TY - JOUR
AU - Tsubaki, Motonari
AB - Abstract Candidate human tumour suppressor gene product, 101F6 protein, is a highly hydrophobic transmembrane protein and a member of cytochrome b561 family. Purified 101F6 protein expressed in Pichia pastoris cells showed visible absorption spectra similar but distinct from those of cytochrome b561. Haem content analysis indicated presence of two haems B per molecule. Midpoint potentials of the purified protein were found as +109 and +26 mV for two haems, slightly lower than those for bovine chromaffin granule or plant Zea mays cytochromes b561. Electron paramagnetic resonance (EPR) spectra in oxidized state at 5 K showed only a highly anisotropic low-spin (HALS) signal at gz = 3.75. However, at 15 and 20 K, another HALS-type signal appeared at gz = 3.65 being overlapped with that of gz = 3.75. The rhombic EPR signal at gz = 3.16 previously seen in other cytochromes b561 was not observed, suggesting distinct haem environments. Absence of the inhibition in the electron transfer from ascorbate by a treatment of 101F6 protein with diethylpyrocarbonate showed a remarkable contrast from those of other cytochromes b561 where the ‘concerted H+/e− transfer mechanism’ at the cytosolic haem centre was blocked by specific Nε-carbethoxylation of haem-coordinating imidazole, suggesting that 101F6 protein might accept electrons via a mechanism distinct from other cytochromes b561. ascorbate, cytochrome b561, electron transfer, EPR, membrane protein The cytochrome b561 family is composed of haem-containing transmembrane electron transfer proteins that are widely distributed in living organisms (1). Bovine cytochrome b561 present in adrenal chromaffin granules (CGs) was found to carry out the electron transfer reaction from ascorbate (AsA) in the cytosol to monodehydroascorbate (MDA) radical inside the vesicles (2–5). Recycling of AsA inside the vesicles would ensure the continuous supply of co-factors for the dopamine β-hydroxylase enzyme that catalyses the synthesis of cathecholamines such as adrenaline and noradrenaline (4,5). The bovine CG cytochrome b561 purified from adrenal glands, with an estimated molecular weight of 28 kDa, was the most characterized one in the cytochrome b561 family (6,7). It contains six transmembrane α-helices and two b-type haems, one on each side of the vesicle membranes (8). The midpoint redox potentials reported have values ranging from +145 to 170 mV for the high potential haem and +30 to 70 mV for the low potential haem (9,10). Electron paramagnetic resonance (EPR) spectra of intact chromaffin granules as well as the purified CG cytochrome b561 showed signals characteristic of the two haems, one with gz = 3.7 for a highly anisotropic low-spin (HALS) species (11) and the other at gz = 3.14 signal, which is similar to that of cytochrome b5 (6,12,13). Tumour suppressor genes are important factors in the development of cancer, as mutations or deletions of these genes can lead to an inactivation or an alteration of the responsible protein or enzyme (14,15). Chromosomal deletions on tumour suppressor genes such as p53, WT1 and RB1 were found to be associated with lung and breast cancers (16). It may be very difficult to estimate the number of tumour suppressor genes that might be involved in human lung carcinogenesis (17). In a review by Kohno and Yokota (17), more than 40 genes from various chromosomal regions were cited as susceptible for homozygous deletions and loss of heterozygosity. On the other hand, for human breast cancer, regions of chromosome 3p such as 3p25, 3p22-24, 3p21.3, 3p21.2-21.3, 3p14.2, 3p14.3 and 3p12 were reported for losses of heterozygosity. It was also cited that chromosome 3p region contains tumour suppressor genes such as retinoid acid receptor β2 (3p24), thyroid hormone receptor β1 (3p24.3), Ras association domain family 1 A (3p21.3) and fragile histidine triad gene (3p14.2) (18). A 630-kb region in 3p21.3 was identified as a critical region for lung cancer homozygous deletions (19). Contiguous candidate tumour suppressor genes within this region had been identified. Six of these genes 101F6, NPRL2, BLU, RASSF1, FUS1, HYAL2 and HYAL1 were used to construct recombinant adenovirus vectors for the transformation of the cancer cells and for animal model studies (20). Upon forced expressions of FUS1, 101F6 and NPRL2 genes in the cultured cancer cells, considerable inhibition of tumour growth by apoptosis and changes in cell cycle processes were observed (20). In experimental lung metastases mouse models, intratumoural injection of the protamine-complexed adenovirus vectors, Ad-101F6, Ad-NPRL2, Ad-FUS1 and Ad-HYAL2, drastically controlled the tumour growth (20). Interestingly, one of these candidate tumour suppressor genes, 101F6 was predicted to code for a protein that is homologous to cytochrome b561 (1). In contrast to normal lung cells, 101F6 protein would be lost in most lung cancers (21). Forced expression of the 101F6 protein via nanoparticle-mediated gene transfer along with subpharmacological doses of AsA resulted in significant suppression of lung cancer cell growth in vitro (21). Cancer cells were observed to have increased uptake of AsA, increased production of hydrogen peroxide, which might cause induction of caspase-independent apoptosis and autophagy (21). In animal model studies, tumour growth in the 101F6-nanoparticle and AsA-treated lung cancer mice was also significantly suppressed (21). The 101F6 protein is ubiquitously expressed in animal tissues, but in higher levels in liver, kidney and lung (22). In cells, 101F6 may be localized in small vesicles, endosomes and endoplasmic reticulum and may possess ferric reductase activity (22). Recombinant mouse tumour suppressor 101F6 protein was recently expressed and purified from Saccharomyces cerevisiae cells (23). Characterization of the recombinant protein revealed several properties similar to CG cytochrome b561, such as AsA reducibility, absorption spectra, slightly lower reduction potentials but possibly slightly different haem binding environments (24). Among the mammalian cytochromes b561, the tumour suppressor protein is considered to be the least homologous to the prototype adrenal CG cytochrome b561 based on the amino acid sequence, which may account for some unique properties of the 101F6 protein (24). In our present study, the candidate human tumour suppressor 101F6 protein was expressed as a histidine-tagged protein in yeast Pichia pastoris cells and was highly purified by metal affinity chromatography. The fusion protein, with a molecular weight of 26 kDa, was found to have characteristic absorption spectra as a member of the cytochrome b561 protein family as well as a high reducibility by AsA. The protein was found to have slightly lower reduction potentials than those of its closest relative, the mouse 101F6 (24). Interestingly, EPR spectra of 101F6 protein in the oxidized state both in the purified form and in the microsomal membrane form were different from those of other members of the cytochromes b561 family, suggesting that members of the E subfamily (1) including 101F6 protein possess two distinct HALS haem species (11). Experimental Procedures Protein expression system and its purification The heterologous expression system for human 101F6 protein was constructed using methylotropic yeast P. pastoris cells and pPICZB vector (Invitrogen) as previously described (25). Briefly, the expression plasmid contained the human 101F6 sequence in the database (AF040704, DDBJ) and additional sequences coding for the thrombin-specific site SALVPRGSSA (underline indicates the sequence for the recognition) and an eight histidine (His8)-tag at the 3′-end. The expression plasmid, named as pPICZB-101F6-H8, was incorporated into the genome of P. pastoris GS115 cells after the linearization with PmeI. The transformation was performed using the EasyComp transformation protocol (Invitrogen Corp., Tokyo, Japan). Following selection procedure was conducted by plating the cells on Yeast Extract Peptone Dextrose Sorbitol Medium (YPDS) agar plates containing 100–400 mg ml−1 Zeocin (Invitrogen Corp.). Culture of the transformed cells was done as previously described (25). Purification of the expressed protein was done as previously described (25), but with slight modifications with omission of a step of DEAE-Sepharose column chromatography and the use of higher concentration of imidazole (50 mM) in the washing buffer during a step of Ni-NTA Sepharose column chromatography. SDS–PAGE was performed using the Laemmli method (26) using 4 and 15% acrylamide concentration for stacking and resolving gels, respectively. Protein bands were visualized with Coomassie Brilliant Blue R-250, while the histidine-tag was detected using a histidine-tag detection reagent (His-Detect In Gel-Stain; Nacalai Tesque, Kyoto, Japan). After SDS–PAGE, the proteins were transferred onto a nitrocellulose membrane. To confirm the presence of the 101F6 protein, a primary antibody (goat anti-CYB561D2 polyclonal IgG antibody that is specific for the COOH-terminal VSNAYLYRKRIQP sequence of the 101F6 protein; IMGENEX, IMG-3051) was used to form the protein–antibody complex on the membrane. Then, the complex was detected using a horseradish peroxidase-conjugated secondary antibody [anti-goat IgG(H+L) HRP-conjugated; RCK #605-703-002] and colour development was achieved using H2O2 and 4-chloro-1-napthol as substrates. UV-Visible absorption spectroscopy UV-visible absorption spectra of the purified 101F6 protein were analysed in the region from 700 to 200 nm using a Shimadzu UV-2400PC spectrophotometer (Shimadzu Corp., Kyoto, Japan). The AsA-reduced form was attained by adding AsA solution to a final concentration of 10 mM, whereas the fully reduced form was achieved by adding small grains of sodium dithionite. Total amounts of the 101F6 protein were estimated as the cytochrome b561 content using the difference in absorbance at 561 and 575 nm for the dithionite-reduced form ‘minus’ oxidized form and 27.7 mM−1 cm−1 as a difference extinction coefficient. MALDI-TOF mass spectrometry Mass spectrometric analyses were conducted with a Voyager DE Pro mass spectrometer (Applied Biosystems, Foster City, CA, USA) as previously described (9,27) using a 20-kV accelerating voltage. The mass spectra were acquired by adding the individual spectrum from 256 laser shots. For peptide analysis, the samples were analysed in a reflector mode. The recombinant 101F6-H8 protein (30 μM) was digested either with TPCK-treated trypsin (0.01 mg ml−1). After 48 h of incubation at room temperature, the peptide solution was diluted 1:9 (v/v) with a matrix solution [α-cyano-4-hydroxycinamic acid (Aldrich, Gillingham, UK), 50 mg ml−1 in 50% acetonitrile in 0.3% TFA]. The mixtures (typically, 1.0 μl) were deposited on a sample plate, allowed to air-dry and analysed. The search of the corresponding fragments in the amino acid sequence of 101F6-H8 was conducted using the program GPMAW (v. 6.11) (Lighthouse Data, Odense M, Denmark). Haem content analysis The purified 101F6-H8 protein was converted to a pyridine haemochrome by the addition of pyridine and NaOH according to the method described previously (28), and modified based on another study (29). An extinction coefficient value for haem B pyridine haemochrome of 34.4 mM−1 cm−1 at 557 nm (in the absolute spectrum) (28) was used. Protein concentration was determined by a modified Lowry method (30) and by a Bradford method (31) (Protein Assay Kit, Bio-Rad). Bovine serum albumin was used as a standard in each method. Concentration of the standard solution was assessed spectrophotometrically using