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Original Studies |
Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
Address all correspondence and requests for reprints to: Dr. C. D. Scott, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia.
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Abstract |
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Introduction |
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The ability of the IGF-II/M6-P receptor to interact with several ligands is reflected by the many functions that have been attributed to it. The receptor has been shown to transport lysosomal enzymes such as ß-D-glucuronidase and ß-D-galactosidase from the Golgi apparatus to the prelysosomes (16, 17). Our group has shown that in rat hepatocytes the soluble receptor inhibits DNA synthesis by modulation of IGF-II action and by an IGF-independent mechanism (18). The importance of the receptor in fetal and tumor development is shown by its ability to bind, internalize, and degrade IGF-II. Disruption of this process in fetal mice leads to cardiac hypertrophy and subsequent perinatal death (19), whereas in tumor cells it has the potential to increase tumor cell proliferation. Hankins and De Souza have presented data suggesting that receptor allelic loss is an early event in breast and hepatocellular tumor etiology (20, 21, 22). Both of these IGF-II-stimulated overgrowth mechanisms rely on the loss of receptor function. TGFß, a potent cytokine, has been shown to require the IGF-II/M6-P receptor for its activation. It is thought that by binding to the latent form of TGFß, the IGF-II/M6-P receptor facilitates TGFß activation (11, 23, 24, 25). Additionally, the receptor has been shown to be involved in the endocytosis of extracellular enzymes (26, 27).
Despite the varied functions of the IGF-II/M6-P receptor, there is currently no simple immunological method for quantitating receptor levels in human biological fluids. We now report the development of a human IGF-II/M6-P receptor enzyme-linked immunosorbent assay (ELISA) and describe its use to study the regulation of the soluble form of the receptor in human serum.
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Materials and Methods |
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D-Mannose 6-phosphate (Ba2+),
ethanolamine, bovine ß-glucuronidase (type B-10), bovine
-globulins (Cohn fraction II), SDS, Triton X-100, Tween-20, BSA (RIA
grade), and DMEM were purchased from Sigma Chemical Co.
(St. Louis, MO). The bicinchoninic acid protein assay kit, the EZ-link
NHS-LC-biotinylation kit, bis(sulfosuccinimidyl) suberate
(BS3), and the Supersignal Chemiluminescence Substrate
Solutions kit for enhanced chemiluminescence were obtained from
Pierce (Rockford, IL). FCS was purchased from Trace
Biosciences (Castle Hill, Australia). Epoxy-activated Sepharose 4B and
protein A-Sepharose were obtained from Pharmacia Biotech
(Uppsala, Sweden). F96 Certmaxisorp Nunc immunoplates were purchased
from Nalge Nunc International (Rochester, NY). Bovine
plasma 
2-macroglobulin and 3,3',5,5'-tetra-methyl
benzidine were obtained from Boehringer Mannheim (Mannheim, Germany).
Aprotinin [10,000 kallikrein inhibitor units (KIU)/mL;
Trasylol] was purchased from Bayer Australia Ltd.
(Sydney, Australia). Streptavidin-horseradish peroxidase, donkey
antirabbit horseradish peroxidase, and Hybond-C Extra were purchased
from Amersham Life Science Australia (Castle Hill, Australia).
Centricon-100 microconcentrators were purchased from Amicon (Beverly,
MA). Recombinant human IGF-II and O-phosphomannan were gifts
from Kabi Peptide Hormones (Stockholm, Sweden), and Dr. M. E.
Slodki (USDA, Peoria, IL), respectively. All other reagents used were
of analytical grade.
M6-P affinity column
Phosphomannan was hydrolyzed and separated into its core phosphomannan and pentamannose phosphate sugars as previously described (28, 29). Epoxy-activated Sepharose 4B was prepared as described by the manufacturers and mixed with the core phosphomannan in 0.1 mol/L sodium phosphate, pH 12, for 22 h at 45 C. The resin was washed extensively as described by the manufacturer, and unbound resin was blocked by incubation with 1 mol/L ethanolamine, pH 8, for 22 h at 45 C. The resin was washed and transferred to a 30 x 1-cm chromatography column.
