Structure–function studies of an IGF-I analogue that can be chemically cleaved to a two-chain mini-IGF-I

Stella Geddes1, Patricia Holst2, Joachim Grotzinger3, Raj Gill1,4, Philip Nugent1, Pierre De Meyts2, Axel Wollmer3, Steve Wood1,4 and Jim Pitts1,5

1 School of Crystallography, Birkbeck College, University of London, Malet Street, London WC1 7HX, UK, 2 Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark, 3 Institut fur Biochemie, Rheinisch-Westfaliche Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, Germany and 4 Department of Biochemistry, School of Biological Sciences, University of Southampton, Southampton SO16 7PX, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The structure and biological activities of two disulphide isomers of a C-region deletion mutant of insulin-like growth factor-I (IGF-I) which has an Asn–Gly link engineered at the junction of the A- and B-regions were studied before and after chemical cleavage. Circular dichroism (CD) spectra and binding affinity to IGF binding protein 3 (IGFBP3) indicated that the treatment with hydroxylamine did not disrupt the overall tertiary fold of the hormones. Cleavage restored some binding affinity for the IGF-I receptor in both isomers and weakly restored the ability to stimulate incorporation of tritiated thymidine into DNA in NIH 3T3 fibroblasts transfected with the human IGF-I receptor. Cleavage also restored metabolic capacity, as measured by the ability of the isomers to promote lipogenesis in isolated rat adipocytes through the insulin receptor. These results are consistent with the theory that binding of IGF-I to the IGF-I receptor requires a conformational change similar to that involved in insulin binding the insulin receptor. The weak affinity for the IGF-I receptor after cleavage is consistent with the belief that residues in the C-region interact with the IGF-I receptor. This structural difference between insulin and IGF-I gives each a higher binding affinity for its own receptor.

Keywords: insulin/insulin-like growth factor-I/receptor binding/structure-function relationships


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Insulin-like growth factor I and II (IGF-I, IGF-II) are single-chain polypeptides which have both growth-promoting and metabolic activity (reviewed in Jones and Clemmons, 1995). IGF-I and -II show close sequence homology with, and are structurally similar to, proinsulin and are similarly processed from a preprohormone. Unlike insulin where the C-region is cleaved from proinsulin to form a disulphide-linked dipeptide, the IGFs retain their shorter C-region.

An unusual property of IGF-I is that it can fold into two disulphide swapped isomers of similar stability (Milleret al., 1993Go). Native-fold IGF-I has disulphide linkages at A20–B18, A6–A11 and A7–B6 (Raschdorf et al., 1988Go; Smith et al., 1989Go). In the swap-fold isomer these become A20–B18, A6–B6 and A7–A11 (Iwai et al., 1989Go; Axelsson et al., 1992Go). These forms have the same amino acid composition yet have different physical and biological properties (Gill et al., 1999Go). The disulphide swap isomer has a much lower binding affinity for both the IGF-I receptor and IGF binding protein (IGFBP, Meng et al., 1988; Hodgkinson et al., 1989; Elliot et al., 1990; Forsberg et al., 1990; Axelsson et al., 1992; Miller et al., 1993). Structural studies have shown significant differences between native- and swap-fold IGF-I (Hober et al., 1992Go; Miller et al., 1993Go; Sato et al., 1993Go).

The biological effects of IGFs and insulin are mediated by structurally related cell surface receptors of the receptor tyrosine kinase family. IGF-I binds with highest affinity to the type I IGF receptor and acts primarily as a growth factor regulating mitogenesis and cell differentiation. It also binds with much lower affinity to the insulin receptor (Massague and Czech, 1982Go; Ullrich et al., 1986Go). The interaction of IGF-I with its receptor is believed to be similar to that of insulin with the insulin receptor (Christoffersen et al., 1994Go; De Meyts, 1994Go; De Meyts et al., 1994Go). Insulin receptor binding is thought to require a conformational change in which the C-terminus of the B-region detaches and exposes residues of the A-region N-terminus. The importance of this flexibility of insulin in receptor binding has been shown in studies using a number of mini-proinsulins cross-linked between A1 Gly and B29 Lys (Brandenburg et al., 1972Go, 1973Go, 1975Go, 1977Go; Brandenburg and Wollmer, 1973Go; Freychet et al., 1974Go; Gliemann and Gammeltoft, 1974Go; Nakagawa and Tager, 1989Go). The affinity of these molecules for the insulin receptor becomes less as the cross-link is shortened. A single chain (des B30) insulin in which A1 Gly and B29 Lys are linked by a peptide bond is inactive yet has a crystal structure isomorphous with that of insulin (Markussen et al., 1985Go; Derewenda et al., 1991Go).