an extinction coefficient of 6.60%−1 cm−1 at 280 nm. Redox titration Spectroscopic redox titrations were performed essentially as described by Dutton (32) and Takeuchi et al. (27) using a Shimadzu UV-2400PC spectrometer equipped with a thermostatted cell holder connected to a low temperature thermobath (NCB-1200, Tokyo Rikakikai Co, Ltd, Tokyo, Japan). A custom anaerobic cuvette (1-cm light path, 5-ml sample volume) equipped with a combined platinum and Ag/AgCl electrode (6860-10C, Horiba, Tokyo, Japan) and a screw-capped side arm was used. Purified 101F6-H8 sample (final, 5 μM) in 50 mM potassium–phosphate buffer (pH 7.0) containing 1.0% (w/v) octyl β-glucoside was mixed with redox mediators (potassium ferricyanide, 60 μM; quinhydrone, 20 μM; 1,2-naphthoquinone, 20 μM; phenazine methosulphate, 20 μM; duroquinone, 40 μM; 2-Hydroxy-1,4-naphtoquinone, 5 μM; riboflavin, 20 μM). After passing through a cellulose acetate filter (DISMIC-25cs, 0.45 μm; Toyo Roshi Kaisha, Ltd, Tokyo, Japan), the sample solution was placed in a cuvette. The sample was kept under a flow of moistened argon gas to exclude dioxygen and was continuously stirred with a small magnetic stirrer (CC-301, SCINICS, Tokyo, Japan) inside. Reductive titration was performed at 20°C by addition of small aliquots of sodium dithionite (5 or 20 mM) solution through a needle in the rubber septum on a side arm; for a subsequent oxidative titration, potassium ferricyanide (5 or 20 mM) was used as the titrant. In an appropriate interval, visible absorption spectra and redox potentials were recorded. The changes in absorbance (A561.0 − A566.8, the isosbestic point of 101F6-H8 protein) were corrected considering the dilution effect and analysed with Igor Pro (v. 6.03A2) by employing Nernst equations with a single redox component,   or two redox components,   in which the parameters, rate1 and rate2, were fixed as 25.619 (mV). EPR spectra measurement The purified 101F6-H8 (∼300 μM) protein in 50 mM potassium phosphate buffer pH 7.0 containing 1% octyl β-glucoside was placed in an EPR tube and frozen in liquid nitrogen. The X-band (9.23 GHz) microwave frequency EPR spectra were recorded on a Varian E-109 EPR spectrometer with 100-kHz field modulation. The microwave frequency was calibrated with a microwave frequency counter (Takeda Riken Co., Ltd, Model TR5212). An Oxford flow cryostat (ESR-900) was used to achieve temperatures down to 4–20 K, as previously described (6,7). Diethylpyrocarbonate treatment of 101F6-H8 The oxidized 101F6- H8 solution was diluted to 6.5 µM with 50 mM potassium phosphate buffer (pH 7.0) containing 1.0% (w/v) octyl β-glucoside. The sample was treated with 0.5 mM (final) diethylpyrocarbonate (DEPC) for 60 min, as previously described (9,27,33). During the DEPC treatment, the difference spectra in UV-visible region were recorded every 5 min with a Shimadzu UV-2400PC spectrophotometer at room temperature. The DEPC-treated samples were gel-filtered through a PD-10 column equilibrated with 50 mM potassium–phosphate buffer (pH 7.0) containing 1.0% (w/v) octyl β-glucoside to remove unreacted DEPC. The sample was then assayed for the electron accepting activity from AsA, using a Shimadzu UV-2400PC spectrophotometer at room temperature. In brief, the oxidized 101F6- H8 (or the DEPC-treated oxidized 101F6- H8) solution was mixed rapidly with AsA (final 10 mM) in an aerobic condition and the absorption change was monitored at 425 nm for 30 min. Results and Discussion Biochemical evaluation of the recombinant human 101F6 protein The candidate human tumour suppressor 101F6 protein, expressed in the methylotropic yeast P. pastoris cells, was purified to homogeneity by metal affinity chromatography. The histidine-tagged fusion protein had a theoretical molecular weight of 25,996 Da. SDS–PAGE analysis of the purified protein showed a single protein band with a molecular size of 26 kDa (Supplementary Fig. S1). Direct staining of the protein in SDS–PAGE gel with a histidine-tag detection reagent confirmed that indeed the 26-kDa protein contained a histidine-tag sequence (Supplementary Fig. S1). Western blot analysis of the purified protein by the 101F6 COOH-terminal sequence-specific antibody identified the 26-kDa protein as the genuine 101F6 protein (Supplementary Fig. S1). Figure 1 shows a part of the MALDI-TOF mass spectrum of the intact purified protein revealing a mass of 25,900 Da, which was slightly lower than the value of our previous report (25) but was very close to the theoretical mass if we consider the expected loss of the initial Met residue, which may occur during the post-translational modification in the heterologous expression system. In the next stage, we conducted a peptide-mapping analysis using MALDI-TOF mass spectrometry. Representative mass spectra for the tryptic (Supplementary Fig. S2) peptides of human 101F6-H8 protein are shown in a region from 6,000 to 8,500 (m/z). Identified peaks for some cleaved products are labelled. In total, of 240 amino acid residues of the 101F6-H8 protein, including the octa-His residue and the thrombin-recognition sequence, we could identify most of the cleaved peptides with a coverage of >99%. We found that there was no post-translational modification occurring on the 101F6-H8 fusion protein. These results confirmed the successful expression and purification of the intact 101F6-H8 protein. We considered this histidine-tagged fusion protein as the native form of human 101F6 protein because presence of a His-tag moiety at the COOH-terminus of Zea may cytochrome b561 did not show any significant effects on its redox activities (34). Fig. 1 View largeDownload slide MALDI-TOF mass spectrum of the purified 101F6-H8 fusion protein. The experimental mass of the protein was found as 25,900.20, which was very close to the theoretical average mass of 