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Cloning, expression, and characterization of a novel guanylate-binding protein, GBP3 in murine erythroid progenitor cells
Biochimica et Biophysica Acta 1384 Ž1998. 373–386
Cloning, expression, and characterization of a novel guanylate-binding protein, GBP3 in murine erythroid progenitor cells Byung Hee Han b
a,1
, Don Jae Park b, Robert W. Lim a , Jeong Hyok Im a , Hyun Dju Kim
a,)
a Department of Pharmacology, UniÕersity of Missouri-Columbia, School of Medicine, Columbia, MO 65212, USA Department of Medicine, DiÕision of Hematology, Vanderbilt UniÕersity Medical Center, NashÕille, TN 37232-6305, USA
Received 14 October 1997; revised 20 February 1998; accepted 25 February 1998
Abstract We report the molecular cloning of a novel guanylate-binding protein ŽGBP., termed mouse GBP3 ŽmGBP3. in Friend virus-induced mouse erythroid progenitor ŽFVA. cells. The 71-kDa mGBP3 belongs to a family of known GBPs that contain the first two consensus motifs, GXXXXGKŽSrT. and DXXG, but lack the third element, ŽNrT.KXD, found in typical GTP-binding proteins. Recombinant mGBP3 protein, expressed using a baculovirus expression system, binds to agarose-immobilized guanine nucleotides ŽGTP, GDP and GMP.. Moreover, mGBP3 has been found to have an intrinsic GTPase activity with K m and Vmax values of 77 " 4 m M and 21 " 0.5 pmol miny1 m gy1 of protein, respectively. The mGBP3 is distinct from the other GBPs, in that it does not have an isoprenylationrmethylation motif CAAX at the carboxyl terminus. The mGBP3 appears to be localized in the cytosol based on immunofluorescence staining. Although the mGBP3 transcript is expressed to a varying degree in numerous mouse tissues, the message is most abundant in FVA cells. The mGBP3 transcript increases in FVA cells undergoing differentiation to a maximum within a few hours and then decreases to an undetectable level by 24 h. These results, taken together, suggest that mGBP3 is a novel member of a family of guanylate-binding proteins, which plays a role in the erythroid differentiation. The nucleotide sequence reported in this paper has been submitted to the GenBanke with accession number U44731. q 1998 Elsevier Science B.V. All rights reserved. Keywords: cDNA sequence; GTPase activity; Gene expression; Subcellular localization; Erythropoietin; Interferon-g
Abbreviations: Epo, Erythropoietin; ERG, Erythropoietin-responsive gene; FBS, Fetal bovine serum; FVA cells, Erythroid progenitor cells isolated from the mouse spleen infected with Friend virus; GBP, Guanylate-binding protein; His-mGBP3, histidine-tagged mouse GBP3; IFN-gc, Interferon gamma; IMDM, Iscoves, modified Dulbecco medium; RACE, Rapid amplification of cDNA ends; RT-PCR, Reverse transcriptase-polymerase chain reaction; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis ) Corresponding author. Fax: q1-573-884-4558; E-mail: [email protected] 1 Present address: Yuhan Research Center, Yuhan Pharmaceutical Co., Seoul, South Korea.
1. Introduction GTP-binding proteins regulate a variety of cellular activities including signal transduction, protein synthesis, protein targeting, and cell motility Žreviewed in Ref. w1x.. Amino acid sequence comparisons and X-ray crystallographic data analysis of GTP-binding proteins have revealed the presence of three hallmark consensus elements, GXXXXGKŽSrT., DXXG, and Ž N rT . K X D w 2 – 6 x . The first tw o m otifs GXXXXGKŽSrT. and DXXG are known to interact
0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 8 . 0 0 0 3 4 - X
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with the phosphate groups of the guanine nucleotides, whereas the third element ŽNrT.KXD is responsible for the specificity of guanine recognition. For example, the modification of Ž NrT.KXD motif by substituting Asp with Asn in p21ras w7,8x and EF-Tu w9,10x leads to a loss of the guanine nucleotide binding specificity. A family of proteins termed the guanylate-binding proteins ŽGBPs. have been originally described as interferon ŽIFN. -inducible guanine nucleotide-binding proteins in fibroblasts and in macrophages w11,12x. Molecular cloning of GBPs has uncovered several characteristic features common to the GBP family w13–16x. All GBPs possess the first two GTP-binding consensus elements GXXXXGKŽSrT. and DXXG, but lack the third ŽNrT.KXD motif. However, these GBPs still retain a high degree of selectivity to guanine nucleotides ŽGTP, GDP, and GMP. over adenine and pyrimidine nucleotides w13,17x. Recently, several GBPs have been shown to be GTPases with slightly different catalytic properties. While the recombinant hGBP1 w17x hydrolyzes GTP predominantly to GMP, the chicken GBP w16x and the hGBP2 w18x also catalyze the conversion of GTP, but GDP is the major product. In addition, most GBPs known to date have an isoprenylationrmethylation modification motif at their carboxyl termini. This posttranslational modification promotes the ability of proteins to associate with the plasma membranes w19–21x. Both hGBP1 w17x and rat p67 GBP w15,22x have been shown to be isoprenylated in vitro. The rat p67 GBP has been reported to be localized in the plasma membranes w15,22x. However, the physiological function of GBPs has not yet been elucidated. Erythropoietin ŽEpo. is a hematopoietic growth factor that regulates cell proliferation, differentiation and apoptosis of erythroid progenitor cells w23–27x. Erythroid progenitor ŽFVA. cells, isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus, have been used as a cell model system for the erythroid differentiationrmaturation w28–34x. FVA cells undergo differentiation to hemoglobin-rich reticulocytes in vitro within 48–72 h upon exposure to Epo. It has been reported that Epo induces early responsive genes such as MYC, MYB, GATA1, BVL1, NFE2, and TAL1r SCL prior to the expression of genes encoding erythrocyte-specific proteins like globins w35–39x.
Recently, we reported the isolation of erythropoietin-responsive genes ŽERGs. from the Epo-treated FVA cells by differential hybridization w38x. In this communication, we present our results in which we cloned and characterized a novel 71-kD guanylate-binding protein, termed mouse GBP3 from the Epostimulated FVA cell cDNA library. The mouse GBP3 protein shows a high sequence identity and GTPase activity similar to those of the other known GBPs. However, unlike other GBP3, the mGBP3 protein, which lacks the isoprenylationrmethylation motif CAAX, appears to be located in the cytosol of FVA cells.
2. Materials and methods 2.1. Cloning and sequencing of mGBP3 cDNA FVA cells were isolated from mouse spleen and cultured as described w40x. The construction of the murine cDNA library from Epo-stimulated FVA cells and the isolation of ERGs have been described previously w38x. One ERG clone, erg11a, appeared to encode part of a 2.6-kb transcript as determined by Northern blot analysis Žsee below. . Using the w a y32 PxdCTP-labeled erg11a cDNA insert as a probe, a random-primed lgt10 cDNA library, prepared from polyŽ A.q RNA of FVA cells treated with 2 Urml Epo for 1 h, was screened to isolate overlapping clones as described w38x. Additional 5X sequence was obtained by using the 5X-rapid amplification of cDNA ends Ž RACE. protocol according to the method of Frohman et al. w41x. Briefly, polyŽ A.q RNA isolated from FVA cells were reverse-transcribed with an erg11-gene specific reverse primer, 5X-AGGGCATTCTCCAGGTACTC-3X corresponding to positions 654–673 of mGBP3 cDNA ŽFig. 1. . The first strand cDNA was tailed with dATP by terminal deoxynucleotidyl transferase ŽUSB. and amplified using an erg11-gene specific primer, 5X-GACAAAGGTGCT GCTCAGAAGCACAG-3X corresponding to positions 418–443, and a dT17-adapter primer with Taq DNA polymerase ŽUSB. in a Amplitrone II thermal cycler ŽBarnsteadrThermolyne. . The PCR product Ž; 0.5 kb. was gel-purified and subcloned into pGEM-T vector ŽPromega. .
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Fig. 1. The cDNA and deduced amino acid sequences of mouse GBP3. Ninety eight nucleotides at the 5X end of the cDNA were obtained by the 5X-RACE PCR method. The rest of the sequence was derived from several overlapping clones containing the mouse GBP3 Ž erg11. cDNA. Amino acid sequence was deduced from the longest open reading frame and indicated by the single letter code. Consensus motifs for nucleotide binding are boxed. Two potential polyadenylation signals AATAAA, in the 3X-untranslated region, are marked by dotted lines. Two repeats of the ATTTA sequence motif found in rapidly degraded messages are underlined. The bold-faced amino acids indicate the residue used to generate polyclonal anti-mGBP3 antibody as described in Section 2.
All cDNA inserts were sequenced on both strands using a Sequenasee Version 2.0 DNA sequencing kit ŽUSB. according to manufacturer’s protocol. Se-
quences were analyzed and compared with those in the GenBanke sequence data banks using a computer program provided by Genetics Computer Group.
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2.2. Northern analysis Total RNA was isolated by the guanidium thiocyanate–phenol method w42x. RNA samples Ž 20 m grlane. were separated by electrophoresis in a 1.1% agarose gel containing formaldehyde, and transferred onto a nylon membrane Ž Amersham. . UVcrosslinked membrane were prehybridized for 2 h at 658C in rapid hybridization buffer ŽAmersham. , and hybridized in the same buffer overnight at 658C with 1 = 10 6 cpmrml of w32 Px-random-primed cDNA probe. Blots were washed twice with 2 = SSC containing 0.1% SDS for 20 min at room temperature and twice with 0.2 = SSC containing 0.1% SDS for 20 min at 658C. Following the washes, blots were exposed to X-ray film ŽKodak. at y708C with intensifying screens. 2.3. RT-PCR Two micrograms of total RNA were reverse-transcribed for 1 h at 378C in a volume of 20 m l with random primers. One tenth of the synthesized cDNA was subjected to PCR amplification for the indicated number of cycles. PCR conditions were as follows: 948C for 1 min, 568C for 1.5 min, and 728C for 1.5 min. The PCR products were separated by electrophoresis in 1.5% agarose gels and visualized by ethidium bromide staining. Under such conditions, the RT-PCR procedure appeared to be quantitative, since the yields of PCR products were proportional to the amount of cDNA input Ždata not shown. . Primers used for PCR were as follows: for mGBP3, 5X-TGGAGGCACCCATTTGTCTGGTG-3X and 5X-GAC AAAGGTGCTGCTCAGAAGCACAG-3X ; for mGBP1 gene, 5X-CAAGCTAGCTGGGAAGAGGACAGG-3X and 5X-GAACTTCCTGATACACAGGCGAGG-3X ; for mouse b-actin, 5X-TCCTATGTGGGTGACGAGGC-3X and 5X-CATGGCTGGGGTGTTGAAGG-3X.
