Biophysical and biological properties of naturally occurring high molecular weight insulin-like growth factor II variants.

A soluble form of the insulin-like growth factor II/mannose 6-phosphate receptor (sIGF-II/MPR) is present in fetal bovine serum and carries mature 7.5-kDa insulin-like growth factor II (IGF-II) and at least 12 different high molecular weight (Mr) IGF-II isoforms (Valenzano, K. J., Remmler, J., and Lobel, P. (1995) J. Biol. Chem. 270, 16441-16448). In this study, we used gel filtration and anion exchange chromatographies to resolve the isoforms into eight fractions that were characterized with respect to their biochemical, biophysical, and biological properties. Each fraction contained one to three major protein species with apparent sizes ranging from 11 to 17 kDa by SDS-polyacrylamide gel electrophoresis. The 11-kDa species contains no post-translational modifications and consists of an extended IGF-II backbone terminating at Gly-87. The remaining high Mr IGF-II isoforms are also composed of an 87-amino acid IGF-II peptide backbone but contain increasing amounts of sialated, O-linked sugars. Plasmon resonance spectroscopy experiments revealed that all the high Mr isoforms and mature 7.5-kDa IGF-II bound to immobilized recombinant soluble human IGF-I receptor, recombinant human IGF-binding protein 1, and sIGF-II/MPR with similar kinetics. In addition, radiolabeled tracer experiments demonstrated that both mature and high Mr IGF-II isoforms have similar binding profiles in fetal bovine serum and have similar affinities for IGF-II-binding proteins secreted from human fibroblasts. Finally, the biological activity of high Mr IGF-II was shown to be similar to or slightly better than mature IGF-II in stimulating amino acid uptake in fibroblasts and in inducing myoblast differentiation.

Insulin-like growth factor II (IGF-II) 1 is a peptide hormone related to insulin that is present at high levels during fetal development. Genetic evidence suggests that IGF-II plays an important role in prenatal growth and that its actions are mediated through the IGF-I receptor and another yet uncharacterized receptor (1)(2)(3). In addition, IGF-II also binds to the IGF-II/mannose 6-phosphate receptor (IGF-II/MPR). This protein has two distinct functions: first, it mediates the biosynthetic targeting of mannose 6-phosphate containing lysosomal enzymes to the lysosome (for review, see Ref. 4); second, it mediates endocytosis of IGF-II, resulting in its delivery to the lysosome and subsequent degradation (5,6).
The availability of IGF-II for interaction with its receptors is regulated by its association with binding proteins. To date, six different IGF-binding proteins have been characterized and have molecular masses ranging from 22.8 to 31.3 kDa (for review, see Refs. 7 and 8). In addition, a soluble form of the IGF-II/MPR (sIGF-II/MPR) has been detected in serum and urine (9 -13). This protein retains IGF-II and Man-6-P binding activities and is abundant (ϳ5 g/ml) in fetal bovine serum (FBS) (14). In FBS, Յ5% of IGF-II is in the free state, with the remainder circulating as high molecular weight (M r ) complexes with the binding proteins and sIGF-II/MPR (14). Complex formation with the binding proteins has been shown to both inhibit and potentiate IGF-II's interactions with its receptors as well as increase the serum half-life of the ligand (for review, see Ref. 8).
IGF-II is synthesized as a preproprotein (15). After cleavage of its signal sequence, the COOH-terminal 88 residues (Epeptide) of the proprotein are removed to yield the 67-residue, mature 7.5-kDa IGF-II. The mature polypeptide can be subdivided into four domains (B, C, A, and D domains) that are homologous to the B and A chains of mature insulin and the B, C, A, and D domains of IGF-I. In addition, the existence of extended forms of IGF-II has been documented (for review, see Refs. 16 -18), although their physiological role in the biology of IGF-II is not clear.
To date, high M r isoforms of IGF-II have been detected in human serum (19 -21), cerebrospinal fluid (22), and malignant tissue extracts (23)(24)(25). Human IGF-II variants that are substituted at Ser-29 and Ser-33 with Arg-Leu-Pro-Gly and Cys-Gly-Asp, respectively, with or without E-peptide extensions, have also been identified (21,26). In addition, two IGF-II species with apparent M r values of 15,000 and 11,500 were purified from human serum Cohn fraction IV 1 and represent 87-88-amino acid polypeptides that are glycosylated at Thr-75 (18).
We recently described the isolation of a high M r IGF-II fraction from FBS (14). In this study we have separated the high M r IGF-II isoforms into eight different fractions. Each fraction is composed of a COOH-terminally extended IGF-II that terminates at Gly-87 and contains variable amounts of O-linked sugars. Our studies indicate that all high M r isoforms are similar to 7.5-kDa IGF-II in terms of both receptor/binding protein interactions and biological activity.
Buffers-HEPES-buffered saline 1 (HBS-1): 150 mM NaCl, 2 mM EDTA, 20 mM HEPES, pH 7.2; HBS-2: 150 mM NaCl, 3.4 mM EDTA, 0.005% Surfactant P20, 10 mM HEPES, pH 7.2; acid buffer: 60 mM ammonium acetate, 340 mM acetic acid, pH 4.0; propanol/acetate: 20% 1-propanol, 8  Isolation and Separation of High M r IGF-II-High M r IGF-II was isolated from FBS using a modification of the procedure described in Ref. 14. Briefly, expired lots of FBS that contained 3-5 mg/liter of soluble insulin-like growth factor II/mannose 6-phosphate receptor (sIGF-II/MPR) were used as source material. Serum (50 or 100 liters) was buffered by the addition of 0.04 volumes of a HEPES stock solution (0.5 M, pH 7.2), filtered through 0.2-m cellulose acetate membranes (Millipore) and loaded onto a phosphomannan-Sepharose CL-6B affinity column (11.3 ϫ 12 cm) at 2-2.5 liters/h. After loading, the column was disassembled, the resin batch was washed with 2 column volumes of HBS-1 (8 -10 times), the column was re-assembled, and the column was further washed at 2-2.5 liters/h until A 280 reached base line. The column was eluted at 200 ml/h with 2.5 mM mannose 6-phosphate in HBS-1. Recovered proteins were concentrated to 20 or 40 ml (for 50-or 100-liter preparations, respectively) by ultrafiltration using a YM-100 membrane (Amicon). The retentate was acidified to pH 4.0 by the addition of 0.1 volumes 10 ϫ acid buffer. After Ն6 h, 10-ml samples were filtered through 0.2-m cellulose acetate membranes (Corning) and chromatographed at 1.0 ml/min on a Superdex 75 column (Pharmacia, 2.6 ϫ 60 cm) equilibrated with 1 ϫ acid buffer. The material was resolved into three peaks that represented sIGF-II/MPR, high M r IGF-II, and mature 7.5-kDa IGF-II, respectively (see Ref. 14 for representative chromatogram). Material eluting in the high M r IGF-II region from two to four individual column runs was pooled, lyophilized, resuspended in 1 ϫ acid buffer (ϳ5 ml), and re-chromatographed at 0.8 ml/min on the Superdex 75 column equilibrated in 1 ϫ acid buffer. This step achieved near base-line separation of the high M r IGF-II fraction from the flanking sIGF-II/MPR and mature IGF-II peaks. All steps were performed at 4°C.
