中华鳖肝金属硫蛋白的分离、纯化及鉴定（英文）

金属硫蛋白(metallothionein,MT)是一类低分子量、富含半胱氨酸的金属结合蛋白.MT几乎广泛分布于所有生物,包括哺乳动物、两栖动物、鱼、植物、真菌和蓝细菌.不同生物金属硫蛋白理化特性和其氨基酸序列及中心片段的比较研究,对研究MT的结构和生物功能及生物的分子进化提供重要依据.哺乳动物MT研究较多,爬行动物鳖MT的研究尚属空白,本文报道鳖肝的金属硫蛋白.中华鳖 (Pelodiscus sinensis) 分别经皮下注射ZnSO4、CuSO4和CdCl2 溶液诱导后,取乙醇沉淀的肝脏无细胞提取液再经Sephadex G-50、DEAE-SepharoseCL-6B 及SephadexG-25凝胶过滤和离子交换柱层析分离,自鳖肝脏中分别获得Zn-MT、Cu-MT和Cd-MT,未经诱导的鳖肝脏中无MT.质谱和HPLC分析其分子量约为6 300 dalton.根据氨基酸组成分析,鳖肝脏MT含61个氨基酸残基,其中MT的典型氨基酸Cys含量占17%.Lys、Glu和Asp含量较高,而芳香族氨基酸和组氨酸含量极低.从紫外光谱特性分析,Zn-MT、Cu-MT、Cd-MT紫外吸收肩分别在220 nm、270 nm和250 nm.表明确为鳖肝脏MT.从氨基酸残基数和分子量看,鳖肝脏MT与哺乳动物MT类似;而从氨基酸组成和结合金属离子的量看,又与低等生物蚯蚓及酵母菌的MT类似.鳖MT的特性介于哺乳动物MT与低等生物MT之间,体现了鳖这种生物进化的特点.     

中华鳖肝金属硫蛋白的分离、纯化及鉴定

金属硫蛋白metallothionein,MT)是一类低分子量、富含半胱氨酸的金属结合蛋白。MT几乎广泛分布于所有生物,包括哺乳动物、两栖动物、鱼、植物、真菌和蓝细菌,不同生物金属硫蛋白理化特性和其氨基酸序列及中心片段的比较研究,对研究MT的结构和生物功能及生物的分子进化提供重要依据。哺乳动物MT研究较多,爬行动物鳖MT的研究尚属空白,本文报道鳖肝的金属硫蛋白,中华鳖（Pelodiscus sinensis)分别经皮下注射ZnSO4,CuSO4和CdCl2溶液诱导后,取乙醇沉淀的肝脏无细胞提取液再经SephadexG-50、DEAE-SepharoseCL-6B及SephadexG-25凝胶过滤和离子交换柱层析分离,自鳖肝脏中分别获得Zn-MT、Cu-MT和Cd-MT,未经诱导的鳖肝脏中无MT,质谱和HPLC分析其分子量约为6300dalton。根据氨基酸组成分析。鳖肝脏MT含61个氨基酸残基,其中MT的典型氨基酸Cys含量占17%。Lys,Glu和Asp含量较高,而芳香族氨基酸和组氨酸含量极低,从紫外光谱特性分析,Zn-MT、Cu-MT、Cd-MT紫外吸收肩分别在220nm、270nm和250nm。表明确为鳖肝脏MT。从氨基酸残基数和分子量看,鳖肝脏MT与哺乳动物MT类似;而从氨基酸组成和结合金属离子的量看,又与低等生物蚯蚓酵母菌的MT类似。鳖MT的特性介于哺乳动物MT与低等生物MT之间,体现了鳖这种生物进化的特点。     

Cloning, expression and characterization of metallothionein from the Antarctic clam Laternula elliptica

