Complete amino acid sequences of five dimeric and four monomeric forms of metallothionein from the edible mussel Mytilus edulis

Abstract

Cadmium-induced metallothioneins from the common sea mussel, Mytilus edulis, were shown to comprise of two groups of isoforms having apparent molecular masses of 10kDa and 20kDa. The 1 0-kDa group was resolved by anion-exchange chromatography into four fractions while the 20-kDa group was resolved into three fractions using this method. After metal removal and Smethylation of the cysteine residues using methyl-p-nitrobenzenesulphonate the complete amino acid sequences were determined. Five isoforms of the 20-kDa group were shown to possess monomeric units consisting of 71 amino acids. These proteins were distinct from the four 72-amino-acid proteins of the 10-kDa group. The FASTA algorithm has been used to compare the degree of similarity between the mussel metallothionein MT-10-IV isoform and other metallothioneins. The mussel MT-10-IV isoform exhibited substantial similarity to other molluscan metallothioneins. Moreover, the mussel metallothionein exhibited more similarity to vertebrate metallothioneins than to those of non-molluscan invertebrates, thus suggesting that the mussel metallothioneins are class I metallothioneins.Despite the discovery of the small cysteine-rich metalbinding-protein metallothionein (MT) more than 30 years ago in equine kidney cortex, the function of this protein is still not fully understood. MT is known to bind metals such as Zn and Cu (which are essential for cell growth and development) and Cd and Hg (which are toxic), which suggests that MT might have more than one function.The search for putative functions for these proteins is exacerbated by the existence of numerous inducers and isoforms. The range of factors which regulate MT synthesis and degradation together with the ubiquity and sequence conservation of MT perhaps suggests that these proteins have a central role in cell metabolism. The proposed functions for MT include the detoxification of heavy metals and the metabolism of essential metals. Zeng et al. (1991) have suggested that thionein may be the active form of the protein and it may be involved in the control of gene expression by virtue of its ability to remove Zn from Zn-finger proteins. Moreover, a tissue-specific isoform of MT in astrocytes (also called growth-inhibitory factor) which inhibits the survival Correspondence to

Isolation and characterisation of metallothionein from the clam Ruditapes decussatus

“…1) demonstrate that all the cadmium was associated with a CdMT fraction with an average apparent molecular weight of 13 700 Da, which was different from the molecular weight of 7328 Da obtained on calibrated gel SDS-PAGE electrophoresis. This discrepancy is largely reported in well-characterised MT proteins from other organisms (Mackay et al, 1993;Olafson et al, 1979;Roesijadi et al, 1989), and is attributed to their flexible (Vasak et al, 1984), nonglobular, prolate ellipsoid shape with a calculated axial ratio of 6 (Kagi et al, 1974).…”

“…All mammals tissues examined thus far usually contain two major class I MT isoforms, designated as MT-I and MT-II (Kagi and Kojima, 1987a). So far, only a few sequences of molluscan MTs have been characterised in the mussels Mytilus edulis (Mackay et al, 1993), Perna viridis (Khoo and Patel, 1999) and the oyster Crassostrea virginica (Roesijadi et al, 1989;Unger et al, 1991).…”

“…In other marine organisms namely the mussel M. edulis (Mackay et al, 1993) two families of MT differing in size, were present with apparent molecular weight of around 10 and 20 kDa. Our results of gel permeation chromatography agree with those for the oyster C. virginica (Roesijadi et al, 1989) and the crab Carcinus maenas (Pedersen et al, 1994) who showed only a single peak at the 10Á/13 kDa position.…”

“…Allelic polymorphism between clams is more likely due to the high degree of heterozygosity in invertebrates than in vertebrates. To date the highest number of isoforms found in aquatic species was in mussel M. edulis MT (at least nine Cd-induced isoforms, Mackay et al, 1993). The CdMT with a NH 2 -terminal blocking moiety might also be a common feature in aquatic invertebrates.…”

“…However, so far, only the primary structure of a few MTs from aquatic invertebrate tissues has been elucidated, either by amino acid sequence analysis or by deduction from MT cDNAs or mRNAs. Among these are the MTs from crabs (Pedersen et al, 1994;Brouwer et al, 1989), mussel (Mackay et al, 1993;Khoo and Patel, 1999) snail (Berger et al, 1995), sea urchin (Wang et al, 1994) and oyster (Roesijadi et al, 1989).…”