25,996.83 Da based on the amino acid sequence of the 101F6-H8 fusion protein. Fig. 1 View largeDownload slide MALDI-TOF mass spectrum of the purified 101F6-H8 fusion protein. The experimental mass of the protein was found as 25,900.20, which was very close to the theoretical average mass of 25,996.83 Da based on the amino acid sequence of the 101F6-H8 fusion protein. The UV-visible absorption spectra of 101F6-H8 protein in oxidized, AsA-reduced and dithionite-reduced states were shown in Fig. 2. The spectrum in oxidized state was similar to those of other members of the cytochrome b561 family and of mouse 101F6 protein expressed in S. cerevisiae cells (24). The spectrum in dithionite-reduced state was also similar to those of other members of the cytochrome b561 family; but, as noticed previously (24), the splitting of the α-band was more enhanced than other cytochromes b561 by showing a clear shoulder at the shorter wavelength side of the peak. Upon addition of AsA, 101F6-H8 protein was partially reduced with a level of ∼65%, which was comparable with, or slightly lower than, other members of the cytochrome b561 family. Fig. 2 View largeDownload slide UV-visible absorption spectra of 101F6-His8 protein in oxidized (trace a), ascorbate (AsA)-reduced (trace b) and dithionite-reduced (trace c) states. Details are described in the text. Inset shows a result of pyridine haemochrome assay of the purified sample, indicating the presence of haem B. Fig. 2 View largeDownload slide UV-visible absorption spectra of 101F6-His8 protein in oxidized (trace a), ascorbate (AsA)-reduced (trace b) and dithionite-reduced (trace c) states. Details are described in the text. Inset shows a result of pyridine haemochrome assay of the purified sample, indicating the presence of haem B. Haem content of the purified recombinant human 101F6 protein was found to be 1.59 (±0.06) mole of haem B/mole protein based on the pyridine haemochrome assay (Fig. 2; inset), in which two different methods (modified Lowry and Bradford methods) for the determination of protein concentration with bovine serum albumin as a standard gave the same results. This value was slightly lower than the value (1.70) for bovine CG cytochrome b561 obtained by a similar method (6). However, a more recent pyridine haemochrome assay using a highly purified sample showed a value of 1.78 mole of haem B/mole protein (Okano et al., unpublished results). It was predicted that as a member of the cytochrome b561 family, 101F6 protein would have two haems B for one protein molecule (1). Present results concluded that indeed there were two haem b moieties per molecule of 101F6 protein. Redox potential Simultaneous potentiometric and spectroscopic titration were conducted to determine the midpoint redox potentials of two haem centres in the purified 101F6-H8 protein. By assuming a single redox centre, the estimated midpoint potential was estimated as +63 mV but the fitted curve was not satisfactory at all. By assuming two one-electron redox centres, the curve-fitting was satisfactory for both the reductive and oxidative titration data in accordance with the results of the haem content analysis. We estimated the midpoint redox potentials as +109 and +26 mV, respectively (Fig. 3). This is in good agreement with our previous redox titration trials with values ranging from +90 to +110 mV for the high potential haem and from +10 to +26 mV for the low potential haem (25). The midpoint potential values for the two haem centres were slightly lower than the reported values for other mammalian cytochromes b561. Bovine CG cytochrome b561 was reported to have midpoint potentials ranging from +145 to +170 mV and from +30 to +70 mV, respectively (9,10,27). On the other hand, the midpoint potentials for plant Z. mays cytochrome b561 were found to be +123 and +15 mV (34). Midpoint potentials for mouse 101F6 were reported to have values of +140 and +40 mV (24). Differences in the measured redox potentials between human and mouse 101F6 may be affected by experimental conditions, such as detergents (octyl β-glucoside versus sucrose monolaurate) and determination method as well as the innate differences in the amino acid sequence and the composition of the proteins (24). Human and mouse 101F6 proteins have the same number of amino acids, almost the same molecular weights and 95% identity (Fig. 4); but the difference in 10 amino acids may have caused these two proteins to differentiate in redox potentials. Fig. 3 View largeDownload slide Determination of midpoint redox potentials of the recombinant human histidine-tagged 101F6 protein. Simultaneous spectroscopic and electrochemical titrations were performed using 5 μM of purified 101F6-H8 protein in 50 mM potassium-phosphate buffer (pH 7.0) containing 1.0% (w/v) octyl β-glucoside in the presence of redox mediators as described in the text. For the analysis assuming a system with two independent, one-electron redox components, the midpoint potentials were estimated as +109 mV and +26 mV. Although for the analysis assuming a system with one redox centre, the midpoint potential was estimated as +63 mV. Fig. 3 View largeDownload slide Determination of midpoint redox potentials of the recombinant human histidine-tagged 101F6 protein. Simultaneous spectroscopic and electrochemical titrations were performed using 5 μM of purified 101F6-H8 protein in 50 mM potassium-phosphate buffer (pH 7.0) containing 1.0% (w/v) octyl β-glucoside in the presence of redox mediators as described in the text. For the analysis assuming a system with two independent, one-electron redox components, the midpoint potentials were estimated as +109 mV and +26 mV. Although for the analysis assuming a system with one redox centre, the midpoint potential was estimated as +63 mV. Fig. 4 View largeDownload slide Comparison of the amino