From the Hannah Research Institute, Ayr KA6 5HL,
Scotland, United Kingdom, the § Dana-Farber Cancer Institute
and the Department of Pathology, Harvard Medical School, Boston,
Massachusetts 02115, and the
Institute of Biomedical and Life
Sciences, University of Glasgow,
Glasgow G12 8QQ, Scotland, United Kingdom
Received for publication, January 17, 2003, and in revised form, March 3, 2003
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ABSTRACT |
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We have previously reported that two highly
conserved amino acids in the C-terminal domain of rat insulin-like
growth factor-binding protein (IGFBP)-5, Gly203 and
Gln209, are involved in binding to insulin-like growth
factor (IGF)-1. Here we report that mutagenesis of both amino acids
simultaneously (C-Term mutant) results in a cumulative effect and an
even greater reduction in IGF-I binding: 30-fold measured by solution
phase IGF binding assay and 10-fold by biosensor analysis. We compared these reductions in ligand binding to the effects of specific mutations
of five amino acids in the N-terminal domain (N-Term mutant), which had
previously been shown by others to cause a very large reduction in
IGF-I binding (1). Our results confirm this as the major IGF-binding
site. To prove that the mutations in either N- or C-Term were specific
for IGF-I binding, we carried out CD spectroscopy and showed
that these alterations did not lead to gross conformational changes in
protein structure for either mutant. Combining these mutations in both
domains (N+C-Term mutant) has a cumulative effect and leads to a
126-fold reduction in IGF-I binding as measured by biosensor.
Furthermore, the equivalent mutations in the C terminus of rat IGFBP-2
(C-Term 2) also results in a significant reduction in IGF-I binding,
suggesting that the highly conserved Gly and Gln residues have a
conserved IGF-I binding function in all six IGFBPs. Finally, although
these residues lie within a major heparin-binding site in IGFBP-5 and
-3, we also show that the mutations in C-Term have no effect on heparin binding.
The actions of insulin-like growth factors
(IGFs)1 are mediated by
binding to and activating type 1 IGF receptors, which are found on the
surface of most cell types. In turn, IGFs are regulated by a family of
six IGF-binding proteins (IGFBPs) that form high affinity complexes
with both IGF-I and IGF-II (reviewed in Ref. 2). Because the affinity
constants of the IGFBPs are between 2- and 50-fold greater for binding
IGFs than that of the IGF type 1 receptor, it is thought that the
IGFBPs are able to regulate the bioavailability of IGFs in different
biological fluids.
IGFBPs are proteins of 216-289 residues, and all share a common
protein structure that can be conceptually divided up into three
domains, where a domain may be defined as a region of a protein that
can fold into a tertiary structure independent of neighboring sequences
(2) (Fig. 1). There is a
particularly high degree of conservation in the N- and C-terminal
domains, which contain 12 and 6 cysteine residues, respectively, in
IGFBPs 1-6 of all species sequenced so far. A nonconserved region
separating the N- and C-terminal domains contains most of the sites for
proteolysis and post-translational modifications. Both the N- and
C-terminal cysteine-rich domains of IGFBPs are believed to participate
in IGF binding. This is supported by work where fragments from the N-terminal cysteine-rich domains of IGFBP-1 (3), IGFBP-3 (4), IGFBP-4
(5), and IGFBP-5 (6, 7) and the C-terminal cysteine-rich domains of
IGFBP-2 (8, 9) and IGFBP-3 (10) were generated and shown to possess
residual IGF binding activity. More recently, several groups have
reported biosensor analysis of N- and C-terminal fragments of bovine
IGFBP-2 and human IGFBP-3 (11-13), and all of these studies indicated
that there were major IGF-binding sites in both the N- and C-terminal
domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Conservation of the C-terminal glycine and
glutamine residues in the IGFBP family. At the top is a
schematic representation of the three structural domains of the IGFBPs.
