Metallothionein from Wild Populations of the African Catfish Clarias gariepinus: From Sequence, Protein Expression and Metal Binding Properties to Transcriptional Biomarker of Metal Pollution

Abstract

Anthropogenic pollution with heavy metals is an on-going concern throughout the world, and methods to monitor release and impact of heavy metals are of high importance. With a view to probe its suitability as molecular biomarker of metal pollution, this study has determined a coding sequence for metallothionein of the African sharptooth catfish Clarias gariepinus. The gene product was recombinantly expressed in Escherichia coli in presence of Zn(II), Cd(II), or Cu, and characterised by Electrospray Ionisation Mass Spectrometry and elemental analysis. C. gariepinus MT displays typical features of fish MTs, including 20 conserved cysteines, and seven bound divalent cations (Zn(II) or Cd(II)) when saturated. Livers from wild C. gariepinus fish collected in all three seasons from four different sites on the Kafue River of Zambia were analysed for their metal contents and for MT expression levels by quantitative PCR. Significant correlations were found between Zn and Cu levels and MT expression in livers, with MT expression clearly highest at the most polluted site, Chililabombwe, which is situated in the Copperbelt region. Based on our findings, hepatic expression of MT from C. gariepinus may be further developed as a major molecular biomarker of heavy metal pollution resulting from mining activities in this region.

Finding High-Quality Metal Ion-Centric Regions Across the Worldwide Protein Data Bank

“…Protein metal binding is part of many biochemical mechanisms including signal transduction, enzyme catalysis, and protein structural integrity [3,4,5]. The local protein structure environment around bound metal ions can provide clues to the biochemical and cellular function of the binding [6,7,8] and reveal how sequence-based structural changes modulates metal binding [9,10]. However, the quality of 3D protein structural data around metal binding sites can vary dramatically from structure to structure, and especially from region to region [8,11].…”

Ag+ Ion Binding to Human Metallothionein-2A Is Cooperative and Domain Specific

“…Kagi and Vallee reported one of the earliest attempts to isolate MT from equine renal cortex, and Kagi and co-workers have reported important applications of both NMR ,, and MS for studies of structure of Cd 7 -MT and cooperativity of metalation. Lobinski and co-workers used both microscale purification techniques (HPLC, capillary electrochromatography) and trace analysis techniques, including ESI-MS, to successfully detect MT isoforms from different systems. − Atrian, Capdevila, and co-workers used ESI-MS to investigate metal binding of MT from different species and to bridge the gap between phytochelatins and metallothions. − Stillman and co-workers have made extensive use of ESI-MS for studies of ion reactivity and kinetics in metalation reactions of MT employing a broad range of metal ions. − Blindauer and co-workers have reported MT expression in different species and dynamics of metal binding and release employing multiple analytical methods including MS. − …”

Genetic Diversity in Insect Metal Tolerance

“…Metallothioneins (MT), small, cysteine-rich, proteins that bind metals including Cu, Zn, and Cd through metal-thiolate bridges, are one of the more thoroughly studied metal-responsive gene families ( Kägi, 1991 ). MT gene expression is up-regulated by metals in a variety of taxa and MT gene expression has been developed as a biomarker of metal contamination in insects and other animals ( Mireji et al, 2010a ; M’kandawire et al, 2017 ). MTs are ubiquitous in eukaryotes, generally present as multi-gene families; e.g., D. melanogaster have at least five ( Atanesyan et al, 2011 ) and mosquitoes ( Anopheles gambiae ) at least two ( Shaw et al, 2007 ).…”