IGF-II/M6-P receptor purification
Human hepatoma cells (HepG2) were grown to confluence in 17
tissue culture flasks (surface area/flask, 525 cm2),
maintained in DMEM supplemented with 5% FCS and 2 mmol/L glutamine.
Cells were harvested and homogenized by six passes with a Teflon-glass
homogenizer in 0.25 mol/L sucrose containing 500 KIU/mL
Trasylol and 5 µg/mL 2-macroglobulin.
Microsomal membranes were isolated by differential centrifugation (30);
resuspended in 25 mmol/L HEPES, pH 7.4, containing 500 KIU/mL
Trasylol, and 5 µg/mL 2-macroglobulin;
and solubilized in 1% Triton X-100. After solubilization, membranes
were diluted 10-fold to give a final Triton X-100 concentration of
0.1%. The membrane solution (140 mL) was loaded onto a M6-P affinity
column at 8.75 mL/h over 16 h at 4 C. The column was washed with 1
L 25 mmol/L HEPES, pH 7.4, containing 0.15 mol/L NaCl, 500 KIU/mL
Trasylol, 5 µg/mL 2-macroglobulin, 0.1%
Triton X-100, and 4 mmol/L D-glucose 6-phosphate followed
by 300 mL 25 mmol/L HEPES, pH 7.4, and 0.1% Triton X-100. The protein
was eluted with 10 mmol/L D-M6-P, 25 mmol/L HEPES (pH 7.4),
and 0.1% (vol/vol) Triton X-100. Fractions were assayed for binding of
[125I]IGF-II (31), and those fractions containing peak
activity were concentrated by ultrafiltration (Centricon-100) and
assayed for purity by SDS-PAGE (nonreducing conditions) and silver
stained (Phast System, Pharmacia Biotech). Membrane and
purified receptor protein concentrations were determined using
the Pierce bicinchoninic acid protein assay kit and
-globulin as standard.
Immunization of rabbit
Purified human membrane IGF-II/M6-P receptor (100 µg) in 0.5 mL 136 mmol/L NaCl containing 2.7 mmol/L KCl, 1.5 mmol/L KH2PO4, and 18.5 mmol/L Na2HPO4, pH 7.4 (PBS), was emulsified with an equal volume of Freund’s adjuvant (0.5 mL) and injected via six sc paralumbal sites into an 8-week-old female New Zealand White rabbit. Two weeks later the rabbit was boosted with 100 µg purified receptor in Freund’s incomplete adjuvant. A second, third, and fourth boost (50 µg each) were injected 3, 8, and 12 weeks later into the hind leg muscles in saline without adjuvant. Test bleeds were taken at 5 and 7 weeks, and serum was assayed by Western immunoblot for immunological activity against purified IGF-II/M6-P receptor (200 ng). The titer was monitored intermittently over the following months. The rabbit was killed at 33 weeks, and blood was collected by cardiac puncture.
SDS-PAGE, Western immunoblotting, and autoradiography
Samples were applied under nonreducing conditions to a 4–15% gradient (Phast Gel System, Pharmacia Biotech) or 6% homogeneous SDS-polyacrylamide gels overlaid with 4% stacking gels and subjected to electrophoresis (32), then electroeluted from the gels at 17 V for 2 h onto nitrocellulose (Hybond-C Extra). The nitrocellulose was blocked with 10 mmol/L Tris, pH 7.4, containing 4.2 mmol/L NaCl, 0.1% Tween-20, and 10 g/L BSA for 22 h at 4 C and then incubated with this buffer containing a 1:1,000 dilution of IGF-II/M6-P receptor antiserum for 22 h at 4 C. The nitrocellulose was washed and incubated with donkey antirabbit IgG conjugated to horseradish peroxidase for 1 h at 22 C, and the immunoreactive species was detected by enhanced chemiluminescence (Pierce) and exposed to film for 0–5 min. Quantitation of developed Western blots was carried out using a Bio-Rad model 620 Video Densitometer (Bio-Rad Laboratories, Inc., Richmond, CA) attached to a chart recorder. Purified human IGF-II/M6-P receptor (0.5 µg) was affinity labeled by incubation with [125I]IGF-II (100,000 cpm; 200 cpm/pg) for 16 h at 4 C in 25 mmol/L HEPES, pH 7.4, containing 1 g/L BSA and 0.1% Triton X-100. The sample was then incubated with 1 mmol/L BS3 for 30 min at 22 C, the reaction was stopped with 1 mol/L Tris (pH 8), and the samples were applied to a 6% SDS-polyacrylamide gel.
