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.