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Endocrine Sciences Group, Department of Medicine, University of Manchester, Manchester, M13 9PT, United Kingdom
Address all correspondence and requests for reprints to: Melissa Westwood, Endocrine Sciences Research Group, Department of Medicine, University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, United Kingdom. E-mail: mwestwoo{at}fs2.scg.man.ac.uk
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Abstract |
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Anion-exchange (IEX) HPLC, nondenaturing electrophoresis, alkaline phosphatase treatment, and ligand-binding studies indicated that the highly phosphorylated IGFBP-1 from HepG2 cells was comparable with IGFBP-1 from plasma. In binding to IGF-I, the plasma phosphoform of IGFBP-1 was found to have a higher affinity (2.3 ± 1.1 x 1010 M-1) than nonphosphorylated IGFBP-1 (2.5 ± 1.7 x 109 M-1, P < 0.002). However, when binding to IGF-II, phosphorylation had no affect on the affinity of IGFBP-1 (3.6 ± 2 x 109 M-1vs. 1.8 ± 3 x 109 M-1, P not significant). Therefore, in the circulation, IGF-I has a considerably higher affinity than IGF-II for IGFBP-1 (P < 0.02). The affinity of phosphorylated IGFBP-1 from plasma (2.3 ± 1.1 x 1010 M-1) also was significantly higher than the affinity of IGFBP-3 for IGF-I (5.6 ± 4.2 x 109 M-1, P < 0.005).
These data suggest that the highly phosphorylated IGFBP-1 in the normal circulation will preferentially bind IGF-I rather than IGF-II, whereas in pregnancy, the affinity of IGFBP-1 for IGF-I will be reduced because of the appearance of non- and lesser-phosphorylated forms. This lends support to the theory that changes in IGFBP-1 phosphorylation may influence the modulatory effects of IGFBP-1 on IGF bioavailability.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown that in the normal adult human circulation, IGFBP-1 is present as a single, highly phosphorylated species; this phosphoform is not found in AF (12). However, circulating IGFBP-1 phosphorylation status can be altered, and this is particularly evident during pregnancy, when non- and lesser-phosphorylated variants increase markedly in the maternal circulation (12). Phosphorylation of IGFBP-1 has been reported to increase affinity for ligand (6); thus, changes in the phosphorylation status of IGFBP-1 may influence the ability of IGF to interact with its cell surface receptors.
In adult human serum, IGFBP-3 is thought to be saturated with IGF-I or -II, forming the 150 kDa complex with the acid-labile glycoprotein (13, 14). In contrast, the low-molecular mass-binding proteins, which form the 40–50 kDa complex, are thought to be unsaturated. Evidence for this comes from cross-linking of iodinated IGF-I to serum, which demonstrates binding to these species but not to IGFBP-3 (15). In addition, gel filtration chromatography of serum from humans injected with radiolabeled IGF-I shows that the labeled IGF-I is initially present in the 40–50 kDa complex (16). This has led some investigators to question the functional importance of IGFBP-1 in terms of regulating IGF bioavailability and actions.
The aim of this study was to determine the significance of changes to IGFBP-1 phosphorylation status by comparing the ligand-binding affinities of the normal circulating phosphoform with those of nonphosphorylated IGFBP-1. Because circulating IGFBP-1 is mainly derived from the liver, we investigated the possibility of using a liver cell line (Hep G2 cells) as a source of the phosphorylated isoform of IGFBP-1 found in plasma. In addition, the affinity of the previously uncharacterized plasma phosphoform of IGFBP-1 was compared with that of IGFBP-3, the main IGF carrier in the circulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture of Hep G2 cells
Hep G2 cells (85011430, passage number 90) obtained from the
European Collection of Animal Cell Cultures (Porton Down, Salisbury,
UK) were cultured in DMEM supplemented with 10% FCS, 1% nonessential
amino acids, 0.04 mM L-glutamine, and 1
mM pyruvate. Biochemical analysis and RIA (see below) of
the growth medium indicated that bovine IGFBP-1 does not cross-react
with the antibodies used in this study. The cells were grown in 5%
carbon dioxide at 37 C until confluent, at which point the medium was
harvested and the cells passaged 1:4 after trypsinization.