Separation of individual high M r IGF-II isoforms was performed using anion exchange chromatography on a Pharmacia SMART system at room temperature. Fractions containing high M r IGF-II from the second acid gel filtration step (eluting between 165 and 250 ml) were consecutively pooled (two to three 6-ml fractions per pool), lyophilized, resuspended in propanol/acetate, and the buffer exchanged to propanol/ acetate using a fast desalting PC 3.2/10 column at 100 l/min. Void volume fractions were pooled, dried, and resuspended with AEX buffer 1A (100 l). Samples were chromatographed on a Mono Q HR 1.6/5 column equilibrated in AEX buffer 1A at a flow rate of 100 l/min. Five minutes after sample injection (when A 280 reached base line), a linear gradient from 0 to 47.5% AEX buffer 1B over 27 min was performed and 50-l fractions collected. Different IGF-II isoforms eluted between 15 and 39% AEX buffer 1B (120 -320 mM NH 4 HCO 3 ) and could be grouped into nine different peaks denoted A through I. Equivalent fractions from different anion exchange runs were pooled, dried, and desalted as described above. Proteins were rechromatographed on the anion exchange column as described above and fractions pooled as indicated under "Results." Some fractions were further purified by anion exchange chromatography using a different buffer system. Here, samples were exchanged into propanol/acetate, dried, and resuspended in AEX buffer 2A (100 l). Anion exchange chromatography was as described above but used a linear gradient of 8 -28% AEX buffer 2B (80 -280 mM NaCl) over 19 min, and 20-l fractions were collected. Appropriate fractions were pooled as indicated under "Results," exchanged into propanol/acetate, and stored at Ϫ80°C.
Radiolabeling of IGF-II-Peaks A-I representing the different bovine IGF-II isoforms and 7.5-kDa rhIGF-II were iodinated using Na 125 I (NEZ-033A, DuPont NEN) and lactoperoxidase (Boehringer Mannheim) as described previously (31) except the reactions were quenched with tyrosine. Radiolabeled high M r IGF-II was separated from free iodine using prepacked G-25 M columns (PD10, Pharmacia) with PBS as the mobile phase. BSA was included as a carrier in the column buffer of early experiments, but later omitted due to its uptake of label. As each of the radiolabeled high M r IGF-II isoforms eluted as a single peak after analytical gel filtration chromatography on a Superose 12 column (1.6 ϫ 85 cm), these were used without further purification (data not shown). Radiolabeled 7.5-kDa IGF-II (peak A and rhIGF-II) was applied to a Sephadex G50 fine column (Pharmacia, 0.7 ϫ 28 cm) using PBS as the mobile phase. Three radioactive peaks were detected, and fractions comprising the second peak (IGF-II monomer) were pooled. The specific activity ranged from 120 to 770 Ci/mmol for high M r IGF-II and was ϳ25 Ci/mmol for rhIGF-II.
Deglycosylation of High M r IGF-II-All digests were carried out at 37°C. For the experiment shown in Fig. 5, samples of iodinated high M r IGF-II and rhIGF-II (ϳ30,000 cpm/reaction) were incubated with either 2.5 milliunits of recombinant neuraminidase (Glyko), 1 milliunit of recombinant O-glycosidase (Glyko), or both enzymes in a total volume of 10 l of glycosidase buffer A. Digests were carried out for 4 h. For double digests, samples were first incubated with neuraminidase for 1 h before addition of O-glycosidase. Digested samples were resolved on a 12.5-17.5% polyacrylamide gel under nonreducing conditions. For the isoelectric focusing experiment shown in Fig. 6, approximately 80,000 cpm of radiolabeled IGFs were used per reaction. Samples were digested with 10 milliunits of neuraminidase (Arthrobacter; Boehringer Mannheim) for 2 h followed by the addition of 2 milliunits of O-glycosidase (Oxford GlycoSystems) and incubation for 18 h. Digests were carried out in a total volume of 48 l of glycosidase buffer B. Both untreated and digested samples were resolved by isoelectric focusing on precast gels (pH 3-7) according to the manufacturer (Novex). Isoelectric gels were fixed in 3.5% sulfosalicylic acid, 11.5% trichloroacetic acid for 30 min prior to staining.
