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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: Robert C. Baxter, Ph.D., D.Sc., Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. E-mail: robaxter{at}med.usyd.edu.au
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Introduction |
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Until recently, one IGFBP, IGFBP-3, was believed to be distinct from the others in its ability to form ternary complexes in the circulation by binding IGF-I or IGF-II and then interacting with a third protein, the acid-labile subunit (ALS) (4). ALS is a liver-derived serum glycoprotein of the leucine-rich repeat family (5). However, we have recently shown that, like IGFBP-3, IGFBP-5 is also able to form ternary complexes with IGFs and ALS, with an apparent binding affinity for ALS comparable to that of the IGFBP-3-ALS interaction (6). Both IGFBP-3 and IGFBP-5 have a highly basic motif in their carboxyl-terminal domain, which appears to be the major site of interaction with ALS (7, 8). The same basic residues are involved with IGFBP-3 and -5 interaction with glycosaminoglycans and the cell surface (7, 9) and transport to the cell nucleus (10). In addition, IGFBP-5 may have a secondary site of interaction with ALS (11) and glycosaminoglycans (12) in its central domain.
An RIA for IGFBP-5 has been reported (13). Using this assay, serum IGFBP-5 was detected in two molecular forms, 30–40 kDa (i.e. free or complexed with IGF-I or IGF-II) and 18 kDa (i.e. partially proteolyzed). The absence of IGFBP-5 in ternary complexes with ALS was surprising, given that it can bind ALS similarly to IGFBP-3 and coelutes with ALS when serum is fractionated by gel chromatography (6). To investigate this discrepancy we have now developed a new RIA for IGFBP-5 and have used it to examine the size distribution of IGFBP-5 in serum from healthy nonpregnant and pregnant subjects. We report that ternary-complexed IGFBP-5 exists in serum from healthy male and female subjects and, to a lesser extent, in pregnancy serum.
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Materials and Methods |
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An 868-bp cDNA encoding human IGFBP-5 was isolated by RT-PCR from U2-OS osteosarcoma cells and subcloned into the adenoviral- mediated expression system, AdEasy (14). Medium (2.5 liters) conditioned by human embryonic retinoblast 911 cells (15) infected with the IGFBP-5 recombinant type 5 adenovirus (16) was applied to a column of IGF-I-Affigel-10, washed in 0.1 M sodium phosphate (pH 6.5), and eluted with 0.5 M acetic acid (pH 3.0) essentially as described for recombinant human IGFBP-3 (17). Eluted fractions were further purified by reverse phase HPLC on a 5-µm, 300 Å C18 column (Symmetry 300, Waters Corp., Milford, MA) using a linear gradient of 15–24% acetonitrile in 0.1% trifluoroacetic acid over 10 min, followed by 24–33% acetonitrile in 0.1% trifluoroacetic acid over 20 min. Peak fractions were pooled and confirmed as human IGFBP-5 by sequencing of the first 10 amino-terminal residues, and the concentration was estimated by quantitative amino acid analysis performed on duplicate samples. The amino-terminal sequencing and quantitative amino acid analysis were performed by the Australian Proteome Analysis Facility (Sydney, Australia).
Preparation of antiserum
An IGFBP-5 antiserum was raised by the Veterinary Services Division at the Institute of Medical and Veterinary Science (Adelaide, Australia). A White Leghorn hen, aged 16–20 wk, was injected sc at multiple sites in the breast with 100 µg recombinant human IGFBP-5 in complete Freund’s adjuvant. Three boosts of 100 µg IGFBP-5 in incomplete Freund’s adjuvant were given at 3-wk intervals, and a final boost was given 5 wk later. Between wk 7–14 of the immunization schedule, 22 eggs were collected. A crude extract of IgY, as a component of water-soluble proteins, was extracted from the hyperimmune egg yolks by a proprietary procedure of Antiven Pty. Ltd. (Adelaide, South Australia). The IgY extract was stored as aliquots at -80 C.