The box below shows a single letter amino acid line up for a
region in the C-terminal domain for all six IGFBPs from several
different species. The residue numbers at the start and end of each
sequence are indicated in parentheses. The glycine and
glutamine residues in question are shown in bold type, which
clearly indicates the complete conservation of Gly203 and
Gln209 in rat IGFBP-5 in all the other IGFBP
sequences.
Work from several laboratories has already identified the specific amino acids in the N-terminal domain of the IGFBP molecule that contribute to IGF binding. Initially it was observed that Tyr60 was protected against iodination upon binding of bovine IGFBP-2 to IGF-II (14), and subsequently, the same group demonstrated that substitution of this amino acid leads to reduced affinities for IGF-I and -II (15). In addition, NMR was carried out on a bacterially expressed N-terminal fragment of IGFBP-5 (residues 40-92), and this clearly demonstrated that a major IGF-binding domain comprises Val49, Tyr50 (equivalent to Tyr60 in IGFBP-2), Pro62, and Lys68-Leu75, where the conserved Leu and Val residues localize in a hydrophobic patch on the surface of the IGFBP-5 protein (7). Subsequently, it was shown that substituting specific hydrophobic residues in this region of IGFBP-5 resulted in an ~1000-fold reduction in affinity for IGF-I, as measured by solution phase ligand binding assays (1). However, there remains a possibility that the effects of these mutations are nonspecific because they may have caused a gross alteration in protein structure, despite the fact that they did not involve the highly conserved cysteine residues.
The NMR study described above also found that the N-terminal fragments of IGFBP-5 had a 10-200-fold lower affinity for IGFs than the full-length protein (7), which agrees well with the reported reduced affinities of other C-terminally truncated fragments of IGFBP-3 (16) and IGFBP-5 (6). This indicated that other residues at the C terminus of the IGFBP proteins must be required in the additional stabilization of IGF complex formation that leads to high affinity binding. Less attention has focused on identifying the C-terminal residues in IGFBPs that are involved in IGF interaction. However, a strong clue as to where some or all of these important residues may lie came from a deletion analysis of the C terminus of bovine IGFBP-2, which identified a critical region between amino acids 222 and 236 of IGFBP-2 involved in binding IGFs (17). This region of bovine IGFBP-2 corresponds to amino acid sequences in both IGFBP-5 and -3, which are rich in basic residues (201-218 in IGFBP-5). Although several of these basic amino acids have been shown to play a critical role in IGFBP-5 binding to both heparin and the extracellular matrix (18-20), it has also been demonstrated that extensive mutagenesis of the basic amino acids alone had no effect on IGF-I binding (21, 22).
Nevertheless, alignment of amino acid sequences for all six binding proteins from several species reveals that there are two completely conserved nonbasic amino acids (Gly203 and Gln209 in rat IGFBP-5) within this region (Fig. 1), and this conservation suggests an important function for these amino acids. We have shown previously that mutation of either Gly203 or Gln209 results in a significant reduction in affinity for binding to IGF-I (7- and 6-fold, respectively) but has no effect on heparin binding (22, 23). This clearly demonstrates that there is overlap of IGF and heparin-binding sites in the C-terminal domain of IGFBP-5.
In the present study, we investigate whether there is a cumulative
effect on the ability of IGFBP-5 to bind to IGF-I (or heparin) when
both Gly203 and Gln209 are mutated
simultaneously (G203A/Q209A; C-Term), and we also extend these
mutations to the corresponding residues in IGFBP-2 (G211A/Q217A; C-Term
2) to determine whether this reflects a conserved IGF-binding site in
IGFBPs. In addition, to ascertain the relative contribution of the N-
and C-terminal domains of IGFBP-5 in binding IGF-I, we have recreated
the mutations described by Imai et al. (1) in the N terminus
(K68N/P69Q/L70Q/L73Q/L74Q; N-Term) and compared IGF-I binding of
C-Term, N-Term, and a combined mutant, N+C-Term, by ligand blotting,
solution phase IGF binding assays, and biosensor real time analysis of
binding kinetics. Furthermore, because it was not previously reported
whether the mutations in the IGFBP-5 C terminus (22, 23) or N terminus
(1) led to a conformational change in the protein structure, we have
carried out CD analysis on both C-Term and N-Term to establish whether these mutations are specific for IGF binding. The implications of these
findings for IGFBP-5 interaction with IGFs and heparin are discussed.
