Structural features involved in the biological activity of insulin and the insulin-like growth...

Vol. 133, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 17, 1985 Pages 423-429

STRUCTURAL FEATURES INVOLVED IN THE BIOLOGICAL ACTIVITY OF INSULIN ANI THE INSULIN-LIKE GROWTH FACTORS: A27,nsu,,n/B,GF-,*

Satish JoshIt, Hiroshi Ogawal, 6. Thompson Burke’, Lucy Y-H. Tseng2, Matthew M. Rechier2 and Panayotis G. Katsoyannislr+

IDepartment of Biochemistry, Mount Sinai School of Medlclne of the City Unfverslty of New York, New York, New York 10029

2Moiecuiar, Cellular and NutrItional Endocrinology Branch, Natlonal Institute of Arthritis, Diabetes, and Dlgestlve and Kidney Diseases,

Natlonal lnstltutes of Health, Bethesda, Maryland 20205

Received October 25, 1985

su)IMARy: A synthetic insulin-like compound consisting of the A-chain of insulin extended at its carboxyi terminus with the hexapeptide nD-domainw of insuiin- llke Growth Factor II, linked via dlsuifide bonds to a B-chain corresponding to the “B-domaIn” of Insulin-like Growth Factor I, has been examined for Insulln- ilke metabolic actlvlty and for mltogenlc actlvlty. The synthetic materlal (A27insuIIn~BiGF-i 1 is less potent than insulin In metabolic assays, and less potent than both insuiln and IGF-I In mftogenic assays. it is proposed that neither the wD-domalnn nor the “B-domain” of the IGFs Is a major contri- butor to mltogenic actlvlty. Thelr presence ln the same molecule does not result In slgniflcant growth-promotfng activity. 0 1985 Academic Press, Inc.

The Insulin-ilke Growth Factors (IGF-I and IGF-ii) are poiypeptides

In humen plasma which show strlking homology with prolnsuiin. The homologous

sequences are ilmited to an “A-domaIn” and a “5domaIn” which correspond to the

A- and B-chains of Insulin, respectlveiy (1). The amino-terminal “B-domaIn” Is

linked vta a W-domain” to the “A-domain”, which in turn Is connected at Its

carboxyi terminus to a SIX- or eight-residue W-domaln”, not present in pro-

insui In. Computer-graphics studies (2,J) indicate that the IGFs can assume a

three-dlmensionai structure sfmllar to the x-ray model of insulfn, and the

*This work was supported In part by the United States Pubilc Health Service. Natlonai institutes of Health Research Grant No. AM 29988.

+ To whom correspondence should be directed.

Abbrevlatlons used: IGF-I, insui in-i Ike Growth Factor I; IGF-I I, lnsui In-i Ike Growth Factor-Iii HPLC, High-Performance Llquld Chromatography.

0006-291x/85 $1.50

423 Copyright 0 1985 by Academic Press, Inc.

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spectrum of blologlcal actlvltles dlsplayed by these compounds Is qualitatively

similar, although the relative potency of the IGFs and insulin vary greatly

depending upon the tissue or cell type used for the assay, and upon the parti-

icular activity being measured. Thus, insulin Is much more potent than the

IGFs in short-term metabolic effects such as the stimulation of glucose trans-

port In adipocytes, while the IGFs are considerably stronger than insulin in

the stimulation of DNA synthesis In ftbroblasts (4,5). We are conducting

studies designed to identify the structural features of insulin and the IGFs

responsible for the quantltatlve differences in their biological activities.

In the present work, we describe the synthesis and biological evaluation of a

hybrid molecule which embodies the A-chain of insulin extended at Its carboxyl

terminus with the hexapaptlde “D-domaIn” of IGF-II, to which a B-chain

corresponding to the wB-domalnn of IGF-I is attached via disulfide bridges.

MATERIALS AM) METHODS

General Aspects of the Synthesls of AP7insulin/BlGF-1:

The synthesis of this compound was carried out by the Interaction of the S-sulfonated forms of the synthetic A- and B-chains in the presence of dlthiothreltol (6). The key Intermediates for this procedure were the protected heptacosapeptide embodying the entlre amino acid sequence of the A-chain of sheep insulin extended at Its carboxyl terminus with the hexapeptide “D-domaIn” of human IGF-I I, and the protected trtacontapeptide embodylng the entire amino acid sequence of the B-domain of human IGF-I. The synthesis of these protected peptides, and the procedures employed in the deblocking, Isolation and purification in the S-sulfonated form, have been described in detail elsewhere (7,8). In a typical experiment, the 27-residue A-chain S-sulfonate (11.9 mg) and the 30-residue B-chain S-sulfonate (6.2 mg) were dlssolved in glycine buffer (O.lM, pH 10.5, 3.5 ml). To this mixture, dlthiothreitoi (1.7 mg) was added, and the mixture was stirred for 36 h at 40c. The product was isolated vta picrate as the hydrochloride.

