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J Biol Chem, Vol. 273, Issue 44, 28791-28798, October 30, 1998
,
From the Kolling Institute of Medical Research,
University of Sydney, Royal North Shore Hospital, St. Leonards,
New South Wales 2065, Australia, the § Chiron Corporation,
Emeryville, California 94608, and the ¶ Division of Endocrinology
and Metabolism, Internal Medicine, University Hospital, 8091 Zurich,
Switzerland
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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We have recently shown that insulin-like growth
factor (IGF)-binding protein 5 forms ternary complexes with IGF-I or
IGF-II and the acid-labile subunit (ALS) (Twigg, S. M., and
Baxter, R. C. (1998) J. Biol. Chem. 273, 6074-6079). Because IGF-binding protein 3 (IGFBP-3) binds to ALS
through its basic carboxyl-terminal domain, we tested whether a
homologous region present in IGFBP-5 is involved in IGFBP-5 binding to
ALS. Chimeric peptides were generated by carboxyl-terminal domain
interchange between recombinant human IGF-BP-5 and IGFBP-6,
producing two IGFBP peptides designated 5-5-6 and 6-6-5. Determined by
immunoprecipitation and by Superose chromatography, 6-6-5 formed
ternary complexes, albeit less potently than IGF-BP-5. In contrast,
5-5-6, like IGFBP-6, did not form ternary complexes by these methods.
Whereas 6-6-5, like IGFBP-6, had a marked preference for binary complex
formation with IGF-II rather than IGF-I, it formed ternary complexes
more efficiently with IGF-I, like IGF-BP-5. The glycosaminoglycans
heparin and heparan sulfate bind to IGFBP-5 through its basic
carboxyl-terminal domain. At high concentrations, these
glycosaminoglycans inhibited ALS binding to binary complexed
IGF-BP-5. In addition, in the absence of IGFs, IGFBP-5, a synthetic
peptide representing the basic carboxyl-terminal sequence
IGFBP-5(201-218), and the corresponding IGFBP-3 basic sequence
IGFBP-3(215-232), competed weakly for ALS binding to covalent
IGF-IGFBP-5 complex, as did a random-sequence synthetic peptide with
the same composition as IGFBP-5(201-218). These findings are
consistent with the basic carboxyl-terminal domain on IGFBP-5 being the
principal site in IGFBP-5 that binds to ALS.
The insulin-like growth factors
IGF-I1 and IGF-II are
anabolic peptides, which regulate cell and tissue growth and glycemic control. They bind to six well characterized high affinity IGF-binding proteins (IGFBP-1 to IGFBP-6) (1-3). The IGFBPs can be considered to
have three domains on the basis of amino acid sequence comparisons (4,
5). They share common structural features with relatively conserved
amino- and carboxyl-terminal domains, including 10-12 conserved
cysteine residues at the amino terminus and six conserved cysteine
residues at the carboxyl terminus (6). The midregion domain of each
IGFBP is more variable in length and amino acid composition.
We recently reported that IGFBP-5, like IGFBP-3, forms ternary
complexes with IGF-I or IGF-II and ALS (7). IGFBP-1, -2, -4, and -6 do
not form ternary complexes (7). IGFBP-5 and IGFBP-3 share a highly
homologous 18-amino acid region in their carboxyl-terminal domain,
IGFBP-5(201-218) and IGFBP-3(215-232) respectively, of which 10 amino
acids are basic and none are acidic (6). This region is known to be of
major importance for IGFBP-3 binding to ALS (8), as well as
cell-surface binding and nuclear localization (9). IGFBP-3 mutagenesis
studies involving substitution of residues 228-232 (KGRKR) with the
corresponding residues of IGFBP-1 (MDGEA), have shown an order of
magnitude loss of affinity for ALS in the presence of IGF-I or IGF-II
(8). The highly basic carboxyl-terminal region in IGFBP-5 is a known
site of interaction with glycosaminoglycans (GAGs) (10, 11), which bind
to IGFBP-5 via the only functional IGFBP-5 heparin binding motif, at
amino acids 206-211 (KRKQCK) (10).