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MATERIALS AND METHODS |
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Site-directed Mutagenesis--
The full-length cDNAs for rat
IGFBP-5 and -2 in pGEM®-7zf (Promega, Madison, WI),
containing both initiator and signal peptide, was kindly provided by
Dr. S. Guenette (John Wayne Cancer Institute, Los Angeles, CA).
Site-directed mutagenesis was carried out using the
QuikChangeTM system (Stratagene, La Jolla, CA), following
the protocol provided by the manufacturer. The rat and mouse protein
sequence of IGFBP-5 only differs by a single amino acid at position 188 (Asp and Glu, respectively). Site-directed mutagenesis was employed to
convert wt rat to wt mouse sequences (D188E) using the
oligonucleotides: 5'-GAC AGG AAT CTG AAC AAG GCC CCT GCC-3' and 5'-GGC
AGG GGC CTT GTT CAG ATT CCT GTC-3'. The oligonucleotide used to make
Q209A was described previously (21). Q209A was used as a template to
make C-Term (G203A/Q209A) using the oligonucleotides 5'-AAC TGT GAC CGC
AAA GCT TTC TAC AAG AGA AAG-3' and 5'-CTT TCT CTT GTA GAA AGC TTT GCG
GTC ACA GTT-3'. N-Term (K68N/P69Q/L70Q/L73Q/L74Q) was made using the
oligonucleotides 5'-GGA TGA GGA GAA CCA GCA GCA CGC CCA GCA GCA CGG CCG
CGG-3' and 5'-CCG CGG CCG TGC TGC TGG GCG TGC TGC TGG TTC TCC TCA
TCC-3'. The N+C-Term composite mutant was made by using the unique
SacII restriction endonuclease site in the IGFBP-5 cDNA
sequence to cut and ligate N-Term and C-Term sequences together. The wt
rat IGFBP-2 cDNA was mutated to make C-Term 2 (G211A/Q217A) using
the oligonucleotides 5'-AAC TGT GAC AAG CAT GCT CTG TAC AAC CTC AAA
GCT-3' and 5'-CAG AGA CAT CTT GCA AGC TTT GAG GTT GTA CAG AGC-3'. Our
previously reported "heparin minus" (Hep) mutant is defective in
its ability to bind to heparin (22). All of the oligonucleotides were
synthesized by MWG BioTech (Milton Keynes, UK). Following site-directed
mutagenesis, automated DNA sequencing (MWG BioTech) was carried out on
all clones to confirm that the correct mutations had been made.
Bacterial Expression-- Expression of recombinant IGFBP-5 proteins was carried out using conditions identical to those described previously (24). Briefly, mouse wt IGFBP-5 and the various IGFBP-5 mutants were cloned without the signal peptide-encoding sequence into the pGEX 6P-1 vector (Amersham Biosciences), so that the proteins would have an N-terminal glutathione S-transferase (GST) tag. The proteins were expressed in the Origami B (DE3) pLysS strain of Escherichia coli (Novagen, Madison, WI), and following cell lysis the GST-IGFBP-5 fusion proteins were extracted from the soluble fraction only and subsequently subjected to two rounds of purification. The cell lysates were loaded onto glutathione-Sepharose columns (Amersham Biosciences), and the purified IGFBP proteins were then eluted following removal of the GST tag with PreScission protease (Amersham Biosciences). Subsequently, the untagged IGFBP proteins were purified on IGF-I affinity columns and quantified as described previously (20). Commercial mouse IGFBP-5 expressed in the mouse myeloma cell line NSO was purchased from R&D Systems, Inc. (Minneapolis, MN). Rat wt IGFBP-2 and C-Term 2 were expressed untagged using the pET-15b vector (Novagen) in Origami B cells, using the same methodology as that used for the IGFBP-5 proteins, except the cell lysate supernatant was used for subsequent analyses.
