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' Constituent Subunit
* Department of Pharmacological Sciences, and
Department of Agrofood Molecular Sciences, University of Milan, 20133 Milan, Italy and
** Department of Medicine, Division of Clinical Chemistry, Albert Ludwigs-University, 79106 Freiburg, Germany
2To whom correspondence should be addressed. E-mail: mariarosa.lovati{at}unimi.it.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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' constituent subunit from 7S globulin in the modulation of LDL catabolism. The results indicated a main fluorescein isothiocyanate-tagged 7S globulin (FITC-7S) component in the cytosolic fraction, that was not present in the nuclear compartment. The electrophoretic mobility of this tagged component suggested either a dissociation of the 7S oligomer or its partial intracellular degradation. Interactions of soybean 7S globulin with FITC-thioredoxin 1 and FITC-cyclophilin B, HepG2 cell membrane proteins, were demonstrated in in vitro assays. In a separate experiment with HepG2 cells, the ability of the ' subunit purified from soybean 7S globulin to modulate the activity of the LDL receptors was evaluated by tracking the uptake and degradation of labeled LDL. The up-regulation of LDL receptors by the ' subunit, as further confirmed by a LDL receptor promoter assay, was significantly greater than that found in the control cells. In conclusion, this study, while confirming our previous indirect evidence of the key role of ' subunit on the cell cholesterol homeostasis, reveals a potentially interesting association of soybean 7S globulin with proteins, such as thioredoxin 1 and cyclophilin B, that are involved in cell protection against oxidative stress.
KEY WORDS: • soybean 7S globulin • capillary electrophoresis • subcellular localization • LDL receptors • cholesterol homeostasis
Soybean proteins are widely accepted as an effective dietary tool in improving the lipid profile in hypercholesterolemic subjects (1,2). Among the mechanisms proposed to explain the cholesterol-lowering properties of soybean proteins, up-regulation of LDL receptors has been suggested by our group, on the basis of results obtained in both hypercholesterolemic type II patients (3) and animal models (4,5), as well as in cell cultures (6,7). In vitro data suggested, in fact, that the 7S globulin, among all storage proteins in soybean seeds, is responsible for the up-regulation of LDL receptors (6) and that this activation is induced by the + ' subunits of 7S, whereas the ß subunit is ineffective (8). The molecular mechanism underlying this biological response is currently under investigation as is its association with the augmented mRNA expression of LDL receptors previously detected after exposure of HepG2 cells to increasing concentrations of 7S globulin (9). The metabolic fate of 7S globulin has been investigated in the same cell model, suggesting a specific uptake and degradation system for this plant protein (10), but no data are presently available on the subcellular localization of soybean 7S globulin in human liver cell cultures or on the protein component(s) involved in the specific interaction between soybean globulin and cell membranes. Recent data concerning the effect of soybean 7S globulin subunits on the up-regulation of LDL receptors indirectly identify the ' subunit as the candidate responsible for this biological effect (8). With a recently developed separation technique (11), it became possible to purify the ' subunit from the other 7S globulin components, the and ß subunits, thus allowing us to evaluate whether this subunit alone is responsible for the up-regulation of LDL receptors, as previously suggested (6,7).
