Human metallothionein MT-I and MT-II processed genes

Two intronless pseudogenes, corresponding to the human metallothionein MT-I and MT-II processed genes, have been isolated from a human genomic library. MT-I processed gene has accumulated a number of mutations including a nonsense mutation giving rise to a termination codon at amino acid position 21, and a single base deletion at amino acid position 47 causing a shift in the reading frame. MT-II processed gene is a full-length perfect copy of its corresponding mRNA except for a few mutations. Most of the mutations in MT-II processed gene are silent except that the amino acid glycine (GGT) at position 10 is changed to serine (AGT) due to a transition. Both MT-I and MT-II processed genes possess poly(A) sequences of 21 and 17 nucleotides, respectively, 3' to the consensus AATAAA sequence. While these genes are quite similar in their sequences at the 3'-untranslated region, they show less than 50% homology in the 5'-untranslated sequences. Two direct repeats of 16 and 18 nucleotides in length define the limits of the MT-I and MT-II processed genes, respectively, and have been confirmed by S1 nuclease mapping analysis. In both MT-I and MT-II processed genes these direct repeats towards the 5' end of the gene start with an AhaIII (TTTAAA) restriction site. Our studies suggest that these direct repeats are the results of the insertion site duplication.     

Characterization of metallothionein cDNAs induced by cadmium in the nematode Caenorhabditis elegans.

cDNAs of metallothioneins (MTs) in the nematode Caenorhabditis elegans were characterized. The MT-II clone encodes 62 amino acid residues and the predicted Mr is 6462. The MT-I clone contains an additional 12 residues at the C-terminal end, and the predicted Mr is 7959. There is a considerable similarity between MT-I and MT-II. Both of these proteins are cysteine-rich and, with a few exceptions, show a good alignment of cysteine residues. No obvious sequence relationship in the coding region was discernible between C. elegans MTs and mammalian MTs, aside from Cys-Cys, Cys-Xaa-Cys, and Cys-Xaa-Xaa-Xaa-Cys segments. However, 3'-untranslated region of cDNAs of C. elegans MT-I and -II have some consensus sequences found in mammalian MT cDNAs, suggesting that these regions may have some roles in the regulation of MT-gene expression.     

Regulation, linkage, and sequence of mouse metallothionein I and II genes.

The mouse metallothionein II (MT-II) gene is located approximately 6 kilobases upstream of the MT-I gene. A comparison of the sequences of mouse MT-I and MT-II genes (as well as those of other mammals) reveals that the coding regions are highly conserved even at "silent" positions but that the noncoding regions and introns are extremely divergent between primates and rodents. There are four blocks of conserved sequences in the promoters of mouse MT-I, mouse MT-II, and human MT-IIA genes; one includes the TATAAA sequence, and another has been implicated in regulation by heavy metals. Mouse MT-I and MT-II mRNAs are induced to approximately the same extent in vivo in response to cadmium, dexamethasone, or lipopolysaccharide. Mouse MT-I and MT-II genes are regulated by metals but not by glucocorticoids after transfection into HeLa cells.     

Purification, characterization, and cDNA cloning of a novel metallothionein-like, cadmium-binding protein from Caenorhabditis elegans