Immunoaffinity chromatography
Immunoaffinity chromatography was used to isolate IGFBP-1 from
normal plasma (NP) or Hep G2-CM. Monoclonal antibody (MAb) 6303 (a
generous gift of Medix Biochemica, Kauniainen, Finland), which
recognizes all IGFBP-1 variants (12), was coupled to Sephacryl S-300
(11) at 1 mg/ml to form the immunoaffinity matrix. A 10-ml column was
equilibrated for 24 h at 4 C by the application of PBS/0.25%
BSA/0.1% Tween 20 at a flow rate of 5 ml/h.
Next, 250 ml plasma or 500 ml Hep G2 CM was recirculated through the column for 72 h at a flow rate of 3.75 ml/h, the column was washed with 100 ml Tris buffer, pH 8.0 (50 mM Tris/0.5 M NaCl/0.1% Tween 20), and the bound peptide was eluted by application of 0.1 M hydrochloric acid. Then, 10 x 1-ml fractions were collected into tubes containing 200 µl 1 M Tris pH 9.0 and analyzed for IGFBP-1 by RIA (see below). Fractions containing more than 100 µg/l IGFBP-1 were pooled and concentrated by centrifugation through Centricon 10 filters (Amicon, Stonehouse, Gloucestershire, UK). The particular IGFBP-1 isoforms contained within the concentrate were assessed by n-octyl glucoside (n-OG) electrophoresis and Western ligand blotting with 125I-IGF-I (see below).
HPLC
One hundred microliters of concentrate obtained from the 6303
immunoaffinity column or 1 µg rhIGFBP-1 (kindly donated by Amgen,
Boulder, Co) dissolved in 0.02 M Tris/0.1 M
NaCl/2% isopropanol, (pH 9.5) were applied to a 4.6 x 150 mm
Hema Mono Q column (Alltech, Camforth, UK). Sample was eluted
isocratically for 5 min with 0.02 M Tris/0.1 M
NaCl/20% isopropanol followed by a linear gradient to 0.02
M Tris/0.3 M NaCl/20% isopropanol over 20 min.
The flow rate was 1.5 ml/min, and absorbance was monitored at 220 nm.
Then, 50 x 30s fractions were collected and analyzed for IGFBP-1
by RIA and n-OG electrophoresis/Western ligand blot (see below).
IGFBP-1 RIA
IGFBP-1 levels in the fractions from the immunoaffinity and IEX
columns were determined using our previously reported RIAs, RIA 6303
and RIA 6305 (12). The assays use rhIGFBP-1 (a kind gift of Dr. L.
Fryklund, Pharmacia, Stockholm, Sweden), for standards (1–250 µg/l)
and radiolabel, and either MAb 6303 or 6305 (generously provided by
Medix Biochemica, Kauniainen, Finland). RIA 6303 recognizes all
isoforms of IGFBP-1, including the phosphoform characteristic of NP,
whereas MAb 6305 does not recognize the circulating phosphoform, and
therefore, the 6305 RIA only detects the non- and lesser-phosphorylated
isoforms (12).
Biochemical characterization of IGFBP-1 isoforms
The IGFBP-1 isoforms in the NP, Hep G2 CM, and the fractions
from the immunoaffinity and IEX columns were characterized by
immunoprecipitation, n-OG electrophoresis, and Western ligand blotting,
as previously described (6, 12). Samples were incubated at 4 C
overnight with 250 µl MAb 6303 or 6305 (1:1000 dilution).
Precipitating antibody (250 µl antimouse coated cellulose suspension
(Sac-Cel; IDS, Tyne and Wear, Boldon, UK) was then added and incubated
for 1 h at 37 C. Bound antibody was separated by centrifugation at
1000 x g for 10 min. The precipitated proteins were
washed (x3) by the addition of 1 ml PBS/0.25% BSA/0.1% tween 20 and
centrifuged at 1000 x g for 10 min before resuspending
in 100 µl gel loading buffer [170 mM
Tris.HPO4 pH 5.5/90 mM n-OG (Sigma, Poole,
Dorset, UK)/40% glycerol/0.008% bromophenol blue]. All samples were
boiled for 5 min before loading onto a stacking gel of 4% acrylamide,
which, like the resolving gel (15% acrylamide), contained the nonionic
detergent n-OG at 20 mM. The gels were run at a constant
voltage of 175V for approximately 6 h. After transfer onto
nitrocellulose membranes, the proteins were incubated with 150,000
cpm/ml 125I-IGF-I for 4 h at 25 C, washed, and
visualized by autoradiography (5 days exposure).
