Beneficial or detrimental effects of carotenoids contained in food: cell culture models.

Epidemiological studies have suggested a correlation between consumption of carotenoid-rich food and incidence of chronic diseases. In this review chemical structure, bioavailability and mechanisms of action of carotenoids most represented in human diet, mainly beta-carotene and lycopene, are reported, with focus on results obtained with cells in culture.

Copper regulated synthesis, secretion and degradation of ceruloplasmin in a mouse immortalized hepatocytic cell line.

Ceruloplasmin, a blue copper oxidase circulating in serum of all vertebrates, is a glycoprotein synthesized mainly in hepatocytes and secreted into plasma with six tightly bound atoms of copper per molecule. Many aspects of the mechanisms by which synthesis and secretion of this protein are regulated by copper are still not known. In HepG2 hepatocarcinoma cells this fine regulation is not maintained; we have then utilized Met-murine-hepatocytes (MMH), isolated from the liver of transgenic mice expressing a truncated form of c-Met (hepatocyte growth factor receptor), that are immortalized but not transformed. Copper deficiency was induced by treatment of cells with bathocuproine disulphonate. Experiments of metabolic labeling with 35S-methionine-cysteine and of Western blotting followed by immunostaining, demonstrated that maturation and secretion of ceruloplasmin but not its synthesis are affected by copper availability. In this paper we have shown that in copper deficiency ceruloplasmin accumulates in a pre-Golgi compartment, in which the protein is still in a Endo H sensitive form, and where presumably copper binding to the apo-protein takes place. Moreover, we found that treatment of copper-deficient cells with the proteasomal inhibitor lactacycstin leads to immature ceruloplasmin accumulation in the cell. We have optimized conditions to induce in vitro copper deficiency and found that MMH-D3 cells represent a suitable model to study in detail the molecular mechanism of copper-regulated ceruloplasmin synthesis, secretion and degradation.

Vitamin A transport: in vitro models for the study of RBP secretion.

Retinol-binding protein (RBP) is the specific plasma carrier of retinol, encharged of the vitamin transport from the liver to target cells. Ligand binding influences the RBP affinity for transthyretin (TTR), a homotetrameric protein involved in the RBP/TTR circulating complex, and the secretion rate of RBP. In fact, in vitamin A deficiency, the RBP release from the hepatocytes dramatically decreases and the protein accumulates in the cells, until retinol is available again. The mechanism is still not clear and new cellular models are needed to understand in detail how the soluble RBP can be retained inside the cell. In fish, a vitamin A transport system similar to that of higher vertebrates is emerging, although with significant differences.

Isolation, expression and characterization of carp retinol-binding protein.

Vitamin A alcohol and its precursors carotenoids are introduced in the organism with the diet, transported to the liver and from there as retinol to target tissues by a specific carrier, the retinol-binding protein (RBP). RBP, isolated and characterized in many vertebrates, shows very high homology among the species investigated; however, very little is known in fish. In the present work RBP cDNA isolated from a carp liver library was transcribed and translated in vitro and the corresponding protein characterized. Carp RBP amino acid sequence and tertiary structure are highly conserved, but the protein shows two peculiar and unique characteristics: the signal sequence is not processed by the ER signal peptidase and two N-glycosylations are present at the N-terminus portion of the protein. It was also demonstrated that RBP glycosylation is not a feature common to all teleosts. Transfection experiments show that the green fluorescent protein (GFP) can be directed into the secretory pathway by the carp RBP N-terminal region, both in fish and in mammal cells, demonstrating that the sequence, although not processed, is recognized as a secretory signal in different species. Results obtained from different investigators indicated that in fish plasma RBP circulates without interacting with transthyretin (TTR) or other proteins, suggesting that the complex with TTR, whose postulated function is to hamper easy kidney filtration of circulating RBP, has evolved later in the evolutionary scale. This hypothesis is reinforced by the finding that carp RBP, as well as trout and other lower vertebrates in which circulating complex has never been demonstrated, lacks a short C-terminal sequence that seems to be involved in RBP-TTR interaction. In carp, carbohydrates could be involved in the control of protein filtration through the kidney glomeruli. Moreover, experiments of carp RBP expression in Cos-1 cells and in the yeast Saccharomyces cerevisiae show that glycosylation is necessary for protein secretion; in particular, additional in vitro experiments have shown it is involved in protein translocation through ER membranes.

Unique biochemical nature of carp retinol-binding protein. N-linked glycosylation and uncleavable NH2-terminal signal peptide.

Retinol transport and metabolism have been well characterized in mammals; however, very little is known in fish. To study the mechanism by which fish retinol-binding protein (RBP) is able to remain in plasma besides its small molecular size, we isolated RBP cDNA from a carp liver cDNA library. Comparison of the deduced amino acid sequence with that of known vertebrate RBPs showed that carp RBP has high homology to the other cloned vertebrate RBPs, but it lacks the COOH-terminal tetrapeptide, RNL(S)L, which is most likely involved in the interaction with transthyretin in mammalian RBPs. In addition, the primary structure of carp RBP contains two consensus N-linked glycosylation sites that represent a unique feature. We have obtained experimental evidence, by in vitro and in vivo expression experiments, that both sites are indeed glycosylated. We have also characterized the protein as a complex type N-linked glycoprotein by lectin binding assay, neuraminidase and endoglycosidase H and F digestion. Inhibition of glycosylation by tunicamycin treatment of transfected cells caused a great reduction of RBP secretion. Since kidney filtration of anionic proteins is less than half that of neutral protein of the same size, this finding strongly suggests that the amount of carp RBP filtration through kidney glomeruli may be reduced by a glycosylation-dependent increase in the molecular size and negative charge of the protein. A second unique feature of carp RBP as secretory protein is the presence of a nonconserved NH(2)-terminal hydrophobic domain, which functions as an insertion signal but is not cleaved cotranslationally and remains in the secreted RBP.

