©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Soluble Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor Carries Multiple High Molecular Weight Forms of Insulin-like Growth Factor II in Fetal Bovine Serum (*)

Kenneth J. Valenzano (§) , Jill Remmler , Peter Lobel (¶)

From the (1)Center for Advanced Biotechnology and Medicine and the Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have characterized a soluble form of the insulin-like growth factor II/mannose 6-phosphate receptor (sIGF-II/MPR) and bound ligands from bovine serum. Fetal serum contained 2-8 mg/liter sIGF-II/MPR. Affinity-purified receptor isolated by adsorption to phosphomannan-agarose and elution with mannose 6-phosphate contained nearly stoichiometric amounts of bound 7.5-kDa IGF-II. In addition, at least 12 distinct 12-20-kDa proteins immunologically related to IGF-II also copurified with receptor. Receptor was separated from its associated ligands by acidification and gel filtration chromatography. Sequence analysis revealed that the 12-20-kDa proteins have the same amino termini as mature 7.5-kDa IGF-II. Protease and glycosidase treatments revealed that the different high molecular weight IGF-II species contain an identical COOH-terminal extension that is differentially glycosylated with O-linked sugars. Radiolabeled tracer experiments demonstrated that the sIGF-II/MPR carries of the IGF-II in fetal bovine serum. These results support a significant role for sIGF-II/MPR in the transport of circulating IGF-II isoforms during development.


INTRODUCTION

The insulin-like growth factor II/mannose 6-phosphate receptor (IGF-II/MPR)()is an integral membrane protein that binds two distinct classes of ligands: mannose 6-phosphate (Man-6-P) containing proteins and insulin like-growth factor II (IGF-II) (for review, see Kornfeld(1992) and Hoflack and Lobel (1993)). The Man-6-P modification on N-linked oligosaccharides of multiple newly synthesized lysosomal enzymes allows them to bind to intracellular IGF-II/MPRs. The complexes then exit the Golgi and travel to an acidic prelysosomal compartment (endosome) where the low pH promotes dissociation of ligand from receptor. Free receptors recycle back to the Golgi to repeat the process or to the cell surface where they mediate the endocytosis and lysosomal targeting of extracellular phosphorylated lysosomal enzymes. The function of the receptor in binding IGF-II is controversial. Mature IGF-II is a nonphosphorylated, nonglycosylated 67-amino acid protein related to insulin that is present at high levels during fetal development. It has been reported that the receptor functions in signal transduction upon IGF-II binding (for review, see Nishimoto et al.(1991)). In contrast, other studies indicate that the IGF-II/MPR functions in clearance rather than signal transduction and that IGF-II exerts its effects through the IGF-I receptor and another uncharacterized receptor (Filson et al., 1993; Liu et al., 1993; Baker et al., 1993). Regardless, the IGF-II/MPR clearly mediates endocytosis of IGF-II, resulting in its delivery to the lysosome and subsequent degradation (Oka et al., 1985; Kiess et al., 1987a). Thus, the receptor targets at least two classes of protein to the lysosome.

The extracytoplasmic ligand-binding region of the receptor contains 15 repeating units that have an average length of 147 amino acids and are 16-38% identical (Lobel et al., 1988). The receptor contains one IGF-II and two Man-6-P binding sites (Tong et al., 1989), and it is likely that each binding site is formed by a different repeating unit (Dahms et al., 1993, 1994; Garmroudi and MacDonald, 1994). One fascinating question is why the receptor contains 15 repeating domains. While it is possible that three units bind ligands and the other 12 are structural components, another possibility is that the receptor functions in lysosomal targeting of other ligands not yet identified. One of the purposes of this study was to search for endogenous non-Man-6-P-containing ligands of the receptor.

A soluble form of the IGF-II/MPR has been detected in serum and urine (Kiess et al., 1987b; Gelato et al., 1988; Causin et al., 1988; Gelato et al., 1989; Li et al., 1991). This protein retains IGF-II and Man-6-P binding activities and, based on its size, contains nearly the entire extracytoplasmic domain. Both the membrane-bound and soluble forms of the receptor are present at high levels during fetal development. In this study, we isolated and characterized the soluble receptor and its bound ligands from fetal bovine serum. While we did not detect any new classes of ligands, we found that in addition to mature 7.5-kDa IGF-II, the receptor binds at least 12 different high molecular weight forms of IGF-II. These proteins have apparent molecular weights of 12,000-20,000 and contain a common peptide backbone modified with different amounts of O-linked sugars. Furthermore, we demonstrate that the soluble receptor carries a significant fraction of IGF-II in fetal bovine serum.


