Raman study of in vivo synthesized Zn(II)-metallothionein complexes: structural insight into metal clusters and protein folding.

Metallothioneins (MTs) are metal-chelating peptides that play an active role in zinc homeostasis. The participation of metal ligands other than cysteines and the presence of secondary structure elements in metal-MT complexes are fairly unknown, especially in nonvertebrate MTs. Here, four Zn(II) complexes of invertebrate MTs (mollusc, insect, nematode, and echinoderm) and the Zn(II)-MT complex of the mammalian MT1 isoform, heterologously synthesized in E. coli, were studied by analytic and spectroscopic techniques. By Raman and circular dichroism spectroscopy, new structural informations were obtained. The five analyzed MT isoforms consist largely of beta-turns with the near exclusion of alpha-helical segments. Raman spectroscopy was revealed as an useful tool, providing information about the state of the cysteine sulfur atoms (metal coordinated and oxidized), the participation of histidine in metal coordination, and the molecular environment of tyrosine residues. In all the five Zn(II)-MT studied samples, acid-labile sulfide anions were found as nonproteic ligands, since sulfide-containing and sulfide-devoid species coexisted in the corresponding preparations. Significantly, Raman bands useful as markers of sulfide bridging ligands were identified. Overall, this work illustrates how the combination of analytical and spectroscopic techniques can be a very informative approach for the analysis of in vivo-synthesized metal-MT complexes, providing new data on the metal binding behavior of MTs from the most diverse organisms.

Evidence for noncooperative metal binding to the alpha domain of human metallothionein.

In the present study, we investigated the metal-binding reactivity of the isolated alpha domain of human metallothionein isoform 1a, with specific emphasis on resolving the debate concerning the cooperative nature of the metal-binding mechanism. The metallation reaction of the metal-free alpha domain with Cd2+ was unequivocally shown to proceed by a noncooperative mechanism at physiologic pH by CD and UV absorption spectroscopy and ESI MS. The data clearly show the presence of intermediate partially metallated metallothionein species under limiting Cd2+ conditions. Titration with four molar equivalents of Cd2+ was required for the formation of the Cd4alpha species in 100% abundance. The implications of a noncooperative metal-binding mechanism are that the partially metallated and metal-free species are stable intermediates, and thus may have a potential role in the currently undefined function of metallothionein.

Structure of the (113)Cd(3)beta domains from Homarus americanus metallothionein-1: hydrogen bonding and solvent accessibility of sulfur atoms.

The three-dimensional structures of the isolated Cd(3)beta domains from Homarus americanus metallothionein have been determined by NMR methods in order to establish a set of beta-domain structures for comparative analysis. First, it was determined that the Cd-cysteine connectivities forming the Cd(3)S(9) metal center were identical to those observed for the beta(N) domain in the native holoprotein. Time- and temperature-dependence studies of the (113)Cd and (1)H 1D-NMR spectra indicated that the beta(N) domain undergoes slow conformational changes before reaching an equilibrium structure. In addition to structural information provided by the metal-to-cysteine connectivities, Phi, chi(1) and chi(2) angle constraints, three H(N...)S hydrogen bond interactions were also determined from a long-range optimized (1)H(N)-(113)Cd HMQC experiment. A simulated annealing protocol was applied to the distance and angle constraints obtained from the 2D-NMR experiments to calculate the three-dimensional structure of the synthetic Cd(3)beta(N) domain of lobster metallothionein. Structure-reactivity relationships are proposed for the reactions of Cd(3)beta domains with 5,5'-dithiobis(2-nitrobenzoate), based on comparisons of surface exposure of sulfur atoms of the lobster and rabbit Cd(3)beta domain structures. Finally, the surface exposure of the beta domains of lobster is compared with beta domains from mammalian metallothioneins.

Metallothionein induction and condition index of dogwhelks Nucella lapillus (L.) exposed to cadmium and hydrogen peroxide.

It has been suggested that metallothionein (MT) not only can regulate essential metals and detoxify toxic metals, but that MT can also play a significant role as an antioxidant and can be induced by oxidative stresses other than metals. This study is aimed at investigating the effect of hydrogen peroxide (H2O2), and the combined effect of H2O2 and cadmium (Cd) on MT induction and condition index (CI) in dogwhelks Nucella lapillus. Adult male dogwhelks (27 +/- 1 mm in shell length) were exposed for 20 days to (1) control (filtered natural seawater only); (2), 0.50 ppm Cd; (3) 2.0 ppm H2O2 + 0.50 ppm Cd; (4) 1.0 ppm H2O2 + 0.25 ppm Cd; (5) 2.0 ppm H2O2; (6) 1000 ppm H2O2 or (7) 1000 ppm H2O2 + 0.50 ppm Cd. The concentration of MT in the Leiblein gland of N. lapillus was quantified using the silver saturation method. MT or MT-like proteins in the animals were induced by Cd (0.5 ppm), H2O2 (2.0 ppm) or Cd + H2O2, indicating that MT in this gastropod species can be induced by either metal or oxidative stresses. Exposure to high H2O2 (1000 ppm) alone or combined with Cd, and exposure to Cd (0.50 ppm) or H2O2 (2.0 ppm), resulted in significant weight loss, indicated by a reduction of CI. However, CIs of groups (3) and (4) were similar to that of the control suggesting that Cd antagonistically reduces toxicity caused by H2O2 since Cd-induced MT may have a protective function against hydroxyl radicals.

Recombinant metallothionein-conjugated streptavidin labeled with 188Re and 99mTc.

