Metallothionein diversity and its ecological service functions of invertebrates: an overview

The fundamental principles and methods of ecological function regionalization were defined in this paper based on field investigation in the estuarine wetland of Shenzhen River and Xinzhou River. The regionalization scope of the two rivers estuary wetland was delineated, and the main ecological service functions, such as climate regulation, water purification, material production, biodiversity conservation, wildlife habitats, education, recreation and tourism etc., were analyzed and summarized in accordance with the integrity of the eco-system and the administrative divisions status quo. According to the proposed regionalization principles and methods, the two rivers estuary wetland was divided into 6 ecological function units combined with its service functions and distribution characteristics, which were urban construction control area, terrestrial forest ecological belt, futian national mangrove reserves, mangrove restoration area, off-shore tidal flat reservation and estuary waters comprehensive treatment area. For each ecological function units, the leading service functions, Status quo and existing problems were elaborated, the ecological protection, repairation and restoration directions were pointed out, and the strengthening measures of ecological service function were put forward.

Mammalian metallothionein-4 (MT-4) was found to be specifically expressed in stratified squamous epithelia where it plays an essential but poorly defined role in regulating zinc or copper metabolism. Here we report on the organization, stability, and the pathway of metal-thiolate cluster assembly in MT-4 reconstituted with Cd(2+) and Co(2+) ions. Both the (113)Cd NMR studies of (113)Cd(7)MT-4 and the spectroscopic characterization of Co(7)MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd(4)Cys(11) and Cd(3)Cys(9) clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands. Moreover, we have demonstrated that the cluster formation in Cd(7)MT-4 is cooperative and sequential, with the Cd(4)Cys(11) cluster being formed first, and that a distinct single-metal nucleation intermediate Cd(1)MT-4 is required in the cluster formation process. Conversely, the absorption and circular dichroism features of metal-thiolate clusters in Cd(7)MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd(7)MT-1/2. The biological implication of our studies as to the role of MT-4 in zinc metabolism of stratified epithelia is discussed.

Metallobiologists have, at large, neglected soil dwelling invertebrates; exceptions are the nematode (Caenorhabditis elegans) and snails (Helix pomatia and Cantareus aspersus). This review aims to compare and contrast the molecular, protein and cellular mechanisms of the multifunctional nematode and snail metallothioneins (MTs). The C. elegans genome contains two MT genes, mtl-1, which is constitutively expressed in the pharynx and likely to act as an essential and/or toxic metal sensor, and mtl-2, which plays a negligible role under normal physiological conditions but is strongly induced (as mtl-1) in intestinal cells upon metal exposure. It has been possible to follow the intricate phenotypic responses upon the knockdown/knockout of single and multiple MT isoforms and we have started to decipher the multifunctional role of C. elegans MTs. The snails have contributed to our understanding regarding MT evolution and diversity, structure and metal-specific functionality. The H. pomatia and C. aspersus genomes contain at least three MT isoform genes. CdMT is responsible for cadmium detoxification, CuMT is involved in copper homeostasis and Cd/CuMT is a putative ancestral MT possibly only of minor importance in metal metabolism. Further investigations of nematode, snail and other invertebrate MTs will allow the development of alternative biomarker approaches and lead to an improved understanding of metallobiology, protein evolution and toxicogenomics.

Identification, Cloning and Characterisation of a Novel Copper-Metallothionein in Tetrahymena pigmentosa. Sequencing of cDNA and Expression

Background and objectivesUrban scenic forests are a main component of forest parks, and a quantitative study of the landscape color of urban scenic forests can provide a scientific reference for the planning of forest parks and the design of scenic forest management measures. The purpose of the study is to find the quantitative correlation between autumn landscape color and ecological service functions and to obtain the important influencing factors.Materials and methodsThis study focuses on Purple Mountain National Forest Park in Nanjing as a case study area and uses forest resource survey data from Purple Mountain National Forest Park in 2020, autumn landscape color photograph data from Purple Mountain National Forest Park in 2020, and digital elevation model data as the main information sources. The correlation between two ecological functions of above-ground biomass (AGB), tree species diversity (TSD), and influencing factors (including color factors, stand factors, and terrain factors) were both analyzed by Pearson correlation analysis. Then, multiple linear regression (MLR) and random forest (RF) methods were used to perform the quantitative relationship between the functions.ResultsThe results show that, in the established quantitative models of AGB, with TSD as the dependent variable, the correlation coefficients of the MLR model are both above 0.784, while the correlation coefficients of the RF model are all above 0.872. Moreover, the brightness value of the main color (BRI), the number of yellow-green blocks (NYG), and the number of yellow blocks (NY) have important effects on the two ecological service functions.ConclusionIn conclusion, there are complex non-linear relationships between the ecological service functions of AGB, TSD, and influencing factors, and the landscape color can reflect the ecological function of the scenic forest to some extent. In addition, stand factors and color factors have important effects on the ecological function of AGB. Color factors and terrain factors have important effects on the ecological function of TSD. BRI, NYG, and NY have important effects on the two ecological functions. Finally, this quantitative method has universal applicability in the temperate zone, warm temperate zone, and subtropical zone of China.