CD Spectroscopy and Fluorescence-- To compare the secondary structures of wt IGFBP-5, C-Term, and N-Term proteins, CD spectropolarimetry data were recorded using a JASCO J-600 spectropolarimeter in cells with path lengths of 0.02 cm (far-UV) using protein concentrations of 0.5 mg/ml for both wild type and mutant IGFBP-5 samples (time constant, 2 s; scan speed, 10 nm/min). The secondary structure contents of wild type, C-Term, and N-Term mutants were estimated using the CONTIN procedure of Provencher and Glöckner (25) and the SELCON procedure of Sreerama and Woody (26). Secondary structure estimates were obtained using data values down to 190 nm in both the wild type and N-Term proteins. In the case of the C-term mutant, the signal to noise ratio was not favorable below 197 nm, thus precluding the use of data below this wavelength. Fluorescence spectra were recorded using a Perkin Elmer LS50B fluorimeter in 1-cm-path length cells using protein concentration of 0.1 mg/ml for both wild type and mutant samples; (excitation, 290 nm; slits of 5-nm band pass).
IGF-I Ligand Blotting and Western Immunoblotting-- The proteins were electrophoresed on 12% acrylamide SDS-PAGE gels under nonreducing conditions and subsequently transferred to nitrocellulose membranes. The ligand blots were performed according to the method described previously (27), using IGF-I (GroPep Limited, Adelaide, Australia) radiolabeled to a specific activity of ~100 µCi/µg. Western blots with either an "in-house" sheep anti-rat IGFBP-5 antiserum or a commercial rabbit anti-bovine IGFBP-2 antibody (Upstate Biotechnology, Inc., Lake Placid, NY) were performed as described previously (23) and were used to monitor protein loading during ligand and heparin blotting experiments. The method used for heparin ligand blotting was also described previously (22).
Solution Phase IGF Binding Assay-- Binding affinities for IGF-I were assayed in solution by the method described previously by Conover et al. (28). The assays were performed using the PreScission protease eluate fractions obtained after expression of wt IGFBP-5 and the C-Term mutant and the supernatant bacterial lysates containing recombinant wt IGFBP-2 and the C-Term 2 mutant. The optimal amount of each protein preparation was calculated following initial saturation binding assays containing 25,000-30,000 dpm (15-18 fmol) [125I]IGF-I. The affinities were then assessed by Scatchard analysis of binding curves obtained in the presence of increasing concentrations of unlabeled IGF-I. Only the affinities for the wt IGFBP-5 and C-Term are reported because binding to the N-Term and N+C-Term mutants was too low to be measured by this method.
Biosensor Analysis--
Biosensor studies were performed using
the Biacore 3000 instrument. Human IGF-I (10 µg/ml in 10 mM sodium acetate, pH 5) was immobilized into flow cell 2 of three separate CM5 biosensor chips at ligand densities of 50, 100, and 200 response units. To provide a control binding surface,
insulin was immobilized in flow cell 1 of the three biosensor chips at
the same level of ligand density. Both IGF-I and insulin were
immobilized using amine-coupling chemistry according to protocols
provided by the manufacturer. For kinetic studies, purified proteins
were injected at five different concentrations and at a flow rate of 30 µl/min in 10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20. All of the experiments were performed at ambient temperature. The protein concentrations were
as follows: commercial mouse IGFBP-5 and wt mouse IGFBP-5 (in-house):
1.56, 3.125, 6.25, 12.5, and 25 nM; C-Term, 3.125, 6.25, 12.5, 25, and 50 nM; and N-Term and N+C-Term, 25, 50, 100, 200, and 400 nM. The association and dissociation phases of
the binding curves were 3 and 15 min, respectively, for the commercial protein and 5 and 15 min, respectively, for all others. Following binding the surface of the chip was regenerated by two 30-s pulses of
50 mM NaOH, 1 M NaCl. Protein samples and a
buffer blank were injected in duplicate and in random order using each
of the three biosensor chips. The data were analyzed assuming a 1:1
stoichiometry of interaction between IGF-I and IGFBP-5 using the
Langmuir model provided in the BiaEvaluation 3.1 software and employing
the global data analysis option.