Consideration is now being given to the use of avidin (or streptavidin) and biotin for radiotherapy of tumor. Accordingly, the goal of this study was to radiolabel a mouse metallothionein-streptavidin fusion protein with 188Re and to compare its properties to those of the same fusion protein radiolabeled with 99mTc. A recombinant metallothionein-streptavidin fusion protein was radiolabeled by transchelation with 99mTc- and 188Re-glucoheptonate. Labeling efficiency, which was not optimized for either radionuclide, was approximately 60% for 99mTc and 20% for 188Re. Radiochemical purity was demonstrated by size exclusion HPLC both by nearly quantitative shifts of the 188Re label to higher molecular weight upon the addition of biotinylated antibody and by the absence of a shift with biotinsaturated 188Re-metallothionein-streptavidin. Stability of the labels in 37 degrees C serum was evaluated by comparing the HPLC radiochromatograms of serum samples both before and after the addition of biotinylated antibody. The 188Re label behaved like 99mTc in that the same peaks were evident, including one prominent peak due to labeled cysteine. Recoveries during HPLC analysis of serum samples showed that oxidation rates to perrhenate and pertechnetate were identical. However, instability to cysteine challenge was greater for 188Re; for example, the loss of label to cysteine after 24 h under one set of conditions was 41% for 188Re and 22% with 99mTc. Analysis by HPLC of liver and kidney homogenates from mice administered the labeled antibodies were qualitatively and, in large measure, quantitatively independent of label. Biodistributions at 5 h in normal mice were statistically identical between the two labels in blood and in most tissues. In conclusion, streptavidin may be radiolabeled with radiorhenium using recombinant mouse metallothionein as a bifunctional chelator, and under one set of labeling conditions at least, 188Re showed similar in vitro and in vivo behavior to that of 99mTc labeled to the same fusion protein.

Amino acids and peptides. XXXIII. Synthesis of N-terminal epitope peptides of mammalian metallothioneins (MTs).

In order to determine the fine structure of the mammalian metallothionein (MT) epitope to a monoclonal anti-rat Zn-MT-II antibody (MT 189-14-7), N-terminal peptides of various lengths of mammalian metallothioneins (MTs) were synthesized by a conventional solution method using the newly developed beta-2-adamantylaspartate, and their immunological properties were examined. It was found that the N-terminal acetyl group was indispensable for the reaction with the monoclonal antibody and the N-terminally acetylated pentapeptide, Ac-Met-Asp-Pro-Asn-Cys-OH, was the smallest peptide which exhibited a significant reactivity with the antibody.

Metalloselenonein, the selenium analogue of metallothionein: synthesis and characterization of its complex with copper ions.

We used an automated peptide synthesizer to produce a peptide, metalloselenonein, that contains selenocysteine residues substituted for all cysteine residues in Neurospora crassa copper metallothionein. Metalloselenonein binds 3 mol of Cu(I) per mol. This adduct shows a broad absorption band between 230 and 400 nm and a fluorescence band at 395 nm, which can be attributed to copper-selenolate coordination. The circular dichroism spectrum of the copper-metalloselenonein complex shows a positive band around 245 nm attributable to asymmetry in metal coordination.

Amino acids and peptides. XXVI. Synthesis of Agaricus bisporus metallothionein and related peptides and examination of their heavy metal-binding properties.

A pentacosapeptide corresponding to the entire amino acid sequence of Agaricus bisporus metallothionein (MT) and related cysteine-containing peptides were prepared by the conventional solution method and their heavy metal-binding properties were examined. The Cu2(+)- or Cu(+)-binding activities of various peptides were not greatly dependent on the peptide structure, so far as examined, although the pentacosapeptide, A. bisporus MT, exhibited slightly higher binding activity than the other peptides. On the contrary, the Cd2(+)-binding activities of these peptides were fairly structure-dependent.

Amino acids and peptides. XXIV. Synthesis of Neurospora crassa metallothionein and related cysteine-containing peptides and examination of their heavy metal-binding properties.

A pentacosapeptide corresponding to the entire amino acid sequence of Neurospora crassa metallothionein and several related cysteine-containing peptides were synthesized by the conventional solution method and their heavy metal-binding properties were examined. The Cu2+- or Cu+-binding properties of the various peptides were similar to each other, whereas the Cd2+-binding properties of these peptides were fairly structure-dependent.

Cadmium-induced enteropathy: comparative toxicity of cadmium chloride and cadmium-thionein.

In protecting the body against the noxious effects of dietary cadmium ions, cadmium is bound to metallothionein in the proximal intestine, and subsequently excreted into the lumen with desquamation of the epithelium. The purpose of this study was to determine the extent to which cadmium in the form of intestinal cadmium-thionein is absorbed from the intestinal lumen and to appraise the toxicity of cadmium-thionein on the intestinal mucosa. With open-ended duodenal perfusion, equivalent amounts of cadmium administered as CdCl2 or cadmium-thionein entered the mucosa, but significantly less cadmium from the perfusate of cadmium-thionein passed into the body. Exposure of the mucosa to CdCl2 for 1 hr led to minor abnormalities in the form of broadening of villi with pseudostratification of epithelium, and swelling of mitochondria, whereas cadmium-thionein produced extensive necrosis of absorptive cells. The results suggest that cadmium-thionein may play a paradoxical role, providing protection against the cadmium ion in the intracellular milieu, but promoting cadmium toxicity when it is present in sufficient amounts in the lumen of the intestine.