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Binding Sites and Binding Properties of Binary and Ternary Complexes of Insulin-like Growth Factor-II (IGF-II), IGF-binding Protein-3, and Acid-labile Subunit

1997; Elsevier BV; Volume: 272; Issue: 44 Linguagem: Inglês

10.1074/jbc.272.44.27936

1083-351X

Ryuji Hashimoto, Mayumi Ono, Hiroyuki Fujiwara, N. Higashihashi, Makoto Yoshida, Tomoko Enjoh-Kimura, Katsu-ichi Sakano,

Metabolism, Diabetes, and Cancer

We have examined regions of rat IGF-binding protein-3 (IGFBP-3) important for complex formations using two kinds of deletion mutants, three kinds of chimera molecules between rat IGFBP-3 and rat IGFBP-2, and a synthetic peptide (41 residues, Glu52-Ala92) derived from rat IGFBP-3. Solid-phase binding assays using 96-well microtiter plates were designed to quantitate the relative binding affinities. It was found that not only the IGFBP-3 derivatives with the amino-terminal, cysteine-rich domain (N domain) but also the synthetic peptide maintained affinity for IGF-II. Ternary complex formation was observed with full-length IGFBP-3 and chimera IGFBP, the carboxyl-terminal cysteine-rich domain (C domain) of which was derived from IGFBP-3, unlike the mutants lacking the C domain and the chimera IGFBPs, the C domain of which was derived from IGFBP-2. These results were confirmed by affinity cross-linking experiments. Furthermore, the IGFBP-3 derivatives that possessed the C domain of IGFBP-3 bound to the acid-labile subunit, even in the absence of IGFs. Finally, we observed sites in IGF-II important for the ternary complex formation using various IGF-II mutants. These IGF-II mutants, which contained a substitution of Tyr27 for Leu, had extremely reduced activity. These results strongly suggest that: 1) the N domain, containing at least Glu52-Ala92, of rat IGFBP-3 is important for binding to IGF-II; 2) the C domain of IGFBP-3 is essential for binding to the acid-labile subunit both in the presence and absence of IGF-II; and 3) Tyr27 of IGF-II is important for the ternary complex formation. We have examined regions of rat IGF-binding protein-3 (IGFBP-3) important for complex formations using two kinds of deletion mutants, three kinds of chimera molecules between rat IGFBP-3 and rat IGFBP-2, and a synthetic peptide (41 residues, Glu52-Ala92) derived from rat IGFBP-3. Solid-phase binding assays using 96-well microtiter plates were designed to quantitate the relative binding affinities. It was found that not only the IGFBP-3 derivatives with the amino-terminal, cysteine-rich domain (N domain) but also the synthetic peptide maintained affinity for IGF-II. Ternary complex formation was observed with full-length IGFBP-3 and chimera IGFBP, the carboxyl-terminal cysteine-rich domain (C domain) of which was derived from IGFBP-3, unlike the mutants lacking the C domain and the chimera IGFBPs, the C domain of which was derived from IGFBP-2. These results were confirmed by affinity cross-linking experiments. Furthermore, the IGFBP-3 derivatives that possessed the C domain of IGFBP-3 bound to the acid-labile subunit, even in the absence of IGFs. Finally, we observed sites in IGF-II important for the ternary complex formation using various IGF-II mutants. These IGF-II mutants, which contained a substitution of Tyr27 for Leu, had extremely reduced activity. These results strongly suggest that: 1) the N domain, containing at least Glu52-Ala92, of rat IGFBP-3 is important for binding to IGF-II; 2) the C domain of IGFBP-3 is essential for binding to the acid-labile subunit both in the presence and absence of IGF-II; and 3) Tyr27 of IGF-II is important for the ternary complex formation. Insulin-like growth factor (IGF) 1The abbreviations used are: IGF, insulin-like growth factor; rIGF, recombinant IGF; IGFBP, IGF-binding protein; ALS, acid-labile subunit; HRP, horseradish peroxidase; PhoA, phosphatase A; pAb, polyclonal antibody. -I and -II contain three disulfide bonds and have both amino acid sequence and tertiary structural homology with each other and with insulin (1Humbel R.E. Eur. J. Biochem. 1990; 190: 445-462Crossref PubMed Scopus (679) Google Scholar, 2Dodson E.J. Dodson G.G. Hodgkin D.C. Reynolds C.D. Can. J. Biochem. 1979; 57: 469-479Crossref PubMed Scopus (98) Google Scholar, 3Cooke R.M. Harvey T.S. Campbell I.D. Biochemistry. 1991; 30: 5484-5491Crossref PubMed Scopus (164) Google Scholar, 4Terasawa H. Kohda D. Hatanaka H. Nagata K. Higashihashi N. Fujiwara H. Sakano K. Inagaki F. EMBO J. 1994; 13: 5590-5597Crossref PubMed Scopus (46) Google Scholar). Unlike insulin, IGFs bind to a family of specific IGF-binding proteins (IGFBPs) that regulate IGF function in blood and body fluids (5Clemmons D.R. Growth Regul. 