IGF-II/M6-P receptor ELISA
IGF-II/M6-P receptor antibody from the terminal bleed was affinity purified using protein A-Sepharose. Biotinylated receptor antibody was prepared by incubating affinity-purified antibody (2 mg) with 74 µg sulfo-NHS-LC-biotin (Pierce) for 30 min at 22 C, according to the manufacturer’s instructions. The unreacted biotin was separated via extensive dialysis against PBS. Affinity-purified antibody (0.6 µg/well) in 10 mmol/L sodium carbonate, pH 9.6, was absorbed to the 96-well immunoplates by a 22-h incubation at 4 C. The unbound antibody was removed, and the wells were blocked by incubation with PBS, 0.1% (vol/vol) Triton X-100 (buffer B) containing 10 g/L BSA for 35 min at 37 C, then washed four times with buffer B. Purified solubilized membrane IGF-II/M6-P receptor in sample dilution buffer (buffer B containing 2 g/L BSA) was used to generate standard curves. Standard and samples (100 µL/well) were incubated for 22 h at 4 C. The plate was washed, then incubated with biotinylated receptor antibody (80 ng/well) for 22 h at 4 C. After washing, the plate was incubated with streptavidin horseradish-peroxidase (1:500) for 30 min at 22 C, followed by substrate [0.1 g/L 3,3',5,5'-tetramethyl benzidine in 0.2 mol/L sodium acetate, pH 6, containing 0.06% (wt/wt) H2O2] for 30 min at 22 C. The reaction was stopped by the addition of 2 mol/L H2SO4, and the absorbance was measured at 450 nm using a Bio-Tek microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). Assay data were analyzed using the Multi Calc program (Wallac Oy, Turku, Finland).
Superose-12 gel chromatography
Serum samples (100 µL) were loaded onto a Superose-12 gel
permeation column (Pharmacia Biotech) eluting at 1 mL/min
in PBS. Fractions (0.5 mL) were collected, and the column was washed
between runs. The column was calibrated with a purified
[125I]IGF-II/M6-P receptor isolated from rat liver (31)
peaking in fractions 19–20, bovine -globulins (150 kDa) peaking in
fractions 22–23, and BSA (67 kDa) peaking in fraction 24. The recovery
of IGF-II/M6-P receptor was typically 64.4 ± 2.8% (mean ±
SEM; n = 10).
Serum, urine, and amniotic fluid samples
Blood and urine samples and amniotic fluid collected for previous studies were obtained for analysis, with ethics committee approval. All samples were stored at -20 C before use. Serum IGF-II levels (in duplicate) were determined by RIA as previously described (33).
Statistical analysis
Each sample was assayed in duplicate. The data presented represent the mean ± SD for n samples. Statistics were calculated using the StatView 4.02 software package (Abacus Concepts, Inc., Berkeley, CA). Group comparisons were calculated by factorial ANOVA followed by Fisher’s protected least significant difference test. P < 0.05 was considered significant. Linear regression coefficients were calculated using a bivariate regression test.
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Results |
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), with occasional additional bands of
approximately 300 and 80 kDa (not shown). Antiserum raised from
purified receptor reacted with a single 220-kDa species in 100 ng
purified receptor and with a single 200 kDa band in 5 µL adult human
serum (Fig. 1B).
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). Confirmation of the [125I]IGF-II binding
specificity was observed by the abolition of radiolabeled ligand
binding with IGF-II (10 µg). A minor effect was observed at high
doses of insulin (10 µg; >15,000 times physiological concentration),
which may be due to a low degree of IGF contamination in the
preparation (Fig. 1C).
