Increasing the length and hydrophobicity of the C‐terminal sequence of transthyretin strengthens its binding affinity to retinol binding protein

Transthyretin (TTR) is a transporter for thyroid hormone (TH) and retinol, the latter via binding with retinol binding protein (RBP). Both the N‐terminal and C‐terminal regions of theTTRsubunit are located in close proximity to the central binding channel for ligands. During the evolution of vertebrates, these regions changed in length and hydropathy. The changes in the N‐terminal sequence were demonstrated to affect the binding affinities forTHs andRBP. Here, the effects of changes in the C‐terminal sequence were determined. Three chimericTTRs, namely pigC/huTTR(humanTTRwith the C‐terminal sequence changed to that ofSus scrofaTTR), xenoN/pigC/huTTR(humanTTRwith the N‐terminal and C‐terminal sequences changed to those ofXenopus laevisandS. scrofa, respectively), and pigC/crocTTR(Crocodylus porosusTTRwith the C‐terminal sequence changed to that ofS. scrofaTTR), were constructed and their binding affinities for humanRBPwere determined at lowTTR/RBPmolar ratio using chemiluminescence immunoblotting. The binding dissociation constant (Kd) values of pigC/huTTR, xenoN/pigC/huTTRand pigC/crocTTRwere 3.20 ± 0.35, 1.53 ± 0.38 and 0.31 ± 0.04 μm, respectively, and theKdvalues of human andC. porosusTTRwere 4.92 ± 0.68 and 1.42 ± 0.45 μm, respectively. These results demonstrate chimericTTRs boundRBPwith a higher strength than wild‐typeTTRs, and the changes in the C‐terminal sequence ofTTRhad a positive effect on its binding affinity forRBP. In addition, changes to the N‐terminal and C‐terminal sequences showed comparable effects on the binding affinity.