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; h owever, 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 REP cDNA from a carp liver cDNA library. Compa rison of the deduced amino acid sequence with that of known vertebrate RBPs showed that carp REP has high homology to the other cloned vertebrate RBPs , but it lacks the COOH-terminal tetrapeptide, RNL(S)L, which is most likel y involved in the interaction with transthyretin in mammalian RBPs, In addi tion, the primary structure of carp REP contains two consensus N-linked gly cosylation sites that represent a unique feature. We have obtained experime ntal evidence, by in vitro and in vivo expression experiments, that both si tes are indeed glycosylated, We have also characterized the protein as a co mplex type N-linked glycoprotein by lectin binding assay, neuraminidase and endoglycosidase H and F digestion. Inhibition of glycosylation by tunicamy cin treatment of transfected cells caused a great reduction of REP secretio n. Since kidney filtration of anionic proteins is less than half that of ne utral protein of the same size, this finding strongly suggests that the amo unt of carp REP filtration through kidney glomeruli may be reduced by a gly cosylation-dependent increase in the molecular size and negative charge of the protein. A second unique feature of carp REP as secretory protein is th e presence of a nonconserved NH2-terminal hydrophobic domain, which functio ns as an insertion signal but is not cleaved cotranslationally and remains in the secreted RBP.