[18]
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hate between the two ligands. Mink lung epithelial-like cells (MvlLu, ATCC CCL64) are grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and I% nonessential amino acids. Cells are seeded at 2 x 105/well in 96-well tissue culture plates with removable wells (Dynatech, Alexandria, VA), for 18 to 24 hr prior to assay. Immediately before the assay the cells are washed twice with DMEM containing 10 g/liter BES [N,N-bis(2-hydroxyethyl)-aminoethanesulfonic acid] buffer, pH 7.0 (Sigma), and 1 g/liter BSA (dilution buffer). The assay samples are serially diluted in the same buffer, and 100-~1 aliquots are added to each well. EGF is labeled with 125I by the chloramine-T method to a specific activity of 80-100/zCi//zg. One hundred microliters containing 10 6 counts/min (cpm) of 125I-labeled EGF diluted in the same dilution buffer is added to each well, and the plates are incubated at 22 ° for 70 min. The wells are washed 4 times with Hanks' balanced salt solution and are then counted with a y-counter. Under these conditions, 4-9% of the lZSI-labeled EGF binds to the cells, and 4 × 10-10 M EGF is needed to displace 50% of the specific binding, as analyzed by Scatchard analysis. Conclusion The development of antibodies and assays for transforming growth factor a has been difficult. In particular, the production of antibodies of sufficient titer and specificity, and especially of neutralizing antibodies, was only possible with the availability of larger quantities (recombinant) of the protein in the proper configuration. In spite of these difficulties, reagents of high quality were produced, and reliable procedures were developed for the measurement of TGF-a.
[18] M o l e c u l a r a n d B i o c h e m i c a l A p p r o a c h e s to Structure-Function Analysis of Transforming Growth Factor a
By
DEBORAH DEFEO-JONES,
JOSEPH Y. TAI, and ALLEN OL1FF
Introduction Transforming growth factor a (TGF-a) is a 50 amino acid peptide hormone. Medical interest in TGF-a stems from its biological activity as a stimulator of tumor cell growth. Transfection of nontransformed murine fibroblasts with a molecular clone of TGF-a cDNA converts these cells to METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by AcademicPress, Inc. All rights of reproductionin any form reserved.
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EGF, TGF-c~, AND RELATEDFACTORS
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tumorogenic cell lines. 1 In addition, many human tumor cell lines secrete TGF-ot, and many cancer patients exhibit elevated levels of TGF-ot in the blood and urine. 2-5 These observations suggest a role for TGF-o~ in the pathophysiology of human malignancies and imply that antagonists of the biological activity of TGF-ot may have utility as anticancer agents. 6,7 The biological effects of TGF-ot are mediated via attachment of TGF-t~ to epidermal growth factor (EGF) receptors on the surface of susceptible cells. 8 This biochemical interaction offers an obvious target for the identification of TGF-ot antagonists. As a first step in the design and isolation of TGF-o~-EGF receptor binding antagonists, a structure-function analysis of the receptor binding properties of TGF-a is desirable. We have used four independent methods for analyzing the contribution of specific regions of TGF-a to the receptor binding activity of the entire hormone. First, synthetic peptides representing discrete segments of TGF-a were prepared. These peptides were tested for their ability to inhibit hormone attachment to the EGF receptor and to block TGF-astimulated mitogenesis in mammalian cells. Second, the same peptides were coupled to thyroglobulin, and the peptide-thyroglobulin conjugates were used to raise antibodies that recognized specific regions of TGF-a. The antibodies were tested for the ability to block the attachment of whole TGF-ot to EGF receptors. Third, a synthetic human TGF-a gene was constructed and introduced into bacteria for the production and isolation of recombinant human TGF-a. The synthetic gene was also modified using techniques of site-directed mutagenesis to create a series of TGF-a derivatives with defined changes in the primary amino acid sequences. These derivatives were analyzed for their EGF receptor binding and mitogenic activities. Fourth, a series of recombinant TGF-a derivatives was J A. Rosenthal, P. B. Lindquist, T. S. Bringman, D. V. Goeddel, and R. Derynck, Cell (Cambridge, Mass.) 46, 301 (1986). 2 B. Gusterson, G. Cowley, J. Mclihinney, B. Ozanne, C. Fisher, and B. Reeves, Int. J. Cancer 36, 689 (1972). 3 R. Derynck, D. V. Goeddel, A. Ullrich, J. U. Gutterman, R. D. Williams, T. S. Bringman, and W. H. Berger, Cancer Res. 47, 707 (1987). 4 T. A. Libermann, H. R. Nusbaum, N. Razon, R. Kris, I. Lax, H. Soreq, N. Whittle, M. D. Waterfield, A. Ullrich, and J. Schlessinger, Nature (London) 313, 144 (1985). 5 S. A. Sherwin, D. R. Twardzik, W. H. Bohn, K. D. Cockley, and G. J. Todaro, Cancer 43, 403 (1983). 6 j. j. Nestor, S. R. Newman, B. M. DeLustro, and A. B. Schreiber, in "Peptides Structure and Function" (C. M. Deber, V. J. Hruby, and K. D. Koppel, eds.), p. 39. Pierce Chemical Co., Rockford, Illinois, 1985. 7 j. j. Nestor, Jr., S. R. Newman, B. M. DeLustro, G. J. Todaro, and A. B. Schreiber, Bioehem. Biophys. Res. Commun. 129, 226 (1985). 8 j. Massague, J. Biol. Chem. 258, 13614 (1983).
