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Prediction of heparin binding of mutated short sequences of rat thyroglobulin

Journal of Endocrinological Investigation volume 44, pages 1237–1241 (2021)Cite this article

Abstract

Background

Binding of thyroglobulin (Tg) to heparin is involved in Tg transcytosis via megalin. Rat Tg (rTg) binds to heparin through an exposed carboxyl terminal region (RELPSRRLKRPLPVK, Arg2489-Lys2503) rich in positively charged residues. This region is not entirely conserved in human Tg (hTg) (Arg2489-Glu2503, REPPARALKRSLWVE), resulting in lower affinity binding. Here, we developed a score to predict to what extent secondary structure modifications affect the heparin-binding ability of rTg.

Methods

We designed eight synthetic peptides, including one with the Arg2489-Lys2503 sequence of rTg (rTgP), one with the corresponding sequence of hTg (hTgP), and six “mutant” peptides, each carrying a point mutation obtained by replacing one amino acid residue of rTgP with the corresponding residue of hTgP. Heparin binding was assessed in solid-phase assays. The Bmax and the constants of dissociation (Kd) were calculated.

Results

Using a no-fee online service, we obtained predictions of peptide secondary structures and developed a scoring system to estimate to what extent mutations are expected to modify rTg secondary structure. The score was designated as Probability of Secondary Structure Change (PSSC) and it significantly correlated with the BMax (R = 0.942, P < 0.001) and the Kds (R = − 0.744, P < 0.01) of heparin binding of hTgP and of the “mutant” peptides.

Conclusions

The PSSC score allows predicting to what extent point mutations are likely to affect the heparin-binding ability of short sequences of proteins: in this case rTg, regardless of whether mutations affect charge of the sequence. The secondary structure of Tg is likely to play a role in heparin binding.

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Acknowledgements

The study was funded by the University of Pisa (Fondi di Ateneo, to MM).

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  1. S. Lisi

    Present address: Bio@SNS Laboratory, Scuola Normale Superiore, Pisa, Italy

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  1. Department of Clinical and Experimental Medicine, Endocrinology Unit II, University of Pisa and University Hospital of Pisa, Via Paradisa 2, 56124, Pisa, Italy

    S. Lisi & M. Marinò

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  1. S. Lisi
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  2. M. Marinò
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Correspondence to M. Marinò.

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Lisi, S., Marinò, M. Prediction of heparin binding of mutated short sequences of rat thyroglobulin. J Endocrinol Invest 44, 1237–1241 (2021). https://doi.org/10.1007/s40618-020-01411-5

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Keywords

  • Thyroid
  • Thyroglobulin
  • Heparin