Hormone affinity and fibril formation of piscine transthyretin: the role of the N-terminal.
Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd=10.6+/-1.7nM) and T4 (Kd=9.8+/-0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3+/-15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.
Morgado
Isabel
I
CCMAR, CIMAR, Laboratório Associado, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal. morgado@enzyme-halle.mpg.de
Melo
Eduardo P
EP
Lundberg
Erik
E
Estrela
Nídia L
NL
Sauer-Eriksson
A Elisabeth
AE
Power
Deborah M
DM
D000682:Amyloid / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004586:Electrophoresis; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D011228:Prealbumin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D021541:Sea Bream; D013974:Thyroxine / Q000378:metabolism*; D014284:Triiodothyronine / Q000378:metabolism*
D000682:Amyloid; D029941:Fish Proteins; D011228:Prealbumin; D011994:Recombinant Proteins; D014284:Triiodothyronine; D013974:Thyroxine