We tested whether the basic carboxyl-terminal domain in IGFBP-5 binds
to ALS using two complementary approaches. We first determined whether
this domain is involved in ALS binding with the use of IGFBP chimeras
in which the carboxyl-terminal domains of IGFBP-5 and IGFBP-6 are
interchanged. Second, to further localize the IGFBP-5 region that binds
to ALS, we have used reagents that are known to bind to or potentially
compete with the basic carboxyl-terminal domain of IGFBP-5 as
inhibitors of IGFBP-5 binding to ALS. These studies demonstrate that
IGFBP-5 binds to ALS through its basic carboxyl-terminal domain.
Reagents--
Recombinant human (rh) IGFBP-5 and rhIGFBP-6 were
derived from yeast expression systems as described previously (12).
Natural human IGFBP-3 (13) and ALS (14) were purified as described. Human IGF-I and IGF-II were generous gifts from Genentech (South San
Francisco, CA) and Kabi Peptide Hormones (Stockholm, Sweden), respectively. The rabbit anti-human ALS antiserum AL2/2 was raised by
immunization with serum-derived ALS (7). Disuccinimidyl suberate was
purchased from Pierce. The glycosaminoglycans heparin, heparan sulfate,
dermatan sulfate, chondroitin sulfate A, and bovine serum albumin were
purchased from Sigma. The following radiolabeled iodinated tracers were
prepared as described previously: ALS (14), IGF-I (7), IGF-II (7), and
IGF-I-IGFBP-5 and IGF-I-IGFBP-3 cross-linked tracers (7).
Synthesis of IGFBP-5 and IGFBP-6 Carboxyl-terminal Domain
Chimeras--
The chimeras were constructed based on the designated
domains of the IGFBPs as described previously (5). The structural domains of IGFBP-5 and IGFBP-6 used as a basis for production of the
chimeras, are shown in Fig.
1A. For generation of the
chimeras, pBS24Ub-IGFBP-5 and pBS24Ub-IGFBP-6 (12) were digested with BamHI and SalI. The pBS24 plasmid was
gel-purified and saved for the final ligation step, as described below.
The ~2-kilobase DNA fragments encoding ubiquitin fused to IGFBP-5 or
IGFBP-6 were also gel-purified and ligated to
BamHI/SalI-digested pUC18 to generate
pUC18Ub-IGFBP-5 and pUC18Ub-IGFBP-6, respectively. These plasmids
were subsequently digested with ApaI and SalI,
which released ~250-base pair DNA fragments encoding the third domain of each IGFBP. Both domain 3-encoding DNA fragments and the pUC18 plasmids were gel-purified and reciprocally ligated to generate plasmids encoding ubiquitin fused to 1) the first two domains of
IGFBP-5 and the third domain of IGFBP-6, termed
pUC18Ub-IGFBP-5-5-6, and 2) the first two domains of IGFBP-5 and the
third domain of IGFBP-5, termed pUC18Ub-IGFBP-6-6-5. These resulting
plasmids were then digested with BamHI and SalI.
The ~2-kilobase DNA fragments encoding ubiquitin fused to the IGFBP
chimeras were gel-purified and ligated to
BamHI/SalI-digested pBS24 (purified in the first step of the construction) to yield pBS24Ub-IGFBP-5-5-6 and
pBS24Ub-IGFBP-6-6-5. These plasmids were used for yeast transformation,
and the chimeric IGFBPs were expressed as fusion proteins with
ubiquitin. The ubiquitin was removed by proteolysis, and the IGFBPs
were purified as described (12).
Synthetic Peptide Generation--
Synthetic 18-amino acid
peptides were generated corresponding to the carboxyl-terminal basic
regions at IGFBP-5(201-218) (RKGFYKRKQCKPSRGRKR) and IGFBP-3(215-232)
(KKGFYKKKQCRPSKGRKR) and the corresponding amino acids of
IGFBP-6(168-185) (HRGFYRKRQCRSSQGQRR). These peptides are designated
Peptide 5, Peptide 3, and Peptide 6, respectively. A control, nonsense
peptide, termed Peptide ns5, was produced using the same amino acid
content as Peptide 5, but as a randomly generated amino acid sequence
(KCRSKFRRPKYKGQRKGR). Peptides were synthesized by Chiron Technologies
Pty. Ltd. (Clayton, Victoria, Australia) by the solid phase Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry method, cleaved
into a trifluroroacetic acid/scavenger mixture, dried down, and
purified by reverse-phase high pressure liquid chromatography, using a
gradient of increasing acetonitrile for elution. After lyophilization,
the identity of the purified peptide was confirmed by ion spray mass
spectrometry and the purity by analytical reverse-phase high pressure
liquid chromatography.
