Proc. Natl. Acad. Sci. USA Vol. 75, No. 10, pp. 4675-4678, October 1978
Biochemistry
Brain-derived fibroblast growth factor: Identity with a fragment of the basic protein of myelin (wound healing/myelin basic protein)
FRED C. WESTALL*, VANDA A. LENNON*t, AND DENIS GOSPODAROWICZt *The Salk Institute, San Diego, California 92112; and tCancer Research Institute, University of California Medical Center, San Francisco, California 94143
Communicated by Robert W. Holley, June 16, 1978
ABSTRACT Fibroblast growth factors (FGF) isolated from bovine brain have been identified chemically and immunologically as components of the myelin basic protein. The intact bovine basic protein molecule (170 residues), prepared by the standard acidlextraction procedure, lacked mitogenic activity (tested at concentrations up to 10 ,g/ml). However, the polypeptide FGF-2, identified as residues 44-153 of the basic protein, was maximally mitogenic for fibroblasts at 10 ng/ml and polypeptide 44-166 (FGF-1) was maximally active at 100 ng/ml. Pituitary-derived FGF is as potent a growth factor as FGF-2, but appears to be biochemically and immunologically distinct from brain-derived FGF. FGF released in the centra or peripheral nervous system as a consequence of myelin damage and basic protein proteolysis could provide a physiological stimulus for wound healing and myelin repair.
in water, dialyzed, and applied to a column of carboxymethyl-Sephadex C-50 equilibrated with 0.1 M sodium phosphate, pH 6.0. The column was sequentially washed with 0.1 M sodium phosphate (pH 6.0) followed by stepwise addition of NaCl (0.15 M and 0.5 M). The 0.5 M NaCI fraction was applied to a Sephadex G-75 column equilibrated with 0.1 M ammonium carbonate (pH 8.5). The mitogenic activity was recovered in two ultraviolet-absorbing peaks. The two mitogenic preparations were lyophilized separately, and each was dissolved in 0.2 M ammonium formate (pH 6.0) and applied to a column of carboxymethyl-cellulose (CM 52) equilibrated with 0.2 M ammonium formate (pH 6.0). After washing, a linear gradient of ammonium formate (0.2-0.4 M) was applied.
Mitogenic activity from each preparation eluted at 0.35 M ammonium formate (pH 6.0). The fractions were named FGF-1 and FGF-2. A third mitogenic preparation, named FGF-3, eluted at 0.35 M ammonium formate after the FGF-2 fraction. Pituitary FGF was prepared as described (5). Homogeneity was established by polyacrylamide gel electrophoresis at pH 4.5 and 8.5 and by sodium dodecyl sulfate gel elec-
Much evidence points to the importance of a class of mitogenic polypeptides termed growth factors in the control of animal cell proliferation. Fibroblast growth factor (FGF) stimulates in vitro proliferation of a variety of mesoderm-derived cells, including fibroblasts, vascular and corneal endothelial cells, steroidproducing cells, amniotic cells, chondrocytes, and myoblasts (1, 2). FGF has been purified from bovine whole brain (3, 4) and pituitary (5). The brain factor has been isolated as two basic polypeptides of molecular weights 13,000 (FGF-1) and 11,700 (FGF-2), and the pituitary factor as a basic polypeptide of molecular weight 13,400 (3-5). Computer-aided comparison of the amino acid compositions of these substances with other known proteins (kindly performed by Roger Burgus of The Salk Institute) revealed that the brain factors are similar in composition to the myelin basic protein. We report here immunological and biochemical evidence that the larger brain factor corresponds to residues 44-166 of bovine myelin basic protein, while the smaller factor corresponds to residues 44-153.
trophoresis (3-5). Myelin basic protein was prepared from bovine and porcine spinal cords and human brains by the procedure of Diebler et al. (6). The protein was similarly prepared from rabbit, monkey, rat, and guinea pig frozen brains as follows: After the sample was defatted with chloroform/methanol (2:1 vol/vol), the residue was homogenized in cold distilled water and the pH was adjusted to 2.0-2.3 with 6 M HC1. The mixture was stirred at 40 overnight and then adjusted to pH 7.0 with 6 M NH40H. After centrifugation, the precipitate was discarded. Ammonium sulfate was added to the supernate (350 g/liter) while the pH was maintained at 7.0. After 2 hr the suspension was centrifuged and the supernate discarded. The residue was dissolved in distilled water, dialyzed at 40 for 18 hr against water, lyophilized to reduce its volume, and applied to a Cellex P column (Bio-Rad Chemical Co., Richmond, CA). The sample was eluted with a stepwise gradient of ammonium acetate (0.1 M 0.4 M 1.6 M) at pH 7. The basic protein eluted with 1.6
MATERIALS AND METHODS Preparation of FGF and Myelin Basic Protein. The major differences in preparation of myelin basic protein and FGF was the use of chloroform/methanol in defatting the brain tissue for myelin basic protein and the initial pH of extraction. In purifying FGF, the brain tissue was briefly exposed to mild acid (pH 4.5) after homogenization. In preparing myelin basic protein, neural tissue was exposed to pH 2.5 overnight. FGF were purified from frozen bovine brain and pituitary as described elsewhere (3-5). In brief, the tissues were homogenized at 40 in 0.15 M (NH4)2SO4. The pH was adjusted to 4.5 with HC1. The suspension was then centrifuged and the supernate was adjusted to a pH of 6.5-7.0. A first (NH4)2SO4 cut (200 g/liter) gave a precipitate that had no activity. A second (NH4)2SO4 cut (250 g/liter) gave a precipitate that contained most of the FGF activity. That fraction was dissolved
M ammonium acetate. Tryptic Digestion. FGF-1, FGF-2, FGF-3, and myelin basic protein were each trypsinized (1:50) in 0.2 M triethylamine/ carbonate buffer, pH 8.0 for 24 hr. The tryptic digests were spotted on Whatman 3 MM paper (42 X 18 inches; 107 X 46 cm) and separated by electrophoresis (pyridine/acetic acid/ butanol/H20 1:1.2:36; pH 4.7), followed by ascending chromatography. Peptides were detected by staining the chromatograms first with ninhydrin (50 mg/200 ml of acetone) followed by fluorescamine staining for more sensitive detection (7). After elution, the individual peptides were hydrolyzed in constant boiling HCl for 24 hr. Tryptophan was determined
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviation: FGF, fibroblast growth factor. t Present address: Neuroimmunology Laboratory, Department of Neurology, Mayo Clinic, Rochester, MN 55901.