The versatility, increasing use and interesting properties of soybean proteins have promoted the development of different methods for their analysis. Recently, capillary zone electrophoresis was applied to analyze, resolve and quantitate soybean proteins present in different dietary preparations (12). In this work, we utilized this analytical methodology to determine the subcellular localization of fluorescein-tagged 7S globulin in HepG2 cells and to study the in vitro interaction of 7S globulin with tagged thioredoxin 1 (Trx1) and cyclophilin B (CyPB). These two proteins, which are present on the plasma membrane of mammalian cells, are putatively involved in the specific binding of soybean 7S globulin, as previously reported (10). Due to the relevance of these proteins in protecting against reactive oxygen species (13), as well as their multiple effects on cellular physiology (14), it seemed of primary importance to assess the interaction of soybean 7S globulin with Trx1 or with CyPB, to substantiate a relationship between soybean protein intake and the biological responses already described (3,6). Subsequently, experiments with HepG2 cells, exposed to the purified ' subunit of 7S globulin, were carried out with the aim of monitoring the effect of this subunit on the LDL receptors through the evaluation of both uptake and degradation of iodinated LDL and LDL receptor promoter activity.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The established human hepatoma cell line (HepG2) was obtained from American Type Culture Collection (Manassas, VA). Eagle’s minimum essential medium (MEM), fetal calf serum (FCS), trypsin-EDTA (1X), penicillin (105 U/L), streptomycin (100 g/L), tricine buffer (1 mmol/L, pH 7.4) and nonessential amino acid solutions (100X) were from GIBCO (Madison, WI). The protease inhibitor cocktail for use with mammalian cell and tissue extracts was from Sigma-Aldrich (Milan, Italy). Petri dishes were from COSTAR (Cambridge, MA). Filters were from Millipore (Bedford, MA). The Protein Coomassie Plus Protein Assay kit was purchased from Pierce (Rockford, IL). 125Iodine, carrier free, in 100 mmol/L NaOH, was from Perkin Elmer Life Sciences (Boston, MA). Sephadex G25 columns (PD10) were from Pharmacia Biotech (Uppsala, Sweden). Defatted soybean flour was purchased from Cargill BV (Amsterdam, The Netherlands). Human recombinant Trx1 from Escherichia coli was purchased from IMCO AB (Stockholm, Sweden). Cyclophilin B was a gift from Novartis Farma S.p.A. (Origgio, Italy). Dual luciferase assay was from Promega (Madison, WI). Acrylamide monomers and catalysts, a Protean II vertical electrophoresis chamber and a 1000/500 power supply were from BioRad (Hercules, CA). All other chemicals were of analytical grade from Merck (Darmstadt, Germany).
Preparative extraction of 7S globulin and its subfractions from defatted soybean flour.
The separation of 7S globulin from defatted soybean flour was performed as previously described (6,7). For the separation of the ' from the + ß subunits, another 7S globulin preparation was used. The method was a modification of that described by Wu et al. (15), based on the selective solubilization of the 7S globulin with respect to the 11S globulin. This method allowed excellent purification of the 7S globulin, whereas it was less effective for the 11S globulin; however, we did not use the latter in this work. With this 7S globulin preparation, the separation of the constituent subunits was achieved under denaturing conditions with 8 mol/L urea by using a chromatographic approach. The details of this procedure are currently under patent review (11) and will be made available in the scientific literature as soon as possible. The + ' subunits, which were tested for their activity on LDL receptor modulation, were separated from whole 7S soybean globulin as previously described (8).
Fluorescein isothiocyanate (FITC)-tagged proteins.
The 7S globulin, Trx1 and CyPB were incubated overnight at 4°C in conjugation buffer (0.5 mmol/L carbonate/bicarbonate, pH 9.5) in the presence of FITC (30 µg/mg protein) under stirring. Unreacted dye was removed using a PD 10 column by elution with PBS. The integrity of tagged proteins was verified by SDS gradient gel electrophoresis (7.5–17.5%).
Capillary electrophoresis.
The capillary electrophoresis instrument was a Beckman Coulter (Fullerton, CA) P/ACE System 2100 equipped with a Beckman 488-nm emission wavelength laser-induced fluorescence (LIF) module 488. Data acquisition and analysis were performed with a Gold System (Beckman Coulter). The bare silica capillary column (length 50 cm to the detector x 75 µm i.d. and 360 µm o.d.), was obtained from Beckman Coulter. The temperature was kept constant at 30°C, and 20 kV were applied. Injection was performed by pressure (67.73 kPa for 5 s). The separations were performed in 0.05 mmol/L phosphate buffer, pH 8.0, for the subcellular localization of isolated 7S soy globulin and in 0.1 mmol/L phosphate buffer, pH 7.4, for the interaction of isolated 7S soy globulin with Trx 1 or CyPB.
Gel electrophoresis.
Nondenaturing gradient gel electrophoresis was performed on a 7.5–17.5% polyacrylamide gel using a Mini Protean II cell (BioRad). Different amounts of isolated FITC-tagged 7S (FITC-7S; 1, 5, 10 µg) and total cell lysate (50, 100, 200 µg) were applied to the gel. Electrophoresis was carried out at 30 mA/slab. The protein bands in the gel were detected by UV Transilluminator Graphic Lite D 5000 Standard viewer (Uvitec Limited, Cambridge, U.K.).