5X-CGGAATTCCTCGAGGCACCCATTTGTCTGGTG-3X Žcorresponding to positions 89-112. and 5X-GCGGATCCTCTAGACTAACTACTTAGTGAGCC-3X Ž corresponding to positions 1923–1940. . PCR-generated fragment was cloned into the pGEM vector to form pGEM-mGBP3. The XhoIrSalI fragment of the pGEM-mGBP3 was excised and ligated into the corresponding site of pBlueBacHis2 Ž Invitrogen. . The resulting recombinant construct, pBlueBacHis2-mGBP3 expresses a fusion peptide containing mouse GBP3 amino acid residue 2–620 fused to the histidine tag at its N-terminus. Spodoptera frugiperda Ž Sf9. cells were co-transfected with the baculovirus Autographa california ŽAcMNPV. DNA and the recombinant construct pBlueBacHis2mGBP3. The recombinant baculovirus was isolated by plaque assay according to manufacture’s protocol ŽInvitrogen.. Sf9 cells Ž2 = 10 6 cellsrml. were infected with the recombinant baculovirus at a multiplicity of infection of 5 plaque-forming unit per cell for 72 h. His-mGBP3 was purified by nickel-chelation affinity chromatography according to manufacturer’s protocol ŽQiagen.. Briefly, infected Sf9 cells were lysed by sonication in binding buffer Ž20 mM Tris–HCl, pH 7.9, 500 mM NaCl, 10% glycerol, 5 mM imidazole, 0.5% Triton X-100, 10 mM b-mercaptoethanol, 1 mM PMSF, 20 m grml of leupeptin and trypsin inhibitor, and 5 m grml of pepstatin A.. Soluble protein was loaded onto Ni–NTA resins ŽQiagen. and washed with 15 bed volumes of the binding buffer. His-mGBP3 was finally eluted with a step-wise gradient of imidazole of 10 to 100 mM. Protein samples were then applied to Q sepharose anion-exchange chromatography column Ž Pharmacia. equilibrated with a buffer containing 20 mM Tris–HCl, pH 8.0 and 0.1% Triton X-100. Proteins retained on the column were eluted with a step-wise gradient of NaCl of 10–300 mM. 2.5. Nucleotide binding assay
2.4. Expression and purification of histidine-tagged mGBP3 Histidine-tagged mGBP3 Ž His-mGBP3. was expressed using a baculovirus expression system. First, the coding region of mouse GBP3 gene was generated by PCR using the following oligonucleotides:
The purified His-mGBP3 fusion protein Ž; 5 m g. was incubated with 30- m l bed volume of nucleotide–agarose resins Ž Sigma. for 30 min at 48C in 1-ml binding buffer as described previously w13x. Resin-bound proteins were eluted with 20 m l SDSPAGE sample buffer containing 2% SDS by boiling
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for 5 min. The eluted proteins were separated by SDS-PAGE in 10% polyacrylamide gels and stained with Coomassie Blue.
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tion fluid and the amount of radioactivity was determined using a scintillation counter ŽBeckman..
2.6. GTPase assay
2.7. Generation of polyclonal antibody and Western blot
GTPase assay was performed as described w17x in a buffer containing 20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 80 nM w a y32 PxGTP Ž 3000 Cirmmol. and unlabeled GTP Ž concentration as indicated in figures. at 378C. The reaction was terminated with 1 mM EDTA and 0.5% SDS. Samples were spotted on polyethyleneiminecellulose thin layer chromatography plates Ž Selecto Scientific. and separated in solution containing 1.5 M LiCl, 0.5 M acetic acid, and 0.025 M citric acid. The dried plates were autoradiographed. Radioactive regions on the TLC plates were scraped into scintilla-
Polyclonal antibody was raised by immunization of rabbits with a synthetic peptide corresponding to the mouse GBP3 amino acid residues 513–541. Of the 29 residues present in this peptide, only 10–12 residues are conserved in other known GBPs Ž see Fig. 2.. Rabbits were immunized with 100 m g of the keyhole limpet hemocyanin-coupled peptide mixed with complete Freund’s adjuvant, and boosted twice with incomplete Friend’s adjuvant at a month interval. For analysis of mouse GBP3 protein, protein samples were separated by SDS-PAGE Ž 10% polyacrylamide gel. , transferred to nitrocellulose mem-
Fig. 2. Alignment of mouse GBP3 with other guanylate-binding proteins. The predicted amino acid sequence of mouse GBP3 gene was aligned with that of mouse GBP1rmag1 w13,14x, mouse GBPmarmag2 ŽGenBanke accession number M81128., human GBP1 w13x, and human GBP2 w13x. Gray area indicates that the corresponding amino acid is identical to that of mouse GBP3. Gaps Žy. are introduced to maximize alignment. The boxed residues indicate the conserved GTP-binding consensus motifs, GXXXXGKŽSrT. and DXXG.
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branes, and preincubated with TBS Ž 20 mM Tris– HCl, pH 7.5, 150 mM NaCl. containing 3% bovine serum albumin. The blocked membranes were subsequently incubated with a 1:2000 dilution of polyclonal anti-mGBP3 antiserum, horseradish peroxidase-conjugated goat anti-rabbit IgG, and visualized with an enhanced chemiluminescence system Ž Pierce. . 2.8. Immunofluorescence staining FVA cells were cultured in IMDM with 2 Urml recombinant Epo. Twelve hours after the initiation of the culture, the cells were rinsed twice with phosphate-buffered saline, fixed with 4% paraformaldehyde for 1 h at room temperature, and attached onto coverslips. Fixed cells were permeabilized with phosphate-buffered saline containing 0.1% Triton X100 for 15 min, and nonspecific reactive sites were blocked with 10% goat serum. Cells were subjected to sequential incubation with a 1:500 dilution of polyclonal anti-mGBP3 antiserum, biotinylated goat anti-mouse IgG antibody, followed by streptavidinconjugated Cy5e Ž Zymed Laboratory.. Immunofluorescence image was analyzed by confocal fluorescence microscopy ŽBio-Rad model 600..
Sequence comparison reveals that the putative protein encoded by erg11 clone bears resemblance to a family of guanylate-binding proteins ŽGBPs. as shown in Fig. 2. The overall sequence identity between the protein encoded by the erg11 clone and the other known GBPs varies from 52% to 62%. The protein encoded by the erg11 clone is referred hereafter to as mouse GBP3. The sequence conservation between mouse GBP3 and the other GBPs is highest in the region surrounding two nucleotide binding motifs, the first GTP-binding motif GXXXXGKŽSrT., which is also known as the Walker A motif, located at amino acid residue 39–46, and the second GTP-binding motif, DXXG at residue 91–94. Like the other GBPs, the mouse GBP3 apparently lacks the third downstream motif, ŽNrT. KXD which is found in typical GTP-binding proteins. However, unlike the other GBPs, the predicted mouse GBP3 does not have a CAAX motif at its C-terminus. Instead, the mouse GBP3 has a hydrophobic 30-amino acid stretch at the C-terminus not found in either GBP1 or GBP2. The
3. Results 3.1. Isolation of a mGBP3 cDNA clone We screened a lgt10 cDNA library generated with mRNA from Epo-treated FVA cells and isolated several overlapping clones using the cDNA insert of erg11a as a probe. Nucleotide sequences of the composite erg11 cDNA were obtained from these overlapping clones with a total length of 2458 nucleotides, in which 98 nucleotides at the 5X end were generated by the 5X-RACE method. Fig. 1 shows the nucleotide and deduced amino acid sequences of the composite erg11 cDNA. The amino acid sequence encoded by the longest open reading frame of the cDNA predicts a protein of 620 amino acids with a calculated molecular mass of 70,720 Da. In the 3X-untranslated region, there are two repeats of polyadenylation signal AATAAA and two repeats of the ATTTA motif, which is commonly found in rapidly degraded mRNA species.
Fig. 3. Northern blot analysis of mouse GBP3 transcript. Twenty micrograms of total RNA isolated from FVA cells were separated by electrophoresis in a 1.1% agarose-formaldehyde gel, blotted onto nitrocellulose membrane, and hybridized to w a y32 PxdCTPlabeled probe containing mouse GBP3 cDNA residue 99-2458. The hybridized membrane was exposed to X-ray films at y708C. Lane 1 shows the ethidium bromide staining of rRNAs. Lane 2 represents the autoradiograph from the Northern analysis following hybridization.
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hydrophobicity profile of mGBP3, plotted by the algorithm of Kyte and Doolittle w43x, is similar to that of hGBP1 except for the hydrophobic region at the C-terminus Ž data not shown..
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contrast, mGBP3 transcript was not detectable by Northern analysis in two erythroleukemia cell lines, HCD-57 and SREI-2, and various mouse tissues including brain, lung, heart, spleen, kidney, liver, and intestine Ždata not shown. .