Caenorhabditis elegans adapted for survival in high concentrations of Cd(II) express a heavy metal binding protein designated C. elegans metallothionein-like protein or MT-Ce. This protein was purified to homogeneity and characterized. MT-Ce binds 6 mol of Cd(II)/mol protein. The sequence of 39 amino-terminal residues in MT-Ce was determined. A radiolabeled 41-mer oligonucleotide, designed from the partial MT-Ce sequence, was used in conjunction with sucrose gradient centrifugation to obtain size-fractionated poly(A+) RNA enriched in MT-Ce sequences. Subsequently, cloned cDNAs, corresponding to MT-Ce mRNA sequences, were isolated from a lambda ZapII cDNA library prepared from the enriched template mRNA. cDNA and protein sequence analysis revealed that MT-Ce comprises 62 amino acid residues and has a predicted Mr of 6462. Seventeen of the 18 Cys residues in the nematode cadmium-binding protein are included in Cys-X-Cys and X-Cys-Cys-X motifs that are characteristic of mammalian metallothioneins (MTs). However, the resemblance of MT-Ce to mammalian MTs is superficial. The amino acid sequence of MT-Ce is unique, and neither its putative alpha and beta domains nor its Cys residues can be readily aligned with the corresponding regions of other eukaryotic MTs. This suggests that MT-Ce is an example of convergent evolution. The MT-Ce mRNA level in nematodes that were selected and grown with Cd(II) concentrations that are lethal for wild-type worms, was 55-fold higher than the level of MT-Ce mRNA in wild-type C. elegans. Comparison of the sequences of MT-Ce cDNAs revealed the occurrence of two types of MT-Ce mRNA. Each contains an identical coding region, but the cDNAs diverge markedly in their 5'-untranslated regions. This suggests the possibilities of regulation by alternative splicing and/or the presence of multiple MT-Ce genes encoding a single protein, but controlled by different regulatory elements.     

Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice.

DNA regions of 10 and 7 kb that flank the mouse metallothionein II (MT-II) and MT-I genes, respectively, were combined with a minimally marked MT-I (MT-I*) gene and tested in transgenic mice. This construct resulted in (i) position-independent expression of MT-I* mRNA and copy number-dependent expression, (ii) levels of hepatic MT-I mRNA per cell per transgene that were about half that derived from endogenous MT-I genes, (iii) appropriate regulation by metals and hormones, and (iv) tissue distribution of transgene mRNA that resembled that of endogenous MT-I mRNA. These features were not observed when MT-I* was tested without the flanking regions. These MT-I flanking sequences also improved the expression of rat growth hormone reporter genes, with or without introns, that were under the control of the MT-I promoter. Moreover, they enhanced expression from two of four heterologous promoters/enhancers that were tested. Deletion analysis indicated that regions known to have DNase I-hypersensitive sites were necessary but not sufficient for high-level expression. These data suggest that the DNA regions flanking the mouse MT-I and MT-II genes have functions like the locus control regions described for other genes.     

Induction of hepatic metallothioneins determined at isoprotein and messenger RNA levels in glucocorticoid-treated rats.

Induction of metallothionein-I (MT-I) and metallothionein-II (MT-II) by glucocorticoids was determined by h.p.l.c. analysis of proteins and Northern-blot analysis of MT mRNAs. Rats were injected with dexamethasone (0.03-10 mumol/kg) and hepatic concentrations of MTs were determined 24 h later. In control rats, only MT-II was detected (9.4 +/- 2.5 micrograms/g of liver), whereas the hepatic concentration of MT-I was below the detection limit (5 micrograms of MT/g). Dexamethasone did not increase MT-I above the detection limit at any dosage tested, but MT-II increased to 2.5 times control values at dosages of 0.30 mumol/kg and higher. Time-course experiments indicated that MT-II reached a maximum at 24 h after a single dosage of dexamethasone and returned to control values by 48 h. To determine whether dexamethasone increased MT-I in liver, samples were saturated with 109Cd, after which the amount of 109Cd in MT-I and MT-II was determined. Results indicated that, by this approach, MT-I and MT-II could be detected in control rats, and there was approx. 1.8 times more 109Cd in MT-II than in MT-I. At 24 h after administration of dexamethasone (1 mumol/kg), there was a small increase in the amount of 109Cd bound to MT-I, whereas the amount of 109Cd bound to MT-II increased to more than 2 times control values. Northern-blot hybridization with mouse cRNA probes indicated that MT-I and MT-II mRNAs increased co-ordinately after administration of dexamethasone. Thus, although glucocorticoids increase both MT-I and MT-II mRNAs, MT-II preferentially accumulates after administration of dexamethasone.     