For purif icatt’on,, the hydrochloride salt was dissolved In acetate buffer (0.024M Nat, pH 3.3, 2.0 ml), and applied to a column of CM-cellulose (21.5 x 0.9 cm), equilibrated with the same acetate buffer. The coiumn was washed with 50 ml of the buffer, and eluted with an exponential gradient extending from 0 to 0.35M NaCl In the same buffer. Conductivity was monitored using a Radiometer conductivity meter, and absorbance at 278 nm was measured with an ISCO absorbance monitor. The elution profile Is shown in Figure 1. Material eluting between 96 ml and 130 ml was concentrated to 5-6 ml, and isolated via picrate as the hydrochloride. Yield 1.15 mg.

Upon isoelectric focusing in a mfxture of.pH 4-6 and pH 3-10 ampholytes, the product focused into a single band (data not shown). A sample of the product was subjected to reversed-phase HPLC under the conditions described (8). The elution proflle, shown in Figure 2, revealed the presence of a single

424


F Chromatography of the compound obtalned by ccnnbinatlon of the -suIfonated A-chain of sheep A 271nsulln and the S-sulfonated human IGF-I

B-chain domaln on a 21.5 x 0.9 cm W-cellulose column with an acetate buffer (0.024M Na+, pH 3.3) and an exponential NaCl gradient. The compound (96-130 ml of the effluent) was recovered vla picrate as the hydrochloride.

Reversed-phase HPLC of the purlfled hybrid compound on a uBondapak at 2 ml/mln wlth an acetonltrlle gradient, O-638 over 30 min.

major species. Amlno acid analysls after acid hydrolysis gave the followlng composition, expressed In molar ratios: ASP5.5 Thr3.z Ser2.5 GlU8.6 -3.3 Gly6.7 Ala3.g Yal3.0 Ileo. Leu5.5 Tvr3.3 PW.3 L~sz.2 Argoso Theory: Ser3 Glug Pro3 Tyrg Phe3 “:F$ AZ;:? Cys not determlned.

GlY6 Ala4 Val4 I lel Leq

lnsu-l -Assavs: The abl I Ity of the synthetic hybrld compound to compete wlth C'25ll

lnsulln for lnsulln receptors In rat Ilver plasma membranes and Its actlvlty In stlmulatlng the tncorporatlon of [3H]glucose Into organic-extractable materlal In rat adlpocytes were assayed by methods recently described In detail (9). lnsullnm

Cross-reactlvlty to antlbodles ralsed against human lnsulln was measured uslng a commercial klt supplied by Amersham. Reagents were reconstituted as dlrected In the klt, and lmnune preclpltates were collected on cellulose- acetate filters (Sartorlus Type 11107, Hayward, CA). Other details of thls assay are given In (9). !zcQHth-Factor AiTisws:

The ability of the hybrld compound to compete wlth C~~~IIIGF-I for IGP receptors In chick-embryo flbroblasts, lncorporatlon of [3H]thymldlne

and Its actlvlty In stlmulatlng the Into the DNA of serum-starved chick-embryo

flbroblasts was assayed by methods described elsewhere (10,lZ).

RESULTS

A27 lnsulln/RLGP-l displayed the ablllty to compete with [125l]

Insulin for fnsulln receptors In rat liver plasma membranes. Prellmlnary

425


CONCENTRATION OF INSULIN OR INSULIN-LIKE COMPOUND (np/mLl

Effect of lnsulfn W and Az7~nsulln/81~-l (0) on I tpogenesIs ad I pocytes . Stlmulatlon of Ilpogenesls, expressed as percent of

max Imum, Is plotted as a function of the concentration of lnsulln or hybrid cunpound. The data are taken from a representative experiment that was performed 4 times. The average maximum stlmulatlon of Iipogenesls due to Insulin was 6-fold over the basal activity.

experlments lndlcated a potency of less than 0.3% relative to bovine lnsulln

(data not shown13 In order to conserve materlal for more sensltlve assays, full

dose-response curves were not performed. Figure 3 shows the effect of bovine

Insulin and of Az71nsuIln /BlGF-I on the stlmulatlon of Ilpogenesls In rat

ad 1 pocytes . The hybrld Is a full agonist, reaching the same maxlmum stimula-

tlon as seen wfth lnsulln, and producing paralllel dose-response curves. The

concentration of synthettc materlal required to produce half-maximal stlmula-

tlon of Iipogenesfs lndlcates a potency of 0.26 + .07$ relative to bovine

lnsul In.

In Insulin radtolmmunoassay, the synthetic hybrid gave good straight line

plots, the slopes of which indicated a potency of 0.05% relative to bovine

insulin (data not shown).

The ablllty of IGF-I, lnsulln, and A271nsulln/BlGF-l to compete

with [T25~l~~-~ for IGF receptors In chick embryo ffbroblasts Is shown In

Figure 4. The concentration of synthetic hybrid required to displace 502 of

the label indicates a potency of 0.41 relative to IGF-I (17.32 relative to

lnsul In). The synthetic hybrid Is less potent than Insulin, and much less

potent than IGF-I In this assay.