EXPERIMENTAL PROCEDURES

Materials

The following buffers were used: physiological binding buffer (PBB), 100 mM NaCl, 25 mM NaHCO, 4 mM KCl, 1.2 mM CaCl, 1.2 mM NaHPO, 0.6 mM MgCl, 0.3 mM MgSO, 0.12 mM citric acid, 17 µM CuSO, 10 µM NaF, 6 µM AlCl, 22 nM NaI, 5 nM CoCl, 45 nM MnCl, 18 µM ZnSO, 5.7 mM NaOH, 5 mM -glycerophosphate, 20 mM HEPES, pH 7.4, at 4 °C; acid buffer, 150 mM ammonium acetate, 250 mM acetic acid, pH 4.5; immunoprecipitation (IP) buffer, 50 mM Tris, pH 7.4, 0.5% bovine serum albumin, 0.02% NaN; modification buffer, 6 M guanidine-HCl, 2 mM EDTA, 50 mM Tris, pH 8.1; Lys-C buffer, 1 mM EDTA, 5% acetonitrile, 25 mM Tris, pH 8.5; glycosidase buffer, 10 mM CaCl, 20 mM sodium cacodylate, pH 6.5; PBS, 137 mM NaCl, 2.7 mM KCl, 10.1 mM NaHPO, 1.76 mM KHPO, pH 7.2; PBST, PBS with 0.05% Tween 20; PBSB, PBS with 0.25% bovine serum albumin. When indicated, a protease inhibitor mixture (PIC) was included at a final concentration of 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 1 mM pepstatin. Fetal bovine serum was obtained from Sigma, Hyclone, and Inovar. Bovine amniotic fluid, allantoic fluid, and urine were generously provided by Drs. Darryl Byerley and Calvin Ferrell (U.S. Department of Agriculture (USDA), Clay City, NE). Yeast phosphomannan was very kindly provided by Dr. M. E. Slodki (USDA, Peoria, IL). Phosphomannan affinity resin was prepared by coupling cyanogen bromide-activated Sepharose CL-6B (Pharmacia Biotech Inc.) to phosphomannan core as described previously (Sahagian et al., 1982). Recombinant human IGF-II was generously provided by Dr. Yitzhak Stabinsky (PeproTech Inc., Rocky Hill, NJ). Nitrocellulose was from Schleicher and Schuell. All other chemicals were reagent grade.

Protein Purification

Either low quality or expired lots of tissue culture grade fetal bovine serum were used for purification of sIGF-II/MPR (see ). Typically, we used lots that contained 4 µg of receptor/ml, but similar elution profiles were obtained using lots that ranged from 2 to 6 µg of receptor/ml. Serum (5 liters) was diluted with an equal volume of PBB/PIC, filtered through 0.2-µm cellulose acetate membranes (Millipore), and loaded onto a phosphomannan-Sepharose CL-6B affinity column (5 10 cm) at 500 ml/h. After loading, the column was disassembled, the resin batch was washed with 2 column volumes of PBB (5 times), the column was reassembled, and the column was washed at 500 ml/h until A reached base line. In early preparations, the column was mock eluted at 30 ml/h with 2.5 mM glucose 6-phosphate in PBB/PIC to release nonspecifically bound substances; this step was subsequently omitted as negligible amounts of protein were released. The column was then eluted at 30 ml/h with 2.5 mM Man-6-P in PBB/PIC. Recovered proteins were concentrated by ultrafiltration using a YM-100 membrane (Amicon Inc.). Gel filtration chromatography at neutral pH was performed on affinity-purified protein using two columns connected in series (Superose 12, Pharmacia; 1.6 85 cm; Superdex 75, Pharmacia; 1.6 60 cm) equilibrated with 0.1 mM Man-6-P/PBB and run at 1.0 ml/min. Selected fractions (e.g. eluting between 96-150 ml; Fig. 1) from the neutral gel filtration chromatography step were concentrated as above, acidified to pH 4.5 by the addition of 0.1 volumes of 10 acid buffer, and incubated overnight. Acidified protein was rechromatographed at 0.25 ml/min on the gel filtration columns equilibrated with 1 acid buffer. All steps were performed at 4 °C.


Figure 1: Separation of sIGF-II/MPR and bound ligands. A, gel filtration chromatography was carried out as described under ``Experimental Procedures.'' Upper part, pH 7.4, neutral pH chromatography on the Man-6-P eluate from the phosphomannan column; lower part, pH 4.5, acidic pH chromatography on pooled fractions (96-150 ml) from the neutral column. Absorbance at 280 nm was continuously monitored and recorded at 1 (dashedline) and 50 (solidline) scale expansion. The minor A absorbing peak eluting after 240 ml in both chromatograms is likely to be salts since it was of variable intensity in different runs and did not contain any detectable protein. Positions of molecular mass standards run on the neutral gel filtration columns are marked with arrowsabovetoppart (from left to right: thyroglobulin, 669 kDa; apoferritin, 443 kDa; myosin, 200 kDa; alcohol dehydrogenase, 150 kDa; bovine serum albumin, 67 kDa; carbonic anhydrase, 29 kDa; ribonuclease A, 13.7 kDa; and aprotinin, 6.5 kDa). B, SDS-PAGE analysis of gel filtration chromatography fractions. Samples (18 µl) of 6-ml column fractions were resolved on a 5-20% polyacrylamide gel under reducing conditions. Volume refers to the elution position of each fraction. The gel was stained using Coomassie Blue, photographed, and then stained using ammoniacal silver. Positions of molecular mass standards are marked.