[18]
TGF-a STRUCTURE--FUNCTIONANALYSIS
193
10
30 NH2
40
O
5O
HO-I
FIG. 1. Amino acid sequence of mature human TGF-a. Note the three loop structures (A, B, and C) formed by three intrachain disulfide bonds. Six synthetic peptides were prepared corresponding to the followingresidues: 1-7, 8-21,8-32, 16-32, 34-43, and 44-50. Disulfide bonds were formed in each peptide containing cysteine residues. Elimination of free sulfhydrylgroups followingdisulfidebond formation was assessed with Ellman's reagent.
created containing a single lysine residue at various positions in the primary structure of TGF-c~. The mutated T G F - a proteins were isolated and modified by covalent attachment of a p-hydroxybenzimidyl group to the e-amino group of the lysine residues. This postsynthetic modification of TGF-o~ generated a series of TGF-o~ derivatives whose biological activities were evaluated both before and after conjugation with the methyl p - h y d r o x y b e n z i m i d a t e "blocking g r o u p . " Synthetic Peptide Analysis The primary amino acid sequence and location of disulfide bonds of mature human T G F - a are presented in Fig. 1. One method of conceptualizing how T G F - a functions is to hypothesize that a discrete segment of the molecule is responsible for binding to the E G F receptor while a separate segment is responsible for triggering the biological activity of T G F - a . This view of peptide hormone structure-function relationships underlies the dissection of T G F - a by synthesis of defined peptide segments. Individual peptides representing the N terminus, C terminus, and
194
EGF, TGF-a, AND RELATEDFACTORS
[18]
each of the disulfide-constrained loop structures or combinations of loop structures were synthesized. The peptides were tested in radiolabeled EGF receptor-binding inhibition studies 9 and in [3H]thymidine incorporation assays of the mitogenic activity of TGF-a. 1° In order to identify weakly active species, it is generally necessary to test the peptides at concentrations up to 1000 times the dose of TGF-a required to illicit a maximal biological response. Synthetic peptides were prepared by standard methods of solid-phase synthesis, TM12using an Applied Biosystems Model 430A automated peptide synthesizer. Purification of the newly synthesized peptides was performed by reversed-phase HPLC.13 Antipeptide Antibodies Antibodies raised against whole TGF-a block the attachment of TGF-a to the EGF receptor. The antibodies recognize multiple regions of the TGF-a molecule. It is unclear which, if any, single epitope on TGF-a is responsible for elliciting the receptor-blocking antibodies. One method of defining the relative contribution of different regions of TGF-a to the generation of receptor-blocking antibodies is to raise antisera against specific TGF-a peptides that represent discrete segments of the TGF-a molecules. Antipeptide antibodies can be purified from the antisera and tested for the ability to block the binding of whole TGF-a to EGF receptors. The synthetic peptides described above are excellent candidates for this type of analysis. Unfortunately, small synthetic peptides are frequently only weakly immunogenic. To ensure an adequate immune response, we first couple the peptides to thyroglobulin and then inject the thyroglobulin-peptide conjugates into rabbits. High titer peptide-specific antisera are consistently produced in this manner.
Preparation of Peptide-Thyroglobulin Conjugates 1. Equal amounts (5-10 mg) of the TGF-a peptides and bovine thyroglobulin are dissolved in 0.1 M phosphate buffer, pH 7.0 (20 mg/ml total protein, final concentration). 9 S. Cohen, H. Ushiro, C. Stoscheck, and M. Chinkers, J, Biol. Chem. 257, 1523 (1982). 10 M. W. Riemen, R. J. Wegrzyn, A. E. Baker, W. M. Hurni, C. D. Bennett, A. 0lift, and R. B. Stein, Peptides 8, 877 (1987). 11 R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963). ~2j. M. Steward and J. D. Young, in "Solid Phase Peptide Synthesis," (J. M. Stewart and J. D. Young, eds.), 2nd Ed. Pierce Chemical Co., Rockford, Illinois, 1984. ~3j. Rivier, R. McClintock, R. Galyean, and H. Anderson, J. Chromatogr. 288, 303 (1984).
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2. The protein solution is cooled to 4° on ice. 3. Twenty-five percent (w/v) glutaraldehyde is diluted 1 : 20 with water to give a 0.125 M glutaraldehyde solution. Two hundred fifty microliters of the diluted glutaraldehyde is added slowly to the protein solution over a period of 15-20 min with stirring. 4. The mixture is incubated with stirring at 4 ° for 18 hr and then dialyzed extensively against phosphate-buffered saline.
Immunization with Peptide-Thyroglobulin Conjugates I. New Zealand white rabbits are injected intramuscularly with 250/xg of the peptide-thyroglobulin conjugate emulsified in Freund's complete adjuvant. 2. Thirty-five days later the rabbits are bled and then injected subcutaneously with 250/xg of conjugate emulsified in Freund's incomplete adjuvant. 3. On day 49 the rabbits are bled and then injected subcutaneously with 250/xg of conjugate emulsified in Freund's incomplete adjuvant. 4. On day 63 the rabbits are bled out and sacrificed; alternatively, a sublethal bleeding is performed so that further immunizations and bleedings can be continued.
