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Originally published In Press as doi:10.1074/jbc.M606543200 on October 26, 2006

J. Biol. Chem., Vol. 281, Issue 52, 39852-39859, December 29, 2006
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Human Erythrocyte Membranes Contain a Cytochrome b561 That May Be Involved in Extracellular Ascorbate Recycling*

Dan Su{ddagger}, James M. May§, Mark J. Koury§, and Han Asard{ddagger}1

From the {ddagger}Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 and the §Department of Medicine, Vanderbilt University School of Medicine and Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee 37232

Received for publication, July 10, 2006 , and in revised form, September 27, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human erythrocytes contain an unidentified plasma membrane redox system that can reduce extracellular monodehydroascorbate by using intracellular ascorbate (Asc) as an electron donor. Here we show that human erythrocyte membranes contain a cytochrome b561 (Cyt b561) and hypothesize that it may be responsible for this activity. Of three evolutionarily closely related Cyts b561, immunoblots of human erythrocyte membranes showed only the duodenal cytochrome b561 (DCytb) isoform. DCytb was also found in guinea pig erythrocyte membranes but not in erythrocyte membranes from the mouse or rat. Mouse erythrocytes lost a majority of the DCytb in the late erythroblast stage during erythropoiesis. Absorption spectroscopy showed that human erythrocyte membranes contain an Asc-reducible b-type Cyt having the same spectral characteristics as recombinant DCytb and biphasic reduction kinetics, similar to those of the chromaffin granule Cyt b561. In contrast, mouse erythrocytes did not exhibit Asc-reducible b-type Cyt activity. Furthermore, in contrast to mouse erythrocytes, human erythrocytes much more effectively preserved extracellular Asc and transferred electrons from intracellular Asc to extracellular ferricyanide. These results suggest that the DCytb present in human erythrocytes may contribute to their ability to reduce extracellular monodehydroascorbate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ascorbate (Asc)2 plays an important role as a molecular antioxidant and cofactor to several metabolic enzymes (1). A variety of species, including humans, higher primates, and guinea pigs, has lost the ability to synthesize Asc because they lack a functional L-gulonolactone oxidase, the final enzyme of the Asc biosynthetic pathway in mammals (2). Thus, efficient mechanisms for Asc absorption, transport, and recycling are important in these species. Asc is delivered to tissues through the blood, in which it is believed to be the primary antioxidant (3, 4). Reaction with an oxidant converts Asc to its oxidized forms, which leads to loss of Asc in the blood. Asc oxidation is a two-step reaction in which monodehydroascorbate (MDHA) and dehydroascorbate (DHA) are produced by loss of one and two electrons, respectively. Two molecules of MDHA can also react with each other to form one molecule each of Asc and DHA. DHA is unstable and undergoes irreversible hydrolysis to 2,3-diketo-L-gulonic acid (5, 6), which results in decreased levels of the vitamin. Therefore, in addition to dietary intake, efficient Asc regeneration systems are needed. Most of the systems identified so far that are capable of re-reducing MDHA and DHA to Asc are located within cells. For example, MDHA can be reduced by NADH- and NADPH-dependent oxidoreductases (7–9), and DHA can be recycled to Asc via direct reduction by glutathione (GSH) (10, 11), by GSH-dependent enzymes such as glutaredoxin and protein-disulfide isomerase (12), and by NADPH-dependent thioredoxin reductase (8, 13).

In recent years, the mechanisms by which Asc is maintained in blood have been studied. Erythrocytes, as the most abundant cells in blood, are likely to play a crucial role in recycling Asc in blood plasma. Erythrocytes can take up extracellular DHA through the GLUT1 glucose transporter for subsequent intracellular reduction to Asc (14). However, because of the slow release of Asc back into the plasma, this mechanism is not very efficient in maintaining blood Asc levels. Recently, redox enzymes in the erythrocyte plasma membrane were found to directly reduce extracellular DHA and MDHA, using intracellular NADH and/or Asc as electron donors (15–18). Because MDHA is the initial product of Asc oxidation, and the final oxidation product (DHA) is unstable, Asc recycling from MDHA is generally considered to be more efficient and economical than recycling of DHA. An unknown redox system in human erythrocyte membranes was reported to reduce extracellular MDHA, using intracellular Asc as an electron donor (16, 18). The possibility of small electron carriers transferring electrons and mediating this reduction was excluded (19). The biochemical nature of this Asc regenerating system remains a mystery.

