Characterization of Structural Domains of Human Osteoclastogenesis Inhibitory Factor
1998; Elsevier BV; Volume: 273; Issue: 9 Linguagem: Inglês
10.1074/jbc.273.9.5117
ISSN1083-351X
AutoresKyoji Yamaguchi, Masahiko Kinosaki, Masaaki Goto, F Kobayashi, Toshio Suda, Tomonori Morinaga, Kanji Higashio,
Tópico(s)Cell Adhesion Molecules Research
ResumoOsteoclastogenesis inhibitory factor (OCIF) is a heparin-binding secretory glycoprotein that belongs to the tumor necrosis factor receptor (TNFR) family. OCIF is present both as a ∼60-kDa monomer and a disulfide-linked homodimer. We attempted to characterize the seven structural domains of OCIF by determining the capabilities of various OCIF mutants to inhibit osteoclastogenesis, to interact with heparin, and to form dimers. We also examined a potential of domains 5 and 6, death domain homologous regions (DDHs), for inducing cell death by expressing OCIF/Fas fusion proteins. Our results show that: (i) the N-terminal portion of OCIF containing domains 1–4, which have structural similarity to the extracellular domains of the TNFR family proteins, is sufficient to inhibit osteoclastogenesis; (ii) a heparin-binding site is located in domain 7, and affinity for heparin does not correlate with the inhibitory activity; (iii) Cys-400 in domain 7 is the residue responsible for dimer formation; and (iv) the C-terminal portion containing domains 5 and 6, DDHs, has a high potential for mediating a cytotoxic signal when it is expressed in cells as an OCIF/Fas fusion protein in which the transmembrane region of Fas is inserted in front of DDHs. Osteoclastogenesis inhibitory factor (OCIF) is a heparin-binding secretory glycoprotein that belongs to the tumor necrosis factor receptor (TNFR) family. OCIF is present both as a ∼60-kDa monomer and a disulfide-linked homodimer. We attempted to characterize the seven structural domains of OCIF by determining the capabilities of various OCIF mutants to inhibit osteoclastogenesis, to interact with heparin, and to form dimers. We also examined a potential of domains 5 and 6, death domain homologous regions (DDHs), for inducing cell death by expressing OCIF/Fas fusion proteins. Our results show that: (i) the N-terminal portion of OCIF containing domains 1–4, which have structural similarity to the extracellular domains of the TNFR family proteins, is sufficient to inhibit osteoclastogenesis; (ii) a heparin-binding site is located in domain 7, and affinity for heparin does not correlate with the inhibitory activity; (iii) Cys-400 in domain 7 is the residue responsible for dimer formation; and (iv) the C-terminal portion containing domains 5 and 6, DDHs, has a high potential for mediating a cytotoxic signal when it is expressed in cells as an OCIF/Fas fusion protein in which the transmembrane region of Fas is inserted in front of DDHs. In the vertebrate, homeostasis and remodeling of bone are by strictly controlled by mostly unrevealed mechanisms. Much effort has been made to clarify the mechanisms, and several protein factors were found to participate in bone homeostasis (1Chambers T.J. Hall T.J. Vitam. Horm. 1991; 46: 41-86Google Scholar, 2Suda T. Takahashi N. Martin J. Endocr. Rev. 1992; 13: 66-80Google Scholar, 3Suda T. Udagawa N. Nakamura I. Miyaura C. Takahashi N. Bone. 1995; 17: 87S-91SGoogle Scholar). Recently, we have isolated one such factor termed osteoclastogenesis inhibitory factor (OCIF) 1The abbreviations used are: OCIF, osteoclastogenesis inhibitory factor; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; DDH, death domain homologous region; TRAP, tartaric-resistant acid phosphatase; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; FBS, fetal bovine serum; PBS, phosphate-buffered saline; IMDM, Iscove's modified Dulbecco's medium; FPLC, fast protein liquid chromatography; X-gal, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside; bp, base pair(s); kb, kilobase pair(s). from the conditioned medium of human embryonic lung fibroblasts, IMR-90 (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). Both a ∼60-kDa monomer and a disulfide-linked homodimer are present in the conditioned medium, and the two forms have similar specific activity in inhibition of osteoclast formation in vitro (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). However, the mechanism by which OCIF inhibits osteoclastogenesis is not yet known. Based on the partial amino acid sequence, cDNA for human OCIF was molecularly cloned. The amino acid sequence deduced from the nucleotide sequence of OCIF cDNA predicted that it consists of 401 amino acid residues, including a putative 21-amino acid residue signal sequence (5Yasuda H. Shima N. Nakagawa N. Mochizuki S.-I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; (in press)Google Scholar). The nucleotide sequence analysis has revealed that OCIF is identical to osteoprotegerin (6Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.-S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Google Scholar). OCIF has seven major domains (domains 1–7) and has overall similarity to proteins of the tumor necrosis factor receptor (TNFR) family, although OCIF lacks an apparent transmembrane region (5Yasuda H. Shima N. Nakagawa N. Mochizuki S.-I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; (in press)Google Scholar, 6Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.-S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Google Scholar). Domains 1–4 are cysteine-rich structures with a characteristic of extracellular domains of the TNFR family proteins. Domains 5 and 6 share structural features with “death domains” of TNFR 1, Fas, DR 3 (also designated as Apo 3, Wsl 1, and TRAMP), the TRAIL receptor, and the several recently identified cytoplasmic proteins mediating apoptosis (5Yasuda H. Shima N. Nakagawa N. Mochizuki S.-I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; (in press)Google Scholar, 7Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Google Scholar, 8Itoh N. Nagata S. J. Biol. Chem. 1993; 268: 10932-10937Google Scholar, 9Tartaglia L.A. Ayres T.M. Wong G.H.W. Goeddel D.V. Cell. 1993; 74: 845-853Google Scholar, 10Cleveland J.L. Ihle J.M. Cell. 1995; 81: 479-482Google Scholar, 11Chinnaiyan A.M. O'Rourke K. Yu G.-L. Lyons R.H. Garg M. Duan D.R. Xing L. Gentz R. Ni J. Dixit V.M. Science. 1996; 274: 990-992Google Scholar, 12Marsters S.A. Sheridan J.P. Donahue C.J. Pitti R.M. Gray C.L. Goddard A.D. Bauer K.D. Ashkenazi A. Curr. Biol. 1996; 6: 1669-1676Google Scholar, 13Kitson J. Raven T. Jiang Y.-P. Goeddel D.V. Giles K.M. Pun K.-T. Grinham C.J. Brown R. Farrow S.N. Nature. 1996; 384: 372-375Google Scholar, 14Bodmer J.-L. Burns K. Schneider P. Hofmann K. Steiner V. Thome M. Bornard T. Hahne M. Schroter M. Becker K. Wilson A. French L.E. Browning J.L. Macdonald H.R. Tschopp J. Immunity. 1997; 6: 79-88Google Scholar, 15Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit M. Science. 1997; 276: 111-113Google Scholar, 16Nagata S. Cell. 1997; 88: 355-365Google Scholar, 17Duan H. Dixit V.M. Nature. 1997; 385: 86-89Google Scholar). However, unlike previously characterized death domains, two death domain homologous regions (DDHs), domains 5 and 6 of OCIF, exist in extracellular environments, because OCIF is secreted into conditioned medium. Domain 7, which does not resemble any protein motifs characterized thus far, consists of 50 amino acid residues and has a relatively high net positive charge; it contains eight basic amino acid residues (Lys and Arg) and only one acidic residue (Glu). To determine which residue(s) or domain(s) is/are involved in the in vitro biological activity, binding to heparin, and dimer formation, we generated and characterized various mutants of OCIF. We also examined the potential of domains 5 and 6 for mediating cell death by overexpressing chimeric proteins in which portions containing the transmembrane domain derived from Fas were inserted into OCIF. Escherichia coli DH5α (Life Technologies, Inc.) was used to propagate and amplify plasmids. 