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

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

Methyl donor deficiency affects fetal programming of gastric ghrelin cell organization and function in the rat

Methyl donor deficiency (MDD) during pregnancy influences intrauterine development. Ghrelin is expressed in the stomach of fetuses and influences fetal growth, but MDD influence on gastric ghrelin is unknown. We examined the gastric ghrelin system in MDD-induced intrauterine growth retardation. By using specific markers and approaches (such as periodic acid-Schiff, bromodeoxyuridine, homocysteine, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunostaining, reverse transcription-polymerase chain reaction), we studied the gastric oxyntic mucosa cellular organization and ghrelin gene expression in the mucosa in 20-day-old fetuses and weanling pups, and plasma ghrelin concentration in weanling rat pups of dams either normally fed or deprived of choline, folate, vitamin B6, and vitamin B12 during gestation and suckling periods. MDD fetuses weighed less than controls; the weight deficit reached 57% at weaning (P < 0.001). Both at the end of gestation and at weaning, they presented with an aberrant gastric oxyntic mucosa formation with loss of cell polarity, anarchic cell migration, abnormal progenitor differentiation, apoptosis, and signs of surface layer erosion. Ghrelin cells were abnormally located in the pit region of oxyntic glands. At weaning, plasma ghrelin levels were decreased (-28%; P < 0.001) despite unchanged mRNA expression in the stomach. This decrease was associated with lower body weight. Taken together, these data indicate that one mechanism through which MDD influences fetal programming is the remodeling of gastric cellular organization, leading to dysfunction of the ghrelin system and dramatic effects on growth

Homocysteinylation of neuronal proteins contributes to folate deficiency-associated alterations of differentiation, vesicular transport, and plasticity in hippocampal neuronal cells.

International audienceDespite the key role in neuronal development of a deficit in the methyl donor folate, little is known on the underlying mechanisms. We therefore studied the consequences of folate deficiency on proliferation, differentiation, and plasticity of the rat H19-7 hippocampal cell line. Folate deficit reduced proliferation (17%) and sensitized cells to differentiation-associated apoptosis (+16%). Decreased production (-58%) of S-adenosylmethionine (the universal substrate for transmethylation reactions) and increased expression of histone deacetylases (HDAC4,6,7) would lead to epigenomic changes that may impair the differentiation process. Cell polarity, vesicular transport, and synaptic plasticity were dramatically affected, with poor neurite outgrowth (-57%). Cell treatment by an HDAC inhibitor (SAHA) led to a noticeable improvement of cell polarity and morphology, with longer processes. Increased homocysteine levels (+55%) consecutive to folate shortage produced homocysteinylation, evidenced by coimmunoprecipitations and mass spectrometry, and aggregation of motor proteins dynein and kinesin, along with functional alterations, as reflected by reduced interactions with partner proteins. Prominent homocysteinylation of key neuronal proteins and subsequent aggregation certainly constitute major adverse effects of folate deficiency, affecting normal development with possible long-lasting consequences

Figure 5

<p><b>(A)</b> 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>(B and C)</b> 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.</p

Figure 3

<p><b>(A)</b> 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>(B)</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.</p

Figure 6

<p><b>(A) Intracellular conversion of exogenous [⁵⁷Co]-labeled B12 (cyano-cobalamin, CN-Cbl)</b> into methyl-cobalamin (Me-Cbl) and ado-cobalamin (Ado-Cbl) in the cytosolic and mitochondria enriched fractions. <b>(B)</b> Homocysteine (Hcy) and methylmalonic acid (MMA) concentrations. <b>(C)</b> S-adenosylmethionine/S-Adenosylhomocysteine ratio (SAM/SAH) in TO and wild type NIE-115 cells. <b>(D)</b> 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.</p

Figure 2

<p><b>(A)</b> 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 <i>Sf</i>9 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>(B)</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).</p