A two-site ELISA was established in which affinity- purified
IGF-II/M6-P receptor polyclonal antibody is used to both capture
and detect the receptor. The ELISA had an analytical range of 0.56–28
ng purified membrane receptor (Fig. 2).
Storage of purified receptor (standard) at 4 C for more than 1 month
resulted in a 90% loss of activity, whereas freeze-dried standard was
stable over at least a 1-yr period. Determined in 31 assays, a
half-maximal response (ED50) was seen at 7.25 ± 1.38
mg/L.
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). Serum sample volumes greater than 10 µL caused an
increase in background signal due to high levels of serum proteins
(results not shown). For three quality control samples covering the
range of the assay, the interassay means and SDs from 10
replicate assays were 13.8 ± 1.23, 5.20 ± 0.80, and
0.64 ± 0.10 ng/well, respectively. The intraassay coefficients of
variation in 10 replicate assays on the same serum samples were 9%,
13%, and 15%, respectively. Rat, mouse, marsupial (wallaroo), ovine,
and bovine serum were assayed for cross-reactivity and were found to
have no detectable activity. The ELISA was tested for interference by
both receptor ligands, and at concentrations of up to 1 µg/mL IGF-II
and 1 µg/mL ß-glucuronidase, the ELISA exhibited no signal
interference (data not shown).
The size of the soluble receptor detected by the ELISA was determined
by two methods. First, 100 µL nonpregnancy and pregnancy sera were
fractionated on a Superose 12 column, and eluted fractions were assayed
by ELISA for IGF-II/M6-P receptor. The detected soluble receptor
displayed an elution profile similar to that of rat membrane
IGF-II/M6-P receptor (Fig. 3A). Further
confirmation of the size and immunoreactivity of the eluted proteins
was obtained by Western immunoblotting the eluted fractions (Fig. 3B).
Two proteins were detected, the soluble IGF-II/M6-P receptor
comigrating with the 200-kDa standard and a smaller receptor fragment
migrating to approximately 120 kDa. Comparison of ELISA and
densitometry of the immunoblot showed a similar distribution of
receptor across the fractions (Fig. 3C), with the bulk of detected
protein being full-sized soluble receptor.
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Samples obtained from healthy infant (age, 0.46 ± 0.70 yr; n
= 44), prepubertal (age, 10.1 ± 0.64 yr; n = 28), and
adolescent (age, 14.7 ± 0.46 yr; n = 27) subjects were
assayed for soluble receptor levels. Infant (1.12 ± 0.28 mg/L)
and prepubertal (1.18 ± 0.6 mg/L) receptor levels were
significantly increased (P < 0.0001) above healthy
adult values, whereas in adolescence, levels (0.73 ± 0.61 mg/L)
were similar to adult levels (Fig. 4A).
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). Preeclampsia (1.67 ± 0.26 mg/L) had no effect on soluble
receptor levels during pregnancy (Fig. 4B). Additionally, heparinized
plasma from these subject groups displayed receptor levels similar to
those in serum (data not shown). A highly significant association was
seen between gestational age and the level of soluble receptor, as
shown in Fig. 5 (r = 0.947;
P < 0.0001).
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).
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Amniotic fluid (n = 13) was assayed for soluble receptor and contained 6.89 ± 6.21 µg/L. Saliva (n = 3) and urinary (n = 4) soluble receptor levels were detectable, but were below the lowest quantifiable level of 5.6 µg/L. Serum IGF-II levels were determined for healthy adult (n = 10), infant (n = 10), diabetic (n = 10), end-stage renal failure (n = 5), pregnancy (15 weeks), and preeclamptic subject groups, and no correlation observed between IGF-II and IGF-II/M6-P receptor serum levels (data not shown).
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Discussion |
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Immunization of a rabbit with purified human membrane IGF-II/M6-P receptor resulted in the production of a highly specific polyclonal antibody, which is immunoreactive to both membrane and soluble forms of the receptor. In this study we have developed a species specific two-site sandwich ELISA using a polyclonal antibody, with an analytical range from 0.56–28 ng purified human membrane receptor. Furthermore, the ELISA has been shown to detect soluble IGF-II/M6-P receptor.