acid sequences of bovine CG cytochrome b561 (CGb561), Z. mays cytochrome b561 (Zmb561) and human and mouse 101F6 proteins (h101F6 and m101F6) in the central domain region. The central region of the cytochrome b561 protein family contains four transmembrane helices (light gray background). Within the region, four heme-coordinating His residues (dark grey background), in which the first and third His residues coordinate intravesicular heme center, whereas the second and the fourth His residues bind to cytosolic heme center. Putative AsA-binding and putative MDA-binding sequences are shown with a single underline. Additionally, a Lys residue (34,35) and an Arg residue (36), which were shown to have some important roles in AsA-binding, are indicated with a double underline. Fig. 4 View largeDownload slide Comparison of the amino acid sequences of bovine CG cytochrome b561 (CGb561), Z. mays cytochrome b561 (Zmb561) and human and mouse 101F6 proteins (h101F6 and m101F6) in the central domain region. The central region of the cytochrome b561 protein family contains four transmembrane helices (light gray background). Within the region, four heme-coordinating His residues (dark grey background), in which the first and third His residues coordinate intravesicular heme center, whereas the second and the fourth His residues bind to cytosolic heme center. Putative AsA-binding and putative MDA-binding sequences are shown with a single underline. Additionally, a Lys residue (34,35) and an Arg residue (36), which were shown to have some important roles in AsA-binding, are indicated with a double underline. We demonstrated previously that the purified 101F6-H8 protein was reducible with AsA (25). Such reducibility with AsA was also confirmed for the mouse 101F6 protein (24). The estimated redox potentials were in good agreements with AsA as a possible electron donor. Human embryonic kidney HEK293T cells transfected with the mouse 101F6 gene construct were observed to have the ability to reduce extracellular ferric ions (22). Mizutani et al. (22) postulated that mouse 101F6 might have ferric reductase activity, although it was not clear whether this activity involved an intracellular reductant and a transmembrane electron transfer reaction. AsA which functions as an electron donor for many physiological processes in living cells may also be a direct electron donor for the 101F6 protein, as for other members of cytochrome b561 protein family, as far as the redox potentials are concerned. EPR spectra For bovine adrenal CG cytochrome b561, two major EPR signals at gz = 3.7 and gz = 3.14 are indicative of the presence of two types of low-spin ferric haems (6). It was proposed that the signal at gz = 3.7 with a HALS character corresponds to the haem species with a low midpoint potential. This haem was proposed to be located on the cytosolic side and believed to accept an electron equivalent from cytosolic AsA (6,7). Accordingly, the gz = 3.14 signal was assigned to the high-potential intravesicular haem (6,7). This haem might have similar environments with those of cytochrome b5. This view was challenged recently as that the gz = 3.7 signal was assignable to the intravesicular haem and vice versa (37–39). In this new topological model, however, the electron transport occurs against the ∼100 mV gradient of the redox potentials of the two haem centres (37). In the present study, EPR spectra of the recombinant human 101F6-H8 showed a sharp signal of gz = 3.65 and a weak one at gy = 2.27 at 15 and 20 K, and a sharp signal of gz = 3.75 at 5 K (Fig. 5A). As the signals around g ∼3.7 seemed to show a temperature-dependent peak shift from g = 3.65 to g = 3.75 (Supplementary Fig. S3), we speculated that these signals might arise from two distinct haem species with slightly different gz-values (at 3.65 and 3.75) having different temperature dependency. Therefore, we conducted a power dependency analysis for the EPR intensities at g = 3.73 and 3.65 at 5 K (Fig. 6). The result indicated that two EPR signals at g = 3.75 and 3.65 had distinct power dependency and were, therefore, derived from two distinct haem centres. We conducted the deconvolution of the broad signal at g ∼ 3.7. Temperature difference spectra (i.e. 5 K − 20 K and 5 K − 15 K) showed that the broad signal at g = 3.7 was actually composed of two distinct components (gz3.75 species with a peak at gz = 3.75) and (gz3.65 species with a peak at gz = 3.65) (Fig. 5B). Based on the gz values and classifications of various ferric-type low-spin haems by Walker (11,40), presence of these two EPR signals could indicate that the human 101F6 protein contains two HALS haems. On the other hand, the EPR signals at gz = 3.14, as previously observed in CG membranes (41), bovine CG cytochrome b561 (6), human Dcytb (42) and Arabidopsis (43) and Z. mays (34) cytochromes b561, were not observed in the spectra of human 101F6 protein. This fact raised a speculation that a cytochrome b5-like (rhombic) haem species in the classical cytochromes b561 where two planes of the axial His ligands being oriented in parallel to each other might have been converted to a HALS species where the planes oriented in quasi-perpendicular to each other in the human 101F6 protein. On the other hand, recombinant mouse 101F6 protein showed EPR spectra having signals at gz = 3.61, gz = 2.96 and gy = 2.26 at 5 K, 10 K and 15 K (24). Although the former gz = 3.61 signal seems very close to that observed in the present study (i.e., the gz=3.65 HALS species), other two EPR signals are significantly different from our present result (gz = 2.96 was missing and gy = 2.26 is much stronger in intensity than in our gy = 2.27 signal) and are also inconsistent with the results for other cytochromes b561. Thus, the recombinant mouse 101F6 protein seemed to contain one low-spin haem