4675
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Biochemistry: Westall et al.
4676
Proc. Natl. Acad. Sci. USA 75 (1978)
153-155
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FIG. 1. Line tracings of two-dimensional chromatograms. Trypsin digests of (A) FGF-1 (residues 44-166); (B) FGF-2 (residues 44-153); (C) FGF-3 (residues 91-153); and (D) bovine myelin basic protein.
by a spectral procedure (8). Amino acid analysis was performed on each hydrolyzed peptide by a Beckman 121 automatic amino acid analyzer. Test for Sugar. The sugar content of the FGF preparations was determined by the procedure of Schoenenberger et al. (9). Assay of Encephalitogenicity. Outbred female guinea pigs weighing 350-500 g were injected intradermally in four distal skin sites with graded doses of FGF preparations dissolved in isotonic saline and emulsified with an equal volume of complete
Freund's adjuvant (Difco Clostridium butyricum supplemented with Mycobacterium tuberculosis, H37Rv Eli Lilly, 2.0 mg/ml). Control guinea pigs received 50 ,tg of myelin basic protein in complete Freund's adjuvant. Female Lewis rats (approximately 10 weeks old) were injected as described (10) with 100,gg of FGF-3 in complete Freund's adjuvant and with Bordetella pertussis. Radioimmunoassays. Sera were tested for antibodies to 125I-labeled bovine myelin basic protein by gel filtration radioimmunoassay (11). Positive guinea pig sera were pooled according to groups, and the antigen-binding capacities of the pools were measured. RESULTS Biochemical characterization Tryptic fragments of FGF-1, FGF-2, and FGF-3 were compared with tryptic fragments of myelin basic protein (Fig. 1). Pituitary-derived FGF was relatively resistant to trypsin digestion in our experimental conditions-i.e., large, diffuse, poorly stained smears were observed on the chromatogram. All the spots on the tryptic "maps" of FGF-1, FGF-2, and FGF-3 were identifiable as portions of myelin basic protein (Fig. 1). There was no evidence of glutamine or asparagine deamidation. The NH2-terminal phenylalanine of FGF-1 and FGF-2 was identified as residue 44 of myelin basic protein. The COOHterminal proline of FGF-1 corresponded to residue 166 and the COOH-terminal isoleucine of FGF-2 corresponded to residue
153. The NH2-terminal lysine of FGF-3 corresponded to residue 91 and its COOH-terminal isoleucine to residue 153. Amino acid sequences deduced for the three fragments are shown in Fig. 2. Sugar content of brain and pituitary FGF preparations was negligible (less than 1 mole per mole of protein). Mitogenicity of brain and pituitary FGF and myelin basic protein
Brain and pituitary FGF fractions were compared with myelin basic protein for their capacity to stimulate initiation of DNA synthesis in serum-starved BALB/c 3T3 cells (12). FGF-2 was the most potent of the three brain FGF fractions (Fig. 3). Pituitary FGF was slightly more potent than FGF-2 on a weight basis. Bovine myelin basic protein lacked significant mitogenicity over the range of concentrations tested (1-10,000 ng/ml). Myelin basic protein of six other species were tested: human, rabbit, monkey, porcine, rat, and guinea pig. Only the guinea pig protein was mitogenic (at 10-100 ,g/ml). Immunologic crossreactivity of FGF and myelin basic protein When injected with adjuvants into guinea pigs and rats, brain-derived FGF preparations induced clinical and histological signs of experimental autoimmune encephalomyelitis, but pituitary-derived FGF was not encephalitogenic (Table 1). Severe encephalomyelitis occurred in guinea pigs injected with 10 ug of FGF-2; rats injected with 100Mg of FGF-3 exhibited hind-limb and tail paresis. Antibodies reacting with bovine myelin basic protein were induced by FGF-1 and FGF-2 in guinea pigs and FGF-3 in rats (Table 1). No antibodies against basic protein were detectable in animals inoculated with pituitary-derived FGF. The antigen-binding capacity of the guinea pig antiserum against FGF-1 was less than that of the pool of antiserum against FGF-2, which was in the same range as pooled sera from guinea pigs immunized with myelin basic protein. Histologic lesions characteristic of experimenal autoimmune encephalomyelitis were found in both gray and white matter of the central nervous system of guinea pigs and rats and were most prominent in the spinal cord.