In the rat and bovine systems, the IGF-II/M6-P receptor has been shown to be developmentally regulated (41, 42, 43). Fetal rat serum receptor levels increase during gestation, peak at term, then decrease dramatically in the neonate and remain very low through adulthood (42). It has been postulated that fetal IGF-II/M6-P receptor acts as an IGF-II sink, by binding and internalizing excess IGF-II to prevent fetal overgrowth (19). To date, regulation of the human IGF-II/M6-P receptor has not been studied extensively. Funk et al. (39) studied the gene expression of human IGF-II/M6-P receptor in various fetal tissues and found it to be developmentally regulated, but to a much lesser degree than in the rat. A recent study looking at serum and amniotic fluid soluble receptor by semiquantitative Western immunoblot (44) concluded that soluble IGF-II/M6-P receptor was developmentally regulated in the fetus, and that amniotic fluid contained low levels of receptor.
Our studies examined the developmental regulation of serum soluble IGF-II/M6-P receptor by specific ELISA. We found that infant and prepubertal serum receptor levels were approximately twice healthy adult levels and dropped by 40% during adolescence, confirming previous semiquantitative findings by Western immunoblotting that expression of soluble receptor is down-regulated in human adult serum (44). We found no GH effect on the soluble IGF-II/M6-P receptor as exhibited by the normal receptor levels in acromegalic patient serum. Maternal soluble receptor was gestationally regulated, with a strong positive correlation between gestational age and receptor levels. Additionally, receptor levels were detected in amniotic fluid, but were below quantifiable levels in urine or saliva.
From our findings the concentration of human adult soluble IGF-II/M6-P receptor is approximately 700 µg/L or 3.5 nmol/L, contrasting with a circulating IGF-II concentration of 50–100 nmol/L (33). This huge excess of ligand would rule out soluble receptor as a significant carrier of total IGF-II, most of which is known to be complexed to IGF-binding protein-3 and acid-labile subunit (45). However, soluble receptor may still be important in carrying the fraction of IGF-II not associated with IGF-binding protein-3 and may thus prevent free IGF-II circulating in the blood.
Other possible roles for the receptor may involve its ability to bind ligands such as lysosomal enzymes. To investigate this, we have examined clinical conditions in which plasma lysosomal enzyme levels are known to be elevated. Studies have shown that the IGF-II/M6-P receptor binds lysosomal enzymes (46), including ß-D-glucuronidase (47, 48, 49, 50) and ß-D-galactosidase (6, 36, 51, 52, 53) with high affinity. Lysosomal enzymes such as ß-D-glucuronidase have been shown to be elevated in pregnancy and preeclampsia sera (54, 55, 56). Furthermore, it has been reported that lysosomal enzymes, including ß-D-glucuronidase and ß-D-galactosidase, are elevated in diabetes (IDDM), and this increase is not affected by complications associated with diabetes, such as retinopathy (57, 58). A variety of explanations for the increase in lysosomal enzyme levels have been suggested, including a variation in enzyme activity in the arterial walls (54) and a loss of glycemic control (hyperglycemia) (57). Our study has determined that soluble IGF-II/M6-P receptor is similarly increased in pregnancy (normal and preeclamptic) and diabetes (with or without complications) above healthy nonpregnant adult levels, suggesting that receptor levels may correlate with lysosomal enzyme levels and play a role in transporting them in the circulation. Future studies into disease states with lysosomal enzyme level abnormalities may help to further establish this relationship.
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Acknowledgments |
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Footnotes |
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Received June 8, 1998.
Revised October 27, 1998.
Accepted November 9, 1998.
