Báo cáo khoa học: Fish and molluscan metallothioneins A structural and functional comparison

Fish and Molluscan Metallothioneins: A Structural and Functional Comparison

Tác giả: Laura Vergani, Myriam Grattarola, Cristina Borghi, Francesco Dondero, Aldo Viarengo

Lĩnh vực: Khoa học Nông nghiệp, Khoa học Sinh học, Hóa sinh, Sinh học Phân tử, Sinh học Cấu trúc, Khoa học Môi trường

Nội dung tài liệu:

Nghiên cứu này so sánh hai loại metallothionein (MT) từ các sinh vật thủy sinh khác nhau về cấu trúc và chức năng: MT A từ cá hồi vân (Oncorhynchus mykiss) và MT 10 từ trai Mytilus galloprovincialis. Cả hai protein tái tổ hợp đều được biểu hiện trong E. coli và được phân tích bằng điện di gel. Các MT này được thử nghiệm về khả năng phản ứng với các tác nhân alkyl hóa và khử, cũng như phân tích cấu trúc bậc hai bằng quang phổ CD. Kết quả cho thấy mặc dù cả hai protein đều có hàm lượng cadmium và khả năng liên kết kim loại tương tự, chúng có sự khác biệt đáng kể về cấu trúc bậc hai, độ ổn định nhiệt và khả năng giải phóng kim loại. MT 10 từ trai cho thấy độ bền nhiệt cao hơn và phản ứng mạnh hơn trong việc giải phóng cadmium khi có mặt H2O2 so với MT A từ cá. Những khác biệt này được cho là do sự khác biệt trong trình tự axit amin, đặc biệt là ở miền alpha, ảnh hưởng đến cấu trúc và chức năng của chúng, có thể liên quan đến môi trường sống khác nhau của hai loài.

Mục lục chi tiết:

