Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588, Japan1
Author for correspondence: Hideaki Matsuoka. Tel: +81 423 88 7029. Fax: +81 423 87 1503. e-mail: bio-cell{at}cc.tuat.ac.jp
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ABSTRACT |
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Keywords: Candida albicans, copper-binding protein, protein purification and characterization, amino-terminal sequence, metallothionein
Abbreviations: CBB, Coomassie brilliant blue; MT, metallothionein; SDTC, sodium diethyldithiocarbamate
a Present address: Natural Products Research Institute, Seoul National University, 28 Yungun-dong, Jongro-ku, Seoul 110-460, Korea.
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INTRODUCTION |
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Candida albicans is the most pathogenic and medically important yeast in the genus Candida (Madhani & Fink, 1998 ). It has been known for some time that clinical isolates of C. albicans and Candida glabrata exhibit high levels of resistance to both copper and cadmium salts, although the molecular basis of this resistance is not known (Malavasic & Cihlar, 1992
; Mehra & Winge, 1991
). Recent studies with C. glabrata have revealed that this yeast employs differing mechanisms to detoxify cadmium and copper salts (Liu & Thiele, 1997
). Cadmium salts stimulate the production of (
-EC)nG peptides, whereas copper salts induce the synthesis of a family of MTs. Several fungi that are pathogenic in humans, including C. albicans, have been screened for the presence of DNA sequences homologous to the Saccharomyces cerevisiae MT gene. Southern blot and restriction enzyme analysis showed that one of the C. albicans strains examined by Butt & Ecker (1987
) contained DNA sequences which hybridize with S. cerevisiae MT. However, detailed analysis of the putative MT locus of C. albicans has yet to be performed. The cloned MT-like gene could be useful as a selectable marker and for regulated gene expression studies in pathogenic fungi. This paper focuses primarily on the purification of a novel copper-binding protein from a clinical isolate of C. albicans. The amino-terminal sequence of the purified protein was determined, and binding of copper was studied.
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METHODS |
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Purification of the copper-binding protein.
Samples (10 g) of freeze-dried yeast cells were physically disrupted with a cold mortar and pestle for 20 min. They were then extracted with 100 ml 20 mM Tris/HCl (pH 7·5) buffer containing: MgCl2 . 2H2O, 5 mM; KCl, 50 mM; glycerol, 5% (w/v); PMSF, 1 mM; DTT, 3 mM; pepstatin A, 1 µg ml-1; and leupeptin, 0·5 µg ml-1. Cell extracts were centrifuged at 28000 g for 30 min to obtain a clarified supernatant. The supernatant was concentrated by lyophilization and suspended in 12 ml N2-saturated 10 mM Tris/HCl (pH 7·4) containing 0·2% ß-mercaptoethanol and then applied to a column of Sephadex G-75 (3x40 cm, Pharmacia Biotech) equilibrated in the same buffer. The copper-binding-protein fractions were identified by copper analysis using atomic absorption spectrophotometry (model AA6600G, Shimadzu). The pooled fractions were concentrated by lyophilization. After resuspension in the same buffer, the sample was ultrafiltered (2000 g, 4 °C, 20 h) using an Ultrafree-4 centrifugal filter unit equipped with a high-flux Biomax membrane (nominal molecular-mass cutoff, 30000 Da; Nihon Millipore). The resulting filtrate containing copper-binding protein was applied to a reverse-phase HPLC µ-Bondapak C18 column (3·9x150 mm; Nihon Waters) connected to a Hewlett Packard HPLC system (HP 1100 series). Adsorbed material was eluted with a linear gradient of 010% solution B (60% acetonitrile in solution A) in solution A (10 mM Tris/HCl containing 0·2% ß-mercaptoethanol, pH 7·4) at a flow rate of 1 ml min-1. The major copper-containing peaks were pooled, 4 vols ice-cold acetone were added and the samples incubated overnight at -80 °C. The precipitate was collected by centrifugation at 28000 g for 15 min and stored anaerobically at -80 °C with Tris/HCl buffer (pH 7·4). This solution was used as purified copper-binding protein for subsequent studies.
PAGE.
Nondenaturing PAGE was carried out at pH 8·9 using a 15% acrylamide gel. Protein was measured by the method of Bradford (1976 ) using bovine serum albumin as a standard. After completion of electrophoresis, the gel was cut into two strips longitudinally. One strip was used for protein staining with Coomassie brilliant blue (CBB) R-250 as follows. The gel was soaked in 10% acetic acid containing 0·25% CBB and 45% methanol for 1 h. It was then destained with 10% acetic acid containing 45% methanol for 1 h and stored in 7% acetic acid containing 5% methanol. The second strip was used for copper staining with SDTC by incubation with 0·2% SDTC solution overnight (Naiki & Yamagata, 1976
). During incubation, the brown band corresponding to the copper-binding protein appeared against a transparent background.
Molecular mass of the copper-binding protein.