3.2. Detection of mGBP3 mRNA by Northern analysis Northern analysis confirmed the expression of mouse GBP3 gene in freshly harvested FVA cells ŽFig. 3.. The mGBP3 cDNA probe corresponding to nucleotide residues 99–2458 detected an approximately 2.6-kb transcript Žlane 2. in these cells. In
3.3. Determination of mouse GBP3 gene expression by RT-PCR Mouse GBP3 gene expression was examined further by RT-PCR. Fig. 4A describes the changes in the
Fig. 4. RT-PCR analysis of mGBP3 expression in various mouse cells and tissues. Total RNA isolated from various sources were reverse transcribed and amplified by PCR for various numbers of cycles indicated below. PCR products were separated by electrophoresis in a 1.5% agarose gel and visualized by ethidium bromide staining. RT-PCR of the constitutively expressed b-actin transcript was used as a control for normalization of RNA input. ŽA. The GBP3 expression during FVA cell differentiation. RT-PCR was performed with total RNA isolated from freshly isolated FVA cells Žlane 1. and cultured FVA cells in the presence of 2 Urml Epo for the indicated number of hours Žlane 2–8.. The mGBP3 and b-actin transcripts were amplified for 27 cycles and 25 cycles, respectively. ŽB. The mGBP3 gene expression in cell lines. mGBP3 gene expression was determined in FVA cells treated with 2 Urml Epo for 1 h Žlane 1., Epo-treated erythroleukemia cell lines HCD-57 Žlane 2. and SREI-2 Žlane 3., thrombopoietin-treated megakaryocytes FDC-P2 Žlane 4., macrophages RAW264.7 Žlane 5. and skeletal muscle cells C 2 C 12 Žlane 6.. mGBP3: 30 amplification cycles, b-actin: 25 amplification cycles. ŽC. Tissue distribution of mGBP3 transcript. mGBP3 and b-actin transcripts were amplified for 30 cycles and 25 cycles, respectively. ŽD. The mGBP3 gene induction by IFN-g . FVA cells Žlane 1–2. and macrophages Žlane 3–4. were treated with 10 Urml IFN-g for 1 h Žlane 2 and 4. or left untreated Žlane 1 and 3., and gene expression was determined by RT-PCR. For mGBP1 and mGBP3: 27 amplification cycles in FVA cells Žlane 1 and 2., 35 cycles in macrophages Žlane 3 and 4.; for b-actin: 25 amplification cycles.
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mGBP3 mRNA expression levels in FVA cells undergoing differentiation with Epo. It was found that mGBP3 transcript expression was readily detectable in freshly harvested FVA cells at the time of their isolation from the spleen. In the course of erythroid differentiation, the message levels steadily increased to a maximum at 3 h, followed by a decrease to a very low level by 24 h. By contrast, the expression level of the constitutively expressed b-actin transcript remained unchanged for up to 24 h. In FVA cells maintained without Epo, the mGBP3 transcript expression levels also increased transitorily but to a lesser extent compared to the message levels seen in FVA cells with Epo Ž data not shown.. Fig. 4B shows the expression levels of mGBP3 transcript determined in various cell lines. It is evident that the Epo-treated FVA cells displayed by far the highest level of mGBP3 transcript expression. The mGBP3 transcript was also detectable in the thrombopoietin-stimulated megakaryocytes, FDC-P2. On the other hand, the mGBP3 mRNA was either expressed at low levels or not detectable in other cell lines, including the erythroleukemia cell lines HCD57 and SREI-2, the macrophages RAW264.7, and the skeletal muscle cells C 2 C 12 . In addition, the mGBP3 transcript was expressed to a varying degree in nu-
merous mouse tissues including the lung, spleen, heart and kidney ŽFig. 4C. . Since IFN-g has been known to induce other GBP transcripts, it was of interest to determine whether IFN-g can also induce mGBP3 gene expression. Fig. 4D shows that, in addition to mGBP3 transcript, FVA cells also expressed mGBP1 transcript to a high basal level. The treatment of FVA cells with IFN-g resulted in a small induction of both transcripts. In contrast, the basal levels of expression of both GBP transcripts were low in the macrophages RAW264.7, and required increased number of PCR amplification cycles for their detection Ž Fig. 4D, lane 3.. Upon treatment with IFN-g , both GBP transcripts were highly induced Ž Fig. 4D, lane 4. . 3.4. Expression and purification of His-mGBP3 To investigate biochemical properties of mouse GBP3, we isolated recombinant mGBP3 protein using a baculovirus expression system. Infection of Sf9 cells with the recombinant baculovirus carrying the His-mGBP3 insert resulted in an expression of histidine-tagged mGBP3 protein. The fusion protein consisted of mGBP3 amino acid residue 2–620 and additional 38 amino acids at the N-terminus, includ-
Fig. 5. Expression and purification of histidine-tagged mouse GBP3 in Sf9 cells. ŽA. Sf9 cells were infected with recombinant baculovirus carrying the histidine-tagged mGBP3 for 72 h. Aliquots from various stages of purification by Ni-chelation affinity column were analyzed by SDS-PAGE and Coomassie Blue staining. Lane 1, soluble protein fraction from non-infected Sf9 cells; lane 2, soluble protein fraction from infected Sf9 cells; lane 3, Ni-chelation column flow through; lane 4, fraction washed with binding buffer; lane 5, fraction eluted with 5 mM imidazole; lane 6, fraction eluted with 30 mM imidazole; lane 7, eluted with 100 mM imidazole; lane 8, molecular mass markers. ŽB. SDS-PAGE analysis of His-mGBP3 Ž; 5 m g. purified by Q sepharose anion-exchange chromatography Žlane 1..
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ing the histidine tag which is necessary for the protein purification by Ni-chelation affinity chromatography. Optimal His-mGBP3 expression was observed when Sf9 cells were infected with the recombinant baculovirus at the multiplicity of infection of 5 plaque-forming unit per cell for 72 h. Fig. 5A displays SDS-PAGE of the proteins obtained from various steps during the fusion protein purification. Total soluble protein fractions prepared from non-infected and infected SF9 cells are shown in lane 1 and lane 2, respectively. The bulk of the soluble proteins from the infected Sf9 cells were not retained by the Ni-chelation affinity column Ž lane 3. . Increasing the imidazole concentration in the elution buffer differentially eluted proteins of varying sizes Žlane 4–6.. A highly pure His-mGBP3 protein was obtained by elution with 100 mM imidazole Ž lane 7.. The yield of His-mGBP3 was approximately 5–10 mgrl of Sf9 cell culture. To obtain His-mGBP3 of higher purity, the affinity-purified protein was subsequently further purified by anion-exchange chromatography. His-mGBP3 was retained on the anionexchange column after loading and washing, and was eluted with buffer containing 300 mM NaCl Ž Fig. 5B, lane 1.. The purified protein showed an estimated molecular weight of 75 kDa, and was immmunologically reactive to the polyclonal anti-mGBP3 antibody Ždata not shown. . 3.5. Nucleotide binding actiÕity of His-mGBP3 Fig. 6 shows the binding activity of His-mGBP3 to various nucleotide-coupled agarose resins. Equal
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Fig. 7. Determination of kinetics for GTPase activity of HismGBP3. His-mGBP3 Ž0.2 m gr m l. was incubated at 378C for 20 min with various concentrations of GTP. GTP hydrolysis was analyzed by polyethyleneimine cellulose thin layer chromatography and autoradiography. The double-reciprocal plot of the substrate vs. GTP hydrolysis rate is shown. Data represent the mean"S.D. from three independent experiments. The calculated K m and Vmax values are 77"4 m M and 21"0.5 pmol miny1 m gy1 of protein, respectively.
aliquots of the affinity purified His-mGBP3 were incubated with nucleotide–agarose resins, and the retained proteins were eluted with the SDS-containing buffer and analyzed by SDS-PAGE. Fig. 6 lane 2 shows Coomassie Blue staining of the aliquot of purified mGBP3 used as control in the absence of nucleotide–agarose resin. It is evident that a sizable fraction of the initial starting mGBP3 was bound to agarose-immobilized guanine nucleotides GTP, GDP, or GMP. By contrast, protein retention to agarose-immobilized adenine nucleotides ŽATP, ADP, and AMP. and pyrimidine nucleotides ŽCDP, CMP, UDP, and UMP. was negligible. The His-mGBP3 also did not bind to the protein A-agarose resin.
Fig. 6. Nucleotide binding assay of His-tagged mGBP3. Purified His-mGBP3 protein was incubated with the nucleotide-agarose resins indicated for 30 min at 48C. The resins were washed with the binding buffer, and the bound proteins were eluted with PAGE sample buffer containing 2% SDS. Samples were analyzed by SDS-PAGE and Coomassie Blue staining. Lane 1, molecular mass markers; Lane 2, the total amount of His-mGBP3 protein used as starting material in the binding reaction; Lane 3, protein retained by protein-A agarose resin, lane 4–13, protein bound to indicated nucleotide–agarose resins.
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3.6. GTPase actiÕity of His-mGBP3
Fig. 8. GTP hydrolysis by His-mGBP3 in the presence of various nucleotides. His-mGBP3 Ž0.18 m gr m l. was incubated at 378C in a buffer containing 80 nM w a y32 PxGTP, 50 m M GTP, and 2 mM competitor nucleotides indicated. Hydrolysis of the radiolabeled GTP was analyzed by polyethyleneimine cellulose thin layer chromatography and autoradiography. Data represent the mean"S.D. from three independent experiments.
We found that the purified His-mGBP3 has GTPase activity that catalyzes conversion of GTP to GDP Ždata not shown. . Maximal GTP hydrolysis activity was correlated with the peak of His-mGBP3 elution during anion-exchange chromatography, showing that GTPase activity measured is likely to have stemmed from His-mGBP3. The GTP hydrolysis was dependent on magnesium and the optimum pH of the reaction was 8.0. To determine K m and Vmax , the GTPase reaction was carried out in the presence of varying GTP from 15 to 180 m M. At these substrate concentrations, GTP hydrolysis was linear up to 40 min Ždata not shown. . Fig. 7 displays double-reciprocal plot of GTP concentration vs. GTP hydrolysis rate. The K m and Vmax values for the
Fig. 9. Immunofluorescence localization of mouse GBP3 protein in FVA cells. Panels A and C show bright-field images of FVA cells cultured for 12 h with erythropoietin. Panel B and D show confocal immunofluorescence images of the same fields corresponding to A and C, respectively. Cells were treated with pre-immune serum Žpanels A and B. or polyclonal anti-mouse GBP3 antiserum Žpanels C and D., followed by incubation with biotinylated goat anti-mouse IgG and Cy5-conjugated streptavidin ŽOriginal image= 600..