293-EBNA (CLONTECH), a human fetal kidney cell line, was grown in Iscove's modified Dulbecco's medium (IMDM) containing 10% fetal bovine serum (FBS) and 250 μg/ml geneticin (Sigma). A mouse bone marrow-derived stromal cell line, ST2 (Riken Cell Bank RCB0224, Japan) was grown in minimum essential medium-α containing 10% FBS. Mammalian expression plasmid pCEP4 (CLONTECH) was used for expression of OCIF mutants, Fas, and OCIF-Fas chimeric proteins. Full-length OCIF cDNA was subcloned into the XhoI and BamHI sites of pCEP4 to yield pCEP4-OCIF in which the cDNA is expressed under the control of the cytomegalovirus promoter. Human Fas cDNA (18Itoh N. Yonehara S. Ishii A. Yonehara M. Mizushima S.-I. Sameshima M. Hase A. Seto Y. Nagata S. Cell. 1991; 66: 233-243Google Scholar) was amplified by PCR using Ex Taq polymerase (Takara Shuzo) and primers 5′-TCTTTCACTTCGGAGGATTG-3′ (sense) and 5′-TCTAGACCAAGCTTTGGATTTC-3′ (antisense). A human activated T cell cDNA library (CLONTECH) was used as a template for the PCR. Mutagenesis and genetic fusions were performed according to a method called “recombinant polymerase chain reaction” (19Higuchi R. PCR Protocols. Academic Press, New York1990: 177-183Google Scholar). To generate the expression vector for OCIF mutants, DNA fragments amplified by PCR were digested with appropriate restriction enzymes (DNA fragments for ΔD1, ΔD2, and ΔD3 byXhoI/NdeI; ΔD4 byXhoI/SphI; ΔD5, C195S, C202S and C277S byNdeI/SphI; ΔD6 and C319S byNdeI/BstEII; ΔCL and C400S bySphI/BstEII; ΔD7 bySphI/BamHI; and ΔD67 and ΔD567 byNdeI/BamHI) and were substituted for the corresponding region of OCIF cDNA in pCEP4-OCIF. The plasmids thus constructed were designated pCEP4-ΔD1, pCEP4-ΔD2, pCEP4-ΔD3, pCEP4-ΔD4, pCEP4-ΔD5, pCEP4-C195S, pCEP4-C202S, pCEP4-C277S, pCEP4-ΔD6, pCEP4-C319S, pCEP4-ΔCL, pCEP4-C400S pCEP4-ΔD7, pCEP4-ΔD67, and pCEP4-ΔD567, respectively. To construct a vector expressing OCIF-Fas, DNA fragment coding for domains 1–4 of OCIF (amplified using primers 5′-TGACAAATGTCCTCCTGGTA-3′ and 5′-AGATCTCGATCCATCTATTCCACATTTTTGAGTTG-3′) and that for the transmembrane domain through the intracellular region of Fas (amplified using primers 5′-TGTGGAATAGATGGATCGAGATCTAACTTGGGGTGGCTT-3′ and 5′-CCGGATCCTCTAGACCAAGCTTTGGATTTC-3′) were fused as described previously (19Higuchi R. PCR Protocols. Academic Press, New York1990: 177-183Google Scholar). The fused fragment was digested with NdeI and BamHI, and the resultant fragment was substituted for the NdeI/BamHI fragment of OCIF cDNA in pCEP4-OCIF to generate pCEP4-OCIF-Fas. To generate pCEP4-TM-OCIF, first, a DNA fragment encoding the transmembrane region of Fas was amplified by PCR with primers 5′-GGCTCGAGTCTTTCACTTCGGAGGATTG-3′ (sense) and 5′-CCTCCGGAACCTTGGTTTTCCTTTCTGTG-3′ (antisense), and the human activated T cell cDNA library as a template. Next, the amplified fragment was digested with BspEI, and the ∼700-bp fragment produced was ligated with the 0.4-kbBspEI/SphI fragment of OCIF cDNA. Then, the ligated DNA fragment was digested with BglII and SphI, and a ∼550-bp fragment was isolated. Finally, a ∼700-bp BglII/XhoI fragment from pCEP4-OCIF-Fas, the ∼550-bp fragment, and an ∼11-kbXhoI/SphI fragment from pCEP4-OCIF were ligated. To construct pCEP4-TM-OCIFΔD567, a DNA sequence encoding domains 5 to 7 was deleted from pCEP4-TM-OCIF by recombinant PCR (19Higuchi R. PCR Protocols. Academic Press, New York1990: 177-183Google Scholar). Mutations were confirmed by DNA sequencing. For the production of ΔD1, ΔD2, ΔD3, ΔD4, ΔD5, ΔD6, ΔD7, ΔD67, ΔD567, ΔCL, C195S, C202S, C277S, C319S, and C400S, 293-EBNA cells were seeded on 24-well plates at a cell density of ∼2 × 105/well, and the cells in each well were transfected with 1 μg of the respective expression vector on the following day using LipofectAMINE (Life Technologies, Inc.). Five hours after transfection, the DNA precipitate was removed, and the cells were cultured in serum-free medium for 40 h. Cells transfected with pCEP4-ΔD567 were cultured in IMDM containing 10% FBS and 200 μg/ml hygromycin B (Wako) to establish stable transformants. An ELISA employing rabbit anti-OCIF polyclonal antibody