IGF Binding Assay--
This was performed essentially as
described previously (15). In brief, IGFBP samples were incubated with
IGF-I or IGF-II radiolabeled tracers at ~50 pg/tube for 2 h at
21 °C in binding buffer (50 mM sodium phosphate buffer,
pH 6.5, with 0.25 g/liter bovine serum albumin) in a final volume of
300 µl. Bound radioactivity was separated from free by the addition
of 1 ml of cold charcoal suspension (10 g/liter) in binding buffer plus
0.2 g/liter protamine sulfate, incubating for 4 min at 4 °C, and
then centrifuging for 10 min at 2000 rpm. For competitive binding
studies, either rhIGFBP-5 (1 ng/tube for IGF-I tracer and IGF-II
tracer), rhIGFBP-6 (10 ng/tube for IGF-I tracer and 1 ng/tube for
IGF-II tracer), 5-5-6 (1 ng/tube for IGF-I and IGF-II tracer), or 6-6-5 (25 ng/tube for IGF-I and IGF-II tracer) was incubated with IGF tracer
in the presence of unlabeled IGF-I or IGF-II in the range 0.01-100 ng
per 300-µl reaction volume, and unbound tracer was removed by
adsorption to charcoal as described above.
Immunoprecipitation Complex Formation Assay--
This was
performed essentially as described previously (7). Increasing amounts
of rhIGFBP-5 (0.05-10 ng) were added to IGF-I or IGF-II tracer (each
~50 pg/tube) and incubated at 22 °C for 2 h with or without
25 ng of ALS per tube, in a total of 300 µl of buffer as described
previously (7). Affinity-purified rabbit anti-human ALS antiserum AL2/2
was added to precipitate the complex. Precipitating goat anti-rabbit
immunoglobulin and then polyethylene glycol were sequentially added,
tubes were centrifuged, and the precipitated complex was counted in a
gamma counter (7). For competition studies involving IGF-I tracer,
rhIGFBP-5 or 6-6-5 (0.1-10 ng/tube) was added to IGF-I tracer (~50
pg/tube) and ALS at 25 ng/tube. Heparin, 25 µg/ml, was added in some
tubes. The mixture was incubated in a total of 300 µl of the
phosphate buffer as used in the ternary complex formation assay. The
precipitation and counting protocol used was exactly the same as for
the immunoprecipitation assay described above. For competition assays
involving cross-linked tracer, IGF-I tracer cross-linked to IGFBP-5 (7)
was added at a constant amount of 10,000 cpm/tube to 20 ng of ALS. When an IGFBP was used in competition, increasing amounts were added over
the range of 0.05-250 ng/tube (to 1000 ng/tube for IGFBP-3), with or
without IGF-I or IGF-II (100 ng/tube). In equivalent formats, either
heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate A, or
one of the synthetic peptides (Peptide 3, Peptide 5, Peptide 6, or
Peptide ns5) was added to the reagents described above at the
concentrations shown in the respective figures. The mixture was
incubated in a total of 300 µl of the phosphate buffer as used in the
ternary complex formation assay. The conditions used for precipitation
of the ternary complex and subsequent counting were exactly the same as
for the immunoprecipitation assay described above.
Size Fractionation Experiments on Superose-12--
The method
used is described in the original description of rhIGFBP-5 ternary
complex formation (7). The Superose-12 column was calibrated with IGF-I
tracer (7.6 kDa, peaking in fraction 34); IGF-I tracer cross-linked to
IGFBP-3 (approximately 50 kDa), which mainly eluted in fractions
26-27, peaking in fraction 27; and IGFBP-3 in ternary complex with
IGF-I tracer and ALS, which eluted mainly in fractions 23-25, peaking
in fraction 24.
