Tomas M et al. (2013), The sea urchin metallothionein system: Comparat...

ECB-ART-47948

FEBS Open Bio 2013 Jan 22;3:89-100. doi: 10.1016/j.fob.2013.01.005.

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The sea urchin metallothionein system: Comparative evaluation of the SpMTA and SpMTB metal-binding preferences.

Tomas M , Domènech J , Capdevila M , Bofill R , Atrian S .

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Metallothioneins (MTs) constitute a superfamily of ubiquitous metal-binding proteins of low molecular weight and high Cys content. They are involved in metal homeostasis and detoxification, amongst other proposed biological functions. Two MT isoforms (SpMTA and SpMTB) have been reported in the echinoderm Strongylocentrotus purpuratus (sea urchin), both containing 20 Cys residues and presenting extremely similar sequences, although showing distinct tissular and ontogenic expression patterns. Although exhaustive information is available for the Cd(II)-SpMTA complex, this including the full resolution of its 3D structure, no data has been reported concerning either SpMTA Zn(II) and Cu(I) binding properties, or the characterization of SpMTB at protein level. In this work, both the SpMTA and SpMTB isoforms, as well as their separate α and β domains, have been recombinantly synthesized in the presence of Zn(II), Cd(II) or Cu(II), and the corresponding metal complexes have been analyzed using electrospray mass spectrometry, and CD, ICP-AES and UV-vis spectroscopies. The results clearly show a better performance of isoform A when binding Zn(II) and Cd(II), and of isoform B when coordinating Cu(I). Thus, our results confirm the differential metal binding preference of SpMTA and SpMTB, which, together with the reported induction pattern of the respective genes, highlights how also in Echinodermata the MT polymorphism may be linked to the evolution of different physiological roles.

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Genes referenced: LOC100887844 LOC594349 LOC594566 thrb

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	Fig. 1. (A) Amino acid sequence alignment of recombinant SpMTA and SpMTB. Note that the N-term Gly–Ser residues derive from the GST-fusion system used for recombinant synthesis and purification. (B) 3D structures of Cd4-αSpMTA (top) and Cd3-βSpMTA (bottom) generated from PDB files 1QJK and 1QJL, respectively, with VMD software (http://www.ks.uiuc.edu/Research/vmd/) [Humphrey W, Dalke A, Schulten K. VMD – visual molecular dynamics. J. Molec. Graphics 1996;14:33–38]. Color code: random coil, silver; turn, cyan; extended beta, orange; Cd(II) ions, purple; Cys residue backbone and Cys-Cd connectivities, yellow. N-term and C-term ends of SpMTA sequence are indicated, and an arrow marks the position of the insertion of a Thr residue in SpMTB. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 2. Representative charge states for the ESI-MS spectra recorded at pH 7.0 of recombinant Zn-SpMTA (A), Zn-αSpMTA (B), Zn-βSpMTA (C), Zn-SpMTB (D), Zn-αSpMTB (E) and Zn-βSpMTB (F). The observed species are collected in Table 2.
	Fig. 3. CD spectra corresponding to the entire MT (A) and the separate constitutive α (B) and β (C) domains of SpMTA (black) and SpMTB (red) recombinantly synthesized in Zn-supplemented media. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 4. Representative charge states for the ESI-MS spectra recorded at pH 7.0 of recombinant Cd-SpMTA (A), Cd-αSpMTA (B), Cd-βSpMTA (C), Cd-SpMTB (D), Cd-αSpMTB (E) and Cd-βSpMTB (F). The observed species are collected in Table 2.
	Fig. 5. CD spectra corresponding to the entire MT (A) and the separate constitutive α (B) and β (C) domains of SpMTA (black) and SpMTB (red) recombinantly synthesized in Cd-supplemented media. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 6. CD spectra recorded during the titration of Zn4-αSpMTA with Cd(II) at pH 7 (A) and superimposition of CD spectra of Cd4-αSpMTA obtained by Zn/Cd substitution over Zn4-αSpMTA (in vitro; black) and by recombinant synthesis in Cd-enriched media (in vivo; red) (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 7. CD spectra recorded during the titrations of Zn-βSpMTA (A, B) and Zn-βSpMTB (C, D) with Cd(II) at pH 7. The effect of the addition of S2− to βSpMTB is also shown (F), as well as the CD superimposition of in vivo (red) and in vitro (black) Cd-βSpMTA (E) and Cd-βSpMTB (G) species obtained after addition of 3 Cd(II) equivalents to Zn-βSpMTA or after an acidification/reneutralization process of Cd-βSpMTB plus addition of 3 S2− equivalents. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 8. Representative charge states for the ESI-MS spectra recorded at pH 7.0 and pH 2.5 of recombinant SpMTA (A, B) and SpMTB (E, F) synthesized in Cu-supplemented E. coli media at normal aeration (N.A.) conditions, and of recombinant Cu-SpMTA (C, D) obtained at low aeration (L.A.) conditions. The observed species are collected in Tables 3 and 4.
	Fig. 9. CD spectra corresponding to the entire MT (A, B) and the separate constitutive α (C, D) and β (E, F) domains of SpMTA (black) and SpMTB (red) recombinantly synthesized in Cu-supplemented media at normal aeration (N.A.) and low aeration (L.A.) conditions (left and right, respectively). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 10. Representative charge states for the ESI-MS spectra of recombinant αSpMTB (A, D, F), βSpMTA (B, G) and βSpMTB (C, E) synthesized in Cu-supplemented E. coli media at normal (N.A.) and low (L.A.) aeration conditions. M-MT species where M = Zn + Cu are assigned in spectra recorded at pH 7.0 (A–C, G), while Cu-MT species are assigned when recorded at pH 2.5 (D–F). The observed species are collected in Tables 3 and 4.
	Fig. 11. CD spectra recorded during the titration of Zn-βSpMTA (A–C) and Zn-βSpMTB (D–F) with Cu(I) at pH 7. Also, a comparison with the CD spectrum of the corresponding recombinant Cu-βMT samples obtained at normal aeration conditions (in red) is shown (G, H). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

References [+] :

Bofill, A new insight into the Ag+ and Cu+ binding sites in the metallothionein beta domain. 1999, Pubmed