; Proteins ; Red fluorescent protein ; Transcobalamins ; Transcobalamins - metabolism ; Vitamin B ; Vitamin B 12 ; Vitamin B 12 - metabolism ; Vitamin B12 ; Vitamin deficiency ; Vitamins</subject><ispartof>PloS one, 2009-07, Vol.4 (7), p.e6325-e6325</ispartof><rights>COPYRIGHT 2009 Public Library of Science</rights><rights>2009 Pons et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><rights>Pons et al. 2009</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c696t-56a484f7e04e2adfae3f9888b50c3f2db33818989065bd0b8b8e311b816dd54d3</citedby><orcidid>0000-0002-0971-3774 ; 0000-0002-2786-9633 ; 0000-0002-5881-6251</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2710007/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2710007/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19623264$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.science/hal-00559010$$DView record in HAL$$Hfree_for_read</backlink></links><search><contributor>Tomé, Daniel</contributor><creatorcontrib>Pons, Laurent</creatorcontrib><creatorcontrib>Battaglia-Hsu, Shyue-Fang</creatorcontrib><creatorcontrib>Orozco-Barrios, Carlos Enrique</creatorcontrib><creatorcontrib>Ortiou, Sandrine</creatorcontrib><creatorcontrib>Chery, Celine</creatorcontrib><creatorcontrib>Alberto, Jean-Marc</creatorcontrib><creatorcontrib>Arango-Rodriguez, Martha Ligia</creatorcontrib><creatorcontrib>Dumas, Dominique</creatorcontrib><creatorcontrib>Martinez-Fong, Daniel</creatorcontrib><creatorcontrib>Freund, Jean-Noel</creatorcontrib><creatorcontrib>Gueant, Jean-Louis</creatorcontrib><title>Anchoring secreted proteins in endoplasmic reticulum by plant oleosin: the example of vitamin B12 cellular sequestration by transcobalamin</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Oleosin is a plant protein localized to lipid droplets and endoplasmic reticulum of plant cells. Our idea was to use it to target functional secretory proteins of interest to the cytosolic side of the endoplasmic reticulum of mammalian cells, through expressing oleosin-containing chimeras. We have designed this approach to create cellular models deficient in vitamin B12 (cobalamin) because of the known problematics associated to the obtainment of effective vitamin B12 deficient cell models. This was achieved by the overexpression of transcobalamin inside cells through anchoring to oleosin.
chimera gene constructs including transcobalamin-oleosin (TC-O), green fluorescent protein-transcobalamin-oleosin (GFP-TC-O) and oleosin-transcobalamin (O-TC) were inserted into pAcSG2 and pCDNA3 vectors for expression in sf9 insect cells, Caco2 (colon carcinoma), NIE-115 (mouse neuroblastoma), HEK (human embryonic kidney), COS-7 (Green Monkey SV40-transfected kidney fibroblasts) and CHO (Chinese hamster ovary cells). The subcellular localization, the changes in vitamin B12 binding activity and the metabolic consequences were investigated in both Caco2 and NIE-115 cells.
vitamin B12 binding was dramatically higher in TC-O than that in O-TC and wild type (WT). The expression of GFP-TC-O was observed in all cell lines and found to be co-localized with an ER-targeted red fluorescent protein and calreticulin of the endoplasmic reticulum in Caco2 and COS-7 cells. The overexpression of TC-O led to B12 deficiency, evidenced by impaired conversion of cyano-cobalamin to ado-cobalamin and methyl-cobalamin, decreased methionine synthase activity and reduced S-adenosyl methionine to S-adenosyl homocysteine ratio, as well as increases in homocysteine and methylmalonic acid concentration.