• Fish and molluscan metallothioneins: A structural and functional comparison
• Keywords
• Correspondence
• 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 β domain that binds three bivalent metal ions and an α 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 Oncorhynchus 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.
• Metallothioneins (MTs) are cytosolic polypeptides found in almost all organisms, including vertebrates, invertebrates, plants and bacteria [1]. They do not appear to be essential for life, even though they are involved in many pathways, such as sequestration of toxic (Cd, Hg) or essential (Zn, Cu) metals, scavenging of oxyradicals, inflammation, and infection [2]. MTs exhibit unusual primary sequence, lacking histidines and aromatic residues, and their 3D structure is unique [3,4]. Cysteines represent one-third of the total amino acids and are distributed in typical motifs consisting of CC, CXC or CXYC sequences [5]. The behaviour of MTs is dominated by the nucleophilic thiol group reacting with electrophilic compounds, including many alkylating agents and radical species [6]. Vertebrate MTs have a monomeric dumbbell shape, composed of two globular domains connected by a flexible linker consisting of a Lys-Lys segment. Each domain contains a ‘mineral core’ enclosed by two large helical turns of the polypeptidic chain. The N-terminal
• Abbreviations
• MT, metallothionein; GST, glutathione S-transferase; PAR, 4-(2-pyridylazo)resorcinol.
• Comparison between two MTs from aquatic organisms
• right-handed β domain binds three bivalent metal ions. The C-terminal α domain is left-handed and binds four bivalent ions. Zinc is preferentially located in the β domain and cadmium in the α domain. Therefore, the β domain would regulate zinc and copper homeostasis, whereas the α domain may play a central role in heavy metal detoxification [7]. The loosely structured β domain is responsible for metal-bridge dimerization, whereas the α domain is involved in oxidative dimerization. Metal bridge dimerization is reversed by dilution or addition of chelating agents, whereas oxidative dimers are reduced by reducing compounds [8]. As reported previously [9], oxidative dimerization may also occur in vivo under conditions of stress, such as exposure to toxic metals and reactive oxygen species and in neurological disorders (e.g. Alzheimer’s disease). A different susceptibility to oxidation may be important for the physiological role of the protein. Despite the high homology among vertebrate MTs, fish and mammalian MTs exhibit significant differences at the level of primary structure, i.e. displacement of one cysteine and fewer lysines [10]. Compared with vertebrates, invertebrate MTs show unusual features in their primary structure. The sequences of only a few MTs from aquatic invertebrates (crab, mussel, sea urchin, snail and oyster) have so far been elucidated [11-17]. As in mammals and fish [18], echinoderm MTs contain two globular domains binding four and three bivalent ions [19]. On the other hand, in crab (Scylla serrata and Cancer pagurus) the two domains bind three bivalent metals each [20-22]. In comparison with mammalians, molluscan MTs usually have higher glycine content (≈ 15% in mussels), randomly distributed throughout the sequence. Despite the differences, molluscan MTs appear to be more closely related to vertebrate MTs than those from other invertebrate phyla [23,24]. In this study we focused on two MTs from different aquatic organisms which we had widely investigated in previous work [25,26]: MT A from Oncorhynchus mykiss and MT 10 from Mytilus galloprovincialis were selected
• as representative of vertebrates and invertebrates, respectively. Although MT 10 is longer than MT A, both have a similar number of cysteine residues and identical cadmium content. Both recombinant MTs were tested for reactivity to alkylating and reducing agents, to evaluate their susceptibility to oxidative and metal-bridge dimerization. Secondary conformation was analyzed in both the metal-free protein and Cd7-MTs. After metal binding, significant differences between the two forms were observed. The altered secondary structure influenced the physicochemical properties of the proteins, with MT 10 being more thermostable than MT A. When the redox-induced metal transfer from Zn7-MT or Cd7-MT to the specific acceptor 4-(2-pyridylazo)resorcinol (PAR) was measured, MT 10 was much more reactive in terms of cadmium release. This observation is interesting because the redox control of metal bioavailability seems to be an important physiological function of MTs [27].
• Results
• Analysis of primary sequence
• When the primary sequence of mussel MT 10 was compared with that of fish MT A (Fig. 1) with Needleman-Wunsch global alignments [28], a low identity was observed (39%). Because the first extra amino acid number is similar in the two recombinant MTs, we assumed that they affect the two proteins in a similar way. Accordingly, experiments using atomic absorption spectroscopy estimated 7 mol cadmium bound per mol recombinant MT in both samples. The β domain of MT A has nine cysteines distri- buted in classic Cys motifs. In MT 10, this domain is two residues longer, but it has only eight cysteines with a similar arrangement of the CXC motifs. Major differences between the two proteins occur at the level of the α domain, which is longer in MT 10 than in MT A (42 vs. 29 residues) and has two additional cysteines. Moreover, the cysteines are organized differently in
• Fig. 1. Sequence alignment of fish MT A and molluscan MT 10. The sequences of the two recombinant MTs were aligned with the program Needleman-Wunsch global alignments. This program uses the Needleman-Wunsch global alignment algorithm [28] to find the optimum alignment (including gaps) of two sequences when considering their entire length.
• terms of both the CXC or CXYC sequence and Cys motif arrangement. In summary, MT A has six CXC, one CXYC and four CXYWC sequences, whereas mussel MT 10 has nine CXC, one CXYC and five CXYWC sequences. In MT 10, the last α domain Cys motif is CXXXCC, instead of CXCC, which is typical of other vertebrates. This feature has been reported in the MT from the Antarctic fish Notothenia coriiceps [18], but not in mussels. In conclusion, this comparative analysis localizes the major differences between the fish and molluscan MTs at the level of the α domain. Because this cluster is mainly involved in cadmium binding and oxidative dimerization, these differences may reflect functional differences in the two MTs with regard to Cd release and oxidation. A further difference is the reduced number of lysines in MT 10 compared with MT A (5 vs. 7), corresponding to 6.8% of the amino-acid composition for the mussel protein and 11.5% for the fish one. In spite of the fewer lysines, MT 10 contains one more CK motif (5 vs. 4), whereas mammalian MTs have seven. The arrangement of the CK motifs also differs between mussel and fish proteins: the four CK motifs of MT A are equally distributed between the α and β domain, whereas in MT 10, four are at the C-terminus and only one at the N-terminus. As previously reported [29], the hydropathic index (a parameter that is inversely proportional to flexibility) is lower in fish MTs than in mammalian