Biochemistry:
Westall et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
4677
Phe Gly Ser Asp Arg Gly Ala Pro Lys Arg Gly Ser Gly Lys Asp 44 50 Gly His His Ala Ala Arg Thr Thr His Tyr Gly Ser Leu Pro Gin Lys 60
Ala Gln Gly His Arg Pro Gln Asp Glu Asn Pro Val Val His Phe Phe 80 90 Lys Asn lle Val Thr Pro Arg Thr Pro Pro Pro Ser Gln Gly Lys Gly ________________________________________________________________
100 Arg Gly Leu Ser Leu Ser Arg Phe Ser Trp Gly Ala Glu Gly Gin Lys _______________________________________________________________
110
120
Pro Gly Phe Gly Tyr Gly Gly Arg Ala Ser Asp Tyr Lys Ser Ala His _______________________________________________________________
130 Lys Gly Leu Lys Gly His Asp Ala Gln Gly Thr Leu Ser Lys lie Phe ___________________________________________________________
140
150
153
Lys Leu Gly Gly Arg Asp Ser Arg Ser Gly Ser Pro
160
166
FIG. 2. FGF-1 is composed of residues 44-166; FGF-2, of residues 44-153 (solid line); and FGF-3, of residues 91-153 (broken line).
DISCUSSION The present study has revealed that FGF derived from bovine brain is not only similar to part of the myelin basic protein in its amino acid composition but also crossreacts antigenically with myelin basic protein. Analysis of peptides derived from tryptic digestion revealed that the most potent brain-derived growth factor, FGF-2, encompasses residues 44-153 of the basic protein. The less active factors, FGF-1 and FGF-3, encompass residues 44-166 and 91-153, respectively. Cleavage of myelin
Proteins, ng/ml
FIG. 3. DNA synthesis in 3T3 cells exposed to graded amounts of pituitary FGF (A), brain FGF-1 (0), -2 (0), -3 (3), or bovine myelin basic protein (-). BALB/c 3T3 cells were plated and maintained as described (5). Incorporation of [3H]thymidine into DNA was determined as described (5). The mitogenicities of the brain FGF and pituitary FGF were compared to that of myelin basic protein; control values were 160 cpm + 14. Every point was done in triplicate, and the SD did not exceed 10% of the mean.
basic protein between residues 43-44,189-90, and 90-91 is effected by an endogenous proteinase of brain that is active at acid pH (13). Therefore, fragments with FGF activity were most likely generated from myelin basic protein by proteolysis during the purification procedure. Pituitary FGF has no biochemical homology with the basic protein and since no antigenic relationship was detected in the present study, it is likely that pituitary FGF is a separate entity from brain FGF. Immunization with FGF-1 and FGF-2 induced clinical and histologic signs of experimental autoimmune encephalomyelitis and antibodies that crossreacted with myelin basic protein. FGF-2 was the more potent immunogen in guinea pigs. FGF-3, at a dose of 100 ,ug induced in rats both encephalomyelitis and antibodies crossreacting with the basic protein. The fragment corresponding to residues 114-122 of myelin basic protein (Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys) contains the sole tryptophan of the basic protein and is the major encephalomylitis-inducing determinant for guinea pigs (14). This peptide is inactive in rats (15). The bovine myelin basic protein contains within residues 90-120 a determinant that is encephalitogenic for rats (16). This would explain why FGF-3 was encephalitogenic for rats. Our results suggest that at least a fragment of the myelin basic protein has FGF activity. Intact basic protein prepared by an acid procedure from bovine spinal cord lacked mitogenic activity. However, Sheffield and Kim (17) reported that myelin basic protein prepared from guinea pig spinal cord was mitogenic for lymphocytes and astrocytes. In our assay for mitogenicity, guinea pig myelin basic protein was the only one of six species tested that had any mitogenic activity, and that was at a protein concentration 103 times greater than FGF-2.
4678
Biochemistry: Westall et al.
