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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.
Ahmad,
Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production.
2014, Pubmed
Ahmad,
Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production.
2014,
Pubmed
Aldred,
Evolution of shorter and more hydrophilic transthyretin N-termini by stepwise conversion of exon 2 into intron 1 sequences (shifting the 3' splice site of intron 1).
1997,
Pubmed
Berni,
The bovine plasma retinol-binding protein. Amino acid sequence, interaction with transthyretin, crystallization and preliminary X-ray data.
1990,
Pubmed
Berry,
Transthyretin blocks retinol uptake and cell signaling by the holo-retinol-binding protein receptor STRA6.
2012,
Pubmed
Cereghino,
Heterologous protein expression in the methylotrophic yeast Pichia pastoris.
2000,
Pubmed
Chang,
Evolution of thyroid hormone binding by transthyretins in birds and mammals.
1999,
Pubmed
Folli,
The interaction between retinol-binding protein and transthyretin analyzed by fluorescence anisotropy.
2010,
Pubmed
Hamilton,
The x-ray crystal structure refinements of normal human transthyretin and the amyloidogenic Val-30-->Met variant to 1.7-A resolution.
1993,
Pubmed
INGBAR,
Pre-albumin: a thyroxinebinding protein of human plasma.
1958,
Pubmed
Kanai,
Retinol-binding protein: the transport protein for vitamin A in human plasma.
1968,
Pubmed
Kopelman,
The interaction between retinol-binding proteins and prealbumins studied by fluorescence polarization.
1976,
Pubmed
LOWRY,
Protein measurement with the Folin phenol reagent.
1951,
Pubmed
Leelawatwattana,
Effect of the N-terminal sequence on the binding affinity of transthyretin for human retinol-binding protein.
2011,
Pubmed
,
Xenbase
Malpeli,
Retinoid binding to retinol-binding protein and the interference with the interaction with transthyretin.
1996,
Pubmed
Manzon,
Molecular cloning, tissue distribution, and developmental expression of lamprey transthyretins.
2007,
Pubmed
Mita,
Cloning and sequence analysis of cDNA for human prealbumin.
1984,
Pubmed
Monaco,
Three-dimensional structure of the transthyretin-retinol-binding protein complex.
2002,
Pubmed
Monaco,
The transthyretin-retinol-binding protein complex.
2000,
Pubmed
Morgado,
Hormone affinity and fibril formation of piscine transthyretin: the role of the N-terminal.
2008,
Pubmed
Naylor,
The structure of human retinol-binding protein (RBP) with its carrier protein transthyretin reveals an interaction with the carboxy terminus of RBP.
1999,
Pubmed
Noy,
Interactions of retinol with binding proteins: studies with retinol-binding protein and with transthyretin.
1992,
Pubmed
OUCHTERLONY,
Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diffusion.
1953,
Pubmed
Prapunpoj,
The evolution of the thyroid hormone distributor protein transthyretin in the order insectivora, class mammalia.
2000,
Pubmed
Prapunpoj,
Crocodile transthyretin: structure, function, and evolution.
2002,
Pubmed
,
Xenbase
Prapunpoj,
Evolutionary changes to transthyretin: structure-function relationships.
2009,
Pubmed
Prapunpoj,
Change in structure of the N-terminal region of transthyretin produces change in affinity of transthyretin to T4 and T3.
2006,
Pubmed
Prapunpoj,
Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin.
2000,
Pubmed
,
Xenbase
Richardson,
Tweaking the structure to radically change the function: the evolution of transthyretin from 5-hydroxyisourate hydrolase to triiodothyronine distributor to thyroxine distributor.
2014,
Pubmed
Robbins,
Thyroxine transport proteins of plasma. Molecular properties and biosynthesis.
1978,
Pubmed
Rostom,
Dissection of multi-protein complexes using mass spectrometry: subunit interactions in transthyretin and retinol-binding protein complexes.
1998,
Pubmed
Tsuzuki,
Structure of the human prealbumin gene.
1985,
Pubmed
Yamauchi,
Purification and characterization of a 3,5,3'-L-triiodothyronine-specific binding protein from bullfrog tadpole plasma: a homolog of mammalian transthyretin.
1993,
Pubmed
Zanotti,
Plasma retinol-binding protein: structure and interactions with retinol, retinoids, and transthyretin.
2004,
Pubmed
Zanotti,
Structural and mutational analyses of protein-protein interactions between transthyretin and retinol-binding protein.
2008,
Pubmed