Metallothionein characterization

Munoz, A., Laib, F., Petering, D.H. and Shaw, C.F. Ill (1999) Characterization of the cadmium complex of peptide 49-61 a putative nudeation center for cadmium-induced folding in rabbit liver metallothionein IIA. Journal of Biological Inorganic Chemistry, 4, 495—507. 

Chassaigne H, and Lobinski R (1998) Characterization of horse kidney metallothionein isoforms by electrospray MS and reversed-phase HPLC-electrospray MS. Analyst 123 2125- 2130. Chemosphere (1999) Special issue - Sources of error in methylmercury determination during sample preparation, derivatisation and detection. Chemosphere 39 1037-1224. 

Thomas, P., K.N. Baer, and R.B. White. 1994. Isolation and characterization of metallothionein in the liver of the red-eared turtle (Trachemys scripta) following intraperitoneal administration of cadmium. Comp. Biochem. Physiol. 107C 221-226. 

Metallothionein was first discovered in 1957 as a cadmium-binding cysteine-rich protein (481). Since then the metallothionein proteins (MTs) have become a superfamily characterized as low molecular weight (6-7 kDa) and cysteine rich (20 residues) polypeptides. Mammalian MTs can be divided into three subgroups, MT-I, MT-II, and MT-III (482, 483, 491). The biological functions of MTs include the sequestration and dispersal of metal ions, primarily in zinc and copper homeostasis, and regulation of the biosynthesis and activity of zinc metalloproteins. 

A brief historical note on the structure of the iron-sulfur clusters in ferredoxins is relevant. After the first analytical results revealed the presence of (nearly) equimolar iron and acid-labile sulfur, it was clear that the metal center in ferredoxins did not resemble any previously characterized cofactor type. The early proposals for the Fe S center structure were based on a linear chain of iron atoms coordinated by bridging cysteines and inorganic sulfur (Blomstrom et al., 1964 Rabino-witz, 1971). While the later crystallographic analyses of HiPIP, PaFd, and model compounds (Herskovitz et al., 1972) demonstrated the cubane-type structure of the 4Fe 4S cluster, the original proposals have turned out to be somewhat prophetic. Linear chains of sulfide-linked irons are observed in 2Fe 2S ferredoxins and in the high-pH form of aconitase. Cysteines linked to several metal atoms are present in metallothionein. The chemistry of iron-sulfur clusters is rich and varied, and undoubtedly many other surprises await in the future. 

Mehra, R.K., Garey, J.R., Butt, T.R., Gray, W.R. Winge, D.R. (1989). Candida glabrata metallothioneins, cloning and sequence of the genes and characterization of proteins. Journal of Biological Chemistry 264, 19747-53. 

Professor Ebadi discovered and characterized brain metallothioneins isoforms in 1983 and subsequently showed that they are able to scavenge free radicals implicated in Parkinson s disease. In addition, he showed that metallothionein averts a-synuclein nitration, enhances the elaboration of coenzyme Q10, increases the activity of complex I, enhances the synthesis of ATP, and as an antioxidant is fifty times more potent than glutathione. His research programs have been supported in the past and currently by the National Institute on Aging (AG 17059-06), the National Institute... 

Metallothioneins (MTs) are a superfamily of low-molecular-weight (<7000-dalton) intracellular metal-binding proteins, which, in many species, play a critical role in (a) the detoxification of nonessential metals such as Cd2+ and Hg2+ and (b) the regulation of intracellular concentrations of essential metals such as Zn2+ and Cu+. In 1957, Kagi and Vallee first purified and characterized MT as a cadmium-binding protein in equine kidney. 

Metallothioneins are evolutionarily conserved in that they contain a high cysteine content and lack of aromatic amino acids. However, few invertebrate MTs have been characterized, and these can exhibit wide variation in noncysteine amino acid residues. Initially, MTs were classified according to their structural characteristics. Class I MTs consist of polypeptides with highly conserved cysteine residue sequences and closely resemble the equine renal MT. Mammalian MTs consist of 61-68 amino acids residues and the sequence is highly conserved with respect to the position of the cysteine residues (e.g., cys-x-cys, cys-x-y-cys, and cys-cys sequences, where x and y are noncysteine, non-aromatic amino acids). Class II MTs have less conserved cysteine residues and are distantly related to mammalian MTs. Class III MTs are defined as atypical and consist of enzymatically synthesized peptides such as phy-tochelatins and cadystins. This former classification scheme has been replaced by a more complex system to include the increasing number of identified isoforms. 

