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• W2053649957 abstract "A heme-binding protein with a molecular mass of 22 kDa, termed p22 HBP, was purified from mouse liver cytosol, using blue Sepharose CL-6B. We identified a cDNA encoding p22 HBP, and sequence analysis revealed that p22 HBP comprises 190 amino acid residues (M r 21,063) and has no homology to any other known heme-binding protein. The p22 HBP mRNA (∼1.0 kilobases) is ubiquitously expressed in various tissues and is extremely abundant in the liver. cDNA allows for expression of active p22 HBP, with a high affinity for 55Fe-hemin, with aK d of 26 ±1.8 nm. TheB max of hemin binding to p22 HBP was 0.55 ± 0.021 mol/mol of protein, a value consistent with one heme molecule binding per molecule of protein. The order of potency of different ligands to compete against 55Fe-hemin binding to p22 HBP was hemin = protoporphyrin IX > coproporphyrin III > bilirubin > palmitic acid > all-trans-retinoic acid. Treatment of mouse erythroleukemia (MEL) cells with dimethyl sulfoxide or hemin resulted in an increase in p22 HBP mRNA. The immunoblot analysis showed that p22 HBP increased with time in dimethyl sulfoxide- and hemin-induced MEL cells. Conversely, transfer of antisense oligonucleotides to p22 HBP cDNA resulted in a decrease of p22 HBP in dimethyl sulfoxide-treated MEL cells, and the heme content in these cells decreased to 66–71% of sense oligonucleotides-transferred cells. Thus, this newly identified heme-binding protein, p22 HBP, may be involved in heme utilization for hemoprotein synthesis and even be coupled to hemoglobin synthesis during erythroid differentiation. A heme-binding protein with a molecular mass of 22 kDa, termed p22 HBP, was purified from mouse liver cytosol, using blue Sepharose CL-6B. We identified a cDNA encoding p22 HBP, and sequence analysis revealed that p22 HBP comprises 190 amino acid residues (M r 21,063) and has no homology to any other known heme-binding protein. The p22 HBP mRNA (∼1.0 kilobases) is ubiquitously expressed in various tissues and is extremely abundant in the liver. cDNA allows for expression of active p22 HBP, with a high affinity for 55Fe-hemin, with aK d of 26 ±1.8 nm. TheB max of hemin binding to p22 HBP was 0.55 ± 0.021 mol/mol of protein, a value consistent with one heme molecule binding per molecule of protein. The order of potency of different ligands to compete against 55Fe-hemin binding to p22 HBP was hemin = protoporphyrin IX > coproporphyrin III > bilirubin > palmitic acid > all-trans-retinoic acid. Treatment of mouse erythroleukemia (MEL) cells with dimethyl sulfoxide or hemin resulted in an increase in p22 HBP mRNA. The immunoblot analysis showed that p22 HBP increased with time in dimethyl sulfoxide- and hemin-induced MEL cells. Conversely, transfer of antisense oligonucleotides to p22 HBP cDNA resulted in a decrease of p22 HBP in dimethyl sulfoxide-treated MEL cells, and the heme content in these cells decreased to 66–71% of sense oligonucleotides-transferred cells. Thus, this newly identified heme-binding protein, p22 HBP, may be involved in heme utilization for hemoprotein synthesis and even be coupled to hemoglobin synthesis during erythroid differentiation. d-aminolevulinic acid synthase 22-kDa heme-binding protein 23-kDa macrophage stress protein 23-kDa heme-binding protein fatty acid-binding protein mouse erythroleukemia cells Dulbecco's modified Eagle medium dimethyl sulfoxide polyacrylamide gel electrophoresis glutathione S-transferase polymerase chain reaction kilobase pair(s) rapid amplification of cDNA ends. Heme regulates protein syntheses transcriptionally and translationally. The first enzyme of heme biosynthetic pathway, ALAS1 consists of two gene products. The housekeeping-type ALAS (ALAS-1) was down-regulated at levels of transcription and translocation into the mitochondrion by hemin (1Yamamoto M. Kure S. Engel J.D. Hiraga K. J. Biol. Chem. 1988; 263: 15973-15979Abstract Full Text PDF PubMed Google Scholar, 2May B.K. Bhasker C.R. Bawden M.J. Cox T.M. Mol. Biol. Med. 1990; 7: 405-421PubMed Google Scholar). Translocation of the erythroid-type ALAS (ALAS-2) into the mitochondrion is also inhibited by heme (3Lathrop J.T. Timko M.P. Science. 