Isolation of Antipeptide Immunoglobulins Anti-TGF-a peptide IgG is isolated from whole antisera by protein A-agarose affinity column chromatography. 1. One milliliter of rabbit serum is diluted with 1 ml of agarose affinity binding buffer (Pierce Chemical Co., Rockford, IL) and applied to a 1-ml, 0.9 by 2.5 cm diameter protein A-agarose column. 2. The column is washed with 15 ml of binding buffer, and IgG is eluted with 5 ml of agarose affinity column elution buffer. Both the binding and elution buffers are prepared as specified by the manufacturer of the agarose affinity column. 3. The IgG fraction is dialyzed extensively against phosphate-buffered saline. 4. The final IgG preparation is analyzed by SDS-PAGE to assess its purity. Site-Directed Mutagenesis of Recombinant Transforming
Growth Factor a Several aspects of the structure-function relationship of TGF-a can only be examined in the context of the entire TGF-a molecule. For example, proper cysteine pairings and formation of correct disulfide bonds are
EGF, TGF-a,
196
AND RELATED FACTORS
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1
L.
NH2
Fie. 2. Amino acid sequence of recombinant human TGF-o~ made in Escherichia coli. The first three and last three residues (cross-hatched) were derived from the precursor of human TGF-ot. The codons for these residues were added to the synthetic gene used to produce TGF-~ in bacteria. Nonconservative substitutions using alanine were introduced at the diagonally shaded residues 8, 16, 21, 22, 32, 34, 38, 43, 44, 47, and 49. Semiconservative substitutions using lysines were introduced at the diagonally shaded residues 4, 18, 35, and 45. A conservative substitution using lysine was introduced at the diagonally shaded residue 22. Both conservative and nonconservative or semiconservative and nonconservative substitutions were introduced at the blackened residues 12,15, 29, and 42,
critical to producing fully active TGF-a. Elimination of disulfide bonds by substituting alanines for the cysteine residues in TGF-a dramatically reduces both receptor binding and mitogenic activity. 14Experiments yielding information on the contribution of individual amino acid residues to the biological activity of TGF-o~ were performed by site-directed mutagenesis of the gene encoding TGF-ot and subsequent expression and isolation of the mutated TGF-ot species in bacteria. Conservative, semiconservative, or nonconservative amino acid changes were introduced either singly or as multiple changes in all regions of TGF-a. Figure 2 illustrates the sitespecific mutations that we have introduced into TGF-a and analyzed. This 14 D. Defeo-Jones, J. Y. Tai, R. J. Wegrzyn, G. A. Vuocolo, A. E. Baker, L. S. Payne, V. M. Oarsky, A, Oliff, and M. W. Riemen, Mol. Cell. Biol. 8, 2999 (1988).
[18]
TGF-cz STRUCTURE-FUNCTIONANALYSIS
197
analysis has identified critical amino acids at several positions in TGF-a, including residues 15, 38, 42, and 48. Nonconservative and in some cases conservative amino acid substitutions at these positions dramatically reduced the receptor binding and mitogenic activities of TGF-ct. 15
Site-Directed Oligonucleotide Mutagenesis 1. The human TGF-a gene is subcloned in M13MP19, and singlestranded DNA is prepared as a template for mutagenesis.16 2. Specific, short [ - 1 8 base pairs (bp)] oligonucleotides encoding the changes are synthesized using an Applied Biosystems Model 380A DNA synthesizer (Applied Biosystems Inc., Foster City, CA). 3. The specific oligonucleotide is phosphorylated in a reaction mixture containing: 1 × kinase buffer, 0.8/zg of each oligonucleotide, 1 mM ATP, and 2.5 units of T4 kinase (New England Biolabs, Beverly, MA) in a total reaction volume of 25/zl. The reaction is carried out at 37 ° for 40 min and stopped by heating to 65 ° for 15 min. The kinased oligonucleotide is next annealed to the single-stranded template DNA using 1 to 2/xg of singlestrand DNA, 0.16 to 0.2 ~g of kinased oligonucleotide, and 1 x hybridization buffer in a total reaction volume of 30/.d. All enzyme buffers are prepared as specified by the enzyme manufacturer. Hybridization buffer is 20 mM Tris-HCl, pH 7.5, 10 mM MgCI2, 0.5 M NaCI. 4. The reaction mixture is incubated at 55 ° for 20 min, then shifted to room temperature for 1 hr. 5. Using the annealed oligonucleotide as a primer, a second-strand synthesis is performed using DNA polymerase (Klenow fragment) under the following conditions: to the 30-/xl annealing reaction are added all four deoxynucleotide triphosphates to a final concentration of 0.5 mM, 0.1 M 2-mercaptoethanol, 1 mM ATP, 5 units of T4 ligase, and 5 units DNA polymerase (Klenow fragment). The reaction is adjusted to 1 x hybridization buffer conditions and is incubated for 3 hr at room temperature. 6. The reaction is stopped by raising the temperature to 65 ° for 10 min and then extracting with an equal volume of phenol/chloroform (1 : 1). After extraction the reaction is precipitated with 2 volumes of 95% ethanol. 7. The mutagenized DNA is resuspended in water and digested with an appropriate restriction enzyme to isolate the mutant gene fragment, which is then ligated into the specific vector DNA required. The resulting plasmid DNA is then transfected into bacteria. 15 D. Defeo-Jones, J. Y. Tai, G. A. Vuocolo, R. J. Wegrzyn, T. L. Schofield, M. W. Riemen, and A. Oliff, Mol. Cell. Biol. 9, 4083 (1989). 16 p. H. Schien and R. Corese, J. Mol. Biol. 129, 169 (1979).
