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• W2024865764 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 ~¼ 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. 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 ~¼ 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. The insulin-like growth factor II/mannose 6-phosphate receptor (IGF-II/MPR)1( 1The abbreviations used are: IGF-II/MPRinsulin-like growth factor II/mannose 6-phosphate receptorsIGF-II/MPRsoluble IGF-II/MPRrhIGF-IIrecombinant human IGF-IIMan-6-Pmannose 6-phosphatePBBphysiological binding bufferIPimmunoprecipitationPBSphosphate-buffered salinePICprotease inhibitor mixtureDTTdithiothreitolLys-Cendoproteinase Lys-CPAGEpolyacrylamide gel electrophoresisFBSfetal bovine serum.) 1The abbreviations used are: IGF-II/MPRinsulin-like growth factor II/mannose 6-phosphate receptorsIGF-II/MPRsoluble IGF-II/MPRrhIGF-IIrecombinant human IGF-IIMan-6-Pmannose 6-phosphatePBBphysiological binding bufferIPimmunoprecipitationPBSphosphate-buffered salinePICprotease inhibitor mixtureDTTdithiothreitolLys-Cendoproteinase Lys-CPAGEpolyacrylamide gel electrophoresisFBSfetal bovine serum. 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 36Nishimoto I. Murayama Y. Okamoto T. Spencer E.M. Modern Concepts of Insulin-like Growth Factors. Elsevier Science Publishing Co., New York1991: 517-528Google Scholar). 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 (14Filson A.J. Louvi A. Efstratiadis A. Robertson E.C. Development. 1993; 118: 731-736Crossref PubMed Google Scholar; 32Liu J.-P. Baker J. Perkins A.S. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2563) Google Scholar; 32Liu J.-P. Baker J. Perkins A.S. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2563) Google Scholar). Regardless, the IGF-II/MPR clearly mediates endocytosis of IGF-II, resulting in its delivery to the lysosome and subsequent degradation (37Oka Y. Rozek L.M. Czech M.P. J. Biol. Chem. 1985; 260: 9435-9442Abstract Full Text PDF PubMed Google Scholar; 24Kiess W. Haskell J.F. Lee L. Greenstein L.A. Miller B.E. Aarons A.L. Rechler M.M. Nissley S.P. J. Biol. Chem. 1987; 262: 12745-12751Abstract Full Text PDF PubMed Google Scholar). Thus, the receptor targets at least two classes of protein to the lysosome. insulin-like growth factor II/mannose 6-phosphate receptor soluble IGF-II/MPR recombinant human IGF-II mannose 6-phosphate physiological binding buffer immunoprecipitation phosphate-buffered saline protease inhibitor mixture dithiothreitol endoproteinase Lys-C polyacrylamide gel electrophoresis fetal bovine serum. insulin-like growth factor II/mannose 6-phosphate receptor soluble IGF-II/MPR recombinant human IGF-II mannose 6-phosphate physiological binding buffer immunoprecipitation phosphate-buffered saline protease inhibitor mixture dithiothreitol endoproteinase Lys-C polyacrylamide gel electrophoresis fetal bovine serum. 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 (33Lobel P. Dahms N.M. Kornfeld S. J. Biol. Chem. 1988; 263: 2563-2570Abstract Full Text PDF PubMed Google Scholar). The receptor contains one IGF-II and two Man-6-P binding sites (42Tong P.Y. Gregory W. Kornfeld S. J. Biol. Chem. 1989; 264: 7962-7969Abstract Full Text PDF PubMed Google Scholar), and it is likely that each binding site is formed by a different repeating unit (8Dahms N.M. Rose P.A. Molkentin J.D. Zhang Y. Brzycki M.A. J. Biol. Chem. 1993; 268: 5457-5463Abstract Full Text PDF PubMed Google Scholar, 9Dahms N.M. Wick D.A. Brzycki-Wessell M.A. J. Biol. Chem. 1994; 269: 3802-3809Abstract Full Text PDF PubMed Google Scholar; 16Garmroudi F. MacDonald R.G. J. Biol. Chem. 1994; 269: 26944-26952Abstract Full Text PDF PubMed Google Scholar). 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 (24Kiess W. Haskell J.F. Lee L. Greenstein L.A. Miller B.E. Aarons A.L. Rechler M.M. Nissley S.P. J. Biol. Chem. 1987; 262: 12745-12751Abstract Full Text PDF PubMed Google Scholarb; 18Gelato M.C. Rutherford C. Stark R.I. Daniel S.S. Endocrinology. 1989; 124: 2935-2943Crossref PubMed Scopus (26) Google Scholar; 5Causin C. Waheed A. Braulke T. Junghans U. Maly P. Humbel R.E. von Figura K. Biochem. J. 1988; 252: 795-799Crossref PubMed Scopus (75) Google Scholar; 18Gelato M.C. Rutherford C. Stark R.I. Daniel S.S. Endocrinology. 1989; 124: 2935-2943Crossref PubMed Scopus (26) Google Scholar; 31Li M. Distler J.J. Jourdian G.W. Glycobiology. 1991; 1: 511-517Crossref PubMed Scopus (9) Google Scholar). 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. The following buffers were used: physiological binding buffer (PBB), 100 mM NaCl, 25 mM NaHCO3, 4 mM KCl, 1.2 mM CaCl2, 1.2 mM NaH2PO4, 0.6 mM MgCl2, 0.3 mM MgSO4, 0.12 mM citric acid, 17 μM CuSO4, 10 μM NaF, 6 μM AlCl3, 22 nM NaI, 5 nM CoCl2, 45 nM MnCl2, 18 μM ZnSO4, 5.7 mM NaOH, 5 mM β-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% NaN3; 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 CaCl2, 20 mM sodium cacodylate, pH 6.5; PBS, 137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.76 mM KH2PO4, 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 (39Sahagian G.G. Distler J.J. Jourdian G.W. Methods Enzymol. 1982; 83: 392-396Crossref PubMed Scopus (43) Google Scholar). 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. 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 A280 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. 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 Na125I (DuPont NEN NEZ-033A) and IODO-GEN (Pierce) as described previously (43Valenzano K.J. Kallay L.M. Lobel P. Anal. Biochem. 1993; 209: 156-162Crossref PubMed Scopus (41) Google Scholar). 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. 