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GTPase activity of His-mGBP3 were estimated to be 77 " 4 m M and 21 " 0.5 pmol miny1 m gy1 of protein, respectively, from three experiments using a single His-mGBP3 preparation. The specific activities of GTPase, however, varied in different preparations. To determine the substrate specificity of HismGBP3, GTPase assay was performed with HismGBP3 in the presence of 80 nM w a y32 PxGTP, 50 m M unlabeled GTP, and 40-fold molar excess of competitor nucleotides Ž Fig. 8. . Hydrolysis of the radiolabeled GTP was inhibited by guanine nucleotides in the decreasing order of GTP, GMP, and GDP. By contrast, ATP and CTP had little effect on GTP hydrolysis by His-mGBP3. 3.7. Subcellular distribution of mGBP3 Fig. 9 displays immunofluorescence localization of mGBP3 in FVA cells cultured for 12 h with Epo. Consistent with previous observation w33x, the FVA cells appear to consist of a large prominent nucleus surrounded by a small region of cytoplasm between the nucleus and the plasma membrane Ž Fig. 9A,C.. The immunofluorescence staining by anti-mGBP3 antiserum showed a preferential distribution of mGBP3 to this non-nuclear compartmental edge, presumably the cytosol ŽFig. 9D. . Moreover, the immunostaining appears to be more prominent in the larger, less matured erythroid progenitor cells than in the smaller, more differentiated cells. No specific immunofluorescence could be detected in FVA cells when the primary antiserum was substituted with pre-immune serum Ž Fig. 9B. . The localization of mGBP3 protein was also examined by SDS-PAGE and immunoblot assay on subcellular fractions. Anti-mGBP3 antiserum recognized exclusively a protein of approximately 70 kDa in all cellular fractions including membrane, cytosol and nucleus Ž data not shown. . The absence of cross-reaction to proteins of other molecular sizes suggests that the antiserum is specific to GBP3 and does not recognize other known GBPs. In addition, the immunoblot data confirm that at least a majority of mGMP3 is present in the cytosol of the FVA cells. However, since we have not determined to what extent the membrane and nuclear fractions might be contaminated by cytosol, the possible presence of mGBP3 in membrane and nuclear compartments should be viewed with caution.
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4. Discussion GBPs were originally identified as a group of proteins with a strong binding activity to GMPagarose resins w11,12x. The stimulation of human fibroblasts and murine macrophages by interferons resulted in the synthesis of GBP1 along with other minor GBPs. The molecular cloning and sequencing of two members of genes encoding GBPs have revealed salient features common to GBP family proteins w13–16x. All members of this GBP family have the first two of three consensus elements found in typical GTP-binding proteins. Despite the lack of the third consensus GTP-binding motif Ž NrT. KXD, which is believed to be essential for the guanine recognition, GBPs exhibits the nucleotide binding specificity to guanine nucleotides Ž GTP, GDP, and GMP. . In addition, GBPs exhibit unusual GTPase activities that hydrolyze GTP to GMP or GDP w15– 18x. Although GBPs are now a rapidly growing family of GTP-binding proteins, their physiologic role has yet to be elucidated. In the present study, we have cloned the cDNA encoding a novel GBP termed mouse GBP3 in FVA cells. Like the other known GBPs, the mouse GBP3 possesses only the first two GTP-binding consensus motifs GXXXXGKŽSrT. and DXXG but devoid of the third consensus element Ž NrT.KXD. The hydrophobicity profile of mouse GBP3 is similar to that of hGBP1 except for the C-terminus, which has an hydrophobic stretch of 30 amino acid residue. Taken together, these findings suggest that mouse GBP3 with a predicted molecular mass of 71 kDa is a new member of the GBP family. In response to stimulation by IFNs, mouse embryonic and splenic cells are known to express multiple GBPs with molecular masses of 65 kDa Ž which was identified as mGBP1. , 70 kDa, and 71 kDa w12x. Inasmuch as amino acid sequence information for the 70- and 71-kDa GBP is currently unavailable, it is not known whether the mouse GBP3 we cloned is identical to the 70- and 71-kDa proteins. Moreover, based on the partial sequence of human GBP3 currently available w44x, mGBP3 is unlikely to be the murine homolog of the human GBP3. To characterize the biochemical property of mouse GBP3, we initially expressed His-GBP3 fusion protein using a bacterial expression system. Although the
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recombinant His-mGBP3 protein could be expressed, it was unstable undergoing extensive degradation during its expression and purification Ždata not shown.. In contrast, the expression of the His-mGBP3 protein in the Sf9 insect cells using a baculovirus expression system allowed the isolation of the highly purified fusion protein by nickel-chelation affinity chromatography and by anion-exchange chromatography. HismGBP3 is similar to the other GBPs in that HismGBP3 strongly binds only to guanine nucleotides ŽGTP, GDP, and GMP.. Schwemmle et al. w16x postulated that the TVRD sequence, which is conserved in other known GBPs, might be responsible for retaining the guanine nucleotide-binding specificity. In mouse GBP3, the sequence corresponding to TVRD is substituted by AVRD at amino acid residue of 173–176. Thus, the substitution of Thr 173 to Ala173 does not seem to affect the nucleotide binding specificity of mouse GBP3. The mouse GBP3 differs from the other GBPs in that the C-terminal CAAX motif, known for the post-translational modification signal via isoprenylationrmethylation, is absent. It is now known that the isoprenylationrmethylation modification plays a crucial role in enhancing the association of the p21ras family proteins and the g-subunits of the G-proteins with the plasma membranes w19–21x. Both hGBP1 w17x and rat p67 GBP w15,22x have been shown to be isoprenylated in vitro at the carboxyl terminal Cys residue. Isoprenylated p67 GBP appeared to be predominantly associated with the cell membranes in rat smooth muscle cells w15x. By contrast, despite being isoprenylated, human GBP1 has been recently reported to be localized in the cytosol of the promyelocytic cell line HL-60 w45x. We also found the presence of mGBP3 in the cytosol of FVA cells based on immunofluorescence staining using a polyclonal anti-mGBP3 antibody Ž Fig. 9. and by the subcellular fractionation and immunoblot analysis Ž unpublished observation. . Since the cDNA encoding mouse GBP3 was initially identified in Epo-stimulated FVA cells, we examined the expression of mouse GBP3 transcript in the course of erythroid differentiation. FVA cells represent the colony-forming unit-erythroid Ž CFU-E. stage of erythroid development, requiring Epo for proliferation and differentiation into enucleated reticulocytes or erythrocytes. We found that the mouse
GBP3 transcript is already expressed at a high level in FVA cells at the time of their isolation from the spleen. In point of fact, mGBP3 expression level is higher in FVA cells than in other cells we tested thus far ŽFig. 4.. Although mouse GBP3 mRNA is transiently increased within 3 h, and then down-regulated thereafter in FVA cells undergoing differentiation in the presence of Epo, the induction of the mouse GBP3 transcript is not strictly Epo-dependent. A transient increase in mouse GBP3 transcript is also observed in FVA cells cultured in Epo-deprived culture medium, but to a lesser extent than in Epo-exposed cells Ždata not shown.. The reasons for the high ‘basal’ expression of mGBP3 in FVA cells is not yet known. It is possible that in FVA cells, the Epo receptor signaling cascades already have been activated by the Friend spleen focus-forming virus ŽSFFV. -encoding glycoprotein gp55, which associates with the Epo receptors w46,47x. Thus, Epo-responsive genes could have already been activated in FVA cells despite the absence of Epo in vitro culture. Another possibility is that cytokines released from the FVA cells in culture may induce mouse GBP3 gene in an autocrine or paracrine manner. The mechanism responsible for the induction of mGBP3 in FVA cells remains to be explored. However, its possible role in erythroid differentiation is suggested by the high basal level of the gene expression, which undergoes a transitory change in the course of FVA cell differentiation in culture. By contrast, erythroleukemia cell lines HCD-57 and SREI-2 lacking the capability to differentiate into hemoglobin-rich reticulocytes w48x have exceedingly low basal levels of mGBP3, which remain unchanged during culture with Epo Ždata not shown.. Mouse GBP3 transcript is also highly induced by IFN-g in macrophages RAW264.7. This suggests that like other GBPs, the mouse GBP3 gene product may be involved in mediating IFN-g ’s action during macrophage activation. It is of interest that IFNs have been reported to inhibit normal erythropoiesis by counteracting the proliferative effect of Epo without affecting the ability of the hormone to induce terminal differentiation w49–51x. In light of the findings that mGBP3 is not only transiently induced during erythropoiesis but also responsive to IFN-g , it is tempting to suggest that mouse GBP3 gene product may play a role in the early molecular events for the
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initiation of the erythroid differentiation rather than in the proliferative response to Epo. The recombinant His-mGBP3 exhibits an intrinsic GTPase activity that hydrolyzes GTP predominantly to GDP Ždata not shown.. While GMP is the major product hydrolyzed by recombinant hGBP1 w17x, GDP rather than GMP is the predominant product of hydrolysis reaction catalyzed by the hGBP2 w18x and the chicken GBP w16x. In this regard, the mouse GBP3 has a catalytic activity similar to that of the hGBP2 and of the chicken GBP. This suggests that although there are high degrees of similarities in peptide sequences and in guanine nucleotides-binding activities, there may be differences in profile of GTPase activities between members of the family of guanylate-binding-proteins.
Acknowledgements We thank S.T. Sawyer for gifts of HCD-57 and SREI-2 cell lines, Shivendra Shukla for FDC-P2 cell line, Mary Kim, Jane Burnett and Elisabeth Norton for their technical assistance. This work was supported in part by National Institute of Health Grant AR40682 and by National Science Foundation Grant MCB-9218686.