Cyts b561 are transmembrane proteins that mediate Asc-driven transmembrane electron transport (20, 21). One member of this protein family, the chromaffin granule Cyt b561 (CGCytb), has been shown to reduce MDHA, at least in vitro, thereby regenerating Asc (22, 23). This protein would be an obvious candidate for the extracellular MDHA reduction observed in erythrocytes. However, Van Duijn et al. (24), demonstrated that CGCytb is not present in erythrocyte membranes and that there is no CGCytb mRNA in erythrocyte progenitor cells. However, with the recent identification of other Cyt b561 family members, the question arises whether any of these isoforms might play a role in the erythrocyte-mediated Asc regeneration. Among five mammalian Cyt b561 family members, three are evolutionarily closely related, suggesting that they may share functional similarities. These are CGCytb, an isoform located in the duodenal brush border cell plasma membrane (DCytb) (25), and a recently identified isoform localized in lysosomal membranes (LCytb) (26). Genetic and biochemical studies support a role for DCytb in iron metabolism, where it functions as a ferrireductase (25). Although the MDHA reductase activity of CGCytb has been documented for many years, we and others have shown that this protein also displays ferrireductase activity when expressed in yeast (27) or Xenopus oocytes (28). It therefore remains an open question whether these Cyts b561 can have both MDHA reductase and ferrireductase activities in vivo or whether one of these activities is favored under physiological conditions.

We hypothesized that one of the Cyts b561 is present in human erythrocyte membranes and that it can function as a transmembrane MDHA reductase using intracellular Asc as an electron donor. In this study, we show that DCytb, but not CGCytb or LCytb, is indeed localized in the human erythrocyte membrane. We also provide evidence to support the possibility that DCytb is involved in extracellular Asc recycling of human erythrocytes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Gene Cloning, Protein Expression in Yeast, and Mammalian Cell Line—cDNAs of CGCytb and LCytb were amplified from mouse kidney RNA; cDNA of DCytb was amplified from mouse duodenal RNA. They were cloned into a pESC-His vector (Stratagene, La Jolla, CA) using EcoRI and SpeI restriction sites. Yeast cells (Saccharomyces cerevisiae, strain YPH499, ura3-52 lys2-801amber ade2-101achre trp1-{Delta}63 his3-{Delta}200 leu2-{Delta}1) were transformed and grown according to the manufacturer's instructions (Stratagene). The transformed yeast cells were grown in synthetic dextrose dropout medium lacking His (SD-His). Protein expression was induced by transferring to synthetic galactose dropout medium lacking His (SG-His). DCytb was cloned into the pcDNA3.1(–) vector (Invitrogen) using XbaI and EcoRI restriction sites. Human embryonic kidney-293 cells (HEK-293 cells; Invitrogen) were transfected using Lipofectamine 2000 (Invitrogen).

Preparation of Erythrocytes and Erythrocyte Ghosts—Human erythrocytes were obtained from freshly drawn heparinized blood from normal volunteers. Mouse erythrocytes were prepared from mouse whole blood with either heparin or EDTA as anticoagulant. Guinea pig erythrocytes were prepared from guinea pig whole blood in Alsever's solution (Rockland Immunochemicals, Gilbertsville, PA). Rat erythrocytes were prepared from rat whole blood with EDTA as anticoagulant (rat whole blood was provided by Dr. Andrea Cupp, University of Nebraska, Lincoln). Erythrocytes were washed three times in 10 volumes of phosphate-buffered saline (PBS), which consisted of deionized water containing 140 mM NaCl and 12.5 mM Na2HPO4, pH 7.4. The buffy coat of white cells was removed with each wash. The unsealed ghosts were prepared from intact cells as described previously (29).

Protein Extraction, Membrane Preparation, and Stripping—Yeast cells were collected when the A600 reached 0.8. Cells were washed with ice-cold homogenization buffer (50 mM Mops-KOH, pH 7.0, 5 mM EDTA, 100 mM KCl, and 100 mM sucrose) and broken in a bead beater (Biospec Products, Bartlesville, OK). Yeast protein extraction and membrane preparation were done as reported previously (30).