The genes for two apparent subtypes of metallothionein (MT) isoform were isolated from the Antarctic clam Laternula elliptica. Determination of the nucleotide sequence showed that the gene consists of 222 bp that code a 73-amino acid protein. The comparison between MT cDNA sequences of L. elliptica and other bivalves showed strong homologies on positions of cysteine residues, which are important for their metal binding abilities. The gene for the MT was inserted into a pET vector and overexpressed as a carboxyl terminal extension of glutathionein-S-transferase (GST) in Escherichia coli. After the GST fusion proteins had been purified by glutathione-Sepharose affinity chromatography column and digested with enterokinase, the MT was purified with gel filtration and analyzed for its biochemical properties. Recombinant MTs were reconstituted with Cd, Cu, and Zn, and kinetic studies of the reactions with electrophilic disulphide, DTNB, were investigated to explore their metal binding ability. It is revealed that the Cd-MT and Zn-MT react with DTNB biphasically, and that Zn-MT reacts with DTNB more rapidly, and with a significantly greater pseudo-first-order rate constant. Cu-MT reacts monophasically and releases metal slowly from MT.     

Isolation and characterization of a self-sufficient one-domain protein. (Cd)-metallothionein from Eisenia foetida.

Earthworms have been shown to accumulate trace elements in general, and particularly high amounts of metal ions such as cadmium, copper and zinc. The earthworm's response to metal contamination has been linked to the induction and expression of metallothionein (MT) proteins, a detoxification strategy analogous to that found in other biological systems. The present study focuses on an inducible Cd-MT isolated from the compost-dwelling brandling worm Eisenia foetida (Savigny). A full characterization of the protein (including protein induction, MT cDNA, amino-acid sequence and metal stoichiometry) revealed a new dimension of knowledge to the molecular genetic information available to date. Whereas the elucidated cDNA codes for a putative protein which possesses 80 amino-acid residues, the characterized protein bears only 41 amino acids. The isolated product has evidently attained its size and shape by cleavage near the N-terminal site and at the linker region between the two putative metal-binding domains of the translated product, yielding a small MT moiety which contains 12 Cys residues (including one triple Cys-motif) binding four cadmium ions. It can be shown that the isolated MT molecule represents a self-sufficient one-domain MT which is stable in vitro. The isolation of the single-domain MT peptide raises the question about the method of formation and significance in vivo of such small MT moieties from tissues of E. foetida and possibly other terrestrial invertebrates. In this respect, two hypotheses are discussed: firstly, the possibility of formation of small MT peptides due to enzymatic cleavage of the intact protein during the process of preparation and isolation; and secondly, the possibility of deliberate post-translational processing of the translated gene product to yield functional one-domain MT moieties.     

Two new members of the Tetrahymena multi-stress-inducible metallothionein family: Characterization and expression analysis of T. rostrata Cd/Cu metallothionein genes.

We report the cloning and characterization of two new metallothionein (MT) genes (TrosMTT1 and TrosMTT2), isolated as cDNAs, from the ciliated protozoa Tetrahymena rostrata. The TrosMTT1 inferred protein has been identified as a CdMT and included into the 7a subfamily of Tetrahymena MTs, while TrosMTT2 has been identified as a CuMT (including it into 7b subfamily), due to its similarity to TpigMT-2 and its significant induction by copper. TrosMTT1 protein sequence reveals a remarkably regular and hierarchical modular organization, as it is known for other Tetrahymena CdMTs, showing a bi-modular structure. TrosMTT2 presents a structural organization based on CKCX(2-5)CKC repeats, like it occurs in other Tetrahymena CuMTs, indicating that an evolutionary history based on intra-gene duplications might be also possible. Both are also multi-stress-inducible genes because they are induced by other heavy metals and stressors, as it has been shown by quantitative real-time RT-PCR. It is the first time that the gene expression of a putative Tetrahymena CuMT is analyzed by quantitative PCR, confirming it as a CuMT. These two new Tetrahymena MTs complete, at present, the actual view of this protein superfamily, and corroborate the unique features of ciliate MTs. Furthermore, both, a comparative analysis of relative gene expression values obtained by quantitative RT-PCR on other Tetrahymena MT genes and an analysis of the different Tetrahymena MTs based on the different Cys clusters of these proteins are carried out, which show an update view of Tetrahymena MT gene family.     

Molecular cloning of chicken metallothionein. Deduction of the complete amino acid sequence and analysis of expression using cloned cDNA.

