Recombinant Transthyretin Purification and Competitive Binding with Organohalogen Compounds in Two Gull Species (Larus argentatus and Larus hyperboreus)

• Published in: Toxicological Sciences
• Main Authors: Ucán-Marín, Francisco, Arukwe, Augustine, Mortensen, Anne, Gabrielsen, Geir W., Fox, Glen A., Letcher, Robert J.
• Format: Text
• Language: English
• Published: Oxford University Press 2009
• Subjects: IN VITRO TOXICOLOGY AND ALTERNATIVE TESTING; Norway; Svalbard; Glaucous Gull; Larus hyperboreus
• Online Access: http://toxsci.oxfordjournals.org/cgi/content/short/107/2/440; https://doi.org/10.1093/toxsci/kfn240

Glaucous gulls ( Larus hyperboreus ) from Svalbard, Norway (marine), and herring gulls ( Larus argentatus ) from the Laurentian Great Lakes (freshwater) of North America are differentially exposed to persistent and bioaccumulative anthropogenic contaminants, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ether (PBDE) flame retardants and metabolic products. Such compounds can potentially perturb hormone transport via binding interactions with proteins such as transthyretin (TTR, prealbumin). In this present study, we isolated, cloned and sequenced TTR cDNA from the brain and liver of two species (herring and glaucous gull), which, to our knowledge, is the first report describing the TTR nucleic acid and amino acid sequences from any gull species. Identical TTR nucleotide and amino acid sequences were obtained from both gull species (liver and brain). Recombinant TTR (rTTR) was expressed and purified, and determined as a monomer of 18 kDa and homodimer of 36 kDa that putatively is comprised of the two protein monomers. Concentration dependent, competitive TTR-binding curves with each of the natural TTR ligands 3,5,3′-triiodothyronine (T 3 ) and thyroxine (T 4 ) were generated as well as by treatment with a range of concentrations (10−3–105nM) of 2,2′,3,4′,5,5′,6-heptaCB (CB187), 2,2′,4,4′-tetrabromoDE (BDE47), and hydroxyl- (OH) and methoxyl (MeO)-containing analogs (i.e., 4-OH-CB187, 6-OH-BDE47, 4′-OH-BDE49, 4-MeO-CB187, and 6-MeO-BDE47). Relative to the nonsubstituted BDE47 and CB187 and their MeO-substituted analogs, the OH-substituted analogs all had lower K i and K d values, indicating greater affinity and more potent competitive binding to both T 3 and T 4 . The OH-substitution position and/or the diphenyl ether substitution of the four bromine atoms resulted in more potent, greater affinity, and greater relative potency for 4′-OH-BDE49 relative to 6-OH-BDE47. CB187 was more comparable in binding potency and affinity to 4-OH-CB187, then was 6-OH-BDE47 and 4′-OH-BDE49 relative to BDE47 ...