Metallothioneins (MTs) are ubiquitous, low molecular mass and cysteine-rich proteins that play important roles in maintaining intracellular metal homeostasis, eliminating metal toxification and protecting the cells against oxidative damages. MTs are able to bind metal ions through the thiol groups of their cysteine residues. Plants have several MT isoforms which are classified into four types based on the arrangement of cysteine residues. In the present study, a rice (Oryza sativa) gene encoding type 1 MT isoform, OsMTI-1b, was inserted in vector pET41a and overexpressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST). The recombinant protein GST-OsMTI-1b was purified using affinity chromatography and its ability to bind with Ni(2+), Cd(2+), Zn(2+) and Cu(2+) ions was analyzed. The results demonstrated that this isoform has ability to bind Ni(2+), Cd(2+) and Zn(2+) ions in vitro, whereas it has no substantial ability to bind Cu(2+) ions. From competitive reaction with 5,5'-dithiobis(2-nitrobenzoic acid), DTNB, the affinity of metal ions for recombinant form of GST-OsMTI-1b was as follows: Ni(2+)/Cd(2+) > Zn(2+) > Cu(2+).

BackgroundThe degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu+ or Cd2+. They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion.ResultsHpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd2+, Zn2+ or Cu2+ and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd6-HpCdMT and Cu12-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions.ConclusionThe Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

Natural Selection, the Costs of Reproduction, and a Refinement of Lack's Principle

Metallothioneins (MTs) are small proteins present in all kingdoms of life. Their high cysteine content enables them to bind metal ions, such as Zn2+, Cd2+, and Cu+, providing means for detoxification and metal homeostasis. Three MT isoforms with distinct metal binding preferences are present in the Roman Snail Helix pomatia. Here, we use nuclear magnetic resonance (NMR) to follow the evolution of Cd2+ and Cu+ binding from the reconstructed ancestral Stylommatophora MT to the three H. pomatia MT (HpMT) isoforms. Information obtained from [15N,1H]-HSQC spectra and T2 relaxation times are combined to describe the conformational stability of the MT-metal complexes. A well-behaved MT-metal complex adopts a unique structure and does not undergo additional conformational exchange. The ancestor to all three HpMTs forms conformationally stable Cd2+ complexes and closely resembles the Cd2+-specific HpCdMT isoform, suggesting a role in Cd2+ detoxification for the ancestral protein. All Cu+-MT complexes, including the Cu+-specific HpCuMT isoform, undergo a considerable amount of conformational exchange. The unspecific HpCd/CuMT and the Cu+-specific HpCuMT isoforms form Cu+ complexes with comparable characteristics. It is possible to follow how Cd2+ and Cu+ binding changed throughout evolution. Interestingly, Cu+ binding improved independently in the lineages leading to the unspecific and the Cu+-specific HpMT isoforms. C-terminal domains are generally less capable of coordinating the non-cognate metal ion than N-terminal domains, indicating a higher level of specialization of the C-domain. Our findings provide new insights into snail MT evolution, helping to understand the interplay between biological function and structural features toward a comprehensive understanding of metal preference.

Quantitation of Human Metallothionein Isoforms in Cells, Tissues, and Cerebrospinal Fluid by Mass Spectrometry.

The fine‐tuning of heavy metals in mycorrhizal fungi

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.

Metallothioneins (MTs) from various eukaryotic species were subjected to capillary zone electrophoresis (CZE) performed under the following conditions: Capillary: untreated polyimide-clad fused silica 75 (im I.D. × 94 cm; Loading and Running Voltage: 30 kV; Electrode Buffer: 50 mM Tris-HCI pH 9.1; Sample Buffer: 10 mM Tris-HCI pH 9.1; Sample Volume: 250 μl; Detection: UV absorbance at 214 nm; Loading Method: electrokinetic migration; Loading Times: 1–6 seconds. Complete separation of MT-1 and MT-2 isoforms was achieved in less than 6 min. Using a rabbit liver Cd, Zn-MT standard (0.5 mg/ml), a linear relationship was found to exist between the voltage used to load the MT sample and the integrated peak area of the individual MT isoforms. Similarly, the integrated peak area of the separated MT isoforms was a linear function of the time used to load the sample. These findings suggest that with appropriate standardization CZE is capable of both qualitative and quantitative determinations of MT isoforms. In CZE, the concentration of MT in solution determines the limit of sensitivity. MT could be accurately detected at a concentration as low as 10 μg/ml. CZE was applied to assess thermal and proteolytic degradation of rabbit liver MT. CZE represents a rapid analytical technique for the characterization of MT isoforms that requires very little sample yet provides excellent resolution of individual MT isoforms.

Metallothioneins in aquatic invertebrates: Their role in metal detoxification and their use as biomarkers

A Liquidambar formosana plantation is a kind of fast-grown forest in the subtropical region, providing a variety of ecosystem services such as superior wood, carbon fixation and oxygen release, and biodiversity maintenance. However, the ecological service function value of Liquidambar formosana plantations is not clear. To gain insights into the characteristics and importance of its ecological and economic benefits, the Liquidambar formosana plantation in the Tianjiling Forest Farm of Changsha City was taken as the specific research object in this paper. The ecological service function evaluation index system for Liquidambar formosana plantations was established based on the relevant research worldwide and the actual situation. The market value method, shadow engineering method, carbon tax method, and other environmental economics methods were used to estimate the value of seven ecological service functions (including organic matter production, carbon fixation and oxygen release, water conservation, soil conservation, soil improvement, air purification, and biodiversity maintenance) of the forest of Liquidambar formosana. The results indicated that the total economic value of ecological service function provided by the Liquidambar formosana plantation of Changsha was 103,277.82 RMB/(hm2·a), and the indirect economic value was 8.47 times that of the direct economic value. Among the seven ecological service functions, the value of carbon fixation and oxygen release was the highest (36,703.33 RMB·hm-2·a-1), thus suggesting that the Liquidambar formosana plantation had strong photosynthesis and significant carbon fixation. This study directly reflects the value of forest ecological service function in the form of currency, which is beneficial to provide more insights into forest ecological service function so as to provide basic data and a scientific basis for the protection, construction, and promotion of the sustainable utilization and development of urban forest resources.