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EGF, TGF-a, AND RELATEDFACTORS
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8. Mutant clones are selected by hybridizing to a radiolabeled oligonucleotide probe containing the mutation. Posthybridization washings [2 × SSC and 0.1% sodium dodecyl sulfate (SDS)] are performed at a temperature 20-3 ° below the determined melt-out temperature for the oligonucleotide used in the mutagenesis procedure. These conditions ensure that only clones carrying the desired change will hybridize to the oligonucleotide probe. Isolation and refolding of recombinant TGF-a and mutant derivatives of TGF-a proteins are performed as described by Defeo-Jones et al. ~4with the following modification of the final purification step. Partially purified TGF-a proteins are loaded on a 2.2 × 25 cm Vydac preparative C4 column developed with a linear gradient of 30 to 50% acetonitrile-water in 0.1% trifluoroacetic acid for 60 min. Fractions from the reversed-phase HPLC column containing the purified TGF-a proteins are identified by spectrophotometric absorption at 210 nm. It should be noted that the synthetic gene employed for producing TGF-a in bacteria encodes three additional N-terminal and three additional C-terminal amino acids derived from the precursor of mature TGF-a. These additional residues appear to stabilize the TGF-a species, enabling consistently high yields of intact proteins to be isolated from bacteria.
Postsynthetic Modification of Recombinant Transforming Growth Factor a In addition to analyzing the biological activity of peptide fragments of TGF-a and TGF-a substitution mutants, we examined the effect of adding chemical "blocking groups" to mature TGF-a at various sites in the molecule. Methyl p-hydroxybenzimidate can be covalently coupled to the e-amino group of lysine residues in TGF-a, creating a bulky side group on the lysine. Human TGF-a normally has only one lysine residue at position 29. However, by using the techniques of site-directed mutagenesis described above, the lysine at position 29 may be converted to an arginine. The TGF-a Lys-29 to Arg mutant exhibited full receptor binding and mitogenic activities. The TGF-a Lys-29 to Arg mutant was further modified to create a series of TGF-a derivatives that possessed unique lysine residues at various positions around the TGF-a protein. The location of lysine in five of these substitution mutants is indicated in Fig. 2. Each of the mutant TGF-a species was isolated and assayed for receptor-binding activity both before and after coupling with methyl p-hydroxybenzimidate. The addition of the p-hydroxybenzimidyl group to the lysine residues at some positions (e.g., 12, 18, and 35) significantly reduced receptor-binding
[18]
TGF-c~ STRUCTURE--FUNCTIONANALYSIS
199
activity. At other positions conjugation of the lysine residue to methyl phydroxybenzimidate had little or no effect on the biological activity of TGF-a. Preparation of Amidinated Mutant Proteins 1. One-half milligram of purified mutant TGF-a is dissolved in 0.5 ml of 0.2 M sodium borate buffer, pH 9.5. 2. Fifteen milligrams of methyl p-hydroxybenzimidate-HC1 (Pierce) is suspended in 100 /zl of dimethyl sulfoxide and solubilized in 400 tzl of water. 3. The methyl p-hydroxybenzimidate solution is titrated to pH 9.5 with 10 N NaOH and added to the TGF-~ protein solution. The mixture is incubated at room temperature for 18 hr. 4. At the end of this time, the reaction mixture is applied directly to a reversed-phase HPLC semipreparative C4 column, 1.0 x 25 cm (Vydac, The Nest Group, Southboro, MA) equilibrated in 0. I% trifluoroacetic acid and 30% acetonitrile and water. The amidinated mutant TGF-o~ proteins are purified with a linear gradient of 30 to 50% acetonitrile-water in 0.1% trifluoroacetic acid at a flow rate of 4 ml/min for 50 min. 5. The amidinated protein peaks are identified by the spectrophotometric absorbance at 210 nm, collected, lyophilized, and stored at - 20° until needed for bioassays. The amidinated protein was characterized by analytical reversed-phase HPLC (C4, 0.46 x 15 cm). The native and amidinated proteins have distinctly different profiles using this HPLC system. The amidinated protein was further characterized by the disappearance of the single lysine residue in each TGF-o~ species as determined by amino acid composition analysis using a Beckman System 6300 amino acid analyzer (Beckman Instruments, Fullerton, CA). 17 Conclusions
We have employed several approaches in analyzing the structure-function relationships of human TGF-o~. Each method has provided complementary data that support the following conclusions: (1) Biological activity resides in the entire TGF-a molecule. Peptide segments of TGF-~ possess little or no receptor-binding or mitogenic activity. (2) Amino acid residues critical to the biological activity of TGF-~ exist in each of the disulfideconstrained loops and in the C terminus of the molecule. An important 17 F. T. Wood, M. M. Wu, and J. C. Gerhart, Anal. Blochem. 69, 339 (1975).