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RESULTS |
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N-terminal sequence analysis of the recombinant wt IGFBP-5 protein showed the expected sequence (GPLGSLGSFVHCEPCDDEK) derived from pGEX 6P-1 and the mature IGFBP-5 sequence minus signal peptide, indicating that there had been proper processing by PreScission protease.2
It can be seen from the CD results (Fig.
2A) that despite the
apparently significant differences in amplitude, the secondary structure estimates (given in Table I)
are broadly similar, exhibiting a strong negative band with a minimum
close to 200 nm in each case. Secondary structure estimates of these
spectra indicate a low percentage of -helix and a greater proportion
of
-sheet and
-turns for each protein. The fluorescence emission
spectra (Fig. 2B) show that wt IGFBP-5, C-Term, and N-Term
have very similar emission maxima (~345 nm) with considerable
differences in intensity. In view of the fact that intensity depends on
the relative magnitudes of the rates of various processes that lead to
the decay of the excited state, the emission maximum is generally
regarded as a more reliable indicator of the polarity of the Trp
environments in a protein (29). On this basis, the solvent exposure of
the Trp side chains is very similar in the three proteins, indicating that little or no overall change in tertiary structure has
occurred.
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Following on from this, we compared [125I]IGF-I binding
of the mutant IGFBP-5 proteins, N-Term, C-Term, and N+C-Term, to that of the wt protein using ligand blotting (Fig.
3, upper panel). The same
filter was also subject to Western analysis with the appropriate
antiserum to verify equivalent protein loading (Fig. 3, lower
panel). Although all three mutant proteins displayed reduced IGF-I
binding, we note that relative to wt IGFBP-5, the greatest effect on
IGF-I binding was with N-Term and N+C-Term, which display no apparent
binding on ligand blots (Fig. 3).
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We also examined the affinity of these mutant proteins in a solution
phase analysis of the IGF-I-IGFBP interaction (Fig.
4). Scatchard analysis could only be
derived from binding curves for wt IGFBP-5 and C-Term
(KD values of 0.13 and 3.54 nM, respectively), which indicated that C-Term had a 30-fold lower affinity
than the wt protein for binding IGF-I. We also included a commercial
mouse wt IGFBP-5 protein from R & D Systems to compare IGF-I binding
of wt IGFBP-5 expressed in either mammalian cells or bacteria. Our data
demonstrate that the commercial and in-house wt IGFBP-5 proteins have
very similar affinities or KD values for binding to
IGF-I, with 0.102 and 0.13 nM, respectively. However, in
support of our ligand blotting data, binding of IGF-I to the N-Term and
N+C-Term mutants was too low to be able to derive Scatchard data,
although Imai et al. (1) were able to measure an
~1000-fold reduction with the equivalent N-Term mutations. This
apparent discrepancy could possibly be the result of differences in the
solution phase assay techniques employed by the two groups. Nevertheless, both agree that amino acid substitutions in the N-terminal domain of IGFBP-5 result in very large reductions in IGF-I
binding.
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As a further confirmation of the IGF binding properties of these
IGFBP-5 mutant proteins, we undertook a biosensor analysis of binding
kinetics of the proteins to IGF-I immobilized to a biosensor surface
(Fig. 5). In agreement with the solution
phase data above, the commercial and in-house wt IGFBP-5 proteins had almost exactly the same KD values, with 0.22 and
0.21 nM, respectively. However, the KD
values for N-Term, C-Term, and N+C-Term were 12.5, 2.1, and 25.9 nM, respectively. For N-Term and C-Term this demonstrates
60- and 10-fold reductions in affinity for binding IGF-I compared with
the wt protein and a cumulative effect of the N+C-Term mutant, which
displays a 126-fold reduction in binding. This type of analysis also
gives details of on and off rate kinetic constants, from which
equilibrium constants can be derived (Table
II). Closer inspection of the biosensor data revealed that although the N- and C-Term mutants displayed similar
changes to their Koff or rate of dissociation
values, the overall larger reduction in IGF-I affinity observed for
N-Term was primarily due to a substantially slower
Kon or rate of association compared with C-Term.