Surface Plasmon Resonance Spectroscopy-A Pharmacia BIAcore system was used to monitor interaction of the high M r IGF-II isoforms with the sIGF-II/MPR, sIGF-IR, and IGFBP-1. Details of the instrumentation and analytical methods have been described in several recent reports (32,33). Briefly, standard amine coupling procedures were used for the immobilization of ligand (sIGF-II/MPR, sIGF-IR, or IGFBP-1) to the dextran matrix of the sensor chip surface (34). Surfaces were activated with a 35-l injection of a 0.05 M N-hydroxysuccinimide, 0.2 M N-ethyl-NЈ-[ (3-dimethylamino)propyl]carbodiimide hydrochloride mixture according to the manufacturer using a flow rate of 5 l/min HBS-2. sIGF-II/MPR and IGFBP-1 were diluted to a final concentration of 5 g/ml in 20 mM sodium acetate, pH 4.7, before injection over the activated dextran matrix. sIGF-IR was diluted to a final concentration of 30 g/ml in 0.2 M acetic acid before injection. Typically 2000 -4000 resonance units of sIGF-II/MPR or sIGF-IR or 300 -600 resonance units of IGFBP-1 were immobilized per experiment. Unreacted carboxyl groups on the dextran matrix were blocked with a 35-l injection of a 0.2 M solution of ethanolamine. Sensorgrams were generated using a flow rate of 10 l/min. Typically, four 2-fold dilutions of analyte (rhIGF-I, rhIGF-II, and different high M r IGF-II isoforms) were used in each experiment. The concentrations ranged from 10 to 80 nM for analyte passed over sIGF-II/MPR and IGFBP-1 surfaces. For sIGF-IR surfaces, 25-200 nM or 12.5-100 nM of the IGF-II isoforms or of rhIGF-I, respectively, were used. Analytes were applied for 3 min to measure association kinetics and then washed out using HBS-2 to measure dissociation kinetics for an additional 3 min. The surface was regenerated after each round with a 1-min injection of either 10 mM HCl for sIGF-II/MPR and IGFBP-1 surfaces or 50 mM NaHCO 3 /1 M NaCl for sIGF-IR surfaces.
Kinetic constants were determined using the BIAevaluation software (version 2.0). Both the association and dissociation phases of the binding curves were fit with simple bimolecular interaction assumptions, using the resonance unit signal as a relative measure of the ligandanalyte complex concentration with respect to time. The association phase of different sensorgrams was fit directly with a nonlinear least squares iterative curve fitting function. The association constants obtained with the four different analyte concentrations were averaged. Dissociation constants were calculated from the highest analyte concentrations used to minimize signal contributions due to rebinding of analyte to ligand at the surface.
Analysis of IGF-II-binding Proteins in FBS-Binding of radiolabeled bovine 7.5-kDa (peak A) or the different high M r IGF-II isoforms (peaks B-I) to serum-binding proteins was determined as described previously (14). Briefly, the different radiolabeled IGFs (ϳ30,000 cpm) were each incubated with FBS (Hyclone; 1 ml). Time course experiments indicated that a 24-h incubation at room temperature was sufficient for both mature and high M r IGF-II radiotracer to reach equilibrium with serum-binding proteins (see Ref. 14 and data not shown), thus reflecting the distribution of endogenous IGF-II. The serum was applied to a Superose 12 column (1.6 ϫ 85 cm) and eluted at 1 ml/min with PBST at room temperature as indicated. Fractions (2 ml) were counted for 1 min in a Packard Cobra auto ␥-counter.
AIB Uptake Studies-Fibroblasts from a normal 18-year-old man (WUMS1) were isolated at Washington University School of Medicine, St. Louis, MO (35). Uptake of AIB in human fibroblasts was measured as described previously (36) using an assay volume of 500 l.
C2I Myoblast Differentiation-The C2I subclone of C2 myoblasts (37) that requires only insulin or IGF-I to differentiate in vitro was kindly provided by Dr. Peter Rotwein at Washington University School of Medicine, St. Louis, MO. Differentiation in C2I myoblasts was induced as described by Rotwein et al. (38) except that assays were performed in 24-well clusters, using an assay volume of 1.0 ml. After ϳ70 h, cells were washed and lysed by incubation with 100 l of TrisT for 15-20 min at room temperature. Creatine kinase activity was determined as described (38) and normalized for total protein content, using the BCA protein assay (Pierce). IGF-binding proteins secreted during C2I myoblast differentiation were analyzed by ligand blotting as described previously (35).
Competition Binding Studies-To compare binding of IGF-I, IGF-II, and the different high M r IGF-II isoforms to the IGF-binding proteins secreted by human fibroblasts, conditioned buffer was collected from WUMS1 fibroblasts after incubation in AIB assay buffer for 3 h and clarified by centrifugation. Competition binding studies were performed as described previously (35) with minor modifications. Briefly, fibroblast-conditioned buffer was incubated for 1 h at 37°C with 125 I-labeled IGF-I and increasing amounts of unlabeled IGF-I, IGF-II, or high M r IGF-II in AIB assay buffer (final volume, 250 l). AIB assay buffer rather than 0.1 M HEPES, pH 6.0, with 44 mM NaH 2 PO 4 , 0.01% Triton X-100, 1 mg/ml BSA, and 0.02% NaN 3 (35) was used in order to reproduce more closely the conditions under which IGFs and secreted binding proteins interact during the AIB uptake assay. Complexes of binding protein and IGF were precipitated by the addition of 750 l of 25% polyethylene glycol, 4.0 mg/ml bovine ␥-globulin (2:1). Binding data were analyzed by LIGAND, a computer program developed by Munson and Rodbard (39).
To compare binding of IGF-I, IGF-II, and the different high M r IGF-II isoforms to the human IGF-I receptor, IGF-I receptor was purified from human placenta using wheat germ agglutinin-Sepharose chromatography, insulin affinity chromatography, and IGF-I affinity chromatography (30). Competition binding studies were performed as described previously (30). Briefly, IGF-I receptor was incubated overnight at 4°C with 125 I-labeled IGF-I and increasing amounts of unlabeled IGF-I, IGF-II, or high M r IGF-II in imidazole binding buffer. Receptor-bound IGF-I was precipitated by the addition of 900 l of 33.3% polyethylene glycol, 1.5 mg/ml bovine ␥-globulin (1:1). Binding data were analyzed by LIGAND.