1992; 2: 80-87PubMed Google Scholar, 6Rechler M.M. Vitam. Horm. 1993; 47: 1-114Crossref PubMed Scopus (447) Google Scholar). There are six kinds of IGFBPs, designated IGFBP-1 to IGFBP-6, which have related primary structures (7Shimasaki S. Ling N. Prog. Growth Factor Res. 1991; 3: 243-266Abstract Full Text PDF PubMed Scopus (649) Google Scholar). Except for IGFBP-6, all human and rat IGFBPs contain 18 homologous cysteine residues in the amino-terminal and the carboxyl-terminal conserved domains (7Shimasaki S. Ling N. Prog. Growth Factor Res. 1991; 3: 243-266Abstract Full Text PDF PubMed Scopus (649) Google Scholar). Recently, it was reported that the mac25 gene encodes a preprotein (named IGFBP-7) of 277 amino acids, which contain the common IGFBP motif in the amino-terminal domain. In this domain, 11 of the usual 12 cysteines are conserved (8Swisshelm K. Ryan K. Tsuchiya K. Sager R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4472-4476Crossref PubMed Scopus (198) Google Scholar), and IGFBP-7 binds IGFs with specificity (9Oh Y. Nagalla S.R. Yamanaka Y. Kim H.-S. Wilson E. Rosenfeld R.G. J. Biol. Chem. 1996; 271: 30322-30325Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Although IGFBP-7 contains a total of 18 cysteines, consistent with the IGFBP-1 to IGFBP-5 molecules, the carboxyl-terminal domain contains only one homologous cysteine position. When IGFs are bound to IGFBP-3, an acid-labile subunit (ALS) can bind to the IGF/IGFBP-3 complex to form an IGF/IGFBP-3/ALS 150-kDa ternary complex (10Baxter R.C. Martin J.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6898-6902Crossref PubMed Scopus (304) Google Scholar, 11Baxter R.C. Martin J.L. Beniac V.A. J. Biol. Chem. 1989; 264: 11843-11848Abstract Full Text PDF PubMed Google Scholar). A recent study also identified a binary complex of IGFBP-3/ALS in rat serum (12Lee C.Y. Rechler M.M. Endocrinology. 1995; 136: 668-678Crossref PubMed Google Scholar, 13Lee C.Y. Rechler M.M. Endocrinology. 1995; 136: 4982-4989Crossref PubMed Google Scholar). Through binding analyses using various IGFs mutants, it was reported that Glu6, Phe26, Phe48, Arg49, and Ser50 of human IGF-II were important for binding to IGFBPs (14Lüthi C. Roth B.V. Humbel R.E. Eur. J. Biochem. 1992; 205: 483-490Crossref PubMed Scopus (17) Google Scholar, 15Francis G.L. Aplin S.E. Milner S.J. McNeil K.A. Ballard F.J. Wallace J.C. Biochem. J. 1993; 293: 713-719Crossref PubMed Scopus (75) Google Scholar, 16Bach L.A. Hsieh S. Sakano K. Fujiwara H. Perdue J.F. Rechler M.M. J. Biol. Chem. 1993; 268: 9246-9254Abstract Full Text PDF PubMed Google Scholar). There is also information about binding regions of IGFBPs to IGFs derived from fragments of IGFBPs that exist in human and rat serum (6Rechler M.M. Vitam. Horm. 1993; 47: 1-114Crossref PubMed Scopus (447) Google Scholar). For example, amino-terminal 30-kDa fragments of IGFBP-3 obtained from human (17Zapf J. Kiefer M. Merryweather J. Musiarz F. Bauer D. Born W. Fischer J.A. Froesch E.R. J. Biol. Chem. 1990; 265: 14892-14898Abstract Full Text PDF PubMed Google Scholar) and rat (18Zapf J. Born W. Chang J.-Y. James P. Froesch E.R. Fischer J.A. Biochem. Biophys. Res. Commun. 1988; 156: 1187-1194Crossref PubMed Scopus (59) Google Scholar, 19Shimonaka M. Schroeder R. Shimasaki S. Ling N. Biochem. Biophys. Res. Commun. 1989; 165: 189-195Crossref PubMed Scopus (55) Google Scholar) serum were able to bind IGFs. Furthermore, an amino-terminal 15-kDa fragment derived from human plasma IGFBP-3 was detected by ligand blotting using125I-labeled IGF-I (20Sommer A. Maack C.A. Spratt S.K. Mascarenhas D. Tressel T.J. Rhodes E.T. Lee R. Roumas M. Tatsuno G.P. Flynn J.A. Gerber N. Taylor J. Cudny H. Nanney L. Hunt T.K. Spencer E.M. Spencer E.M. Modern Concepts of Insulin-like Growth Factors. Elsevier Science Publishing Co., NY1991: 715-728Google Scholar). These natural fragments corresponding to the amino-terminal domain of IGFBP-3 were isolated without disulfide reduction, suggesting that these domains are not linked by disulfide bonds (6Rechler M.M. Vitam. Horm. 1993; 47: 1-114Crossref PubMed Scopus (447) Google Scholar). In the case of the IGFs/IGFBP-3/ALS ternary complex formation, Tyr24 and the D-domain (residues 63–70) of human IGF-I are thought to be important (21Baxter R.C. Bayne M.L. Cascieri M.A. J. Biol. Chem. 1992; 267: 60-65Abstract Full Text PDF PubMed Google Scholar). However, the properties of IGFBP-3 and ALS for ternary complex formation are not understood well. We previously characterized IGFs binding to their receptors and biological activities of IGFs using various recombinant IGF-II mutants (22Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Abstract Full Text PDF PubMed Google Scholar, 23Hashimoto R. Fujiwara H. Higashihashi N. Enjoh-Kimura T. Terasawa H. Fujita-Yamaguchi Y. Inagaki F. Perdue J.F. Sakano K. J. Biol. Chem. 1995; 270: 18013-18018Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) and