A cDNA library was constructed using RNA isolated from the livers of chickens which had been treated with zinc. This library was screened with a RNA probe complementary to mouse metallothionein-I (MT), and eight chicken MT cDNA clones were obtained. All of the cDNA clones contained nucleotide sequences homologous to regions of the longest (376 bp) cDNA clone. The latter contained an open reading frame of 189 bp, and the deduced amino acid sequence indicates a protein of 63 amino acids of which 20 are cysteine residues. Amino acid composition and partial amino acid sequence analyses of purified chicken MT protein agreed with the amino acid composition and sequence deduced from the cloned cDNA. Amino acid sequence comparisons establish that chicken MT shares extensive homology with mammalian MTs, but is more closely related to the MT-II than to the MT-I isoforms from various mammals. The nucleotide sequence of the coding region of chicken MT shares approximately 70% homology with the consensus sequence for the mammalian MTs. Southern blot analysis of chicken DNA indicates that the chicken MT gene is not a part of a large family of related sequences, but rather is likely to be a unique gene sequence. In the chicken liver, levels of chicken MT mRNA were rapidly induced by metals (Cd2+, Zn2+, Cu2+), glucocorticoids and lipopolysaccharide. MT mRNA was present in low levels in embryonic liver and increased to high levels during the first week after hatching before decreasing again to the basal levels found in adult liver. The results of this study establish that MT is highly conserved between birds and mammals and is regulated in the chicken by agents which also regulate expression of mammalian MT genes. However, in contrast to the mammals, the results suggest the existence of a single isoform of MT in the chicken.     

Molecular characterization of two metallothionein isoforms in avian species: evolutionary history, tissue distribution profile, and expression associated with metal accumulation

To characterize avian MTs, MT cDNAs were cloned from liver of cormorant (Phalacrocorax carbo) and mallard (Anas platyrhynchos). Expression profiles of MT isoforms and relationships between metal accumulation and MT mRNA expression in tissues were also investigated. We succeeded in cDNA cloning of MT1/2 from cormorant and MT1 in mallard. DNA sequence of chicken MT1 was obtained from chicken (Gallus gallus) genomic database. Considering previous reports on avian MTs, birds possess at least two distinct MT isoforms. Comparison of genomic synteny among vertebrates and phylogenetic analysis of MT amino acid sequences revealed that avian MT1/2 are evolutionarily close to mammalian MT3. Messenger RNAs of both MT isoforms were detected in all the tissues/organs in cormorant and mallard. Liver was the primary organ for cormorant MT1/2, and mallard MT2, whereas MT1 was dominant in mallard heart. Interspecies comparison of tissue distribution of MT mRNA expression between cormorant and mallard indicated that MT2 profile was similar, but MT1 was not. Significant positive correlations of mRNA expression levels between MT1 and MT2 were observed in the liver and kidney of cormorants, whereas no correlation was found in mallards. Expression levels of cormorant MT1/2 showed significant positive correlations with hepatic Cu and Zn concentrations, suggesting that both MT isoforms were induced by Cu and Zn in livers. Cormorant MT2 expression level exhibited a significant positive correlation with hepatic Ag, and a negative correlation with Rb, indicating that Ag and Rb concentrations depend on the expression of MT2 by Cu and Zn. In mallard, MT1 had no correlation with any metal concentration, and MT2 expression was positively correlated only with Cu, even though hepatic Cu and Zn concentrations in mallard were much higher than in cormorant. This may indicate that cormorant is a more susceptible species than mallard in terms of MT induction. These findings suggest tissue-, species-, and MT isoform-specific responses to metal stresses in these aquatic birds.     

Copper-metallothionein induction in the liver of LEC rats.

Recently, copper (Cu) was found to be unusually accumulated, suggesting the induction of metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis. Thus, the direct relationship between the unusual Cu accumulation and the induction of Cu-MT was investigated by giving LEC rats Cu-overloaded or Cu-deficient diets. Results based on the determinations of Cu and MT levels in several organs, as well as the gel-filtration profiles of the cytosols of liver homogenates, showed that dietary Cu induced Cu-MT and development of hepatic injury associated with jaundice.     