Other Methods-sIGF-II/MPR concentrations were determined by a two-antibody sandwich enzyme-linked immunosorbent assay as described previously (40). Protein concentrations were determined with BSA standards using the Lowry method (41) adapted to microtiter plates. Molar concentrations were calculated using the following protein M r values: rhIGF-I, 7639; rhIGF-II, 7475; 7.5-kDa bovine IGF-II (peak A), 7532; high M r IGF-II (peaks B-I), 9578. SDS-PAGE was performed as described (42). Gels were stained with 0.2% Coomassie Brilliant Blue R-250 (Bio-Rad) and/or silver using the Novex Silver-Xpress silver staining kit as indicated. Dried radioactive gels were exposed to a phosphor storage screen, scanned, and quantitated using a Molecular Dynamics PhosphorImager 400 and ImageQuant 3.15 software. The molecular mass of the different IGF-II isoforms was determined using both a 252Cf plasma desorption time-of-flight mass spectrometer Bio-Ion 20 (Applied Biosystems, Gothenburg, Sweden) and a VG Quattro electrospray mass spectrometer (Fisons Instruments, Altrincham, United Kingdom). For laser desorption mass spectrometry, samples were dissolved in 0.1% trifluoroacetic acid, applied to nitrocellulose-coated foil (Applied Biosystems), dried, and analyzed in a positive-ion mode for 1 h with an acceleration voltage of 18 kV. Samples for electrospray mass spectrometry were applied in 50% methanol, 1% acetic acid at a flow rate of 5 l/min. Amino acid analyses were performed at the Cornell Biotechnology Analytical/Synthesis Facility (Ithaca, NY). Amino-terminal sequencing was performed using automated Edman degradation on a Hewlett-Packard G1000A protein sequencer or a Milligen Biosearch Prosequencer type 6600. Predicted isoelectric points were calculated from amino acid compositions using the University of Wisconsin GCG program (43). The contribution of glycosylation to pI (isoelectric point) was estimated using a pK a of 2.6 for sialic acid (44), and ionizable cysteines were excluded from the analysis as all six cysteines present in IGF-II are disulfide bonded.

RESULTS
Isolation and Separation of High M r IGF-II-We previously reported the co-purification of sIGF-II/MPR and bound IGF-II from FBS (14). In addition to mature 7.5-kDa IGF-II, we also isolated ϳ12 different high M r IGF-II isoforms that were associated with the soluble receptor in low abundance. In order to investigate the properties of this high M r IGF-II fraction in more detail, we modified and scaled-up our original purification scheme to isolate milligram quantities of these IGF-II isoforms (see "Experimental Procedures"). Large-scale isolation of sIGF-II/MPR and bound IGF-II from 50-and 100-liter lots of FBS was achieved by affinity adsorption of receptor to phosphomannan-agarose and elution with mannose 6-phosphate. Mildly acidic gel filtration chromatography was employed to dissociate bound IGF-II from receptor and to resolve high M r IGF-II species from sIGF-II/MPR and 7.5-kDa IGF-II. Finally, pooled fractions from the high M r IGF-II peak were re-applied to the acidic gel filtration column to remove contaminating sIGF-II/MPR and 7.5-kDa IGF-II. Using this strategy, we have processed ϳ1000 liters of FBS yielding ϳ1.5 mg of total high M r IGF-II.
The different high M r IGF-II isoforms were subfractionated by anion exchange chromatography at pH 8.0. For each 50-or 100-liter preparation, the high M r IGF-II peak from the second acid gel filtration step was divided into five to eight fractions, and each was chromatographed on a Mono Q column using an ammonium bicarbonate gradient (Fig. 1). The elution profiles of the different chromatograms were similar, although in general, when comparing early with late gel filtration fractions (larger versus smaller proteins), the earlier eluting gel filtration fractions tended to be biased toward the later (more acidic) eluting ion exchange fractions. The chromatogram depicted in Fig. 1 is of material eluting late in the acidic gel filtration column, and all the peaks of interest are well represented. The anion exchange column fractions were analyzed by SDS-PAGE and Coomassie staining on 14% gels. The regions labeled A-I contained visualizable protein and sequence analysis on selected peaks (A, B, D, and H) revealed that all contained proteins with the amino-terminal residues AYRPS, identical to 7.5-kDa bovine IGF-II (45).
For further purification, peaks A-I from different anion exchange runs were pooled and rechromatographed (Fig. 2) as described above; fractions were pooled as indicated. In addition, Peaks A, B, D, and H were chromatographed on a Mono Q column at pH 6.0 and eluted with a gradient of sodium chloride (Fig. 3); fractions were pooled as indicated.
Chemical Characterization of High M r IGF-II-The purity of peaks A-I was assessed using SDS-PAGE and silver staining (Fig. 4). Peak A consisted of a single protein species that ran identically to the rhIGF-II standard. Subsequent analysis by mass spectrometry and isoelectric focusing (see below) showed that this species represented mature 7.5-kDa bovine IGF-II that was not resolved from the high M r IGF-II peak by gel filtration chromatography. The remaining peaks contained one to three different protein species with apparent sizes ranging from 11 to 17 kDa.
We previously examined the nature of the increased M r seen in the unfractionated high M r IGF-II pool using a combination of protease and glycosidase sensitivity assays (14). We demonstrated that all proteins comprising this pool contain a common peptide backbone that extends beyond Glu-67 and that this extension is modified with various amounts of sialated, Olinked sugars resulting in the pool's heterogeneity. To confirm and extend these earlier studies, the fractionated high M r isoforms in peaks B-I were iodinated and then digested with neuraminidase and/or O-glycosidase to remove terminal sialic acids and O-linked core disaccharides, respectively (Fig. 5). No shift in the electrophoretic mobility of peak B (Fig. 5) or of rhIGF-II standard (data not shown) was detected. In contrast, digestion of peaks C-I using a combination of neuraminidase and O-glycosidase caused a mobility shift in all the isoforms, resulting in the appearance of a single major species that ran identically to both undigested and digested peak B (arrow). These data suggest that peak B represents an extended, nonglycosylated isoform of high M r IGF-II and that the species contained in peaks C-I have the same peptide backbone as peak B but are further modified with various amounts of Olinked sugars. Interestingly, double digestion of peak F demonstrated the presence of two bands, one that ran identically to peak B and another that migrated slightly slower. This behavior may represent multiple IGF-II species with slight differences in their peptide backbones (i.e. longer extensions and/or variant internal sequences) or the presence of other post-translational modifications.