Finally, the highest reduction in IGF-I affinity observed for the
N+C-Term mutant was largely the result of a cumulative effect on its
Koff, which was approximately twice as fast as
that of either N- or C-Term. We also note that the absolute
KD values for wt IGFBP-5 and C-Term varied by up to
2-fold between biosensor and solution phase assays and that C-Term
displayed a greater reduction in binding relative to the wt protein in
the latter system (30-fold versus 10-fold).
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Because Gly203 and Gln209 lie within the
heparin-binding site between amino acids 201 and 218 in the C-terminal
domain of IGFBP-5, experiments were also carried out to investigate the
heparin binding properties of the C-Term mutant. Using a heparin
blotting methodology we have previously reported that wt IGFBP-5 and
the two single mutants, G203A and Q209A, bind to heparin in this
system, whereas the Hep mutant, which contains mutations at four
basic residues in this region (R201L, K202E, K206Q, and R214A) binds
heparin very poorly (22). We have now used this system to compare
heparin binding between wt IGFBP-5, C-Term (G203A/Q209A combined) and Hep
(Fig. 6, upper panel).
Although wt IGFBP-5 and C-Term display comparable binding to heparin in
this system, there is no evidence of binding for Hep
. As with the IGF
ligand blotting, corresponding Western blots were carried out to verify
equivalent protein loading (Fig. 6, lower panel). Therefore,
the residues Gly203 and Gln209 within the
201-218 region are involved in IGFBP-5 binding to IGF-I but appear to
play no part in binding to heparin.
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Finally, because Gly203 and Gln209 in IGFBP-5
are completely conserved in all six IGFBPs from all species sequenced
to date, we investigated whether these residues were also involved in
IGF-I binding in a different IGFBP species, IGFBP-2. Ligand Western analysis and solution phase IGF binding assays were carried out on wt
IGFBP-2 and the C-Term 2 mutant, which contains the corresponding amino
acid substitutions (G211A/Q217A) in the rat IGFBP-2 sequence (Fig.
7). IGF ligand blotting demonstrated that
there was a very significant reduction in C-Term 2 binding to
[125I]IGF-I relative to wt IGFBP-2, where only a faint
band was detectable for C-Term 2 (Fig. 7A, upper
panel). We note that Western blot analysis of the same filter
using an anti-IGFBP-2 antiserum demonstrated equivalent protein loading
of wt IGFBP-2 and C-Term 2 (Fig. 7A, lower
panel). Further support of this conclusion comes from the observation that the solution phase binding curves also demonstrate compromised IGF-I binding for C-Term 2 relative to the wt protein (Fig.
7B). Scatchard analysis derived from binding curves for wt
IGFBP-2 and C-Term 2 (KD values of 0.14 and 0.61 nM, respectively) indicated that C-Term 2 had a 4.5-fold
lower affinity than the wt protein for binding IGF-I. Taken together,
these results clearly demonstrate that Gly211 and
Gln217 in the IGFBP-2 sequence are involved in binding to
IGF-I.
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DISCUSSION |
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At the outset, we considered it essential to establish whether the
effects of our amino acid substitutions in the IGFBP-5 sequence on
IGF-I binding were specific or were simply the effect of a gross
conformational change in protein structure. CD spectra were obtained to
compare the secondary structures of wt IGFBP-5, C-Term, and N-Term, and
it was observed that the overall shapes of the spectra were similar for
all three proteins. Interestingly, similar CD spectra have also been
shown for other proteins, including oxidized ribonuclease and a
subclass of all- proteins (termed
-II proteins) such as
-chymotrypsin, elastase, and soy bean trypsin inhibitor protein.