confirmed the three-dimensional structure of recombinant IGF-II (4Terasawa H. Kohda D. Hatanaka H. Nagata K. Higashihashi N. Fujiwara H. Sakano K. Inagaki F. EMBO J. 1994; 13: 5590-5597Crossref PubMed Scopus (46) Google Scholar). In this study, we describe new solid-phase binding assays that can be performed easily using 96-well microtiter plates. Using these assays, we evaluated the binding properties of various mutants, chimeras, and a synthetic peptide derived from rat IGFBP-3 to analyze IGFs/IGFBP-3 and IGFs/IGFBP-3/ALS structure-function relationships. A rat pancreas cDNA (λgt 11) library of adult Sprague-Dawley females was purchased fromCLONTECH Laboratories, Inc. (Palo Alto, CA). The prokaryotic expression vector pTrc99A, pGEX-2T, DEAE-Sepharose CL-6B resin, SP-Sepharose F.F. resin, and NHS-activated HiTrap affinity column were from Pharmacia Biotech, Inc. (Uppsala, Sweden). Protein A affinity resin (PROSEP-A) was from Bioprocessing, Ltd. (Durham, United Kingdom) and DEAE-5PW 75 × 7.5 mm inside diameter was from Tosoh (Tokyo, Japan). A YMC-Pack Protein-RP column (250 × 4.6 mm inside diameter) was from YMC Co., Ltd. (Kyoto, Japan). A maleimide-activated horseradish peroxidase kit and disuccinimidyl suberate were purchased from Pierce. 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) peroxidase substrate and microtiter plates (96-well) were obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD) and Costar (Cambridge, MA), respectively. Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (anti-rabbit IgG-HRP), a biotinylation kit, and125I-labeled human recombinant IGF-II (2000 Ci/mmol) were obtained from Amersham Corp. All other chemicals were of the highest quality commercially available. Recombinant human IGF-II and IGF-II mutants of [Leu27]rIGF-II, [Leu43]rIGF-II, and [Arg54, Arg55]rIGF-II were expressed inEscherichia coli MC1061 using constructs and purification procedures as described previously (22Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Abstract Full Text PDF PubMed Google Scholar). A double mutant of [Leu27, Leu43]rIGF-II was constructed by identical procedures using synthetic oligonucleotides. Oligonucleotides were synthesized by an Applied Biosystems model 380A synthesizer, purified as described previously (24Marumoto Y. Sato Y. Fujiwara H. Sakano K. Saeki Y. Agata M. Furusawa M. Maeda S. J. Gen. Virol. 1987; 68: 2599-2606Crossref PubMed Scopus (28) Google Scholar), and sequences were confirmed by the dideoxynucleotide chain termination method (25Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52653) Google Scholar) using a 7-DEAZA sequencing kit (Takara Shuzo, Kyoto, Japan). Cloning of IGFBP-3 and IGFBP-2 cDNA was performed by polymerase chain reaction with reference to known cDNA sequences (26Shimasaki S. Koba A. Mercado M. Shimonaka M. Ling N. Biochem. Biophys. Res. Commun. 1989; 165: 907-912Crossref PubMed Scopus (138) Google Scholar, 27Brown A.L. Chiariotti L. Orlowski C.C. Mehlman T. Burgess W.H. Ackerman E.J. Bruni C.B. Rechler M.M. J. Biol. Chem. 1989; 264: 5148-5154Abstract Full Text PDF PubMed Google Scholar) using rat pancreas cDNA library (λgt 11) as a template. Considering the significant homology among all rat IGFBPs, IGFBP-3 contained the three major domains: an amino-terminal (Gly1-Cys89) and carboxyl-terminal domain (Cys187-Gln265), rich in cysteine residues; and an linkage-domain (Ala90-Pro186) with no significant homology. IGFBP-3 mutants designated N+L domain (Gly1-Pro186) and N domain (Gly1-Ser93) were constructed by polymerase chain reaction methods using the rat IGFBP-3 cDNA as a template. A chimera protein whose N, L, and C domain were derived from IGFBP-3, IGFBP-2, and IGFBP-2, respectively, was named as chimera-322. In this way, chimera proteins designated chimera-322, chimera-323, and chimera-332 were constructed by mixing polymerase chain reaction-derived fragments from the N, L, and C domains using appropriate rat IGFBP-3 and rat IGFBP-2 cDNA clones as templates. All IGFBP-3 derivatives are illustrated in Fig.1. For the expression of IGFBP-3, N+L domain, chimera-322, chimera-323, and chimera-332 in E. coli, the phosphatase A (PhoA) signal sequence was chemically synthesized and introduced into theNcoI-HindIII site of pTrc99A, upstream of these clones, which were devoid of their own signal sequences. These expression vectors were transformed into E. coli UT5600. The cells were grown at 37 °C in an LB medium supplemented with 50 μg/ml ampicillin. After isopropyl-1-thio-β-d-galactopyranoside induction (final concentration, 0.3 mm), the cells were cultured an additional 1 h and then collected by centrifugation at 6000 rpm for 20 min. The periplasm prepared by osmotic shock (28Nossal N.G. Heppel L.A. J. Biol. Chem. 1966; 241: 3055-3062Abstract Full Text PDF PubMed Google Scholar) from the cells