Precipitation of IGF-II

Samples were taken from a single fraction of the A2 and A3 peaks (equivalent to the 200- and 235-ml elution volumes, respectively, of the acidic gel filtration chromatogram in Fig. 1A) and radiolabeled using NaI (DuPont NEN NEZ-033A) and IODO-GEN (Pierce) as described previously (Valenzano et al., 1993). Approximately 150,000 cpm each of labeled peak A2 and A3 proteins were incubated with either 40 ng of anti-IGF-II monoclonal antibody (Amano) or 10 µl of sIGF-II/MPR-Sepharose (0.9 mg of sIGF-II/MPR per ml of resin) in a final volume of 400 µl of IP buffer. Samples precipitated with immobilized receptor had 10 mM Man-6-P included in the IP buffer. All samples were mixed overnight at 4 °C in the absence or presence of 60 ng of recombinant human IGF-II (rhIGF-II). Protein A-Sepharose (Pharmacia) was added to the samples containing anti-IGF-II and mixed at room temperature for an additional 30 min. After washing 5 times with IP buffer, pellets were resuspended in 80 µl of sample buffer and analyzed by SDS-PAGE.

Reduction and Alkylation

Samples in 1 acid buffer were dried and resuspended to 0.1-0.3 µg/µl protein in modification buffer. Samples were reduced with 20 mM DTT and alkylated in the dark with either 40 mM iodoacetic acid or iodoacetamide. Incubations were performed for 3 h at 50 °C under a N atmosphere with continuous stirring. For radiolabeling, samples were initially reduced with 2 mM DTT and alkylated with 4 mM iodo[2-C]acetic acid (50 µCi; Amersham Corp.) as above followed by 20 mM DTT and 40 mM iodoacetic acid to ensure complete alkylation. Reactions were quenched with 150 mM -mercaptoethanol for 15 min at room temperature. Reaction mixtures were diluted with an equal volume of water and applied to 0.1% trifluoroacetic acid-equilibrated Sep-Pak C18 cartridges (Waters). After washing with 10 ml of 0.1% trifluoroacetic acid, protein was eluted with 70% acetonitrile, 20% 1-propanol, 0.1% trifluoroacetic acid. Specific activities for radiolabeled high M and 7.5-kDa IGF-II were 250 Ci/mol assuming complete recovery of protein.

Analysis of IGF-II Binding Proteins in Fetal Bovine Serum

Mature bovine IGF-II from peak A3 was iodinated using lactoperoxidase as described previously (Daughaday, 1987). Radiolabeled protein was purified on a Sephadex G50 fine column (Pharmacia, 0.7 28 cm) using PBSB as the mobile phase. Three radioactive peaks were detected, and fractions comprising the second peak (IGF-II monomer) were pooled. The specific activity was 10 Ci/mmol assuming 50% recovery of IGF-II in the monomer peak. Radiolabeled IGF-II (10 ng) was incubated with fetal bovine serum (1 ml) for 24 h at room temperature. The serum was applied to a Superose 12 column (1.6 85 cm) and eluted at 1 ml/min with PBST at 4 °C. Fractions (2 ml) were counted for 1 min in a Packard Cobra auto--counter.

Other Procedures

sIGF-II/MPR concentrations in both purified and crude samples were determined by a two-antibody sandwich enzyme-linked immunosorbent assay as described previously (Chen et al., 1993). Protein concentrations were determined with bovine serum albumin standards using the dye-binding assay (Bradford, 1976) adapted to microtiter plates. SDS-PAGE was performed as described (Laemmli, 1970). Gels were stained with 0.2% Coomassie Brilliant Blue R-250 (Bio-Rad) and/or silver (Wray et al., 1981; Merril et al., 1981) as indicated. Dried radioactive gels were either imaged on Kodak X-Omat RP film or exposed to a phosphor storage screen, scanned, and quantitated using a Molecular Dynamics PhosphorImager 400 and ImageQuant 3.15 software. Sequencing was performed using automated Edman degradation on an Applied Biosystems Inc. model 475A gas phase sequencer with a 120A on-line phenylthiohydantoin-derivative analyzer and a 900A data control analysis module. The chromatograms were recorded and the results analyzed using an Applied Biosystems 475A report generator.


RESULTS

Purification of sIGF-II/MPR and Bound Ligands

This study was initiated to identify non-Man-6-P-containing ligands that are associated with the receptor in vivo. A suitable source of sIGF-II/MPR was identified by screening different bovine body fluids including serum, allantoic fluid, amniotic fluid, and urine. The results are summarized in . The richest source was fetal serum, with receptor levels in 23 different lots ranging from 1.8 to 7.9 µg/ml. Interestingly, serum classified as unacceptable for tissue culture by different suppliers (``low quality'') had lower receptor concentrations than tissue culture grade serum ().