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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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hate between the two ligands. Mink lung epithelial-like cells (MvlLu, ATCC CCL64) are grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and I% nonessential amino acids. Cells are seeded at 2 x 105/well in 96-well tissue culture plates with removable wells (Dynatech, Alexandria, VA), for 18 to 24 hr prior to assay. Immediately before the assay the cells are washed twice with DMEM containing 10 g/liter BES [N,N-bis(2-hydroxyethyl)-aminoethanesulfonic acid] buffer, pH 7.0 (Sigma), and 1 g/liter BSA (dilution buffer). The assay samples are serially diluted in the same buffer, and 100-~1 aliquots are added to each well. EGF is labeled with 125I by the chloramine-T method to a specific activity of 80-100/zCi//zg. One hundred microliters containing 10 6 counts/min (cpm) of 125I-labeled EGF diluted in the same dilution buffer is added to each well, and the plates are incubated at 22 ° for 70 min. The wells are washed 4 times with Hanks' balanced salt solution and are then counted with a y-counter. Under these conditions, 4-9% of the lZSI-labeled EGF binds to the cells, and 4 × 10-10 M EGF is needed to displace 50% of the specific binding, as analyzed by Scatchard analysis. Conclusion The development of antibodies and assays for transforming growth factor a has been difficult. In particular, the production of antibodies of sufficient titer and specificity, and especially of neutralizing antibodies, was only possible with the availability of larger quantities (recombinant) of the protein in the proper configuration. In spite of these difficulties, reagents of high quality were produced, and reliable procedures were developed for the measurement of TGF-a.
[18] M o l e c u l a r a n d B i o c h e m i c a l A p p r o a c h e s to Structure-Function Analysis of Transforming Growth Factor a
By
DEBORAH DEFEO-JONES,
JOSEPH Y. TAI, and ALLEN OL1FF
Introduction Transforming growth factor a (TGF-a) is a 50 amino acid peptide hormone. Medical interest in TGF-a stems from its biological activity as a stimulator of tumor cell growth. Transfection of nontransformed murine fibroblasts with a molecular clone of TGF-a cDNA converts these cells to METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by AcademicPress, Inc. All rights of reproductionin any form reserved.
192
EGF, TGF-c~, AND RELATEDFACTORS
[18]
tumorogenic cell lines. 1 In addition, many human tumor cell lines secrete TGF-ot, and many cancer patients exhibit elevated levels of TGF-ot in the blood and urine. 2-5 These observations suggest a role for TGF-o~ in the pathophysiology of human malignancies and imply that antagonists of the biological activity of TGF-ot may have utility as anticancer agents. 6,7 The biological effects of TGF-ot are mediated via attachment of TGF-t~ to epidermal growth factor (EGF) receptors on the surface of susceptible cells. 8 This biochemical interaction offers an obvious target for the identification of TGF-ot antagonists. As a first step in the design and isolation of TGF-o~-EGF receptor binding antagonists, a structure-function analysis of the receptor binding properties of TGF-a is desirable. We have used four independent methods for analyzing the contribution of specific regions of TGF-a to the receptor binding activity of the entire hormone. First, synthetic peptides representing discrete segments of TGF-a were prepared. These peptides were tested for their ability to inhibit hormone attachment to the EGF receptor and to block TGF-astimulated mitogenesis in mammalian cells. Second, the same peptides were coupled to thyroglobulin, and the peptide-thyroglobulin conjugates were used to raise antibodies that recognized specific regions of TGF-a. The antibodies were tested for the ability to block the attachment of whole TGF-ot to EGF receptors. Third, a synthetic human TGF-a gene was constructed and introduced into bacteria for the production and isolation of recombinant human TGF-a. The synthetic gene was also modified using techniques of site-directed mutagenesis to create a series of TGF-a derivatives with defined changes in the primary amino acid sequences. These derivatives were analyzed for their EGF receptor binding and mitogenic activities. Fourth, a series of recombinant TGF-a derivatives was J A. Rosenthal, P. B. Lindquist, T. S. Bringman, D. V. Goeddel, and R. Derynck, Cell (Cambridge, Mass.) 46, 301 (1986). 2 B. Gusterson, G. Cowley, J. Mclihinney, B. Ozanne, C. Fisher, and B. Reeves, Int. J. Cancer 36, 689 (1972). 3 R. Derynck, D. V. Goeddel, A. Ullrich, J. U. Gutterman, R. D. Williams, T. S. Bringman, and W. H. Berger, Cancer Res. 47, 707 (1987). 4 T. A. Libermann, H. R. Nusbaum, N. Razon, R. Kris, I. Lax, H. Soreq, N. Whittle, M. D. Waterfield, A. Ullrich, and J. Schlessinger, Nature (London) 313, 144 (1985). 5 S. A. Sherwin, D. R. Twardzik, W. H. Bohn, K. D. Cockley, and G. J. Todaro, Cancer 43, 403 (1983). 6 j. j. Nestor, S. R. Newman, B. M. DeLustro, and A. B. Schreiber, in "Peptides Structure and Function" (C. M. Deber, V. J. Hruby, and K. D. Koppel, eds.), p. 39. Pierce Chemical Co., Rockford, Illinois, 1985. 7 j. j. Nestor, Jr., S. R. Newman, B. M. DeLustro, G. J. Todaro, and A. B. Schreiber, Bioehem. Biophys. Res. Commun. 129, 226 (1985). 8 j. Massague, J. Biol. Chem. 258, 13614 (1983).