• MTs. A higher flexibility should facilitate conformational changes in organisms living at low temperatures. When the hydropathic index was calculated for the trout MT A and the mussel MT 10 using the PROTPARAM tool [30], MT A yielded a negative value (-0.110), similar to that recorded for N. coriiceps [29], whereas MT 10 gave a positive value (0.199) consis- tently higher than that for mammalian MTs (0.098). This points to mussel MT having a lower flexibility than either the fish or mammalian counterparts.
• Oxidative and metal bridge polymerization
• After chromatographic purification and enzymatic removal of the glutathione S-transferase (GST) tail, proteins were analyzed by SDS/PAGE (15% gel). As expected, MT A showed a lower molecular mass than MT 10, but also more marked smearing at high molecular mass than MT 10. This effect is due to the presence of polymeric forms typical of native MTs (Fig. 2). When both MTs were alkylated with N-ethyl- maleimide, a unique band at a lower molecular mass appeared, representing the monomeric form, and no differences in mass between the two MTs could be observed. Moreover, alkylation of the thiol group of MT A resulted in disappearance of the smearing at high molecular mass. A similar effect on the aggre- gates was observed when MT A was reduced with dithiothreitol, which caused the appearance of a single
• Fig. 2. Electrophoretic comparisons between fish MT A and mussel MT 10. MT A (A) and MT 10 (B) were electrophoresed on SDS/15% polyacrylamide gel before (lane 1) and after the addition of an alkylating agent (N-ethylmaleimide) at two different concentrations: 40 and 80 mm (lanes 2 and 3) for 3 h. MTs were also treated for the same period with a reducing agent (dithiothreitol) at 40 and 80 mm (lanes 4 and 5). To reduce aggregation, MT samples were handled in anaerobic conditions under nitrogen atmosphere. Molecular markers (lane M) from the top: BSA, 66 kDa; chicken egg ovalbumin, 45 kDa; bovine chymotrypsinogen, 25 kDa; lysozyme, 14.3 kDa; ribonuclease A, 11.9 kDa; bovine lung aprotinin 6.5 kDa.
• band at ≈ 12 kDa, corresponding to the dimeric form of the protein. In contrast, no changes occurred in MT 10 when exposed to the reducing agent, indicating lower susceptibility of the mussel protein to oxidation. These results suggest that the smearing at high molecular mass (12-18 kDa) is due to oxidative polymeriza- tion of the MT molecules, whereas dimer formation (≈ 12 kDa) is probably due to metal bridge effects. The marked differences between the α domain sequences of the two MTs described above is in line with the reduced sensitivity to oxidation exhibited by MT 10, as oxidative polymerization occurs mainly in the α domain.
• Characterization of structure and metal binding
• UV absorption spectra of both MTs were recorded on addition of increasing equivalents of cadmium ions to the metal-free apoprotein, at neutral pH. After Cd(II) titration, a shoulder peak appeared at 254 nm, reflect- ing the charge-transfer interaction of the cadmium- thiolate clusters. In both curves (Fig. 3) absorption at 254 nm increased steadily, until saturation was reached at seven metal equivalents. The slope of the curve was almost identical for the two MTs, indicating no signifi- cant differences with respect to cadmium-binding prop- erties. This agrees with the data acquired by atomic absorption spectroscopy, which estimated 7 mol cad- mium bound per mol MT. On analysis of the CD spectra of the MTs, both metal-free thioneins showed a strong negative band at
• Fig. 3. Spectrophotometric titration following the binding of Cd(II) to the apo-MTs. The Cd-induced contribution to the absorption spectrum at 254 nm is plotted against the number of Cd equiva- lents added, from 0.3 to 8 ratio for both fish MT A (▲) and mussel MT 10 (■). Each curve is representative of at least three independ- ent sets of measurements.
• ≈230 nm (Fig. 4), typical of proteins in random coil conformation [31]. This confirms that both apo-MTs were unfolded in the absence of metals, and only after binding of the correct number of cadmium equivalents did they assume a stable secondary structure. When complexed to the metal, both MTs showed a strong positive ellipticity band above 250 nm, but the peak was red-shifted in the MT 10 spectrum compared with that of MT A. The major differences were evident in the region below 250 nm. In fact, both the negative band at 245 nm and the positive one at 228 nm, char- acteristic of the fish MT A, were lost in the MT 10 spectrum. Considering the spectral peculiarities, we can infer that mussel MT 10 has an atypical secondary conformation, which is probably due to the differences in primary sequence.
• Fig. 4. CD analysis. CD spectra were acquired in the near-UV region (from 190 to 290 nm) for fish MTA (A) and molluscan MT 10 (B) for both Cd7-MT forms and the apoproteins. The metal- free protein was obtained by acidification with HCl. The measure- ments were performed on three different MT preparations.
• Thermal stability
• We examined whether the differences in primary and secondary structure affected the thermal stability of the two MTs. UV absorption spectra were acquired for fish and mussel Cd7-MTs, after exposure to a thermal gradient. A254 was plotted as a function of tem- perature. As expected, in both cases we observed a decrease in percentage absorbance with temperature increase (Fig. 5). The absorbance of fish MTA declined steadily starting at 30 °C, with a marked change in slope above 50 °C, similar to the description of D’Auria et al [18]. The thermal profile of MT 10 showed a similar trend, but the slope change occurred at a higher temperature (above 60 °C). Moreover, MT 10 maintained higher percentage absorbance at all temperatures than the fish protein (0.7 vs. 0.5 at 90 °C, respectively). These results suggest that the mussel MT is much more thermostable at high temperatures than the fish protein. This is in line with the greater rigidity suggested by the hydropathic index.
• Fig. 5. Thermal stability of fish and mussel Cd7-MTs. Absorption UV spectra were acquired for fish MT A (▲) and mussel MT 10 (■) as a function of the temperature increase from 20 to 90 °C. The absorbance decrease at 254 nm was reported as a fraction of the standard absorbance (absorbance at room temperature) in order to compare the denaturation profile of the Cd-thiolate chromophore of the two MTs. Each curve is representative of four independent sets of measurements.
• Kinetics of metal release
• Cysteine residues can be oxidized in vitro by mild cellu- lar oxidants and release metals during the process [32-34]. It has been suggested that oxidoreductive mechanisms may also modulate in vivo the affinity of cysteines for metal ions and regulate the bioavailability of bivalent metals [35]. In the presence of the glutathione redox couple (GSH/GSSG), we observed zinc release from both
• recombinant MTs. The kinetics