The second step in zinc absorption involves the intracellular interaction of zinc with various compounds which may enhance or impede absorptive processes. In 1969, Starcher noted that radioactive copper, given orally, associated with a low molecular weight protein (25). Subsequently, this mucosal protein was isolated and characterized by Richards and Cousins, who classified it as a metallothionein (26), and who further showed that it was induced in response to zinc administration (5). The appearance of this metallothionein, with properties similar to those described for both rat (27) and human (28) liver metallothionein, appears to be related to changes in both dietary zinc status and plasma zinc levels (5). The synthesis of mucosal metallothionein has been shown to be under transcriptional control (29,30). Menard al. reported that dietary zinc administration resulted in enhancement of metallothionein mRNA transcription and its subsequent translation, to yield nascent metallothionein polypeptides(31). The intestinal metallothionein appearance was correlated to both an increase in mucosal zinc content primarily associated with the protein and with a decrease in serum zinc levels. In addition. Smith e al., using the isolated, vascularly perfused intestinal system, reported an inverse relationship between the synthesis of metallothionein and zinc transfer to the portal system, confirming earlier studies (32). 

Element-specific detection by ICP-MS has been widely used in the characterization of metallothioneins (MTs). The biological importance of these proteins is due to their role in homeostatic regulation of essential heavy metals like Cu and Zn. On the other hand, MT protects the cells from harmful chemicals, like nonessential and excessive essential heavy metals, reactive oxygen species, radicals, and alkylating agents. Fararello et reviewed different chromatographic approaches with ICP-MS detection for the multielemental speciation in MTs and MT-like proteins. 

H. Chassaigne, R. Lobinski, Characterization of horse kidney metallothionein isoforms by ESI-MS and RPLC-ESI-MS, Analyst, 123 (1998) 2125. 

Metallothioneins are a unique and widely distributed group of proteins. They are characterized by their low molecular weight (—6000), high cysteinyl content, and the ability to bind substantial numbers of metal ions (43). The proteins bind copper and zinc, thereby providing a mobile pool as part of the normal metabolism of these elements, and offer protection from the invasion of inorganic forms of the toxic elements cadmium, lead, and mercury. In addition, other metals, such as iron and cobalt, can be induced to bind. XAS is ideally suited to probe the environment of these different metal atoms (see Fig. 1), and the structural interpretations obtained from an analysis of the EXAFS data obtained in several such studies are summarized in Table 1(44). Thus, in each case, the data are consistent with the primary coordination of the metal deriving from the cysteinyl residues. 

Capillary electrophoresis has proven to be useful in characterizing different molecular forms of various metalloproteins like metallothionein, transferrin, and conalbumin [2-5]. Molecular forms arise from differences in the amino acid sequence of proteins (isoforms), differences in the amount or type of metal bound (metalloforms), or from differences in the type and amount of carbohydrate side chains linked to the protein (glycoforms). CZE was used to follow the formation of the oligomeric iron core and its incorpora-... 

Slice, L.W., Freedman, J.H. and Rubin, C. (1990) Purification, characterization, and cDNA cloning of a novel metallothionein-like, cadmium-binding protein from Caenorhabditis elegans. Journal of Biological Chemistry, 265, 256-263. 

Stiirzenbaum, S.R., Kille, P. and Morgan, A.J. (1998c) The identification, cloning and characterization of earthworm metallothionein. FEBS Letters, 431, 437-442. 

Zafarullah, M., K. Bonham and L. Gedamu. Structure of the rainbow trout metallothionein B gene and characterization of its metal-responsive region. Mol. Cell Biol. 8 4469-4476, 1988. 

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