Proc. Nati. Acad. Sci. USA 75 (1978) Table 1. Encephalitogenicity of FGFs
1251-Labeled Dose,
Preparation
jig
Experimental autoimmune encephalomyelitis Clinical Histologic
Mortality
Antibody to basic protein
basic protein bound,* ,gg/ml serum
In guinea pigs
3/4 4/8 5/6 1.46 3/4 3/4 2/2 FGF-1 3/5 3/8 5/5 0.205 2/4 0/4 0/4 3/4 0/4 0/4 0/4 FGF-3 4/4 3/4 0/2 FGF (pituitary) 0/4 0/4 Bovine basic protein NDt 2/4 2/4 1.12 In Lewis rats FGF-3 100 2/4 1/1 0/4 4/4 Bovine basic protein 100 2/4 3/4 0/4 4/4 * Antigen-binding capacities determined on pooled positive sera obtained later than 30 days after inoculation. t Not determined. FGF-2
100 10 50-100 10 5 1 100 100 50
5/8 3/4 6/8 1/4 0/8 0/4 4/4 0/4 3/4
There are at least two possible explanations for the intact basic protein's lack of mitogenic activity. First, the mitogenicity of FGF is acid labile (inactivated at pH 3.5). Acid inactivation might result from a conformational change in the molecule or the cleavage of a covalent bond. For example, an acid-labile phosphate group could be hydrolyzed or an active amide could be deamidated. Smith et al. (18) have reported the presence of acid-labile phosphate residues in myelin basic protein. A second possible explanation for the intact basic protein's lack of mitogenic activity is that proteolysis of the protein gives rise to an activity which is quiescent in the intact molecule. Proteolysis of myelin basis protein in vio may have physiological significance. For example, in multiple sclerosis, brain tissue with evidence of active demyelination possesses large amounts of acid proteinase (19). Furthermore, in the active phase of multiple sclerosis, basic protein and peptides, including the 45-90 fragment, are released into the cerebrospinal fluid (20). It is conceivable that FGF released in these situations could trigger the astrocyte proliferation, which is characteristic of multiple sclerosis lesions, and oligodendroglial cell division, which would at least partially repair myelin. Since most, if not all, tissues are innervated by myelinated nerve fibers, generation of peptides with FGF activity from myelin basic protein released from injured peripheral myelin may have profound implications for initiating and localizing the wound-healing process. As a consequence of nerve injury, the process of Wallerian degeneration is initiated, during which myelin is fragmented. Increased proteolytic activity during the first 8-12 days after trauma would readily generate FGF molecules. This may be the most elegant way for the organism to deliver a growth factor that has been shown to be mitogenic for the two most important cell types involved in wound healing-fibroblasts, which form the ground substance, and vascular endothelial cells, from which capillaries are formed. We thank Cynthia Sommers, Mildred Newberry, and Sue Peterson for excellent technical assistance. This work was supported by Grants NS 12391, NS 11719 from the National Institutes of Health, and the Kroc Foundation, Santa Ynez, CA.
1. Gospodarowicz, D., Greenburg, G., Bialecki, H. & Zetter, B. (1978) In Vitro 14, 85-118. 2. Gospodarowicz, D., Vlodavsky, I., Fielding, P. & Birdwell, C. R. (1978) in The Fifth International Conference of Birth Defects, ed. Peerenboom, T. (Excerpta Medica, Amsterdam, The Neth-
erlands). 3. Gospodarowicz, D., Bialecki, H. & Greenburg, G. (1978) J. Biol. Chem., in press. 4. Gospodarowicz, D., Moran, J. & Bialecki, H. (1976) in Third International Symposium on Growth Hormone and Related
Peptides (Excerpta Medica, Amsterdam, The Netherlands), Vol. 5. 6.
7. 8. 9. 10. 11. 12.
13. 14.
15.
381, pp. 141-155. Gospodarowicz, D. (1975) J. Biol. Chem. 250,2515-2520. Diebler, G. E., Martenson, R. E. & Kies, M. W. (1972) Prep. Biochem. 2, 139-145. Vanderkerckoue, J. & Van Montague, M. (1974) Eur. J. Biochem. 44,279-288. Godwin, T. W. & Morton, R. A. (1946) Biochem. J. 40, 628635. Schoenenberger, M., Kellner, H., Sudhof, H. & Kaupt, H. (1957) Koppe-Seyler's Physiol. Chem. 309, 145-157. Lennon, V. A., Westall, F. C., Thompson, M. & Ward, E. (1976) Eur. J. Immunol. 6,805-810. Lennon, V. A., Whittingham, S., Carnegie, P. R., McPherson, T. A. & Mackay, I. R. (1971) J. Immunol. 107,56-62. Gospodarowicz, D. & Moran, J. S. (1975) J. Cell Biol. 66,451457. Einstein, E. R., Csejtey, J. & Marks, N. (1968) FEBS Lett. 1, 191-196. Westall, F. C., Robinson, A. B., Caccam, F., Jackson, F. C. & Eylar, E. H. (1971) Nature (London) 229,22-25. Lennon, V. A. (1972) Dissertation (University of Melbourne,
Melbourne, Australia). 16. Westall, F. C. & Thompson, M. (1977) Immunol. Commun. 6, 13-21. 17. Sheffield, W. D. & Kim, S. V. (1977) Brain Res. 132,580-584. 18. Smith, L. S., Kern, C. W., Hlalpern, R. C. & Smith, R. A. (1976) Biochem. Biophys. Res. (ommun. 71, 459-465. 19. Adams, C. W. M. (1968) in Macromolecules and the Formation of the Neuron, eds. Loden, Z. & Rose, S. P. R. (Excerpta Medica,
Amsterdam, The Netherlands), pp. 111-119.