Purification of the IGFBP Chimeras
By nonreducing SDS-polyacrylamide gel electrophoresis, the IGFBPs
were seen as silver-stained bands at ~32 kDa for rhIGFBP-5 and ~27
kDa for rhIGFBP-6, both as previously reported (12), and at ~31 kDa
for the 5-5-6 chimera and ~28 kDa for the 6-6-5 chimera, averaged
from duplicate gels (Fig. 2). In all
lanes, lightly staining bands were also present at higher molecular
mass, consistent with dimerization. Dimerization has previously been described for rhIGFBP-5 and rhIGFBP-6 (12) and for other IGFBPs (16, 17) using these methods.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Synthesis of the IGFBP-5 and IGFBP-6 chimeras
and the carboxyl-terminal synthetic peptides. A, human
IGFBP-5 and IGFBP-6 (6) are each shown schematically with their three
previously designated domains (5). The boundaries between the middle
and carboxyl-terminal domains in IGFBP-5 (at amino acids 169-170) and
IGFBP-6 (at amino acids 133-134) are indicated by the vertical
arrows. The cDNAs coding for the carboxyl-terminal domains of
IGFBP-5 and IGFBP-6 were interchanged and fused with amino-terminal and
middle domain cDNAs to generate the 6-6-5 chimera and the 5-5-6 chimera, as described under "Experimental Procedures."
B, the highly basic carboxyl-terminal regions in
IGFBP-5(201-218) and IGFBP-3(215-232) and the corresponding region in
IGFBP-6(161-178) are shown. Basic amino acids are in
boldface. The 18-amino acid synthetic peptides, Peptide 5, Peptide 3, and Peptide 6, were generated using the amino acid sequences
of these regions from IGFBP-5, IGFBP-3, and IGFBP-6, respectively. The
amino acids at 228-232 in IGFBP-3, which were mutated in a previous
study (8) and which resulted in 90% loss of IGFBP-3 affinity for ALS
binding, are shown in italics. The classical heparin binding
domain sequences of IGFBP-3, -5, and -6 are underlined in
the middle of these carboxyl-terminal regions.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (89K):
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Fig. 2.
Silver stain of the purified IGFBPs. For
each IGFBP, 200 ng was run on 12.5% SDS-polyacrylamide gels under
nonreducing conditions. Lane 1, rhIGFBP-6; lane
2, the 6-6-5 chimera; lane 3, rhIGFBP-5; lane
4, the 5-5-6 chimera. Molecular mass markers are shown to the
left of the figure. The estimated molecular masses of the
IGFBPs are as follows: rhIGFBP-6, 27 kDa; 6-6-5, 28 kDa; rhIGFBP-5, 32 kDa; and 5-5-6, 31 kDa.
Binary Complex Formation
Because IGF binding is necessary for high affinity ALS binding to IGFBP-3 (14), we first compared IGF-I and IGF-II tracer binding to that by rhIGFBP-5, rhIGFBP-6, and the chimeric proteins (Fig. 3, A and B). rhIGFBP-5 and rhIGFBP-6 bound IGF-I and IGF-II with relative potencies consistent with previous studies (12, 18). The 5-5-6 chimera bound IGF-I and IGF-II with high potency, with some preference for IGF-II binding. The 6-6-5 chimera bound both IGFs less well than native rhIGFBP-5 and rhIGFBP-6. A marked preference for IGF-II binding over IGF-I binary complex formation was seen for the 6-6-5 chimera, more so even than was seen for rhIGFBP-6. Competitive binding curves (Fig. 3, C and D) with IGF tracer and IGFBP were generated in the presence of increasing IGF-I (for IGF-I tracer) or IGF-II (for IGF-II tracer), and the 50% effective concentration for tracer displacement (EC50) for each IGFBP was determined as shown in Table I. The EC50 values for all the IGFBPs tested were lower for IGF-II binding than for IGF-I, with the greatest difference seen for the 6-6-5 chimera, for which a value could not be determined for IGF-I.
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Ternary Complex Formation
Immunoprecipitation Assay-- In this assay, IGF-I or IGF-II radiolabeled tracers were added in the presence of ALS and increasing concentrations of IGFBP. Ternary complexes formed were precipitated by the addition of ALS antiserum, and then radioactivity in the precipitated complex was counted. By this method, only rhIGFBP-5 and the 6-6-5 chimera formed ternary complexes using either IGF tracer (Fig. 4, A and B). No ternary complex formation was seen for the 5-5-6 chimera or for rhIGFBP-6 at the highest concentration tested (10 ng/0.3 ml). The 6-6-5 chimera was significantly less potent than rhIGFBP-5 using either tracer.