HEK-293 cells were collected 24 h after transfection and were rinsed twice with ice-cold PBS. The cell lysate was prepared with lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, pH 7.0) supplemented with freshly added protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 1 µg ml–1 of each of the following: aprotinin, pepstatin, leupeptin, antipain, and chymostatin), and 0.5% Triton X-100. The cell lysates were centrifuged at 3,000 x g for 10 min, and the supernatant was stored at –80 °C before gel loading.

Yeast membrane fractions and erythrocyte ghost membranes were "stripped" to remove soluble proteins trapped inside the vesicles and loosely bound proteins. The membrane fractions were made up to 10 ml by adding storage buffer (20 mM Mes-Tris, pH 6.8, 2% glycerol, and 1 mM Asc). Triton X-100 was added to a final concentration of 0.025% (w/v). Samples were incubated at room temperature on a rocker for 15 min, followed by addition of 10 ml of 2 M KCl and incubation on a rocker for another 15 min. Subsequently, 20 ml of deionized water was added to each sample, and samples were again incubated for 15 min on a rocker. Stripped membranes were collected by centrifugation at 75,000 x g for 75 min at 4 °C. The stripped membrane fractions were then kept at –80 °C in storage buffer until use.

Preparation of Mouse Erythroblast Samples—To obtain erythroid cell samples at various stages of differentiation, an in vitro system of differentiation of proerythroblasts isolated from the spleens of Friend virus-infected mice was used (31). In this system, developmentally synchronized proerythroblasts differentiate in culture over 2 days becoming reticulocytes, and then the reticulocytes formed in vitro are isolated and re-cultured for an additional 3 days (32). At various times of culture, erythroblasts or reticulocytes were harvested from culture, washed in PBS, and lysed in Laemmli sample buffer. The erythroid cell lysates were separated by SDS-PAGE and transferred onto polyvinylidene difluoride sheets as described below. Because of significant decreases in cell size during terminal differentiation and the extreme predominance of hemoglobin among proteins in the late erythroblasts and reticulocytes, the erythroid cells lysates were standardized by cell number so that each lane contained the lysate of 2.5 x 106 cells.

Production and Purification of Antibodies—Polyclonal antibodies against three mouse Cyts b561 were generated by injecting rabbits with synthetic C-terminal peptides (20 amino acids) (Cocalico Biologicals, Reamstown, PA). The peptides used to raise antibodies are: [Cys]-KRPDPGALTDRQPLLHDRE for LCytb, [Cys]-EQALSMDFKTLTEGDSPSPQ for CGCytb, and [Cys]-EGAARKRTLGLADSGQRSTM for DCytb. The peptides were coupled to keyhole limpet hemocyanin through the addition of an N-terminal cysteine residue. Antibodies were purified using Sulfolink coupling gel (Pierce) according to the manufacturer's instructions. The isoform specificities of antibodies were verified by immunoblotting with three Cyts b561 expressed in yeast cells. The peptide antibodies specifically detect each Cyt b561 homolog, and each band was located at the predicted molecular mass (results not shown).

Protein Gels and Western Blot—Proteins were resolved by SDS-PAGE using 12% acrylamide gels. Samples were not heated or boiled before loading on the gels, because this caused Cyt b561 aggregation and prevented them from penetrating the gel (30, 33). After SDS-PAGE, proteins were transferred onto a polyvinylidene difluoride membrane with a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). The Cyts b561 were detected using the purified peptide antibodies as primary antibodies and the horseradish peroxidase-conjugated anti-rabbit antibody as a secondary antibody (ECL detection kit; Amersham Biosciences). The dilution of the primary antibodies was 1:250.

Assessment of Ability of Erythrocytes to Preserve Extracellular Asc—After incubations of human or mouse erythrocytes as noted, aliquots of mixed cells and buffer were removed from the incubations and were quickly centrifuged at 3 °C for 1 min in a Sorvall Microspin 24 centrifuge to pellet the cells, where present. Aliquots of the supernatant were taken for assay of Asc.