Receptor and non-Man-6-P-containing ligands were isolated using a three-step scheme (see ``Experimental Procedures''). First, purification on a Man-6-P affinity resin (phosphomannan-agarose) was used to isolate receptor and to remove endogenous Man-6-P-containing ligands. Second, gel filtration chromatography at neutral pH was used to separate receptor and bound ligands from low molecular weight contaminants. As the receptor contains two Man-6-P binding sites (Tong et al., 1989), Man-6-P was included in this step to guard against the unlikely possibility that some Man-6-P-containing ligands remained associated with the receptor during affinity purification. Third, gel filtration chromatography was performed at mildly acidic pH to separate receptor from ligands. The final step capitalizes on the functional properties of this recycling receptor, which binds ligands at the near neutral pH of the Golgi or extracellular compartment and releases ligands into the acidic endosome.

Initial affinity purification of receptor was quite efficient: enzyme-linked immunosorbent assay on the starting material and run through of the phosphomannan column demonstrated that in a typical preparation, >95% of the receptor adsorbed to the resin. The material eluted with Man-6-P was concentrated by ultrafiltration and fractionated by gel filtration chromatography at neutral pH (Fig. 1A, upperpart). SDS-PAGE and Coomassie staining showed that both the 250-kDa receptor and a 6-kDa protein eluted near the void volume (Fig. 1A, peakN1; Fig. 1B, Coomassie, pH 7.4), while silver staining revealed that several additional proteins were present in the peak (Fig. 1B, silver, pH 7.4, 108 and 120ml). The lower molecular weight proteins were tightly associated with the receptor, since an identical staining pattern was observed for the material eluted from the phosphomannan column, the retentate from the ultrafiltration step, and the fractions in peak N1. In contrast, no protein was detected in the ultrafiltrate or later eluting fractions from the neutral gel filtration columns (data not shown).

A specific subset of the lower molecular weight species represents ligands that copurify with the sIGF-II/MPR. After acidification, gel filtration chromatography resolved peak N1 into three fractions (Fig. 1A, lowerpart). The major peak eluted near the void volume (peak A1), while two minor peaks eluted in the included volume (peaks A2 and A3). The centers of peaks A2 and A3 eluted as 18- and 7-kDa globular proteins, respectively. SDS-PAGE and Coomassie staining showed that peak A1 contained the 250-kDa receptor, while peak A3 contained the 6-kDa protein (Fig. 1B, Coomassie, pH4.5, 120 and 228ml, respectively). In addition, silver staining revealed that peak A2 contained some of the 6-kDa protein and a series of other proteins with apparent molecular weights in the range of 12,000-20,000 (Fig. 1B, silver, pH4.5, 168-216 ml). Note that compared with peak N1, peak A1 exhibits increased staining of multiple bands (25-200 kDa). However, this smear probably represents nicked fragments of the sIGF-II/MPR generated by prolonged acidification. The important finding is that compared with peak N1, peak A1 exhibits decreased staining of the 6- and 12-20-kDa bands that are now present in peaks A3 and A2, respectively. This suggests that these proteins represent true ligands of the receptor. In contrast, sequence analysis demonstrated that the 66-kDa band seen in peaks N1, A1, and some of A2 represents contaminating bovine serum albumin.

Characterization of Ligands

Protein blotting experiments using an I-labeled sIGF-II/MPR probe demonstrated that the 6- and 12-20-kDa bands are specifically recognized by the receptor (Fig. 2). Furthermore, addition of Man-6-P does not abolish binding, while IGF-II does. In contrast to their Coomassie and silver staining intensities, there is a greater signal seen for the 12-20-kDa bands than for the 6-kDa band. This is misleading, since control experiments using iodinated tracers demonstrate that significant amounts of all species are washed off the membrane during the assay, with the 6-kDa band being lost preferentially. In addition, the 6-kDa band has precisely the same mobility as the 7.5-kDa rhIGF-II standard (Fig. 2). In a complementary experiment using radiolabeled protein, the bands present in peaks A2 and A3 were all precipitated by immobilized sIGF-II/MPR, and this was inhibited by rhIGF-II (Fig. 3). In addition, all of these radiolabeled bands were specifically recognized by an anti-IGF-II monoclonal antibody (Fig. 3). Finally, sequence analysis showed that >90% and >99% of the material present in representative fractions of peaks A2 and A3, respectively, had the amino terminus AYRPS. This is identical to that reported for mature bovine IGF-II (Honegger and Humbel, 1986). Taken together, these data indicate that the 12-20-kDa bands represent high molecular weight forms of IGF-II and that the 6-kDa band represents mature 7.5-kDa bovine IGF-II.


Figure 2: Activity blotting of gel filtration column fractions. Peak fractions from the neutral and acidic gel filtration columns were sampled (peaksN1 and A1, 0.1% of total; peaksA2, A2`, and A3, 0.3% of total), and fractionated on 5-20% polyacrylamide gels under nonreducing conditions. Protein was transferred to 0.2-µm nitrocellulose and probed with 5 nMI-labeled sIGF-II/MPR (Valenzano et al., 1993) in the presence or absence of 2.5 mM Man-6-P or 1 µM human IGF-II as indicated. The PhosphorImager printouts shown represent a 16-h exposure and a color range of 1-1500 machine counts. Only the relevant portions of the membranes are shown, since no signal was detected in the higher molecular weight regions. Brackets and arrows represent the migration of high M and 7.5-kDa IGF-II, respectively.