The high M r forms of IGF-II were also digested with the two glycosidases separately (Fig. 5). Treatment with neuraminidase alone caused a small but significant increase in the mobility of all bands indicating the presence of sialic acid. In contrast, treatment with O-glycosidase alone had no effect on any of the high M r IGF-II isoforms. As O-glycosidase only removes the unmodified disaccharide Gal␤1-3GalNAc from threonine or serine residues, these data suggest that essentially all of the O-linked sugars in each of the high M r IGF-II isoforms are protected with sialic acid.
The contribution of oligosaccharide to the net charge of high M r IGF-II was investigated using isoelectric focusing. The measured pI of recombinant human IGF-II was ϳ6.65 (Fig. 6,  upper panel), close to the pI of 6.80 predicted from its composition. (The minor band in the rhIGF-II lane likely represents contaminating BSA introduced from the column buffer as ascertained by comparison of the iodinated preparation with authentic BSA by SDS-PAGE and isoelectric focusing.) Peak A gave a pI of ϳ6.85 (data not shown), similar to the pI of 6.84 predicted for unmodified mature 7.5-kDa bovine IGF-II. Peak B gave a pI of ϳ4.85 (Fig. 6, upper panel), which is consistent with the isoelectric point of 4.81 predicted for an unmodified 87-residue extended IGF-II. The remaining bands displayed increasingly acidic pI's, ranging from ϳ4.6 to 3.5, respectively (Fig. 6, upper panel). Each sialic acid added to the 87-residue peptide is expected to decrease its isoelectric point 0.12 to 0.23 pH units. Thus, the decreasing isoelectric points of peaks C-I are consistent with increasing numbers of sialic acid residues decorating the core O-linked disaccharides. Treatment with both neuraminidase and O-glycosidase reduced all of the high M r IGF-II isoforms to a major species that had a pI of ϳ4.85 (Fig. 6, lower panel), identical to both undigested and digested peak B (Fig. 6, upper and lower panels, respectively). In addition, we noted that the deglycosylated samples contained minor species with pI values of approximately 5.4, 4.7, and 4.5 (Fig. 6,  lower panel). These minor species may represent experimentally (iodination)-induced modifications in the peptide backbone as similar variability was not detected by SDS-PAGE of native or radiolabeled samples (see above), Coomassie staining of isoelectric focusing gels (data not shown) or mass spectrometry (see below). Regardless of the identity of the minor species, the important point is that all of the high M r IGF-II isoforms contain an identical peptide backbone and that peak B repre-sents non-glycosylated, extended bovine IGF-II.
We next determined the molecular mass of the high M r IGF-II isoforms using mass spectrometry (Table I). Recombinant human IGF-II isoforms were used for method validation. Mass determinations of 7465 and 9802 for rhIGF-II and rhIGF-IIE 88 standards, respectively, were within ϳ0.1% of those predicted from the cDNA (28,29). Peak A had a M r of 7527, strongly supporting its assignment as the 67-residue mature 7.5-kDa bovine IGF-II. The molecular weights of peaks B and D were 9572 and ϳ10,850, respectively. This difference is presumably due to the presence of sugar as deglycosylated peak D appeared identical to peak B by both SDS-PAGE and isoelectric focusing (see above). The measured mass of 9572 for peak B is again consistent with this protein terminating at Gly-87, which would give a predicted mass of 9578. Precise mass determinations on the other high M r IGF-II peaks were not possible due to their large oligosaccharide component and microheterogeneity within the individual peaks (Table I and data not shown). However, all values measured had molecular masses of ϳ13,000 or less, providing an upper limit toward the size and degree of glycosylation of the IGF-II isoforms.
We performed quantitative amino acid analyses as an additional test to determine the peptide composition of peaks B and D. The lysine content of these species is consistent with the COOH terminus being Gly-87: the analyses indicated the presence of a single lysine for both peaks B and D, while two were observed for rhIGF-IIE 88 (data not shown). Based on the cDNA, IGF-II with an E-peptide extension to position 88 would contain two lysines (Lys-65 and Lys-88), while IGF-II terminating before position 88 would contain only one as seen for peaks B and D. In addition, values obtained for the remaining amino acids are close to those predicted for an IGF-II polypeptide extended to Gly-87 (data not shown). These results, taken together with that presented above, indicate that nearly all of the high M r IGF-II isoforms have a common peptide backbone that is extended 20 amino acids beyond that of the 67-residue, mature IGF-II and that the heterogeneity observed in the different peaks is due to differences in O-linked glycosylation.
Surface Plasmon Resonance Studies-The interactions of high M r IGF-II with sIGF-II/MPR, a soluble fragment of recombinant human IGF-I receptor (sIGF-IR), and recombinant hu-man IGF-binding protein 1 (IGFBP-1) were compared with those of mature IGF-II by measuring their association and dissociation rate constants using the BIAcore system (see "Experimental Procedures"). Either the individual receptors or binding protein were covalently attached to the dextran matrix of the sensor chip and different concentrations of the IGF-II isoforms (analyte) passed through the flow cell. The signal is proportional to the amount of IGF-II bound to the immobilized sIGF-II/MPR. The regions of the sensorgrams following introduction or washout of the analyte were used to determine the association and dissociation rate constants, respectively. Kinetic rate constants for all high M r IGF-II isoforms with the two receptors and binding protein are presented in Tables II and III. In addition, equilibrium dissociation constants (K d ) were determined from the kinetic data and are presented in Table IV.