Treatment of purified plasma and Hep G2 phosphoform with alkaline
phosphatase
The highly phosphorylated IGFBP-1 isoform, obtained from IEX
HPLC of plasma and Hep G2 CM, was incubated with 1 U calf intestinal
alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN) for 2
h at 37 C. Samples were then subjected to immunoprecipitation and
Western ligand blotting, as described above.
Assay of IGFBP binding to IGFs
Ligand-binding assays were performed with 125I IGF-I
or -II in the presence of unlabeled IGF-I or IGF-II to determine the
relative affinities of rhIGFBP-1, the highly phosphorylated IGFBP-1
isoform, purified from plasma and Hep G2 CM and rhIGFBP-3 (recombinant
IGFBP-3 was used in these studies because Sommer et al. (18)
and Mukku et al. (19) have shown that posttranslational
modifications do not affect its affinity for ligand.). The
concentration yielding 25% specific binding was first determined for
each IGFBP preparation from dilution curves established with
125I IGF-I or -II.
125I-labeled rhIGF-I (18, 500 cpm; specific activity 185
µCi/µg) or 125I-rhIGF-II (25,000 cpm; specific activity
255 µCi/µg) was incubated with 10 µg/liter IGFBP-1or 1 µg/liter
IGFBP-3 in the presence of unlabeled IGF-I or -II (final concentration
0–40 ng/ml). The reactions were performed in triplicate overnight at 4
C in 0.25 ml 0.1 M HEPES/44 mM
NaH2PO4/0.01% Triton X-100/0.25% BSA/0.02%
sodium azide, pH 6.0). Bound label was separated from free by adding
250 µl 1% human 
-globulin and 500 µl 25% polyethylene glycol
(Mr 8000; Sigma) and centrifugation at 1000 x
g for 15 min. The pellet was washed with 1 ml 6.25%
polyethylene glycol and the final pellet counted in a spectrometer.
Nonspecific binding was determined by measuring the amount of
125I-IGF-I/-II that could be precipitated in the presence
of 1.0 µg/l unlabeled IGF.
To confirm the effect of phosphorylation on the affinity of IGFBP-1 for IGF, the dephosphorylated plasma phosphoform was subjected to ligand-binding assays, as described above.
Statistical analysis
The Simfit program (20), kindly provided by Dr. W. G.
Bardsley, University of Manchester, Manchester, UK, was used to analyze
the ligand-binding data, and the Mann-Whitney U test for nonparametric
data was used to compare the mean affinities of the various IGFBP
preparations for their ligands.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Only MAb 6303 recognizes all IGFBP-1 variants. MAb
6305 does not detect the isoform found in the circulation (lane 4), and
this highly phosphorylated form of IGFBP-1 was not present in AF (lane
2). Hep G2 cells do produce the IGFBP-1 phosphoform found in the normal
circulation; however, multiple lesser-phosphorylated isoforms and
nonphosphorylated IGFBP-1 also were present (lane 5).
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. The upper line represents the elution
profile and the lower line, a fitted baseline used for
calculation of peaks. A species (peak a) with identical retention time
to rh (nonphosphorylated) IGFBP-1 (Fig. 2B) was apparent, and in
addition, the IGFBP-1 prepared from Hep G2 CM separated into several
other discrete peaks (peaks b–e) with increased retention times,
suggesting the separation of variantly phosphorylated isoforms.
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. All fractions
corresponding to chromatographic peaks contained IGFBP-1. With
rhIGFBP-1, both assays measured approximately the same level of IGFBP-1
in each of the fractions (Fig. 3A); however, this was not true for the
fractions obtained from IEX-chromatographic analysis of IGFBP-1
purified from HepG2 CM. Unlike RIA 6303, RIA 6305 failed to detect any
IGFBP-1 in the fraction corresponding to the peak e, suggesting that
this fraction contained the circulating IGFBP-1 phosphoform; because
this is the most highly phosphorylated variant of IGFBP-1, this species
would be expected to have the greatest retention time. To support this
data, an aliquot of the IGFBP-1 purified from plasma by immunoaffinity
chromatography also was analyzed by IEX HPLC (Fig. 3C). A single peak
of immunoreactivity, which coincided with peak e in the Hep G2
chromatogram, was observed when the fractions were analyzed with RIA
6303; however, as anticipated, RIA 6305 did not detect any IGFBP-1 in
any of the fractions.
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).
The phosphoform characteristic of NP was found in the fractions,
coinciding with the immunoreactivity peak (peak e) detected by RIA 6303
but not RIA 6305.