Membrane phospholipid reorganization differentially regulates metallothionein and heme oxygenase by heme-hemopexin

Heme-hemopexin coordinately regulates genes encoding protective proteins including metallothionein-I (MT-I) and heme oxygenase 1 (HO-1). Hexamethylene-bisacetamide (HMBA), which induces differentiation and activates protein kinase C (PKC), synergistically augments the induction of both MT-I and MT-II mRNAs in response to heme-hemopexin, but attenuates the induction of HO-1. HMBA also augments the increase in MT mRNA in response to cobalt protoporphyrin-hemopexin, a hemopexin (HPX) receptor ligand that activates signaling cascades without tetrapyrrole uptake. Unlike the PKC-activating phorbol esters that induce MT-I and HO-1, HMBA has minimal effects on MT-I or HO-1. HMBA is an amphipathic molecule, and is shown here to interact physically with lipids in model membranes using differential scanning calorimetry (DSC). The data are consistent with a stabilization of the lipid bilayer and an HMBA-induced segregation of lipids into separate domains each relatively enriched in one of the lipids. HMBA also perturbs membrane-protein interactions, and causes a loss of PKC and G-protein subunits from plasma membranes in vitro. Taken together, these observations reveal an additional level of complexity in the regulation of protective proteins induced by HPX, and which may take place in vivo in response to natural compounds that reorganize membrane phospholipids. A model is proposed whereby a reorganization of lipids by HMBA alters signaling pathways and fusion events considered to be the etiology of the differential response of the MT-1 (and MT-II) and the HO-1 genes to HMBA and heme-HPX.     

Separation and quantitation of metallothioneins by high-performance liquid chromatography coupled with atomic absorption spectrophotometry

A rapid, reproducible, and sensitive high-performance liquid chromatography (HPLC) method for the determination of the concentrations of metallothionein-I (MT-I) and metallothionein-II (MT-II) in rat liver has been developed. Metallothioneins (MTs) were separated and quantitated by anion-exchange high-performance liquid chromatography coupled with atomic absorption spectrophotometry (AAS). Purified rat liver MT-I and MT-II, used as standards for developing the method, were easily resolved, eluting at 7.5 and 10.4 min, respectively. To establish standard curves, protein concentrations of solutions of the purified MTs were determined by the Kjeldahl method for the determination of nitrogen, after which the standards were saturated with Cd (final concentration of 50 ppm Cd). Rat liver cytosols obtained from untreated and Cd- or Zn-treated rats were prepared for HPLC-AAS analysis by saturation with Cd (50 ppm Cd) followed by heat denaturation (placing in a boiling water bath for 1 min). Based on the method of standard additions, recovery of MTs exceeded 95% and repeated injection of a sample yielded a coefficient of variance of approximately 2%. A detection limit of 5 micrograms MT/g liver was established for the method. Only MT-II was detected in untreated rats, whereas following exposure to Cd or Zn, both forms of MTs were detected. Concentrations of total MTs in liver of untreated and Cd- or Zn-treated rats were also determined by the Cd/hemoglobin radioassay (which fails to distinguish MT-I from MT-II) and indicated that results obtained with the HPLC-AAS method compared favorably to the Cd/hemoglobin radioassay. Thus, the HPLC-AAS method for quantitating MT-I and MT-II offers the advantage of determining the concentrations of both proteins in tissues and should be useful for studying the regulation of MT-I and MT-II.     