1993; 259: 522-525Crossref PubMed Scopus (248) Google Scholar). Heme as well as iron mediate the interaction of the iron-responsive element-binding protein with its target mRNAs, including those of ferritin, ALAS-2, and transferrin receptors (4Lin J.-J. Daniels-McQueen S. Patino M.M. Gaffield L. Walden W.E. Thach R.E. Science. 1990; 247: 74-77Crossref PubMed Scopus (79) Google Scholar, 5Theil E.C. J. Biol. Chem. 1990; 265: 4771-4774Abstract Full Text PDF PubMed Google Scholar, 6Melefores O. Goosen B. Johansson H.E. Stripecke R. Gray N.K. Hentze M.W. J. Biol. Chem. 1993; 268: 5974-5978Abstract Full Text PDF PubMed Google Scholar). In addition, an increase in free heme in cells leads to transcriptional induction of heme oxygenase-1 (7Shibahara S. Muller R. Taguchi H. Yoshida T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7865-7869Crossref PubMed Scopus (430) Google Scholar, 8Maines M.D. Trakshel G.M. Kutty R.K. J. Biol. Chem. 1986; 261: 411-419Abstract Full Text PDF PubMed Google Scholar). Cells seem to maintain adequate heme levels by a combination of synthetic and degradative mechanisms. Cells are equipped with a sensing system to monitor changes in size of the uncommitted heme pool. Although such a system has not been identified, identification of uncommitted heme is one approach to elucidate cellular regulation by heme. Conditions of uncommitted heme may be linked to cytosolic heme-binding proteins because heme is not so soluble in aqueous solutions at body pH and tends to form large aggregates at concentrations as low as 10−7m (9Granick S. Beale S.I. Adv. Enzymol. Relat. Areas Mol. Biol. 1978; 46: 33-203PubMed Google Scholar). Several heme-binding and heme-transport proteins from the cytosol have been isolated. FABP constituting 3–5% of the total cytosolic proteins has an affinity for heme that is 10-fold higher than that for oleic acid (9Granick S. Beale S.I. Adv. Enzymol. Relat. Areas Mol. Biol. 1978; 46: 33-203PubMed Google Scholar, 10Vincent S.H. Müller-Eberhard U. J. Biol. Chem. 1985; 260: 14521-14528Abstract Full Text PDF PubMed Google Scholar). GSTs constituting 3–5% of total cytosol proteins not only catalyze the conjugation of glutathione with xenobiotics but also bind organic anions, such as bilirubin and bile acid (11Bhargava M.M. Arias I.M. Trends Biochem. Sci. 1981; 6: 131-133Abstract Full Text PDF Scopus (10) Google Scholar, 12Boyer T.D. Hepatology. 1989; 9: 486-496Crossref PubMed Scopus (198) Google Scholar), and heme (13Harvey J.W. Beutler E. Blood. 1982; 60: 1227-1230Crossref PubMed Google Scholar). HBP23 has been purified from rat liver cytosol, using chromatography on hemin-agarose (14Iwahara S. Satoh H. Song D.-X. Webb J. Burlingame A.L. Nagae Y. Müller-Eberhard U. Biochemistry. 1995; 34: 13398-13406Crossref PubMed Scopus (134) Google Scholar). cDNA cloning revealed that HBP23 was a homologue of MSP23, the mouse macrophage 23-kDa stress-induced protein that may function as an antioxidant (15Ishii T. Yamada M. Sato H. Matsue M. Taketani S. Nakayama K. Sugita Y. Bannai S. J. Biol. Chem. 1993; 268: 18633-18636Abstract Full Text PDF PubMed Google Scholar, 16Ishii T. Kawane T. Taketani S. Bannai S. Biochem. Biophys. Res. Commun. 