IGFBP-5 RIA
Approximately 2–4 x 108 cpm 125I-labeled IGF-I (160–200 µCi/µg) (18) were incubated with 10 µg IGFBP-5 in 50 mM sodium phosphate, 1 g/liter BSA, and 0.2 g/liter sodium azide, pH 7.0, at 22 C for 2 h. The sample was then covalently cross-linked with 0.25 mM (final concentration) disuccinimidyl suberate (Pierce Chemical Co., Rockford, IL) at 22 C for 30 min, and the reaction was terminated by the addition of 50 mM (final concentration) Tris-HCl, pH 7.8. The tracer was separated from unincorporated 125I-labeled IGF-I by chromatography on a Sephadex G-100 column in 0.5 M acetic acid (pH 3) containing 0.1 M NaCl and 2.5 g/liter BSA. The tracer was stored as aliquots of 2 x 106 cpm at -80 C.
Samples or standards (50 µl), IGFBP-5 antibody (100 µl of a 1:100 dilution), 125I-labeled IGF-I cross-linked to IGFBP-5 tracer (100 µl; 
10,000 cpm), and IGF-I (10 ng) were set up in duplicate in a total volume of 500 µl 0.1 M Tris-HCl buffer (pH 7.5) containing 2.5 g/liter BSA, 0.1 ml/liter Triton X-100, and 0.2 g/liter sodium azide. The standard curve covered the range of 0.25–50 ng IGFBP-5. Serum samples were measured at 5 and 10 µl, diluted to 50 µl in assay buffer. After 18–20 h of incubation at 4 C, 2 µl sheep anti-IgY antiserum and 0.5 µl normal chicken serum were added together in 25 µl buffer to each tube. The tubes were further incubated for 1 h at 4 C before adding 1 ml cold polyethylene glycol (60 g/liter in 0.15 M NaCl). After 20–30 min at 4 C, the bound radioactivity was precipitated by centrifugation. Supernatants were decanted, and the radioactivity in the pellets was determined by a 
-counter.
Other assays
Human IGFBP-3 and ALS were measured by specific RIA methods as previously described (19, 20), except that the IGFBP-3 assays used the high titer rabbit antiserum R-100 at a 1:100,000 final dilution.
SDS-PAGE analysis, blotting, and affinity labeling
Proteins for analysis were reconstituted in 20 µl Laemmli sample buffer, heated at 95 C for 5 min, fractionated under nonreducing conditions on a 10% SDS-polyacrylamide gel, and electroblotted onto nitrocellulose. For immunoblot analyses, the nitrocellulose was incubated with 50 g/liter skim milk powder in Tris-buffered saline (TBS) at 37 C for 2 h, followed by incubation with the IGFBP-5 antibody (1:100 dilution) in 50 g/liter skim milk powder in TBS at 4 C for 16 h. The blot was then washed twice for 10 min each time with TBS containing 0.05% Nonidet P-40 and four times with TBS. After incubation with rabbit antichicken IgY-horseradish peroxidate conjugate (1:1000 dilution) in 50 g/liter skim milk powder in TBS at 22 C for 2 h, the blot was washed as described above, then developed in 25 ml TBS containing 150 µl 30% hydrogen peroxide mixed with 5 ml 17 mM 4-chloro-1-naphtol (made up in methanol) for 20 min. The blot was then washed in water.