Figure 3: Precipitation of high M and 7.5-kDa IGF-II. Iodinated samples of peaks A2 and A3 were incubated with either anti-IGF-II monoclonal antibody/protein A-Sepharose or sIGF-II/MPR-coupled Sepharose CL-6B in the absence or presence of rhIGF-II as described (see ``Experimental Procedures''). Pellets were resuspended in reducing SDS-PAGE sample buffer and resolved on a 5-20% polyacrylamide gel. Proteins were visualized by autoradiography after a 24-h exposure. The positions of high M and 7.5-kDa IGF-II are indicated.



In the course of characterizing these IGF-II species, we noticed that their migration during SDS-PAGE was dramatically influenced by different reduction and alkylation treatments. We systematically investigated this using rhIGF-II (Fig. 4A). Both nonreduced and DTT-treated rhIGF-II migrated with the 6-kDa standard (lanes1 and 4, respectively), while carboxyamidation with iodoacetamide slightly retarded migration (lane3). In contrast, carboxymethylation with iodoacetic acid greatly decreased migration, with the modified protein now running just behind the 14-kDa standard (lane2). The major band in peak A3 also exhibited this shift, precisely comigrating with rhIGF-II under all conditions (Fig. 4B, lanes1, 2, 4, and 5, and data not shown). These observations further confirm that peak A3 contains 7.5-kDa bovine IGF-II. The bands in peak A2 also show this characteristic mobility shift upon iodoacetic acid modification (Fig. 4B, lanes3 and 6, and data not shown). This supports our conclusion that these bands represent different IGF-II species. The markedly decreased migration of the carboxymethylated proteins may be a consequence of reduced SDS binding or altered conformation due to introduction of the negatively charged functional groups. Similar behavior has been reported previously for other chemically modified or phosphorylated proteins (Banker and Cotman, 1972; McCumber et al., 1976; Deibler et al., 1975). In addition, iodoacetic acid treatment is useful in increasing resolution of the bands by SDS-PAGE: analysis on 12.5-17.5% acrylamide gels revealed that there were at least 12 distinct high M IGF-II proteins (Fig. 4C). This complexity was also evident using ion exchange chromatography on the native proteins.()


Figure 4: Electrophoretic mobilities of high M and 7.5-kDa IGF-II. A, rhIGF-II was modified as described (see ``Experimental Procedures''). Lane1, DTT-reduced; lane2, DTT + iodoacetic acid; lane3, DTT + iodoacetamide; lane4, nonreduced. B, rhIGF-II (lanes1 and 4), bovine 7.5-kDa IGF-II (lanes2 and 5), and bovine high M IGF-II (lanes3 and 6) modified with iodoacetic acid (lanes1-3) or iodoacetamide (lanes4-6). C, high M IGF-II modified with iodoacetic acid. PanelsA and B represent Coomassie-stained 16% Novex gels. PanelC represents a 12.5-17.5% polyacrylamide gel stained with silver. The high M IGF-II in panelB was prepared by rechromatography of peak A2 on the Superdex 75 column to remove 7.5-kDa IGF-II. Chromatography conditions were identical to the acidic gel filtration step described in Fig. 1. Two fractions from the purified high M IGF-II peak were pooled and used for modification. The high M IGF-II in panelC was prepared by sampling all peak A2 fractions to obtain an accurate representation of the high M IGF-II proteins and mature 7.5-kDa IGF-II. The molecular mass standards for each panel are shown.



To investigate factors that contribute to the increased molecular weight and size heterogeneity of the high M IGF-II proteins, we adapted the strategy of protease and glycosidase treatments used by Hudgins and colleagues (Hudgins et al., 1992). Pooled fractions of high M IGF-II and rhIGF-II standard were radiolabeled at Cys-9, -21, -46, -47, -51, and -60 by reduction and alkylation with iodo[2-C]acetic acid. The proteins were digested with Lys-C which is predicted to cleave pro-IGF-II after Lys-65, -88, -96, -120, -129, and -151 (see Fig. 5). The rhIGF-II standard, which terminates at Glu-67, showed a slight shift in mobility by SDS-PAGE, consistent with the loss of its two COOH-terminal amino acids (Fig. 6). Incubation with the protease converted all the high M IGF-II species into a single band that had the same mobility as digested rhIGF-II. This demonstrates that the increased molecular weight and size heterogeneity seen among the high M IGF-II proteins is due to variations beyond Lys-65.


Figure 5: Schematic of prepro-IGF-II. Bovine IGF-II is synthesized as a 179-amino acid preproprotein. After removal of the signal sequence (hatchedregion), further processing within the E peptide (stippledregion) occurs, ultimately yielding the mature 67-residue protein. The enlarged region shows potential sites for O-linked glycosylation (asterisks). The positions of the six lysine residues (65, 88, 96, 120, 129, and 151) are marked with arrows (see Fig. 6).