was used to quantify OCIF mutants in the conditioned medium. The polyclonal antibody was prepared as follows. OCIF produced in 293-EBNA harboring pCEP4-OCIF was purified to homogeneity as described previously (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). JW rabbits were immunized with the purified OCIF. Anti-OCIF antibody was purified from serum of the rabbits using protein G-Sepharose (Pharmacia Biotech Inc.). Horseradish peroxidase-labeled anti-OCIF antibody was prepared using a maleimide-activated peroxidase kit (Pierce). Osteoclastogenesis inhibitory activity was determined by observing the suppression of osteoclast-like cell formation. Osteoclast-like cell formation was evaluated by measuring tartaric acid-resistant acid phosphatase (TRAP) activity in co-culture of mouse spleen cells and ST2 cells after cultivating for 1 week in the presence of 10 nm 1,25-dihydroxyvitamin D3 and 100 nm dexamethasone concomitant with various concentrations of OCIF mutants as described previously (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). TRAP activity is expressed in absorbance at 405 nm as described previously (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). Mutant ΔD567 was purified from conditioned medium of a stable 293-EBNA transfectant using rabbit anti-OCIF polyclonal antibody-immobilized HiTrap NHS-activated column (Pharmacia). The conditioned medium (∼100 ml) was applied to the column at a flow rate of 0.5 ml/min. After washing the column with 21.5 ml of 50 mm Tris-HCl buffer (pH 7.5) containing 1m NaCl and 0.1% CHAPS (Sigma) and subsequently with 8 ml of 0.15 m NaCl containing 0.1% CHAPS, proteins were eluted from the column with 0.2 m acetic acid (pH 2.5) containing 0.15 m NaCl and 0.1% CHAPS at a flow rate of 0.5 ml/min. Fractions containing ΔD567 were determined by an ELISA employing anti-OCIF polyclonal antibody, collected, and applied to a HiTrap Blue (Pharmacia) column equilibrated with 10 mm phosphate buffer (pH 6.0) containing 0.1% CHAPS. After washing the column with the same buffer, proteins bound to the column were eluted with a linear gradient from 0 to 2 m NaCl at a flow rate of 0.5 ml/min. Fractions containing ΔD567 (∼10 μg) were concentrated and desalted using Centricon 10 (Amicon). The affinity of wild-type OCIF and OCIF mutants for heparin was determined by FPLC on HiTrap heparin column (Pharmacia). Conditioned medium (∼1 ml) containing each OCIF mutant was applied to the column equilibrated with 50 mm Tris-HCl (pH 7.0) containing 0.1% CHAPS. The column was developed with a 40-min linear gradient of 0 to 1 m NaCl in equilibration buffer at a flow rate of 1.0 ml/min, and fractions (1.0 ml) were collected. The concentration of the mutant in each fraction was determined by ELISA employing rabbit anti-OCIF polyclonal antibody. Proteins were separated on SDS-polyacrylamide gel (10 or 13%) electrophoresis. Rainbow-colored molecular weight markers (Bio-Rad) were used as standards. Proteins were blotted onto ProBlott (Perkin-Elmer Corp.) using a semidry-type electroblotter (Bio-Rad). OCIF or OCIF mutants were detected using horseradish peroxidase (HRP)-conjugated rabbit anti-OCIF polyclonal antibody, and the membrane was exposed to x-ray film using enhanced chemiluminescence system (ECL, Amersham Corp.). Total RNA was isolated from cells 24 h posttransfection using Trizol (Life Technologies, Inc.). Reverse transcriptase-PCR was performed with Super Script preamplification system (Life Technologies, Inc.) using 1 μg of the total RNA. Primers for the reverse transcriptase-PCR were 5′-ATGAACAACTTGCTGTGCTGCGCGCT-3′ (sense) and 5′-CAAACTGTATTTCGCTCTGG-3′ (antisense). The primers were designed to amplify a 424-bp fragment corresponding to the putative signal peptide and domains 1–3 of OCIF. Size of the amplified products was determined by 1% agarose gel electrophoresis. 