Similar studies of the interaction of IGF-I with the IGF-I receptor and insulin receptor have used `mini' IGF-I analogues. Substitution of C2 Tyr with Ala resulted in a 6-fold loss of IGF-I receptor binding, while substitution of positively charged arginine residues at C36 and C37 led to a 15-fold loss (Bayne et al., 1990Go; Zhang et al., 1994Go). A single chain IGF-I (mini-IGF-I) in which the C-region is deleted was shown to have no affinity for either the IGF-I receptor or insulin receptor, nor to have any biological activity. In the same study the substitution of the C-region of IGF-I with a four-residue glycine bridge gave a 100-fold loss of affinity for the IGF-I receptor and a 10-fold loss of affinity for the insulin receptor (Gill et al., 1996Go).

In this work, we characterized both disulphide isomers of a two-chain mini-IGF-I completely lacking a C-region. An Asn–Gly site engineered at the junction of the A- and B-regions allowed cleavage of the molecule at this site by hydroxylamine.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Construction, expression and purification of cleavable mini-IGF-I mutant (C-mini-IGF-I)

C-mini-IGF-I was constructed by site-directed mutagenesis using the Sculptor in vitro mutagenesis system (Amersham International). DNA sequences were confirmed by the dideoxy chain termination method (Sanger et al., 1977Go). C-mini-IGF-I was expressed in Saccharomyces cerevisiae, purified and disulphide isomers separated as described previously (Gill et al., 1996Go, 1999Go).

Hydroxylamine cleavage

A 1 mg amount of protein was incubated at 45°C for 4 h in 2 M hydroxylamine, 0.2 M Tris–HCl, pH 9.2 and the pH was adjusted to <6.0 with concentrated HCl. The protein was then desalted by washing with eight volumes of doubly distilled water in a Centricon 3 (Amicon). Cleavage at the junction of the A- and B-chains was confirmed by N-terminal sequencing of the reduced products after Tricine–SDS–polyacrylamide gel electrophoresis and electroblotting (Schagger and von Jagow, 1987Go). The resulting product is referred to as cleaved C-mini-IGF-I.

Cell culture

A stable NIH 3T3 mouse fibroblast cell line previously transfected with a wild-type IGF-I receptor clone WT2 (Grønborg et al., 1993Go) was grown in Dulbecco's Minimal Essential Medium (DMEM) (Gibco, Roskilde, Denmark) supplemented with 10% newborn calf serum (NCS), penicillin, streptomycin, sodium pyruvate and 2 mM glutamax. These cells express approximately 2x106 receptors per cell (Grønborg et al., 1993Go).

Competition binding of [125I]-IGF-I

Tracer binding to membrane-bound IGF-I receptors overexpressed in WT2 cells was carried out in the presence of increasing concentrations of wild-type IGF-I, native- and swap-fold intact and cleaved C-mini-IGF-I as described previously (Gill et al., 1996Go) with some modifications. Subconfluent monolayers of WT2 cells were washed once with HBSS and incubated with 25 mM EDTA in HBSS. The cells in suspension were then centrifuged, washed with HBSS and resuspended in HEPES binding buffer. Cell suspensions (1.2x105 cells/ml) were incubated for 3 h at 4°C with [125I]-IGF-I and increasing concentrations of ligand in a total volume of 500 µl. Bound [125I]-IGF-I was measured after centrifugation. Competition curves were drawn and numerical analysis was done in Microsoft Excel or using a Unix workstation. Data were fitted by a non-linear curve-fitting algorithm (Press et al., 1986Go) using a one-site binding model.