1995; 216: 970-975Crossref PubMed Scopus (44) Google Scholar) and belongs to thioredoxin peroxidase family (17Jin D.-Y. Chae H.Z. Rhee S.G. Jeang K.-T. J. Biol. Chem. 1997; 272: 30952-30961Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). However, as studies on functions of these proteins are few, their participation in cellular regulation by heme and the intracellular transport of heme has remained poorly understood. We earlier reported that ferrochelatase, the terminal enzyme of heme biosynthesis, bound tightly to blue Sepharose (18Taketani S. Tokunaga R. J. Biol. Chem. 1981; 256: 12748-12753Abstract Full Text PDF PubMed Google Scholar, 19Taketani S. Tokunaga R. Eur. J. Biochem. 1982; 127: 443-447Crossref PubMed Scopus (50) Google Scholar). Once the enzyme bound to the blue dye, it was not released from the dye even after washing with solution containing more than 1 m NaCl and nonionic detergents such as Triton X-100 and Nonidet P-40. There are reports that heme-binding proteins (20Hrkal Z. Cabart P. Kalousek I. Biomed. Chromatogr. 1992; 6: 212-214Crossref PubMed Scopus (12) Google Scholar, 21Cornego J. Beale S.I. J. Biol. Chem. 1988; 263: 11915-11921Abstract Full Text PDF PubMed Google Scholar) and enzymes of chlorophyll biosynthesis (22Schon A. Krupp G. Gough S. Berry-Lowe S. Kannangara C.G. Soll D. Nature. 1986; 322: 281-284Crossref PubMed Scopus (200) Google Scholar, 23Walker C.J. Castelfranco P.A. Whyte B.J. Biochem. J. 1991; 276: 691-697Crossref PubMed Scopus (40) Google Scholar) can bind to blue dye. Although mechanisms involved in such binding to proteins interacted with heme and porphyrins are unclear, it is considered that chromatography on blue Sepharose would be useful for purifying proteins interacting with heme and porphyrins. We purified three major proteins from the cytosol of mouse liver. Although two of them have been reported (24Xu X. Stambrook P.J. J. Biol. Chem. 1994; 269: 30268-30273Abstract Full Text PDF PubMed Google Scholar, 25Mainwaring G.W. Williams S.M. Foster J.R. Tugwood J. Green T. Biochem. J. 1996; 318: 297-303Crossref PubMed Scopus (75) Google Scholar), the third was a heretofore unrecognized heme-binding protein with a molecular mass of 22 kDa, termed as p22 HBP. Molecular cloning and functional expression showed that p22 HBP binds heme, protoporphyrin, and coproporphyrin, with relatively high affinities. Induction of the protein in Me2SO- or hemin-treated MEL cells undergoing erythroid differentiation was also noteworthy. [α-32P]dCTP (3000 Ci/mol),55FeSO4 (60 mCi/mg) and nylon membranes were obtained from Amersham. 55Fe-hemin was prepared, as described previously and dissolved in Me2SO (26Taketani S. Kohno H. Furukawa T. Tokunaga R. J. Biochem. 1995; 117: 875-880Crossref PubMed Scopus (111) Google Scholar). The specific radioactivity of 55Fe-heme was 1.06 × 106 dpm/nmol heme. Restriction endonuclease and other nucleic acid-modifying enzymes were from Toyobo Co. and Takara Shuzo Co. A 5′-RACE cDNA library from mouse liver mRNA was fromCLONTECH. Blue Sepharose CL-6B, EAH-Sepharose CL-6B, and pGEX-4T-1 were from Amersham Pharmacia Biotech. Protoporphyrin IX and coproporphyrin III were from Porphyrin Products Co. Hemin-bound Sepharose were prepared by incubation of hemin with EAH-Sepharose CL-6B by the method of Olsen (27Olsen K.W. Anal. Biochem. 