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CHEMICAL SYNTHESIS OF BOVINE TRANSFORMING GROWTH FACTOR-a: SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL ACTIVITY Jacob S. Tou, Mike F. McGrath, Mark E. Zupec, John C. Byatt, Bernie N. Violand, Larry A. Kaempfe and Billy D. Vineyard Monsanto Company St. Louis, Missouri 63198 Received
January
16,
1990
Bovine transforming growth factor-a (bTGF-a) is a 50 amino acid polypeptide with three disulfide linkages. In order to evaluate the biological function of this peptide, bTGF-a was synthesized via an automatic synthesizer and purified to homogeneity in high yield. The integrity of this synthetic peptide was confirmed by chemical analyses and bioassays. In a bovine liver radioreceptor assay, bTGF-a competes with radiolabeled EGF and has activity comparable to mEGF and hTGF-a. Compared to hEGF, bTGF-a elicits 01990 Academic Press, Inc. a greater response in a bovine mammary cell proliferation. Transforming growth factor-a is a secreted polypeptide which was originally discovered in tissue culture medium conditioned with 3T3 fibroblasts (1). The mature form of the molecule contains 50 amino acids and structurally is grouped into a family of factors which include EGF (2). TGF-a competes for the EGF receptor (3) and elicits similar biological effects on numerous cell types. Originally, TGF-a was thought to be intimately associated with cells expressing a transformed phenotype. However, it is now known that TGF-a can be extracted from normal tissues (4) most notably those of fetal or embryonic
origin (5). TGF-a transcripts have also been detected in early mouse (6) and rat (7) embryos indicating
that it may play a physiological
role during normal fetal development.
Previously TGF-a from two different species, human and rat, was characterized and chemically synthesized (8,9). Recently, the bovine sequence was successfully deduced from genomic clones (10). It differs from the human sequence by two amino acids and from the rat sequence by four. Since bTGF-a shares a high degree of sequence homology with h- and rTGF-a’s, the three cysteinyl disulfide linkages of bTGF-a are believed to be conserved (Fig. 1). This report describes the chemical synthesis of this complex molecule by an automatic peptide synthesizer. The characterization of this novel peptide by chemical analyses and its biological
activities are also reported.
Abbreviations: TGF-a, transforming growth factor-a; EGF, epidermal growth factor; b, bovine; m, mouse; h, human; r, rat; Boc, butyloxycarbonyl; BOM, pi-benzyloxymethyl; ABI, Applied Biosystems; DMS, dimethyl sulfide; TFA, trifluoroacetic acid; HPLC, High Pressure Liquid Chromatography; AcCN, acetonitrile; HF, hydrogen fluoride; D’IT, dithiothreitol; BME, bovine mammary epithelial. 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
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human
VVSH
FN
BOVINE
VVSH
FN
rat
VVSH
FN
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I (disulfide
linkages)
Figure 1: Sequence homology of bTGF-cr, hTGF-a and rTGF-a. Residues which are different from the bovine sequence are shown in dotted lines.
MATERIALS
AND
METHODS
Peptide synthesis: The following side-chain protected Boc-amino acids were used throughout the synthesis: Asp and Glu, cyclohexyl; Cys, 4-methylbenzyl; Thr and Ser, benzyl; Arg, p-toluenesulfonyl; Lys, 2-chloro-benzyloxycarbonyl; His, BOM; Tyr, 2-bromobenzyl. All the Boc-amino acids except His (Bachem), Asp and Glu (Permisula Lab) were purchased from ABI. Boc-Ala-OCH,-Pam resin with 0.76 mmol/gm loading was obtained from ABI. Peptide was synthesized by solid phase synthesis technique and was performed on ABI-430A automatic synthesizer (11). Amino acids were sequentially incorporated to BocAla-OCH,-Pam resin (0.44 mmol). The standard ABI coupling cycles were used except for His which was coupled with the same protocol as Arg. In order to ensure the completion of coupling, all the amino acids underwent double-couple cycle. Dimethyl sulfide was added to the TFA solution (0.1%) as a scavenger in the Boc-deprotection step. HPLC: For all the HPLC work, the following binary solvent system was used: A, water containing 5% AcCN and 0.1% TFA, B, AcCN containing 25% A with 0.1% TFA. The following eluting program was used for all analytical runs (Vydac 218TP54) reported here: O-2 min, 22% B; 2-22 min, a linear gradient from 22% B to 70% B. The elution was run at 1.1 ml/mm and monitored at 215 nm. Final product was purified by preparative HPLC on a Waters Delta Prep 3000 which was equipped with a 48 mm X 30 cm, 100 A, 15 pm of C-18 Delta Pack. Cleavage: Peptide was cleaved from the solid support by