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To compare the ability of the IGFBPs to compete for ALS binding in the absence and presence of excess unlabeled IGFs, a tracer of iodinated IGF-I covalently cross-linked to IGFBP-5 was added to a fixed amount of ALS and increasing IGFBP concentrations from 1 to 250 ng, with excess IGF-I or IGF-II (100 ng) in some tubes (Fig. 4, C-E). These results show that the 6-6-5 chimera competed for ternary complex formation more effectively in the presence of IGF-I than IGF-II (Table II). This is in contrast to the binary complex studies, in which 6-6-5 showed a clear preference for binding to IGF-II rather than IGF-I. The 6-6-5 chimera in this competitive assay had 40% of the potency of rhIGFBP-5. This relatively high activity is likely to be due to the presence of excess IGFs in this format, in contrast to the positive complex formation assay (Fig. 4, A and B), in which the tracer IGF concentration is limiting.
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In the absence of added IGFs (Fig. 4E), though at much higher concentrations than in the presence of IGFs, rhIGFBP-5 and the 6-6-5 chimera were able to compete with the cross-linked tracer for binding to ALS. In the absence of IGFs, the 6-6-5 chimera was more potent than rhIGFBP-5, in contrast to the greater activity of IGFBP-5 when IGFs were present. The concentrations of these IGFBPs required for 50% inhibition of cross-linked tracer binding to ALS are shown in Table II. Using this format neither rhIGFBP-6 nor the 5-5-6 chimera had any effect on IGFBP-5 ternary complex formation in the presence or absence of IGFs.
Size Fractionation Chromatography-- A second method of assessing ternary complex formation demonstrated equivalent results to those seen by the immunoprecipitation method. By fast protein liquid chromatography on a column of Superose-12 (Amersham Pharmacia Biotech), of the two chimeras, only 6-6-5 could form ternary complexes (Fig. 5, A and C), as was also seen for IGFBP-5 (Fig. 5, B and D). In the presence of ALS, the 5-5-6 chimera formed binary complexes only with each of the IGF tracers (Fig. 5, A and C). A similar result was seen (Fig. 5, B and D) for IGFBP-6, as was reported previously (5). The broad-peaked distribution of the IGF tracer across fractions, which is seen particularly in the presence of the 6-6-5 chimera, may indicate some dissociation of previously formed complexes during fast protein liquid chromatography and a dynamic equilibrium between ternary complexes, binary complexes, and free IGF tracer. Such dissociation is well recognized in complex formation studies using fast protein liquid chromatography (19).
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Inhibition of IGFBP-5 Binding to ALS by Glycosaminoglycans
Because GAGs bind IGFBP-5 in its basic carboxyl-terminal domain (10, 11), they were used in the ALS immunoprecipitation format to determine whether they affected IGFBP-5 binding to ALS. Binding of IGFBP-5 to ALS in the presence of excess IGF-I was markedly inhibited by the addition of heparin sulfate (25 µg/ml). The effect on 6-6-5 binding to ALS was even greater with binding entirely abolished by 25 µg/ml heparin sulfate (Fig. 6A). Similar results were seen in the presence of IGF-II (not shown). Because GAGs are reported to inhibit IGFBP-5 binding to IGFs (10, 20), the alternative assay format using IGF-I-IGFBP-5 cross-linked tracer and ALS was used to determine whether IGFBP-5 binary complex binding to ALS was inhibited by these substances. Even under conditions where IGF-I could not be displaced from IGFBP-5, increasing concentrations of heparan sulfate and heparin inhibited IGFBP-5 ternary complex formation (Fig. 6B), whereas under the study conditions, dermatan sulfate and chondroitin sulfate A did not (not shown). In comparison with IGFBP-3 ternary complex formation using an equivalent assay format, these GAGs inhibited IGFBP-5 ternary complex more potently. Because GAGs are known to bind to the basic carboxyl-terminal region of IGFBP-5, these results are consistent with the involvement of this region in ALS binding.