1980; 109: 250-254Crossref PubMed Scopus (10) Google Scholar). Amino acid sequencing was carried out using a Hewlett Packard Protein Sequencer G1000A. All other chemicals used were of analytical grade. All procedures for the purification of p22 HBP was done at 4 °C. Solution containing 10 mm Tris-HCl buffer, pH 8.0, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 150 mm NaCl was designated as Solution A. Fresh mice livers (20 g) were minced and homogenized in 6 volumes of Solution A with 6 strokes of a Teflon pestle in a glass homogenizer. After crude lysates were centrifuged at 10,000 × g for 10 min to remove nuclei and mitochondria, cytosol was separated from the postmitochondrial supernatant by ultracentrifugation at 105,000 ×g for 1 h. Then, to the obtained cytosolic fraction, 10% Triton X-100 solution was added at the final concentration of 1%. The resulting samples (865 mg of protein) were applied to a column of blue Sepharose CL-6B (1.5 × 5.0 cm) that was equilibrated with Solution A containing 1% Triton X-100. The column was washed with 500 ml of Solution A containing 1% Triton X-100 and 1 m NaCl at a flow rate of 30 ml/h. The proteins were eluted with Solution A containing 1% sodium cholate and 1 m NaCl. Fractions containing proteins were collected, concentrated using an Amicon YM-10 membrane, and dialyzed with Solution A for 16 h. Amounts of protein in the final preparation were 0.23 mg. For chromatography on hemin-Sepharose CL-6B, eluates of blue Sepharose were applied to a small column of hemin-Sepharose (1.0 × 2.0 cm) equilibrated with Solution A containing 0.5% Tween 20, after which the column was washed with 100 ml of Solution A containing 1 m NaCl. The proteins were eluted from the column with 50 mm sodium acetate, pH 5.0. Fractions containing proteins were collected and dialyzed against Solution A for 16 h. Protein concentration was determined by the method of Lowry et al. (28Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). To identify the N-terminal amino acid sequence of the obtained proteins, the proteins were analyzed by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad), as described (29Taketani S. Yoshinaga T. Furukawa T. Kohno H. Tokunaga R. Nishimura K. Inokuchi H. Eur. J. Biochem. 1995; 230: 760-765Crossref PubMed Scopus (37) Google Scholar). After staining the proteins, parts of the filter corresponding to molecular masses of 26, 24, and 22 kDa (see Fig. 1) were cut out and directly sequenced. A primer (5′-GT-CTC-CAC-(G/A)CT-(G/A/T/C)CC-(G/A)AA-3′) complementary to amino acid sequence (FGSVET) was prepared, and PCR was carried out using as a template 5′-RACE mouse liver cDNA library. The PCR products were separated by agarose gel electrophoresis, purified, and ligated to pGEM-T vector (Promega), and then the insert was examined by DNA sequencing (30Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5436-5467Crossref Scopus (52865) Google Scholar). To isolate full-length cDNA, the MEL cell cDNA library in λgt11 (29Taketani S. Yoshinaga T. Furukawa T. Kohno H. Tokunaga R. Nishimura K. Inokuchi H. Eur. J. Biochem. 1995; 230: 760-765Crossref PubMed Scopus (37) Google Scholar) was screened with the radiolabeled partial fragment of p22 HBP cDNA. Five positive plaques were isolated from 1.5 × 105 plaques, and the longest insert (1.0 kbp) of clone 5 was cut out and subcloned into a pBluescript vector for sequencing. The cDNA corresponding to mouse p22 HBP was subcloned into BamHI-NotI site of an expression vector pGEX-4T-1 in the correct reading frame. p22 HBP was expressed as GST fusion protein in Escherichia coli JM109 and purified on glutathione-Sepharose beads, according to the method of Self and Hall (31Self A.J. Hall A. Methods Enzymol. 