20. Whittaker, J. (1977) Neurology 27,911-920.
Biochemistry
Brain-derived fibroblast growth factor: Identity with a fragment of the basic protein of myelin (wound healing/myelin basic protein)
FRED C. WESTALL*, VANDA A. LENNON*t, AND DENIS GOSPODAROWICZt *The Salk Institute, San Diego, California 92112; and tCancer Research Institute, University of California Medical Center, San Francisco, California 94143
Communicated by Robert W. Holley, June 16, 1978
ABSTRACT Fibroblast growth factors (FGF) isolated from bovine brain have been identified chemically and immunologically as components of the myelin basic protein. The intact bovine basic protein molecule (170 residues), prepared by the standard acidlextraction procedure, lacked mitogenic activity (tested at concentrations up to 10 ,g/ml). However, the polypeptide FGF-2, identified as residues 44-153 of the basic protein, was maximally mitogenic for fibroblasts at 10 ng/ml and polypeptide 44-166 (FGF-1) was maximally active at 100 ng/ml. Pituitary-derived FGF is as potent a growth factor as FGF-2, but appears to be biochemically and immunologically distinct from brain-derived FGF. FGF released in the centra or peripheral nervous system as a consequence of myelin damage and basic protein proteolysis could provide a physiological stimulus for wound healing and myelin repair.
in water, dialyzed, and applied to a column of carboxymethyl-Sephadex C-50 equilibrated with 0.1 M sodium phosphate, pH 6.0. The column was sequentially washed with 0.1 M sodium phosphate (pH 6.0) followed by stepwise addition of NaCl (0.15 M and 0.5 M). The 0.5 M NaCI fraction was applied to a Sephadex G-75 column equilibrated with 0.1 M ammonium carbonate (pH 8.5). The mitogenic activity was recovered in two ultraviolet-absorbing peaks. The two mitogenic preparations were lyophilized separately, and each was dissolved in 0.2 M ammonium formate (pH 6.0) and applied to a column of carboxymethyl-cellulose (CM 52) equilibrated with 0.2 M ammonium formate (pH 6.0). After washing, a linear gradient of ammonium formate (0.2-0.4 M) was applied.
Mitogenic activity from each preparation eluted at 0.35 M ammonium formate (pH 6.0). The fractions were named FGF-1 and FGF-2. A third mitogenic preparation, named FGF-3, eluted at 0.35 M ammonium formate after the FGF-2 fraction. Pituitary FGF was prepared as described (5). Homogeneity was established by polyacrylamide gel electrophoresis at pH 4.5 and 8.5 and by sodium dodecyl sulfate gel elec-
Much evidence points to the importance of a class of mitogenic polypeptides termed growth factors in the control of animal cell proliferation. Fibroblast growth factor (FGF) stimulates in vitro proliferation of a variety of mesoderm-derived cells, including fibroblasts, vascular and corneal endothelial cells, steroidproducing cells, amniotic cells, chondrocytes, and myoblasts (1, 2). FGF has been purified from bovine whole brain (3, 4) and pituitary (5). The brain factor has been isolated as two basic polypeptides of molecular weights 13,000 (FGF-1) and 11,700 (FGF-2), and the pituitary factor as a basic polypeptide of molecular weight 13,400 (3-5). Computer-aided comparison of the amino acid compositions of these substances with other known proteins (kindly performed by Roger Burgus of The Salk Institute) revealed that the brain factors are similar in composition to the myelin basic protein. We report here immunological and biochemical evidence that the larger brain factor corresponds to residues 44-166 of bovine myelin basic protein, while the smaller factor corresponds to residues 44-153.
trophoresis (3-5). Myelin basic protein was prepared from bovine and porcine spinal cords and human brains by the procedure of Diebler et al. (6). The protein was similarly prepared from rabbit, monkey, rat, and guinea pig frozen brains as follows: After the sample was defatted with chloroform/methanol (2:1 vol/vol), the residue was homogenized in cold distilled water and the pH was adjusted to 2.0-2.3 with 6 M HC1. The mixture was stirred at 40 overnight and then adjusted to pH 7.0 with 6 M NH40H. After centrifugation, the precipitate was discarded. Ammonium sulfate was added to the supernate (350 g/liter) while the pH was maintained at 7.0. After 2 hr the suspension was centrifuged and the supernate discarded. The residue was dissolved in distilled water, dialyzed at 40 for 18 hr against water, lyophilized to reduce its volume, and applied to a Cellex P column (Bio-Rad Chemical Co., Richmond, CA). The sample was eluted with a stepwise gradient of ammonium acetate (0.1 M 0.4 M 1.6 M) at pH 7. The basic protein eluted with 1.6
MATERIALS AND METHODS Preparation of FGF and Myelin Basic Protein. The major differences in preparation of myelin basic protein and FGF was the use of chloroform/methanol in defatting the brain tissue for myelin basic protein and the initial pH of extraction. In purifying FGF, the brain tissue was briefly exposed to mild acid (pH 4.5) after homogenization. In preparing myelin basic protein, neural tissue was exposed to pH 2.5 overnight. FGF were purified from frozen bovine brain and pituitary as described elsewhere (3-5). In brief, the tissues were homogenized at 40 in 0.15 M (NH4)2SO4. The pH was adjusted to 4.5 with HC1. The suspension was then centrifuged and the supernate was adjusted to a pH of 6.5-7.0. A first (NH4)2SO4 cut (200 g/liter) gave a precipitate that had no activity. A second (NH4)2SO4 cut (250 g/liter) gave a precipitate that contained most of the FGF activity. That fraction was dissolved
M ammonium acetate. Tryptic Digestion. FGF-1, FGF-2, FGF-3, and myelin basic protein were each trypsinized (1:50) in 0.2 M triethylamine/ carbonate buffer, pH 8.0 for 24 hr. The tryptic digests were spotted on Whatman 3 MM paper (42 X 18 inches; 107 X 46 cm) and separated by electrophoresis (pyridine/acetic acid/ butanol/H20 1:1.2:36; pH 4.7), followed by ascending chromatography. Peptides were detected by staining the chromatograms first with ninhydrin (50 mg/200 ml of acetone) followed by fluorescamine staining for more sensitive detection (7). After elution, the individual peptides were hydrolyzed in constant boiling HCl for 24 hr. Tryptophan was determined
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviation: FGF, fibroblast growth factor. t Present address: Neuroimmunology Laboratory, Department of Neurology, Mayo Clinic, Rochester, MN 55901.