For ligand blot, the nitrocellulose was incubated with 10 g/liter BSA in TBS at 37 C for 2 h. This was followed by an incubation with 1 x 106 cpm of either [125I]IGF-I or [125I]IGF-II in TBS containing 10 g/liter BSA and 0.05% Nonidet P-40 at 4 C for 16 h. The blot was washed twice with TBS containing 0.05% Nonidet P-40 and four times with TBS (10 min/wash) and then exposed to film. For the affinity labeling experiment, approximately 2 x 105 cpm 125I-labeled IGF-I cross-linked to IGFBP-5 (described above) were incubated with 1 µg ALS, in a total volume of 300 µl 50 mM sodium phosphate (pH6.5) containing 2.5 g/liter BSA, at 22 C for 2.5 h. Complexes were cross-linked with 0.25 mM disuccinimidyl suberate (final concentration) at 22 C for 30 min. The reaction was then terminated by the addition of 15 µl 1 M Tris-HCl (pH 7.8). Part (2 x 104 cpm) of each reaction was reconstituted in Laemmli sample buffer, heated to 95 C for 5 min, and separated under nonreducing conditions on a 6% SDS-polyacrylamide gel. The gel was stained with Coomassie blue, dried, and then exposed to film.
Fractionation of serum samples
Sera from healthy men, nonpregnant women, and women in the first trimester of pregnancy were obtained from staff and patients at Royal North Shore Hospital with the approval of the institutional human ethics committee. Sera were fractionated by running 200-µl aliquots on a Superose-12 column (Amersham Pharmacia Biotech, Piscataway, NJ) at 0.5 ml/min at 22 C in 50 mM sodium phosphate and 100 mM NaCl, pH 7.5. To inactivate ALS, some samples were adjusted to pH 2.5–3.0 with dilute HCl, incubated for 1 h at 22 C, then reneutralized with dilute NaOH before fractionation. Fractions of 0.5 ml were collected, and fractions 20–33 were assayed in duplicate for IGFBP-5 (200 µl) and IGFBP-3 (25 µl of a 1:4 dilution).
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shows samples of 25, 50, and 100 ng IGFBP-5 expressed in the human embryonic retinoblast 911 cell line, which appear as a characteristic doublet of approximately 30 kDa. By ligand blot, the recombinant IGFBP-5 reacted more strongly with radiolabeled IGF-II than IGF-I (Fig. 1B). The cross-linked IGF-I-IGFBP-5 tracer used in the RIA is shown in Fig. 1C. Affinity labeling pure human ALS with this tracer yielded a characteristic doublet ternary complex of approximately 120–130 kDa, as seen previously with a yeast-derived human IGFBP-5 preparation (6). The smaller band is assumed to represent the complex containing partially degraded IGFBP-5.
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shows the cross-reactivity of the IGFBP-5 RIA for other human IGFBPs. Only IGFBP-6 showed any reactivity, estimated to be approximately 0.3%. Sera from a variety of primate and nonprimate species showed cross-reactivity, illustrated in Fig. 2B for the primates lemur, chimpanzee, and human, and the nonprimates horse, rat, goat, and cat.
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). The mean for 15 males, aged 16–18 yr (180 ± 33 ng/ml), was slightly lower than that for 26 males, aged 23–53 yr (224 ± 85 ng/ml), whereas for nonpregnant females there was no difference between mean values for 16 subjects, aged 16–18 yr, and 21 subjects, aged 22–49 yr (P = 0.853). Figure 3B shows the relationship between age and serum IGFBP-5 for all 89 subjects.
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, in both men and women approximately 55% of immunoreactive IGFBP-5 was found in fractions corresponding to approximately 130 kDa, with 45% corresponding to free or binary-complexed IGFBP-5. In contrast, as previously described (21), almost 90% of immunoreactive IGFBP-3 was in high molecular mass forms of approximately 150 kDa, peaking one fraction earlier than IGFBP-5. We have previously shown that the size distribution of IGFBP-3 in pregnancy serum is identical to that in nonpregnancy despite the fact that pregnancy IGFBP-3 appears heavily proteolyzed when analyzed electrophoretically (22). IGFBP-5, however, showed a markedly altered serum profile in pregnancy, with a shift in the size distribution toward that expected for free and binary-complexed forms. As shown for five fractionated pregnancy sera, a mean of only approximately 35% of immunoreactive IGFBP-5 was found in high molecular mass peaks (Fig. 5A). When sera were transiently acidified and reneutralized before size fractionation, essentially all of the IGFBP-5 appeared at a size corresponding to free or binary-complexed protein, eluting slightly later than IGFBP-3 in the same acidified sera (Fig. 5B).