Figure 6: Lys-C digestion of high M and 7.5-kDa IGF-II. Duplicate samples (1 µg) of reduced and C-alkylated high M and 7.5-kDa rhIGF-II were dried and resuspended in 26 µl of Lys-C buffer. Endoproteinase Lys-C (0.4 µg; Boehringer Mannheim) was added to each sample and incubated at 37 °C for 20 h. Digested samples were resolved on a 12.5-17.5% polyacrylamide gel and visualized by autoradiography after a 2-week exposure. Positions of molecular mass standards are indicated. High M IGF-II was separated from 7.5-kDa IGF-II as described in Fig. 4.



Pro-IGF-II has no consensus N-linked glycosylation sites; however, numerous potential sites for O-linked glycosylation are present (Fig. 5). To determine if variability among the high M IGF-II species arises from this modification, the mixture was digested with neuraminidase and O-glycanase to remove terminal sialic acids and O-linked core disaccharides, respectively (Fig. 7). Treatment with both enzymes led to a collapse of nearly all high M IGF-II species into a single band that had the same mobility as the smallest high M IGF-II and likely represents extended, nonglycosylated IGF-II (arrow). These data, together with the Lys-C data above, indicate that most of the high M forms of IGF-II contain an identical COOH-terminal extension with variable O-linked glycosylation. In addition, the high M forms of IGF-II were also digested with the two glycosidases separately. Treatment with neuraminidase alone converts many of the upper bands into lower molecular weight species, demonstrating that these proteins contain sialic acid. In contrast, treatment with O-glycanase alone has little effect on the higher molecular weight forms, while the band running just above the deglycosylated form displays an increased intensity. As O-glycanase only removes the unmodified disaccharide Gal1-3GalNAc from threonine or serine residues, this suggests that at least some of the high M IGF-II species contain both O-glycanase-sensitive and resistant linkages, indicating glycosylation at two or more sites.


Figure 7: Glycosidase treatment of high M IGF-II. Samples (0.8 µg) of reduced and C-alkylated high M IGF-II were dried and incubated with either 10 milliunits of neuraminidase (Arthrobacter; Boehringer Mannheim), 2 milliunits of O-glycanase (Genzyme), or both enzymes in a total volume of 30 µl of glycosidase buffer. Digests were carried out at 37 °C for 20 h. For double digests, samples were first incubated with neuraminidase for 2 h at 37 °C before addition of O-glycanase. Digested samples were resolved on a 12.5-17.5% polyacrylamide gel and visualized by autoradiography after a 2-week exposure. Positions of molecular mass standards are indicated. High M IGF-II was separated from 7.5-kDa IGF-II as described in Fig. 4.



sIGF-II/MPR as an IGF-II Binding Protein

A large proportion of the affinity-purified receptor contains bound IGF-II. In a typical preparation from 5 liters of serum, we recover 18, 0.19, and 0.27 mg of protein in peaks A1, A2, and A3, respectively. (These values were obtained using the dye-binding assay, but similar results were found using quantitative amino acid analysis). Based on our recoveries from peaks A1 and A3, at least 50% of the receptor contained bound IGF-II, assuming that the protein molecular weights of sIGF-II/MPR and mature IGF-II are 250,000 and 7,500, respectively, and that the binding stoichiometry of IGF-II and receptor is 1:1 (Tong et al., 1988). This is a conservative estimate, since it ignores the contribution of mature IGF-II and high M IGF-II present in peak A2.

In addition to soluble receptor, there are at least six other IGF binding proteins that have molecular weights ranging from 25 to 150 kDa (for review, see Lamson et al.(1991) and Clemmons(1993)). To estimate the fraction of IGF-II carried by the sIGF-II/MPR, fetal bovine serum was incubated with iodinated bovine IGF-II and applied to a gel filtration column (White et al., 1982; Arner et al., 1989). Time course experiments indicated that 24-h incubation at room temperature was sufficient to approach equilibrium. This occurred in a biphasic process. At both early and late time points nearly all tracer bound to serum proteins, with 5% eluting as the uncomplexed form (Fig. 8, upperpanel, peak4; also see below). However, tracer redistributes among the different peaks as a function of time, shifting from low to high M species (Fig. 8, upperpanel, compare peak3 with peak1). While biphasic binding is also seen at 4 °C, the slow redistribution process has not reached equilibrium even after 48 h (Fig. 8, middlepanel and inset).


Figure 8: Effects of time and temperature on the association of I-labeled IGF-II with serum proteins. Upper and middlepanels, serum was incubated with radiolabeled IGF-II tracer as indicated and fractionated by gel filtration chromatography (see ``Experimental Procedures''). Insets, radioactivity eluting in peak1 is expressed as percentage of the total recovered radioactivity. Lower panel, serum was preincubated as indicated and then incubated with radiolabeled IGF-II tracer for 24 h at 4 °C. Positions of molecular mass standards are marked with arrows above the toppanel (from left to right: sIGF-II/MPR, 250 kDa; alcohol dehydrogenase, 150 kDa; aprotinin, 6.5 kDa).