Metal dealing at the origin of the chordata phylum: the metallothionein system and metal overload response in amphioxus

Non-vertebrate chordates, specifically amphioxus, are considered of the utmost interest for gaining insight into the evolutionary trends, i.e. differentiation and specialization, of gene/protein systems. In this work, MTs (metallothioneins), the most important metal binding proteins, are characterized for the first time in the cephalochordate subphylum at both gene and protein level, together with the main features defining the amphioxus response to cadmium and copper overload. Two MT genes (BfMT1 and BfMT2) have been identified in a contiguous region of the genome, as well as several ARE (antioxidant response element) and MRE (metal response element) located upstream the transcribed region. Their corresponding cDNAs exhibit identical sequence in the two lancelet species (B. floridae and B. lanceolatum), BfMT2 cDNA resulting from an alternative splicing event. BfMT1 is a polyvalent metal binding peptide that coordinates any of the studied metal ions (Zn, Cd or Cu) rendering complexes stable enough to last in physiological environments, which is fully concordant with the constitutive expression of its gene, and therefore, with a metal homeostasis housekeeping role. On the contrary, BfMT2 exhibits a clear ability to coordinate Cd(II) ions, while it is absolutely unable to fold into stable Cu (I) complexes, even as mixed species. This identifies it as an essential detoxification agent, which is consequently only induced in emergency situations. The cephalochordate MTs are not directly related to vertebrate MTs, neither by gene structure, protein similarity nor metal-binding behavior of the encoded peptides. The closest relative is the echinoderm MT, which confirm proposed phylogenetic relationships between these two groups. The current findings support the existence in most organisms of two types of MTs as for their metal binding preferences, devoted to different biological functions: multivalent MTs for housekeeping roles, and specialized MTs that evolve either as Cd-thioneins or Cu-thioneins, according to the ecophysiological needs of each kind of organisms.     

异常汉逊酵母BD102金属硫蛋白的分离纯化和鉴定

从异常汉逊酵母中分离出拮抗Cu²⁺、Cd²⁺等重金属、并经铜、镉诱导产生金属硫蛋白的异常汉逊酵母 (Hansenulaanomala)BD1 0 2。无细胞抽提液经SephadexG 50、DEAESepharoseCL 6B、SephadexG 2 5三次凝胶及阴离子交换柱层析分离纯化 ,Cu²⁺诱导得到Cu MTs两个亚型 ,Cd²⁺诱导得到Cd MT一个亚型。Mr分别约为 7kD和 7 5kD ,由 60和 61个氨基酸组成 ,其中半胱氨酸含量各为 6.8%和 1.0 %。每分子金属硫蛋白 (Cu MTs或Cd MT)可结合 4个铜或镉原子     

Two metal-binding peptides from the insect Orchesella cincta (Collembola) as a result of metallothionein cleavage

Metallothionein (MT) is an ubiquitous heavy metal-binding protein which has been identified in animals, plants, protists, fungi and bacteria. In insects, primary structures of MTs are known only for Drosophila and the collembolan, Orchesella cincta. The MT cDNA from O. cincta encodes a 77 amino acid protein with 19 cysteines. Isolations of the protein itself have demonstrated the presence of two smaller metal-binding peptides, whose amino acid sequences correspond to parts of the cDNA, and which apparently result from cleavage of the native protein. The present study was undertaken to complete the picture of cleavage sites within the MT protein by applying protein isolation techniques in combination with mass spectrometry and N-terminal sequence analysis. Further, recombinant expression allowed us to study the intrinsic stability of the MT and to perform in vitro cleavage studies. The results show that the MT from O. cincta is specifically cleaved at two sites, both after the amino acid sequence Thr-Gln (TQ). One of these sites is located in the N-terminal region and the other in the linker region between two putative metal-binding clusters. When expressed in Escherichia coli, the recombinant O. cincta MT can be isolated in an uncleaved form; however, this protein can be cleaved in vitro by the proteolytic activity of O. cincta. In combination with other studies, the results suggest that the length of the linker region is important for the stability of MT as a two domain metal-binding protein.     

Fish and molluscan metallothioneins

Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a beta domain that binds three bivalent metal ions and an alpha domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S-transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal-binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7-MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7-MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms.     

Induction, regulation, degradation, and biological significance of mammalian metallothioneins

MTs are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homology. Mammalian MTs are 61 or 62 amino acid polypeptides containing 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biological demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 years of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biological role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biological role of MTs. This article reviews the current knowledge on the biochemistry, induction, regulation, and degradation of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biological roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.