was adjusted to pH 7.2 with phosphate-buffered saline and applied to an IGF-II coupled affinity resin column prepared from an NHS-activated HiTrap affinity column according to the manufacturer's recommendations. Each IGFBP-3 derivative was eluted with 0.5m acetic acid and lyophilized, dissolved in 0.1% trifluoroacetic acid, and then further purified by reverse-phase high-performance liquid chromatography (YMC-Pack Protein-RP 250 × 10 mm inside dimater) with a linear gradient from 25 to 55% acetonitrile in 0.1% trifluoroacetic acid. The mutant constructed of only the IGFBP-3 N domain was expressed inE. coli BL-21 as a fusion protein with glutathioneS-transferase. The fusion protein, into which a cyanogen bromide-cleavable methionine was introduced, was constructed using expression vector pGEX-2T. The fusion protein was purified from cell lysate on a glutathione Sepharose-4B affinity resin using a batchwise method according to the manufacturer's recommendations (Pharmacia Biotech). The fraction, eluted with 25 mm reduced glutathione, was dialyzed against water and lyophilized. The N domain was released from the glutathione S-transferase fusion protein by treatment with cyanogen bromide in 70% (v/v) formic acid and lyophilized. The obtained N domain was refolded as described previously (29Marumoto Y. Teruuchi T. Enjoh T. Numata F. Sakano K. Biosci. Biotech. Biochem. 1992; 56: 13-16Crossref PubMed Scopus (3) Google Scholar) and further purified using the IGF-II coupled affinity column and reverse-phase high-performance liquid chromatography as described above. Two of the six disulfide bond pairs included in the N domain of IGFBP-3 were determined by peptide mapping procedures that involved trypsin digestion, reverse-phase high-performance liquid chromatography, and amino-terminal amino acid sequence analysis of isolated peptide fragments (data not shown). By referring to the identified disulfide bonds of Cys56-Cys69 and Cys63-Cys89, a 41-residue peptide derived from positions Glu52-Ala92 was synthesized by an Applied Biosystems model 431A peptide synthesizer using the selective S-S formation procedures (30Nakagawa S.H. Tager H.S. J. Biol. Chem. 1991; 266: 11502-11509Abstract Full Text PDF PubMed Google Scholar). Eighty ml of rat serum were dialyzed against 50 mm Tris-HCl, pH 8.2, and chromatographed on a DEAE-Sepharose CL-6B column and an IGF-II/IGFBP-3 complex affinity column essentially as described previously with some modifications (11Baxter R.C. Martin J.L. Beniac V.A. J. Biol. Chem. 1989; 264: 11843-11848Abstract Full Text PDF PubMed Google Scholar, 31Baxter R.C. Dai J. Endocrinology. 1994; 134: 848-852Crossref PubMed Scopus (62) Google Scholar). For this study, IGF-II was conjugated to an NHS-activated HiTrap affinity column, and recombinant IGFBP-3 was used instead of serum-derived IGFBP-3. Finally, the ALS was purified on a DEAE-5PW column (75 × 7.5 mm inside dimater) using a linear gradient from 0.1 to 0.5 m NaCl in 10 mm sodium phosphate buffer, pH 8.0. Fractions containing ALS were collected and dialyzed against 50 mm sodium phosphate buffer, pH 6.5, by ultrafiltration. The purity of each purified IGFBP-3, N+L domain, N domain, chimera-322, chimera-323, chimera-332, the 41-residue peptide, and ALS was confirmed by SDS-polyacrylamide gel electrophoresis in the Laemmli buffer system (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207165) Google Scholar) under reducing conditions followed by Coomassie Brilliant Blue staining. All of the protein concentrations were determined using an AccQ-Tag amino acid composition analysis column according to the manufacturer's instructions (Waters, Milford, MA). Amino acid sequences of all purified proteins were also confirmed by sequencing using an Applied Biosystems 476A gas phase sequencer. HRP-IGF-II was prepared using a maleimide-activated HRP kit according to the manufacturer's recommendations (Pierce). Briefly, IGF-II was reacted withN-succinimidyl-S-acetylthioacetate to introduce a free sulfhydryl into the primary amines and was then conjugated with maleimide-activated HRP. ALS was biotinylated on primary amines using a biotinylation kit according to the manufacturer's recommendations (Amersham Co.). Five injections (0.4 mg each) of IGFBP-3 or the 41-residue peptide were administered s.c. to five male rabbits at 2-week intervals. Each polyclonal antibody (anti-BP-3 pAb and anti-Pep pAb, respectively) was purified from the antisera using a PROSEP-A affinity column according to the manufacturer's recommendations and dialyzed against 50 mm sodium phosphate buffer, pH 6.5. All solid-phase binding assays described were carried out with 96-well microtiter plates using binding components as illustrated in Fig. 2. For all assays, the plates were coated with proteins in 50 mmsodium phosphate buffer, pH 6.5, and