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 N2 atmosphere with continuous stirring. For radiolabeling, samples were initially reduced with 2 mM DTT and alkylated with 4 mM iodo[2-14C]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 β-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 Mr and 7.5-kDa IGF-II were ~250 Ci/mol assuming complete recovery of protein. 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-γ-counter. sIGF-II/MPR concentrations in both purified and crude samples were determined by a two-antibody sandwich enzyme-linked immunosorbent assay as described previously (6Chen H.J. Remmler J. Delaney J.C. Messner D.J. Lobel P. J. Biol. Chem. 1993; 268: 22338-22346Abstract Full Text PDF PubMed Google Scholar). 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 (45Wray W. Boulikas T. Wray V.P. Hancock R. Anal. Biochem. 1981; 118: 197-203Crossref PubMed Scopus (2499) Google Scholar; 35Merril C.R. Goldman D. Sedman S.A. Ebert M.H. Science. 1981; 211: 1437-1438Crossref PubMed Scopus (2103) Google Scholar) 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. 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 Table I. 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 (Table I).Table:sIGF-II/MPR content of bovine body fluids Open table in a new tab 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, upper part). SDS-PAGE and Coomassie staining showed that both the 250-kDa receptor and a 6-kDa protein eluted near the void volume (Fig. 1A, peak N1; 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 120 ml). 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, lower part). 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, pH 4.5, 120 and 228 ml, 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, pH 4.5, 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. Protein blotting experiments using an 125I-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 (21Honegger A. Humbel R.E. J. Biol. Chem. 1986; 261: 569-575Abstract Full Text PDF PubMed Google Scholar). 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 3:Precipitation of high Mr 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 Mr and 7.5-kDa IGF-II are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (lanes 1 and 4, respectively), while carboxyamidation with iodoacetamide slightly retarded migration (lane 3). In contrast, carboxymethylation with iodoacetic acid greatly decreased migration, with the modified protein now running just behind the 14-kDa standard (lane 2). The major band in peak A3 also exhibited this shift, precisely comigrating with rhIGF-II under all conditions (Fig. 4B, lanes 1, 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, lanes 3 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 (3Banker G.A. Cotman C.W. J. Biol. Chem. 1972; 247: 5856-5861Abstract Full Text PDF PubMed Google Scholar; 34McCumber L.J. Clem L.W. Biochim. Biophys. Acta. 1976; 446: 536-541Crossref PubMed Scopus (5) Google Scholar; 12Deibler G.E. Martenson R.E. Kramer A.J. Kies M.W. Miyamoto E. J. Biol. Chem. 1975; 250: 7931-7938Abstract Full Text PDF PubMed Google Scholar). 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 Mr IGF-II proteins (Fig. 4C). This complexity was also evident using ion exchange chromatography on the native proteins.2( 2K. J. Valenzano and P. Lobel, unpublished data.) To investigate factors that contribute to the increased molecular weight and size heterogeneity of the high Mr IGF-II proteins, we adapted the strategy of protease and glycosidase treatments used by Hudgins and colleagues (22Hudgins W.R. Hampton B. Burgess W.H. Perdue J.F. J. Biol. Chem. 1992; 267: 8153-8160Abstract Full Text PDF PubMed Google Scholar). Pooled fractions of high Mr IGF-II and rhIGF-II standard were radiolabeled at Cys-9, −21, −46, −47, −51, and −60 by reduction and alkylation with iodo[2-14C]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 Mr 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 Mr IGF-II proteins is due to variations beyond Lys-65.Figure 6:Lys-C digestion of high Mr and 7.5-kDa IGF-II. Duplicate samples (1 μg) of reduced and 14C-alkylated high Mr 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 Mr IGF-II was separated from 7.5-kDa IGF-II as described in Fig. 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 Mr 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 Mr IGF-II species into a single band that had the same mobility as the smallest high Mr 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 Mr forms of IGF-II contain an identical COOH-terminal extension with variable O-linked glycosylation. In addition, the high Mr 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 Galβ1-3GalNAc from threonine or serine residues, this suggests that at least some of the high Mr IGF-II species contain both O-glycanase-sensitive and resistant linkages, indicating glycosylation at two or more sites. 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 (41Tong P.Y. Tollefsen S.E. Kornfeld S. J. Biol. Chem. 1988; 263: 2585-2588Abstract Full Text PDF PubMed Google Scholar). This is a conservative estimate, since it ignores the contribution of mature IGF-II and high Mr 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 29Lamson G. Giudice L.C. Rosenfel" @default.
• W2024865764 created "2016-06-24" @default.
• W2024865764 creator A5005292388 @default.
• W2024865764 creator A5034399581 @default.
• W2024865764 creator A5035296804 @default.
• W2024865764 date "1995-07-01" @default.
• W2024865764 modified "2023-10-10" @default.
• W2024865764 title "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" @default.
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