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Competition for IGFBP-5 Binding to ALS by Carboxyl-terminal Basic Region Synthetic Peptides
Increasing amounts of a synthetic fragment representing the basic sequence IGFBP-5(201-218) (designated Peptide 5) were added in a competitive immunoprecipitation assay using IGF-I-IGFBP-5 cross-linked tracer and ALS (Fig. 7A). This peptide inhibited IGFBP-5 ternary complex formation in a dose-dependent manner. A synthetic basic region IGFBP-3 peptide (215-232), designated Peptide 3, inhibited IGFBP-5 ternary complex with potency equivalent to that of Peptide 5. A nonsense peptide (designated Peptide ns5), with the same amino acid content of Peptide 5 but with a random amino acid sequence, inhibited cross-linked tracer binding to ALS to the same extent as Peptide 5 and Peptide 3. The synthetic peptide generated from the equivalent IGFBP-6 carboxyl-terminal region IGFBP-6(168-185) (designated Peptide 6) also inhibited complex formation, albeit less potently than the other synthetic peptides. Similar results were seen in the presence of 100 ng/tube IGF-I (not shown). Taken together, these synthetic peptide results suggest that charge is important for ALS binding, at least for these short synthetic peptides. The results are not specific to the amino acid sequence in the carboxyl-terminal IGFBP-5 basic region, as Peptide ns5 showed inhibition equivalent to that seen for Peptide 5.
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In contrast to the equipotent effect of synthetic Peptides 3 and 5 in
inhibiting cross-linked tracer binding to ALS, differences were found
for the native IGFBPs. Whereas full- length rhIGFBP-5, at high
concentrations, did compete for cross-linked tracer binding to ALS in
the absence of IGFs (Fig. 7B and Table II), full-length IGFBP-3 (to 1 µg/tube) showed no competition in the absence of IGFs,
confirming our previous observations (14). This result suggests that
IGFBP-5 binds weakly to ALS in the absence of IGFs.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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IGF-I and IGF-II are carried in serum predominantly in a growth hormone-dependent ternary complex (21, 22), with the ALS (14, 23) and IGFBP-3. We have recently shown that similar complexes can form with IGFBP-5 instead of IGFBP-3 (7), although the role of these complexes in serum IGF transport has not been demonstrated. The ternary complexes appear to function as a circulating reservoir of IGFs and limit the rate of efflux of IGFs to tissues, predominantly due to the stabilizing effect of ALS (24, 25). With the recent recognition of the ability of IGFBP-5 to form ternary complexes (7), and its presence in complexes with ALS in human serum (7), the region in IGFBP-5 that binds to ALS assumes biological importance.
IGFBP chimeras in which the carboxyl-terminal domains of IGFBP-5 and IGFBP-6 are interchanged were used in this study to localize the ALS binding domain in IGFBP-5. Whereas the native rhIGFBP-6 did not form ternary complexes, the 6-6-5 chimera was able to form such complexes, as seen for native rhIGFBP-5. These findings are consistent with the hypothesis that the carboxyl-terminal domain of IGFBP-5 contains a site that binds to ALS. In a recent study, IGFBP-3 and IGFBP-2 chimeras were used to assign a region in IGFBP-3 that binds to ALS (26), and site-directed mutagenesis studies have implicated IGFBP-3 residues 228-232 in this interaction (8).
We have previously observed that both intact IGFBP-5 (7) and IGFBP-3 (22) form ternary complexes more efficiently in the presence of IGF-I than in the presence of IGF-II. In contrast, binary complex formation between these IGFBPs and IGFs is somewhat more efficient with IGF-II than with IGF-I (12, 13, 27). This study demonstrates this phenomenon even more clearly. The 6-6-5 chimera has a much higher potency for binary complex formation with IGF-II than IGF-I. However, in the presence of excess IGFs or with IGF labeled tracers, this chimera was more potent in forming ternary complexes with IGF-I rather than with IGF-II. In contrast, studies in rodent serum have implicated a proteolyzed form of IGFBP-3, which binds preferentially with IGF-II, as a factor that may form part of an IGF-II preferring ternary complex (28). The mechanism by which IGFs influence the binding of IGFBPs to ALS remains to be determined. Previous studies suggest that the IGF in IGFBP-3 ternary complex may be closely apposed to ALS during complex formation (22, 29).