blocked with the same buffer containing 1% (w/v) bovine serum albumin. All washes were done with the same phosphate buffer containing 0.03% (v/v) Tween 20. Binding proteins were diluted in the phosphate buffer containing 0.25% bovine serum albumin and 0.03% (v/v) Tween 20. After coating, all steps were performed at room temperature. For the HRP-IGF-II/IGFBP-3 competitive binding assay (Fig.2 A), 50 μl of 150 ng/ml IGFBP-3 were immobilized at 4 °C overnight. The wells were washed and blocked for 2 h at room temperature. The wells were washed, and 25 μl of a 1:6000 dilution of HRP-IGF-II and 25 μl of each competitor were added simultaneously to each well. The plates were incubated for 2 h and washed, and 100 μl of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate (Kirkegaard & Perry Laboratories) were added. After 20 min, the absorbance of the reaction product at 405 nm was read using aV max microplate reader (Molecular Devices, Menlo Park, CA). To study complex formation with ALS, indirect immobilization procedures using biotinylated ALS were performed. Wells were precoated with 50 μl of 1 μg/ml streptavidin, and unbound sites were blocked. After washing, 50 μl of 200 or 50 ng/ml biotinylated ALS were added and incubated for 2 h to immobilize ALS indirectly. To observe the relative affinities of the IGFBP-3 derivatives to form ternary complexes (Fig. 2 B; the HRP-IGF-II/IGFBP-3/ALS solid-phase binding assay), 50 μl of 200 ng/ml biotinylated ALS were used. After indirect immobilization of ALS, 25 μl of a 1:1000 dilution of HRP-IGF-II and 25 μl of various concentrations of IGFBP-3 derivatives were incubated for 2 h to form the ternary complex. The quantity of the formed complex in the well was measured as described above. To detect the binding of IGFBP-3 derivatives to ALS in the absence of IGFs (Fig. 2 C; the IGFBP-3/ALS solid-phase binding assay), various concentrations of IGFBP-3 derivatives were incubated with the immobilized biotinylated ALS. After washing the wells, the quantity of the formed complex was detected by incubation with 50 μl of 3 μg/ml anti-BP-3 pAb for 2 h, followed by washing and 50 μl of a 1:1000 dilution of anti-rabbit IgG-HRP for 2 h. To observe the ability of the IGF-II mutants to form the ternary complexes (Fig. 2 D; the IGFs/IGFBP-3/ALS solid-phase binding assay), 50 μl of 50 ng/ml biotinylated ALS were immobilized indirectly to avoid detection of the IGFBP-3/ALS complex in the absence of IGFs. After indirect immobilization of ALS, 25 μl of various concentrations of IGF-II mutants and 25 μl of 50 ng/ml IGFBP-3 were incubated for 2 h. The formed ternary complexes were detected using the anti-BP-3 pAb and anti-rabbit IgG-HRP system as described above. Each IGFBP-3 mutant or the 41-residue peptide was incubated at 4 °C overnight in 270 μl of 25 mm sodium phosphate buffer, pH 6.5, containing 0.05% Tween 20, 0.1% sodium azide, and 60,000 cpm of125I-labeled IGF-II in the absence (for the binary complex) or in the presence (for the ternary complex) of 0.6 μg of ALS. Following an overnight incubation, 30 μl of 1.5 mmdisuccinimidyl suberate were added, and the reaction mixture was further incubated on ice for 15 min. The cross-linking reaction was terminated by adding 60 μl of 1 m Tris-HCl buffer, pH 8.5. Proteins were precipitated by trichloroacetic acid at a final concentration of 20% and applied to a 12% SDS-polyacrylamide gel electrophoresis gel under reducing conditions, followed by autoradiography. IGFBP-3, two kinds of deletion mutants, three kinds of chimera IGFBPs, the 41-residue peptide, and rat serum ALS were prepared as described under “Experimental Procedures,” and their purity was confirmed by reverse-phase high-performance liquid chromatography (data not shown) and by SDS-polyacrylamide gel electrophoresis (Fig. 3). The chimera IGFBPs were expressed using the same expression vector as the IGFBP-3, but all chimera IGFBPs showed doublet bands. The doublet bands were also detected by Western blotting using anti-BP-3 pAb and by far Western ligand blotting using HRP-IGF-II (data not shown). From the results of amino-terminal amino acid sequence analyses, it was found that all chimera IGFBPs contained the expected amino-terminal sequence of Gly-Ala-Gly-Ala-Val-Gly-Ala as the primary sequence and an unexpected minor Gly-Ala-Val-Gly-Ala sequence. The minor sequence is probably due to different processing of the PhoA signal sequence, resulting in the doublet bands. Recombinant rat IGFBP-3 expressed inE. coli did not show such doublet bands nor the minor sequence, however, it is possible that IGFBP-3 may have contained the minor sequence at undetectable levels. Using the HRP-IGF-II/IGFBP-3 solid-phase competitive binding assay, the relative affinities of five IGFBP-3 mutants and the 41-residue peptide to IGF-II were calculated from their displacement curves (Fig.4) and compared with that of IGFBP-3, which was set to 100% (Table I). The chimera IGFBPs retained over 40% of the relative affinity, but the isolated N+L domain or N domain showed much lower affinity. A three-fold difference in affinity was observed between N+L domain and N domain. In the case of the 41-residue peptide, although it showed only 0.008% affinity relative to IGFBP-3, an anti-Pep pAb inhibited HRP-IGF-II binding to immobilized IGFBP-3. To verify the IGFBP-3 mutants and the 41-residue peptide binding to IGF-II, affinity cross-linking procedure was performed using 125I-labeled IGF-II. The IGFBP-3, N+L domain, N domain, and the 41-residue peptide all showed bands consistent with cross-linking specifically to125I-labeled IGF-II (Fig. 5). The negative control using only ALS did not form a complex band. These results are consistent with the conclusion that the N domain of IGFBP-3, and at least a portion of the 41-residue peptide (position Glu52-Ala92), is important for binding to IGF-II.Table IRelative binding affinities of IGFBP-3 derivatives and 41-residue peptide for IGF-IIIGFBP-3 derivativesRelative affinity1-aRelative affinities were determined from the competitive binding inhibition data shown in Fig. 4. (% of IGFBP-3)N + L domain12.5N domain4.241-residue peptide0.008Chimera-32240Chimera-32363Chimera-3321001-a Relative affinities were determined from the competitive binding inhibition data shown in Fig. 4. Open table in a new tab Figure 5Characterization of the binding of IGFBP-3 deletion mutants to 125I-labeled IGF-II by affinity cross-linking. Radioactive complexes formed by cross-linking 60,000 cpm of 125I-labeled IGF-II and lane 1,IGFBP-3 (1.5 ng); lane 2, N+L domain (30 ng); lane 3, N domain (30 ng); and lane 4, 41-residue peptide (3 μg). Lane 5 shows a negative control of cross-linking 60,000 cpm of 125I-labeled IGF-II and ALS (0.6 μg).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Using the HRP-IGF-II/IGFBP-3/ALS solid-phase binding assay, activities for the ternary complex formation of IGFBP-3 mutants and the 41-residue peptide could be compared. In this assay, indirect immobilization of ALS was adopted because direct immobilization of ALS or biotinylated ALS did not give a measurable absorbance using 25 ng/ml of IGFBP-3 (data not shown). IGFBP-3 showed concentration-dependent ternary complex formation between 0.1 and 10 nm, the same range as the competitive binding assay shown in Fig. 4. However, neither the N+L domain, N domain, nor the 41-residue peptide, which all had low affinity for IGF-II, were observed to form ternary complexes (Fig.6 A). Of the chimeras, only chimera-323 showed ternary complex formation (Fig. 6 B). The activities of the chimera IGFBPs for the ternary complex formation were also examined by affinity cross-linking experiments using125I-labeled IGF-II and ALS (Fig.7). The bands derived from the binary complex with 125I-labeled IGF-II were detected with IGFBP-3 and all chimera IGFBPs, whereas bands derived from the ternary complex were detected only with IGFBP-3 and chimera-323. Because chimera-332, which contains only the C domain from IGFBP-2, did not show activity, this suggests that the L domain is not involved for ternary complex formation.Figure 7Characterization of chimera IGFBPs activities in ternary complex formation by affinity cross-linking.Radioactive complexes formed by cross-linking 60,000 cpm of125I-labeled IGF-II and lanes 1 and2, IGFBP-3 (1.5 ng); lanes 3 and 4,chimera-322 (1.5 ng); lanes 5 and 6, chimera-323 (1.5 ng); lanes 7 and 8, chimera-332 (1.5 ng) with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) ALS (0.6 μg). Bands derived from the ternary complex (A), the binary complex (B), and 125I-labeled IGF-II (C) are shown with arrowheads.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To detect the IGFBP-3/ALS binary complex, the indirect immobilization procedure was adopted, similar to the HRP-IGF-II/IGFBP-3/ALS solid-phase binding assay. Various concentrations of IGFBP-3 were added to the wells, and the IGFBP-3/ALS binary complex formed was detected by anti-BP-3 pAb followed by anti-rabbit IgG-HRP. Preliminary experiments confirmed that the anti-BP-3 pAb could bind to all chimera IGFBPs equally by Western blot analysis (data not shown). As shown in Fig.8, IGFBP-3 and chimera-323 bound to ALS in a concentration-dependent manner. The other chimera IGFBPs (chimera-322 and chimera-332) had essentially no binding to ALS. These results strongly suggested that the C domain of IGFBP-3 plays an important role for the binding of IGFBP-3 to ALS in the presence and absence of IGFs. The relative affinities of four IGF-II mutants to IGFBP-3 were determined by the HRP-IGF-II/IGFBP-3 solid-phase competitive binding assay from their displacement curves shown in Fig.9 A