4675
~ 92-7
Biochemistry: Westall et al.
4676
Proc. Natl. Acad. Sci. USA 75 (1978)
153-155
A 106-1
130c
140-1420 C
156159
49-52
136-139 54-57
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Q75-91 O
0
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0 160-162
0
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58-64
+- + Origin
- v- + Origin
0131-135
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114-130
92-97 106-1130Q ,13o65-74 0 0043-48
98-105
136-139
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C)75-91 98-105
49-52 136-139
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0140-142 91
0_114-130
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143-152
58-64
C
92-97
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140-1420 156- 0 C)75-91 013-24
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FIG. 1. Line tracings of two-dimensional chromatograms. Trypsin digests of (A) FGF-1 (residues 44-166); (B) FGF-2 (residues 44-153); (C) FGF-3 (residues 91-153); and (D) bovine myelin basic protein.
by a spectral procedure (8). Amino acid analysis was performed on each hydrolyzed peptide by a Beckman 121 automatic amino acid analyzer. Test for Sugar. The sugar content of the FGF preparations was determined by the procedure of Schoenenberger et al. (9). Assay of Encephalitogenicity. Outbred female guinea pigs weighing 350-500 g were injected intradermally in four distal skin sites with graded doses of FGF preparations dissolved in isotonic saline and emulsified with an equal volume of complete
Freund's adjuvant (Difco Clostridium butyricum supplemented with Mycobacterium tuberculosis, H37Rv Eli Lilly, 2.0 mg/ml). Control guinea pigs received 50 ,tg of myelin basic protein in complete Freund's adjuvant. Female Lewis rats (approximately 10 weeks old) were injected as described (10) with 100,gg of FGF-3 in complete Freund's adjuvant and with Bordetella pertussis. Radioimmunoassays. Sera were tested for antibodies to 125I-labeled bovine myelin basic protein by gel filtration radioimmunoassay (11). Positive guinea pig sera were pooled according to groups, and the antigen-binding capacities of the pools were measured. RESULTS Biochemical characterization Tryptic fragments of FGF-1, FGF-2, and FGF-3 were compared with tryptic fragments of myelin basic protein (Fig. 1). Pituitary-derived FGF was relatively resistant to trypsin digestion in our experimental conditions-i.e., large, diffuse, poorly stained smears were observed on the chromatogram. All the spots on the tryptic "maps" of FGF-1, FGF-2, and FGF-3 were identifiable as portions of myelin basic protein (Fig. 1). There was no evidence of glutamine or asparagine deamidation. The NH2-terminal phenylalanine of FGF-1 and FGF-2 was identified as residue 44 of myelin basic protein. The COOHterminal proline of FGF-1 corresponded to residue 166 and the COOH-terminal isoleucine of FGF-2 corresponded to residue
153. The NH2-terminal lysine of FGF-3 corresponded to residue 91 and its COOH-terminal isoleucine to residue 153. Amino acid sequences deduced for the three fragments are shown in Fig. 2. Sugar content of brain and pituitary FGF preparations was negligible (less than 1 mole per mole of protein). Mitogenicity of brain and pituitary FGF and myelin basic protein
Brain and pituitary FGF fractions were compared with myelin basic protein for their capacity to stimulate initiation of DNA synthesis in serum-starved BALB/c 3T3 cells (12). FGF-2 was the most potent of the three brain FGF fractions (Fig. 3). Pituitary FGF was slightly more potent than FGF-2 on a weight basis. Bovine myelin basic protein lacked significant mitogenicity over the range of concentrations tested (1-10,000 ng/ml). Myelin basic protein of six other species were tested: human, rabbit, monkey, porcine, rat, and guinea pig. Only the guinea pig protein was mitogenic (at 10-100 ,g/ml). Immunologic crossreactivity of FGF and myelin basic protein When injected with adjuvants into guinea pigs and rats, brain-derived FGF preparations induced clinical and histological signs of experimental autoimmune encephalomyelitis, but pituitary-derived FGF was not encephalitogenic (Table 1). Severe encephalomyelitis occurred in guinea pigs injected with 10 ug of FGF-2; rats injected with 100Mg of FGF-3 exhibited hind-limb and tail paresis. Antibodies reacting with bovine myelin basic protein were induced by FGF-1 and FGF-2 in guinea pigs and FGF-3 in rats (Table 1). No antibodies against basic protein were detectable in animals inoculated with pituitary-derived FGF. The antigen-binding capacity of the guinea pig antiserum against FGF-1 was less than that of the pool of antiserum against FGF-2, which was in the same range as pooled sera from guinea pigs immunized with myelin basic protein. Histologic lesions characteristic of experimenal autoimmune encephalomyelitis were found in both gray and white matter of the central nervous system of guinea pigs and rats and were most prominent in the spinal cord.