Our data demonstrate that all of the high M r IGF-II isoforms bind with similar affinities as those measured for mature IGF-II (both bovine and human) and reflect nearly identical association and dissociation rates. Whereas binding to the sIGF-II/MPR and IGFBP-1 was in the low nanomolar range for all species, interaction with the sIGF-IR was of considerably lower affinity (K d Ͼ 35 nM). In contrast, rhIGF-I displayed 6 -10-fold higher affinity for the sIGF-IR than any of the IGF-II isoforms. These data suggest that E-peptide extensions and glycosylation do not influence binding of IGF-II to the sIGF-II/ MPR, sIGF-IR, or IGFBP-1.    88 , and peaks A, B, and D are presented as the mean Ϯ standard deviation for the major peak over n determinations. The range shown for peaks D, G, and I represents the values of multiple peaks observed in a single analysis (see text). At least one determination was performed using electrospray mass spectrometry for rhIGF-II, rhIGF-IIE 88 , and peaks A and B; all others were performed using laser desorption time-of-flight mass spectrometry. The deduced masses of rhIGF-II and rhIGF-IIE 88 are based on the human cDNA; the deduced masses of peaks A and B are based on the bovine cDNA, assuming COOH termini at Glu-67 and Gly-87, respectively. NA, not applicable, only single peak detected. Serum Binding Profiles of High M r IGF-II-Radiolabeled tracer experiments were performed to: 1) estimate the fraction of high M r IGF-II carried by the sIGF-II/MPR in FBS and 2) compare the endogenous distribution of high M r and mature IGF-II among different serum-binding proteins in FBS. Serum was incubated with each of the eight different iodinated high M r IGF-II isoforms or mature IGF-II under conditions that allow approach to equilibrium (see Ref. 14; similar kinetics were observed for mature and high M r IGF-II) and fractionated by gel filtration chromatography as described under "Experimental Procedures." Comparison of mature 7.5-kDa bovine IGF-II (peak A) and two high M r IGF-II isoforms (peaks B and E, representing extended, nonglycosylated and extended, glycosylated isoforms, respectively) demonstrated similar binding profiles with essentially no tracer eluting as the free polypeptide (Fig. 7, upper three panels). In addition, ϳ35% of the tracer in each case was associated with a peak that elutes identically to sIGF-II/MPR standard (Fig. 7, upper three panels, region 1). Likewise, values ranging from 31-39% for radiotracer association with the sIGF-II/MPR peak were determined for the other high M r IGF-II isoforms (data not shown). Most of this IGF-II binding activity represents authentic sIGF-II/MPR as receptordepleted serum has greatly diminished activity (Fig. 7, lower   panel, region 1). Interestingly, while enzyme-linked immunosorbent assay analysis demonstrated complete removal of receptor (data not shown), 1.5-5% of the IGF-II tracer still migrated in this region when the different high M r IGF-II isoforms were tested (Fig. 7, lower panel and data not shown). Thus, while another protein makes a minor contribution to IGF-II binding in this region, these results clearly demonstrate that the endogenous sIGF-II/MPR carries ϳ1/3 of the total IGF-II (both mature and high M r isoforms) in FBS.
The different radiolabeled IGF-II isoforms also eluted in two other regions. Region 2 represents tracer associated with a binding protein(s) that elutes similarly to the 150-kDa standard and may represent ternary complex formation between tracer, IGFBP-3, and the acid labile subunit (8). Region 3 represents tracer association with any of the five remaining IGF-binding proteins that have apparent sizes ranging from 25 to 40 kDa. The similarity in the binding profiles for the different high M r IGF-II isoforms and mature bovine IGF-II suggests that these molecules distribute similarly among different IGFbinding proteins in FBS. It is worth noting that peak E appears to have diminished binding activity in region 2 compared with the other IGF-II isoforms (Fig. 7). While this could be interpreted as reflecting differences in affinity for binding proteins, the apparent decreased magnitude of region 2 may simply represent incomplete resolution of the different peak E-binding protein complexes in regions 2 and 3 on the gel filtration column.
Biological Activity of High M r IGF-II-It has been demonstrated that uptake of the amino acid analog, ␣-aminoisobutyric acid (AIB), is stimulated by IGF-I in human fibroblasts (36). Fig. 8A, shows a representative experiment comparing stimulation of AIB uptake by rhIGF-I, mature bovine IGF-II, and high M r IGF-II isoforms C-H. In six experiments, the ED 50 of rhIGF-I-stimulated AIB uptake in human fibroblasts was 0.29 Ϯ 0.03 nM, as reported previously (36). Stimulation of AIB uptake by mature and high M r IGF-II is summarized in Fig.  8B. The ED 50 of IGF-II-stimulated AIB uptake was 1.41 Ϯ 0.05 nM, 4.8-fold higher than that of IGF-I. Surprisingly, the ED 50 values for stimulation of AIB uptake by high M r IGF-II ranged from 0.59 Ϯ 0.04 nM to 0.96 Ϯ 0.09 nM, suggesting that they were slightly more effective than mature IGF-II in this system. Maximal stimulation of AIB uptake (mean Ϯ S.E.) by rhIGF-I (2.42 Ϯ 0.12-fold, n ϭ 6), mature IGF-II (2.36 Ϯ 0.12-fold, n ϭ 5), and high M r IGF-II (2.35 Ϯ 0.16-fold to 2.59 Ϯ 0.10-fold, n ϭ 5) did not differ significantly in these experiments.
The small, but reproducible, differences between mature and high M r IGF-II in stimulating AIB uptake in fibroblasts might be explained by reduced affinity of high M r IGF-II for binding proteins secreted by the fibroblasts, resulting in greater availability for IGF receptor binding. To investigate this possibility, binding of rhIGF-I, mature bovine IGF-II, and high M r IGF-II isoforms C-H to the IGF-binding proteins secreted by fibroblasts was investigated. The major IGF-binding protein present in fibroblast-conditioned buffer is IGFBP-3 (35). Fig. 9 shows displacement curves from a representative study. The mean dissociation constant derived from LIGAND analysis of IGF-I binding to secreted binding proteins in two independently-performed experiments was 0.21 nM, slightly higher than reported previously (35). IGF-II bound with a very similar affinity, whereas high M r IGF-II bound with a similar or slightly higher affinity. These results indicate that the enhanced effectiveness of high M r IGF-II in stimulating AIB uptake cannot be explained by reduced affinity for fibroblast IGF-binding proteins.
