PUBLICATION

In vitro characterization of human, mouse and zebrafish MCT8 orthologues

Authors
Groeneweg, S., Kersseboom, S., van den Berge, A., Dolcetta-Capuzzo, A., van Geest, F.S., van Heerebeek, R.E.A., Arjona, F.J., Meima, M., Peeters, R., Visser, W.E., Visser, T.J.
ID
ZDB-PUB-190823-8
Date
2019
Source
Thyroid : official journal of the American Thyroid Association   29(10): 1499-1510 (Journal)
Registered Authors
Arjona, F.J.
Keywords
none
MeSH Terms
  • Animals
  • COS Cells
  • Cell Line, Tumor
  • Chlorocebus aethiops
  • Diiodothyronines/metabolism
  • Disease Models, Animal
  • Gene Knockdown Techniques
  • Humans
  • Immunoblotting
  • Immunohistochemistry
  • In Vitro Techniques
  • Mental Retardation, X-Linked/genetics
  • Mental Retardation, X-Linked/metabolism
  • Mice
  • Mice, Knockout
  • Models, Molecular
  • Monocarboxylic Acid Transporters/metabolism*
  • Muscle Hypotonia/genetics
  • Muscle Hypotonia/metabolism
  • Muscular Atrophy/genetics
  • Muscular Atrophy/metabolism
  • Sequence Alignment
  • Symporters/metabolism*
  • Thyroxine/metabolism
  • Triiodothyronine/metabolism
  • Triiodothyronine, Reverse/metabolism
PubMed
31436139 Full text @ Thyroid
Abstract
Background - Mutations in the thyroid hormone (TH) transporter MCT8 cause MCT8 deficiency, characterized by severe intellectual and motor disability and abnormal serum thyroid function tests. Various Mct8 knock-out mouse models as well as mct8 knock-out and knockdown zebrafish models are used as a disease model for MCT8 deficiency. Although important for model eligibility, little is known about the functional characteristics of the MCT8 orthologues in these species. Therefore, we here compared the functional characteristics of mouse (mm) MCT8 and zebrafish (dr) Mct8 to human (hs) MCT8. Methods - We performed extensive transport studies in COS-1 and JEG-3 cells transiently transfected with hsMCT8, drMct8 and mmMCT8. Protein expression levels and subcellular localization were assessed by immunoblotting, surface biotinylation and immunocytochemistry. Sequence alignment and structural modelling were used to interpret functional differences between the orthologues. Results - hsMCT8, drMct8 and mmMCT8 all facilitated the uptake and efflux of 3,3'-T2, rT3, T3, and T4, although the initial uptake rates of drMct8 were 1.5-4.0-fold higher than for hsMCT8 and mmMCT8. drMct8 exhibited 3-50-fold lower apparent IC50 values than hsMCT8 and mmMCT8 for all tested substrates, and substrate preference of drMct8 (3,3'-T2, T3>T4>rT3) differed from hsMCT8 and mmMCT8 (T3>T4>rT3, 3,3'-T2). Compared to hsMCT8 and mmMCT8, cis-inhibition studies showed that T3 uptake by drMct8 was inhibited at a lower concentration and by a broader spectrum of TH metabolites. Total and cell surface expression levels of drMct8 and hsMCT8 were equal and both significantly exceeded those of mmMCT8. Structural modeling located most non-conserved residues outside the substrate pore, except for H192 in hsMCT8 which is replaced by a glutamine in drMct8. However, a H192Q substituent of hsMCT8 did not alter its transporter characteristics. Conclusion - Our studies substantiate the eligibility of mice and zebrafish models for human MCT8 deficiency. However, differences in the intrinsic transporter properties of MCT8 orthologues may exist, which should be realized when comparing MCT8 deficiency in different in vivo models. Moreover, our findings may indicate that the protein domains outside the substrate channel may play a role in substrate selection and protein stability.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping