Assessment of biological activity of synthetic fragments of transforming growth factor α

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EGF, TGF-a, AND RELATEDFACTORS

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question that remains unanswered is whether changes at these critical residues affect the conformation of TGF-a or are directly involved as contact points between TGF-a and the EGF receptor. Clarification of this issue will require a detailed structure analysis of native TGF-a as well as several mutant versions of TGF-a by NMR or X-ray crystallography techniques.

[19] A s s e s s m e n t o f B i o l o g i c a l A c t i v i t y o f S y n t h e t i c Fragments of Transforming Growth Factor a

By GREGORY SCHULTZ and DANIEL TWARDZIK Introduction Transforming growth factor a (TGF-a) is a mitogenic hormone which appears to play important roles in normal fetal development, tissue regeneration, and tumor growth. 1Mature TGF-o~ is a 50 amino acid, single-chain polypeptide (Fig. 1) belonging to the family of structurally related peptide growth factors that includes epidermal growth factor (EGF), 2 vaccinia virus growth factor (VGF), 3 and amphiregulin. 4 TGF-a has substantial (-40%) sequence similarity with the other growth factors in the EGF-like family and, in particular, shares similar placement of three conserved intrachain disulfide bonds. All four structurally related growth factors bind and activate the tyrosine kinase activity of a common 170-kDa membrane receptor. 5 The sequence of the gene for TGF-a suggests that it is synthesized as part of a larger, single-chain, transmembrane glycoprotein of 160 amino acids. The mature 50 amino acid polypeptide hormone is apparently cleaved from the precursor between Ala and Val residues at both the N and C terminals. 6 A major biochemical objective in studying growth factor-receptor systems is the identification of amino acid sequences which form the specific I R. Derynck, Cell (Cambridge, Mass.) 54, 593 (1988). 2 C. R. Savage, Jr., T. Inagami, and S. Cohen, J. Biol. Chem. 247, 7612 (1972). 3 j. p. Brown, D. R. Twardzik, H. Marquardt, and G. J. Todaro, Nature (London) 313, 491 (1985). 4 M. Shoyab, V. L. McDonald, J. G. Bradley, and G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 85, 6528 (1988). 5 C. S. King, J. A. Cooper, B. Moss, and D. R. Twardzik, Mol. Cell. Biol. 6, 332 (1986). 6 G. J. Todaro, D. C. Lee, N. R. Webb, T. M. Rose, and J. P. Brown, Cancer Cells 3, 51 (1985).

METHODS IN ENZYMOLOGY, VOL. 198

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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BIOLOGICAL ACTIVITY OF TGF-t~ FRAGMENTS

201

Io

.vs

F A

TI

P

I

NHz

0/

40

FIG. 1. Primary amino acid sequence of rat TGF-c~. TGF-a has substantial sequence similarity with EGF, VGF, and amphiregulin, including the alignment of the three disulfide bonds.

receptor-binding domains of the growth factor. Once the sequences have been identified, a more rational approach can be used to select peptide sequences of the growth factor which might have agonist or antagonist activities, or sequences which might be useful in generating specific antibodies that would neutralize the action of the growth factor. As part of our studies on the role of TGF-a in various biological systems, we utilized standard solid-phase techniques to synthesize a series of peptides that encompassed the entire primary sequence of the mature TGF-a 50-mer. 7 These peptides and several peptides purchased from commercial sources were evaluated in five different assays for TGF-a activity. Previous volumes of this series (Volumes 146 and 147) have dealt extensively with the purification of TGF-a from transformed cells, biochemical assays for TGF-a, identification of receptor proteins for TGF-a, and techniques for solid-phase synthesis of mature TGF-a. We have drawn on many of the techniques discussed in those volumes, and in this chapter we discuss how we used and modified these techniques. Several studies on fragments of TGF-a and EGF also helped to guide our work, and recent publications on the partial solution structures of TGF-o~ and EGF provide additional insight into putative receptor-binding domains of TGF-a. 7 K. Darlak, G. Franklin, P. Woost, E. Sonnerfeld, D. Twardzik, A. Spatala, and G. Schultz, J. Cell. Biochem. 36, 341 (1988).

