Glycosylation is essential for translocation of carp retinol-binding protein across the endoplasmic reticulum membrane.
Retinoid transport is well characterized in many vertebrates, while it is still largely unexplored in fish. To study the transport and utilization of vitamin A in these organisms, we have isolated from a carp liver cDNA library retinol-binding protein, its plasma carrier. The primary structure of carp retinol-binding protein is very conserved, but presents unique features compared to those of the correspondent proteins isolated and characterized so far in other species: it has an uncleavable signal peptide and two N-glycosylation sites in the NH(2)-terminal region of the protein that are glycosylated in vivo. In this paper, we have investigated the function of the carbohydrate chains, by constructing three mutants deprived of the first, the second or both carbohydrates. The results of transient transfection of wild type and mutant retinol-binding protein in Cos cells followed by Western blotting and immunofluorescence analysis have shown that the absence of both carbohydrate moieties blocks secretion, while the presence of one carbohydrate group leads to an inefficient secretion. Experiments of carp RBP mRNA in vitro translation in a reticulocyte cell-free system in the presence of microsomes have demonstrated that N-glycosylation is necessary for efficient translocation across the endoplasmic reticulum membranes. Moreover, when Cos cells were transiently transfected with wild type and mutant retinol-binding protein (aa 1-67)-green fluorescent protein fusion constructs and semi-permeabilized with streptolysin O, immunofluorescence analysis with anti-green fluorescent protein antibody revealed that the double mutant is exposed to the cytosol, thus confirming the importance of glycan moieties in the translocation process.
Devirgiliis
Chiara
C
National Research Institute on Food and Nutrition, Via Ardeatina 546, 00178 Roma, Italy.
Gaetani
Sancia
S
Apreda
Marianna
M
Bellovino
Diana
D
D000818:Animals; D019556:COS Cells; D002347:Carps / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D012177:Retinol-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D054839:Retinol-Binding Proteins, Plasma
D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins; D054839:Retinol-Binding Proteins, Plasma