293-EBNA cells were inoculated into each well in a 96-well plate at a cell density of ∼2 × 104cells/200 μl/well. The cells in each well were transfected on the following day with 250 ng of empty vector, pCEP4-Fas, pCEP4-OCIF-Fas, pCEP4-TM-OCIF, or pCEP4-TM-OCIFΔD567 using LipofectAMINE. Twenty hours after transfection, cells were fixed with 1% glutaraldehyde (Wako) for 10 min at room temperature. After washing the cells twice with 200 μl of phosphate buffered saline (PBS), HRP-conjugated anti-OCIF polyclonal antibody was added to the wells, and the plate was incubated for 2 h at room temperature. After washing the cells five times with 200 μl of PBS, 100 μl of substrate solution (0.4 mg/ml o-phenylenediamine dihydrochloride (Sigma) and 0.006% H2O2 in 0.1 m citrate-phosphate buffer, pH 4.5) was added to the cells, and the incubation was continued for additional 3 min at room temperature. The reaction was stopped by adding 50 μl of 6 nH2SO4 to each well, and the absorbance at 490 nm was measured. 293-EBNA cells were inoculated into each well in a 24-well plate at a cell density of ∼5 × 104 cells/2 ml/well. The cells in each well were transfected on the following day with 1 μg of empty vector, pCEP4-Fas, pCEP4-OCIF-Fas, pCEP4-TM-OCIF, or pCEP4-TM-OCIFΔD567, together with 0.2 μg of pCH110, an expression plasmid for β-galactosidase (Pharmacia), using LipofectAMINE. Five hours after transfection, the DNA precipitate was removed, and IMDM containing 10% FBS (500 μl/well) was added to each well. After 15 h of cultivation, cells were fixed by treating with 1% glutaraldehyde for 5 min at room temperature, washed twice with 1 ml of PBS, and stained with X-gal (Wako). Cell morphology was examined under a phase-contrast microscope, and the percentage of round-shaped blue cellsversus total blue cells was calculated. For the assay of lactate dehydrogenase activity in the medium, aliquots of conditioned medium were removed before fixing the cells. Lactate dehydrogenase activity was measured using a colorimetric kit (Shino-test, Tokyo, Japan). DNA fragmentation assay was performed as described previously (20Hertmann M. Lorenz H.-M. Voll R. Grunke M. Woith W. Kaldin J.R. Nucleic Acids Res. 1994; 22: 5506-5507Google Scholar) using cells cultivated for 20 h after transfection. A schematic representation of OCIF and mutants of OCIF used in this work is shown in Fig. 1. We constructed a series of expression vectors and transfected them into 293-EBNA cells to produce deletion mutants, ΔD1, ΔD2, ΔD3, ΔD4, ΔD5, ΔD6, and ΔD7. We also constructed expression vector for C-terminal truncation mutants, ΔD67 and ΔD567. Furthermore, to identify a residue responsible for dimer formation of OCIF, a series of Cys to Ser mutants, C195S, C202S, C277S, C319S, and C400S, in which each Cys residue in domains 5, 6, and 7 was replaced with Ser, and a deletion mutant, ΔCL, which lacks two C-terminal amino acid residues, Cys-400 and Leu-401, were prepared. All but two mutants (ΔD1 and ΔD2) were detected in the conditioned medium by Western blot analysis (see below) employing anti-OCIF polyclonal antibody. OCIF mutants were quantified by ELISA using anti-OCIF polyclonal antibody. ΔD1 and ΔD2 were not secreted at levels detectable by Western blotting or ELISA. The concentration of most of the mutants in their conditioned media ranged from 200 ng/ml to 2 μg/ml. Accurate determination of inhibitory activity of ΔD6 was impossible due to poor productivity (∼50 ng/ml). Fig. 2 A shows the osteoclastogenesis inhibitory activity of the deletion mutants measured based on the inhibition of TRAP-positive osteoclast-like cell formation in the cocultures. Although wild-type OCIF inhibited the osteoclast-like cell formation in a dose range of 5 to 40 ng/ml (half-maximal inhibitory dose (ID50) of ∼6 ng/ml), ΔD3 and ΔD4 failed to inhibit the osteoclast-like cell formation at concentrations 35 and 40 ng/ml, respectively. In contrast, ΔD5 retained the inhibitory activity with an ID50 of ∼15 ng/ml. Furthermore, ΔD7 had a specific activity comparable to that of wild-type OCIF. A C-terminal truncation mutant ΔD67, which lacks domains 6 and 7, possessed the osteoclastogenesis inhibitory activity, although potency in the inhibitory activity was considerably