The marked preference of the 6-6-5 chimera to form binary complexes with IGF-II compared with IGF-I parallels that described for IGFBP-6 (12, 15). It has been suggested that the lack of two cysteine residues in the amino-terminal end of IGFBP-6, which are conserved in all the other IGFBPs, may confer this preferential IGF-II binding (30). Compared with the 6-6-5 chimera, the difference in binding affinities of the 5-5-6 chimera for IGF-I and IGF-II was minimal. In contrast, two recent reports suggest that for IGFBP-2, the preferential IGF-II binding domain is in the carboxyl-terminal region (31, 32) indicating that at least for some IGFBPs, the carboxyl-terminal IGF binding site may also be important for IGF binding preference.
We have shown that heparin and its related O-linked sulfated glycosaminoglycan, heparan sulfate, inhibit IGFBP-5 ternary complex formation, but dermatan sulfate and chondroitin sulfate do not. The ability of heparin and related GAGs to bind to IGFBP-5 has been extensively studied (10, 11, 20, 33). Consensus amino acid sequences have been identified that are common to proteins that bind heparin, and these have been implicated in protein-glycosaminoglycan interactions (34). These sequences are XBBXBX and XBBBXXBX, where B is a basic amino acid and X is a nonbasic amino acid. IGFBP-5 has two putative classical heparin binding motifs (10, 11), one in its midregion (119-124), PKHTRI, and a second sequence in its basic carboxyl-terminal domain (205-212), YKRKQCKP. Studies using synthetic peptides from these regions (10) and others using IGFBP-5 peptides with substitutions at these putative heparin binding motifs (20) have shown that only the carboxyl-terminal basic region of IGFBP-5 is able to bind heparin to any detectable extent.
The more important amino acids in the carboxyl-terminal basic domain of IGFBP-5 that bind heparin have been reported to be Lys201, Arg202, Lys206, and Arg214 (20). Of these, only Lys206 forms part of the classical heparin binding motif in IGFBP-5. Arg214 corresponds to the first residue in the ALS and cell binding motif defined in IGFBP-3 (8). Mutation of residues 217-218 of rhIGFBP-5 did not affect heparin binding to IGFBP-5 (20). It is likely that the GAG molecule inhibits IGFBP-5 binding to ALS by itself binding to a region adjacent to or overlapping with the ALS binding motif on IGFBP-5. The presence of the GAG inhibitory effect on ternary complex formation that we have described is consistent with an ALS binding site existing in the basic carboxyl-terminal region of IGFBP-5 in the vicinity of the heparin binding motif.
Further studies with heparin have shown that after binding to IGFBP-5, a 17-fold reduction in the affinity of IGFBP-5 binding to IGFs occurs (20). We used covalently cross-linked IGF-I-IGFBP-5 and IGF-I-IGFBP-3 radiolabeled tracers in our studies to avoid the effect that GAGs would be expected to have on IGFBP-5 and IGF binary complex formation. The concentration of the GAGs required to inhibit the formation of ternary complex was greater than that previously required for inhibition of binary complex between IGF-I or IGF-II and IGFBP-5 (20) or for IGFBP-3 ternary complex (35). It may be that the generation of covalently cross-linked IGF-IGFBP-5 tracer reduces the ability of GAGs to compete for IGFBP-5 and IGFBP-3 ternary complex.
The mechanism of IGFBP-5 ternary complex dissociation to binary complex or free peptides in vivo is yet to be determined. This study suggests that one mechanism may be by competition for binary complexed IGFBP-5 binding to ALS by circulating or endothelial cell bound GAGs, as has been previously proposed for IGFBP-3 in ternary complexes (35, 36). An alternate or additional mechanism is by proteolysis of IGFBP-5. We have observed in fractionated human serum that proteolyzed IGFBP-5 is present in fractions containing ALS (7). The basic carboxyl-terminal region (201-218) in IGFBP-5 has also been identified as the sequence in IGFBP-5 that binds to tissue matrix factors, such as plasminogen activator-inhibitor type 1 (37), vitronectin (37), tenascin (20), and hydroxyapatite (38). Whether these factors interact with ALS binding to IGFBP-5 is not known.