Additional control experiments indicate that the redistribution process is not caused by preferential degradation of the low M binding proteins over time. Serum was preincubated at either 4 °C for 4 h or 22 °C for 24 h, chilled to 4 °C, and then incubated with tracer for 24 h. Both conditions yielded nearly identical binding profiles (Fig. 8, lowerpanel). This strongly suggests that no appreciable degradation occurs during the 24-h incubation at 22 °C.

Several different models could explain the biphasic binding of tracer to serum proteins. For instance, this may represent rapid binding of tracer to unoccupied low M binding proteins that have relatively low affinity for IGF-II, followed by a slow equilibration to higher affinity high M binding proteins. Alternatively, the initial binding could reflect the forward rate constants of the different binding proteins, while the final distribution would reflect both the forward and reverse rate constants. Regardless of the mechanism, the process appears to plateau after 24 h at 22 °C (Fig. 8, upperpanel, inset), and the equilibrium value should reflect the relative concentration and affinities of the different binding proteins and hence the underlying distribution of endogenous IGF-II (Frost and Pearson, 1953).

Binding experiments using different lots of serum indicated that 23-32% of the tracer was in a peak that eluted identically to sIGF-II/MPR standard (Fig. 9A, shadedregion; ). Furthermore, the fraction of tracer associated with this peak was proportional to endogenous receptor levels (). Most of this IGF-II binding activity represents authentic sIGF-II/MPR, since receptor-depleted serum has greatly diminished activity (Fig. 9B, shadedregion; ). Interestingly, while enzyme-linked immunosorbent assay () and Western blotting analysis using polyclonal antisera (data not shown) demonstrated complete removal of receptor, 4-5% of the IGF-II tracer still migrated in this region. This binding is specific, since excess unlabeled rhIGF-II resulted in elution of tracer as the free 7.5-kDa protein (Fig. 9C). Thus, while another protein makes a minor contribution to IGF-II binding in this region, these results clearly show that the endogenous sIGF-II/MPR carries of the total IGF-II.


Figure 9: Contribution of sIGF-II/MPR as an IGF-II binding protein. A, untreated serum. B, serum depleted of sIGF-II/MPR by phosphomannan-agarose chromatography. C, nonspecific binding. Untreated FBS was co-incubated with tracer IGF-II and a 1000-fold excess (1.3 µM) of cold rhIGF-II. (All incubations with tracer were for 24 h at 22 °C.)




DISCUSSION

These studies were initiated to identify ligands that bind to the IGF-II/MPR at regions distinct from the Man-6-P binding sites. Our approach was to isolate receptor and bound ligands on a phosphomannan resin, reasoning that Man-6-P-containing proteins would be displaced without disrupting interactions at other binding sites. We purified a soluble form of the receptor from fetal serum, eliminating the need for detergent solubilization of membrane fractions that could potentially disrupt receptor-ligand complexes. In addition, we used a buffer that reflected the total ionic composition of human plasma (Lentner, 1984) to supply possible cofactors required for ligand binding. Using this strategy, we successfully isolated both mature 7.5-kDa IGF-II and a fraction containing at least 12 different higher molecular weight ligands. Given the complexity of the latter fraction, we used multiple approaches to determine its composition. Activity blotting with soluble receptor, affinity and immunoprecipitation experiments using immobilized receptor, and an anti-IGF-II monoclonal antibody, respectively, and amino-terminal sequencing indicate that all the proteins isolated represent high molecular weight forms of IGF-II.

There are several possible reasons why this strategy would not detect other, yet unidentified, classes of ligands that may bind to the IGF-II/MPR. For instance, this approach requires that there be no steric or allosteric interactions that prevent copurification of receptor and ligand. It has been shown that Man-6-P and IGF-II do not compete with each other for binding to the receptor, while the phosphorylated lysosomal enzyme -galactosidase and IGF-II do, despite having different microscopic binding sites (Kiess et al., 1989). Similarly, potential new ligands bound to the serum receptor could be displaced by the phosphomannan agarose and/or endogenous IGF-II. In addition, potential new ligands may be present in tissues other than serum. To address these possibilities, we screened different tissue extracts for non-Man-6-P-containing ligands.()Two approaches were used. First, extracts were fractionated by SDS-PAGE, transferred to nitrocellulose, and probed with the radiolabeled soluble receptor as described in Fig. 2. Second, extracts were applied to immobilized sIGF-II/MPR columns in the presence of Man-6-P to enrich for non-Man-6-P containing-proteins. Bound material was eluted with an acidic buffer, fractionated by SDS-PAGE, and screened as above. Both approaches yielded negative results. Thus, although we cannot exclude the possibility that other ligands exist, despite extensive effort, we have not detected them.