Asc-dependent Plasma Membrane Ferricyanide (FeCN) Reductase Activity of Erythrocytes—Mouse or human erythrocytes at a 5% packed cell volume were incubated with gentle mixing at 37 °C in PBS that contained 5 mM D-glucose and DHA at 0, 0.1, 0.2, 0.5, 1, or 2 mM. After 30 min, aliquots of the cells were removed for assay of intracellular Asc by high pressure liquid chromatography as described previously (34), except that 1 mM tetrapentylammonium bromide was used as the ion pairing reagent in the mobile phase. The remaining cell suspension was incubated for another 30 min with mixing at the same temperature in the presence of 1 mM FeCN and 20 mM cytochalasin B (to prevent further uptake of any remaining DHA into the cells). The cells were pelleted by centrifugation, and aliquots of the supernatant were removed for assay of ferrocyanide by the method of Avron and Shavit (35) as described previously (36). The cells were rinsed three times by centrifugation to remove residual FeCN and then taken for assay of intracellular Asc. Results for intracellular Asc are expressed in millimolar, based on an intracellular cytosolic space of 70% of the packed cell volume (37). Results for FeCN reduction are expressed per ml of packed cell volume.

Absorption Spectroscopy—Absorption spectra were recorded under continuous stirring in a dual wavelength mode (from 500 to 600 nm and reference at 601 nm) using an SLM-Aminco DW2000 spectrophotometer with a 1 nm slit-width, 0.5 nm s–1 scan rate. The fully oxidized spectra were obtained by addition of 0.5 mM FeCN to the samples. The Asc-reduced and Asc + dithionite-reduced spectra were obtained after sequential addition of Asc and sodium dithionite to the samples to concentrations of 25 and 2 mM, respectively. The difference spectra were obtained by subtracting the FeCN-oxidized spectra from the Asc or (Asc + dithionite)-reduced spectra. Multiple scans were averaged to improve the signal-to-noise ratio.


Figure 1
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  		FIGURE 1.
DCytb expression in erythrocyte membranes. Panel A, Cyts b561 in erythrocyte membranes of human and mouse. Immunoblots with antibodies against CGCytb, LCytb, and DCytb are shown. The 1st lanes in each blot are 10 µg of whole protein extraction from yeast cells transformed with the mouse CGCytb, LCytb, and DCytb genes. The 2nd and 3rd lanes are 20 µg of erythrocyte membrane fractions from mouse (Mm-erythrocyte) and human (Hs-erythrocyte), respectively. Panel B, loss of DCytb during the mouse erythropoiesis process. Immunoblot with antibodies against DCytb. Lane 1, 20 µg of mouse erythrocyte membrane fraction. Lanes 2–4, three erythroblast developmental stages (0, 24, and 44 h during progression in culture). Lanes 5–8, four reticulocyte developmental stages (0, 20, 40, and 86 h in culture). Lane 9, mouse DCytb expressed in the HEK-293 cell line (15 µg of whole protein extraction). Panel C, DCytb in erythrocyte membranes from other animals. Immunoblot with antibodies against DCytb. Lanes 1–4, erythrocyte membrane fractions from guinea pig (3 µg, lane 1), mouse (15 µg, lane 2), rat (40 µg, lane 3, and human (15 µg, lane 4). Lane 5, 1 µg of stripped membrane fraction from yeast cells expressing mouse DCytb. Lane 6, 15 µg of whole protein extraction from HEK-293 cells expressing mouse DCytb.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
DCytb Expression in Erythrocyte Membranes—To determine whether erythrocytes express one or more Cyts b561, we carried out immunoblotting experiments (Fig. 1). Isoform-specific Cyt b561 antibodies were used to evaluate the presence of each of these three Cyts b561 in mouse and human erythrocyte membranes (Fig. 1, panel A). No protein band was recognized by the CGCytb and LCytb antibodies in either mouse or human erythrocyte membrane fractions. However, a band with a size similar to the predicted mass of the mouse DCytb was detected in membranes derived from human erythrocytes but not in those from mouse erythrocytes. The predicted molecular weights of mouse DCytb and human DCytb are very similar (31.84 kDa for mouse DCytb and 31.64 kDa for human DCytb). In addition, the C-terminal sequences of each pair of human and mouse Cyts b561 are also very similar: 100% identity for CGCytb, 85% identity for LCytb, and 70% identity (and 85% similarity) for DCytb. Hence, the antibodies against mouse Cyts b561 should be able to recognize human Cyts b561. Thus, it can be concluded that only DCytb is present in human erythrocyte membranes and that mouse erythrocytes lack this protein.