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). There was no significant association between IGFBP-3 and IGFBP-5 levels.
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Discussion |
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IGFBP-5 is also capable of forming ternary complexes with IGFs and ALS (6). As in the case of IGFBP-3, basic residues in the carboxyl-terminal domain of IGFBP-5 appear responsible for the interaction with ALS (8, 16). Given that IGFBP-5-containing ternary complexes appear to form with a similar affinity as complexes containing IGFBP-3 (6), it might be expected that, like IGFBP-3, the majority of IGFBP-5 would circulate complexed to ALS. However, the only study to date that has used a quantitative immunoassay for IGFBP-5 to examine this question concluded that the majority of IGFBP-5 in human serum was between 25–43 kDa (i.e. free or complexed to IGFs), with a small proportion in forms of lower molecular mass, presumably due to proteolysis (13). In an attempt to reconcile this observation with our in vitro data on ALS binding by IGFBP-5, we therefore developed reagents to examine the size distribution of circulating immunoreactive IGFBP-5 by expressing recombinant human IGFBP-5 in a human cell line and establishing a specific immunoassay.
After several failed attempts to raise human IGFBP-5 antisera in rabbits, an antibody was raised in chickens and harvested from egg yolks. Unlike all of the human IGFBP antisera previously raised in this laboratory, and the majority reported in the literature, this IgY antibody showed a broad specificity among both primate and nonprimate species, making it a valuable resource for studies in both humans and experimental animals. Undetectable cross-reactivity in the RIA with all other IGFBPs apart from IGFBP-6 (<1% cross-reactivity) allows the assay to be used with confidence under all experimental conditions.
We found no difference in serum IGFBP-5 levels between healthy men and women, and no age effect between 16 and 50 yr, but the mean levels of approximately 200 ng/ml are only half the levels previously reported by Mohan et al. (13). A variety of differences between the two assays might contribute to this discrepancy: the source or calibration of standard IGFBP-5, the different IGFBP-5 tracers [iodo-IGF-I cross-linked to IGFBP-5 in this study vs. iodo-IGFBP-5 in the study by Mohan et al. (13)], or the different source of antibodies (egg-yolk vs. guinea pig serum), which might distinguish minor differences, such as glycosylation or phosphorylation, between the form of IGFBP-5 used as an immunogen and standard, and the form in the circulation. More striking is the difference in IGFBP-5 size distribution found in our study compared with the previous report, with about 55% of total immunoreactive IGFBP-5 corresponding to ternary complexes in both men and nonpregnant women. This contrasts with about 90% of IGFBP-3 in ternary complexes. The high molecular mass IGFBP-5 complexes were, like those containing IGFBP-3, acid-labile, with all immunoreactive IGFBP-5 detectable at a size corresponding to free or binary complexed protein, when analyzed after acidification and reneutralization.
Heterotrimers containing IGF-IGFBP-ALS are remarkably stable in the circulation, and IGF-I bound in this form has been reported to have a circulating half-life of 12–15 h (26), whereas IGFs bound in binary complexes exit the circulation within minutes (26, 27). The availability of IGFs to the tissues will therefore be influenced by the degree to which they are distributed between ALS-containing complexes and binary complexes. This is also true for the IGFBPs, which are greatly stabilized in the circulation complexed to ALS (27). Thus, mice with a deleted ALS gene have barely detectable serum IGFBP-3 levels despite normal IGFBP-3 mRNA levels due to their inability to stabilize the protein in high molecular mass complexes (28). Similarly, GH administration to humans over 5 d increases serum IGFBP-3 levels without any change in hepatic IGFBP-3 mRNA, presumably the result of increased IGF-I and ALS stabilizing IGFBP-3 in the circulation (29).