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Cloning, expression, and characterization of a novel guanylate-binding protein, GBP3 in murine erythroid progenitor cells Byung Hee Han b
a,1
, Don Jae Park b, Robert W. Lim a , Jeong Hyok Im a , Hyun Dju Kim
a,)
a Department of Pharmacology, UniÕersity of Missouri-Columbia, School of Medicine, Columbia, MO 65212, USA Department of Medicine, DiÕision of Hematology, Vanderbilt UniÕersity Medical Center, NashÕille, TN 37232-6305, USA
Received 14 October 1997; revised 20 February 1998; accepted 25 February 1998
Abstract We report the molecular cloning of a novel guanylate-binding protein ŽGBP., termed mouse GBP3 ŽmGBP3. in Friend virus-induced mouse erythroid progenitor ŽFVA. cells. The 71-kDa mGBP3 belongs to a family of known GBPs that contain the first two consensus motifs, GXXXXGKŽSrT. and DXXG, but lack the third element, ŽNrT.KXD, found in typical GTP-binding proteins. Recombinant mGBP3 protein, expressed using a baculovirus expression system, binds to agarose-immobilized guanine nucleotides ŽGTP, GDP and GMP.. Moreover, mGBP3 has been found to have an intrinsic GTPase activity with K m and Vmax values of 77 " 4 m M and 21 " 0.5 pmol miny1 m gy1 of protein, respectively. The mGBP3 is distinct from the other GBPs, in that it does not have an isoprenylationrmethylation motif CAAX at the carboxyl terminus. The mGBP3 appears to be localized in the cytosol based on immunofluorescence staining. Although the mGBP3 transcript is expressed to a varying degree in numerous mouse tissues, the message is most abundant in FVA cells. The mGBP3 transcript increases in FVA cells undergoing differentiation to a maximum within a few hours and then decreases to an undetectable level by 24 h. These results, taken together, suggest that mGBP3 is a novel member of a family of guanylate-binding proteins, which plays a role in the erythroid differentiation. The nucleotide sequence reported in this paper has been submitted to the GenBanke with accession number U44731. q 1998 Elsevier Science B.V. All rights reserved. Keywords: cDNA sequence; GTPase activity; Gene expression; Subcellular localization; Erythropoietin; Interferon-g
Abbreviations: Epo, Erythropoietin; ERG, Erythropoietin-responsive gene; FBS, Fetal bovine serum; FVA cells, Erythroid progenitor cells isolated from the mouse spleen infected with Friend virus; GBP, Guanylate-binding protein; His-mGBP3, histidine-tagged mouse GBP3; IFN-gc, Interferon gamma; IMDM, Iscoves, modified Dulbecco medium; RACE, Rapid amplification of cDNA ends; RT-PCR, Reverse transcriptase-polymerase chain reaction; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis ) Corresponding author. Fax: q1-573-884-4558; E-mail: [email protected] 1 Present address: Yuhan Research Center, Yuhan Pharmaceutical Co., Seoul, South Korea.
1. Introduction GTP-binding proteins regulate a variety of cellular activities including signal transduction, protein synthesis, protein targeting, and cell motility Žreviewed in Ref. w1x.. Amino acid sequence comparisons and X-ray crystallographic data analysis of GTP-binding proteins have revealed the presence of three hallmark consensus elements, GXXXXGKŽSrT., DXXG, and Ž N rT . K X D w 2 – 6 x . The first tw o m otifs GXXXXGKŽSrT. and DXXG are known to interact
0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 8 . 0 0 0 3 4 - X
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with the phosphate groups of the guanine nucleotides, whereas the third element ŽNrT.KXD is responsible for the specificity of guanine recognition. For example, the modification of Ž NrT.KXD motif by substituting Asp with Asn in p21ras w7,8x and EF-Tu w9,10x leads to a loss of the guanine nucleotide binding specificity. A family of proteins termed the guanylate-binding proteins ŽGBPs. have been originally described as interferon ŽIFN. -inducible guanine nucleotide-binding proteins in fibroblasts and in macrophages w11,12x. Molecular cloning of GBPs has uncovered several characteristic features common to the GBP family w13–16x. All GBPs possess the first two GTP-binding consensus elements GXXXXGKŽSrT. and DXXG, but lack the third ŽNrT.KXD motif. However, these GBPs still retain a high degree of selectivity to guanine nucleotides ŽGTP, GDP, and GMP. over adenine and pyrimidine nucleotides w13,17x. Recently, several GBPs have been shown to be GTPases with slightly different catalytic properties. While the recombinant hGBP1 w17x hydrolyzes GTP predominantly to GMP, the chicken GBP w16x and the hGBP2 w18x also catalyze the conversion of GTP, but GDP is the major product. In addition, most GBPs known to date have an isoprenylationrmethylation modification motif at their carboxyl termini. This posttranslational modification promotes the ability of proteins to associate with the plasma membranes w19–21x. Both hGBP1 w17x and rat p67 GBP w15,22x have been shown to be isoprenylated in vitro. The rat p67 GBP has been reported to be localized in the plasma membranes w15,22x. However, the physiological function of GBPs has not yet been elucidated. Erythropoietin ŽEpo. is a hematopoietic growth factor that regulates cell proliferation, differentiation and apoptosis of erythroid progenitor cells w23–27x. Erythroid progenitor ŽFVA. cells, isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus, have been used as a cell model system for the erythroid differentiationrmaturation w28–34x. FVA cells undergo differentiation to hemoglobin-rich reticulocytes in vitro within 48–72 h upon exposure to Epo. It has been reported that Epo induces early responsive genes such as MYC, MYB, GATA1, BVL1, NFE2, and TAL1r SCL prior to the expression of genes encoding erythrocyte-specific proteins like globins w35–39x.
Recently, we reported the isolation of erythropoietin-responsive genes ŽERGs. from the Epo-treated FVA cells by differential hybridization w38x. In this communication, we present our results in which we cloned and characterized a novel 71-kD guanylate-binding protein, termed mouse GBP3 from the Epostimulated FVA cell cDNA library. The mouse GBP3 protein shows a high sequence identity and GTPase activity similar to those of the other known GBPs. However, unlike other GBP3, the mGBP3 protein, which lacks the isoprenylationrmethylation motif CAAX, appears to be located in the cytosol of FVA cells.
2. Materials and methods 2.1. Cloning and sequencing of mGBP3 cDNA FVA cells were isolated from mouse spleen and cultured as described w40x. The construction of the murine cDNA library from Epo-stimulated FVA cells and the isolation of ERGs have been described previously w38x. One ERG clone, erg11a, appeared to encode part of a 2.6-kb transcript as determined by Northern blot analysis Žsee below. . Using the w a y32 PxdCTP-labeled erg11a cDNA insert as a probe, a random-primed lgt10 cDNA library, prepared from polyŽ A.q RNA of FVA cells treated with 2 Urml Epo for 1 h, was screened to isolate overlapping clones as described w38x. Additional 5X sequence was obtained by using the 5X-rapid amplification of cDNA ends Ž RACE. protocol according to the method of Frohman et al. w41x. Briefly, polyŽ A.q RNA isolated from FVA cells were reverse-transcribed with an erg11-gene specific reverse primer, 5X-AGGGCATTCTCCAGGTACTC-3X corresponding to positions 654–673 of mGBP3 cDNA ŽFig. 1. . The first strand cDNA was tailed with dATP by terminal deoxynucleotidyl transferase ŽUSB. and amplified using an erg11-gene specific primer, 5X-GACAAAGGTGCT GCTCAGAAGCACAG-3X corresponding to positions 418–443, and a dT17-adapter primer with Taq DNA polymerase ŽUSB. in a Amplitrone II thermal cycler ŽBarnsteadrThermolyne. . The PCR product Ž; 0.5 kb. was gel-purified and subcloned into pGEM-T vector ŽPromega. .
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Fig. 1. The cDNA and deduced amino acid sequences of mouse GBP3. Ninety eight nucleotides at the 5X end of the cDNA were obtained by the 5X-RACE PCR method. The rest of the sequence was derived from several overlapping clones containing the mouse GBP3 Ž erg11. cDNA. Amino acid sequence was deduced from the longest open reading frame and indicated by the single letter code. Consensus motifs for nucleotide binding are boxed. Two potential polyadenylation signals AATAAA, in the 3X-untranslated region, are marked by dotted lines. Two repeats of the ATTTA sequence motif found in rapidly degraded messages are underlined. The bold-faced amino acids indicate the residue used to generate polyclonal anti-mGBP3 antibody as described in Section 2.
All cDNA inserts were sequenced on both strands using a Sequenasee Version 2.0 DNA sequencing kit ŽUSB. according to manufacturer’s protocol. Se-
quences were analyzed and compared with those in the GenBanke sequence data banks using a computer program provided by Genetics Computer Group.
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2.2. Northern analysis Total RNA was isolated by the guanidium thiocyanate–phenol method w42x. RNA samples Ž 20 m grlane. were separated by electrophoresis in a 1.1% agarose gel containing formaldehyde, and transferred onto a nylon membrane Ž Amersham. . UVcrosslinked membrane were prehybridized for 2 h at 658C in rapid hybridization buffer ŽAmersham. , and hybridized in the same buffer overnight at 658C with 1 = 10 6 cpmrml of w32 Px-random-primed cDNA probe. Blots were washed twice with 2 = SSC containing 0.1% SDS for 20 min at room temperature and twice with 0.2 = SSC containing 0.1% SDS for 20 min at 658C. Following the washes, blots were exposed to X-ray film ŽKodak. at y708C with intensifying screens. 2.3. RT-PCR Two micrograms of total RNA were reverse-transcribed for 1 h at 378C in a volume of 20 m l with random primers. One tenth of the synthesized cDNA was subjected to PCR amplification for the indicated number of cycles. PCR conditions were as follows: 948C for 1 min, 568C for 1.5 min, and 728C for 1.5 min. The PCR products were separated by electrophoresis in 1.5% agarose gels and visualized by ethidium bromide staining. Under such conditions, the RT-PCR procedure appeared to be quantitative, since the yields of PCR products were proportional to the amount of cDNA input Ždata not shown. . Primers used for PCR were as follows: for mGBP3, 5X-TGGAGGCACCCATTTGTCTGGTG-3X and 5X-GAC AAAGGTGCTGCTCAGAAGCACAG-3X ; for mGBP1 gene, 5X-CAAGCTAGCTGGGAAGAGGACAGG-3X and 5X-GAACTTCCTGATACACAGGCGAGG-3X ; for mouse b-actin, 5X-TCCTATGTGGGTGACGAGGC-3X and 5X-CATGGCTGGGGTGTTGAAGG-3X.