Figure 2
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  		FIGURE 2.
Extracellular Asc preservation by human but not by mouse erythrocytes. Incubations in PBS at 37 °C contained 5 mM D-glucose, 20 milliunits/ml Asc oxidase, 80 µM Asc, and one of the following: 40% packed cell volume of mouse erythrocytes (circles), 40% packed cell volume of human erythrocytes (squares), or no cells (triangles). At the times indicated, the incubations were mixed, and aliquots were removed for assay of Asc in the supernatant as described under "Experimental Procedures." Results are shown from four experiments.

 
Because DCytb is not present in the cell membrane of mature mouse erythrocytes, the question arises whether DCytb is present in earlier stages of erythropoiesis. To address this question, we used a culture model of developmentally synchronized nascent murine proerythroblasts that mature into erythrocytes in vitro (31, 32). The immunoblot of Fig. 1, panel B, shows that DCytb was easily detected in the proerythroblasts and early stage erythroblasts, but the large majority of DCytb was lost from the late stage erythroblasts by 44 h of culture. A slight residual DCytb in nascent reticulocytes was lost by 20 h of their maturation. DCytb was also heterologously expressed in yeast cells and HEK-293 cells, although in these cells another band of ~52 kDa was observed. In fact, a band of 50–55 kDa is often observed for each Cyt b561 and is believed to represent an aggregation product produced by freezing and thawing of samples (30, 38).

We also examined erythrocyte membranes from guinea pigs and rats for the presence of DCytb. As shown in Fig. 1, panel C, DCytb was present in erythrocyte membranes from guinea pig but not in those from rat. Neither LCytb nor CGCytb was found in either erythrocyte membrane from these species (data not shown).


Figure 3
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  		FIGURE 3.
Asc-dependent FeCN reductase activity of human and mouse erythrocytes. Panel A, rates of FeCN reduction in Asc-loaded human (squares) and mouse (circles) erythrocytes were measured as described under "Experimental Procedures." The results from five experiments are expressed relative to the measured intracellular Asc concentrations at the start of each experiment. Panel B, initial (open symbols) and post-FeCN treatment (closed symbols) of intracellular Asc concentrations in human (squares) and mouse (circles) erythrocytes measured in the experiments shown in panel A.

 
Evidence That DCytb Is an MDHA Reductase in Human Erythrocytes—If DCytb contributes to trans-plasma membrane electron transport in human erythrocytes, then this function should be decreased or absent in mouse erythrocytes, which lack this protein. We first compared these two cell types with regard to their ability to maintain extracellular Asc in the presence of Asc oxidase, which generates MDHA from Asc outside the cells. As shown in Fig. 2, human erythrocytes (squares) at a 40% packed cell volume effectively preserved extracellular Asc compared with the same packed cell volume of mouse erythrocytes (circles). In fact, the rate of Asc loss because of the action of Asc oxidase was similar in mouse cells and control buffer (Fig. 2, triangles). This result shows that, in contrast to human erythrocytes, mouse erythrocytes lack the ability to preserve extracellular Asc.

