Diversity of metallothioneins in the American oyster, Crassostrea virginica, revealed by transcriptomic and proteomic approaches

Abstract

Metallothioneins are typically low relative molecular mass (6000-7000), sulfhydryl-rich metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-X n -Cys) and a prolate ellipsoid shape containing single a-and b-domains. While functionally diverse, they play important roles in the homeostasis, detoxification and stress response of metals. The originally reported metallothionein of the American oyster, Crassostrea virginica showed the canonical molluscan ab-domain structure. Oyster metallothioneins have been characterized as cDNA and as expressed proteins, and here it is shown that the previously reported metallothionein is a prototypical member of a subfamily (designated as CvMT-I) of ab-domain metallothioneins. A second extensive subfamily of oyster metallothioneins (designated as CvMT-II) has apparently arisen from (a) a stop mutation that truncates the protein after the a-domain, and (b) a subsequent series of duplication and recombination events that have led to the development of metallothionein isoforms containing one to four a-domains and that lack a b-domain. Analysis of metallothioneins revealed that certain CvMT-I isoforms showed preferential association either with cadmium or with copper and zinc, even after exposure to cadmium. These data extend our knowledge of the evolutionary diversification of metallothioneins, and indicate differences in metal-binding preferences between isoforms within the same family.Keywords: cadmium; gene expression; MALDI-TOF; metallothionein; oyster.Metallothioneins (MTs) are a superfamily of ubiquitously expressed metal-binding proteins that can be upregulated by metal exposure, oxidative stress and immune challenge. Typical MTs are low relative molecular mass (M r ) (6000-7000) proteins of high thiol content that lack histidine and aromatic amino acids [1,2]. While they are functionally diverse, they play major roles in metal homeostasis and detoxification. The defining characteristic of MTs is the high cysteine content ( 30%) and conserved Cys-X n -Cys motifs, where X can be any amino acid other than cysteine. The proteins typically have a one-or two-domain structure and bind multiple mono-and divalent metal ions. The structure of MTs, and the nature of their metal-binding, reveal extensive evolutionary diversification. While fungi and early diverged metazoans have small, single-domain MT proteins capable of binding up to eight monovalent metal ions [3][4][5][6], most MTs are comprised of two domains, designated a and b, which are capable of binding metals independently and are separated by a short linker region [7,8]. The a-domain typically contains 11 or 12 cysteines, binds four divalent metal cations, and is believed to convey structure and stability to the protein [9]. In contrast, the b-domain contains nine cysteines, binds three divalent metal cations and participates in metal exchange reactions involving glutathione-shuttling with zinc-and copper-requiring apoproteins [10][11][12]. Some crustacean MTs deviate from this canonical struc...

Copper and zinc detoxification in Gammarus pulex (L.)

“…Through the calibration of the column, the apparent mass of this protein was calculated to be 7.5 kDa. The maximum peak had an absorbance value of 1.2 at 254 nm and 0.8 at 280 nm (A 254 /A 280 ratio of 1.5), suggestive F. R. Khan, N. R. Bury and C. Hogstrand of an amino acid composition that is rich in cysteine (Morris et al, 1999;Jenny et al, 2004). Both Cu and Zn were shown to elute with the 7.5 kDa MTLP.…”

“…Forty fractions of 3ml were collected, from which 1ml was used to measure absorbance at 254nm and 280nm (Cecil 3000 Spectrophotometer, Cecil Instruments, Cambridge, UK). Measurement at 280nm is used for proteins that contain aromatic amino acids, such as tryptophan and tyrosine, whereas 254nm is more specific to cysteine-rich proteins as it monitors metal thiolate bonds (Morris et al, 1999;Jenny et al, 2004).…”

Cloning and Characterization of a New Multi-Stress Inducible Metallothionein Gene in Tetrahymena pyriformis

“…comm.). Cases of MT gene duplication associated with heavy metal resistant capability have been described from invertebrate organisms (Jenny et al 2004;Maroni et al 1987) to higher mammalian eukaryotes (Tio et al 2004). It has also been proposed that different MT isoforms may carry out distinct functions during the processes of metal homeostasis and detoxification (Dondero et al 2005;Sadhu and Gedamu 1988).…”

“…In many invertebrates, various MT isoforms are also present with a high degree of complexity in the gene organization and expression patterns (Dondero et al 2005;Gruber et al 2000;Jenny et al 2004;Leignel et al 2005;Tanguy and Moraga 2001). Functional diversities among those isoforms were hypothesized to be a consequence of evolutionary adaptation, driven by fluctuating conditions in their living environments.…”

Diversification and domain evolution of molluskan metallothioneins: a mini review

“…Accordingly, the exploitation of genetic determinants of molluskan MTs has been a progressively growing domain in the field of MT researches. To date, a number of previous literatures have claimed that molluskan species should represent a great structural diversity of MT proteins (Jenny et al 2004;Jenny et al 2006;Leignel and Laulier 2006). Moreover, some mollusk species particularly including American oyster Crassostrea virginica have shown extraordinarily large-sized MT isoforms comprising of over 100 amino acid (aa) residues (Jenny et al 2004;Jenny et al 2006;, which have not been usually observable in vertebrate orthologs (Blindauer and Leszczyszyn 2010;Serén et al 2014).…”