The biological activity of high M r IGF-II was also investigated using the C2I subclone of C2 myoblasts selected by FIG. 7. Serum binding profiles of high M r IGF-II. FBS was incubated with different radiolabeled IGF-II tracers as indicated (peak A, 7.5-kDa bovine IGF-II; peaks B and E, high M r IGF-II without and with glycosylation, respectively) and fractionated by gel filtration chromatography at room temperature (see "Experimental Procedures"). In the lower panel, tracer was incubated with serum depleted of sIGF-II/MPR by phosphomannan chromatography. The small arrows in each panel represent the elution volume of the respective free radiotracer. Positions of molecular mass standards are marked with arrows above the top panel (left-to-right: sIGF-II/MPR, 250 kDa; alcohol dehydrogenase, 150 kDa; aprotinin, 6.5 kDa). Pinset et al. (37). This cell line requires only insulin or IGF-I to differentiate in vitro. In preliminary experiments, we found that human or bovine IGF-II also induced terminal differentiation, as evidenced by equivalent maximal induction of creatine kinase activity and myotube formation. Fig. 10 shows a representative experiment comparing the dose-dependent induction of creatine kinase activity in C2I myoblasts by rhIGF-I, mature bovine IGF-II, and high M r IGF-II isoforms D through G. In three experiments, the ED 50 of IGF-II-induced differentiation was 2.2-2.8-fold higher than that of rhIGF-I. The high M r IGF-II isoforms were at least as or slightly more effective than mature IGF-II in this system. Rotwein et al. (38) have shown that the expression of IGFBP-5 is stimulated during C2I myoblast differentiation. To examine IGF-binding protein expression during differentiation, media were collected from C2I myoblasts after incubation for ϳ70 h with rhIGF-I, mature bovine IGF-II, or high M r IGF-II isoforms C-H and analyzed by ligand blotting. As shown in Fig. 11, the major IGF-binding protein stimulated during differentation migrated with an apparent molecular mass of 33.3 kDa. We confirmed by immunoblot analysis that this binding protein was IGFBP-5 (data not shown). Similar amounts of IGFBP-5 were present in conditioned media from myoblasts incubated with rhIGF-I, IGF-II, and high M r IGF-II. An IGF-binding protein with an apparent molecular mass of 27.6 kDa was present in conditioned media from myoblasts incubated without IGF or insulin and did not change in amount during differentiation.

Interaction of High M r IGF-II with Intact IGF-I Recep-
tor-We next compared binding of rhIGF-I, mature bovine IGF-II, and high M r IGF-II isoforms F and G to intact IGF-I receptor purified from human placenta. Fig. 12 shows displacement curves from this study. The mean dissociation constant derived from LIGAND analysis of rhIGF-I binding to the human IGF-I receptor in two independently performed experiments was 0.37 nM, very similar to that reported previously (30). The difference between this and the equilibrium dissociation constant obtained by surface plasmon resonance spectroscopy probably reflects the different receptor preparations employed and suggests that the extracellular portion of the receptor binds IGFs with a lower affinity than the intact receptor. The mean concentration of mature IGF-II which inhibited 50% of 125 I-labeled IGF-I binding to the receptor was 1.78 nM. Interestingly, the high M r IGF-II isoforms bound to the purified IGF-I receptor FIG. 9. Binding of IGF-I, IGF-II, and high M r IGF-II isoforms to secreted fibroblast IGF-binding proteins. Binding of IGF-I, IGF-II, or high M r IGF-II isoforms C through H, as indicated, to the IGFbinding proteins secreted by human fibroblasts was compared in competition binding studies as described under "Experimental Procedures." In this study, binding assays contained 19,900 cpm of 125 I-labeled IGF-I with 25.6 -31.1% of the 125 I-labeled IGF-I bound in the absence of unlabeled ligand. Data are presented as B/Bo, the fraction of maximum bound. Nonspecific binding has been subtracted. 125 I-Labeled IGF-I binding was dependent on the amount of fibroblast-conditioned buffer in the assay. The experimental results presented in this study were those obtained using an amount of conditioned buffer (100 l) at which the dose-dependent relationship was linear.
FIG. 10. Induction of differentiation in C2I myoblasts by IGF-I, IGF-II, and the high M r IGF-II isoforms. C2I myoblasts were incubated for ϳ70 h in serum-free medium with 60 nM transferrin and increasing amounts of IGF-I, IGF-II, or high M r IGF-II isoforms D through G, as indicated, to induce differentiation. Creatine kinase activity in C2I cell extracts was then determined and normalized to total protein content. Each point represents the mean of duplicate determinations. The data are presented as the percent of maximal stimulation observed for each dose-response curve. In this experiment, creatine kinase activity (basal) ranged from 72-92 units/g of protein and increased to 630 -1114 units/g of protein during differentiation. Incubation of C2I myoblasts in serum-free medium with 1.6 M insulin resulted in a similar maximal induction of creatine kinase activity (636 -998 units/g of protein) and myotube formation (not shown).

FIG. 8. Stimulation of AIB uptake in human fibroblasts by IGF-I, IGF-II, and the high M r IGF-II isoforms.
Human fibroblasts were incubated for 3 h with increasing amounts of IGF-I, IGF-II, or high M r IGF-II isoforms C-H, as indicated. Uptake of 30 M AIB containing 2 Ci/ml [ 3 H]AIB in 20 min was then measured as described previously (36). A, a representative experiment. Each point represents the mean of triplicate determinations. The data are presented as the percent of maximal stimulation observed for each dose-response curve. B, ED 50 values for stimulation of AIB uptake. The data are expressed as the mean Ϯ standard error. Experiments were independently performed six times with IGF-I and five times with IGF-II and the high M r IGF-II isoforms.
with slightly higher affinity than mature IGF-II, a result which may explain their slightly enhanced biological activity. DISCUSSION These studies were initiated to characterize individual high M r IGF-II isoforms. Preliminary analysis of the unfractionated high M r IGF-II pool isolated from fetal bovine serum revealed that it contained at least 12 different IGF-II isoforms and that all species contained an identical peptide backbone extended beyond the COOH terminus of mature 7.5-kDa IGF-II (14). In addition, this pool contained a significant amount of sialated, O-linked sugars. In this study, the high M r IGF-II pool was fractionated into nine distinct fractions to facilitate analysis. Taken together, results from amino-terminal sequence analysis, amino acid analysis, mass spectrometry, glycosidase digestions, isoelectric focusing, and SDS-PAGE experiments demonstrate that all fractions predominantly contain IGF-II species with a 20-amino acid COOH-terminal extension to Gly-87 that is modified with different amounts of sialated, O-linked oligosaccharides.