Cell cultures.
HepG2 cells were grown in monolayers in 90 mm-diameter Petri dishes, and maintained at 37°C in a humidified atmosphere of 95% air, 5% CO2 in MEM supplemented with 10% FCS, nonessential amino acid solution (10 g/L, v/v), penicillin (105 U/L), streptomycin (0.1 g/L), tricine buffer (20 mmol/L, pH 7.4), NaHCO3 (24 mmol/L) and sodium pyruvate (0.11 g/L). For experiments designed to evaluate the subcellular localization of FITC-7S, as well as the LDL receptor modulation, cells were seeded in 35-mm plastic dishes (3–5 x 105 cells) and used just before reaching confluence. In all cell culture experiments, the medium was changed every 2–3 d.
Lipoproteins and lipoprotein-deficient serum.
LDL (1.019 < d < 1.063 kg/L) were isolated by sequential preparative ultracentrifugation (16) from the plasma of clinically healthy normolipidemic volunteers. Lipoproteins were labeled according to the method of McFarlane as modified by Bilheimer et al. (17), as previously described (7). 125I-LDL were sterilized by filtration (Millipore filters, 0.45-µm pore size) and stored at 4°C until use (<10 d after preparation). Human lipoprotein-deficient serum (LPDS) was prepared according to Brown et al. (18).
Subcellular localization of isolated soybean 7S globulin by capillary electrophoresis.
HepG2 cells, grown as described above, were incubated for 24 h in MEM plus 5% LPDS containing 0.5 g/L of FITC-7S, a concentration at which an up-regulation of LDL receptors was previously reported (6). After incubation, cells were washed by standard techniques, and then each dish received 10 mL of 50 mmol/L NaCl, 10 mmol/L (pH 7.4) buffer containing 10 g/L of heparin and was incubated for another hour at 4°C as previously described (10). Heparin, a negatively charged molecule, is widely used in binding experiments as a displacer, e.g., for LDL (17) or 7S soybean globulin (10), from their binding sites. The ability of heparin to release 7S globulin from the cell surface permitted us to analyze its localization into the cells. Nuclei were separated from the cytosolic fractions before processing by capillary electrophoresis. After heparin treatment, HepG2 cells were washed with PBS, harvested and centrifuged for 5 min at 500 x g at 4°C. The cell pellet was treated with Nonidet P-40 (NP-40) lysis buffer [10 mmol/L Tris · Cl, pH 7.4, 10 mmol/L NaCl, 3 mmol/L MgCl2 and 0.5% (v/v) NP-40, containing a protease inhibitor cocktail]. A few microliters of cell lysate were examined with a phase-contrast microscope to ensure that cells had uniformly lysed and nuclei were free of cytoplasmic material. Lysed cells were centrifuged for 5 min at 500 x g at 4°C. The supernatant represented the cytosolic fraction. The nuclear pellet was resuspended in NP-40 lysis buffer and centrifuged again for 5 min at 500 x g at 4°C. The supernatant was discarded and the final pellet represented the washed nuclei. Isolated tagged 7S globulin, cytosol and nuclei fractions of HepG2 cells were processed by LIF-CE.
In vitro interaction of soybean 7S globulin with Trx1 and CyPB by capillary electrophoresis.
The in vitro interaction of isolated 7S globulin with FITC-Trx1 and FITC-CyPB was analyzed by a standard electrophoretic technique and LIF-CE with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. To study the interaction of isolated 7S globulin with the two proteins under investigation, different molar ratios of 7S globulin to the ligands were chosen. Riboflavin (0.7 g/L in 0.1 mmol/L phosphate buffer, pH 7.4) was used as internal standard, allowing us to calculate the corrected protein electrophoretic mobility (µcorr) for each run. The µcorr values were calculated by subtracting the mobility of the electroendoosmotic flow (EOF) (µeo) from the observed protein mobility (µobs) as follows:
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in which veo is the velocity of the EOF, E is the electric field, ld is the distance from the injection point to the LIF detector, trf is the riboflavin retention time, 
V is the electric potential difference, lt is the distance between positive and negative electrodes, van is the measured velocity and tan is the measured retention time of the tagged protein.