[18]
TGF-a STRUCTURE--FUNCTIONANALYSIS
193
10
30 NH2
40
O
5O
HO-I
FIG. 1. Amino acid sequence of mature human TGF-a. Note the three loop structures (A, B, and C) formed by three intrachain disulfide bonds. Six synthetic peptides were prepared corresponding to the followingresidues: 1-7, 8-21,8-32, 16-32, 34-43, and 44-50. Disulfide bonds were formed in each peptide containing cysteine residues. Elimination of free sulfhydrylgroups followingdisulfidebond formation was assessed with Ellman's reagent.
created containing a single lysine residue at various positions in the primary structure of TGF-c~. The mutated T G F - a proteins were isolated and modified by covalent attachment of a p-hydroxybenzimidyl group to the e-amino group of the lysine residues. This postsynthetic modification of TGF-o~ generated a series of TGF-o~ derivatives whose biological activities were evaluated both before and after conjugation with the methyl p - h y d r o x y b e n z i m i d a t e "blocking g r o u p . " Synthetic Peptide Analysis The primary amino acid sequence and location of disulfide bonds of mature human T G F - a are presented in Fig. 1. One method of conceptualizing how T G F - a functions is to hypothesize that a discrete segment of the molecule is responsible for binding to the E G F receptor while a separate segment is responsible for triggering the biological activity of T G F - a . This view of peptide hormone structure-function relationships underlies the dissection of T G F - a by synthesis of defined peptide segments. Individual peptides representing the N terminus, C terminus, and
194
EGF, TGF-a, AND RELATEDFACTORS
[18]
each of the disulfide-constrained loop structures or combinations of loop structures were synthesized. The peptides were tested in radiolabeled EGF receptor-binding inhibition studies 9 and in [3H]thymidine incorporation assays of the mitogenic activity of TGF-a. 1° In order to identify weakly active species, it is generally necessary to test the peptides at concentrations up to 1000 times the dose of TGF-a required to illicit a maximal biological response. Synthetic peptides were prepared by standard methods of solid-phase synthesis, TM12using an Applied Biosystems Model 430A automated peptide synthesizer. Purification of the newly synthesized peptides was performed by reversed-phase HPLC.13 Antipeptide Antibodies Antibodies raised against whole TGF-a block the attachment of TGF-a to the EGF receptor. The antibodies recognize multiple regions of the TGF-a molecule. It is unclear which, if any, single epitope on TGF-a is responsible for elliciting the receptor-blocking antibodies. One method of defining the relative contribution of different regions of TGF-a to the generation of receptor-blocking antibodies is to raise antisera against specific TGF-a peptides that represent discrete segments of the TGF-a molecules. Antipeptide antibodies can be purified from the antisera and tested for the ability to block the binding of whole TGF-a to EGF receptors. The synthetic peptides described above are excellent candidates for this type of analysis. Unfortunately, small synthetic peptides are frequently only weakly immunogenic. To ensure an adequate immune response, we first couple the peptides to thyroglobulin and then inject the thyroglobulin-peptide conjugates into rabbits. High titer peptide-specific antisera are consistently produced in this manner.
Preparation of Peptide-Thyroglobulin Conjugates 1. Equal amounts (5-10 mg) of the TGF-a peptides and bovine thyroglobulin are dissolved in 0.1 M phosphate buffer, pH 7.0 (20 mg/ml total protein, final concentration). 9 S. Cohen, H. Ushiro, C. Stoscheck, and M. Chinkers, J, Biol. Chem. 257, 1523 (1982). 10 M. W. Riemen, R. J. Wegrzyn, A. E. Baker, W. M. Hurni, C. D. Bennett, A. 0lift, and R. B. Stein, Peptides 8, 877 (1987). 11 R. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963). ~2j. M. Steward and J. D. Young, in "Solid Phase Peptide Synthesis," (J. M. Stewart and J. D. Young, eds.), 2nd Ed. Pierce Chemical Co., Rockford, Illinois, 1984. ~3j. Rivier, R. McClintock, R. Galyean, and H. Anderson, J. Chromatogr. 288, 303 (1984).
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2. The protein solution is cooled to 4° on ice. 3. Twenty-five percent (w/v) glutaraldehyde is diluted 1 : 20 with water to give a 0.125 M glutaraldehyde solution. Two hundred fifty microliters of the diluted glutaraldehyde is added slowly to the protein solution over a period of 15-20 min with stirring. 4. The mixture is incubated with stirring at 4 ° for 18 hr and then dialyzed extensively against phosphate-buffered saline.
Immunization with Peptide-Thyroglobulin Conjugates I. New Zealand white rabbits are injected intramuscularly with 250/xg of the peptide-thyroglobulin conjugate emulsified in Freund's complete adjuvant. 2. Thirty-five days later the rabbits are bled and then injected subcutaneously with 250/xg of conjugate emulsified in Freund's incomplete adjuvant. 3. On day 49 the rabbits are bled and then injected subcutaneously with 250/xg of conjugate emulsified in Freund's incomplete adjuvant. 4. On day 63 the rabbits are bled out and sacrificed; alternatively, a sublethal bleeding is performed so that further immunizations and bleedings can be continued.