1995; 258: 3-10Crossref Scopus (167) Google Scholar). To prepare heme-free GST fusion protein, the protein solution, adjusted to pH 5.0 by adding 1 m acetic acid, was passed through DEAE-cellulose (DE52, Whatman Co.) (1.5 × 5.0 cm) equilibrated with 50 mm sodium acetate, pH 5.0. The resulting solution containing the proteins was dialyzed against 10 mm Tris-HCl buffer, pH 8.0, containing 150 mmNaCl and 0.3% Tween 20 (TBS-T) at 4 °C for 16 h. The amount of bound55Fe-hemin to the purified GST-p22 HBP was determined as follows. The GST (2.5 μg) or GST-p22 HBP (2.5 μg) in TBS-T containing 1 mg/ml rabbit γ-globulin was incubated with 0–300 nm55Fe-hemin at 4 °C for 20 min, after which the sample was mixed with 50 μl of glutathione-Sepharose beads. After washing the beads five times with TBS-T, the proteins were eluted with TBS-T containing 20 mm glutathione. To determine the degree of nonspecific binding, parallel experiments were carried out with a 100-fold excess of nonradioactive hemin. The radioactivities in the eluted samples were counted in a Packard liquid scintillation spectrophotometer. The native p22 HBP was also expressed in E. coli BL21(DE3) strain as follows. The cDNA for p22 HBP was subcloned intoNdeI-EcoRI site of an expression vector pAR-HF, as described (32Furukawa T. Kohno H. Tokunaga R. Taketani S. Biochem. J. 1995; 310: 533-538Crossref PubMed Scopus (57) Google Scholar). p22 HBP was purified using blue Sepharose, as described above. For binding of hemin to p22 HBP, the purified p22 HBP (100 μg) in 10 mm Tris-HCl buffer, pH 8.0, was incubated with 20 μm hemin at 4 °C for 20 min. The sample was loaded onto a column of DEAE-cellulose (1.0 × 2.0 cm) equilibrated with 10 mm Tris-HCl buffer, pH 8.0, the column was washed with 10 ml of the above solution and the protein was eluted with 10 mm Tris-HCl buffer, pH 8.0, containing 150 mm NaCl. MEL cells (clone 745A) were grown in DMEM supplemented with 7% fetal calf serum and antibiotics. To induce erythroid differentiation, cells at an initial density of 1 × 105/ml were grown for 3 days in medium containing 2% Me2SO or 100 μm hemin. Mouse macrophage RAW 264.7 cells were grown in RPMI 1640 medium containing 10% fetal calf serum, as described (32Furukawa T. Kohno H. Tokunaga R. Taketani S. Biochem. J. 1995; 310: 533-538Crossref PubMed Scopus (57) Google Scholar). A mouse multiple tissue Northern blot membrane was purchased from CLONTECH Co. Total RNA was isolated from MEL cells and RAW264.7 cells, as described previously (29Taketani S. Yoshinaga T. Furukawa T. Kohno H. Tokunaga R. Nishimura K. Inokuchi H. Eur. J. Biochem. 1995; 230: 760-765Crossref PubMed Scopus (37) Google Scholar). 10 μg of RNA were applied to a 1% agarose-formaldehyde gel, electrophoresed, and transferred onto a nylon membrane (Amersham Pharmacia Biotech Hybond N+) for hybridization with DNA probes. The filters were hybridized and washed as described (29Taketani S. Yoshinaga T. Furukawa T. Kohno H. Tokunaga R. Nishimura K. Inokuchi H. Eur. J. Biochem. 1995; 230: 760-765Crossref PubMed Scopus (37) Google Scholar). The mRNA concentration was quantified by densitometry using an Advantec DMC-33c densitometer. Antibodies against p22 HBP were prepared by injecting a rabbit with 0.5 mg of the purified GST-p22 HBP fusion protein in Freund's complete adjuvant. After three subsequent injections at 2-week intervals, the resulting antisera were collected and antibodies were purified as described (29Taketani S. Yoshinaga T. Furukawa T. Kohno H. Tokunaga R. Nishimura K. Inokuchi H. Eur. J. Biochem. 1995; 230: 760-765Crossref PubMed Scopus (37) Google Scholar). Antibodies for ferrochelatase and MSP23 were prepared, as described (16Ishii T. Kawane T. Taketani S. Bannai S. Biochem. Biophys. Res. Commun. 1995; 216: 970-975Crossref PubMed Scopus (44) Google Scholar, 29Taketani S. Yoshinaga T. Furukawa T. Kohno H. Tokunaga R. Nishimura K. Inokuchi H. Eur. J. Biochem. 