The ratio between µcorr calculated for increasing molar ratios of 7S globulin and µcorr in the absence of 7S globulin (µ%) is reported using a Cartesian diagram vs. the molar ratio of 7S globulin to the ligands. Variation in the values of FITC-CyPB or FITC-Trx1 µcorr induced by co-incubation with 7S globulin reflects the complex formation between these proteins.
Uptake and degradation of 125I-LDL.
Monolayers of cells were preincubated for 24 h at 37°C in MEM supplemented with 5% LPDS to up-regulate the LDL receptors (19), in the presence/absence of isolated 7S globulin or its different subunits (', + ') at concentrations listed in Table 1 or 1 µmol/L simvastatin. A fixed concentration (7.5 mg/L) of 125I-LDL protein was then added to the medium and the incubation continued for a further 4 h at 37°C. Specific uptake (binding + internalization) and degradation of 125I-LDL were evaluated as previously reported (7).
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HepG2 cells were plated in 48-well polystyrene plates. Cells were transiently transfected with the reporter gene constructs (0.5 µg per well) by lipofection (Tfx50, Promega) for 4 h. To normalize for transfection efficacy, a control plasmid harboring the Renilla luciferase gene driven by the viral SV40 promoter (pRL-SV40, Promega) was cotransfected, as reported (20). Transfected cells were treated with low serum medium containing the purified ' subunit at different concentrations for 24 h and lysed. Firefly and Renilla luciferase activities were determined using the Dual Luciferase Assay (Promega) on a luminometer (Lumat LB9501, EG&G Bertold). In the construct pLDLR1563, the luciferase gene was placed under control of a LDL-R promoter fragment extending from nucleotides –1563 through –58 of the LDL-R promoter. This fragment harbors repeats 1 through 3, known to be essential for the regulation of LDL-R gene transcription (21). The level of LDL-R promoter activity, relative to transfection, of control cells was set as 1.
Statistical analyses.
Differences in cell uptake and degradation of LDL after incubation with isolated 7S globulin or its subunits (' and + '), and in LDL-R promoter assays in HepG2 cells exposed to the purified ' subunit were determined by ANOVA (SYSTAT 5.2, running on an Apple Macintosh LC 630). Values are expressed as means ± SD. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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After incubation with isolated FITC-7S globulin for 24 h, HepG2 cells were processed as described in Materials and Methods. The capillary electropherograms were carried out on the isolated FITC-7S, and on the cytosolic and nuclear HepG2 fractions. The LIF-CE profile of isolated FITC-7S globulin indicated a heterogeneous composition, likely due to the several heterotrimers resulting from the variable aggregation of the three different subunits that constitute the whole 7S soybean globulin (, ' and ß) (Fig. 1A). Cytosolic (Fig. 1B) and nuclear (Fig. 1C) fraction LIF-CE profiles showed that 7S globulin is taken up by HepG2 cells and processed uniquely in the extranuclear compartment. The presence of a more homogeneous component in the cytosolic matrix (Fig. 1B) with a different retention time (25 min) than the isolated FITC-7S globulin (7–15 min) likely consisted of either one of the 7S subunits, possibly the ß subunit according to Lovati et al. (7), or one resistant major fragment arising from the intracellular proteolytic degradation of the soybean globulin. This component was totally absent in the nucleus, as depicted in Figure 1C.
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The in vitro interaction of isolated 7S globulin with FITC-Trx1 or FITC-CyPB was studied by LIF-CE. 7S globulin solutions are characterized by high viscosity, and the protein is likely to interact with bare silica capillaries, with an ensuing modification of the EOF. Therefore, riboflavin, a small fluorescent vitamin, which does not interact with soybean storage globulins, was co-injected in all runs. Its mobility (µeo) was then used to calculate the EOF values for each run. This procedure allowed us to correct the mobilities of the 7S globulin-containing samples (µcorr) and to compare the relative mobilities of the 7S + FITC-Trx1 or 7S + FITC-CyPB complexes. The mobilities of the mixtures with respect to the tagged ligands alone changed significantly and that mobility was affected by the increased molar ratios between 7S globulin and the ligands (Fig. 3). The association of both proteins with 7S globulin was unaffected even under the high electric fields used in this study, thus suggesting that both ligand proteins interact strongly with the 7S globulin.