5X-CGGAATTCCTCGAGGCACCCATTTGTCTGGTG-3X Žcorresponding to positions 89-112. and 5X-GCGGATCCTCTAGACTAACTACTTAGTGAGCC-3X Ž corresponding to positions 1923–1940. . PCR-generated fragment was cloned into the pGEM vector to form pGEM-mGBP3. The XhoIrSalI fragment of the pGEM-mGBP3 was excised and ligated into the corresponding site of pBlueBacHis2 Ž Invitrogen. . The resulting recombinant construct, pBlueBacHis2-mGBP3 expresses a fusion peptide containing mouse GBP3 amino acid residue 2–620 fused to the histidine tag at its N-terminus. Spodoptera frugiperda Ž Sf9. cells were co-transfected with the baculovirus Autographa california ŽAcMNPV. DNA and the recombinant construct pBlueBacHis2mGBP3. The recombinant baculovirus was isolated by plaque assay according to manufacture’s protocol ŽInvitrogen.. Sf9 cells Ž2 = 10 6 cellsrml. were infected with the recombinant baculovirus at a multiplicity of infection of 5 plaque-forming unit per cell for 72 h. His-mGBP3 was purified by nickel-chelation affinity chromatography according to manufacturer’s protocol ŽQiagen.. Briefly, infected Sf9 cells were lysed by sonication in binding buffer Ž20 mM Tris–HCl, pH 7.9, 500 mM NaCl, 10% glycerol, 5 mM imidazole, 0.5% Triton X-100, 10 mM b-mercaptoethanol, 1 mM PMSF, 20 m grml of leupeptin and trypsin inhibitor, and 5 m grml of pepstatin A.. Soluble protein was loaded onto Ni–NTA resins ŽQiagen. and washed with 15 bed volumes of the binding buffer. His-mGBP3 was finally eluted with a step-wise gradient of imidazole of 10 to 100 mM. Protein samples were then applied to Q sepharose anion-exchange chromatography column Ž Pharmacia. equilibrated with a buffer containing 20 mM Tris–HCl, pH 8.0 and 0.1% Triton X-100. Proteins retained on the column were eluted with a step-wise gradient of NaCl of 10–300 mM. 2.5. Nucleotide binding assay
2.4. Expression and purification of histidine-tagged mGBP3 Histidine-tagged mGBP3 Ž His-mGBP3. was expressed using a baculovirus expression system. First, the coding region of mouse GBP3 gene was generated by PCR using the following oligonucleotides:
The purified His-mGBP3 fusion protein Ž; 5 m g. was incubated with 30- m l bed volume of nucleotide–agarose resins Ž Sigma. for 30 min at 48C in 1-ml binding buffer as described previously w13x. Resin-bound proteins were eluted with 20 m l SDSPAGE sample buffer containing 2% SDS by boiling
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for 5 min. The eluted proteins were separated by SDS-PAGE in 10% polyacrylamide gels and stained with Coomassie Blue.
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tion fluid and the amount of radioactivity was determined using a scintillation counter ŽBeckman..
2.6. GTPase assay
2.7. Generation of polyclonal antibody and Western blot
GTPase assay was performed as described w17x in a buffer containing 20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 80 nM w a y32 PxGTP Ž 3000 Cirmmol. and unlabeled GTP Ž concentration as indicated in figures. at 378C. The reaction was terminated with 1 mM EDTA and 0.5% SDS. Samples were spotted on polyethyleneiminecellulose thin layer chromatography plates Ž Selecto Scientific. and separated in solution containing 1.5 M LiCl, 0.5 M acetic acid, and 0.025 M citric acid. The dried plates were autoradiographed. Radioactive regions on the TLC plates were scraped into scintilla-
Polyclonal antibody was raised by immunization of rabbits with a synthetic peptide corresponding to the mouse GBP3 amino acid residues 513–541. Of the 29 residues present in this peptide, only 10–12 residues are conserved in other known GBPs Ž see Fig. 2.. Rabbits were immunized with 100 m g of the keyhole limpet hemocyanin-coupled peptide mixed with complete Freund’s adjuvant, and boosted twice with incomplete Friend’s adjuvant at a month interval. For analysis of mouse GBP3 protein, protein samples were separated by SDS-PAGE Ž 10% polyacrylamide gel. , transferred to nitrocellulose mem-
Fig. 2. Alignment of mouse GBP3 with other guanylate-binding proteins. The predicted amino acid sequence of mouse GBP3 gene was aligned with that of mouse GBP1rmag1 w13,14x, mouse GBPmarmag2 ŽGenBanke accession number M81128., human GBP1 w13x, and human GBP2 w13x. Gray area indicates that the corresponding amino acid is identical to that of mouse GBP3. Gaps Žy. are introduced to maximize alignment. The boxed residues indicate the conserved GTP-binding consensus motifs, GXXXXGKŽSrT. and DXXG.
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branes, and preincubated with TBS Ž 20 mM Tris– HCl, pH 7.5, 150 mM NaCl. containing 3% bovine serum albumin. The blocked membranes were subsequently incubated with a 1:2000 dilution of polyclonal anti-mGBP3 antiserum, horseradish peroxidase-conjugated goat anti-rabbit IgG, and visualized with an enhanced chemiluminescence system Ž Pierce. . 2.8. Immunofluorescence staining FVA cells were cultured in IMDM with 2 Urml recombinant Epo. Twelve hours after the initiation of the culture, the cells were rinsed twice with phosphate-buffered saline, fixed with 4% paraformaldehyde for 1 h at room temperature, and attached onto coverslips. Fixed cells were permeabilized with phosphate-buffered saline containing 0.1% Triton X100 for 15 min, and nonspecific reactive sites were blocked with 10% goat serum. Cells were subjected to sequential incubation with a 1:500 dilution of polyclonal anti-mGBP3 antiserum, biotinylated goat anti-mouse IgG antibody, followed by streptavidinconjugated Cy5e Ž Zymed Laboratory.. Immunofluorescence image was analyzed by confocal fluorescence microscopy ŽBio-Rad model 600..
Sequence comparison reveals that the putative protein encoded by erg11 clone bears resemblance to a family of guanylate-binding proteins ŽGBPs. as shown in Fig. 2. The overall sequence identity between the protein encoded by the erg11 clone and the other known GBPs varies from 52% to 62%. The protein encoded by the erg11 clone is referred hereafter to as mouse GBP3. The sequence conservation between mouse GBP3 and the other GBPs is highest in the region surrounding two nucleotide binding motifs, the first GTP-binding motif GXXXXGKŽSrT., which is also known as the Walker A motif, located at amino acid residue 39–46, and the second GTP-binding motif, DXXG at residue 91–94. Like the other GBPs, the mouse GBP3 apparently lacks the third downstream motif, ŽNrT. KXD which is found in typical GTP-binding proteins. However, unlike the other GBPs, the predicted mouse GBP3 does not have a CAAX motif at its C-terminus. Instead, the mouse GBP3 has a hydrophobic 30-amino acid stretch at the C-terminus not found in either GBP1 or GBP2. The
3. Results 3.1. Isolation of a mGBP3 cDNA clone We screened a lgt10 cDNA library generated with mRNA from Epo-treated FVA cells and isolated several overlapping clones using the cDNA insert of erg11a as a probe. Nucleotide sequences of the composite erg11 cDNA were obtained from these overlapping clones with a total length of 2458 nucleotides, in which 98 nucleotides at the 5X end were generated by the 5X-RACE method. Fig. 1 shows the nucleotide and deduced amino acid sequences of the composite erg11 cDNA. The amino acid sequence encoded by the longest open reading frame of the cDNA predicts a protein of 620 amino acids with a calculated molecular mass of 70,720 Da. In the 3X-untranslated region, there are two repeats of polyadenylation signal AATAAA and two repeats of the ATTTA motif, which is commonly found in rapidly degraded mRNA species.
Fig. 3. Northern blot analysis of mouse GBP3 transcript. Twenty micrograms of total RNA isolated from FVA cells were separated by electrophoresis in a 1.1% agarose-formaldehyde gel, blotted onto nitrocellulose membrane, and hybridized to w a y32 PxdCTPlabeled probe containing mouse GBP3 cDNA residue 99-2458. The hybridized membrane was exposed to X-ray films at y708C. Lane 1 shows the ethidium bromide staining of rRNAs. Lane 2 represents the autoradiograph from the Northern analysis following hybridization.
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hydrophobicity profile of mGBP3, plotted by the algorithm of Kyte and Doolittle w43x, is similar to that of hGBP1 except for the hydrophobic region at the C-terminus Ž data not shown..
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contrast, mGBP3 transcript was not detectable by Northern analysis in two erythroleukemia cell lines, HCD-57 and SREI-2, and various mouse tissues including brain, lung, heart, spleen, kidney, liver, and intestine Ždata not shown. .