Selective and tandem amplification of a member of the metallothionein gene family in Candida glabrata

Metallothioneins constitute a multigene family in the yeast Candida glabrata. Two genes, designated metallothionein-I (MT-I) and one member of the metallothionein-II family (MT-II), were cloned and sequenced previously (Mehra, R. K., Garey, J. R., Butt, T. R., Gray, W. R., and Winge, D. R. (1989) J. Biol. Chem. 264, 19747-19753). Southern analysis of the genomic DNA samples from different wild-type isolates indicated that the MT-I gene was always present as a single copy but multiple (3-9) and tandemly arranged copies of one MT-II gene were present in different strains. Strains of C. glabrata highly resistant to copper salts were obtained by repeated culturing of wild-type isolates in medium containing increasing concentrations of copper sulfate. These strains showed further stable chromosomal amplification (greater than 30 copies) of the MT-II gene. The MT-I gene remained as a single copy. Amplified copies of the MT-II gene were always arranged tandemly. One of the copper-resistant strains acquired more copies of the MT-II gene by apparent duplication of the chromosome carrying this gene. The size of the amplification unit was 1.25 kilobases. The principal MT-I and -II genes of C. glabrata were shown to map to different chromosomes by electrophoretic karyotypic analysis. The length of chromosome carrying MT-II gene increased appreciably in strains exhibiting the highest amplification of this gene. Northern analysis showed increased basal levels of MT-II mRNA in strains having highly amplified MT-II locus.     

Cloning and sequence analysis of two monkey metallothionein cDNAs

We have isolated two metallothionein (MT) cDNA clones copied from the RNA of cadmium-resistant monkey kidney cells. The complete DNA sequences of these clones show that they encode two distinct MTs. One clone appears to represent monkey MT-II, as shown by its close homology to the human MT-II sequence, whereas the second may correspond to monkey MT-I or a related variant metallothionein. Conserved sequences were identified in both the 5′ and 3′ untranslated regions of these clones.     

Metallothionein Induction in Neonatal Rat Primary Astrocyte Cultures Protects Against Methylmercury Cytotoxicity

Abstract: Metallothionein (MT) protein and mRNA levels were monitored following exposure of rat neonatal primary astrocyte cultures to methylmercury (MeHg). MT-I and MT-II mRNAs were probed on northern blots with an [α-³²P]dCTP-labeled synthetic cDNA probe specific for rat MT mRNA. MT-I and MT-II mRNAs were detected in untreated cells, suggesting constitutive MT expression in these cells. The probes hybridize to a single mRNA with a size appropriate for MT, ∼550 and 350 bp for MT-I and MT-II, respectively. Expression of MT-I and MT-II mRNA in astrocyte monolayers exposed to 2 × 10⁻⁶ M MeHg for 6 h was increased over MT-I and MT-II mRNA levels in controls. Western blot analysis revealed a time-dependent increase in MT protein synthesis through 96 h of exposure to MeHg. Consistent with the constitutive expression of MTs at both the mRNA level and the protein level, we have also demonstrated a time-dependent increase in MT immunoreactivity in astrocytes exposed to MeHg. The cytotoxic effects of MeHg were measured by the rate of astrocytic d -[³H]aspartate uptake. Preexposure of astrocytes to CdCl₂, a potent inducer of MTs, completely reversed the inhibitory effect of MeHg on d -[³H]aspartate uptake that occurs in MeHg-treated astrocytes with constitutive MT levels. Associated with CdCl₂ treatment was a time-dependent increase in astrocytic MT levels. In summary, astrocytes constitutively express MTs; treatment with MeHg increases astrocytic MT expression, and increased MT levels (by means of CdCl₂ pretreatment) attenuate MeHg-induced toxicity. Increased MT expression may represent a generalized response to heavy metal exposure, thus protecting astrocytes and perhaps also, indirectly, juxtaposed neurons from the neurotoxic effects of heavy metals.     