species with HALS type (type I; corresponding to gz = 3.61 species) and one low-spin haem species with cytochrome b5-like character (type II; corresponding to the gz = 2.96 and gy = 2.26 species), although the quality of the EPR spectra were somewhat ambiguous (24). However, shifts, appearance or disappearance of signals in the EPR spectra of the cytochrome b561 family were reported to occur occasionally when they undergo various processing or treatments. Oakhill et al. (42) reported that the EPR spectra of recombinant Dcytb protein expressed in Sf9 cells had varying amounts of three haem species depending on the preparation conditions. They observed that, for the sonication-treated sample, the gz = 3.7 signal was more prominent. On the other hand, the sample without a sonication step had a more prominent EPR signal at gz = 2.95. Takeuchi et al. (44) observed a conversion of the gz = 3.1 species to a gz = 2.94 species by the treatment of CG cytochrome b561 with a cysteine-modifying reagent. Fractionation by centrifugation of the membrane extracts from CG cytochrome b561-expressing Sf9 cells resulted in the disappearance of an EPR signal previously present when the membrane fractions were centrifuged to include heavier components (12). It is possible that certain purification steps or reagents used in the preparation of the mouse 101F6 protein might cause the EPR signals to vary from the native ones as observed for other cytochromes b561. It must be stressed further that according to our recent study, there was no indication of the gz = 3.14 signal nor gz = 2.96 signal in the EPR spectra of human 101F6 protein in Pichia microsomal membranes (Fig. 7), ruling out the possibility that absence of the gz = 3.14 signal or gz = 2.96 signal was not due to the artefact produced during the solubilization and purification steps. Further, a harsh treatment of the Pichia microsomal membranes caused an appearance of a rhombic species with gz = 2.98 and gy = 2.25 (spectra, data not shown). Since the human 101F6 protein has the least sequence identity with other members of the cytochrome b561 protein family (Fig. 4), it is more likely that human 101F6 protein has unique haem centres distinct from other classical members of the cytochrome b561 protein family. Being consistent with this view, our recent EPR study on the purified human CYB561D1 protein (cytochrome b561 domain-containing protein 1) being expressed similarly in P. pastoris cells indicated that there were two HALS-type haem signals being overlapped around gz = 3.6 but no EPR signals around the gz = 3.14 or gz = 2.96 (Asada et al., unpublished observation). As the human CYB561D1 protein and 101F6 protein belong to the same E subfamily (1), it might be very likely that presence of two HALS-type haem species (and therefore the absence of EPR signals around the gz = 3.14 or gz = 2.96) is a common property of this protein subfamily. Fig. 5 View largeDownload slide EPR spectra of the purified recombinant histidine-tagged human 101F6 protein (101F6-H8) in air-oxidized state. [Upper (A)] Wide-scan spectra of 101F6-H8 measured at 5, 15 and 20 K. The protein concentration was ∼300 μM in 50 mM potassium phosphate buffer (pH 7.0) containing 1.0% octyl β-glucoside. [Bottom (B)] Deconvolution of the broad g = 3.7 signal by taking the difference between a lower temperature spectrum and the higher temperature spectrum, which when multiplied by an indicated factor showed two components (i.e., gz3.75 and gz3.65 species, thick lines). Asterisks in the 5 K spectrum in upper panel indicate the signals from impurities attached on the EPR sample tube. Conditions; modulation frequency, 100-kHz; modulation amplitude, 10 G; microwave power, 10 mW. Fig. 5 View largeDownload slide EPR spectra of the purified recombinant histidine-tagged human 101F6 protein (101F6-H8) in air-oxidized state. [Upper (A)] Wide-scan spectra of 101F6-H8 measured at 5, 15 and 20 K. The protein concentration was ∼300 μM in 50 mM potassium phosphate buffer (pH 7.0) containing 1.0% octyl β-glucoside. [Bottom (B)] Deconvolution of the broad g = 3.7 signal by taking the difference between a lower temperature spectrum and the higher temperature spectrum, which when multiplied by an indicated factor showed two components (i.e., gz3.75 and gz3.65 species, thick lines). Asterisks in the 5 K spectrum in upper panel indicate the signals from impurities attached on the EPR sample tube. Conditions; modulation frequency, 100-kHz; modulation amplitude, 10 G; microwave power, 10 mW. Fig. 6 View largeDownload slide Power dependency of the EPR intensities for recombinant human 101F6-H8 protein measured at the X-band (9.23 GHz) microwave frequency at 5K. The fusion protein (∼300 μM) was suspended in 50 mM potassium phosphate buffer, pH 7.0 containing 1% (w/v) octyl β-glucoside. Power-dependent EPR intensities were measured with magnetic field strength at 175 mT (corresponding to gz = 3.75) and at 183 mT (corresponding to gz = 3.65). Fig. 6 View largeDownload slide Power dependency of the EPR intensities for recombinant human 101F6-H8 protein measured at the X-band (9.23 GHz) microwave frequency at 5K. The fusion protein (∼300 μM) was suspended in 50 mM potassium phosphate buffer, pH 7.0 containing 1% (w/v) octyl β-glucoside. Power-dependent EPR intensities were measured with magnetic field strength at 175 mT (corresponding to gz = 3.75) and at 183 mT (corresponding to gz = 3.65). Fig. 7 View largeDownload slide EPR spectra of microsomal membranes of P. pastoris cells expressing recombinant human 101F6-H8 protein measured in air-oxidized state with the X-band (9.23 GHz) microwave frequency at 5 and 15 K.Pichia microsomal fraction containing ∼90 µM of human 101F6-H8 protein in air-oxidized state in 50 mM potassium phosphate buffer, pH 7.0 was measured. Fig. 7 View largeDownload slide