and compared with that of IGF-II, which was set to 100%. The affinities of [Leu27]rIGF-II, [Leu43]rIGF-II, [Arg54, Arg55]rIGF-II, and [Leu27, Leu43]rIGF-II for IGFBP-3 were 100, 108, 25, and 100%, respectively, relative to IGF-II. The values from the first three mutants are similar to those reported previously,i.e., 70, 100, and 37%, respectively (16Bach L.A. Hsieh S. Sakano K. Fujiwara H. Perdue J.F. Rechler M.M. J. Biol. Chem. 1993; 268: 9246-9254Abstract Full Text PDF PubMed Google Scholar). The ability to form ternary complexes with these IGF-II mutants were examined with the IGFs/IGFBP-3/ALS solid-phase binding assay (Fig. 9 B). The [Leu43]rIGF-II and [Arg54, Arg55]rIGF-II showed almost the same activities relative to IGF-II. However, the two mutants with substitution of Tyr27 to Leu showed markedly less ternary complex formation, suggesting that Tyr27 of IGF-II contributes to the binding energy in the ternary complex. To ensure optimal folding of the recombinant IGFBP-3 and the mutant, the expression system with PhoA signal sequence was used with the exception of the N domain molecule. All of the expressed proteins were purified by IGF-II coupled affinity column chromatography, a binding property that requires correct folding and permitted comparison of relative affinities. The IGFBP-3 derivatives used in this study were highly purified except for chimera IGFBPs. Each chimera IGFBP contained a mature form and a minor percentage with two amino acids deleted from the amino terminus. Although the reason is unclear, it is possible that the Ala2 of each chimera IGFBP was recognized as an alternative processing site because the PhoA signal sequence is processed after Ala. On the other hand, IGFBP-3 and the N+L domain were also expressed with the same PhoA signal sequence, and they showed only Gly-Ala-Gly-Ala-Val-Gly-Ala sequence. We cannot rule out the possibility that IGFBP-3 and the N+L domain may also be processed at the secondary site, albeit at undetectable levels. From the HRP-IGF-II/IGFBP-3 solid-phase binding assay and affinity cross-linking experiments, the deletion mutants of IGFBP-3, the N+L domain, and the N domain showed 12.5 and 4.2% affinities for IGF-II, respectively, relative to full-length IGFBP-3. Previous studies showed that a 30-kDa IGFBP-3 in pregnancy plasma has 20-fold lower affinity for IGF-I than normal plasma IGFBP-3 (33Binoux M. Hossenlopp P. Lassarre C. Segovia B. Spencer E.M. Modern Concepts of Insulin-like Growth Factors. Elsevier Science Publishing Co., NY1991: 329-336Google Scholar), and a carboxyl-terminal truncated 31-kDa IGFBP-3 from rat serum also has lower affinity for IGF-I (34Schmid C. Rutishauser J. Schläpfer I. Froesch E.R. Zapf J. Biochem. Biophys. Res. Commun. 1991; 179: 579-585Crossref PubMed Scopus (114) Google Scholar). The truncated IGFBP-3 resulted from proteolysis by IGFBP-3 proteases and reduced its affinity for IGFs, thereby facilitating dissociation of the complexes and hence increasing the bioavailability of the IGFs (33Binoux M. Hossenlopp P. Lassarre C. Segovia B. Spencer E.M. Modern Concepts of Insulin-like Growth Factors. Elsevier Science Publishing Co., NY1991: 329-336Google Scholar). Results obtained from the binding of IGFBP-3 deletion mutants to IGF-II are in good agreement with that of proteolyzed and truncated form of IGFBP-3. Furthermore, we found that the 41-residue peptide (position Glu52-Ala92) could bind to IGF-II. In a previous study, the amino-terminal 88 amino acids of human IGFBP-3 showed low affinity for IGF-I (20Sommer A. Maack C.A. Spratt S.K. Mascarenhas D. Tressel T.J. Rhodes E.T. Lee R. Roumas M. Tatsuno G.P. Flynn J.A. Gerber N. Taylor J. Cudny H. Nanney L. Hunt T.K. Spencer E.M. Spencer E.M. Modern Concepts of Insulin-like Growth Factors. Elsevier Science Publishing Co., NY1991: 715-728Google Scholar). The 41-residue peptide is the smallest fragment of the IGFBP-3 derivatives found to bind to IGFs. This peptide was designed to obtain two S-S bonds, which were identified by peptide mapping procedures. The remaining four S-S bonds in the N domain could not be determined because of the cysteine-rich region at positions Cys42-Gly-Cys44-Cys45-Leu-Thr-Cys48. Therefore, we prepared one peptide that contained the S-S bonds. Although the 41-residue peptide showed 1/500th of the affinity for IGF-II relative to the N domain, the 41-residue peptide whose disulfide bonds were reduced and alkylated did not show any binding affinity (data not shown). Thus, the structure maintained by the S-S bonds are thought to be important for the binding affinity. However, compared to the relative affinities of chimera-332 (100%) and the N+L domain (12.5%) for IGF-II, the presence of only the N domain is important but not sufficient for full binding. For example, the L and C domains possibly play important