The sIGF-II/MPR has been detected in human (Causin et al., 1988), primate (Gelato et al., 1988), rodent (Kiess et al., 1987b), ovine (Gelato et al., 1989), and bovine sera (Yang et al., 1991; Li et al., 1991) and in human urine (Causin et al., 1988). The circulating form is developmentally regulated, with the highest levels occurring in fetal serum (Kiess et al., 1987b; Gelato et al., 1988; Gelato et al., 1989; Li et al., 1991). The sIGF-II/MPR from different species carries variable amounts of circulating IGF-II; 3, 20, and 50% of the total circulating IGF-II in fetal sera from rat (Kiess et al., 1987b), monkey (Gelato et al., 1988), and sheep (Gelato et al., 1989), respectively, was associated with a high molecular weight protein that was excluded from Sephadex G-200. Chemical cross-linking studies with radiolabeled IGF-II indicated that the receptor was the major carrier in these fractions. In addition, receptor purified on pentamannosylphosphate-agarose from fetal bovine serum contained substantial amounts of bound IGF-II, since amino-terminal sequencing yielded equivalent levels of both proteins (Li et al., 1991). Our results confirm and extend these findings, demonstrating that 25% of the total IGF-II in fetal bovine serum is bound to soluble receptor.

An important finding of this study is that at least 12 different high molecular weight forms of IGF-II are associated with the circulating fetal sIGF-II/MPR. Different variant and high molecular weight IGF-IIs have been reported previously (for review, see Humbel(1990) and Rechler(1991); also see Hudgins et al.(1992)). Human IGF-II variants that are substituted at Ser-29 and Ser-33 with RLPG and CGD, respectively, with or without COOH-terminal extensions, have been identified (Zumstein et al., 1985; Hampton et al., 1989). Proteolysis experiments (Fig. 6) indicate that our high M IGF-IIs do not contain these internal substitutions. Recently, two IGF-II species with apparent molecular weights of 15,000 and 11,500 were purified from human serum Cohn fraction IV and represent partially processed forms of the 156-residue pro-IGF-II (Hudgins et al., 1992). In addition, these 87-88-amino acid proteins are glycosylated at Thr-75. It is likely that our glycosylated high M IGF-II species represent additional isoforms of these proteins.

The existence of the sIGF-II/MPR and bound ligands in serum may have important experimental and biological implications. First, fetal serum is frequently used in mammalian cell culture experiments. While it is now appreciated that IGF-II added to serum-containing media can be buffered by binding proteins, the added IGF-II could also displace high M IGF-II ligands, allowing them to exert their effects. Second, the biological function of the high M IGF-II proteins and the purpose of their glycosylation remain intriguing and unanswered questions. It has been suggested that glycosylation is required for proper proteolytic processing of pro-IGF-II to the mature 7.5-kDa form (Daughaday et al., 1993). 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 (Fielder and Simons, 1994). In this case, the sIGF-II/MPR-bound high M IGF-IIs may simply reflect inefficiencies in the processing system. Alternatively, the glycosylated IGF-II species may have unique biological functions. This is not unprecedented, since N-linked glycosylation of several glycoprotein hormones is necessary for receptor activation but not binding (for review, see Sairam(1989)). Similarly, the different glycosylated high M IGF-II species may be targeted to specific destinations or activate specific receptors. Future studies using individually-purified high M IGF-II glycoproteins will be instrumental in determining their pharmacological and biochemical properties.

  
Table: sIGF-II/MPR content of bovine body fluids

Two independent sample dilutions were assayed for sIGF-II/MPR by enzyme-linked immunosorbent assay as described by Chen et al. (1993). The n values represent the number of different serum lots or samples from different animals (other fluids).


  
Table: sIGF-II/MPR as an IGF-II binding protein



FOOTNOTES

*
This work was supported in part by National Science Foundation Grant DCB-9118681 and March of Dimes Birth Defects Foundation Basil O'Connor Starter Scholar Research Award 5-1217. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
K. J. V. was a predoctoral trainee on National Institutes of Health Biotechnology Training Grant GM08339.

To whom correspondence should be addressed: Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854. Tel.: 908-235-5032; Fax: 908-235-5318.

The abbreviations used are: IGF-II/MPR, insulin-like growth factor II/mannose 6-phosphate receptor; sIGF-II/MPR, soluble IGF-II/MPR; rhIGF-II, recombinant human IGF-II; Man-6-P, mannose 6-phosphate; PBB, physiological binding buffer; IP, immunoprecipitation; PBS, phosphate-buffered saline; PIC, protease inhibitor mixture; DTT, dithiothreitol; Lys-C, endoproteinase Lys-C; PAGE, polyacrylamide gel electrophoresis; FBS, fetal bovine serum.

K. J. Valenzano and P. Lobel, unpublished data.

K. J. Valenzano, J. Remmler, and P. Lobel, unpublished data.


ACKNOWLEDGEMENTS

We thank Henry Lackland and Stanley Stein of the Center for Advanced Biotechnology and Medicine Protein Chemistry Laboratory for protein sequencing and amino acid analyses.


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