Purification and Characterisation of Recombinant Sea Urchin Metallothionein Expressed in Escherichia Coli

Abstract

Metallothioneins (MT) are metalloproteins expressed tissue specifically during the development of the sea urchin, Strongylocentrotus pururutus. To explore their structural and functional features and to compare them with those of the evolutionary distant mammalian MTs, one isoform (MTA) was obtained as the cadmium-containing form, from synthetic cDNA heterologously expressed in Escherichia coli. The purified protein was identified as the desired product by a combination of peptide-map analysis, amino acid sequence analysis and ion-spray mass spectroscopy. The existence of seven " T d NMR resonances revealed that the recombinant protein binds seven Cd ions/molecule. The position of the NMR resonances (605 -695 ppm) and the electronic absorption features suggest that the sea urchin MTA, like the mammalian MTs, possesses tetrahedrally coordinated cadmiumthiolate clusters. With its large Stokes' radius, sea urchin MTA resembles the mammalian forms, suggesting a comparable elongated molecular shape. Measurements by spectrophotometric pH titration of cadmium binding by the recombinant protein suggest that it possesses two metal-thiolate clusters of distinctly different stability. At pH 7 the average apparent association constant for Cd2' in the clusters is about 20-times weaker in sea urchin MTA than in rabbit MT-2.Metallothioneins (MT) are widely found low-molecularmass, cysteine-rich proteins distinguished by an exceptionally high content of d' O metal ions (Zn", Cd2+ and Cu'+) (see Roesijadi, 1993, andKagi, 1993, for reviews). MTs have traditionally been thought to be involved in cellular detoxification of metals but more central functions such as an involvement in the regulation of Zn-dependent processes in gene expression (Zeng et al., 1991a and and in development (Nemer et al. 1984;De et al., 1991) have also been suggested.The sea urchin Strongylocentotus pupurutus like many other species, contains a family of MT genes. Two of these genes, designated MTA and MTB,, have been well characterised. These genes show a high degree of identity, but probes specific for their mRNAs have been constructed and used to show that the expression patterns of the genes are quite distinct. Both mRNAs begin to accumulate at the early blastula stage (12 h after fertilization) but the MTB, mRNA becomes localized in the embryonic gut and in the oral ectoderm while the MTA mRNA is spatially restricted to the aboral ectoderm (Nemer et al., 1991). As found for mammalian MTs, both forms are readily inducible by zinc although with unequal thresholds (Wilkinson and Nemer, 1987). This responsiveness to zinc supply is accounted for by the occurrence of at least six metal-regulatory elements (MRE) in the 5' upstream region of both the sea urchin genes (Bai et al., 1993). The two MREs of the MTA gene (designated a and b) nearest to the transcription start site are identical in both sequence and position (relative to the TATA box) to those which give maximal activity in mammalian systems (Harlow et al., 1989). In addition to this conservation of...

Separation and Characterization of the Metal‐Thiolate‐Cluster Domains of Recombinant Sea Urchin Metallothionein

“…As binding of Cd to apoMTA releases 2.86 protons/metal ion, the corrected mass incremenVCd ion was 109.5 Da. On this basis the mass difference between the apo and holo forms indicated the presence of 7.05 Cd ions in the latter in close agreement with the metal composition of the recombinant protein (Wang et al, 1994).…”

“…7030 Da for a major component and 7160 Da for a minor component, were observed. The increment of 130 Da can be attributed to the residual initiator methionine residue which is only partially removed in expression (Wang et al, 1994(Wang et al, , 1995. Fig.…”

“…In a recent study with recombinant Cd-containing MTA from the sea urchin Strongylocentrotus purpurutus, we have found using '13Cd-113Cd COSY and homonuclear decoupling ' "Cd-NMR measurements that like the mammalian forms this invertebrate MT also features both a M:' Cys, and a M:+Cys,, cluster. However, in contrast to the mammalian MT the arrangement of the two clusters in the echinodermal protein is reversed; in the latter the four-metal cluster is associated with the N-terminal portion and the three-metal cluster with the C-terminal portion of the polypeptide chain (Wang et al, 1994(Wang et al, , 1995. In the present study the two clusters were further scrutinized by enzymatically bisecting the recombinant sea urchin protein (Cd,-MTA) and by characterizing the fragments by chromatographic, mass spectrometric and optical spectroscopic techniques.…”

“…Sea urchin holoMTA was prepared as described by Wang et al (1994). The digestion of the holoprotein with subtilisin was carried out in 20 mM Tris/HCl, pH 8.0, for 3 h at 37°C at a protein/protease ratio of 20:l (by mass).…”

Isolation and characterisation of metallothionein from the clam Ruditapes decussatus

“…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).…”