EPR spectra of microsomal membranes of P. pastoris cells expressing recombinant human 101F6-H8 protein measured in air-oxidized state with the X-band (9.23 GHz) microwave frequency at 5 and 15 K.Pichia microsomal fraction containing ∼90 µM of human 101F6-H8 protein in air-oxidized state in 50 mM potassium phosphate buffer, pH 7.0 was measured. Effects of the modification with DEPC Previously, we employed DEPC for analyses on the electron transfer mechanism of bovine CG and Z. mays cytochromes b561 (9,27,33). DEPC is well known as a chemical modification reagent with high selectivity towards a de-protonated nitrogen atom of an imidazole ring of His residues (45). We previously showed that DEPC treatment of bovine CG cytochrome b561 in oxidized state caused a significant inhibition of the electron transfer from AsA both in the final reduction level (30–35%) and in the initial reaction rate (∼1/400) and such inhibition was caused by specific Nδ-carbethoxylation of the haem axial His residue with the bond between the haem and the axial His residue being intact (9,27,33). We proposed further that the haem axial His residue might be responsible for the ‘concerted H+/e− transfer mechanism’ (46) at the cytosolic haem centre to withdraw a proton from the bound AsA molecule to facilitate the electron transfer to the cytosolic haem iron (47). If human 101F6 protein utilized cytosolic AsA as the physiological electron donor for its putative transmembrane electron transfer activity, it is very likely that the ‘concerted H+/e− transfer mechanism’ at the cytosolic haem centre might be operative. To examine such a possibility, we treated the purified recombinant human 101F6 protein with DEPC. Upon addition of DEPC to the 101F6-H8 protein in oxidized state at a high molar ratio (DEPC:101F6-H8 = 77:1), absorbance change in UV region with a positive peak at 240 nm occurred, followed by a gradual change in the Soret region with a negative peak at 415 nm (Fig. 8A). The changes in the UV region indicated that chemical modifications occurred on some His residues exposed to solvents and/or with a higher reactivity. Extent of the N-carbethoxylation of His residues after 30 min of the treatment was calculated based on the absorbance change at 240 nm (ΔA240) using a molar extinction coefficient of 3.2 mM−1 cm−1 for N-carbethoxylated histidine (45), and it was estimated as 8.76 per molecule. Considering the total number of His residues (17, including 8 residues of the His-tag) in the deduced amino acid sequence of human 101F6-H8, the estimated value was much larger than the corresponding values observed for bovine CG cytochrome b561 (9,27) and Z. mays cytochrome b561 (34) and was comparable with those of cytochrome b5 (48) and six-coordinated porcine myoglobin mutant (VHA-Mb) (49) upon the DEPC-treatments under similar experimental conditions. The slight change in absorbance at the Soret band was not concerted with a rapid progress in absorbance at 240 nm (Fig. 8A), indicating that majority of the N-carbethoxylation of His residues occurred at distant sites from the haem centres. This behaviour was also different from bovine CG cytochrome b561 (9,27) and Z. mays cytochrome b561 (34). Fig. 8 View largeDownload slide Effects of DEPC treatment on the UV-visible absorption spectra and its electron accepting ability of purified recombinant human 101F6-H8 protein. (A) Difference spectral changes after the addition of DEPC to the oxidized form of 101F6-H8 (6.5 µM) were monitored every 5 min. Just after the addition of DEPC (final, 0.5 mM), rapid increase in absorbance at 240 nm due to the formation of N-carbethoxyl histidine residues was observed. In a later time-domain, a gradual decrease in absorbance, ∼415 nm was observed. (B) Time courses of absorbance measured at 425 nm of the human 101F6-H8 protein pre-treated with DEPC after mixing with AsA (final, 10 mM) (broken line) aerobically. A control experiment (using untreated 101F6-H8) was shown in a solid line. Fig. 8 View largeDownload slide Effects of DEPC treatment on the UV-visible absorption spectra and its electron accepting ability of purified recombinant human 101F6-H8 protein. (A) Difference spectral changes after the addition of DEPC to the oxidized form of 101F6-H8 (6.5 µM) were monitored every 5 min. Just after the addition of DEPC (final, 0.5 mM), rapid increase in absorbance at 240 nm due to the formation of N-carbethoxyl histidine residues was observed. In a later time-domain, a gradual decrease in absorbance, ∼415 nm was observed. (B) Time courses of absorbance measured at 425 nm of the human 101F6-H8 protein pre-treated with DEPC after mixing with AsA (final, 10 mM) (broken line) aerobically. A control experiment (using untreated 101F6-H8) was shown in a solid line. The DEPC-treated 101F6-H8 protein was, then examined by a spectroscopic mixing assay to measure the electron accepting ability from AsA in the detergent-solubilized state. Surprisingly, the DEPC-treatment did not affect significantly on the electron accepting ability of 101F6-H8 from AsA both in the final reduction level and in the initial quick rise of the reduction level (Fig. 8B and Supplementary Fig. S4). The absence of the inhibition in the electron transfer from AsA by the DEPC treatment was further confirmed by a stopped-flow analysis (results not shown; Recuenco et al., unpublished observation). Further, redox potential measurements of the DEPC-treated 101F6-H8 protein did not show any significant changes (10–15 mV positive shift for both haem moieties) (Supplementary Fig. S5). These unexpected observations suggested that the Nδ-carbethoxylation of axial His residue(s) did not occur for human 101F6 protein and therefore human 101F6 protein might not utilize the ‘concerted H+/e− transfer mechanism’ at the cytosolic haem centre (47) upon the electron