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EGF, TGF-a, AND RELATEDFACTORS

[19]

2-

-2 0

10

20

30

40

50

Residue Number FIG. 2. Hydrophilicity plot of rat TGF-a. TGF-a contains three separate regions of hydrophilicity.

Strategy for Selection of Transforming Growth Factor a Fragment Sequences Since all the members of the EGF family of peptide growth factors bind to a common membrane receptor protein (170 kDa), one might expect that the receptor-binding domain of TGF-a would lie primarily in the region which has the highest degree of conservation of amino acid sequence between the members of the EGF family of growth factors. In addition, one would expect the receptor-binding domain to have a strong hydrophilic character which would favor its orientation to the surface of the peptide where it could interact with the receptor protein. The third disulfide loop region (34-43) of TGF-a has the highest degree of sequence similarity with the other members of EGF family. 3 In addition, hydrophilicity plots of TGF-a (Fig. 2) indicate three discrete sequences which are theoretically exposed on the surface of the peptide and, thus, are potentially able to interact with the TGF-a receptor. Initial studies of the activities of synthetic fragments of TGF-a and EGF indicated that different regions of the related growth factors were active in binding to their common receptor. Nestor e t a l . 8 reported that 8 j. j. Nestor, Jr., S. R. Newman, B. DeLustro, G. J. Todaro, and A. B. Schreiber, Biochem. Biophys. Res. Commun. 129, 226 (1985).

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203

a fragment of rat TGF-a comprising the C-terminal third disulfide loop (residues 34-43) had low affinity (500-fold less potent than EGF) for EGF receptors of human cells. The fragment also blocked the mitogenic effect of TGF-o~ and EGF on human fibroblasts and inhibited vaccinia virus infection purportedly by occupying the EGF receptor of target cells. 9 In contrast, Komoriya e t a l . Jo reported that the C-terminal third disulfide loop of mouse EGF (residues 32-48) had no detectable activity in competing for EGF receptor binding. They concluded that the second disulfide loop region (residues 14-31) constituted a major receptor-binding region based on the results that both the linear and cyclic second disulfide loop region of mouse EGF (residues 14-31) induced several biological responses associated with intact EGF but were 10,000-fold less potent than intact EGF. A third study by Heath and Merrifield lj reported that a fragment comprising both the second and third disulfide loop region of EGF (residues 15-53) was 10,000 times less potent than the mature EGF (residues 1-50) for binding to the EGF receptor and stimulating DNA synthesis. Furthermore, they found that the C-terminal third disulfide loop of mouse EGF (residues 32-53) was 100,000-fold less potent than EGF in receptor binding and did not stimulate DNA synthesis. These initial studies presented a somewhat confusing and contradictory concept of the major receptor-binding domain for TGF-a and EGF. Crystal structures for EGF or TGF-a are currently not available, but partial solution structures for both TGF-a and EGF have recently been reported. The key structures detected for human 12'~3 and for murine 14'~5 EGF are similar and consist of a triple-stranded, antiparallel, fl sheet at the N terminal involving bonding between residues 3 and 23, 5 and 21, and residues 18-23 paired with residues 34-28. A small antiparallel fl-sheet structure also links residues 37-38 with 45-44 at the C terminal, and an interaction of residue 52 with the edge of the 13 sheet of the second loop region tends to restrict the position of the C-terminal segment. In contrast, a cyclic heptadecapeptide of the second loop of human TGF-a (residues 16-32) did not form an antiparallel fl-sheet structure, as observed for this 9 D. A. Eppstein, Y. V. Marsh, A. B. Schreiber, S. R. Newman, G. J. Todaro, and J. J. Nestor, Jr., Nature (London) 318, 663 (1985), 10 A. Komoriya, M. Hortsch, C. Meyers, M. Smith, H. Kanety, and J. Schlessinger, Proc. Natl. Acad. Sci: U.S,A. 81, 1351 (1984). N W. F. Heath and R. B. Merrifield, Proc. Natl. Acad. Sci. U.S.A. 83, 6367 (1986). J2 K. Makino, M. Morimoto, M. Nishi, S, Sakamoto, A. Tamura, H. Inooka, and K. Akasaka, Proc. Natl. Acad. Sci. U.S.A. 84, 7841 (1987). 13 R. M. Cooke, A. J. Wilkinson, M. Baron, A. Pastore, M. J. Tappin, I. D. Campbell, H. Gregory, and B. Sheard, Nature (London) 327, 339 (1987). 14 K. H. Mayo, Biochemistry 24, 3783 (1985). 15 G. T. Montelione, K. Wuthrich, E. C. Nice, A. W. Burgess, and H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 83, 8594 (1986).

204

EGF, TGF-a, AND RELATEDFACTORS TABLE

[19]

I

TRANSFORMING GROWTH FACTOR ix FRAGMENTSa

Peptide number

Sequence

1 2 3 4 5 6 7 8 9 10 11 12 13

TGF-a (34-50) Ac-TGF-a (34-43)-NH2 [S-AcmCysZI'32]TGF-a (21-33) [Alan]TGF-ix (22-43)-NH2 [Alalr]TGF-a (1-21)-NH2 [S-AcmCyslr'32AIa21]TGF-c~ (15-33) [AIa~I]TGF-a (16-33) TGF-a (1-15) [Tyrn]TGF-a (22-32) TGF-a (34-50) TGF-a (34-43) TGF-a (41-50) TGF-a (26-36)

a

Peptides 1 through 7 were prepared by the Merrifield method of solid-phase peptide synthesis, and peptides 8 through 13 were prepared by Peninsula Laboratories. (From Ref. 7.)

sequence of intact EGF, but instead assumed an ellipsoidal conformation. 16 Materials and Methods

Synthesis of Peptide Fragments The primary amino acid sequence of rat T G F - a (residues 1-50) is shown in Fig. I. Fragments of T G F - a evaluated for activity are listed in Table I. Peptides 1-7 are prepared by solid-phase peptide synthesis (SPPS) using double coupling with dicyclohexylcarbodiimide (DCC) active ester formation with a Peptides International Synthor 2000 automated synthesizer (Louisville, KY), and peptides 8-13 are prepared by Peninsula Laboratories (San Carlos, CA). An extensive description of SPPS techniques for preparing mature T G F - a (1-50) have been presented in a previous volume o f this series by Tam.17 The synthetic strategies we use for the T G F - a fragments are similar to that used by Tam with the a-amino groups protected with tert-butyloxycarbonyl (Boc) group and the following side16K. H. Han, C. H. Niu, and P. P. Roller, Biopolymers27, 923 (1988). 17j. p. Tam, this series, Vol. 146, p. 127.

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BIOLOGICAL ACTIVITY OF T G F - a FRAGMENTS

205

chain protecting groups: Arg(Tos), Asp(Cxl), Cys(Acm) or Cys(Meb), Glu(Cxl), His(Bom), Lys(C1-Z), Ser(Bzl), and Tyr(Dcb). Each synthetic cycle consists of (1) 5 and 25 min deprotection with 40% trifluoroacetic acid (TFA) (v/v)/10% anisole (v/v)/50% dichloromethane (v/v), (2) 5 min neutralization with 10% triethylamine in dichloromethane, and (3) double couplings (3 and 16 hr) with Boc-amino acid in the presence of N-hydroxybenzotriazole (HOBt) and DCC both at 2.5-fold mole excess over amino acid on resin. Asn and Gin are coupled as their preformed p-nitrophenyl esters in the presence of HOBt, and Arg(Tos) and Gly are coupled with DCC omitting HOBt. All couplings are monitored with the Kaiser test. Triethylamine is distilled from ninhydrin; dichloromethane is distilled from potassium carbonate; dimethylformamide is distilled under reduced pressure. Other solvents and reagents are of analytical grade. Peptides are deprotected and cleaved from the resin with anhydrous HF (I hr at 0°) in the presence of 5%p-cresol (v/v) and 5% dimethyl sulfide (v/v). After evaporation of HF and scavengers, solid residues are washed with diethyl ether, extracted with 10% acetic acid (v/v), and lyophilized. Residues are dissolved in 30% acetic acid (v/v) and desalted on a Sephadex G-25 column eluted with 30% acetic acid. The major peak of each peptide is pooled, lyophilized, and purified by reversed-phase high-performance liquid chromatography (HPLC) using a Cla column (10 × 250 mm, 300 .~ pore size, Vydac, Hesperia, CA) with a gradient of 15 to 30% acetonitrile/ water containing 0.05% TFA. We have found that the separation of peaks is substantially improved with silica matrices that have large pore sizes, that is, 300 A, compared to resins that have smaller pore sizes such as 75 ~,. In addition, it is extremely important to use new reversed-phase materials to purify fragments of TGF-a. We have found that reversed-phase HPLC columns continue to release small amounts of active TGF-a and EGF even after extensive washing under extremely effective elution conditions. Since the activities of small fragments of TGF-a are very low compared to those of the intact molecule, artifacts can be generated by very low levels of EGF or TGF-a eluting from columns during purification of peptide fragments. Peptides containing cystine disulfide bonds are produced by washing the residues obtained after HF cleavage with diethyl ether containing 1% 2-mercaptoethanol, then extracting with 10% acetic acid under nitrogen. After lyophilization, residues are dissolved in 0.2% acetic acid saturated with nitrogen, and 2 N aqueous ammonia is added gradually to give a final pH of 7.0 to 7.5. Solutions of the peptides are treated with 20 /~M K3Fe(CN)6 until a permanent yellow color is generated and then stirred for an additional 20 min. Solutions are passed through an anion-exchange column (AG 3-X4, acetate form) to remove excess ferri- and ferrocyanide

206

EGF, TGF-a, AND RELATEDFACTORS

[19]

ions and then lyophilized. Residues are desalted by chromatography on a Sephadex G-15 column eluted with 30% acetic acid and then purified by HPLC as described above. Competition o f Epidermal Growth Factor Binding

Peptides are tested for their ability to compete for ~25I-labeled EGF binding using two receptor sources: placental cell microvillus membranes~8 and A431 J9 cells. Placental microvillus membrane is our preferred source of EGF/TGF-a receptor because very large amounts of relatively pure receptor can be obtained rapidly at very low cost. Once a large preparation of placental microvillus membranes has been prepared, it can be stored in small aliquots at - 8 0 ° until thawed with essentially no loss of activity. Placental microvillus membranes are prepared from fresh, normal term human placenta that is immediately cooled to 4° and maintained at this temperature throughout the procedure. Placental membranes are removed and the placental lobes cut into small pieces (1 cm 3 cubes) using crossed scalpel blades. The pieces of placental tissue are washed 3 to 5 times in 0.15 M NaCI containing 50 mM CaC12 until the solution is free of blood. Failure to remove red blood cells results in substantial contamination of microvilli with material that may contribute to nonspecific binding. Washed pieces of tissue are vigorously stirred on a magnetic stirrer for l hr at 4 ° in 1.5 volumes of 0.15 M NaCI. This step sheers off microvilli from the plasma membrane of placental cells while minimally disrupting the cells. Large tissue pieces are removed by filtration through a coarse pore nylon membrane (500/~m pores), and the filtrate is centrifuged at 500 g for 10 min. The supernatant is centrifuged at 25,000 g for 30 min, after which the supernatant is discarded and the pellet resuspended in buffer (150 mM NaCI, 2 mM CaC12, and 50 mM NaH2PO 4, pH 7.4) at a high concentration. The microvillus membrane suspension is frozen in 1-ml aliquots at - 80 ° and thawed one time for use in the receptor-binding assay. Aliquots of placental membrane (100/A containing -50/.~g protein) are incubated in 12 × 75 mm polypropylene tubes with a 100 ~l of J25I-labeled EGF [100,000 counts/min (cpm) at a concentration of -100 pM] and 100 /~1 of buffer containing increasing concentrations of either unlabeled EGF or peptides for 2 hr at 37°. Four milliliters of buffer at 4° is then added, and the placental microvillus membranes are pelleted by centrifugation at 7000 g for 20 rain at 4°, the supernatant removed by aspiration, and pellets counted with a y scintillation counter. 18Ch. V. Rao, N. Ramani,N. Chegini,B. K. Stadig, F. R. Carman,Jr., P. G. Woost, G. S. Schultz, and C. L. Cook, J. Biol. Chem. 260, 1705(1985). 19j. E. DeLarcoand G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 75, 4001 (1978).

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BIOLOGICAL ACTIVITY OF TGF-c~ FRAGMENTS

207

Competition for binding of ~25I-labeled EGF to receptors on A431 cells is performed using fixed monolayers of cells. A431 cells are seeded onto 24-well plates and grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum (v/v) to a density of approximately 10,000 cells/ well, then washed and fixed by a brief 10-min exposure to 10% formalin in phosphate-buffered saline (PBS). Brief fixation by formalin does not destroy EGF binding, and replicate values are more consistant since fixed cells do not slough off plates as easily as do unfixed cells. Using a procedure similar to that described above, 100 ~1 of buffer containing 125I-labeled EGF (final concentration - 3 0 0 pM) is added to each well along with 100 ~1 of buffer containing increasing concentrations of either unlabeled EGF or TGF-~ peptide fragments. After incubation for 1 hr at 37 °, the wells are washed with PBS and the cells solubilized by the addition of 1 ml of 0.5 N NaOH; the radioactivity is measured with a y scintillation counter.

Induction of DNA Synthesis Peptides are tested for the ability to stimulate DNA synthesis using incorporation of radioactive analogs of thymidine, 5-[125I]iodo-2'-deoxyuridine (IdU), or tritiated thymidine into trichloroacetic acid-insoluble material of diploid human foreskin fibroblasts (HFF) or mouse 3T3 fibroblast cells. Cultures of H F F are established from explants of newborn foreskin, and H F F are seeded in 96-well plates and grown to confluency in DMEM containing 10% calf serum and antibiotics. Quiescent cultures of H F F which have been held in 0.2% calf serum for 2 days receive 10 ng/ml of TGF-~ or 100 ng/ml of peptides. After 8 hr, cultures are labeled with IdU (I0/~Ci/ml) for 2 hr, then washed with PBS followed by 10% trichloroacetic acid (TCA). Cells are dissolved in 1 ml of 1 N NaOH, the amount of radioactivity incorporated into TCA-insoluble material is measured by y scintillation counting, and the mean level for triplicate wells is calculated. The effect of the peptides on DNA synthesis in 3T3 fibroblast cells is determined using the procedure described below.

Inhibition of EGF-Induced DNA Synthesis Confluent cultures of the J-2 clone of mouse 3T3 fibroblasts (H. Green, Harvard University) are washed with PBS and held in chemically defined medium (CDM) (equal parts of DMEM, Medium 199, and Ham's F10 and buffered with 25 mM HEPES to pH 7.4) containing 0.5% calf serum for 24 hr, then harvested with trypsin. Twenty-four-well plates are seeded with 30,000 cells/well in 500/xl of CDM containing 0.5% calf serum and tritiated thymidine (1/zCi/ml, Amersham, Arlington Heights, IL, [methyll',2'-3H]thymidine, final specific activity I00 t~Ci/mmol). Five hundred

208

EGF, TGF-a, AND RELATEDFACTORS

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microliters of CDM containing 0.5% calf serum and the indicated levels of serum, EGF, or TGF-a peptides is added and the cells incubated for 72 hr. Culture wells are washed twice with PBS, the DNA is precipitated with 5% trichloroacetic acid (TCA) and washed with methanol, the cells are then dissolved in 1 ml of 1 N NaOH, and the radioactivity is measured with a/3 scintillation counter.

Stimulation of Anchorage-Independent Cell Growth A soft agar colony growth assay is performed using normal rat kidney fibroblasts (NRK), clone 49F.19 Agar plates are prepared in 60-mm petri dishes by first applying a 2-ml base layer of 0.5% agar (w/v) (Difco, Detroit, MI, Agar Noble) in DMEM containing 10% calf serum. Over this basal layer, an additional 2 ml of 0.3% agar in the same medium/calf serum mixture is added which contained 30,000 NRK cells/ml and TGF-a (5 ng/ml) or TGF-a fragment peptides (5 or 50 ng/ml). The cells are incubated at 37 ° in a humidified atmosphere of 5% COz in air. Colonies are measured unfixed and unstained by using a microscope with a calibrated grid. The number of soft agar colonies represents the number of colonies containing a minimum of 20 N R K cells per six random low power fields 10 days after seeding. Plates of N R K cells treated with TGF-O alone or without TGF-/3 did not form colonies.

Growth Factor-Induced Phosphorylation Placental microvillus membranes are isolated as described above, and aliquots (100 /xl containing 50 /zg) are incubated for 10 min at 22 ° in phosphorylation buffer (20 mM MgCI2, 1.5 mM MnC12, 25 mM HEPES, pH 7.4) which contains EGF, TGF-a, or TGF-a peptides at the designated amounts. The membranes are then cooled to 4°, and phosphorylation is initiated by addition of 5/.~M [y-32p]ATP, 100/zM adenyl-5'-yl imidodiphosphate (AMP-PNP), and 10 mM Na2MoO4 (which reduces phosphatase activity). The final reaction mixture is 200 ~1. After incubation for 10 min at 4 °, phosphorylation is stopped by addition of Laemmli 2° sodium dodecyl sulfate (SDS) sample buffer and heating for 15 min at 60°. Samples are chromatographed by SDS-10% (w/v) polyacrylamide gel electrophoresis, the gels are stained with Coomassie blue and dried under vacuum, and autoradiography is performed at - 7 0 ° using Kodak X-Omat AR film and Lightning Plus intensifying screen (Du Pont, Wilmington, DE). Proteins 20 U. K. Laemmli, Nature (London) 227, 680 (1970).

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BIOLOGICAL ACTIVITY OF T G F - a FRAGMENTS

209

of known molecular weights are used as standards to calculate the apparent molecular weight of phosphorylated bands.

Iodination of Epidermal Growth Factor Recombinant human EGF was provided by Dr. Carlos Nascimento of Chiron Corporation (Emeryville, CA). We have used several different methods for radioactively iodinating EGF including lactoperoxidase/glucose oxidase, Iodogen, Bolton-Hunter, and chloramine-T. All these methods produce 125I-labeled EGF which is suitable for the receptor binding assays described here. The main objectives of the iodination are to obtain specific activities in the range of 100 to 400 ~Ci/~g and to retain biological activity of the iodinated EGF sample. We routinely use chloramine-T as the oxidizing agent in the iodination reaction because the reaction is the simplest, least expensive, and most consistent in our hands. However, it is important to recognize that the chloramine-T method is also the most vigorous and potentially damaging method, and it may not be compatible with some applications. For example, the rabbit polyclonal antiserum we prepared to TGF-a peptide 1 did not recognize mature TGF-a iodinated by chloramine-T, but the antiserum bound very well with mature TGF-a iodinated by the Bolton-Hunter reagent. The procedure used to iodinate EGF is modified from Carpenter and Cohen 2~ and is carried out using an approximately equal molar ratio of EGF to Na125I. The reaction is performed in a 15-ml conical centrifuge tube mounted on a magnetic stirring plate with a short piece of paper clip as a stirring bar. Twenty microliters of 50 mM phosphate buffer, pH 7.5, containing 2.5/xg EGF is added to 20/xl of 500 mM phosphate buffer, pH 7.5, containing 1 mCi of carrier-free NaJ25I (13.4 mCi//xgof iodine). Ten microliters of 50 mM phosphate buffer containing 100 p.g of chloramineT is added, and the reaction is stopped after 25 sec by the addition of 100 /A of 50 mM phosphate buffer containing 100/xg of sodium metabisulfite. One hundred microliters of 1% KI (w/v) solution is added followed by 100 /zl of 1% y-globulin (w/v), and the reaction mixture is added to a 1 × 50 cm column of Sephadex G-50 and eluted in 1-ml fractions with column buffer (150 mM NaC1, 1 mM CaCIE, and 50 mM phosphate, pH 7.5). The labeled EGF is pooled and stored at 4°. Specific activities generally range from 100 to 300 ~Ci//~g, and the labeled EGF is usable for 4 to 6 weeks. Rechromatography on Sephadex G-25 column is not usually necessary prior to receptor competition assays. 2t G. Carpenter and S. Cohen, J. Cell Biol. 71, 159 (1976).