1995; 230: 760-765Crossref PubMed Scopus (37) Google Scholar). For immunoblotting, cells were lysed with Laemmli's sample buffer (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207856) Google Scholar). The lysate was then sonicated and boiled for 1 min. After the proteins had been resolved by SDS-PAGE on 12% gel, the protein in the gel were electroblotted onto a polyvinylidene difluoride membrane. Conditions of immunoblotting and detection of the cross-reacted antigen were as described (29Taketani S. Yoshinaga T. Furukawa T. Kohno H. Tokunaga R. Nishimura K. Inokuchi H. Eur. J. Biochem. 1995; 230: 760-765Crossref PubMed Scopus (37) Google Scholar). Phosphorothioate sense (5′-ATGTTGGGCATGATCAGG-3′) oligonucleotides, which were identical to nucleotide positions 58–75 of p22 HBP cDNA, and antisense (5′-CCTGATCATGCCCAACAT-3′) oligonucleotides were synthesized and purified. MEL cells (5 × 105) treated with 2% Me2SO for 16 h were collected and rinsed twice with serum-free DMEM. Phosphorothionate sense or antisense oligonucleotides (5 μm) were transfected into the cells, using a DOTAP Liposomal Transfection Reagent (Boehringer Mannheim GmbH) (34Bertram J. Killian M. Brysch W. Schlingensiepen K.-H. Kneba M. Biochem. Biophys. Res. Commun. 1994; 200: 661-667Crossref PubMed Scopus (46) Google Scholar). After a 6-h transfection, the medium was changed to DMEM containing 7% fetal calf serum, 2% Me2SO, and antibiotics. The cells were further cultured for 24 or 48 h and then collected. The heme content in the cells were estimated as described previously (29Taketani S. Yoshinaga T. Furukawa T. Kohno H. Tokunaga R. Nishimura K. Inokuchi H. Eur. J. Biochem. 1995; 230: 760-765Crossref PubMed Scopus (37) Google Scholar). After blue Sepharose CL-6B chromatography of mouse hepatic cytosolic protein, three major bands, 22, 24, and 26 kDa, appeared on SDS-PAGE (Fig. 1). To determine of these proteins would bind to heme, eluates of blue Sepharose were loaded to hemin-bound Sepharose, the column was washed with Solution A containing 1 m NaCl, and proteins were eluted with the solution at pH 5.0. All proteins were bound to hemin-Sepharose. Thus, three proteins with relatively low molecular weights that were tightly bound to blue dye could bind to heme. We then analyzed the N-terminal amino acid sequences of three proteins. The protein with a molecular mass of 22 kDa had the following amino acid sequence: (NH2)-N-S-L-F-G-S-V-E-T-W-. When we searched for the above sequence in the GenBankTM Data Bank, we found no homology with any known proteins. The 26 and 24 kDa proteins showed the sequence of (NH2)-V-L-E-L-Y-L-D-L-L-S- and (NH2)-P-P-Y-T-I-V-Y-F-P-V-, respectively. The latter sequences perfectly matched with GST-P1 (24Xu X. Stambrook P.J. J. Biol. Chem. 1994; 269: 30268-30273Abstract Full Text PDF PubMed Google Scholar), and the former was with mouse GST-T1 (25Mainwaring G.W. Williams S.M. Foster J.R. Tugwood J. Green T. Biochem. J. 1996; 318: 297-303Crossref PubMed Scopus (75) Google Scholar). We then prepared a primer complementary to amino acid sequence positions 4–9 (FGSVET) and obtained a 112-bp partial cDNA fragment (clone 20) corresponding to a 22-kDa protein by the 5′-RACE PCR amplification method, using a mouse liver 5′-RACE cDNA library. The nucleotide sequence of clone 20 showed no homology to known sequences. We termed this 22-kDa heme-binding protein, p22-HBP. We then isolated the full-length cDNA. p22 HBP cDNA proved to be a 1033-bp insert with an open reading frame of 630 bp, which encodes a polypeptide of 190 amino acids with a molecular weight of 21,063 (Fig. 2). The amino acid sequence of the N terminus of purified p22 HBP coincided with the deduced amino acid sequence at position 7–16. The overall structure of the deduced amino acid sequence suggested that the protein is soluble. A search of the EMBL and GenBankTM Data Banks indicated that the amino acid sequence was not similar to any known proteins. While p22 HBP did not have a heme-regulated motif (3Lathrop J.T. Timko M.P. Science. 