Anchoring secreted proteins in endoplasmic reticulum by plant oleosin: the example of vitamin B12 cellular sequestration by transcobalamin

Abstract

Background: 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.

Methodology: 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.

Principal findings: 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.

Figure 1

(A) Experimental model of vitamin B12 intracellular sequestration by the transcobalamin-oleosin chimeric protein. (B) The schematics of the transcobalamin-oleosin (TC-oleosin) and oleosin-transcobalamin (oleosin-TC) cDNA constructs in the plasmid pAcSG2.

Figure 2

(A) Characterization of the recombinant oleosin (top) and transcobalamin-oleosin (bottom) chimeric proteins; SDS-PAGE (12.5%) (lanes 1–3, 10) and Western blot (WB) (lanes 4–9) of subcellular fractions (pellets and supernatant from differential centrifugation) from Sf9 cells infected with recombinant baculovirus expressing either peanut oleosin or the transcobalamin-oleosin chimeric protein. Lanes 1, 4, 7: 900 g pellet, lanes 2, 5, 8: 100 000 g supernatant, lanes 3, 6, 9: 100 000 g pellet, lane 10: 85 ng purified peanut oleosin and lane 11: autoradiography of [¹⁴C]-leucine-labeled oleosin expressed in rabbit reticulocyte lysate. (B) Saturation curve of vitamin B12 binding of transcobalamin-oleosin (TC-O) in membrane fraction of lysed sf9 insect cells and corresponding Scatchard plot (inlet).

Figure 3

(A) Vitamin B12 binding capacity (cyano[⁵⁷Co]Cbl) of membrane fraction (100 µg proteins per assay) from Caco2 cells, 72 hrs after transient transfection with various recombinant pcDNA3 plasmids expressing transcobalamin (TC), oleosin (O) and the chimeric proteins transcobalamin-oleosin (TC-O) and oleosin-transcobalamin (O-TC). (B) Vitamin B12 ([57Co]-labeled) binding capacity of Caco2 cells of the intact and lyzed (Caco2 cells were lysed prior to the addition of vitamin B12 or used intact), at days 5–15 of culture, after stable transfection with a recombinant pcDNA3 plasmid expressing the chimeric protein transcobalamin-oleosin. Exponential and stationary phases were evaluated at days 5–7 and days 10–15, respectively. Bars from left to right for each time: non-transfected intact cells, non-transfected lysed cells, TC-O transfected intact cells, TC-O transfected lysed cells.

Figure 4

(A) Confocal microscopic examination of the localization of the green fluorescent protein-transcobalamin-oleosin (GFP-TC-O) chimeric protein 24 hrs after transient transfection of Caco2, human embryonic kidney cells (HEK293), Chinese hamster ovary (CHO) and COS-7 cells. In Caco2 cells, GFP-TC-O co-localization was detected with an ER-targeted red fluorescent protein (pDsRED2-ER, Clontech USA) (merged). (B) Left: Transmission electron microscope examination of the chimera detected immunologically by a Gold conjugate (arrows) (left). Calibration bar = 0.1 µm. Abbreviations: mb, membrane, ER, endoplasmic reticulum. Middle: confocal image of immunolabeling of megalin in apical surface of O-TC caco-2 cells. Right: relative fluorescence intensity (low, red; high: blue) of megalin on apical area. Calibration bar = 18 µm. (C) Left and middle: Confocal image of immunolabeling of megalin (Alexa488 coded in green, left channel) and calnexine (Alexa555 coded in cyan, right channel). Right: Merged. Calibration bar = 18 µm.

Figure 5

(A) Confocal microscopic examination of transcobalamin in COS-7 cells transiently transfected with lipofectamine. The cells were transfected with one of the following pCDNA3-plasmids: TC-O coding for transcobalamin-oleosin (TC-O), O-TC coding for oleosin-transcobalamin (O-TC), TC coding for transcobalamin (TC), O coding for oleosin (O), and the empty plasmid (pCDNA3). The immuno-fluorescence was done with a goat polyclonal antibody to TC and a donkey antigoat IgG fluorescin labeled. Cell nuclei were counterstained with Hoechst 33258. Calibration bars = 20 µm. (B and C) Co-localization of the protein GFP-TC-O with endoplasmic reticulum in transient transfected Cos-7 cells using lipofectamine. The cells were transfected with the plasmid GFP-TC-O coding for GFP-transcobalamin-oleosin (GFP-TC-O). The immuno-fluorescence was done with a mouse monoclonal antibody to the human golgin-97 or a rabbit polyclonal antibody to calreticulin. The secondary antibodies were a donkey IgG anti-mouse TRITC labeled or a donkey IgG anti-rabbit TRITC labeled. Cell nuclei were counterstained with Hoechst 33258. Calibration bars = 20 µm.

Figure 6

(A) Intracellular conversion of exogenous [⁵⁷Co]-labeled B12 (cyano-cobalamin, CN-Cbl) into methyl-cobalamin (Me-Cbl) and ado-cobalamin (Ado-Cbl) in the cytosolic and mitochondria enriched fractions. (B) Homocysteine (Hcy) and methylmalonic acid (MMA) concentrations. (C) S-adenosylmethionine/S-Adenosylhomocysteine ratio (SAM/SAH) in TO and wild type NIE-115 cells. (D) Activity of methionine synthase in TO transfected and wild type NIE-115 and Caco2 cells in exponential growth. Mean±S.D. of sextuplet assays are given. ****: p<0.0001, **: p<0.001, *: p<0.001.