Metallothioneins I and II: neuroprotective significance during CNS pathology

Metallothioneins (MTs) constitutes a superfamily of highly conserved, low molecular weight polypeptides, which are characterized by high contents of cysteine (sulphur) and metals. As intracellular metal-binding proteins they play a significant role in the regulation of essential metals. The major isoforms of the protein (MT-I and MT-II) are induced by numerous stimuli and pathogens but most importantly their induction by metals is closely linked to the physiological metabolism of zinc and protection from the toxic affects following heavy metal exposure. Although the preservation of their genetic expression across animal phyla suggests that MTs may play an important physiological role, MT-I, II knock out (KO) mice survive to adulthood. In both central and peripheral nervous tissues, MT-I, II have neuroprotective roles, which are also induced by exogenous MT-I and/or MT-II treatment. Hence, MT-I, II may provide neurotherapeutic targets offering protection against neuronal injury and degeneration.     

Identification and characterization of asporin. a novel member of the leucine-rich repeat protein family closely related to decorin and biglycan

Asporin, a novel member of the leucine-rich repeat family of proteins, was partially purified from human articular cartilage and meniscus. Cloning of human and mouse asporin cDNAs revealed that the protein is closely related to decorin and biglycan. It contains a putative propeptide, 4 amino-terminal cysteines, 10 leucine-rich repeats, and 2 C-terminal cysteines. In contrast to decorin and biglycan, asporin is not a proteoglycan. Instead, asporin contains a unique stretch of aspartic acid residues in its amino-terminal region. A polymorphism was identified in that the number of consecutive aspartate residues varied from 11 to 15. The 8 exons of the human asporin gene span 26 kilobases on chromosome 9q31.1-32, and the putative promoter region lacks TATA consensus sequences. The asporin mRNA is expressed in a variety of human tissues with higher levels in osteoarthritic articular cartilage, aorta, uterus, heart, and liver. The deduced amino acid sequence of asporin was confirmed by mass spectrometry of the isolated protein resulting in 84% sequence coverage. The protein contains an N-glycosylation site at Asn(281) with a heterogeneous oligosaccharide structure and a potential O-glycosylation site at Ser(54). The name asporin reflects the aspartate-rich amino terminus and the overall similarity to decorin.     

Activation of the complete mouse metallothionein gene locus in the maternal deciduum

The mouse metallothionein (MT) gene family consists of four known members (MT-I through IV) clustered on chromosome 8. Studies reported herein examine the expression and regulation of the MT-III and MT-IV genes in specific cell types in the maternal reproductive tract, developing embryo, and fetus known to express the MT-I and -II genes. MT-III and MT-IV mRNAs were absent from the visceral yolk sac, placenta, and fetal liver, tissues with high levels of MT-I and MT-II mRNAs. In contrast, MT-III and MT-IV mRNAs were both abundant in the maternal deciduum, and in experimentally induced deciduoma on 7 and 8 days postcoitum (1 dpc = vaginal plug), as are MT-I and -II mRNAs. The abundance of each of these MT mRNAs increased coordinately during development of the deciduum (6–8 dpc), and in situ hybridization localized MT-I, MT-III, and MT-IV mRNAs to the secondary decidual zone of the antimesometrial region on 8 dpc, where in some regions all of the cells were apparently positive. Thus, all of the known mouse MT genes are co-expressed in at least some of the cells in the secondary decidual zone. Electrophoretic analysis of decidual MT suggested that the MT-I, -II, and -III isoforms are abundant proteins in the secondary deciduum. Bacterial endotoxin-lipopolysaccharide (LPS) and Zn are powerful inducers of MT-I and MT-II gene expression in many adult organs, whereas these agents apparently have little effect on MT-III and MT-IV gene expression. Neither of these agents significantly effected levels of decidual MT-III or MT-IV mRNAs in vivo or in primary cultures of decidual cells in vitro, and only modest effects of Zn on MT-I mRNA levels were noted. During 2 days of in vitro culture, decidual cell MT-I and MT-III mRNA levels remained elevated while MT-IV mRNA levels decreased. Thus, expression of the mouse MT gene locus in the deciduum appears to be developmentally regulated, and in this tissue, the MT genes are refractory to induction by Zn or inflammation. © 1996 Wiley-Liss, Inc.     

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.