MMH cells: An in vitro model for the study of retinol-binding protein secretion regulated by retinol.

The untransformed stable cell line Met murine hepatocytes (MMH), generated from liver explants of transgenic mice expressing a constitutively active truncated form of the human hepatocyte growth factor receptor (cyto-Met), represents an innovative tool for in vitro studies of liver function. In the present report, we show that the MMH-D3 line isolated from the liver of a 3-day-old mouse is a useful model to investigate the regulation of the synthesis and secretion of retinol-binding protein (RBP) by retinol (vitamin A alcohol). Experiments with Northern blot hybridization, metabolic labeling of cellular proteins followed by immunoprecipitation, and Western blot analysis demonstrated that, similarly to the in vivo situation, in MMH-D3 cells the presence of retinol does not affect transcriptional and translational rate of the RBP gene but is essential for regulating the secretion rate of the protein. Unlike HepG2 human hepatocarcinoma cells used thus far in studies of retinoid metabolism, including the synthesis and secretion of RBP, vitamin A deficiency causes, in MMH-D3 cells, the inhibiton of RBP secretion and the protein accumulation in the cell, whereas retinol repletion promptly results in RBP secretion. This model will be very useful in future studies on vitamin A distribution in the organism.

In vitro and in vivo studies on transthyretin oligomerization.

The oligomerization of transthyretin has been studied in vitro and in vivo. The results showed that wild-type transthyretin synthesized in vitro in the absence of microsomes did not form dimers and tetramers, but in the presence of microsomes the mature transthyretin which had been translocated into the microsomal lumen formed dimers and a small amount of tetramers which could be detected only by using a cross-linking reagent. Efficiency of tetramer formation depends upon the source of microsomes; in fact the amount of tetramer formed in liver microsomes was much higher than that in pancreas microsomes. Transthyretin synthesized in HepG2 cells appeared after SDS-PAGE analysis in mostly tetrameric form, while that synthesized in transfected COS-1 cells appeared mainly as dimers. Brefeldin A treatment and pulse-chase experiments in HepG2 cells showed that transthyretin tetramer was formed in the endoplasmic reticulum. These results strongly indicate that transthyretin tetramer is formed most efficiently in the endoplasmic reticulum lumen of hepatocytes. Transthyretin without the signal peptide [S(-)] and transthyretin with a mutation that prevents processing of the signal peptide Msc failed to form dimers and tetramers in vitro. In the transfected COS-1 cells, however, S(-) transthyretin did form dimers while Msc transthyretin failed to oligomerize. These results show that the cleavage of the signal peptide and some cellular factors are required for transthyretin oligomerization.

Only the first and the last hydrophobic segments in the COOH-terminal third of Na,K-ATPase alpha subunit initiate and halt, respectively, membrane translocation of the newly synthesized polypeptide. Implications for the membrane topology.

We studied the topogenic properties of five hydrophobic segments (H5-H9) in the COOH-terminal third of Na,K-ATPase alpha subunit using in vitro insertion of fusion proteins into endoplasmic reticulum membranes. These fusion proteins consisted of several different lengths of truncated alpha subunit starting at Met729 and a reporter protein, chloramphenicol acetyltransferase, that was linked in frame after each hydrophobic segment. We found that membrane insertion of the newly synthesized COOH-terminal third was initiated by H5 and terminated by H9, indicating that here only H5 and H9 have topogenic function. The other three, H6-H8, did not have topogenic function in the native context and were translocated into the endoplasmic reticulum lumen. These results were in striking contrast to the previous models in which four or six hydrophobic segments were proposed to cross the membrane. Furthermore, the findings suggest a novel mechanism for achieving the final membrane topology of the COOH-terminal third of the alpha subunit.

Retinol binding protein and transthyretin are secreted as a complex formed in the endoplasmic reticulum in HepG2 human hepatocarcinoma cells.

Retinol binding protein (RBP), the retinol-specific carrier, circulates in blood as a 1:1 complex with the homotetrameric protein transthyretin (TTR). Both RBP and TTR are synthesized and secreted by the hepatocyte. In this work we have demonstrated, using HepG2 cells as a model system, that the association between the two proteins occurs inside the cell before secretion. The intracellular complex was detected only when metabolically labeled cells were lysed under mild detergent conditions (1.5% octylglucoside), followed by immunoprecipitation and SDS-PAGE. Alternatively, the immunoprecipitates from unlabeled cells lysed with the same buffer were analyzed by Western blotting. This finding was confirmed using the cross-linking agent dithiobis(succinimidyl) propionate before cell lysis. Moreover, we found that in cells treated with brefeldin A to block the exit of proteins from the endoplasmic reticulum (ER), the complex was present in the microsomal fraction. Thus, we can conclude that the RBP-TTR complex is formed inside the cell, more precisely within the ER. As RBP and TTR both lack an ER retention signal, we considered the possible involvement of chaperones in RBP and TTR retention in the ER and in complex formation. We found that calnexin, an ER integral membrane protein which functions as a chaperone, coprecipitates with RBP and TTR when cell lysis and immunoprecipitation are performed under mild conditions (1% Triton X-100). This result strongly suggests that calnexin may be involved in RBP and TTR retention in the ER, in TTR tetramer assembly, and possibly in complex formation.