Biochemistry:
Westall et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
4677
Phe Gly Ser Asp Arg Gly Ala Pro Lys Arg Gly Ser Gly Lys Asp 44 50 Gly His His Ala Ala Arg Thr Thr His Tyr Gly Ser Leu Pro Gin Lys 60
Ala Gln Gly His Arg Pro Gln Asp Glu Asn Pro Val Val His Phe Phe 80 90 Lys Asn lle Val Thr Pro Arg Thr Pro Pro Pro Ser Gln Gly Lys Gly ________________________________________________________________
100 Arg Gly Leu Ser Leu Ser Arg Phe Ser Trp Gly Ala Glu Gly Gin Lys _______________________________________________________________
110
120
Pro Gly Phe Gly Tyr Gly Gly Arg Ala Ser Asp Tyr Lys Ser Ala His _______________________________________________________________
130 Lys Gly Leu Lys Gly His Asp Ala Gln Gly Thr Leu Ser Lys lie Phe ___________________________________________________________
140
150
153
Lys Leu Gly Gly Arg Asp Ser Arg Ser Gly Ser Pro
160
166
FIG. 2. FGF-1 is composed of residues 44-166; FGF-2, of residues 44-153 (solid line); and FGF-3, of residues 91-153 (broken line).
DISCUSSION The present study has revealed that FGF derived from bovine brain is not only similar to part of the myelin basic protein in its amino acid composition but also crossreacts antigenically with myelin basic protein. Analysis of peptides derived from tryptic digestion revealed that the most potent brain-derived growth factor, FGF-2, encompasses residues 44-153 of the basic protein. The less active factors, FGF-1 and FGF-3, encompass residues 44-166 and 91-153, respectively. Cleavage of myelin
Proteins, ng/ml
FIG. 3. DNA synthesis in 3T3 cells exposed to graded amounts of pituitary FGF (A), brain FGF-1 (0), -2 (0), -3 (3), or bovine myelin basic protein (-). BALB/c 3T3 cells were plated and maintained as described (5). Incorporation of [3H]thymidine into DNA was determined as described (5). The mitogenicities of the brain FGF and pituitary FGF were compared to that of myelin basic protein; control values were 160 cpm + 14. Every point was done in triplicate, and the SD did not exceed 10% of the mean.
basic protein between residues 43-44,189-90, and 90-91 is effected by an endogenous proteinase of brain that is active at acid pH (13). Therefore, fragments with FGF activity were most likely generated from myelin basic protein by proteolysis during the purification procedure. Pituitary FGF has no biochemical homology with the basic protein and since no antigenic relationship was detected in the present study, it is likely that pituitary FGF is a separate entity from brain FGF. Immunization with FGF-1 and FGF-2 induced clinical and histologic signs of experimental autoimmune encephalomyelitis and antibodies that crossreacted with myelin basic protein. FGF-2 was the more potent immunogen in guinea pigs. FGF-3, at a dose of 100 ,ug induced in rats both encephalomyelitis and antibodies crossreacting with the basic protein. The fragment corresponding to residues 114-122 of myelin basic protein (Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys) contains the sole tryptophan of the basic protein and is the major encephalomylitis-inducing determinant for guinea pigs (14). This peptide is inactive in rats (15). The bovine myelin basic protein contains within residues 90-120 a determinant that is encephalitogenic for rats (16). This would explain why FGF-3 was encephalitogenic for rats. Our results suggest that at least a fragment of the myelin basic protein has FGF activity. Intact basic protein prepared by an acid procedure from bovine spinal cord lacked mitogenic activity. However, Sheffield and Kim (17) reported that myelin basic protein prepared from guinea pig spinal cord was mitogenic for lymphocytes and astrocytes. In our assay for mitogenicity, guinea pig myelin basic protein was the only one of six species tested that had any mitogenic activity, and that was at a protein concentration 103 times greater than FGF-2.
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Biochemistry: Westall et al.