Accordingly, we confirmed a high correlation between serum IGFBP-3 and ALS levels and found a lower, although still highly significant, correlation between IGFBP-5 and ALS, reflecting the lower percentage of IGFBP-5 stabilized in ALS complexes. A significant association between serum IGFBP-5 and ALS has been reported previously in children with chronic renal failure (30) and was taken as evidence that IGFBP-5 and ALS interact in the circulation, although this was not demonstrated. It is unclear why the extent of complexing with ALS, and hence the correlation with serum ALS levels, is weaker for circulating IGFBP-5 than for IGFBP-3. The recombinant proteins bind ALS similarly when tested in vitro (16), but it is possible that the proteins in the circulation are posttranslationally modified in ways that are undetectable by the methods used in this study, such that their ALS-binding activity in vivo is altered.
IGFBP-3 circulates in adult humans at about 3000 ng/ml (70 nM), of which about 10% (7 nM) is free or in binary complexes. We have now shown that IGFBP-5 circulates at about 200 ng/ml (7 nM), of which about 45% (3 nM) is free or in binary complexes. Therefore, despite the 10-fold lower molar concentration of total IGFBP-5 compared with IGFBP-3, the fraction that is available to transport IGFs to the tissues has a concentration almost half that of the equivalent fraction of IGFBP-3. IGFBP-5 must therefore be considered to potentially play an important role in regulating the tissue availability of IGFs.
Pregnancy was associated with a 50% reduction in total serum IGFBP-5 levels and a substantial shift in size distribution from about 55% in ternary complexes to about 35% in this form. In contrast, total serum immunoreactive IGFBP-3 showed a slight increase, and the size distribution was unchanged, in pregnancy (22). Pregnancy serum is known to contain increased concentrations of proteases that act on IGFBPs (31), and IGFBP-3 in pregnancy appears fully proteolyzed when analyzed by immunoblotting after SDS-PAGE (32). A pregnancy-dependent protease with activity against IGFBP-5 has also been identified (31).
As IGFBP-5 ternary complexes are decreased in pregnancy, it may be speculated that proteolyzed IGFBP-5 associates with ALS less readily than the intact protein, even though this has been shown not to be the case for IGFBP-3 (33). If the carboxyl-terminal domain of IGFBP-5 is lost as a result of partial proteolysis, a weak interaction with ALS might remain, as a central domain binding site for ALS has been demonstrated (11). However, the relatively low affinity for ALS might cause a greater proportion of IGFBP-5 to remain in binary, rather than ternary, complexes. In this way, even though total IGFBP-5 is reduced in pregnancy, the increased proportion in binary complexes might ensure that IGF bioavailability from IGFBP-5 complexes is not decreased.
In conclusion, we have established a specific immunoassay for IGFBP-5 and have shown that a majority of serum IGFBP-5 in healthy adults occurs in ternary complexes with ALS. Pregnancy decreases total IGFBP-5, but increases the proportion that is free or in binary complexes. IGFBP-5 has recently been shown to have growth-promoting activity in vivo (34), so that, whether alone or as a carrier of IGFs, IGFBP-5 that is not complexed with ALS might have preferential access to the tissues, where it could act to stimulate growth. It will be of great interest to determine whether IGFBP-5 and IGFs circulating in IGFBP-5 complexes reach a multitude of tissue sites or are selectively targeted to particular tissues.
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Acknowledgments |
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Footnotes |
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Abbreviations: ALS, Acid-labile subunit; IGFBP, IGF binding protein; TBS, Tris-buffered saline.
Received July 12, 2001.
Accepted October 1, 2001.
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) subunit of the high molecular weight insulin-like growth factor-binding protein complex in normal subjects. J Clin Endocrinol Metab 70:1347–1353This article has been cited by other articles:
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) and IGFBP-5 (•) in representative serum sample after transient acidification to approximately pH 3 and reneutralization. Serum was fractionated on a column of Superose-12, and the concentration of immunoreactive IGFBP-3 or IGFBP-5 was determined in each 0.5-ml fraction.