the heterologous expression of TC-O in mammalian cells can be used as an effective strategy for investigating the cellular consequences of vitamin B12 deficiency. More generally, expression of oleosin-anchored proteins could be an interesting tool in cell engineering for studying proteins of pharmacological interest.</description><subject>5-Methyltetrahydrofolate-homocysteine S-methyltransferase</subject><subject>Analysis</subject><subject>Anchoring</subject><subject>Animal models</subject><subject>Animals</subject><subject>Apoptosis</subject><subject>Base Sequence</subject><subject>Binding</subject><subject>Biochemistry</subject><subject>Biochemistry, Molecular Biology</subject><subject>Biophysics</subject><subject>Biosynthesis</subject><subject>Blotting, Western</subject><subject>Calreticulin</subject><subject>Cell Biology/Gene Expression</subject><subject>Cell culture</subject><subject>Cell Line</subject><subject>Cells (Biology)</subject><subject>Chimeras</subject><subject>Cloning</subject><subject>Colon</subject><subject>Colon cancer</subject><subject>Cyanocobalamin</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA methylation</subject><subject>DNA Primers</subject><subject>DNA, Complementary</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Endoplasmic Reticulum</subject><subject>Endoplasmic Reticulum - metabolism</subject><subject>Expression vectors</subject><subject>Fibroblasts</subject><subject>Fluorescence</subject><subject>Food engineering</subject><subject>Green fluorescent protein</subject><subject>Homocysteine</subject><subject>Humans</subject><subject>Insect cells</subject><subject>Insects</subject><subject>Life Sciences</subject><subject>Localization</subject><subject>Mammalian cells</subject><subject>Mammals</subject><subject>Metabolism</subject><subject>Methionine</subject><subject>Microscopy, Confocal</subject><subject>Nervous system</subject><subject>Neuroblastoma</subject><subject>Neurosciences</subject><subject>Nutrition/Deficiencies</subject><subject>Oleosin</subject><subject>Pharmacology</subject><subject>Physiology</subject><subject>Plant cells</subject><subject>Plant Proteins</subject><subject>Plant Proteins - metabolism</subject><subject>Plasmids</subject><subject>Proteins</subject><subject>Red fluorescent protein</subject><subject>Transcobalamins</subject><subject>Transcobalamins - metabolism</subject><subject>Vitamin B</subject><subject>Vitamin B 12</subject><subject>Vitamin B 12 - metabolism</subject><subject>Vitamin B12</subject><subject>Vitamin deficiency</subject><subject>Vitamins</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk1tv0zAUxyMEYmPwDRBYQprEQ4sviZPwgFQmYJUqTeL2atnJSevKsTM7qbavwKfGWQNrJx5QHmId_87tf3yS5CXBc8Jy8m7rBm-lmXfOwhxjzBnNHiWnpGR0xilmjw_OJ8mzELYYZ6zg_GlyQkpOGeXpafJrYauN89quUYDKQw816rzrQduAtEVga9cZGVpdoXirq8EMLVK3KBptj5wBF7R9j_oNILiRbWcAuQbtdC_b6P6RUFSBMYORPia4HiD0Xvba2TFGPNpQOSXNCD9PnjTSBHgx_c-SH58_fb-4nK2uviwvFqtZxUvezzIu0yJtcsApUFk3ElhTFkWhMlyxhtaKsYIUZVFinqkaq0IVwAhRBeF1naU1O0te7-N2xgUxyRgEoSWJIvKsjMRyT9RObkXndSv9rXBSizuD82shfdTCgFBcQUZANjjjKVVEpU3DacVTkJID5THWhynboFqoK7Cxa3MU9PjG6o1Yu52gOYlTzWOAt_sAmwdul4uVGG1xrFmJCd6RyJ5Pyby701q0Ooz6SwtuCILnGS7yfKzqzQPw3zrM99Raxla1bVyssIpfDfE9xHfX6GhfpDktU85zfF_r5BCZHm76tRxCEMtvX_-fvfp5zJ4fsBuQpt8EZ4bxIYVjMN2DlXcheGj-KkawGNfmT59iXBsxrU10e3U4pHunaU_Yb4D0Fa0</recordid><startdate>20090722</startdate><enddate>20090722</enddate><creator>Pons, 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secreted proteins in endoplasmic reticulum by plant oleosin: the example of vitamin B12 cellular sequestration by transcobalamin</title><author>Pons, Laurent ; Battaglia-Hsu, Shyue-Fang ; Orozco-Barrios, Carlos Enrique ; Ortiou, Sandrine ; Chery, Celine ; Alberto, Jean-Marc ; Arango-Rodriguez, Martha Ligia ; Dumas, Dominique ; Martinez-Fong, Daniel ; Freund, Jean-Noel ; Gueant, Jean-Louis</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c696t-56a484f7e04e2adfae3f9888b50c3f2db33818989065bd0b8b8e311b816dd54d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>5-Methyltetrahydrofolate-homocysteine S-methyltransferase</topic><topic>Analysis</topic><topic>Anchoring</topic><topic>Animal models</topic><topic>Animals</topic><topic>Apoptosis</topic><topic>Base Sequence</topic><topic>Binding</topic><topic>Biochemistry</topic><topic>Biochemistry, Molecular Biology</topic><topic>Biophysics</topic><topic>Biosynthesis</topic><topic>Blotting, Western</topic><topic>Calreticulin</topic><topic>Cell Biology/Gene Expression</topic><topic>Cell culture</topic><topic>Cell Line</topic><topic>Cells (Biology)</topic><topic>Chimeras</topic><topic>Cloning</topic><topic>Colon</topic><topic>Colon cancer</topic><topic>Cyanocobalamin</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA methylation</topic><topic>DNA Primers</topic><topic>DNA, Complementary</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Endoplasmic Reticulum</topic><topic>Endoplasmic Reticulum - metabolism</topic><topic>Expression vectors</topic><topic>Fibroblasts</topic><topic>Fluorescence</topic><topic>Food