210

EGF, TGF-a, AND RELATEDFACTORS

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TABLE II PHYSICOCHEMICAL PROPERTIES OF PEPTIDE FRAGMENTSa

Rf Peptide number

BAW

BAWP

(HPLC)

1 2 3 4 5 6 7

0.08 0.05 0.09 0.02 0.15 0.05 0.16

0.77 0.76 0.78 0.70 0.74 0.78 0.77

2.90 3.96 4.40 3.39 4.55 3.18 3.92

K'

a Purifiedpeptides gave the indicated relative migration in TLC systems: BAW, 1-butanol-acetic acid-water (4 : 1 : 5, v/v, upper phase); BAWP, l-butanol-acetic acid-water-pyridine (15 : 12 : l0 : 3, v/v). Analytical Cj8 reversed-phase HPLC was performed using 20min linear gradient (flow rate 1 ml/min) from 15 to 40% acetonitrile/water (v/v) containing 0.05% TFA.

Results

Chemical Characterization of Peptide Fragments The primary amino acid sequence of rat T G F - a is shown in Fig. 1, and the hydrophilicity plot o f T G F - a is shown in Fig. 2. Three regions contain high indexes o f hydrophilicity and encompass amino acids 8-13, 25-31, and 42-47. These sequences are located essentially within the three disulfide loops. Table I lists the synthetic fragments of T G F - a analyzed. Amino acid compositions o f the T G F - a fragments we synthesized (peptides 1-6) were within experimental error of predicted values for all the peptides. Table II lists the physicochemical properties of the T G F - a fragments (peptides 1-6), and all the peptides were greater than 95% pure by H P L C analysis and gave a single spot in two thin-layer chromatography (TLC) systems.

Competition of Epidermal Growth Factor Binding As shown in Fig. 3, E G F effectively competed for lzSI-labeled E G F binding to human placental membranes, with 50% displacement at approximately 2 n M and 90% of the total binding displaced by I0/~M unlabeled

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BIOLOGICALACTIVITYOF TGF-a FRAGMENTS

211

140 120 "0

c-

100

o

m

LI.

80"

(3

LIJ

60 =

n

4°1 20 O

-' 2

|

-11

|

-10

|

-9

i

-8

!

-7

i

-6

!

-5

i

-4

o3

Log [peptide]

FIc. 3. Competition of EGF binding to human placental membranes. Aliquots of placental membranes (50/zg) were incubated with a constant amount of ~25I-labeledEGF (100 pM) and unlabeled EGF or peptides at the indicated levels. After 2 hr at 37°, tubes were centrifuged and pellets counted. Values are the mean of triplicate samples. Peptides are EGF (C)),peptide 1 (©), peptide 2 (A), peptide 3 (&), peptide 4 (x), peptide 5 ([~), peptide 6 (11), and peptide 7(+).

E G F . In contrast, none of the T G F - a fragments (peptides 1-7) effectively c o m p e t e d for ~25I-labeled E G F binding even at 100 t~M concentrations. Utilizing A431 cells as the E G F / T G F - a receptor source, the same results were obtained: E G F (10 nM) displaced 92% of 125I-labeled E G F binding whereas none of the TGF-o~ fragments (peptides 1-5, 7) c o m p e t e d for JzsIlabeled E G F binding e v e n at 100/zM.

Induction of DNA Synthesis T G F - a (10 ng/ml) stimulated incorporation of [~25I]IdU 7-fold o v e r control cultures of h u m a n foreskin fibroblasts. Fragments of T G F - a (peptides 1-5, 7) tested at 100 ng/ml all failed to stimulate D N A synthesis a b o v e control levels. W h e n 3T3 fibroblasts were used for the mitogenesis assay, E G F (! nM) stimulated thymidine incorporation 2-fold o v e r control cultures and peptides 1 to 4 (100 /zM) again failed to stimulate D N A synthesis.

212

EGF, TGF-a, AND RELATEDFACTORS

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Inhibition of Epidermal Growth Factor-Induced DNA Synthesis 3T3 cells incubated in CDM containing 0.5% calf serum incorporated small amounts of tritiated thymidine. Addition of 10% calf serum increased thymidine incorporation approximately 5-fold, and addition of 1 nM EGF increased thymidine incorporation approximately 2-fold over incorporation in the presence of 0.5% calf serum. Simultaneous addition of 1 nM EGF with each of TGF-a fragments 1 through 4 at 100 /zM failed to significantly reduce thymidine incorporation below the level of stimulation measured with EGF alone.