lower than that of wild-type OCIF (an ID50 of 10 ng/ml) (Fig. 2 A). For the accurate determination of the inhibitory activity of ΔD567, which lacks DDHs entirely, we purified it using an anti-OCIF-antibody-immobilized affinity column. The purified ΔD567 inhibited osteoclastogenesis (Fig. 2 B), indicating that truncation of domains 5–7 (consisting of the C-terminal 204 residues) does not abolish the biological activity. However, the potency of ΔD567 was approximately 10% of that of wild-type OCIF (Fig. 2 B) as estimated from their ID50. These results indicate that the N-terminal portion containing the first four domains is sufficient for exerting the osteoclastogenesis inhibitory activityin vitro. To examine the significance of the binding of OCIF to heparin in the inhibition of osteoclastogenesis, we analyzed the affinity of ΔD7, ΔD67, and ΔD567 for heparin by FPLC on HiTrap heparin column. Conditioned medium of the cells transiently expressing each OCIF mutant was loaded on the column and each mutant was eluted from the column with NaCl-containing buffer. Wild-type OCIF was eluted at NaCl concentrations of 0.55 m and 0.74 m, which correspond to those at which the monomer and the dimer form of OCIF are eluted, respectively (Fig. 3). ΔD7 was eluted as a single peak at an NaCl concentration of 0.24m. Further truncation had only marginal effects on the binding of OCIF to heparin (Fig. 3). These three mutant proteins were eluted as single peaks, probably because they are present as monomers (see below). These results strongly suggest that domain 7, which occupies the C-terminal 50 amino acid residues, contains a heparin-binding site. The fact that deletion of domain 7 did not affect the inhibition of osteoclastogenesis (Fig. 2 A) but significantly decreased the binding of OCIF to heparin (Fig. 3) indicates that binding ability of OCIF to heparin does not correlate with its osteoclastogenesis inhibitory activity in vitro. OCIF from IMR-90-conditioned medium is present as two forms, a monomer with an approximate molecular mass of 60 kDa and a disulfide-linked dimer with an approximate mass of 120 kDa (4Tsuda E. Goto M. Mochizuki S.-i. Yano K. Kobayashi F. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1997; 234: 137-142Google Scholar). To identify (a) domain(s) responsible for the dimer formation, the capability of the domain deletion mutants to form dimers was analyzed using immunoblotting as shown in Fig. 4 A. A protein with a mass of 80–100 kDa was detected for ΔD3, ΔD4, ΔD5, and ΔD6 as a major band. In contrast, ΔD7 is present almost exclusively as a ∼55-kDa protein. Thus, ΔD7 is present mainly as a monomer, while ΔD3, ΔD4, ΔD5, and ΔD6 are present in two forms, a monomer and a dimer (or a multimer) in the conditioned medium, suggesting that domain 7 is involved in the dimer formation. ΔD67 and ΔD567, both lacking domain 7, migrated as monomers as expected (Fig. 4 A). Difference in size between ΔD3 and ΔD4 (Fig. 4 A) is probably due to a different degree of glycosylation. Indeed, there are three potential N-glycosylation sites (Asn-X-Ser/Thr) in domain 3, whereas there is no such site in domain 4 (5Yasuda H. Shima N. Nakagawa N. Mochizuki S.-I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; (in press)Google Scholar, 6Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.-S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Google Scholar). Since there is only one Cys residue (Cys-400) in domain 7 (5Yasuda H. Shima N. Nakagawa N. Mochizuki S.-I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; (in press)Google Scholar, 6Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.