acceptance from cytosolic AsA. Alternatively, this may merely indicate that AsA is not a physiological electron donor for human 101F6 protein at all. Distinct haem environments and putative AsA-binding site structure of 101F6 protein Figure 4 shows the comparison of the amino acid sequences of human and mouse 101F6 proteins with those of bovine CG and Z. mays cytochromes b561, in which only a central part of the four helix bundle is shown. It is apparent that the immediate haem environments for both cytosolic and intravesicular haems are not conserved well in the 101F6 proteins. In our present EPR study, we found that one of the two EPR species in the molecule, a type II (rhombic EPR species) haem, was converted to a type I (HALS-type) haem species in human 101F6 protein, which may be consistent with the lower homology in the amino acid sequence (Fig. 4). As there were two HALS-type EPR signals overlapped in the spectra, we could not assign precisely the haem signals; but the gz = 3.65 species was more likely corresponding to the intravesicular haem. This assignment was based on the temperature dependency of the two HALS-type EPR signals (Supplementary Fig. S3); the gz = 3.75 species was predominant at 4 K, which was more likely corresponding to the gz = 3.7 species locating on the cytosolic side of classic type cytochrome b561 (6). In any case, it might be concluded that the 101F6 protein has clearly different haem environments from those of other members of the cytochrome b561 protein family. This might be partially responsible for the appearance of the splitting of α-band peak and the lowered redox potentials. The absence of the inhibitory effect of the DEPC treatment on the electron transfer activity from AsA was very important in considering the physiological role(s) of 101F6 proteins in living cells. Present result indicated that human 101F6 protein might not utilize the ‘concerted H+/e− transfer mechanism’ at the cytosolic haem centre upon the electron acceptance from cytosolic AsA (47). This property is very different from other classic members of cytochrome b561 protein family. Figure 4 shows that, indeed, conservation of the amino acid sequences corresponding to the putative AsA-binding site was very low. Particularly, the conserved positively charged residues (Lys and Arg, indicated in red), as claimed previously to be important for the interaction with a negatively charged AsA molecule (34–36) are missing in the 101F6 sequence. Instead, it might be noticed that other positively charged residues which are well-conserved among the subfamily of 101F6 proteins are distributed on the cytosolic side. These results suggested that cytosolic AsA might donate its reducing equivalent to the cytosolic haem centre via a mechanism different from other cytochromes b561, provided that human 101F6 proteins use cytosolic AsA as the physiological electron donor. Figure 4 also shows the lower conservation of amino acid sequences for the putative MDA-binding site, indicating some possible differences in the electron transfer reaction to MDA radical from the interavesicular haem centre. This particular subject is now under our current investigation. In cultured human cancer cell studies, the cells transfected with the 101F6 gene nanoparticles exhibited increased uptake of AsA, leading to an accumulation of H2O2 and would undergo caspase-independent apoptosis and autophagy pathways (21). It is possible that cellular signals would activate the expression of 101F6 protein so that there would be an increase of the rate of AsA recycling by donating reducing equivalents to MDA radical very efficiently. Being consistent with this view, our recent study using pulse radiolysis technique, purified recombinant 101F6 protein exhibited a strong electron donating activity towards the pulse-generated MDA radical, several times faster than those of other cytochromes b561 (Recuenco et al., unpublished observation). The AsA that was generated on the lumen side of the small vesicles or endoplasmic reticulum may be used as a co-factor to pathways leading to caspase-independent apoptosis and autophagy, thus, 101F6 protein will act as a tumour suppressor. In conclusion, we found that the haem centres of human 101F6 protein in oxidized state were distinctly different from other members of the cytochrome b561 protein family. The lower homology in the amino acid sequence of human 101F6 to other members of the cytochrome b561 family would account for the difference in the haem environments as observed in the EPR spectra. Further, the absence of the inhibition in the electron transfer activity upon the DEPC treatment of human 101F6 protein suggested strongly that human 101F6 protein might accept reducing equivalents from AsA via a mechanism distinct from those of other cytochromes b561. It is very likely that these unique properties of human 101F6 protein might be responsible for the physiological function of this protein as a putative tumour suppressor during the development of human cancers. Funding Grants-in-Aid for Scientific Research on Priority Areas (System Cell Engineering by Multi-scale Manipulation; 18048030 and 20034034 to M.T.); the Ministry of Education, Culture, Sports, Science and Technology in Japan and by Grant-in-Aid for Scientific Research (C) (22570142 to M.T.); Japan Society for the Promotion of Science. Conflict of interest None declared. 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TI - Functional characterization of the recombinant human tumour suppressor 101F6 protein, a cytochrome b561 homologue
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvs139
DA - 2012-12-11
UR - https://www.deepdyve.com/lp/oxford-university-press/functional-characterization-of-the-recombinant-human-tumour-suppressor-I0XUtODlwc
SP - 233
EP - 242
VL - 153
IS - 2
DP - DeepDyve
ER -