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The addition of soybean proteins (7S globulin, + ' and purified ' subunits) to HepG2 cells produced a significant dose-dependent rise in LDL receptor-mediated uptake and degradation compared with the control (Table 1). LDL receptor modulation was greater with the ' subunit than with 7S globulin or the + ' subunit. The result obtained with the purified ' subunit at the higher concentration, was only slightly lower than that of the positive control, simvastatin. The up-regulation of LDL receptors by the purified ' subunit occurred also at the promoter level of LDL receptors. Preincubation of HepG2 cells with 3.5 µmol/L of the ' subunit resulted, in fact, in a 38% increase in the promoter activity of the LDL receptor. This result suggests that the increased uptake and degradation of LDL in response to cell exposure to soybean polypeptides can be attributed at least in part to stimulation of LDL receptor biosynthesis.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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' subunit on LDL receptor modulation was also examined to elucidate the molecular mechanism(s) underlying the up-regulation of the LDL receptors induced by this protein (6). In this context, LIF-CE, because of its high sensitivity, was very useful for detecting tagged 7S globulin components inside the cell and revealing the interactions of this plant protein with other cell proteins. The results obtained with this technique confirmed our previous two-dimensional gel electrophoresis data (7) that 7S globulin is likely to dissociate into constituent subunits or to be degraded in the cytosol of HepG2 cells. The unique localization of 7S globulin or its peptides in the cytosolic compartment, and not in the nucleus, demonstrated that this protein does not interact directly with nuclear structures and that its biological activity is exerted through a more complex mechanism involving interaction with other nonnuclear cell components. No apoptotic activity was in fact demonstrated in cell cultures exposed to 7S globulin from soybean or its peptides, contrary to what was observed with the isoflavone genistein (22). Moreover, the presence in the HepG2 cytosol of a modified 7S globulin with a different LIF-CE retention time than that of the isolated 7S globulin indicated its possible interaction with cell protein component(s). In addition, on the basis of our previous studies showing a marked degradation of the + ' subunit from 7S globulin by HepG2 cells (7), we suggest that peptide(s) deriving from the intracellular proteolysis of 7S globulin might interact with sterol regulatory element binding proteins, likely SREBP-2 (23), thus up-regulating the LDL receptors. The experiments carried out to investigate the ability of 7S globulin to interact with Trx1 or CyPB in vitro had the following rationale: in previous studies we demonstrated that 7S globulin is metabolized in human cell cultures by a specific uptake and degradation system, and that its interaction with cell membranes occurs through specific interactions with relatively low-molecular-weight protein components (10). The N-terminal amino acid sequence analysis of the proteins involved in this specific interaction resulted in the identification of two putative ligand proteins, i.e., Trx1 and CyPB (Prof. Torsten Sejlitz, Pharmacia AB, Stockholm, Sweden, personal communication). The fundamental role of these proteins in homeostasis is widely acknowledged and confirmed by their remarkable conservation through evolution (13). Cyclophilin B, belonging to the family of peptidylproline cis-trans-isomerases (24), is a cyclosporin A-binding protein (25); it is associated mainly with the secretory pathway (24) and is essential to in vivo protein folding (26). In particular, CyPB accumulates in both the endoplasmic reticulum and in complexes on the plasma membrane (14). Thioredoxin 1, a small multifunctional protein with a redox-active disulfide-dithiol in the conserved active site sequence -Cys-Gly-Pro-Cys- (27), plays a variety of roles, ranging from a scavenger of reactive oxygen intermediates (28) to a regulator of proliferation in several cell types (29,30). Because in vitro interaction of the 7S globulin with tagged Trx1 was observed, we suggest that in our cell system, Trx1 could act as a carrier for 7S globulin into the cells, where its effect on the LDL receptor modulation may occur. These results open a new area of investigation of the biological activities exerted by soybean proteins and may help explain the complex mechanisms involved in the slower progression of atheromatous plaque formation in an animal model of human soft plaque (31) fed soy proteins (unpublished data). Recently, Okuda and colleagues (32) observed an increased expression of glutaredoxin and thioredoxin in autopsy samples of human coronary arteries, thus suggesting the possible involvement of thiol-disulfide oxidoreductases in antioxidant protection in human coronary arteries. The interaction of 7S globulin with CyPB, on the other hand, suggests an involvement of this plant protein in the transport of cholesterol between the endoplasmic reticulum and the cell surface caveolae (33).