Isolation of Antipeptide Immunoglobulins Anti-TGF-a peptide IgG is isolated from whole antisera by protein A-agarose affinity column chromatography. 1. One milliliter of rabbit serum is diluted with 1 ml of agarose affinity binding buffer (Pierce Chemical Co., Rockford, IL) and applied to a 1-ml, 0.9 by 2.5 cm diameter protein A-agarose column. 2. The column is washed with 15 ml of binding buffer, and IgG is eluted with 5 ml of agarose affinity column elution buffer. Both the binding and elution buffers are prepared as specified by the manufacturer of the agarose affinity column. 3. The IgG fraction is dialyzed extensively against phosphate-buffered saline. 4. The final IgG preparation is analyzed by SDS-PAGE to assess its purity. Site-Directed Mutagenesis of Recombinant Transforming
Growth Factor a Several aspects of the structure-function relationship of TGF-a can only be examined in the context of the entire TGF-a molecule. For example, proper cysteine pairings and formation of correct disulfide bonds are
EGF, TGF-a,
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AND RELATED FACTORS
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1
L.
NH2
Fie. 2. Amino acid sequence of recombinant human TGF-o~ made in Escherichia coli. The first three and last three residues (cross-hatched) were derived from the precursor of human TGF-ot. The codons for these residues were added to the synthetic gene used to produce TGF-~ in bacteria. Nonconservative substitutions using alanine were introduced at the diagonally shaded residues 8, 16, 21, 22, 32, 34, 38, 43, 44, 47, and 49. Semiconservative substitutions using lysines were introduced at the diagonally shaded residues 4, 18, 35, and 45. A conservative substitution using lysine was introduced at the diagonally shaded residue 22. Both conservative and nonconservative or semiconservative and nonconservative substitutions were introduced at the blackened residues 12,15, 29, and 42,
critical to producing fully active TGF-a. Elimination of disulfide bonds by substituting alanines for the cysteine residues in TGF-a dramatically reduces both receptor binding and mitogenic activity. 14Experiments yielding information on the contribution of individual amino acid residues to the biological activity of TGF-o~ were performed by site-directed mutagenesis of the gene encoding TGF-ot and subsequent expression and isolation of the mutated TGF-ot species in bacteria. Conservative, semiconservative, or nonconservative amino acid changes were introduced either singly or as multiple changes in all regions of TGF-a. Figure 2 illustrates the sitespecific mutations that we have introduced into TGF-a and analyzed. This 14 D. Defeo-Jones, J. Y. Tai, R. J. Wegrzyn, G. A. Vuocolo, A. E. Baker, L. S. Payne, V. M. Oarsky, A, Oliff, and M. W. Riemen, Mol. Cell. Biol. 8, 2999 (1988).
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TGF-cz STRUCTURE-FUNCTIONANALYSIS
197
analysis has identified critical amino acids at several positions in TGF-a, including residues 15, 38, 42, and 48. Nonconservative and in some cases conservative amino acid substitutions at these positions dramatically reduced the receptor binding and mitogenic activities of TGF-ct. 15
Site-Directed Oligonucleotide Mutagenesis 1. The human TGF-a gene is subcloned in M13MP19, and singlestranded DNA is prepared as a template for mutagenesis.16 2. Specific, short [ - 1 8 base pairs (bp)] oligonucleotides encoding the changes are synthesized using an Applied Biosystems Model 380A DNA synthesizer (Applied Biosystems Inc., Foster City, CA). 3. The specific oligonucleotide is phosphorylated in a reaction mixture containing: 1 × kinase buffer, 0.8/zg of each oligonucleotide, 1 mM ATP, and 2.5 units of T4 kinase (New England Biolabs, Beverly, MA) in a total reaction volume of 25/zl. The reaction is carried out at 37 ° for 40 min and stopped by heating to 65 ° for 15 min. The kinased oligonucleotide is next annealed to the single-stranded template DNA using 1 to 2/xg of singlestrand DNA, 0.16 to 0.2 ~g of kinased oligonucleotide, and 1 x hybridization buffer in a total reaction volume of 30/.d. All enzyme buffers are prepared as specified by the enzyme manufacturer. Hybridization buffer is 20 mM Tris-HCl, pH 7.5, 10 mM MgCI2, 0.5 M NaCI. 4. The reaction mixture is incubated at 55 ° for 20 min, then shifted to room temperature for 1 hr. 5. Using the annealed oligonucleotide as a primer, a second-strand synthesis is performed using DNA polymerase (Klenow fragment) under the following conditions: to the 30-/xl annealing reaction are added all four deoxynucleotide triphosphates to a final concentration of 0.5 mM, 0.1 M 2-mercaptoethanol, 1 mM ATP, 5 units of T4 ligase, and 5 units DNA polymerase (Klenow fragment). The reaction is adjusted to 1 x hybridization buffer conditions and is incubated for 3 hr at room temperature. 6. The reaction is stopped by raising the temperature to 65 ° for 10 min and then extracting with an equal volume of phenol/chloroform (1 : 1). After extraction the reaction is precipitated with 2 volumes of 95% ethanol. 7. The mutagenized DNA is resuspended in water and digested with an appropriate restriction enzyme to isolate the mutant gene fragment, which is then ligated into the specific vector DNA required. The resulting plasmid DNA is then transfected into bacteria. 15 D. Defeo-Jones, J. Y. Tai, G. A. Vuocolo, R. J. Wegrzyn, T. L. Schofield, M. W. Riemen, and A. Oliff, Mol. Cell. Biol. 9, 4083 (1989). 16 p. H. Schien and R. Corese, J. Mol. Biol. 129, 169 (1979).