Stimulation of Anchorage-Independent Cell Growth TGF-a stimulated a large number of colonies of NRK cells in soft agar. In contrast, peptides 1-5 and 7 failed to stimulate colony formation even at 10-fold higher concentration (50 ng/ml).

Growth Factor-Induced Phosphorylation Autoradiography of SDS-polyacrylamide gels of placental microvillus membranes incubated with EGF or TGF-a (2/.~M) and [32p]ATP showed enhanced phosphorylation of the EGF/TGF-a receptor (170,000 daltons) and p35 (35,000 daltons) relative to membranes incubated without the growth factors. Placental microvillus membranes incubated with TGF-a fragments (peptides 1-5, 7) at 200/zM showed no increased phosphorylation of the EGF/TGF-a receptor or p35 above control. Discussion

Analysis of the peptide fragments of TGF-a for activities characteristic of mature TGF-a gave uniformly negative results in five different assays. The peptides did not compete for 125I-labeled EGF binding to receptors of intact A431 cells or human placental membranes even at 1000 times the concentration of EGF that completely displaced the labeled EGF. Concentrations of the peptides up to l0/xM did not stimulate DNA synthesis of human foreskin fibroblasts or 3T3 cells, nor did they stimulate anchorageindependent growth of N R K cells. Also, the peptides did not inhibit EGFinduced stimulation of DNA synthesis or stimulate phosphorylation of the EGF/TGF-a receptor or p35 even when added at l0/xM. In addition to the seven peptides we synthesized, six peptides with sequences overlapping our peptides were synthesized and purified by Peninsula Laboratories. These fragments (peptides 8-13) also uniformly failed to produce any response characteristic of TGF-a in the five assays.

[20]

PURIFICATION

OF AMPHIREGULIN

213

Thus, our results of five different assays testing the 13 peptides consistently indicated that none of the individual sequences tested had the ability to bind significantly to the EGF/TGF-a receptor. Based on these results, we conclude either that the major receptor-binding domain of TGF-a is not contained entirely within any of the single peptide sequences reported here or that the conformation of the peptides in solution is sufficiently different from their conformation in intact TGF-a to prevent effective binding to the receptor. Since the solution structures predicted by NMR suggest that the disulfide loops of TGF-a and EGF are folded over each other, we feel that it is likely that the receptor-binding domain is composed of separate regions of the TGF-a sequence which fold into the correct alignment when the hormone assumes its native conformation rather than being formed from a single segment of the sequences. In addition, linear TGF-a, which has the disulfide bonds reduced, and polymeric EGF molecules have 100- to 1000-fold lower potency, respectively, than the correctly folded hormones, even though both structures encompass the entire primary amino acid sequences.t1'22 There are limited data, however, to support the possibility that the conformation of the TGF-tz fragments differs significantly from structure of the sequences in the mature proteins. The cyclic peptide comprising the second disulfide loop of TGF-a appears to assume an ellipse structure rather than the B-sheet structure observed in intact TGF-a. 14 As the three-dimensional structure of TGF-a and EGF become more defined, it may be possible to synthesize fragments with constrained conformations that will closely model the conformations of the regions in the intact molecules. 22 A. R. Hanauske, J. B. Buchok, L. R. Pardue, V. A. Muggia, and D. D. Von Huff, Cancer Res. 46, 5567 (1986).

[20] P u r i f i c a t i o n o f A m p h i r e g u l i n f r o m S e r u m - F r e e Conditioned Medium of 12-O-Tetradecanoylphorbol 13-Acetate-Treated Cell L i n e s

By MOHAMMED SHOYAB and GREGORY D. PLOWMAN Introduction

We have recently reported the isolation of a novel growth regulatory glycoprotein, termed amphiregulin (AR), from the serum-flee conditioned medium of MCF-7 human breast carcinoma cells that had been treated METHODS IN ENZYMOLOGY, VOL. 198

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