roles for conformational stabilization of the N domain. For ternary complex formation, it was found that only the chimera IGFBP, which possessed the C domain derived from IGFBP-3 (chimera-323), showed ternary complex formation activity in the HRP-IGF-II/IGFBP-3/ALS solid-phase binding assay. Thus, the mutants lacking the C domains, such as the N+L domain, the N domain, and the 41-residue peptide, were not active in the affinity cross-linking experiment, and it was found that the bands derived from the ternary complex were observed using only IGFBP-3 and chimera-323. Although complexes between125I-labeled IGF-II and ALS were not observed (Fig. 5,lane 5), bands derived from the binary complex of125I-labeled IGF-II and ALS were detected in the presence of IGFBP-3 or chimera-323 as shown in Fig. 7. A previous study also showed that such a complex was detected by affinity cross-linking in the presence of IGFBP-3 (10Baxter R.C. Martin J.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6898-6902Crossref PubMed Scopus (304) Google Scholar). It is speculated that IGF-II does not have a significant binding affinity for ALS but may be positioned near ALS in the ternary complex such that the cross-linker can link IGF-II and ALS in the presence of IGFBP-3. From the observation that IGFBP-3 and chimera-323 can form the ternary complex but that the N+L domain and chimera-332 cannot, it is likely that the C domain of IGFBP-3 is necessary for binding to ALS. The results of the IGFBP-3/ALS solid-phase binding assay also suggest that the C domain of IGFBP-3 is important for binding to ALS, even in the absence of IGF-II. In this study, we did not perform an affinity cross-linking experiment to observe chimera IGFBPs binding to ALS. To clarify the binding of IGFBP-3 to ALS, only the C domain expressed in E. colishould be used in the solid-phase binding assay and affinity cross-linking experiments. Previously, we confirmed the three-dimensional structure of recombinant IGF-II (4Terasawa H. Kohda D. Hatanaka H. Nagata K. Higashihashi N. Fujiwara H. Sakano K. Inagaki F. EMBO J. 1994; 13: 5590-5597Crossref PubMed Scopus (46) Google Scholar) and identified the binding sites of IGF-II for their receptors (22Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Abstract Full Text PDF PubMed Google Scholar, 23Hashimoto R. Fujiwara H. Higashihashi N. Enjoh-Kimura T. Terasawa H. Fujita-Yamaguchi Y. Inagaki F. Perdue J.F. Sakano K. J. Biol. Chem. 1995; 270: 18013-18018Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) and for IGFBPs (16Bach L.A. Hsieh S. Sakano K. Fujiwara H. Perdue J.F. Rechler M.M. J. Biol. Chem. 1993; 268: 9246-9254Abstract Full Text PDF PubMed Google Scholar) using various IGF-II mutants. We also observed IGF-II sites important for the ternary complex formation using IGF-II mutants. Previous studies revealed that Tyr27of IGF-II is important for binding to insulin and IGF-I receptors (22Sakano K. Enjoh T. Numata F. Fujiwara H. Marumoto Y. Higashihashi N. Sato Y. Perdue J.F. Fujita-Yamaguchi Y. J. Biol. Chem. 1991; 266: 20626-20635Abstract Full Text PDF PubMed Google Scholar) and not for binding to the IGF-II/cation-independent mannose 6-phosphate receptor, and that the Tyr27 residue is structurally positioned at the opposite side from a binding region for IGFBPs, which include Glu6, Phe48, Arg49, and Ser50 (4Terasawa H. Kohda D. Hatanaka H. Nagata K. Higashihashi N. Fujiwara H. Sakano K. Inagaki F. EMBO J. 1994; 13: 5590-5597Crossref PubMed Scopus (46) Google Scholar). In this study, the results of the IGFs/IGFBP-3/ALS solid-phase binding assay suggest that mutation of Tyr27 to Leu does not support ternary complex formation. Similar results have been obtained by other groups using IGF-I mutants in which Tyr24 substituted with Leu ([Leu24]rIGF-I) reduced the ternary complex formation activity (21Baxter R.C. Bayne M.L. Cascieri M.A. J. Biol. Chem. 1992; 267: 60-65Abstract Full Text PDF PubMed Google Scholar). Tyr24 of IGF-I is also important for binding to insulin and IGF-I receptors (35Cascieri M.A. Bayne M.L. LeRoith D. Raizada M.K. Molecular and Cellular Biology of Insulin-like Growth Factors and Their Receptors. Plenum Publishing Corp., NY1989: 285-297Crossref Google Scholar). Therefore, the Tyr27 residue of IGF-II, which is important for binding to insulin and IGF-I receptors, interacts only weakly with ALS and is on a side opposite to the Glu6, Phe48, Arg49, and Ser50 residues, which bind the amino-terminal domain of IGFBP-3, within positions Glu52-Ala92. Additionally, the carboxyl-terminal domain of IGFBP-3 binds to ALS, even in the absence of IGF-II. We thank Drs. M. Furusawa, J. F. Perdue, and Y. Fujita-Yamaguchi for valuable advice, discussions, and continuous encouragement. We also thank Y. Nagano, M. Nishimura, and K. Sato for excellent technical assistance.