Thymidine incorporation assay

Stimulation of DNA synthesis by wild-type IGF-I, swap- and native-fold intact and cleaved C-mini-IGF-I was measured by incorporation of tritiated thymidine as described previously with minor modifications (Grønborg et al., 1993Go). Subconfluent monolayers of WT2 cells in 96 well plates were starved in DMEM with 2% NCS for 2 days to achieve quiescence. Wild-type IGF-I, swap- and native-fold intact and cleaved C-mini-IGF-I were added in increasing concentrations with 10% FBS as control. After 17 h the medium was aspirated and the cells pulse labeled with 1.0 µCi per well [methyl-1',2',3H]thymidine (Amersham) for 3 h. The cells were solubilized in 0.2 M NaOH and SuperMix scintillation fluid (Perkin Elmer Life Sciences, Finland) was added and allowed to mix for 1 h on a plate shaker then counted for radioactivity.

Free fat cell assay (lipogenesis)

Isolated fat cells were prepared by a modification of the method of Gliemann (1967) from the epididymal fat pads of male Sprague–Dawley rats. After digestion of the adipose tissue in 1 mg/ml collagenase in Krebs–Ringers HEPES buffer (KRH) containing 3.5% (w/v) BSA, 0.2 µM adenosine and 2 mM glucose for 45 min at 37°C, the adipocytes were washed and resuspended in incubation buffer (KRH containing 3.5% BSA, 0.56 mM glucose and 0.2 µM adenosine). Hormone stimulated conversion of glucose into lipids was measured by the method of Moody et al. (1974) after 0.5% (v/v) fat cells were incubated for 2 h at 37°C in 500 µl of incubation buffer containing 0.05 mCi [3-3H]-D-glucose (Amersham) and increasing concentrations of insulin, wild-type IGF-I, swap- and native-fold intact and cleaved C-mini-IGF-I. Incubation was terminated by the addition of 3 ml of BetaMax scintillant (ICN, Costa Mesa) and total lipids were extracted by allowing the sample to stand for 24 h at 4°C. The results are presented as the percentage of the maximum signal obtained with wild-type IGF-I. Potency was calculated by using the half-maximal stimulation with IGF-I and calculating the dose required to achieve the same half-maximal stimulation with the isomers.

Circular dichroism (CD) spectroscopy

CD measurements were carried out on an AVIV (Lakewood, NJ) 62DS CD spectrometer, equipped with a temperature control unit and a Jasco J-600 spectropolarimeter, both calibrated with a 0.1% aqueous solution of D-10-camphorsulphonic acid according to Chen and Yang (1977). The spectral bandwidth was 1.5 nm. The time constant ranged between 1 and 4 s and the cell pathlength between 0.1 and 10 mm. All measurements were carried out at 25°C.

Measurement of binding affinity to IGFBP3 by plasmon resonance

Binding affinity of IGF analogues to immobilized IGFBP3 was measured on an IAsys Manual System instrument (Affinity Sensors, Cambridge, UK) using carboxymethyldextran (CMD) sensor surfaces. Solutions of the different IGF analogues in PBS/T were added to the sensor surfaces with immobilized IGFBP3 at concentrations from 5 to 100 nM and the association kinetics were followed. Afterwards only a slight dissociation could be observed with a PBS/T wash. To remove the IGF analogues completely, incubation with 10% acetic acid was necessary. The concentration-dependent on-kinetics were analysed using IASys Fastfit Kinetics Data Analysis Software (Affinity Sensors). In all cases the association data could be satisfactorily fitted with one exponential. The affinity constants were subsequently calculated from the concentration dependence of the on rates.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Competitive binding to the IGF-I receptor

Competition data for the inhibition of [125I]-IGF-I binding to NIH 3T3 cells overexpressing the IGF-I receptor by the IGF-I analogues are presented in Figure 1Go. The concentration of wild-type IGF-I required for half-maximal inhibition of tracer binding was ~1 nM. Neither of the intact isomers competed for IGF-I binding with wild-type IGF-I tracer. Cleavage restored some ability to compete with half-maximal inhibition of tracer binding of >60 nM for the cleaved native-fold C-mini-IGF-I and >200 nM for the cleaved swap-fold C-mini-IGF-I.



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Fig. 1. Competition curves for the inhibition of [125I]-IGF-I binding the IGF-I receptor on NIH 3T3 cells overexpressing the IGF-I receptor (clone WT2) by increasing concentrations of ({blacktriangleup}) wild-type IGF-I, ({circ}) intact or (•) cleaved swap- and ({square}) intact ({blacksquare}) or cleaved native-fold C-mini-IGF-I. Competitive binding was carried out as described in Materials and methods. The data were analysed and competition curves drawn. The bound/total tracer is plotted as the fraction of tracer alone as a function of the concentration of the competing ligand. Data points are the average of duplicate values from a representative experiment.