Proc. Nati. Acad. Sci. USA 75 (1978) Table 1. Encephalitogenicity of FGFs
1251-Labeled Dose,
Preparation
jig
Experimental autoimmune encephalomyelitis Clinical Histologic
Mortality
Antibody to basic protein
basic protein bound,* ,gg/ml serum
In guinea pigs
3/4 4/8 5/6 1.46 3/4 3/4 2/2 FGF-1 3/5 3/8 5/5 0.205 2/4 0/4 0/4 3/4 0/4 0/4 0/4 FGF-3 4/4 3/4 0/2 FGF (pituitary) 0/4 0/4 Bovine basic protein NDt 2/4 2/4 1.12 In Lewis rats FGF-3 100 2/4 1/1 0/4 4/4 Bovine basic protein 100 2/4 3/4 0/4 4/4 * Antigen-binding capacities determined on pooled positive sera obtained later than 30 days after inoculation. t Not determined. FGF-2
100 10 50-100 10 5 1 100 100 50
5/8 3/4 6/8 1/4 0/8 0/4 4/4 0/4 3/4
There are at least two possible explanations for the intact basic protein's lack of mitogenic activity. First, the mitogenicity of FGF is acid labile (inactivated at pH 3.5). Acid inactivation might result from a conformational change in the molecule or the cleavage of a covalent bond. For example, an acid-labile phosphate group could be hydrolyzed or an active amide could be deamidated. Smith et al. (18) have reported the presence of acid-labile phosphate residues in myelin basic protein. A second possible explanation for the intact basic protein's lack of mitogenic activity is that proteolysis of the protein gives rise to an activity which is quiescent in the intact molecule. Proteolysis of myelin basis protein in vio may have physiological significance. For example, in multiple sclerosis, brain tissue with evidence of active demyelination possesses large amounts of acid proteinase (19). Furthermore, in the active phase of multiple sclerosis, basic protein and peptides, including the 45-90 fragment, are released into the cerebrospinal fluid (20). It is conceivable that FGF released in these situations could trigger the astrocyte proliferation, which is characteristic of multiple sclerosis lesions, and oligodendroglial cell division, which would at least partially repair myelin. Since most, if not all, tissues are innervated by myelinated nerve fibers, generation of peptides with FGF activity from myelin basic protein released from injured peripheral myelin may have profound implications for initiating and localizing the wound-healing process. As a consequence of nerve injury, the process of Wallerian degeneration is initiated, during which myelin is fragmented. Increased proteolytic activity during the first 8-12 days after trauma would readily generate FGF molecules. This may be the most elegant way for the organism to deliver a growth factor that has been shown to be mitogenic for the two most important cell types involved in wound healing-fibroblasts, which form the ground substance, and vascular endothelial cells, from which capillaries are formed. We thank Cynthia Sommers, Mildred Newberry, and Sue Peterson for excellent technical assistance. This work was supported by Grants NS 12391, NS 11719 from the National Institutes of Health, and the Kroc Foundation, Santa Ynez, CA.
1. Gospodarowicz, D., Greenburg, G., Bialecki, H. & Zetter, B. (1978) In Vitro 14, 85-118. 2. Gospodarowicz, D., Vlodavsky, I., Fielding, P. & Birdwell, C. R. (1978) in The Fifth International Conference of Birth Defects, ed. Peerenboom, T. (Excerpta Medica, Amsterdam, The Neth-
erlands). 3. Gospodarowicz, D., Bialecki, H. & Greenburg, G. (1978) J. Biol. Chem., in press. 4. Gospodarowicz, D., Moran, J. & Bialecki, H. (1976) in Third International Symposium on Growth Hormone and Related
Peptides (Excerpta Medica, Amsterdam, The Netherlands), Vol. 5. 6.
7. 8. 9. 10. 11. 12.
13. 14.
15.
381, pp. 141-155. Gospodarowicz, D. (1975) J. Biol. Chem. 250,2515-2520. Diebler, G. E., Martenson, R. E. & Kies, M. W. (1972) Prep. Biochem. 2, 139-145. Vanderkerckoue, J. & Van Montague, M. (1974) Eur. J. Biochem. 44,279-288. Godwin, T. W. & Morton, R. A. (1946) Biochem. J. 40, 628635. Schoenenberger, M., Kellner, H., Sudhof, H. & Kaupt, H. (1957) Koppe-Seyler's Physiol. Chem. 309, 145-157. Lennon, V. A., Westall, F. C., Thompson, M. & Ward, E. (1976) Eur. J. Immunol. 6,805-810. Lennon, V. A., Whittingham, S., Carnegie, P. R., McPherson, T. A. & Mackay, I. R. (1971) J. Immunol. 107,56-62. Gospodarowicz, D. & Moran, J. S. (1975) J. Cell Biol. 66,451457. Einstein, E. R., Csejtey, J. & Marks, N. (1968) FEBS Lett. 1, 191-196. Westall, F. C., Robinson, A. B., Caccam, F., Jackson, F. C. & Eylar, E. H. (1971) Nature (London) 229,22-25. Lennon, V. A. (1972) Dissertation (University of Melbourne,
Melbourne, Australia). 16. Westall, F. C. & Thompson, M. (1977) Immunol. Commun. 6, 13-21. 17. Sheffield, W. D. & Kim, S. V. (1977) Brain Res. 132,580-584. 18. Smith, L. S., Kern, C. W., Hlalpern, R. C. & Smith, R. A. (1976) Biochem. Biophys. Res. (ommun. 71, 459-465. 19. Adams, C. W. M. (1968) in Macromolecules and the Formation of the Neuron, eds. Loden, Z. & Rose, S. P. R. (Excerpta Medica,
Amsterdam, The Netherlands), pp. 111-119.
20. Whittaker, J. (1977) Neurology 27,911-920.