Tam’s two-step method (12). In the low HF treatment, p-thiocresol, p-cresol and DMS were charged to the vessel in a ratio of 10:1:3:26 with a total volume of 20 ml. It was stirred at 0-5°C for 2 hrs. was then washed with Solvents were evaporated to near dryness. Peptide-resin dichloromethane and ethyl acetate. This partially deprotected peptide-resin then underwent high HF treatment in the presence of HF:p-cresol:p-thiocresol (94:4.5:1.5) for one hour at 0-5°C. The resulting mixture was thoroughly washed with 300 ml of ether containing 1% mercaptoethanol and then extracted (5X) with a solution (500 ml) of 8 M urea, 0.1 M TriseHCl, 0.2 M DTT at pH 8.0. The combined extracts were filtered (8 pm) and then subject to folding and purification. Folding and purification: The above solution was sequentially dialyzed (Spectra Por 6, MW cutoff 1000) against a series of the following solutions: 8 M, 4 M and 2 M urea in 0.1 M D’IT, 0.1 M TrisaHCl at pH 8.1. DYT was removed by further dialysis (2X) with 2 M urea in 0.1 M Tris * HCl at pH 8.1. A portion (l/3) of this solution was placed in a silanized beaker at 4°C and then glutathiones, oxidized (0.3 mM) and reduced (0.15 mM), were added. After refolding, peptide buffer was exchanged to 1 M acetic acid by a series of dialyses against 4-liter each of the following solutions: 0.1 M Tris buffer at pH 8.1, water and 1 M acetic acid. This crude peptide solution was filtered (0.45 pm) and introduced onto the prep column. Fractions were collected and analyzed by analytical HPLC before pooling.
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Combined fractions were then lyophilized acetate salt.
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twice from 30% acetic acid and isolated as its
PeDtide hktDDing: Bovine TGF-(U was dissolved at 2 mg/ml in 50 mM Tris - HCI, pH 8.0. Chymotrypsin (Sigma) was added at 0.034 mg/ml and the sample was incubated at 22°C for 15 hrs. The sample was then reduced by adding 0.1 M DTT and incubated for 40 min at 55°C before being analyzed by HPLC. Bioloeical assavs: Bovine TGF-a was tested in the bovine liver receptor assay for its capacity to specifically displace radioiodinated mEGF. The liver membrane preparation was prepared in a similar way as reported by Haro et al. (13). To each assay tube was added in order: assay buffer (200 ul, 100 mM TriseHCl, 10 mM CaCI,, 0.1% BSA, pH 7.6), sample (50 ul) or standard (2-1000 ng/rnl mEGF; Collaborative Research) diluted with assay buffer; radioiodinated mEGF (50 ul, 0.08 rig/tube; Collaborative Research) and liver membranes (100 ul). The assay was incubated at room temperature for 3 hrs. A Micromedic gamma counter was used to measure the counts bound to the membrane pellet. The bioactivity of the synthetic bTGF-cr was tested using BME cells grown in vitro (14). Peptides were tested in the presence of an enriched basal media consisting of Dulbecco’s Modified Eagles Medium:Ham’s F-12 (1:l) plus 3% fetal bovine serum and 76 rig/ml insulin-like growth factor I. Cell proliferation was determined in triplicate following 12 days of culture using a fluorometric method to determine total DNA (15,16). RESULTS
AND DISCUSSION
Peotide svnthesis: The average operating time for each residue assembled into the chain (double coupled) was about 2 hrs. For Asp and Glu, cyclohexyl was chosen as the side chain protecting group in order to prevent the possible succinimide formation (17). For His, BOM group was used for the side chain protection
and it was readily cleaved in the high
HF step. Approximately 0.21 mmol, as determined by amino acid analysis, of partially assembled resins at different stage of couplings were removed for other use. The couplings of the remaining 0.23 mm01 material were continued to the completion of synthesis. The low-high HF procedure should minimize some side reactions which may occur during the traditional
one-step HF treatment
(12). Gradient
dialyses under reducing
conditions were carried out to remove small peptides and organic scavengers in the crude mixture. This crude reduced peptide solution gave a major peak in HPLC (Fig. 2a) which indicated a highly efficient synthesis. After dialyzing in the absence of D’IT, broad peaks formed at the expense of the reduced peak (Fig. 2b). Further oxidation was done by the mixed disulfide method using glutathiones. As monitored by HPLC, a new peak eluting 2 min earlier gradually increased (Fig. 2~). The height of this peak plateaued in about 3 days (Fig. 2d). Pure bTGF-cr (35 mg) was isolated effectively by a single HPLC step (Fig. 3). It accounts for approximately 10% overall yield based on the resin used. In a parallel experiment, glutathiones.