engineering</topic><topic>Green fluorescent protein</topic><topic>Homocysteine</topic><topic>Humans</topic><topic>Insect cells</topic><topic>Insects</topic><topic>Life Sciences</topic><topic>Localization</topic><topic>Mammalian 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(HAL)</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Pons, Laurent</au><au>Battaglia-Hsu, Shyue-Fang</au><au>Orozco-Barrios, Carlos Enrique</au><au>Ortiou, Sandrine</au><au>Chery, Celine</au><au>Alberto, Jean-Marc</au><au>Arango-Rodriguez, Martha Ligia</au><au>Dumas, Dominique</au><au>Martinez-Fong, Daniel</au><au>Freund, Jean-Noel</au><au>Gueant, Jean-Louis</au><au>Tomé, Daniel</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Anchoring secreted proteins in endoplasmic reticulum by plant oleosin: the example of vitamin B12 cellular sequestration by transcobalamin</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2009-07-22</date><risdate>2009</risdate><volume>4</volume><issue>7</issue><spage>e6325</spage><epage>e6325</epage><pages>e6325-e6325</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Oleosin is a plant protein localized to lipid droplets and endoplasmic reticulum of plant cells. Our idea was to use it to target functional secretory proteins of interest to the cytosolic side of the endoplasmic reticulum of mammalian cells, through expressing oleosin-containing chimeras. We have designed this approach to create cellular models deficient in vitamin B12 (cobalamin) because of the known problematics associated to the obtainment of effective vitamin B12 deficient cell models. This was achieved by the overexpression of transcobalamin inside cells through anchoring to oleosin.
chimera gene constructs including transcobalamin-oleosin (TC-O), green fluorescent protein-transcobalamin-oleosin (GFP-TC-O) and oleosin-transcobalamin (O-TC) were inserted into pAcSG2 and pCDNA3 vectors for expression in sf9 insect cells, Caco2 (colon carcinoma), NIE-115 (mouse neuroblastoma), HEK (human embryonic kidney), COS-7 (Green Monkey SV40-transfected kidney fibroblasts) and CHO (Chinese hamster ovary cells). The subcellular localization, the changes in vitamin B12 binding activity and the metabolic consequences were investigated in both Caco2 and NIE-115 cells.
vitamin B12 binding was dramatically higher in TC-O than that in O-TC and wild type (WT). The expression of GFP-TC-O was observed in all cell lines and found to be co-localized with an ER-targeted red fluorescent protein and calreticulin of the endoplasmic reticulum in Caco2 and COS-7 cells. The overexpression of TC-O led to B12 deficiency, evidenced by impaired conversion of cyano-cobalamin to ado-cobalamin and methyl-cobalamin, decreased methionine synthase activity and reduced S-adenosyl methionine to S-adenosyl homocysteine ratio, as well as increases in homocysteine and methylmalonic acid concentration.
the heterologous expression of TC-O in mammalian cells can be used as an effective strategy for investigating the cellular consequences of vitamin B12 deficiency. More generally, expression of oleosin-anchored proteins could be an interesting tool in cell engineering for studying proteins of pharmacological interest.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>19623264</pmid><doi>10.1371/journal.pone.0006325</doi><tpages>e6325</tpages><orcidid>https://orcid.org/0000-0002-0971-3774</orcidid><orcidid>https://orcid.org/0000-0002-2786-9633</orcidid><orcidid>https://orcid.org/0000-0002-5881-6251</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2009-07, Vol.4 (7), p.e6325-e6325 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1291063659 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | 5-Methyltetrahydrofolate-homocysteine S-methyltransferase Analysis Anchoring Animal models Animals Apoptosis Base Sequence Binding Biochemistry Biochemistry, Molecular Biology Biophysics Biosynthesis Blotting, Western Calreticulin Cell Biology/Gene Expression Cell culture Cell Line Cells (Biology) Chimeras Cloning Colon Colon cancer Cyanocobalamin Deoxyribonucleic acid DNA DNA methylation DNA Primers DNA, Complementary Electrophoresis, Polyacrylamide Gel Endoplasmic Reticulum Endoplasmic Reticulum - metabolism Expression vectors Fibroblasts Fluorescence Food engineering Green fluorescent protein Homocysteine Humans Insect cells Insects Life Sciences Localization Mammalian cells Mammals Metabolism Methionine Microscopy, Confocal Nervous system Neuroblastoma Neurosciences Nutrition/Deficiencies Oleosin Pharmacology Physiology Plant cells Plant Proteins Plant Proteins - metabolism Plasmids Proteins Red fluorescent protein Transcobalamins Transcobalamins - metabolism Vitamin B Vitamin B 12 Vitamin B 12 - metabolism Vitamin B12 Vitamin deficiency Vitamins |
| title | Anchoring secreted proteins in endoplasmic reticulum by plant oleosin: the example of vitamin B12 cellular sequestration by transcobalamin |
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