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EGF, TGF-a, AND RELATEDFACTORS
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8. Mutant clones are selected by hybridizing to a radiolabeled oligonucleotide probe containing the mutation. Posthybridization washings [2 × SSC and 0.1% sodium dodecyl sulfate (SDS)] are performed at a temperature 20-3 ° below the determined melt-out temperature for the oligonucleotide used in the mutagenesis procedure. These conditions ensure that only clones carrying the desired change will hybridize to the oligonucleotide probe. Isolation and refolding of recombinant TGF-a and mutant derivatives of TGF-a proteins are performed as described by Defeo-Jones et al. ~4with the following modification of the final purification step. Partially purified TGF-a proteins are loaded on a 2.2 × 25 cm Vydac preparative C4 column developed with a linear gradient of 30 to 50% acetonitrile-water in 0.1% trifluoroacetic acid for 60 min. Fractions from the reversed-phase HPLC column containing the purified TGF-a proteins are identified by spectrophotometric absorption at 210 nm. It should be noted that the synthetic gene employed for producing TGF-a in bacteria encodes three additional N-terminal and three additional C-terminal amino acids derived from the precursor of mature TGF-a. These additional residues appear to stabilize the TGF-a species, enabling consistently high yields of intact proteins to be isolated from bacteria.
Postsynthetic Modification of Recombinant Transforming Growth Factor a In addition to analyzing the biological activity of peptide fragments of TGF-a and TGF-a substitution mutants, we examined the effect of adding chemical "blocking groups" to mature TGF-a at various sites in the molecule. Methyl p-hydroxybenzimidate can be covalently coupled to the e-amino group of lysine residues in TGF-a, creating a bulky side group on the lysine. Human TGF-a normally has only one lysine residue at position 29. However, by using the techniques of site-directed mutagenesis described above, the lysine at position 29 may be converted to an arginine. The TGF-a Lys-29 to Arg mutant exhibited full receptor binding and mitogenic activities. The TGF-a Lys-29 to Arg mutant was further modified to create a series of TGF-a derivatives that possessed unique lysine residues at various positions around the TGF-a protein. The location of lysine in five of these substitution mutants is indicated in Fig. 2. Each of the mutant TGF-a species was isolated and assayed for receptor-binding activity both before and after coupling with methyl p-hydroxybenzimidate. The addition of the p-hydroxybenzimidyl group to the lysine residues at some positions (e.g., 12, 18, and 35) significantly reduced receptor-binding
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199
activity. At other positions conjugation of the lysine residue to methyl phydroxybenzimidate had little or no effect on the biological activity of TGF-a. Preparation of Amidinated Mutant Proteins 1. One-half milligram of purified mutant TGF-a is dissolved in 0.5 ml of 0.2 M sodium borate buffer, pH 9.5. 2. Fifteen milligrams of methyl p-hydroxybenzimidate-HC1 (Pierce) is suspended in 100 /zl of dimethyl sulfoxide and solubilized in 400 tzl of water. 3. The methyl p-hydroxybenzimidate solution is titrated to pH 9.5 with 10 N NaOH and added to the TGF-~ protein solution. The mixture is incubated at room temperature for 18 hr. 4. At the end of this time, the reaction mixture is applied directly to a reversed-phase HPLC semipreparative C4 column, 1.0 x 25 cm (Vydac, The Nest Group, Southboro, MA) equilibrated in 0. I% trifluoroacetic acid and 30% acetonitrile and water. The amidinated mutant TGF-o~ proteins are purified with a linear gradient of 30 to 50% acetonitrile-water in 0.1% trifluoroacetic acid at a flow rate of 4 ml/min for 50 min. 5. The amidinated protein peaks are identified by the spectrophotometric absorbance at 210 nm, collected, lyophilized, and stored at - 20° until needed for bioassays. The amidinated protein was characterized by analytical reversed-phase HPLC (C4, 0.46 x 15 cm). The native and amidinated proteins have distinctly different profiles using this HPLC system. The amidinated protein was further characterized by the disappearance of the single lysine residue in each TGF-o~ species as determined by amino acid composition analysis using a Beckman System 6300 amino acid analyzer (Beckman Instruments, Fullerton, CA). 17 Conclusions
We have employed several approaches in analyzing the structure-function relationships of human TGF-o~. Each method has provided complementary data that support the following conclusions: (1) Biological activity resides in the entire TGF-a molecule. Peptide segments of TGF-~ possess little or no receptor-binding or mitogenic activity. (2) Amino acid residues critical to the biological activity of TGF-~ exist in each of the disulfideconstrained loops and in the C terminus of the molecule. An important 17 F. T. Wood, M. M. Wu, and J. C. Gerhart, Anal. Blochem. 69, 339 (1975).
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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.