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shows that the phosphoform purified from Hep G2
comigrates with the phosphoform purified from plasma and that both the
Hep G2-derived and the plasma-derived IGFBP-1 are recognized by MAb
6303 but not by MAb 6305. To confirm that the Hep G2- and
plasma-derived IGFBP-1 were phosphorylated, both isoforms were
incubated with alkaline phosphatase for 2 h at 37 C. With the Hep
G2-derived IGFBP-1, this resulted in the appearance of an isoform that
comigrated with nonphosphorylated rhIGBP-1 and could be detected by MAb
6305 and MAb 6303. Although a 2-h incubation was insufficient for the
complete dephosphorylation of the plasma-derived IGFBP-1, alkaline
phosphatase did produce an isoform that comigrated with rhIGFBP-1
and was detected by MAb 6305.
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IGF-I. Figure 6 shows the Scatchard analysis of
IGF-I binding to rhIGFBP-1 and the phosphoform isolated from NP (before
and after treatment with alkaline phosphatase). The mean (n = 5)
affinity constant of rhIGFBP-1 was 2.5 ± 1.7 x
109 M-1 (Table 1),
which is in accordance with previous reports (6). However, the affinity
of the highly phosphorylated IGFBP-1 isolated from plasma was
significantly greater at 2.3 ± 1.1 x 1010
M-1 (n = 5; P < 0.002),
as was that of the highly phosphorylated isoform purified from HepG2 CM
(P < 0.007; Table 1). There was no statistical
difference between the affinity of the plasma- and Hep G2-derived
phosphoforms for IGF-I, though the range in affinities determined for
the latter isoform (1.3 x 1010
M-1 to 5.8 x 1011
M-1) resulted in a mean of 1.6 ±
2.8 x 1011 M-1.
Dephosphorylation of the phosphorylated IGFBP-1 from plasma with
alkaline phosphatase, though incomplete, resulted in a significant
decrease in the affinity for IGF-I (2.3 ± 1.1 x
1010 M-1 to 5.4 ± 1.1
x 109 M-1; P <
0.004).
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. The mean (n = 5) affinity constant of
rhIGFBP-1 for IGF-II was 1.8 ± 3.0 x 109
M-1, which was not significantly different
from the affinity constant for IGF-I (Table 1
). The affinity constant
of the highly phosphorylated isoform derived from plasma and Hep G2
medium (3.6 ± 2.0 x 109
M-1 and 5.7 ± 6.3 x
109 M-1, respectively) was no
different from that of rhIGFBP-1. Hence, phosphorylation does not seem
to affect the affinity of circulating IGFBP-1 for IGF-II. Therefore,
the circulating phosphoform has a significantly higher affinity
(P < 0.02) for IGF-I than for IGF-II (Table 1).
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),
though this was not significant. However, there was a significant
difference between the affinity of IGFBP-3 and the circulating isoform
of IGFBP-1 for IGF-I; IGFBP-1 had a 4-fold higher affinity
(P < 0.005) than IGFBP-3, the main IGF carrier in the
circulation, though for IGF-II, the affinity of IGFBP-3 was no
different from that of the highly phosphorylated isoform of
IGFBP-1. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The affinity of the plasma phosphoform for IGF-I was much greater than that reported for the phosphoforms present in AF (6) and was approximately 10-fold greater than nonphosphorylated IGFBP-1 seen in maternal plasma during pregnancy (12); thus, phosphorylation does increase the affinity for IGF-I, as anticipated from other studies (6). Dephosphorylation of IGFBP-1 during pregnancy may result in IGF peptides, particularly IGF-I, being liberated from or more weakly bound to this altered circulating IGFBP-1, leading to increased IGF bioavailability for placental and fetal growth.