 
Mitogenesis

To assess the mitogenic potential of the mutants, stimulation of DNA synthesis in WT2 cells was determined by measuring the incorporation of [3H]thymidine into DNA after exposure of the serum-starved cells to hormone or serum (10% FBS) for 18 h. The results presented in Figure 2Go are expressed as percentage of thymidine incorporation in the presence of hormone relative to stimulation with serum. Half-maximal stimulation with wild-type IGF-I was ~1.5 nM. As shown previously for mini-IGF-I, the intact swap-fold isomer elicited no mitogenic response at concentrations up to 100 nM (Gill et al., 1996Go). A weak mitogenic response to the intact native-fold isomer was seen at concentrations >100 nM. Cleavage of the swap-fold isomer restored the maximal mitogenic signal of the analogue to near that of wild-type IGF-I. The half-maximal response was ~40-fold lower than for wild-type IGF-I. Cleavage of the native-fold gave a half-maximal response 30-fold lower than for wild-type IGF-I and the response did not reach the same maximum.




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Fig. 2. [3H]Thymidine incorporation by serum starved NIH 3T3 cells overexpressing the IGF-I receptor after 18 h of incubation with increasing concentrations of intact or cleaved swap- and native-fold C-mini-IGF-I. (A) Data for ({blacktriangleup}) wild-type IGF-I, (•) intact and (•) cleaved swap-fold C-mini-IGF-I; (B) data for ({blacktriangleup}) wild-type IGF-I, ({square}) intact and ({blacksquare}) cleaved native-fold C-mini-IGF-I. Data are presented as the percentage of maximum obtained with 10% fetal bovine serum as a function of ligand concentration (average of triplicates ± standard deviation from a typical experiment).

 
Lipogenesis

Conversion of [3-3H]-D-glucose into lipids was used to measure the metabolic potential of intact and cleaved native- and swap-fold C-mini-IGF-I (Figure 3Go). In response to insulin, isolated rat adipocytes incorporated labelled glucose into lipids with a maximal stimulation at 40 pM. The half-maximal stimulation of lipogenesis in response to IGF-I was 3.5 nM indicating that IGF-I is acting through the insulin receptor (Christoffersen et al., 1999). Neither intact native-fold nor intact swap-fold C-mini-IGF-I elicited a lipogenic response. Cleavage restored the ability of both native- and swap-fold C-mini-IGF-I to stimulate lipogenesis with potencies of 70 and 40%, respectively, and a maximal response approaching that of wild type IGF-I.




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Fig. 3. Intact and cleaved swap- or native-fold C-mini-IGF-I were compared with wild-type IGF-I and human insulin for their ability to stimulate lipogenesis in primary rat adipocytes after a 2 h incubation at 37°C with increasing concentrations of the ligand. Data are presented as the percentage of maximum [3-3H]glucose incorporation into lipids obtained with wild-type IGF-I. The dose–response curve for insulin is shown for comparison. Data points represent the average of triplicate values from two experiments ± standard deviation. (A) Data for ({blacklozenge}) insulin, ({blacktriangleup}) wild-type, ({circ}) intact and (•) cleaved swap-fold C-mini-IGF-I. (B) Data for ({blacklozenge}) insulin, ({blacktriangleup}) wild-type, for ({square}) intact and ({blacksquare}) cleaved native-fold C-mini-IGF-I.

 
Structural analysis of IGF-I analogues

The near-UV CD spectra of intact and cleaved native- and swap-fold C-mini-IGF-I are presented in Figure 4Go. The spectra of intact native- and swap-fold C-mini-IGF-I are similar to those of native- and swap-fold mini-IGF-I, respectively (Gill, 1994Go). The spectra of both intact isomers show small bands between 260 and 270 nm that are not present in the spectra of the cleaved isomers. These are likely to be due to phenylalanine fine structure and are also seen in mini-IGF-I spectra. The disappearance of these bands upon cleavage of the proteins suggests that the phenylalanine side chains are released from their usual position and now point out into the solvent.