The ability to reduce extracellular FeCN is another transmembrane process that has been linked to Asc in erythrocytes and other cells (36, 37). If DCytb is involved in this activity, it should also be decreased in mouse erythrocytes compared with human cells. Because FeCN reduction is dependent on intracellular Asc, mouse and human erythrocytes were loaded to similar high intracellular Asc concentrations by allowing them to take up and reduce DHA to Asc. A caveat in this experiment is that DHA is taken up on glucose transporters, which are expressed about 200-fold higher in human than in rat erythrocytes (and presumably in erythrocytes from the mouse) (39). Therefore, mouse cells were loaded with 4-fold higher DHA concentrations to generate similar intracellular Asc concentrations. As shown in Fig. 3, panel A, at any given initial intracellular Asc concentration, human cells (squares) reduced extracellular FeCN from 2- to 3-fold more rapidly than did the same packed cell volume of mouse cells (circles). Another caveat in this experiment is the fact that mouse erythrocytes have approximately half the volume of human erythrocytes. Calculations using an equation derived previously (40) show that mouse erythrocytes at the same packed volume as human erythrocytes would have 1.7-fold more membrane surface area. Because FeCN reductase activity is a membrane function, this would suggest an even greater difference between human and mouse erythrocytes. Moreover, the Asc content of human erythrocytes was markedly decreased by FeCN treatment (Fig. 3, panel B, open versus closed squares), whereas it was only slightly decreased at higher Asc concentrations in mouse cells (Fig. 3, panel B, open versus closed circles). This marked loss of Asc in the human cells correlates well with the amount of FeCN reduced compared with mouse cells. It is possible that the observed Asc-dependent FeCN reduction in the mouse cells was due to Asc that had leaked from the erythrocytes or that was released from extremely low levels of hemolysis during the incubation. However, we found no evident hemolysis after 30-min incubations with both mouse and human erythrocytes. The extent to which Asc leakage from erythrocytes could account for FeCN reduction was examined by loading human and mouse erythrocytes to 2–3 mM with Asc under the conditions of Fig. 3, then following appearance of Asc in the incubation medium with time. After 30 min of incubation at 37 °C, extracellular Asc was 9.7 ± 0.2% of total Asc in human erythrocytes (n = 3 determinations), whereas it was 55 ± 5% of total Asc in mouse erythrocytes (n = 3 determinations). For human erythrocytes, this amount of extracellular Asc would account for only 1.8% of the FeCN reduced at 30 min, whereas in mouse erythrocytes, it would account for 12.4%. Thus, even though mouse erythrocytes leaked more Asc than human erythrocytes, this leakage accounted for only a small fraction of total FeCN reduced.

The results of Figs. 2 and 3 suggest that human cells are much more efficient than mouse cells in their ability to preserve extracellular Asc and to transfer electrons from intracellular Asc to extracellular FeCN. To further test whether DCytb might be involved in these activities, its activity in mouse and human ghost membranes was compared.

An Asc-reducible Cyt b in Stripped Human Erythrocyte Membranes—Asc is generally accepted as the physiological electron donor for the Cyts b561 (20). It has been observed that recombinant and native plant and mammalian Cyts b561 have characteristic Asc-reducible spectra (30, 33, 41–44). To confirm this biochemical characteristic for DCytb, the absorption spectra were measured using stripped membrane fractions from yeast cells expressing mouse DCytb (Fig. 4, panel A). Reduced minus oxidized difference spectra demonstrate the presence of an Asc-reducible b-type Cyt with an {alpha}-band near 561 nm. Reduction by Asc (25 mM) reached 79.56 ± 3.97% of the reduction level obtained with dithionite (2 mM)(n = 3), as is also observed for most other Cyts b561 (30, 33). As reported previously (30), no such characteristic peak was detected in stripped membrane fractions from yeast cells transformed with the empty pESC-His vector (data not shown).


Figure 4
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  		FIGURE 4.
Stripped human erythrocyte membrane contains an Asc-reducible Cytb. The difference spectra were obtained by subtracting the FeCN oxidized spectra from Asc (+Asc) or Asc + dithionite (+Asc+Dith)-reduced spectra. 8 mg of a stripped membrane fraction from yeast cells expressing DCytb (panel A), 9 mg of a stripped human erythrocyte membrane fraction (panel B), and 3.5 mg of a stripped mouse erythrocyte membrane fraction (panel C) were used for the measurements.

 
To further identify the presence of DCytb in human erythrocyte membranes, the absorption spectra of stripped human erythrocyte membranes were recorded (Fig. 4, panel B). The spectra demonstrate the presence of an Asc-reducible Cyt b with absorbance maximum near 561 nm. Reduction by Asc reached 93.10 ± 0.33% of the reduction level obtained with dithionite (n = 2). As shown in Fig. 4, panel C, mouse erythrocyte membranes had no detectable Asc-reducible spectra.


Figure 5
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  		FIGURE 5.
Concentration-dependent reduction of membrane fractions of yeast cells expressing DCytb (panel A) and human erythrocytes (panel B). 8 mg of a stripped membrane fraction from yeast cells expressing DCytb (panel A) and 16.7 mg of a stripped human erythrocyte membrane fraction (panel B) were used for the measurements. Results are from one representative experiment of three repetitions. The data were plotted using SigmaPlot, and the curves were calculated based on the estimated affinity constants.

