Effect of the N-terminal sequence on the binding affinity of transthyretin for human retinol-binding protein.

During vertebrate evolution, the N-terminal region of transthyretin (TTR) subunit has undergone a change in both length and hydropathy. This was previously shown to change the binding affinity for thyroid hormones (THs). However, it was not known whether this change affects other functions of TTR. In the present study, the effect of these changes on the binding of TTR to retinol-binding protein (RBP) was determined. Two wild-type TTRs from human and Crocodylus porosus, and three chimeric TTRs, including a human chimeric TTR in which its N-terminal sequence was changed to that of C. porosus TTR (croc/huTTR) and two C. porosus chimeric TTRs (hu/crocTTR in which its N-terminal sequence was changed to that of human TTR and xeno/crocTTR in which its N-terminal sequence was changed to that of Xenopus laevis TTR), were analyzed for their binding to human RBP by native-PAGE followed by immunoblotting and a chemilluminescence assay. The K(d) of human TTR was 30.41 ± 2.03 μm, and was similar to that reported for the second binding site, whereas that of crocodile TTR was 2.19 ± 0.24 μm. The binding affinities increased in croc/huTTR (K(d) = 23.57 ± 3.54 μm) and xeno/crocTTR (K(d) = 0.61 ± 0.16 μm) in which their N-termini were longer and more hydrophobic, but decreased in hu/crocTTR (K(d) = 5.03 ± 0.68 μm) in which its N-terminal region was shorter and less hydrophobic. These results suggest an influence of the N-terminal primary structure of TTR on its function as a co-carrier for retinol with RBP.