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Fig. 4. Far-UV CD spectra of (A) cleaved (labelled C) and intact (labelled U) native-fold C-mini-IGF-I and (B) cleaved (labelled U) and intact (labelled U) swap-fold C-mini-IGF-I in 5% acetic acid. Protein concentrations were normalized for tyrosine content.

 
The far-UV spectra of intact and cleaved native- and swap-fold C-mini-IGF-I are presented in Figure 5Go. The spectra are broadly similar to those of native- and swap-fold mini-IGF-I, respectively, and suggest that the proteins retain their overall structure upon cleavage. The differences between the cleaved and intact proteins are the same in both native- and swap-fold. A decrease in amplitude is seen in the spectra of both cleaved molecules. This may be due to a loss of some helical structure or to a change in orientation of the helices.



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Fig. 5. Near-UV CD spectra of (A) cleaved (labelled C) and intact (labelled U) native-fold C-mini-IGF-I and (B) cleaved (labelled C) and intact (labelled U) swap-fold C-mini-IGF-I in 5% acetic acid. Protein concentrations were normalized for tyrosine content.

 
Measurement of binding affinity to IGF BP3

The binding affinities to BP3 of native- and swap-fold C-mini-IGF-1 before and after hydroxylamine cleavage are shown in Table IGo. The affinities of the cut and uncut proteins showed no significant difference, suggesting that the overall structure is unchanged after hydroxylamine treatment.


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Table I. Binding affinity of IGF-I analogues to IGFBP 3 measured on an IAsys Manual System instrument using CMD sensor surfaces
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Both isomers of C-mini-IGF-I were correctly folded, as demonstrated by the far-UV CD spectra. Hydroxylamine treatment was shown by N-terminal sequencing to cut the proteins as predicted at the junction of the A- and B-regions. The far-UV CD spectra and the ability of the cut proteins to bind to IGFBP3 confirmed that they retained the insulin fold after treatment with hydroxylamine.

Cleavage restored weak binding affinity for the IGF-I receptor in both native- and swap-fold proteins. Cleavage of both isomers was able to restore a near maximal lipogenic response with only moderately reduced potency relative to wild-type IGF-I (41 and 70% for the swap- and native-fold, respectively). In contrast, mitogenic potential, as measured by the stimulation of tritiated thymidine incorporation into DNA, was restored to a much lesser extent, with a 40-fold reduction in potency with the swap-fold isomer and a 30-fold reduction with the native-fold isomer. This agrees with the concept that the C-peptide domain of IGF-I is involved in binding to the IGF-I receptor, but not the insulin receptor. Intact native-fold C-mini-IGF-I was able to elicit a weak mitogenic response at very high concentrations.

The increased affinities and biological activities observed upon cleavage of the isoforms lend support to the hypothesis that the loss of activity in mini-IGF-I is caused by the inability of the B-region ß-strand to move away from the body of the molecule and expose core residues for interaction with the insulin and IGF-I receptors (Gill et al., 1996Go).

The solution structure of native-fold mini-IGF-I shows clear differences from wild-type IGF-I in the ß-strand region B23 Phe–B24 Tyr–B25 Phe–B26 Asn. Owing to the restriction imposed by deletion of the C-region the ß-strand is displaced and the side chains of Phe23 and Phe25 point into the core of the molecule (De Wolf et al., 1996Go). This allows the hydrophobic core of mini-IGF-I to remain shielded from solvent and makes this region unavailable for receptor binding. The disappearance of bands attributed to phenylalanine fine structure in the near-UV spectra of both cleaved native- and swap-fold C-mini-IGF-I suggests that the side chains of the phenylalanine residues are now oriented towards the solvent. This would make this region accessible for receptor interaction and is consistent with the return of biological activity in the cleaved proteins.

The data presented in this paper add further weight to the theory that IGF-I binds its receptor in a manner analogous to that of insulin receptor binding except for the specificity difference conferred by the C-peptide (Christoffersen et al., 1994Go; De Meyts, 1994Go). A conformational change in the ligand appears to be a prerequisite for receptor binding. Differences between the insulin and IGF-I molecules have evolved so that each ligand binds to its own receptor with highest affinity.


    Notes
 
5 To whom correspondence should be addressed Back


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 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received May 25, 2000; revised November 1, 2000; accepted November 9, 2000.





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