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

Abstract

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 lM, and was similar to that reported for the second binding site, whereas that of crocodile TTR was 2.19 ± 0.24 lM. The binding affinities increased in croc ⁄ huTTR (K d = 23.57 ± 3.54 lM) and xeno ⁄ crocTTR (K d = 0.61 ± 0.16 lM) in which their N-termini were longer and more hydrophobic, but decreased in hu ⁄ crocTTR (K d = 5.03 ± 0.68 lM) 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.

Proteolytic activity of Crocodylus porosus transthyretin protease and role of the terminal polypeptide sequences

“…In addition, variations in the length of the C-terminal region of TTRs from pig 3 and microbats 4,5 compared with that from human have also been detected. The changes in length and hydrophobicity were demonstrated to affect the binding affinities of TTR for THs and retinol binding protein (RBP) 6,7 . Recently, the proteolytic activity of TTR was discovered and its participation in the biology of the nervous system and high density lipoprotein (HDL) was demonstrated 8,9 .…”

“…The sizes of the TTR subunits were obtained by comparing their relative mobilities with protein markers: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soya bean trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa). Crossed-reactivity of the recombinant TTRs was determined by western www.scienceasia.org blotting, followed by enhanced chemiluminescence (ECL) detection as previously described 6,7 . The primary and secondary antibodies used were anticrocTTR antibody raised in rabbit (dilution 1:2500) and horseradish peroxidase-conjugated anti-rabbit IgG antibody (dilution 1:10 000), respectively.…”

“…The induction of Pichia clones to produce all the studied recombinant and chimeric TTRs was performed as described previously 6,7 . In brief, the Pichia clones of crocTTR 17 , xenoN/crocTTR 17 and the two new chimeric crocTTRs were induced with 0.5% methanol for 3 days at 28°C in buffered complex glycerol or methanol medium (BMGY/BMMY).…”

“…The nucleotide sequences of the cDNAs were confirmed by sequencing before they were ligated into a Pichia expression vector, pPIC9. The recombinant vectors were transformed into P. pastoris and the transformant clones were selected as described previously 6,7 .…”

“…In addition, changes in the length and hydropathy of the terminal regions of TTR polypeptide have been clearly demonstrated to affect the properties that are related to the functions of TTR, i.e., the affinities of the bindings to THs and RBP 7 . The question arose whether the N-and C-terminal sequences affect the proteolytic activity of C. porosus TTR.…”