The molecular mass of the purified copper-binding protein was measured by HPLC gel filtration on a Shodex Protein KW-802.5 column (8x300 mm, Showa Denko) at a flow rate of 0·3 ml min-1 with 50 mM Tris/HCl containing 0·2 M NaCl (pH 7·5) as an eluent. The molecular mass of the copper-binding protein was measured by comparison with the following calibration standard proteins from the LMW gel filtration calibration kit (Pharmacia Biotech): bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13·7 kDa); and insulin chain B (4 kDa, Sigma).
Preparation of apoprotein and amino-terminal sequence analysis.
The removal of copper ions from the copper-binding protein was accomplished by boiling the protein with a solution of 10 mM EDTA in 7 M guanidinium hydrochloride containing 0·5 M Tris (pH 8·5) for 5 min (Mehra et al., 1988 ). The mixture containing apoprotein was filtered with a 0·22 µm pore-size membrane filter. The resulting filtrate was fractionated by HPLC on a Shodex Protein KW-802.5 column. Adsorbed material was eluted with 0·02% trifluoroacetic acid. The protein peak was localized by measuring absorbance at 214 nm. The copper-free protein peak was pooled and concentrated by lyophilization. The amino-terminal sequence of the copper-binding protein was determined by automated Edman degradation of carboxymethylated apoprotein (Winge et al., 1985
) using a Shimadzu Protein Sequencer PPSQ-10 system (Shimadzu). Cysteine was determined by Edman degradation of S-pyridylethylated cysteine residues.
Reconstitution of the apoprotein with Cu(I).
Copper reconstitution was carried out by the addition of increasing mole equivalents of Cu(I) to the apoprotein. A solution of Cu(I) prepared by the method of Mehra et al. (1988 ) was added to apoprotein (4 nmol) dissolved in 0·02 M HCl to achieve 120 mol eq. copper (mol protein)-1. The samples were neutralized with 200 µl 0·2 M dibasic potassium phosphate and diluted to a final volume of 500 µl with water. The absorbance and luminescence spectra of the reconstituted samples were measured using UV spectrophotometry (model UV-1200, Shimadzu) and spectrofluorometry (model FP-777, Japan Spectroscopic), respectively.
Proton displacement of Cu(I) from the copper-binding protein.
Samples of the native protein (4 nmol) were adjusted to the desired pH by adding 500 µl 0·2 M potassium phosphate preadjusted to that pH. The absorbance spectra of these samples were then recorded. In this experiment, the base line absorbance changed in response to pH change. A base line correction was performed.
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RESULTS |
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DISCUSSION |
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MTs have been described in most vertebrate and invertebrate species as low-molecular-mass proteins with high cysteine content (approx. 30%), a lack of aromatic residues and the presence of 712 heavy metal atoms per molecule (Kagi, 1993 ). Vertebrate MTs constitute a family of highly conserved proteins and the positions of the cysteine residues involved in metal binding are invariant (Hamer, 1986
; Kagi & Kojima, 1988
). Isometallothioneins from the invertebrate Scylla serrata show significant homology to each other as well as to vertebrate MTs (Hamer, 1986
; Kagi & Kojima, 1988
). In contrast, the two MT genes in the invertebrate Drosophila and C. glabrata encode proteins which show little sequence homology to each other (Lastowski-Perry et al., 1985
; Mehra et al., 1989
). In this study, the C. albicans copper-binding protein exhibited the typical MT sequence motif, Cys-Xaa-Cys. The role played by these sequence motifs in the formation of metal clusters in MTs is well recognized (Hamer, 1986
; Kagi & Kojima, 1988
). Thus the C. albicans copper-binding protein is structurally analogous to other well-characterized MTs.
In many cells, MT appears to exist with varying ratios of bound copper. Our reconstitution study suggested that the equivalent extent of Cu(I) binding of the purified C. albicans apoprotein was approximately 14 mol eq. Concentrations of copper in excess of 14 ions per molecule did not alter the absorption properties of the molecule. These results suggested that binding was specific. It is stressed that the value of 14 mol eq. is not rigorous since the UV absorption method provides only an approximation. Substantiation of these results could be obtained using luminescence (Mehra et al., 1989 ). Mammalian MT usually binds seven zinc ions, but it also can contain copper, cadmium and traces of other metals. Although binding stoichiometries and coordination geometry have not been clearly established for metal ions, Cu-MT is one form of the protein that deviates from the usual coordination of seven tetrahedrally bound metal ions per polypeptide (Boulanger et al., 1983
; Nielson & Winge, 1983
). Nielson et al. (1985
) found that 11 or 12 copper ions were bound to MT. The S. cerevisiae CRS5 MT-like protein (Culotta et al., 1994
; Jensen et al., 1996
) and C. glabrata MT (I and II) (Mehra et al., 1989
) bind in excess of 10 copper ions per molecule. It is important to understand the coordination properties and structure of Cu-MT because the protein may function in cellular processes involving copper (Aschner, 1996
; Jasani & Schmid, 1997
). Further studies to elucidate the gene sequence and the cluster structure of the Cu-protein are in progress.
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Received 30 September 1998;
revised 26 April 1999;
accepted 17 May 1999.