In the present study we also evaluated the effect of the purified ' subunit from 7S globulin on the LDL receptor modulation, following both the catabolism of labeled LDL and LDL-R promoter activity in HepG2 cells. In a previous paper, we suggested that this subunit may in fact directly up-regulate the LDL receptors (7) because 7S globulin from Keburi, a soybean mutant lacking the ' subunit (34), did not affect LDL receptor up-regulation (8). In those experiments, on the other hand, this conclusion was drawn only indirectly; at that time, due to the similarities between the two subunits, methods for their separation were not yet available. In this work, we took advantage of a new method, currently under patent approval (11), developed to separate ' from + ß subunits. This procedure allowed us to test the ' subunit directly in our cell system. As a result, we found that the up-regulation of LDL receptors induced by the ' subunit parallels the increased LDL-R promoter activity, tested in the same cell system. To our knowledge, this is the first direct evidence of the role of the ' subunit of 7S globulin in the modulation of LDL receptors in HepG2 cells. For the quantitative aspects of the results (Table 1), we calculated the molar concentrations of the protein samples using the molecular weight of the ' subunit (71 kDa) for the sake of comparison among the samples. In fact, no information exists at present on the levels of association of the protein and its isolated subunits under test conditions. Therefore, the lower effect of the 7S globulin and + ' fraction compared with the purified ' at similar molar concentrations can be attributed to the fact that the average content of ' in the heterotrimer of the 7S globulin is only 
25%, whereas in the + ' fraction, it is estimated to be 40% (35). Consequently, the HepG2 cells were actually exposed to increasing amounts of the ' subunit, and the measured effect increased accordingly. In addition, the effect of the purified ' subunit on the variables cited above may be underestimated because of the low solubility of the isolated denatured subunit in physiologic buffers. Whether and to what extent the polypeptide chain partially refolds, self-assembles and becomes soluble under the assay conditions is currently under investigation.
In conclusion, this study, while confirming our previous data on the effect of ' subunit on LDL receptor up-regulation, revealed a potentially interesting association of 7S globulin with Trx1 and CyPB. This latter finding may possibly help to explain the cardiovascular benefit exerted directly by the protein moiety of soybeans, independent of nonprotein components. Nutritional research on the biological consequences of dietary protein intake is particularly active (36), and the role of specific protein components is increasingly recognized. In fact, soybean proteins have been approved by the U.S. FDA as a powerful dietary tool in the treatment and prevention of cardiovascular diseases (37). Interest is focused particularly on those dietary peptides whose biological activities make them potential pharmacologic agents. Our laboratory wanted to identify the peptide(s) responsible for the cholesterol-lowering properties of soybean proteins. In fact, peptides selected on the basis of amino acid sequence differences between the and ' subunits of 7S globulin also modulate LDL receptors in HepG2 cells (38). These findings could lead to the development of foods with beneficial effects on various diseases, including hypercholesterolemia and cardiovascular disease, to be used as an adjunct to drug therapy, particularly in those patients in whom the pharmacologic approaches may be ineffective or have undesirable side effects.
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FOOTNOTES |
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3 Abbreviations: CE, capillary electrophoresis; CyPB, cyclophilin B; EOF, electroendoosmotic flow; FCS, fetal calf serum; FITC-7S, fluorescein isothiocyanate-tagged 7S; GGE, gradient gel electrophoresis; LDL-R, LDL receptor; LIF-CE, laser-induced fluorescence CE; LPDS, lipoprotein-deficient serum; MEM, minimum essential medium; 7S, soybean 7S globulin; Trx1, thioredoxin 1.
Manuscript received 10 December 2002. Initial review completed 7 February 2003. Revision accepted 18 March 2003.
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LITERATURE CITED |
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) of 7S globulin to the ligands were used.