Figure 5 shows the stimulation of incorporatlon of [3HlthymIdlne into

the DNA of serum-starved chick-embryo ffbroblasts by IGF-I, Insulin and

426


J

. .

p

,:’ d*’

./ Da /, , 0 o 0 2

IO 10.0 100 1000 w CONCENTRATION OFTEST COMPOUND0qlmLI w CONCENTRATION OFTEST COMPOUND

Ing/mL)

Es

Effect of IGF-I 0, tnsulln (0, and A271nsulLn/BtGF-l f& on lndlng of [1251]lGF-l In chick embryo fibroblasts. The lnhlbltlon of

C~~~IIIGF-I blndlng, expressed as percent of maxlmum, Is plotted as a function of the concentration of test compound. The data are taken from a representative exper lment that was performed three times. [‘25l]lGF-I binding In the absence of competitor averaged 4.3% of the Input radloactlvlty.

Fig. 5. Effect of IGF-I 0, lnsulln 0, and A27Lnsulln/BlGF-l (4 on the Incorporatlon of [3HlthymldIne Into the DNA of serum-starved chick embryo flbroblasts. Stlmulatlon of Incorporation, expressed as percent of maxlmum, Is plotted as a function of the concentration of test compound. The data are taken from a representative experiment which was performed twice. The average maxlmum stlmulatlon of [5H]thymfdine was 5-fold over basal actlvlty.

A271nsulin~BiGF-l. in agreement with the binding data, the synthetic

material is the weakest of the three agonists, 0.7% relative to IGF-I (20%

relative to insui in). (These figures show extrapolated potencies for the

hybrid; In view of its low potency, we did not determine an actual ED5O.j

DISCUSSION

in view of the conslderabie homology in primary structure of Insulin and

the IGFs (I) and of the stmilarlty of their three-dimenslonai conflgurations

(2,3), the quailtativeiy similar biological activities displayed by these com-

pounds are not surprising. The fact that quantitative differences are observed

between short-term metabolic activity and mitogenic activity for insulin and

the IGFs indicates that the structural features associated with the two types

of activity are different and possibly dissociable.

We have reported the synthesis of two hybrid molecules, one in which the

“D-dcma in” of IGF-II was added as a C-terminal extension to the A-chain of

insulin (7), and one in which the B-domain of insulin was replaced with a

421


B-chain corresponding to the B-domain of IGF-I (8). A figure showing a sche-

matic representation of these hybrids, as well as of the present molecule, Is

given in reference 11. The biologlcal evaluation of the first hybrid led us to

propose that the uD-domaintt of IGF-Ii plays no special role In the growth-

promoting activity of that factor, but that the “D domain” does diminish

Insulin-like metabolic activity, both ih the hybrid and in IGF-II Itself,

possibly by partially covering the putative insulin “receptor blndlng region”

which is conserved In both insuiln and IGF-Ii (3). The biological evaluation

of the second hybrid led us to propose that the “B-domain” of IGF-I is

important for recognition of IGF carrier proteins and IGF antibodies, but does

not, in itself, play an important role in the mitogenic activity of IGF-I

(8,111. The observation that Ainsuiin/BiGF-l is less potent than insulin in

both metabolic and mitogenic assays (7,121 led us to propose that interactions,

as yet undefined, between the A- and B-chains of insulin, and between the ItA-”

and “B-domains” of the IGFs may be required to confer full activity on either

class of miecules.

The present data confirm that the “D-domain” of IGF-II is deleterious to

insulin-like activity, but does not contribute to growth-promoting activity.

Az7insuiin/BIGF-I is (0.3% as potent as insulin in metabolic assays,

almost IO-fold lower than Ainsulin/BIGF-i* In insulin radioimmunoassay,

it is again very weak, suggesting that whatever immunogenic determinants are

preserved in Ainsuiin/BiGF-i are further compromised by the presence of

the hexapeptlde extension at the carboxyi terminus of the A chain. The present

compound is less potent than either IGF or insulin, and essentially equipotent

to Ainsuiin~BiGF-i in mitogenic assays. This confirms that neither the

I’D-dcma in” of IGF-II nor the “B-dcmafn” of IGF-I is primarily responsible for

mitogenic activity; their presence in the same molecule does not result in

significant growth-factor activity. It is possible that, as we proposed for

Ainsuiin/BiGF-is interact ions between the A- and B-chains which could

confer either insulin-ilke metaboilc activity or mitogenic activity on the

molecule are unfavorable. Alternatively, determinants on the “A-domain” of the

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IGFs or, less I Ikely, the “C-doma In,” may be of crftlcal importance in

trlggerlng this biological activity.

ACKNOULEGEMENT: The authors express their appreclatlon to Mrs. Shlma Josh1 for the amlno acid analysis.

REFERENCES

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Structural features involved in the biological activity of insulin and the insulin-like growth...

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