An Iron-Regulated Ferric Reductase Associated with the Absorption of Dietary Iron
Tóm tắt
The ability of intestinal mucosa to absorb dietary ferric iron is attributed to the presence of a brush-border membrane reductase activity that displays adaptive responses to iron status. We have isolated a complementary DNA, Dcytb (for duodenal cytochrome b), which encoded a putative plasma membrane di-heme protein in mouse duodenal mucosa. Dcytb shared between 45 and 50% similarity to the cytochrome b561 family of plasma membrane reductases, was highly expressed in the brush-border membrane of duodenal enterocytes, and induced ferric reductase activity when expressed in
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Tài liệu tham khảo
Duodenal mucosa from a hpx mouse was used as the target tissue to isolate Dcytb. A subtracted cDNA library was constructed as previously described (10). Using this strategy we isolated a 114–base pair (bp) cDNA fragment (representing base pairs 205 to 319 of Dcytb). Using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) we isolated a 1100-bp cDNA for Dcytb which appeared to encode the entire protein of 284 amino acids and had a predicted molecular mass of ∼30 kD. Further analysis of expressed sequence tags (ESTs) indicated an additional two amino acids (Met and Ala) at the NH 2 -terminus giving a predicted protein of 286 amino acids. A genomic sequence (GenBank accession number ) containing the human Dcytb open reading frame was found by searching the High-Throughput Genomic Sequences (HTGS) database with the mouse sequence. In addition a fully sequenced cDNA clone from a human small intestine library containing the full-length human Dcytb cDNA [pME18S-FL/Dcytb (GenBank accession number )] was found by searching EST databases.
Male mice (6 to 9 weeks old) of the CD-1 strain were used for experimental manipulations of iron status. Hypoxia iron deficiency and iron loading were carried out as described previously (10). Hypotransferrinemic mice were bred and maintained as described previously (16). Animal procedures were approved by the U.K. Home Office. Total RNA was extracted from tissues and Northern blotting was carried out as previously described (10). The above-described 114-bp fragment of Dcytb was used as the probe. We carried out reverse transcription on 10 μg of total RNA from each tissue using 100 U of Superscript II (Gibco) and 100 nM random hexamers (Pharmacia) and following the manufacturer's instructions. PCR was performed in a PTC 200 thermal cycler (MJ Research Waltham MA) with 0.5 U of Taq polymerase (Supertaq HT Biotechnology Cambridge) 200 μM deoxynucleoside triphosphate (Fermentas Hanover MD) and gene-specific primers (sequences available on request) at a concentration of 10 μM. Products were compared after 20 25 and 30 cycles. The products shown in Fig. 2D are after 20 [GAPDH (glyceraldehyde phosphate dehydrogenase) and Dcytb] and 30 cycles (gp91- phox ). The data shown were reproducible in independent experiments on individual mice from each group. In situ hybridizations were carried out as previously described (10).
To generate antisera to Dcytb we synthesized the peptides [Cys]-DAESSSEGAARKRTLGLADSGQRSTM (peptide 1) and [Cys]-KRPREPGSVPLQLNGGNADRME (peptide 2) corresponding to the COOH-terminus and amino acids 223 to 244 respectively of mouse Dcytb (28). Peptides 1 and 2 were then injected into two individual rabbits (coded 834 and 835 respectively) and a mix of both peptides 1 and 2 was injected into a third rabbit (coded 836) (Sigma-Genosys). Western blots were performed with 836 antiserum whereas immunohistochemical and antibody-blocking experiments were performed with 834 antiserum. For both Western blots and immunohistochemistry 834 antiserum was found to work well whereas 836 worked only on Western blots and did not block reductase activity; 835 antiserum was not tested as a function-blocking antibody and worked weakly (compared to 834) in Western blotting and immunostaining experiments. Duodenal protein samples (mucosal scrapes) from hypotransferrinemic mice were prepared with Trisol (Gibco) following manufacturer's instructions. We prepared intestinal brush-border membrane vesicles from mucosa of the duodenum and ileum of normal mice using a well-established procedure that in our hands [(9 29) and references therein] yields highly enriched brush-border membranes. Western blotting was performed as described (10). Membranes were incubated for 1 hour with Dcytb antisera (antiserum 836) at a dilution of 1:100. Detection of the primary antibody was performed with the Westernbreeze system (Invitrogen). For immunohistochemistry sections (5 μm) were cut from frozen tissue blocks at –20°C and placed on glass slides fixed for 10 min at room temperature in acetone and allowed to air-dry. Blocking was performed in 1% bovine serum albumin in phosphate-buffered saline (PBS) for 30 min. Sections were incubated with primary Dcytb antibody (antiserum 834) diluted 1:100 for 1 hour in blocking buffer. After extensive washing in PBS sections were incubated for 30 min with a fluorescein isothiocyanate (FITC)–conjugated anti-rabbit secondary (DAKO Carpinteria CA) and then washed extensively with PBS. Sections were counterstained with propidium iodide and mounted. Images were captured on an MRC1024 confocal microscope (Bio-Rad). As a negative control we processed parallel sections incubated with preimmune sera in the same way. No signal was obtained with preimmune serum.