the other one-third of crude mixture was air oxidized without using However, only 20 mg of pure bTGF-cr was isolated after a similar purification
procedure. 486
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a -
C
d
Figure 2. The refolding progresswas monitored by HPLC: (a) crude mixture after HF cleavage and dialysis under reducing condition; (b) the reduced form (12.13 min.) disappearedafter further dialysisin the absenceof DW, (c) refolding wasin progress(24 hrs); (d) more correctly folded product (10.26min.) formed after 72 hrs.
blical
analvsis: The homogeneity of the isolated product was verified by three
different columns: C-18, C-4 and Phenyl silica materials. By treatment with the reducing guanidine buffer solution, the pure TGF-(r peak was cleanly converted to its reduced form (Fig. 4). The amino acid analysis of the purified bTGF-cr is in agreement with the expected amino acid composition (theoretical values in parentheses), Asx, 4.0 (4); Thr, 1.0 (1); Ser, 3.9 (4); Glx, 4.9 (5); Pro, 2.0 (2); Gly, 3.1 (3); Ala, 4.1 (4); Cys, 6.5 (6); Val, 4.3 (5); Leu, 3.1 (3); Tyr, 1.1 (1); Phe, 3.9 (4); His, 4.9 (5); Lys, 1.0 (1); Arg, 2.0 (2). Mass spectrum indicates a distinct peak at 5547 (M+H)
which corresponds to the calculated molecule
weight of 5546 (Fig. 5). The first 33 residues of the expected sequence were confirmed by gas-phasesequencing. 487
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bTGF-a
Figure 3. HPLC profile during a preparative separation of pure bTGF-cr. After introducing sample onto column, 400 ml of solvent A was eluted before beginning a linear gradient from 25% to 30% of B in 17 min at 40 ml/min. Fractions of the eluent were collected in an isocratic mode (30% of B). Pevtide mamkg: bTGF-a,
peptide
chromatogram
In order to obtain the remainder
mapping was performed
of the chymotryptic
of the amino acid sequence for
using chymotrypsin.
digest of bTGF-o
Figure
after reduction
6 shows a HPLC with
DIT.
a
Figure 4. By treating with DlT, the pure bTGF-cr (a) and a misfolded fraction B12 (b) were converted cleanly to the reduced form (c). 488
The
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554J
18k 99. 61. ;a. E2. sa.
ca. 33. 28 IE 8 see0
5 Slue
5288
5688
5?w
Figure 5’. Mass spectrumof bTGF-cr. Sample was run in a VG ZAB-SE instrument.
5bel
59w
double-focusing
resulting five major peptide fragments were isolated and analyzed by sequencing. From these results, the following sequenceswere obtained for each peptide: 1 (residues 6-15), 2 (residues l-5), 3 (residues 24-38) 4 (residues 39-50) and 5 (residues 16-23). These five peptides account for the entire sequence in bTGF-a and demonstrate that it contains the correct sequence. The three disulfide bond pairings in this synthetic bTGF-a were successfully determined by a peptide mapping procedure using thermolysin. The disulfide linkages are indeed homologous with its rat and human counterparts. Details will be reported in a subsequent publication (18). Receptor binding assay: As can be seen from Figure 7, bTGF-a dilution curves were parallel to both mEGF and hTGF-cu (Pennisula Lab). The activity of the bTGF-a: was
Figure 6. HPLC chromatogram of the chymotryptic digest of synthetic bTGF-a analyzed after reduction with DIT. Sample was injected onto a Nucleosil C-18 column (Alltech) equilibrated in 12 mM TFA and then eluted using a O-35% AcCN gradient over 35 min at 2 ml/min.
489
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BlBo
1
0.8
-
0.6
-
\
0.4
% -
0.2
0 100
10
1000
Concentration +
mEGF
--IS-
bTGF--
10000
(ngiml) @g/ml) +hTGF--
&
8,~
Figure 7. Dilution curves for synthetic bTGF-a, hTGF-a, mEGF and a r&folded bTGF-a peptide B12 in the radioreceptor assay. The displacement of ‘“I mEGF from a crude microsomal membrane preparation of bovine liver was measured. comparable
to that of mEGF
and hTGF-cr. The fact that this synthetic bTGF-cr
competed
with radiolabeled
mEGF and the dilution curves were parallel to mEGF standard suggested
correct refolding
and probable biological potency.