3.2. Detection of mGBP3 mRNA by Northern analysis Northern analysis confirmed the expression of mouse GBP3 gene in freshly harvested FVA cells ŽFig. 3.. The mGBP3 cDNA probe corresponding to nucleotide residues 99–2458 detected an approximately 2.6-kb transcript Žlane 2. in these cells. In
3.3. Determination of mouse GBP3 gene expression by RT-PCR Mouse GBP3 gene expression was examined further by RT-PCR. Fig. 4A describes the changes in the
Fig. 4. RT-PCR analysis of mGBP3 expression in various mouse cells and tissues. Total RNA isolated from various sources were reverse transcribed and amplified by PCR for various numbers of cycles indicated below. PCR products were separated by electrophoresis in a 1.5% agarose gel and visualized by ethidium bromide staining. RT-PCR of the constitutively expressed b-actin transcript was used as a control for normalization of RNA input. ŽA. The GBP3 expression during FVA cell differentiation. RT-PCR was performed with total RNA isolated from freshly isolated FVA cells Žlane 1. and cultured FVA cells in the presence of 2 Urml Epo for the indicated number of hours Žlane 2–8.. The mGBP3 and b-actin transcripts were amplified for 27 cycles and 25 cycles, respectively. ŽB. The mGBP3 gene expression in cell lines. mGBP3 gene expression was determined in FVA cells treated with 2 Urml Epo for 1 h Žlane 1., Epo-treated erythroleukemia cell lines HCD-57 Žlane 2. and SREI-2 Žlane 3., thrombopoietin-treated megakaryocytes FDC-P2 Žlane 4., macrophages RAW264.7 Žlane 5. and skeletal muscle cells C 2 C 12 Žlane 6.. mGBP3: 30 amplification cycles, b-actin: 25 amplification cycles. ŽC. Tissue distribution of mGBP3 transcript. mGBP3 and b-actin transcripts were amplified for 30 cycles and 25 cycles, respectively. ŽD. The mGBP3 gene induction by IFN-g . FVA cells Žlane 1–2. and macrophages Žlane 3–4. were treated with 10 Urml IFN-g for 1 h Žlane 2 and 4. or left untreated Žlane 1 and 3., and gene expression was determined by RT-PCR. For mGBP1 and mGBP3: 27 amplification cycles in FVA cells Žlane 1 and 2., 35 cycles in macrophages Žlane 3 and 4.; for b-actin: 25 amplification cycles.
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mGBP3 mRNA expression levels in FVA cells undergoing differentiation with Epo. It was found that mGBP3 transcript expression was readily detectable in freshly harvested FVA cells at the time of their isolation from the spleen. In the course of erythroid differentiation, the message levels steadily increased to a maximum at 3 h, followed by a decrease to a very low level by 24 h. By contrast, the expression level of the constitutively expressed b-actin transcript remained unchanged for up to 24 h. In FVA cells maintained without Epo, the mGBP3 transcript expression levels also increased transitorily but to a lesser extent compared to the message levels seen in FVA cells with Epo Ž data not shown.. Fig. 4B shows the expression levels of mGBP3 transcript determined in various cell lines. It is evident that the Epo-treated FVA cells displayed by far the highest level of mGBP3 transcript expression. The mGBP3 transcript was also detectable in the thrombopoietin-stimulated megakaryocytes, FDC-P2. On the other hand, the mGBP3 mRNA was either expressed at low levels or not detectable in other cell lines, including the erythroleukemia cell lines HCD57 and SREI-2, the macrophages RAW264.7, and the skeletal muscle cells C 2 C 12 . In addition, the mGBP3 transcript was expressed to a varying degree in nu-
merous mouse tissues including the lung, spleen, heart and kidney ŽFig. 4C. . Since IFN-g has been known to induce other GBP transcripts, it was of interest to determine whether IFN-g can also induce mGBP3 gene expression. Fig. 4D shows that, in addition to mGBP3 transcript, FVA cells also expressed mGBP1 transcript to a high basal level. The treatment of FVA cells with IFN-g resulted in a small induction of both transcripts. In contrast, the basal levels of expression of both GBP transcripts were low in the macrophages RAW264.7, and required increased number of PCR amplification cycles for their detection Ž Fig. 4D, lane 3.. Upon treatment with IFN-g , both GBP transcripts were highly induced Ž Fig. 4D, lane 4. . 3.4. Expression and purification of His-mGBP3 To investigate biochemical properties of mouse GBP3, we isolated recombinant mGBP3 protein using a baculovirus expression system. Infection of Sf9 cells with the recombinant baculovirus carrying the His-mGBP3 insert resulted in an expression of histidine-tagged mGBP3 protein. The fusion protein consisted of mGBP3 amino acid residue 2–620 and additional 38 amino acids at the N-terminus, includ-
Fig. 5. Expression and purification of histidine-tagged mouse GBP3 in Sf9 cells. ŽA. Sf9 cells were infected with recombinant baculovirus carrying the histidine-tagged mGBP3 for 72 h. Aliquots from various stages of purification by Ni-chelation affinity column were analyzed by SDS-PAGE and Coomassie Blue staining. Lane 1, soluble protein fraction from non-infected Sf9 cells; lane 2, soluble protein fraction from infected Sf9 cells; lane 3, Ni-chelation column flow through; lane 4, fraction washed with binding buffer; lane 5, fraction eluted with 5 mM imidazole; lane 6, fraction eluted with 30 mM imidazole; lane 7, eluted with 100 mM imidazole; lane 8, molecular mass markers. ŽB. SDS-PAGE analysis of His-mGBP3 Ž; 5 m g. purified by Q sepharose anion-exchange chromatography Žlane 1..
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ing the histidine tag which is necessary for the protein purification by Ni-chelation affinity chromatography. Optimal His-mGBP3 expression was observed when Sf9 cells were infected with the recombinant baculovirus at the multiplicity of infection of 5 plaque-forming unit per cell for 72 h. Fig. 5A displays SDS-PAGE of the proteins obtained from various steps during the fusion protein purification. Total soluble protein fractions prepared from non-infected and infected SF9 cells are shown in lane 1 and lane 2, respectively. The bulk of the soluble proteins from the infected Sf9 cells were not retained by the Ni-chelation affinity column Ž lane 3. . Increasing the imidazole concentration in the elution buffer differentially eluted proteins of varying sizes Žlane 4–6.. A highly pure His-mGBP3 protein was obtained by elution with 100 mM imidazole Ž lane 7.. The yield of His-mGBP3 was approximately 5–10 mgrl of Sf9 cell culture. To obtain His-mGBP3 of higher purity, the affinity-purified protein was subsequently further purified by anion-exchange chromatography. His-mGBP3 was retained on the anionexchange column after loading and washing, and was eluted with buffer containing 300 mM NaCl Ž Fig. 5B, lane 1.. The purified protein showed an estimated molecular weight of 75 kDa, and was immmunologically reactive to the polyclonal anti-mGBP3 antibody Ždata not shown. . 3.5. Nucleotide binding actiÕity of His-mGBP3 Fig. 6 shows the binding activity of His-mGBP3 to various nucleotide-coupled agarose resins. Equal
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Fig. 7. Determination of kinetics for GTPase activity of HismGBP3. His-mGBP3 Ž0.2 m gr m l. was incubated at 378C for 20 min with various concentrations of GTP. GTP hydrolysis was analyzed by polyethyleneimine cellulose thin layer chromatography and autoradiography. The double-reciprocal plot of the substrate vs. GTP hydrolysis rate is shown. Data represent the mean"S.D. from three independent experiments. The calculated K m and Vmax values are 77"4 m M and 21"0.5 pmol miny1 m gy1 of protein, respectively.
aliquots of the affinity purified His-mGBP3 were incubated with nucleotide–agarose resins, and the retained proteins were eluted with the SDS-containing buffer and analyzed by SDS-PAGE. Fig. 6 lane 2 shows Coomassie Blue staining of the aliquot of purified mGBP3 used as control in the absence of nucleotide–agarose resin. It is evident that a sizable fraction of the initial starting mGBP3 was bound to agarose-immobilized guanine nucleotides GTP, GDP, or GMP. By contrast, protein retention to agarose-immobilized adenine nucleotides ŽATP, ADP, and AMP. and pyrimidine nucleotides ŽCDP, CMP, UDP, and UMP. was negligible. The His-mGBP3 also did not bind to the protein A-agarose resin.
Fig. 6. Nucleotide binding assay of His-tagged mGBP3. Purified His-mGBP3 protein was incubated with the nucleotide-agarose resins indicated for 30 min at 48C. The resins were washed with the binding buffer, and the bound proteins were eluted with PAGE sample buffer containing 2% SDS. Samples were analyzed by SDS-PAGE and Coomassie Blue staining. Lane 1, molecular mass markers; Lane 2, the total amount of His-mGBP3 protein used as starting material in the binding reaction; Lane 3, protein retained by protein-A agarose resin, lane 4–13, protein bound to indicated nucleotide–agarose resins.
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3.6. GTPase actiÕity of His-mGBP3
Fig. 8. GTP hydrolysis by His-mGBP3 in the presence of various nucleotides. His-mGBP3 Ž0.18 m gr m l. was incubated at 378C in a buffer containing 80 nM w a y32 PxGTP, 50 m M GTP, and 2 mM competitor nucleotides indicated. Hydrolysis of the radiolabeled GTP was analyzed by polyethyleneimine cellulose thin layer chromatography and autoradiography. Data represent the mean"S.D. from three independent experiments.
We found that the purified His-mGBP3 has GTPase activity that catalyzes conversion of GTP to GDP Ždata not shown. . Maximal GTP hydrolysis activity was correlated with the peak of His-mGBP3 elution during anion-exchange chromatography, showing that GTPase activity measured is likely to have stemmed from His-mGBP3. The GTP hydrolysis was dependent on magnesium and the optimum pH of the reaction was 8.0. To determine K m and Vmax , the GTPase reaction was carried out in the presence of varying GTP from 15 to 180 m M. At these substrate concentrations, GTP hydrolysis was linear up to 40 min Ždata not shown. . Fig. 7 displays double-reciprocal plot of GTP concentration vs. GTP hydrolysis rate. The K m and Vmax values for the
Fig. 9. Immunofluorescence localization of mouse GBP3 protein in FVA cells. Panels A and C show bright-field images of FVA cells cultured for 12 h with erythropoietin. Panel B and D show confocal immunofluorescence images of the same fields corresponding to A and C, respectively. Cells were treated with pre-immune serum Žpanels A and B. or polyclonal anti-mouse GBP3 antiserum Žpanels C and D., followed by incubation with biotinylated goat anti-mouse IgG and Cy5-conjugated streptavidin ŽOriginal image= 600..