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References |
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-N-acetylglucosaminyl phosphodiesterase. J Biol
Chem. 258:7345–7351.This article has been cited by other articles:
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  A. G. W. Norden, Sharon. C. Gardner, W. van't Hoff, and R. J. Unwin Lysosomal enzymuria is a feature of hereditary Fanconi syndrome and is related to elevated CI-mannose-6-P-receptor excretion Nephrol. Dial. Transplant., September 1, 2008; 23(9): 2795 - 2803. [Abstract] [Full Text] [PDF]
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 J. D. Veldhuis, J. N. Roemmich, E. J. Richmond, and C. Y. Bowers Somatotropic and Gonadotropic Axes Linkages in Infancy, Childhood, and the Puberty-Adult Transition Endocr. Rev., April 1, 2006; 27(2): 101 - 140. [Abstract] [Full Text] [PDF]
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 C Gicquel, J Weiss, J Amiel, V Gaston, Y Le Bouc, and C D Scott Epigenetic abnormalities of the mannose-6-phosphate/IGF2 receptor gene are uncommon in human overgrowth syndromes J. Med. Genet., January 1, 2004; 41(1): e4 - 4. [Full Text] [PDF]
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 L. Duplomb, B. Chaigne-Delalande, P. Vusio, S. Raher, Y. Jacques, A. Godard, and F. Blanchard Soluble Mannose 6-Phosphate/Insulin-Like Growth Factor II (IGF-II) Receptor Inhibits Interleukin-6-Type Cytokine-Dependent Proliferation by Neutralization of IGF-II Endocrinology, December 1, 2003; 144(12): 5381 - 5389. [Abstract] [Full Text] [PDF]
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 A. B. Hassan Keys to the Hidden Treasures of the Mannose 6-Phosphate/Insulin-Like Growth Factor 2 Receptor Am. J. Pathol., January 1, 2003; 162(1): 3 - 6. [Full Text] [PDF]
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 S. Garrone, G. Radetti, M. Sidoti, M. Bozzola, F. Minuto, and A. Barreca Increased Insulin-Like Growth Factor (IGF)-II and IGF/IGF-Binding Protein Ratio in Prepubertal Constitutionally Tall Children J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5455 - 5460. [Abstract] [Full Text] [PDF]
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D. B. O'Gorman, J. Weiss, A. Hettiaratchi, S. M. Firth, and C. D. Scott Insulin-Like Growth Factor-II/Mannose 6-Phosphate Receptor Overexpression Reduces Growth of Choriocarcinoma Cells in Vitro and in Vivo Endocrinology, November 1, 2002; 143(11): 4287 - 4294. [Abstract] [Full Text] [PDF]
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 J. J. Saris, F. H. M. Derkx, R. J. A. De Bruin, D. H. W. Dekkers, J. M. J. Lamers, P. R. Saxena, M. A. D. H. Schalekamp, and A. H. Jan Danser High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1706 - H1715. [Abstract] [Full Text] [PDF]
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 J. J. Saris, F. H. M. Derkx, J. M. J. Lamers, P. R. Saxena, M. A. D. H. Schalekamp, and A. H. J. Danser Cardiomyocytes Bind and Activate Native Human Prorenin : Role of Soluble Mannose 6-Phosphate Receptors Hypertension, February 1, 2001; 37(2): 710 - 715. [Abstract] [Full Text] [PDF]
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K. Ong, J. Kratzsch, W. Kiess, M. Costello, C. Scott, and D. Dunger Size at Birth and Cord Blood Levels of Insulin, Insulin-Like Growth Factor I (IGF-I), IGF-II, IGF-Binding Protein-1 (IGFBP-1), IGFBP-3, and the Soluble IGF-II/Mannose-6-Phosphate Receptor in Term Human Infants J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 4266 - 4269. [Abstract] [Full Text]
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 H. Yu and T. Rohan Role of the Insulin-Like Growth Factor Family in Cancer Development and Progression J Natl Cancer Inst, September 20, 2000; 92(18): 1472 - 1489. [Abstract] [Full Text] [PDF]
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 D. B. O'Gorman, M. Costello, J. Weiss, S. M. Firth, and C. D. Scott Decreased Insulin-like Growth Factor-II/Mannose 6-Phosphate Receptor Expression Enhances Tumorigenicity in JEG-3 Cells Cancer Res., November 1, 1999; 59(22): 5692 - 5694. [Abstract] [Full Text] [PDF]
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220
kDa), 