1993; 259: 522-525Crossref PubMed Scopus (248) Google Scholar), an amino acid segment spanning amino acid positions 73–82 exhibited a hydrophobic region, suggesting that this region may be involved in heme-binding (35Shin W.S. Yamashita H. Hirose M. Biochem. J. 1994; 304: 81-86Crossref PubMed Scopus (26) Google Scholar). Fig. 3 shows RNA blots of p22 HBP in mouse tissues. p22 HBP mRNA was ubiquitously expressed in various tissues. The extremely high level of expression in liver may reflect the requirement for p22 HBP in tissues where heme metabolism is high. Although faint bands were visible with testis or skeletal muscle RNA samples in the exposure given in Fig. 3, extended exposures revealed the same RNA species in these tissues. When the GST-p22 HBP was expressed in E. coli and purified by glutathione-Sepharose, the purified protein had a yellow color. Fig. 4 shows absorption spectrum of the recombinant p22 HBP after incubation with hemin in vitro. The peak in the Soret region of bound hemin to the protein at 408 nm shifted from that of unbound hemin at 397 nm. This spectrum resembled the spectra of major heme-binding proteins including GSTs, albumin, and HBP23 (a rat homologue to MSP23) (14Iwahara S. Satoh H. Song D.-X. Webb J. Burlingame A.L. Nagae Y. Müller-Eberhard U. Biochemistry. 1995; 34: 13398-13406Crossref PubMed Scopus (134) Google Scholar, 36Tipping E. Ketterer B. Christodoulides L. Enderby G. Biochem. J. 1976; 157: 461-467Crossref PubMed Scopus (27) Google Scholar, 37Beaven G.H. Chen S.-H. D'Albis A. Gratzer W.B. Eur. J. Biochem. 1974; 41: 539-546Crossref PubMed Scopus (256) Google Scholar). Scatchard plots revealed that 55Fe-hemin binding to the GST-p22 HBP fusion protein had an affinity of K d = 26 ±1.8 nm. B max of the binding site in the GST-p22 HBP was 0.55 ± 0.021 pmol/pmol of protein (Fig. 5). The parasite GST expressed by pGEX-4T-1, used as control, did not bind hemin. Protoporphyrin IX and coproporphyrin III competed against 55Fe-hemin binding (40 nm) to the GST-p22 HBP with 50% inhibitory concentration of 22 and 75 nm, respectively (Fig. 6). High concentrations (>1 μm) of bilirubin, all-trans-retinoic acid, and palmitic acid were required for the 50% inhibition. These results indicate that porphyrin compounds as well as hemin can bind to p22 HBP.Figure 5Effect of hemin concentration on binding of55Fe-hemin to GST-p22 HBP fusion protein. Purified GST-p22 HBP fusion protein (2.5 μg) was incubated at 4 °C for 20 min with increasing concentrations of 55Fe-hemin (1,060 dpm/pmol heme) prepared as described under “Experimental Procedures.” To isolate the heme-p22 HBP complex, glutathione-Sepharose (50 μl) was added, and the preparation was mixed. After washing the beads, protein was eluted with solution containing 20 mm glutathione. The radioactivities in the eluted samples were determined. Specific binding (•) was calculated by subtracting nonspecific (- - -) from total binding of radioactive hemin (○). The amount of hemin bound to GST expressed by pGEX-4T-1 is also shown (▪). Inset, Scatchard analysis of the binding data. Bars represent S.E. (n = 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Inhibition of 55Fe-hemin binding to p22 HBP. Nonradioactive hemin (▪), protoporphyrin (•), coproporphyrin (○), bilirubin (▴), palmitic acid (▵), and all-trans retinoic acid (×) were tested for inhibition of specific binding of 55Fe-hemin (40 nm) to GST-p22 HBP fusion protein (2.5 μg). Data are means of triplicate experiments. Bars represent S.E. (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT) MEL cells were induced to differentiate with 2% Me2SO, heme biosynthesis increased with time, and then over 90% of the cells synthesized large quantities of hemoglobin at 4 days (29Taketani S. Yoshinaga T. Furukawa T. Kohno H. Tokunaga R. Nishimura K. Inokuchi H. Eur. J. Biochem. 