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FEBS Open Bio
2017 Nov 16;712:1891-1898. doi: 10.1002/2211-5463.12329.
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Increasing the length and hydrophobicity of the C-terminal sequence of transthyretin strengthens its binding affinity to retinol binding protein.
Poodproh R
,
Kaewmeechai S
,
Leelawatwattana L
,
Prapunpoj P
.
???displayArticle.abstract??? 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 the TTR subunit 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 for THs and RBP. Here, the effects of changes in the C-terminal sequence were determined. Three chimeric TTRs, namely pigC/huTTR (human TTR with the C-terminal sequence changed to that of Sus scrofa TTR), xenoN/pigC/huTTR (human TTR with the N-terminal and C-terminal sequences changed to those of Xenopus laevis and S. scrofa, respectively), and pigC/crocTTR (Crocodylus porosus TTR with the C-terminal sequence changed to that of S. scrofa TTR), were constructed and their binding affinities for human RBP were determined at low TTR/RBP molar ratio using chemiluminescence immunoblotting. The binding dissociation constant (Kd) values of pigC/huTTR, xenoN/pigC/huTTR and pigC/crocTTR were 3.20 ± 0.35, 1.53 ± 0.38 and 0.31 ± 0.04 μm, respectively, and the Kd values of human and C. porosus TTR were 4.92 ± 0.68 and 1.42 ± 0.45 μm, respectively. These results demonstrate chimeric TTRs bound RBP with a higher strength than wild-type TTRs, and the changes in the C-terminal sequence of TTR had a positive effect on its binding affinity for RBP. In addition, changes to the N-terminal and C-terminal sequences showed comparable effects on the binding affinity.
Figure 1. The analysis of purified TTRs by native PAGE (A) and SDS/PAGE (B). Aliquots of purified human TTR (1), xenoN/pigC/huTTR (2), pigC/huTTR (3), Crocodylus porosus
TTR (4) and pigC/crocTTR (5) were loaded onto the gels. For SDS/PAGE, the protein sample was boiled in the presence of SDS and β‐mercaptoethanol at final concentrations of 2% and 2.5%, respectively, for 30 min. After analysis, gels were stained with Coomassie Brilliant Blue R‐250. HP, human plasma with an excess amount was included to locate the positions of albumin (ALB) and TTR; M, low molecular mass protein markers. Dimer and monomer positions of TTR are indicated.
Figure 2. Analysis of binding between TTR and human RBP by native PAGE followed by western analysis. Purified TTR (0.5 μm) was incubated with human RBP at various concentrations. Then, free and bound TTRs were separated by native PAGE in duplicate. One of the gels was stained with Coomassie Brilliant Blue R‐250, and another gel was subjected to western blot analysis using antibody specific to human TTR or Crocodylus porosus
TTR, and followed by ECL detection. Purified TTR (0.5 μm; TTR) and human RBP (4 μm; RBP) were included as controls. Free and bound TTRs are indicated by closed and opened arrowheads, respectively.
Figure 3. Scatchard plots of the specific binding with human RBP of human TTR (A), pigC/huTTR (B), xenoN/pigC/huTTR (C), C. porosus
TTR (crocTTR) (D), and pigC/crocTTR (E). Bound and free TTRs were determined by western blot analysis followed by ECL, and the intensities were used to calculate the K
d of the binding. The inset in each Scatchard plot shows a representative ECL blot of the studied TTR.
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