Characterization of little skate (Leucoraja erinacea) recombinant transthyretin: Zinc-dependent 3,3',5-triiodo-l-thyronine binding

Gen Comp Endocrinol. Jun-Jul 2015;217-218:43-53. doi: 10.1016/j.ygcen.2015.04.006. Epub 2015 Apr 9.


Transthyretin (TTR) diverged from an ancestral 5-hydroxyisourate hydrolase (HIUHase) by gene duplication at some early stage of chordate evolution. To clarify how TTR had participated in the thyroid system as an extracellular thyroid hormone (TH) binding protein, TH binding properties of recombinant little skate Leucoraja erinacea TTR was investigated. At the amino acid level, skate TTR showed 37-46% identities with the other vertebrate TTRs. Because the skate TTR had a unique histidine-rich segment in the N-terminal region, it could be purified by Ni-affinity chromatography. The skate TTR was a 46-kDa homotetramer of 14.5kDa subunits, and had one order of magnitude higher affinity for 3,3',5-triiodo-l-thyronine (T3) and some halogenated phenols than for l-thyroxine. However, the skate TTR had no HIUHase activity. Ethylenediaminetetraacetic acid (EDTA) treatment inhibited [(125)I]T3 binding activity whereas the addition of Zn(2+) to the EDTA-treated TTR recovered [(125)I]T3 binding activity in a Zn(2+) concentration-dependent manner. Scatchard analysis revealed the presence of two classes of binding site for T3, with dissociation constants of 0.24 and 17nM. However, the high-affinity sites were completely abolished with 1mM EDTA, whereas the remaining low-affinity sites decreased binding capacity. The number of zinc per TTR was quantified to be 4.5-6.3. Our results suggest that skate TTR has tight Zn(2+)-binding sites, which are essential for T3 binding to at least the high-affinity sites. Zn(2+) binding to the N-terminal histidine-rich segment may play an important role in acquisition or reinforcement of TH binding ability during early evolution of TTR.

Keywords: 5-Hydroxyisourate hydrolase; Divalent cation; Endocrine disruption; Leucoraja erinacea; Thyroid hormone; Transthyretin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidohydrolases / metabolism
  • Animals
  • Binding Sites
  • Carrier Proteins / metabolism
  • Membrane Proteins / metabolism
  • Prealbumin / metabolism*
  • Recombinant Proteins / metabolism*
  • Skates, Fish / physiology*
  • Thyroid Gland / cytology
  • Thyroid Gland / metabolism*
  • Thyroid Hormones / metabolism
  • Thyroxine / metabolism*
  • Triiodothyronine / metabolism*
  • Uric Acid / analogs & derivatives
  • Uric Acid / metabolism
  • Zinc / metabolism*


  • 5-hydroxyisourate
  • Carrier Proteins
  • Membrane Proteins
  • Prealbumin
  • Recombinant Proteins
  • Thyroid Hormones
  • thyroid hormone-binding proteins
  • Triiodothyronine
  • Uric Acid
  • Amidohydrolases
  • hydroxyisourate hydrolase
  • Zinc
  • Thyroxine