 
To further study the substrate binding and kinetics of DCytb, the concentration dependences for Asc reduction of the mouse recombinant DCytb expressed in yeast cells and native DCytb in human erythrocyte membranes were measured (Fig. 5). The data from both measurements fit well to a biphasic hyperbolic plot. The two apparent affinity constants obtained for recombinant mouse DCytb in stripped yeast cell membranes were 0.07 ± 0.02 and 1.82 ± 0.14 mM (n = 3) and for native human DCytb in stripped human erythrocyte membranes were 0.01 ± 0.00 and 0.77 ± 0.23 mM (n = 3). For both recombinant and native DCytb, each phase contributed about 50% to the maximal level of reduction by Asc. This result demonstrates that both recombinant and native DCytb have two apparent binding sites for Asc (see "Discussion"), which is consistent with the results obtained for recombinant and native CGCytb (30).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Human erythrocytes are capable of reducing extracellular MDHA to Asc using intracellular Asc as an electron donor (16, 18). The mechanism(s) of this trans-membrane electron transport is unknown, but could be analogous to a similar system in chromaffin granules of the adrenal gland, in which CGCytb transfers electrons from cytoplasmic Asc to MDHA in the chromaffin granules (22, 23). However, Van Duijn et al. (24) showed, and we confirmed herein, that CGCytb is absent from human erythrocyte membranes. Nonetheless, it is possible that other Cyts b561 could mediate such electron transfer in human erythrocytes. Three evolutionarily closely related Cyts b561 have been identified in mammals (21). Immunoblot analyses demonstrated that only DCytb is present in human but not in mouse erythrocytes. The presence of DCytb was also suggested by a study of the human erythrocyte proteome using ion trap tandem mass spectrometry (45), which identified a 10-amino acid peptide sequence corresponding to the C-terminal sequence of human DCytb.3

Surprisingly, DCytb was not present in erythrocytes from the rat but was present in guinea pig erythrocytes. The other Cyts b561, CGCytb and LCytb, were also absent by immunoblotting in rat and guinea pig erythrocytes. Thus, it appears that there are no other Cyt b561 isoforms that functionally compensate for the lack of DCytb in rat and mouse erythrocytes. By using an in vitro murine erythroid differentiation system, we found that mouse DCytb is present in the erythroblast stage but is lost during the differentiation of erythroblasts and reticulocytes. During erythropoiesis, late stage erythroblasts enucleate and become reticulocytes. In this final step, exosome release leads to a net loss of lipids, other membrane transporters, and the transferrin receptor (46). The reticulocytes subsequently mature into erythrocytes in the blood circulation. Although DCytb is lost during mouse erythroblast and reticulocyte maturation, human erythrocyte maturation does not result in loss of DCytb. One could speculate that, in contrast to humans and guinea pigs, mice and rats are capable of synthesizing Asc and hence may not need this Asc regeneration system.

The only proposed function for DCytb so far is ferrireductase activity (25, 27). However, it is possible that DCytb has the same function as CGCytb in chromaffin granules in that it can recycle Asc by reducing MDHA outside of erythrocytes. Indeed, in addition to its well documented function as an MDHA reductase, CGCytb also showed ferrireductase activity when expressed in yeast (27) and Xenopus oocytes (28). It is possible that these Cyts b561 can mediate both activities, with one activity favored depending on the cellular milieu. Our results provide at least circumstantial evidence for this hypothesis. In contrast to mouse erythrocytes, which lack DCytb, human erythrocytes preserved extracellular Asc, which previous studies have shown to be due to the reduction of extracellular MDHA to Asc (16), using intracellular Asc as an electron donor (18). Furthermore, human erythrocytes reduced extracellular FeCN at much greater rates than did mouse erythrocytes, implying that ferrireductase activity is also present. Although DCytb is not the only protein with differential expression in rodent and human erythrocytes (e.g. the GLUT1 glucose transporter (39)), DCytb is the only candidate for this activity identified to date.

