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
Inserm U954, Faculté de Médecine, Nancy-Université, Vandoeuvre-les-Nancy, France.
PLoS One. 2009 Jul 22;4(7):e6325. doi: 10.1371/journal.pone.0006325.
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.
CONCLUSIONS/SIGNIFICANCE: 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.
油质蛋白是一种植物蛋白，定位于植物细胞的脂滴和内质网。我们的想法是通过表达含油质蛋白的嵌合体，利用它将感兴趣的功能性分泌蛋白靶向到哺乳动物细胞内质网的胞质侧。由于获取有效的维生素B12缺乏细胞模型存在已知问题，我们设计了这种方法来创建维生素B12（钴胺素）缺乏的细胞模型。这是通过将转钴胺素锚定到油质蛋白上在细胞内过表达来实现的。
将包括转钴胺素 - 油质蛋白（TC - O）、绿色荧光蛋白 - 转钴胺素 - 油质蛋白（GFP - TC - O）和油质蛋白 - 转钴胺素（O - TC）的嵌合基因构建体插入pAcSG2和pCDNA3载体中，以在sf9昆虫细胞、Caco2（结肠癌细胞）、NIE - 115（小鼠神经母细胞瘤）、HEK（人胚肾细胞）、COS - 7（绿猴SV40转染的肾成纤维细胞）和CHO（中国仓鼠卵巢细胞）中表达。在Caco2和NIE - 115细胞中研究了亚细胞定位、维生素B12结合活性的变化以及代谢后果。
TC - O中的维生素B12结合显著高于O - TC和野生型（WT）。在所有细胞系中均观察到GFP - TC - O的表达，并且在Caco2和COS - 7细胞中发现其与内质网靶向的红色荧光蛋白和内质网的钙网蛋白共定位。TC - O的过表达导致B12缺乏，表现为氰钴胺向腺苷钴胺和甲基钴胺的转化受损、甲硫氨酸合酶活性降低以及S - 腺苷甲硫氨酸与S - 腺苷同型半胱氨酸比值降低，同时同型半胱氨酸和甲基丙二酸浓度升高。
结论/意义：TC - O在哺乳动物细胞中的异源表达可作为研究维生素B12缺乏的细胞后果的有效策略。更一般地说，油质蛋白锚定蛋白的表达可能是细胞工程中研究具有药理学意义的蛋白质的一个有趣工具。