Cell proliferation
assay:
Synthetic bTGF-a
bovine mammary cell proliferation
consistently
and significantly
increased
(Fig. 8). During the culture period, a significant increase
in cell number was observed when bTGF-cr was present from 1 rig/ml to 100 rig/ml. This response was greater than that observed when hEGF (prepared
as described in Ref. 19) was
provided in the culture media. It is interesting to note that a late-eluting fraction B12 (Fig. 3) isolated in pure form exhibits some, but low activity in the receptor-binding and the cell proliferation assay. As bTGF-aloha
“g DNA
I5
hEGF ---.
lo-
o 0
1
5 CONCENTRATION
10 (rig/ml)
25
100
Figure 8. Comparisonof bTGF-a with hEGF at concentrationsof 1 rig/ml to 100rig/ml (as indicated) on bovine mammarycell proliferation. 490
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compared with bTGF-cu, this peptide contains the correct amino acid sequence. However, the first four cysteines are n&paired,
Cys8-Cys32, Cysl6-Cys21 (18). The high activity of
the correctky folded bTGF-cr demonstrates the importance the bTGF-or molecule for the biological responses.
of a favorable conformation
of
ACKNOWLEDGMENTS We thank Drs. P. Toren and E. Kolodziej for the mass spectra, C. Sweeny, J. Schmuke, M. Jennings and J. Zobel for technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
DeLarco, J.E. & Todaro, G.J. (1978) Proc. Natl. Acad. Sci. USA 75:4001-4005. Defeo-Jones, D., Tai, J.Y., Wegrzyn, R.J., Vuocolo, G.A., Baker, A.E., Payne, L.S., Garsky, V.M., Oliff, A. and Riemen, M.W. (1988) Mol. Cell. Bio. 8:2999-3007. Massague, J. (1983) J. Biol. Chem. 258:13606-13613. Roberts, A.B., Anzano, M.A., Lamb, L.C., Smith, J.M. and Sporn, M.B. (1981) Proc. Natl. Acad. Sci. USA 78:5339-5343. a. Skinner, M.K. & Coffey, R.J. (1988) Endocrinology 123:2632-2638. b. Wilcox, J.N. & Derynck, R. (1988) Mol. Cell. Bio. 8:3415-3422. Rizzino, A. (1985) In Vitro Cell Dev. Biol. 21:.531-536. Lee, D.C., Rochford, R., Todaro, G.J. & Villarreal, L.P. (1985) Mol. Cell Biol. 5:3644-3646. Tam., J.P., Sheikh, M.A., Solomon, D.S. & Ossowski, L. (1986) Proc. Natl. Acad. Sci. USA 83:8082-8086. Tam., J.P., (1987) Int. J. Peptide Protein Res. 29:421-431. Zurfluh, L.L., Bolten, S.L., Byatt, J.C., McGrath, M.F., Tou, J.S. & Zupec, M.E., submitted to J. Biol. Chem. Kent, S. & Clark-Lewis, I. “Synthetic Peptides in Biology and Medicine”, Alitalo, K. & Vaheri, A. (eds), Elsevier, Amsterdam (1986) 29-56. Tam, J.P., Heath, W.F. & Merrifield, R.B. (1983) J. Am. Chem. Sot. 105:6442-6455. Haro, L.S., Collier, R.J. & Talamantes, F.J. (1984) Mol. Cell. Endocrinol. 38:109116. McGrath, M.F. (1987) J. Dairy Sci. 70:1967-1980. Hinegardner, R.T. (1971) Anal. Biochem. 38: 197-201. McGrath, M.F., Palmer, S. & Nandi, S. (1985) J. Cell Physiol. 125:182-191. Tam, J.P., Wong, T.W., Riemen, M., Tjoeng, F.S. & Merrifield, R.B. (1979) Tetr. Jitt. 42:4033-4036. Violand, B.N., Tou, J.S., Vineyard, B.D., Siegel, N.R., Toren, P.C. and Kolodziej, E.W., will be submitted to Int. J. Peptide Protein Res. Smith, J., Cook, E., Fotheringham, I., Pheby, S., Derbyshire, R., Eaton, M., Doel, M., Lilley, D., Pardon, J.F., Patel, T., Lewis, H. and Bell, L.D. (1982) Nucleic Acids Res. 10:4467-4482.
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