X-ray diffraction data have shown that these
-sheet proteins are
either highly distorted or made up of short irregular
strands (30,
31), and this could also hold true for IGFBP-5. The fluorescence
spectra show that wt IGFBP-5, C-Term, and N-Term have very similar
emission maxima, which indicates that the orientation of the Trp side
chains is very similar in the three proteins. However, subtle changes
in the microenvironments of the these side chains could effect the
efficiency of quenching by moieties such as disulfide bonds, and it is
possible that this could account for the significant differences
observed in the intensity of fluorescence spectra for the three
proteins. Nevertheless, these spectra would argue that little or no
overall change in tertiary structure has occurred between the wt
protein and the two mutants. Therefore, taken together our structural
analyses confirm that the amino acid substitutions that we have made in both N-Term and C-Term have not led to gross changes in protein structure.
Bearing this in mind, our experiments confirm the previous work of Imai et al. (1), which showed that these N-Term mutations led to the disruption of a major IGF-I-binding site in IGFBP-5, because both groups report very large decreases in binding with the same amino acid substitutions. However, although we could not detect any binding to N-Term and N+C-Term using solution phase IGF binding assays, Imai et al. (1) reported an ~1000-fold reduction in IGF-I binding for their corresponding N-Term mutant. This apparent discrepancy is likely to be due to minor variations in the solution phase IGF binding protocols employed by the two labs. Another difference is that our analyses were with bacterially expressed proteins, whereas Imai et al. (1) expressed their recombinant proteins in Chinese hamster ovary cells (1). However, we also demonstrate that a commercial mouse wt IGFBP-5 protein, which was expressed in mammalian cells and is therefore likely to be properly post-translationally modified (phosphorylated and glycosylated), has almost exactly the same affinity for binding IGF-I in solution phase assays and biosensor analysis as our bacterially expressed wt protein. This is supported by other work where it was shown that mutagenesis to remove the glycosylation sites or expression in a prokaryotic nonglycosylating system had no effect on the binding of IGFBP-3 to IGF-I (32, 33). Therefore, post-translational modifications of either IGFBP-5 or -3 do not appear to affect IGF-I binding.
In addition to confirming that there is a major IGF-binding site in the N-terminal domain of IGFBP-5, the major findings presented here identify the important residues in the C-terminal domain that are involved in IGF-I binding: glycine 203 and glutamine 209. Mutagenesis of both of these amino acids in the C-Term mutant has led to a cumulative effect over the single mutations reported previously (23), with either a 30- or 10-fold reduction in the affinity of C-Term for IGF-I when measured by either solution phase assays or biosensor analysis respectively. It is possible, of course, that other residues in the C-terminal domain of IGFBP-5 may contribute to IGF-I binding. However, it is interesting to compare our findings with the biosensor analyses of Carrick et al. (11), where they measured the IGF-I binding affinity of recombinant fragments of bovine IGFBP-2. Relative to full-length IGFBP-2, they observed a 5-fold reduction in IGF-I binding with their 1-185 fragment, which contains the entire N-terminal and central domains. This is very close to the 4.5-fold reduction in affinity for IGF-I that we observe for our C-Term 2 mutant relative to wt IGFBP-2, as derived from Scatchard analysis. Therefore, relative to the native proteins, the removal of the entire C-terminal domain in IGFBP-2 leads to a reduction in IGF-I binding very similar to that observed for both our C-Term and C-Term 2 mutants, which only carry substitutions of the C-terminal residues Gly203 and Gln209 or residues Gly211 and Gln217 in IGFBP-5 or -2, respectively. This would imply that these two amino acids are the major, if not the only, residues in the C terminus of both IGFBP-5 and -2 that are involved in IGF-I binding. The fact that they are also highly conserved throughout the IGFBP family (Fig. 1) would also suggest that these residues are specifically involved in binding IGF-I in all six IGFBP proteins.