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GTPase activity of His-mGBP3 were estimated to be 77 " 4 m M and 21 " 0.5 pmol miny1 m gy1 of protein, respectively, from three experiments using a single His-mGBP3 preparation. The specific activities of GTPase, however, varied in different preparations. To determine the substrate specificity of HismGBP3, GTPase assay was performed with HismGBP3 in the presence of 80 nM w a y32 PxGTP, 50 m M unlabeled GTP, and 40-fold molar excess of competitor nucleotides Ž Fig. 8. . Hydrolysis of the radiolabeled GTP was inhibited by guanine nucleotides in the decreasing order of GTP, GMP, and GDP. By contrast, ATP and CTP had little effect on GTP hydrolysis by His-mGBP3. 3.7. Subcellular distribution of mGBP3 Fig. 9 displays immunofluorescence localization of mGBP3 in FVA cells cultured for 12 h with Epo. Consistent with previous observation w33x, the FVA cells appear to consist of a large prominent nucleus surrounded by a small region of cytoplasm between the nucleus and the plasma membrane Ž Fig. 9A,C.. The immunofluorescence staining by anti-mGBP3 antiserum showed a preferential distribution of mGBP3 to this non-nuclear compartmental edge, presumably the cytosol ŽFig. 9D. . Moreover, the immunostaining appears to be more prominent in the larger, less matured erythroid progenitor cells than in the smaller, more differentiated cells. No specific immunofluorescence could be detected in FVA cells when the primary antiserum was substituted with pre-immune serum Ž Fig. 9B. . The localization of mGBP3 protein was also examined by SDS-PAGE and immunoblot assay on subcellular fractions. Anti-mGBP3 antiserum recognized exclusively a protein of approximately 70 kDa in all cellular fractions including membrane, cytosol and nucleus Ž data not shown. . The absence of cross-reaction to proteins of other molecular sizes suggests that the antiserum is specific to GBP3 and does not recognize other known GBPs. In addition, the immunoblot data confirm that at least a majority of mGMP3 is present in the cytosol of the FVA cells. However, since we have not determined to what extent the membrane and nuclear fractions might be contaminated by cytosol, the possible presence of mGBP3 in membrane and nuclear compartments should be viewed with caution.
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4. Discussion GBPs were originally identified as a group of proteins with a strong binding activity to GMPagarose resins w11,12x. The stimulation of human fibroblasts and murine macrophages by interferons resulted in the synthesis of GBP1 along with other minor GBPs. The molecular cloning and sequencing of two members of genes encoding GBPs have revealed salient features common to GBP family proteins w13–16x. All members of this GBP family have the first two of three consensus elements found in typical GTP-binding proteins. Despite the lack of the third consensus GTP-binding motif Ž NrT. KXD, which is believed to be essential for the guanine recognition, GBPs exhibits the nucleotide binding specificity to guanine nucleotides Ž GTP, GDP, and GMP. . In addition, GBPs exhibit unusual GTPase activities that hydrolyze GTP to GMP or GDP w15– 18x. Although GBPs are now a rapidly growing family of GTP-binding proteins, their physiologic role has yet to be elucidated. In the present study, we have cloned the cDNA encoding a novel GBP termed mouse GBP3 in FVA cells. Like the other known GBPs, the mouse GBP3 possesses only the first two GTP-binding consensus motifs GXXXXGKŽSrT. and DXXG but devoid of the third consensus element Ž NrT.KXD. The hydrophobicity profile of mouse GBP3 is similar to that of hGBP1 except for the C-terminus, which has an hydrophobic stretch of 30 amino acid residue. Taken together, these findings suggest that mouse GBP3 with a predicted molecular mass of 71 kDa is a new member of the GBP family. In response to stimulation by IFNs, mouse embryonic and splenic cells are known to express multiple GBPs with molecular masses of 65 kDa Ž which was identified as mGBP1. , 70 kDa, and 71 kDa w12x. Inasmuch as amino acid sequence information for the 70- and 71-kDa GBP is currently unavailable, it is not known whether the mouse GBP3 we cloned is identical to the 70- and 71-kDa proteins. Moreover, based on the partial sequence of human GBP3 currently available w44x, mGBP3 is unlikely to be the murine homolog of the human GBP3. To characterize the biochemical property of mouse GBP3, we initially expressed His-GBP3 fusion protein using a bacterial expression system. Although the
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recombinant His-mGBP3 protein could be expressed, it was unstable undergoing extensive degradation during its expression and purification Ždata not shown.. In contrast, the expression of the His-mGBP3 protein in the Sf9 insect cells using a baculovirus expression system allowed the isolation of the highly purified fusion protein by nickel-chelation affinity chromatography and by anion-exchange chromatography. HismGBP3 is similar to the other GBPs in that HismGBP3 strongly binds only to guanine nucleotides ŽGTP, GDP, and GMP.. Schwemmle et al. w16x postulated that the TVRD sequence, which is conserved in other known GBPs, might be responsible for retaining the guanine nucleotide-binding specificity. In mouse GBP3, the sequence corresponding to TVRD is substituted by AVRD at amino acid residue of 173–176. Thus, the substitution of Thr 173 to Ala173 does not seem to affect the nucleotide binding specificity of mouse GBP3. The mouse GBP3 differs from the other GBPs in that the C-terminal CAAX motif, known for the post-translational modification signal via isoprenylationrmethylation, is absent. It is now known that the isoprenylationrmethylation modification plays a crucial role in enhancing the association of the p21ras family proteins and the g-subunits of the G-proteins with the plasma membranes w19–21x. Both hGBP1 w17x and rat p67 GBP w15,22x have been shown to be isoprenylated in vitro at the carboxyl terminal Cys residue. Isoprenylated p67 GBP appeared to be predominantly associated with the cell membranes in rat smooth muscle cells w15x. By contrast, despite being isoprenylated, human GBP1 has been recently reported to be localized in the cytosol of the promyelocytic cell line HL-60 w45x. We also found the presence of mGBP3 in the cytosol of FVA cells based on immunofluorescence staining using a polyclonal anti-mGBP3 antibody Ž Fig. 9. and by the subcellular fractionation and immunoblot analysis Ž unpublished observation. . Since the cDNA encoding mouse GBP3 was initially identified in Epo-stimulated FVA cells, we examined the expression of mouse GBP3 transcript in the course of erythroid differentiation. FVA cells represent the colony-forming unit-erythroid Ž CFU-E. stage of erythroid development, requiring Epo for proliferation and differentiation into enucleated reticulocytes or erythrocytes. We found that the mouse
GBP3 transcript is already expressed at a high level in FVA cells at the time of their isolation from the spleen. In point of fact, mGBP3 expression level is higher in FVA cells than in other cells we tested thus far ŽFig. 4.. Although mouse GBP3 mRNA is transiently increased within 3 h, and then down-regulated thereafter in FVA cells undergoing differentiation in the presence of Epo, the induction of the mouse GBP3 transcript is not strictly Epo-dependent. A transient increase in mouse GBP3 transcript is also observed in FVA cells cultured in Epo-deprived culture medium, but to a lesser extent than in Epo-exposed cells Ždata not shown.. The reasons for the high ‘basal’ expression of mGBP3 in FVA cells is not yet known. It is possible that in FVA cells, the Epo receptor signaling cascades already have been activated by the Friend spleen focus-forming virus ŽSFFV. -encoding glycoprotein gp55, which associates with the Epo receptors w46,47x. Thus, Epo-responsive genes could have already been activated in FVA cells despite the absence of Epo in vitro culture. Another possibility is that cytokines released from the FVA cells in culture may induce mouse GBP3 gene in an autocrine or paracrine manner. The mechanism responsible for the induction of mGBP3 in FVA cells remains to be explored. However, its possible role in erythroid differentiation is suggested by the high basal level of the gene expression, which undergoes a transitory change in the course of FVA cell differentiation in culture. By contrast, erythroleukemia cell lines HCD-57 and SREI-2 lacking the capability to differentiate into hemoglobin-rich reticulocytes w48x have exceedingly low basal levels of mGBP3, which remain unchanged during culture with Epo Ždata not shown.. Mouse GBP3 transcript is also highly induced by IFN-g in macrophages RAW264.7. This suggests that like other GBPs, the mouse GBP3 gene product may be involved in mediating IFN-g ’s action during macrophage activation. It is of interest that IFNs have been reported to inhibit normal erythropoiesis by counteracting the proliferative effect of Epo without affecting the ability of the hormone to induce terminal differentiation w49–51x. In light of the findings that mGBP3 is not only transiently induced during erythropoiesis but also responsive to IFN-g , it is tempting to suggest that mouse GBP3 gene product may play a role in the early molecular events for the
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initiation of the erythroid differentiation rather than in the proliferative response to Epo. The recombinant His-mGBP3 exhibits an intrinsic GTPase activity that hydrolyzes GTP predominantly to GDP Ždata not shown.. While GMP is the major product hydrolyzed by recombinant hGBP1 w17x, GDP rather than GMP is the predominant product of hydrolysis reaction catalyzed by the hGBP2 w18x and the chicken GBP w16x. In this regard, the mouse GBP3 has a catalytic activity similar to that of the hGBP2 and of the chicken GBP. This suggests that although there are high degrees of similarities in peptide sequences and in guanine nucleotides-binding activities, there may be differences in profile of GTPase activities between members of the family of guanylate-binding-proteins.
Acknowledgements We thank S.T. Sawyer for gifts of HCD-57 and SREI-2 cell lines, Shivendra Shukla for FDC-P2 cell line, Mary Kim, Jane Burnett and Elisabeth Norton for their technical assistance. This work was supported in part by National Institute of Health Grant AR40682 and by National Science Foundation Grant MCB-9218686.
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