This study demonstrates that free IGFBP-5 is able to compete with binary complexed IGFBP-5 for binding to ALS. This is consistent with the ability of IGFBP-5 to form a weak binary complex with ALS in the absence of IGFs. Our previous demonstration that IGFBP-5 co-migrates predominantly with ALS in human serum (7) could be partly accounted for by IGFBP-5 bound to ALS in the absence of IGFs. Although we have not yet determined whether IGFBP-5 is able to bind IGFs after it has complexed to ALS, ALS-IGFBP-5 heterodimers might account for unsaturated IGF binding sites in high molecular weight IGFBP complexes.
The inhibitory studies using the carboxyl-terminal synthetic peptides show weak effects only. That Peptide ns5 inhibited IGFBP-5 complex formation with ALS to the same extent as Peptide 5 suggests that for these synthetic peptides, the charge density of the basic region peptides may account for this inhibitory effect. The lesser inhibition seen for Peptide 6 is also consistent with a charge density effect, because this peptide has only 8 basic amino acids, in comparison with the 10 basic amino acids present in the other 18-mer peptides used. Recent studies using a 201-218 synthetic peptide of IGFBP-5 have shown that it inhibits plasminogen activation (39), potentiates binding of native IGFBP-5 to hydroxyapatite (40), stimulates cell migration (41), and inhibits IGFBP-5 binding to cell surfaces (11). Although control peptides were used in these experiments, our data suggest that the most rigorous method to control for the high charge density in IGFBP-5(201-218) is the use of a control nonsense peptide that has the same amino acid content but a different amino acid sequence.
In contrast to the synthetic peptide results, full-length rhIGFBP-5 and the 6-6-5 chimera are more potent by orders of magnitude in molar terms than Peptide 5 in inhibiting labeled IGF-I-IGFBP-5 tracer binding to ALS, even in the absence of IGFs. This suggests that if this basic sequence in IGFBP-5(201-218) is involved in specific binding to ALS, as is implicated by the results of the glycosaminoglycan studies we have described, then the conformation of these residues as they occur in full-length IGFBP-5 and in the 6-6-5 chimera is important in conferring the ability of the basic region carboxyl-terminal site to bind to ALS.
IGFBP-6 is the most closely related IGFBP in phylogeny (42) and in amino acid sequence (30) to IGFBP-5 and IGFBP-3. A basic region in the carboxyl-terminal site of IGFBP-6 has some homology to the basic regions of IGFBP-5 and IGFBP-3 (6); at residues 168-185 in IGFBP-6, there are 8 basic and no acidic residues. Despite having the same putative heparin binding motif in this region as IGFBP-5 and IGFBP-3, IGFBP-6 binds heparin with relatively much lower potency (11). The structural constraints limiting high affinity IGFBP-6 binding to heparin have not been determined. Consistent with findings in our earlier studies (7, 15), IGFBP-6 with or without IGFs did not bind to ALS in the methods we tested. This further supports the importance of the basic residues IGFBP-5(214-218) and IGFBP-3(228-232) (shown in Fig. 1) in the interaction with ALS.
In conclusion, we have shown using IGFBP chimeric proteins that IGFBP-5
binds to ALS through its carboxyl-terminal domain. The
glycosaminoglycan studies suggest that the site in this domain that
binds ALS is the IGFBP-5 basic region residues 201-218. Binding studies performed in the presence compared with the absence of IGFs and
studies with synthetic peptides suggest that the conformation conferred
on this carboxyl-terminal basic region within the native IGFBP
structure is important in facilitating IGFBP-5 binding to ALS.
Competition by glycosaminoglycans at the carboxyl-terminal basic region
in IGFBP-5 may be involved in regulating the amount of IGFBP-5 that is
complexed to ALS in serum and in the delivery of IGFBP-5 and IGFs to
particular tissue sites.
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FOOTNOTES |
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* This study was supported by the National Health and Medical Research Council, Australia (to S. M. T. and R. C. B.) and by Grant 32-46808.96 from the Swiss National Science Foundation (to J. Z.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel:
61-2-99268486; Fax: 61-2-99268484; E-mail:
robaxter{at}med.usyd.edu.au.
The abbreviations used are: IGF, insulin-like growth factor; ALS, acid-labile subunit; IGFBP, IGF-binding protein; GAG, glycosaminoglycan; rh, recombinant human; ns, nonsense.
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REFERENCES |
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