The presence of O-linked sugars on IGF-II precursors may represent an important modification to promote correct processing to the mature form (46). Mapping of the glycosylation site on an extended IGF-II isoform isolated from human serum indicated that Thr-75 carries this post-translational modification (18). While we did not directly determine the location of the modified residues for the different high M r IGF-II isoforms, our mass spectrometry and isoelectric focusing results strongly suggest that most isoforms are likely to be glycosylated at multiple positions. The E-peptide extension (residues 68 -87) contains five potential O-linked glycosylation sites (two serines and three threonines; Ref. 47). If three sites were modified with a tetrasaccharide consisting of a Gal␤1-3GalNAc core modified with two sialic acids, this would give a predicted size of ϳ12,630 and an isoelectric point of 3.9. In comparison, the major species of peak H had an isoelectric point of 3.7 and the M r range of 10,500 -12,500. Similarly, an 87-residue IGF-II with one site modified with the tetrasaccharide would have a predicted isoelectric point of 4.4 and a M r of 10,600. In comparison, the major species of peak D had an isoelectric point of 4.35 and an M r of 10,854. Thus, the most likely explanation for the heterogeneity of the different high M r isoforms is that they contain 1-3 O-linked oligosaccharides, each of which contains one to two sialic acids.
Binding studies with mature 7.5-kDa IGF-II and the high M r IGF-II isoforms indicate that no substantial differences exist in the association or dissociation rate constants (Tables II and III, respectively) or in the calculated equilibrium dissociation constants (Table IV) for interaction with the sIGF-II/MPR, sIGF-I receptor, or IGFBP-1. In addition, similar distribution profiles among serum-binding proteins (Fig. 7), and similar displacement curves from IGFBP-3 ( Fig. 9) and intact human IGF-I receptor (Fig. 12) were observed for both mature and high M r IGF-II. This suggests that the COOH-terminal extensions do not have adverse effects on the rates or affinities with which these ligands interact with their receptors and binding proteins. This is consistent with other reports. Site-directed mutagenesis studies interpreted in light of the three-dimensional structure of mature IGF-II have implicated three regions of the folded molecule that appear to play important roles in determining protein-protein interactions (48 -50). Whereas residues 27 and 43 of the B and A domains, respectively, play key roles in IGF-I receptor interactions, residues 48 -50 and 54 -55 of the A chain are important in mediating IGF-II/MPR interactions. Likewise, residues 48 -50, together with 6 -7 of the B domain, are important in IGFBP interactions. More importantly, mutations within, or deletion of, the D domain (comprising the carboxyl-terminal six amino acids of mature IGF-II) has no effect on IGFBP interactions (48). Finally, it has been shown that extended forms of human IGF-II display similar binding affinities for the IGF-II/MPR (20) and the IGF-I receptor (51) as compared with mature IGF-II. Taken together, these data suggest that E-peptide extensions, with or without glycosylation, do not supplement or occlude the important contact regions between IGF-II and the sIGF-II/MPR, IGF-I receptor, or the IGFBPs.
An intriguing question still remaining, therefore, is what is the biological significance of the different IGF-II isoforms? One possible answer is that glycosylation is important for proper folding or export of IGF-II. Evidence suggests that glycosylation of proIGF-II is necessary for proper proteolytic processing to the mature 7.5-kDa form (46). If so, the mechanism may involve binding of glycosylated IGF-II to intracellular lectins that retain and present the glycoprotein to the appropriate processing enzymes (52). In this case, the sIGF-II/MPR-bound high M r IGF-IIs may simply reflect inefficiencies in this system resulting in the secretion of processing intermediates. Alternatively, the glycosylated IGF-II species may have unique biological functions. This is not unprecedented as N-linked glycosylation of several glycoprotein hormones is necessary for receptor activation but not binding (for review, see Ref. 53). Although no differences in the binding rates or affinities for mature and high M r IGF-II were observed for the receptors and binding proteins analyzed in this study, this does not preclude FIG. 11. Ligand blot analysis of the IGF-binding proteins secreted by differentiating C2I myoblasts. Media were collected from C2I myoblasts after incubation for ϳ70 h without (Control) or with ϳ26 nM IGF-I, IGF-II, or high M r IGF-II isoforms C-H, as indicated. Aliquots (22.5 l) were analyzed by ligand blotting as described under "Experimental Procedures." The migration of prestained molecular weight standards is indicated.

FIG. 12. Interaction of IGF-I, IGF-II, and high M r IGF-II with purified human IGF-I receptor.
Binding of IGF-I, IGF-II, and high M r IGF-II isoforms F or G, as indicated, to the purified human IGF-I receptor was compared in competition binding studies as described under "Experimental Procedures." In this study, binding assays contained 56,700 cpm of 125 I-labeled IGF-I with 18.0 -19.4% of the 125 Ilabeled IGF-I bound in the absence of unlabeled ligand. Data are presented as B/Bo, the fraction of maximum bound. Nonspecific binding has been subtracted. the possibility that the high M r isoforms have specialized roles in signal transduction. For instance, the extensions and/or saccharides could: 1) serve as a targeting signal to concentrate IGF-II in specific tissues or cell types; 2) interact with other cellular components, either when free or complexed with receptors or binding proteins, and/or; 3) alter the efficacy/potency of receptor activation after ligand binding. Such possibilities are supported from previous studies that demonstrated that a high M r form of IGF-II with an apparent M r of 15,000 was more potent than mature IGF-II in stimulating the replication of fetal dermal fibroblasts (20) and in the clonal expansion and differentiation of peripheral blood granulocyte colony-forming cells (54). Future studies will be required to determine if the high M r IGF-II glycoproteins have other specialized biological activities or pharmacological properties.