title: Glycan-Dependent Mutual and Reversible Sequestration of Two Thyroid Cancer Biomarkers.
abstract: BACKGROUND
  Thyroglobulin (Tg), the major thyroidal protein, plays important roles in thyroid hormone biosynthesis and in autoimmune thyroid diseases (AITD). Tg also serves as a pre- and postoperative biomarker of differentiated thyroid cancer (DTC). The endogenous β-galactoside binding lectin galectin-3 (Gal-3), secreted by transformed thyroid cells, has been shown to be another useful biomarker of DTC. Tg contains covalently linked complex-type glycans that can serve as binding epitopes of Gal-3. The objective of the study is to investigate the interaction between Tg and Gal-3 and discuss its potential consequences.
  
  
  METHODS
  Binding interaction between Tg and Gal-3 was first studied by hemagglutination inhibition assays. Subsequently, a detailed analysis of binding thermodynamics was carried out by isothermal titration calorimetry. Quantitative precipitation was performed to study the complex formation between Tg and Gal-3 and to determine the binding stoichiometry. The concentration-dependent rate and amount of complex formation between Tg and Gal-3 was examined spectrophotometrically. A similar approach was taken to study the effect of free Tg and Gal-3 on preformed Tg-Gal-3 complex.
  
  
  RESULTS
  Quantitative biochemical and biophysical data show that these two biomarkers produced by thyroid cancer cells interact with each other with submicromolar affinity and form an insoluble complex at their stoichiometric concentration. One Tg molecule could bind up to 14 molecules of Gal-3. Such complex formation mutually sequestered both Tg and Gal-3, decreasing the concentration of their freely available forms. Formation of the Tg-Gal-3 complex was reversible as the preformed complex was dissolved by free Tg as well as free Gal-3. While free Tg rapidly dissolved preformed Tg-Gal-3 complex in a concentration-dependent manner, Gal-3 was found to be much less efficient and slowly dissolved only a fraction of the preformed complex at a relatively higher Gal-3 concentration.
  
  
  CONCLUSIONS
  Complex formation between Tg and Gal-3 through high affinity binding and the sensitivity of the complex to free Tg and Gal-3 can potentially influence their biological functions. Interactions between Tg and Gal-3 might also interfere with their clinical detection, the same way Tg autoantibody (TgAb) is reported to interfere with Tg assays. The data support a model of Gal-3-mediated homeostatic process of Tg.
authors.affiliation: 1 Laboratory of Mechanistic Glycobiology, Department of Chemistry, Michigan Technological University , Houghton, Michigan.
authors.forename: Ni
authors.initials: N
authors.lastname: Fan
authors.affiliation: 1 Laboratory of Mechanistic Glycobiology, Department of Chemistry, Michigan Technological University , Houghton, Michigan.
authors.forename: Melanie L
authors.initials: ML
authors.lastname: Talaga
authors.affiliation: 1 Laboratory of Mechanistic Glycobiology, Department of Chemistry, Michigan Technological University , Houghton, Michigan.
authors.forename: Robert K
authors.initials: RK
authors.lastname: Brown
authors.affiliation: 1 Laboratory of Mechanistic Glycobiology, Department of Chemistry, Michigan Technological University , Houghton, Michigan.
authors.forename: Ashli L
authors.initials: AL
authors.lastname: Fueri
authors.affiliation: 2 Department of Biological Sciences, Michigan Technological University , Houghton, Michigan.
authors.forename: Purnima
authors.initials: P
authors.lastname: Bandyopadhyay
authors.affiliation: 1 Laboratory of Mechanistic Glycobiology, Department of Chemistry, Michigan Technological University , Houghton, Michigan.|3 Biotechnology Research Center, Michigan Technological University , Houghton, Michigan.
authors.forename: Tarun K
authors.initials: TK
authors.lastname: Dam
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism*; D002151:Calorimetry; D011232:Chemical Precipitation; D037502:Galectin 3 / Q000378:metabolism*; D006385:Hemagglutination Inhibition Tests; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D013816:Thermodynamics; D013954:Thyroglobulin / Q000378:metabolism*; D013964:Thyroid Neoplasms / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; D037502:Galectin 3; D011134:Polysaccharides; D013954:Thyroglobulin