In this respect, it is important to remember that Gly203 and Gln209 lie within a major heparin-binding site in both IGFBP-5 and -3, which has previously been shown to be involved in binding to heparin and to components of the extracellular matrix (18, 19). Although the work described above clearly indicates a role for Gly203 and Gln209 in binding IGF-I, we also demonstrate that these two amino acids play no part in heparin binding because heparin-ligand analysis demonstrates similar activities for wt IGFBP-5 and C-Term. Therefore, it would appear that within the C terminus of IGFBP-5, and likely also within IGFBP-3, there is an overlap of residues involved in IGF-I and heparin binding. Furthermore, because Gly203 and Gln209 are conserved in all six binding proteins, whereas the heparin-binding site is only common to IGFBP-5 and -3, this must mean that the IGF-I binding function of this region predates heparin binding during evolution. Taken together, this has important implications for the biological actions of these two IGFBPs, because it implies that any of the numerous functions ascribed to this region (amino acids 201-218 in IGFBP-5) may be mutually exclusive to effective IGF-I binding. Other functions ascribed to this region in IGFBP-5 include binding to the acid labile subunit, the putative IGFBP-5 receptor, and plasminogen activator inhibitor-I, in addition to acting as a nuclear localization signal (reviewed recently in Ref. 34). In support of this hypothesis, others have clearly demonstrated that incubation of IGFBP-5 with heparin resulted in a 17-fold decrease in the affinity for IGF-I (35). This lowering of IGF-I affinity when the 201-218 region is otherwise occupied may be critical in obtaining the correct balance between free and bound growth factor.
Our site-directed mutagenesis strategy has also enabled us to determine the differential contributions of the N- and C-terminal domains of IGFBP-5 in binding IGF-I. Independent of the technique used to measure IGF-I binding, it is clear that the amino acid substitutions in the N-terminal domain have led to a significantly greater reduction in IGF-I binding, which would argue that the major IGF-binding site is in this domain. Nevertheless, we note that Gly203 and Gln209 in the C terminus also make a significant contribution to IGF-I binding. Another advantage of our site-directed mutagenesis approach over analysis of N- and C-terminal IGFBP fragments is that we were able to combine the mutations in both termini by making the N+C-Term mutant. This clearly demonstrates that the disruption of IGF-I-binding sequences in both the N and C termini simultaneously has a cumulative effect and leads to a far greater (126-fold) reduction in IGF-I binding as measured by biosensor analysis.
Study of the individual Kon and Koff values from the biosensor analysis allows us to observe that the N-Term has a considerably slower association rate than C-Term (17- and 2.6-fold slower than wt IGFBP-5, respectively). However, the N- and C-Term mutants have comparable increases in dissociation rates, (both have ~3.5-fold faster dissociation rates than wt IGFBP-5). The effect of combining the mutations, as is the case with the N+C-Term mutant, appears to be more pronounced on the dissociation rate (which is now 7-fold faster relative to wt IGFBP-5). Thus, there would appear to be an additive effect of the N- and C-term mutations on dissociation. Taken together, these biosensor results would suggest that although the N-terminal sequences play a major role in the association of IGF-I with IGFBP-5, the C-terminal sequences are more involved in stabilization of the IGFBP/IGF complex, because mutating these sequences increases the speed of dissociation.
In conclusion, we have identified Gly203 and
Gln209 as the important residues in the C-terminal domain
of IGFBP-5 that are involved in IGF-I binding. Although their
substitution leads to a significant loss of ligand binding, it is less
than that observed when several residues in the major IGF-binding site
in the N-terminal domain are mutated. Further advances in this area
will require the determination of the three-dimensional structure of
the IGF-IGFBP complex by x-ray crystallography.
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ACKNOWLEDGEMENTS |
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We thank Prof. Nicholas C. Price (Institute of Biomedical and Life Sciences, University of Glasgow) for help and useful discussions. We also thank Dr. Ian Davidson (Department of Molecular and Cell Biology, University of Aberdeen) for carrying out C-terminal peptide sequencing of our recombinant IGFBP-5 protein.
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FOOTNOTES |
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* This work was funded by the Scottish Executive Rural Affairs Department.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.
¶ Supported by the British Council through the Chevening Scholarship scheme.
** To whom correspondence should be addressed. E-mail: allang@hri.sari.ac.uk.
Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M300526200
2 I. Davidson, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: IGF, insulin-like growth factor; IGFBP, IGF-binding protein; wt, wild type; GST, glutathione S-transferase.
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