-S. Lüthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Boyle W.J. Cell. 1997; 89: 309-319Google Scholar), participation of the residue in the intermolecular disulfide-linkage was suspected. To confirm this, two mutants, one with substitution of a Ser residue for Cys-400 (C400S) and the other with a deletion of the two C-terminal amino acid residues Cys-400 and Leu-401 (ΔCL) were produced. As a control, a series of Cys to Ser mutants, C195S, C202S, C277S, and C319S, in which each Cys residue in domains 5 and 6 was replaced with a Ser residue, were generated. The mutants were transiently expressed in 293-EBNA cells, and the structure of the mutants was analyzed by Western blotting as shown in Fig. 4 B. The results indicate that both C400S and ΔCL exist almost exclusively as a monomer with a mass of ∼60 kDa (Fig. 4 B). No monomer-form OCIF with a mass of ∼60 kDa was detected in the conditioned medium of C195S, C202S, C277S, or C319S (Fig. 4 B). These four mutants migrated even slower than the dimer form OCIF with a mass of ∼120 kDa. The slower migrating bands may represent higher order multimers derived from unusual disulfide bonding. These results demonstrate that Cys-400 is responsible for the dimer formation of OCIF. Thus, we conclude that the 120-kDa protein detected in the conditioned medium of OCIF-producing cells is a homodimer consisting of two 60-kDa monomers linked together by an intermolecular disulfide bond between two Cys-400 residues. C400S and ΔCL, which are present almost exclusively as a monomer in the conditioned medium, were as potent as wild-type OCIF in the inhibition of the in vitro osteoclast formation (Fig. 5). These results provide further evidence that formation of the dimer is not essential for exerting the in vitro osteoclastogenesis inhibitory activity. C195S, C202S, C277S, and C319S, which are present mainly as multimers as shown in Fig. 4 B, maintained the biological activity (data not shown). We next asked whether domains 5 and 6 (DDHs) have a potential for mediating cytotoxic signals. For this purpose, we transfected plasmids encoding various OCIF/Fas fusion proteins together with pCH110, an expression plasmid for β-galactosidase, in 293-EBNA cells. The structure of OCIF, Fas, and their fusion proteins used in this experiment is schematically illustrated in Fig. 6 A. The presence of mRNA derived from each chimeric construct in the transfected cells was confirmed by reverse transcriptase-PCR. Primers were designed to amplify a 424-bp fragment corresponding to the 5′ portion of OCIF mRNA. As shown in Fig. 6 B, reverse transcriptase-PCR using RNA from the cells transfected with pCEP4-OCIF-Fas, pCEP4-TM-OCIF, or pCEP4-TM-OCIFΔD567 generated the 424-bp fragment, but not with pCEP4 or pCEP4-Fas. HRP-labeled anti-OCIF polyclonal antibody specifically bound to the cells transfected with pCEP4-OCIF-Fas, pCEP4-TM-OCIF, or pCEP4-TM-OCIFΔD567, but not with the empty vector or pCEP4-Fas (Fig. 6 C). These results indicate that each chimeric cDNA was efficiently expressed, and the fusion products were translocated to the surface of the transfected cells. These cells were then stained with X-gal to examine the size and the shape of the cells harboring each expression plasmid. Microscopic examination of the cells transfected with pCEP4-Fas, pCEP4-OCIF-Fas, or pCEP4-TM-OCIF revealed that 30–60% of the blue cells were round and shrunken, showing signs of cell death (Fig. 7 A). In contrast, when transfected with the empty vector, pCEP4-OCIF or pCEP4-TM-OCIFΔD567, more than 90% of the blue cells retained the flat and adherent appearance (Fig. 7 A). Lactate dehydrogenase activity in the conditioned medium of the cells transfected with pCEP4-OCIF-Fas or pCEP4-TM-OCIF was significantly higher than that transfected with the empty vector or pCEP4-TM-OCIFΔD567 (Fig. 7 B), showing that overexpression of OCIF-Fas or TM-OCIF induced cell death. The transfection efficiency was almost the same (approximately 40%) in all transfection experiments. Cytotoxic signal induced by OCIF-Fas was apparently stronger than that induced by Fas for a currently unknown reason. To examine whether the cell death was caused by apoptosis, we next analyzed the integrity of DNA in the cells transfected with the empty vector, pCEP4-OCIF-Fas or pCEP4-TM-OCIF. Cells transfected with pCEP4-OCIF-Fas or pCEP4-TM-OCIF showed severe fragmentation of DNA, a clear symptom of
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