Whether the trans-membrane ferrireductase activity in human erythrocytes is of physiologic relevance is unknown. This activity, measured typically as extracellular FeCN reduction (37), uses intracellular Asc as the primary electron donor (47). On the other hand, some of the observed FeCN reductase activity could also be mediated by another erythrocyte plasma membrane redox system, which uses NADH as the electron donor (48–51). The most likely function for this activity in the developing erythroblast is to reduce ferric iron during its uptake and use in hemoglobin synthesis. Because DCytb shows Asc-dependent FeCN reductase activity when expressed in yeast (27), it is possible that it includes part of the erythroblast iron reduction/uptake system. However, all components of the hemoglobin-synthesizing system, as well as the transferrin receptor and the ability to take up iron, are lost during the maturation of reticulocytes (52–56). Furthermore, DMT-1 (divalent metal transporter 1), which works in concert with DCytb for iron uptake (57), was not detected in the membrane fractions of mature human erythrocytes by ion trap mass spectrometry (45). Taken together, our results support the hypothesis that the function of DCytb in the mature human erythrocyte has shifted from that of a ferrireductase to an MDHA reductase.

Little is known about the biochemical properties of DCytb. Sequence analysis suggests that DCytb, similar to other Cyts b561, has six transmembrane domains and four highly conserved His residues, possibly coordinating two heme molecules (21). Two conserved domains, which are predicted for Asc and MDHA binding (58), are conserved in DCytb and located on the electron-accepting and electron-donating sides of the protein, respectively. No biochemical data are available to demonstrate that the gene identified as DCytb effectively codes for a di-heme b-type Cyt. Hence, we generated recombinant mouse DCytb in yeast. The reduced minus oxidized difference spectra of the recombinant protein confirmed its nature as an Asc-reducible b-type Cyt, with an {alpha}-band near 561 nm (Fig. 4, panel A). This spectral characteristic is almost identical to that of CGCytb (30) and one plant Cyt b561 isoform (33). Furthermore, the {alpha}-band in human erythrocytes was reduced by Asc in an identical manner to that of DCytb expressed in yeast. In contrast, mouse erythrocyte membranes had no detectable Asc-reducible b-type Cyt. The Asc reducibility of DCytb further supports its possible role in extracellular Asc preservation and FeCN reduction.

Moreover, the concentration dependence for Asc-mediated reduction of DCytb, showing that both recombinant mouse DCytb in yeast and native DCytb in the human erythrocytes, have two apparent binding sites for Asc. This result is identical to that obtained with both recombinant and native CGCytb (30). The physiological relevance of these two apparent binding constants for the reduction of DCytb and CGCytb is still unclear. It is possible that the two apparent reduction constants represent the sequential reduction of each heme in the Cyts b561. Mutation of a well conserved Arg residue in CGCytb, which was predicted to be critical for Asc binding, abolished high affinity CGCytb reduction by Asc (30). This indicated that the high affinity reduction site may correspond to Asc binding and reduction on the cytoplasmic side of the protein. These common properties of DCytb and CGCytb with respect to Asc reducibility support the idea that they have similar functions.

In conclusion, we provide evidence to support the notion that DCytb is involved in extracellular Asc recycling of human erythrocytes. Although the major function of this Cyt b561 isoform may have been to reduce extracellular iron during erythroblast differentiation, its retention in mature human and guinea pig erythrocytes, but not in mouse or rat erythrocytes, fits with the notion that it may serve to preserve blood Asc in species that can no longer make the vitamin. Proof that DCytb does indeed recycle extracellular MDHA to Asc is not yet available but could be provided by differential expression studies, both in vitro and in vivo.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants 1P20RR17675 and DK050435, National Science Foundation Grant IBN-0416742, and a Merit Review (to M. J. K.) from the Department of Veterans Affairs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Belgium. Tel.: 32-3-2653638; Fax: 32-3-265-3417; E-mail: han.asard{at}ua.ac.be.

2 The abbreviations used are: Asc, ascorbate; MDHA, mono-dehydroascorbate; DHA, dehydroascorbate; Cyts b561, cytochromes b561; CGCytb, chromaffin granule cytochrome b561; DCytb, duodenal cytochrome b561; FeCN, ferricyanide; LCytb, lysosomal cytochrome b561; PBS, phosphate-buffered saline; Mes, 4-morpholineethanesulfonic acid; Mops, 4-morpholinepropanesulfonic acid. Back

3 D. Kakhniashvili, personal communication. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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