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Xenopus oocytes were prepared for injection as previously described (5). To synthesize Dcytb cRNA we linearized the mouse Dcytb/pcDNA3.1 construct with Avr II and cRNA transcribed from the T7 RNA polymerase promoter site using a commercial kit (Ambion Austin TX). Oocytes were injected with 25 ng of cRNA. Controls were injected with the same volume of water or 25 ng of an unrelated cRNA encoding a membrane protein. Reductase assays were carried out on individual oocytes 48 hours after injection. Oocytes were incubated for 10 hours in the dark in 50 μl of either standard Barths solution (pH 7.4) or buffer ND96 [98 mM NaCl 2.0 mM KCl 0.6 mM CaCl 2 1.0 mM MgCl 2 and 10 mM Hepes (pH 6.0) with tris base] containing 100 μM FeNTA 2 (100 μM ferric chloride: 200 μM nitrilotriacetic acid) and 1 mM ferrozine. Formation of ferrous iron was calculated by measuring the optical density of the oocyte incubation buffer at 562 nm. Results were consistent in experiments performed on batches of oocytes harvested from three individual frogs. HuTu-80 and CaCo-2 cells were obtained from the American Type Culture Collection. Both cell types were cultured in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal calf serum and antibiotics [penicillin G (3 mg/liter) and streptomycin (5 mg/liter)] supplemented with nonessential amino acids (Sigma). Human Dcytb was excised from the original vector [pME18S-FL/Dcytb (GenBank accession number )] with Eco RI and Xho I and subcloned into the pcDNA3.1myc/his(+) mammalian expression vector (Invitrogen). Cells grown to ∼40% confluence in 10-cm plates were transfected with ∼2 μg of DNA using the transfection reagent Effectene following manufacturer's instructions (Qiagen Valencia CA). Stable cell lines were obtained by selection in G418 sulfate (50 μg/ml) (Calbiochem Darmstrad Germany) for 10 days. Expression of Dcytb was confirmed by Western blotting. The MTT assay is based on the reduction of a yellow water-soluble monotetrazolium salt MTT to an insoluble purple formazan. HuTu-80 cells (mixed population of Dcytb-expressing cells and untransfected cells) were seeded in 96-well plates (1 × 10 4 /ml) and left to grow for 48 hours. On the day of the assay the medium was removed and cells were incubated with Dcytb antibody (834 antiserum) in PBS or with PBS alone for 15 min. Solutions were removed and replaced with culture medium containing 20 μl of MTT solution (5 mg/ml) and incubated at 37°C for varying times. The medium and MTT solution were then removed and 200 μl of dimethyl sulfoxide was added to each well. Absorbance was read at 540 nm. Data are the average of six wells and the experiment was repeated three times with similar results. Ferric reductase assays were carried out on CaCo-2 cells as follows. Cells were harvested and homogenized in PBS with a glass Dounce-type homogenizer and centrifuged for 10 min at 10 000 g to remove mitochondria nuclei and intact cells. The supernatant (corresponding to microsomes plasma membrane and cytosol) was incubated with 10 μM NADH in 50 mM Hepes (pH 7.4) with 100 μM FeNTA 2 and 1 mM bathophenanthroline at 37°C for up to 2 hours. Absorbance was read at 540 nm versus blanks lacking cells. For antibody-blocking experiments cells were preincubated for 15 min at room temperature with antibody to Dcytb (834 antiserum diluted 1:50). The experiment shown was representative and has been repeated with the same result. For NBT assays the duodenum was removed opened lengthwise and rinsed with 150 mM NaCl. Slices (full width of duodenum by 1 to 2 mm) were taken ∼1 cm from the pylorus and incubated for 5 min at 37°C in 200 μl of 1 mM NBT in incubation buffer [125 mM NaCl 3.5 mM KCl and 16 mM Hepes/NaOH (pH 7.4)]. After incubation tissues were rinsed twice with 150 mM NaCl and photographed with a Polaroid Microcam and a dissecting microscope. Inhibition of NBT reductase was demonstrated by preincubation of the tissue for 15 min with 200 μl of preimmune or antiserum to Dcytb (834 antiserum diluted 1:100 in incubation buffer) rinsing once in 200 μl of incubation buffer then incubating and washing as above. The experiment shown was representative of at least three similar results. The mouse Dcytb construct used in oocyte experiments began MEGYRG whereas in later experiments in cultured cells we used a human Dcytb construct which started MAMEGYRG (28). Both constructs induced reductase activity in transfected cells and gave a 30-kD protein by Western blotting. We do not at present know which methionine is used as the start of translation.
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Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr.
This work was supported by a Medical Research Council Training Fellowship (A.T.M.) and by the Wellcome Trust. The authors gratefully acknowledge the Joint Research Committee of King's College Hospital (D. Barrow) the Personal Assurance Charitable Trust (A.B.) the Royal Society (UK) (G.O.L.-D.) and the government of Libya (G.S.) for additional support. The authors thank A. Pini and R. Hider for support and encouragement and acknowledge C. Gove J. Pizzey and C. McNulty (Randall Institute King's College London) for assistance in performing in situ hybridizations and U. Berger and N. Basora (Harvard Medical School) for advice and assistance with immunohistochemistry.