The affinity of the highly phosphorylated form of IGFBP-1 for IGF-I was also significantly higher than the affinity of IGFBP-3, the main carrier of IGF in the circulation. It is thought that the 150-kDa complex of IGFBP-3, IGF-I, or -II and the acid-labile glycoprotein (13, 14) is saturated with IGFs and serves as their endocrine storage site. In this form, the half-life of the IGF peptides is increased (16), and their access is limited to extravascular spaces (21) because the 150-kDa complex does not readily cross the capillary barrier. The IGF bound to the IGFBP-1 and -2 in the 40- to 50-kDa complex has a serum half-life of 20–30 min (16) because the complex can leave the vascular compartment (22); thereby its IGF may reach and interact with tissue receptors. We and others (23, 24, 25) have observed marked variations in IGFBP-1 levels throughout the day. Some investigators (26, 27, 28) have suggested that such fluctuations in plasma IGFBP-1 levels, particularly in response to insulin, imply a role for IGFBP-1 in glucose homeostasis. However, the significance of this has been questioned (29) because the concentration of IGFBP-1 in normal adult serum is reported to be approximately 10-fold lower than IGFBP-2 and around 100-fold less than that of IGFBP-3 (29); thus, fluctuations of IGFBP-1 occur on top of a high IGFBP background. The current findings indicate that the NP phosphoform of IGFBP-1 has a slightly higher affinity for IGF-I than IGFBP-3 and, indeed, than any of the other IGFBPs (30); this suggests that circulating IGFBP-1 could be fully saturated with IGFs and supports a role for IGFBP-1 in modifying IGF bioavailability and contributing to glucose counterregulation.
In general, the IGFBPs are thought to inhibit IGF actions (31) by preventing or attenuating IGF interaction with their cell surface receptors, which have a lower affinity for IGF (6.7 x 108 M-1; 32). Enhancement of IGF action has been observed with IGFBP-1, -3, and -5. It is thought that this is facilitated by posttranslational modifications of the IGFBPs leading to altered IGF affinities. Association of IGFBP-3 with the cell surface (33) and binding of IGFBP-5 to the extracellular matrix (34) significantly lowers their affinities for IGFs, as compared with the soluble forms of these IGFBPs, therefore favoring IGF/receptor interactions. Similarly, proteolytic cleavage of IGFBP-3 (35, 36) and IGFBP-5 (37) lowers IGF affinities. IGFBP-2 (38) and -4 (39) also are susceptible, though the exact roles of these proteases in controlling the distribution of the IGFs in serum has not been determined. To date, no such protease has been described for IGFBP-1; we and others (12, 31) have suggested that changes in phosphorylation status may represent the mechanism by which IGFs are released by IGFBP-1. This hypothesis is supported by our finding that the normal circulating form of IGFBP-1 has a 10-fold higher affinity for IGF-I than the nonphosphorylated IGFBP-1 that appears during pregnancy. Whether change in IGFBP-1 phosphorylation status (for example, at the cell surface of IGF target tissues) represents a more generalized mechanism for controlling tissue IGF bioavailability remains to be determined, the analogy being that tissue IGFBP-3 proteolytic activity is reported to be 8-fold higher than in serum (40).
All IGFBPs bind both IGF-I and -II with high specific affinity, and Rechler (30) has suggested that the affinity constants of the six IGFBPs are similar for IGF-I and IGF-II, with the exception of IGFBP-6, which has a 20- to 70-fold higher affinity for IGF-II, and that variations of some affinity constants found in the literature may be caused by different temperatures at which Ka values were determined. In the present study, the binding affinity of the plasma form of IGFBP-1 for IGF-I and IGF-II was determined under the same conditions, and phosphorylation was found to affect only the affinity of IGFBP-1 for IGF-I. Although the explanation for this phenomenon is not clear, it may involve different binding sites for IGF-I and -II on IGFBP-1, as has been suggested recently for IGFBP-2 (41). These findings may also explain why Kratz et al. (42) were able to show enhanced proliferative response to IGF-I, but not IGF-II, in the presence of nonphosphorylated (recombinant) IGFBP-1 in human keratinocytes and fibroblasts. Scatchard plots obtained by Roghani et al. (43) also imply two classes of binding site for IGF-I and IGF-II, one of high and one of low affinity, though this is likely to be because their IGFBP-1 preparation was purified from AF and would have contained several different isoforms of IGFBP-1. Our Scatchard data gives linear plots, which would suggest that, when using a homogenous preparation of IGFBP-1, the binding of IGF-I or -II is at a single site.
In summary, we have purified the highly phosphorylated form of IGFBP-1 found in the normal circulation and have shown that this species has a significantly higher affinity for IGF-I in comparison with the nonphosphorylated isoform that appears under some physiological and pathological conditions. These data suggest that normally, IGFBP-1 in the circulation would be inhibitory of IGF actions; however, changes in IGFBP-1 phosphorylation status may permit increased IGF, particularly IGF-I, bioavailability.
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Footnotes |
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Received May 23, 1996.
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).
), IGFBP-1 purified
from plasma (•), and dephosphorylated plasma IGFBP-1 (
). The
slopes of the first order regression lines obtained from five
experiments were used to determine mean Ka values.