title: Change in structure of the N-terminal region of transthyretin produces change in affinity of transthyretin to T4 and T3.
abstract: The relationship between the structure of the N-terminal sequence of transthyretin (TTR) and the binding of thyroid hormone was studied. A recombinant human TTR and two derivatives of Crocodylus porosus TTRs, one with the N-terminal sequence replaced by that of human TTR (human/crocTTR), the other with the N-terminal segment removed (truncated crocTTR), were synthesized in Pichia pastoris. Subunit mass, native molecular weight, tetramer formation, cross-reactivity to TTR antibodies and binding to retinol-binding protein of these recombinant TTRs were similar to TTRs found in nature. Analysis of the binding affinity to thyroid hormones of recombinant human TTR showed a dissociation constant (Kd) for triiodothyronine (T3) of 53.26+/-3.97 nM and for thyroxine (T4) of 19.73+/-0.13 nM. These values are similar to those found for TTR purified from human serum, and gave a Kd T3/T4 ratio of 2.70. The affinity for T4 of human/crocTTR (Kd=22.75+/-1.89 nM) was higher than those of both human TTR and C. porosus TTR, but the affinity for T3 (Kd=5.40+/-0.25 nM) was similar to C. porosus TTR, giving a Kd T3/T4 ratio of 0.24. A similar affinity for both T3 (Kd=57.78+/-5.65 nM) and T4 (Kd=59.72+/-3.38 nM), with a Kd T3/T4 ratio of 0.97, was observed for truncated crocTTR. The obtained results strongly confirm the hypothesis that the unstructured N-terminal region of TTR critically influences the specificity and affinity of thyroid hormone binding to TTR.
authors.affiliation: Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla, Thailand. porntip.p@psu.ac.th
authors.forename: Porntip
authors.initials: P
authors.lastname: Prapunpoj
authors.forename: Ladda
authors.initials: L
authors.lastname: Leelawatwatana
authors.forename: Gerhard
authors.initials: G
authors.lastname: Schreiber
authors.forename: Samantha J
authors.initials: SJ
authors.lastname: Richardson
mesh_terms: D000489:Alligators and Crocodiles; D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011228:Prealbumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D013974:Thyroxine / Q000378:metabolism*; D014284:Triiodothyronine / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D011228:Prealbumin; D011993:Recombinant Fusion Proteins; D014284:Triiodothyronine; D013974:Thyroxine