1995; 230: 760-765Crossref PubMed Scopus (37) Google Scholar). To examine involvement of p22 HBP in heme biosynthesis, total RNA was obtained from MEL cells at various periods after Me2SO treatment, and RNA blots were carried out using p22 HBP cDNA as a probe. As shown in Fig. 7 A, p22 HBP mRNA corresponding to about 1.0 kbp increased gradually with time up to 72 h. Hemin is an inducer of erythroid differentiation of MEL cells (38Beaumont C. Deybach J.-C. Grandchamp B. Da Silva V. De Verneuil H. Nordmann Y. Exp. Cell Res. 1984; 154: 474-484Crossref PubMed Scopus (31) Google Scholar). We next examined the induction of p22 HBP mRNA in MEL cells treated with 100 μm hemin. Treatment of cells with hemin resulted in an increase in hemoglobin synthesis, similar to findings in MEL cells treated with Me2SO. p22 HBP mRNA increased with time, similar to the case of Me2SO-treated cells, but at 72 h the level of the RNA slightly decreased (Fig. 7 B). Thus, the p22 HBP mRNA was induced during MEL cell differentiation. As HBP23, a rat homologue to MSP23, was induced by treatment of rat primary cultured hepatocytes with hemin (39Immenschuh S. Iwahara S. Satoh H. Nell C. Katz N. Müller-Eberhard U. Biochemistry. 1995; 34: 13407-13411Crossref PubMed Scopus (67) Google Scholar), we compared changes in p22 HBP in Me2SO- or hemin-treated MEL cells with those of MSP23. Fig. 8 shows immunoblot analysis of p22 HBP and MSP23 during MEL cell differentiation. The density of p22 HBP with a molecular mass of 22 kDa markedly increased with time by treatment with Me2SO, whereas MSP23 with a molecular mass of 23 kDa remained unchanged in cells for 72 h (Fig. 8 A). An increase in p22 HBP was also observed during hemin-induced differentiation of MEL cells but was absent in case of MSP23 (Fig. 8 B). The magnitudes of the induction of p22 HBP in Me2SO- and hemin-induced MEL cells were about 6-fold and 5-fold, respectively, at 72 h. Ferrochelatase with a molecular mass of 41 kDa is induced with concomitant increases in heme biosynthesis (29Taketani S. Yoshinaga T. Furukawa T. Kohno H. Tokunaga R. Nishimura K. Inokuchi H. Eur. J. Biochem. 1995; 230: 760-765Crossref PubMed Scopus (37) Google Scholar) and was increased prior to the increase of p22 HBP during Me2SO- and hemin-induced differentiation. Treatment of nonerythroid mouse macrophage RAW 264.7 cells with 100 μm hemin did not result in the induction of p22 HBP protein and its mRNA (data not shown). Therefore, p22 HBP seems to be markedly induced during erythroid differentiation. Expression of p22 HBP in MEL cells with oligonucleotides was estimated by immunoblotting. As shown in Fig. 9 A, p22 HBP in cells transfected with sense-oligonucleotides was similar to that in control cells, whereas the protein in cells transfected with antisense oligonucleotides was markedly reduced compared with control cells. We finally examined effects of sense or antisense oligonucleotides on heme content during Me2SO-induced differentiation of MEL cells. The heme contents in cells transfected with antisense oligonucleotides, followed by treatment with 2% Me2SO for 24 and 48 h, decreased to" @default.
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• W2053649957 title "Molecular Characterization of a Newly Identified Heme-binding Protein Induced during Differentiation of urine Erythroleukemia Cells" @default.
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