Anchoring Secreted Proteins in Endoplasmic Reticulum by Plant Oleosin: The Example of Vitamin B12 Cellular Sequestration by Transcobalamin

• DOI: 10.1371/journal.pone.0006325
• Publication Date: 2009-07-21T22:35:22Z
• Authors: Laurent Pons; Shyue-Fang Battag...; Carlos Enrique Or...; Sandrine Ortiou; Celine Chery; Jean-Marc Alberto; Martha Ligia Aran...; Dominique Dumas; Daniel Martinez-Fong; Jean-Noel Freund; Jean-Louis Gueant
• Keywords: Oleosin

Background Oleosin is a plant protein localized to lipid droplets and endoplasmic reticulum of cells. Our idea was use it target functional secretory proteins interest the cytosolic side mammalian cells, through expressing oleosin-containing chimeras. We have designed this approach create cellular models deficient in vitamin B12 (cobalamin) because known problematics associated obtainment effective cell models. This achieved by overexpression transcobalamin inside cells anchoring oleosin. Methodology chimera gene constructs including transcobalamin-oleosin (TC-O), green fluorescent protein-transcobalamin-oleosin (GFP-TC-O) oleosin-transcobalamin (O-TC) were inserted into pAcSG2 pCDNA3 vectors for expression sf9 insect Caco2 (colon carcinoma), NIE-115 (mouse neuroblastoma), HEK (human embryonic kidney), COS-7 (Green Monkey SV40-transfected kidney fibroblasts) CHO (Chinese hamster ovary cells). The subcellular localization, changes binding activity metabolic consequences investigated both Principal findings dramatically higher TC-O than that O-TC wild type (WT). GFP-TC-O observed all lines found be co-localized with an ER-targeted red calreticulin led deficiency, evidenced impaired conversion cyano-cobalamin ado-cobalamin methyl-cobalamin, decreased methionine synthase reduced S-adenosyl homocysteine ratio, as well increases methylmalonic acid concentration. Conclusions/Significance heterologous can used strategy investigating deficiency. More generally, oleosin-anchored could interesting tool engineering studying pharmacological interest.