Proc. Nati. Acad. Sci. USA Vol. 86, pp. 3901-3905, May 1989 Neurobiology Purification and characterization of glia maturation factor f8: A growth regulator for neurons and glia RAMON LIM*, JOYCE F. MILLER, AND ASGAR ZAHEER Department of Neurology, Division of Neurochemistry and Neurobiology, University of Iowa College of Medicine and Veterans Administration Medical Center, Iowa City, IA 52242 Communicated by Hewson Swift, February 17, 1989

ABSTRACT A protein has been isolated from bovine column (5 x 90 cm) and eluted with 0.15 M NaCl containing brains by using a modification of the procedure used to purify the above buffer at 40 ml/hr. The fractions active on astro- glia maturation factor. The method consists of ammonium cytes were pooled (500 ml) and used for the final steps of sulfate precipitation, chromatography with DEAE-Sephacel, purification (see Results). Sephadex G-75, and hydroxylapatite columns, passage through Bioassay and Cell Testing. Bioassay ofGMF activity during a heparin-Sepharose column, and rinally fractionation by purification was performed on cultured astrocytes as de- reverse-phase HPLC with a C4 column. The isolated protein scribed (7). Intact dorsal root ganglia (DRGs) were obtained reacts strongly with the mouse monoclonal antibody G2-09 and from 12-day chicken embryos and cultured on a collagen has a molecular weight of =17,000 and an isoelectric point of surface in Dulbecco's modified Eagle's medium (DMEM) pH 4.9. The N terminus is blocked, but tryptic digestion containing 4.5 g of glucose per liter, 1% glutamine, and 10% releases 28 peptides, 8 of which have been sequenced. The total (vol/vol) heat-inactivated fetal calf serum. Dissociated DRG known residues add up to more than two-thirds of the entire neurons were obtained from 20-day rat fetuses and cultured 140-residue protein, estimated from amino acid composition, on a poly(D-lysine)-coated surface (12-well plastic plates) in and show no sequence homology with any known protein. the above nutrient with supplements. Mouse neuroblastoma Reversible thermal renaturation greatly enhances its biological line clone N-18 (from M. Nirenberg, National Institutes of activity. The purified protein stimulates differentiation of Health) was seeded in 24-well plastic plates and tested in normal neurons as well as glial cells. It inhibits the proliferation RPMI 1640 medium containing 1o (vol/vol) heat-inactivated of the N-18 neuroblastoma line and the C6 glioma line while fetal calf serum. Rat glioma line clone C6 (American Type promoting their phenotypic expression. We designate this Culture Collection) was seeded in 8-well plastic plates and protein glia maturation factor f. tested in F12/DMEM (1:1) containing 5% (vol/vol) fetal calf serum. The trophic function of the nervous system on peripheral Production of Antibodies and Immunoassay. The mouse organs was detected in an in vivo system as early as 1823 (1). monoclonal antibody G2-09 (an IgG2b) against bovine GMF The in vitro counterpart of this experiment was conducted in was produced as described (8). Rabbit polyclonal antibodies 1939 (2-4), showing the mitogenic effect of brain homoge- were obtained by immunizing rabbits with 200 ,ug of GMF-P nates on cultured fibroblasts. However, it was not until 1972t in 0.5 ml of complete Freund's adjuvant by injections into that an autoregulatory role ofbrain-derived growth factors on eight hind toe pads. Three weeks later, each rabbit was given brain cells was first demonstrated in this laboratory (5, 6). a booster injection with 600 ,ug of GMF-18 in 2 ml of incom- This factor was named glia maturation factor (GMF), based plete Freund's adjuvant by intradermal injections at four sites on the bioassay system in which the activity was first on the back. Antisera were collected after another week. The observed. Since then this laboratory has engaged in an serum designated 88-02 was used in the current experiment. intensive search for the molecule responsible for this impor- ELISA were carried out by using a peroxidase-labeled sec- tant function. The effort culminated in the isolation of a brain ond antibody and 2,2'-azinobis(3-ethylbenzothiazoline- protein with a unique amino acid sequence documented 6-sulfonic acid), diammonium salt, (ABTS) as substrate (9); below. results were read at 415 nm from 96-well microtiter plates by using a Bio-Tek microplate reader. Amino Acid Composition. Protein samples were hydro- MATERIALS AND METHODS lyzed at 110°C in 6 M HCl under argon for 24, 48, and 72 hr Preliminary Purification of GMF-j3. The published proce- and derivatized with phenylisothiocyanate. The phenylthio- dure (7, 8) through the Sephadex G-75 step was followed with carbamyl amino acids were separated with HPLC, using a slight modifications. Briefly, four beef brains [1.0 kg (total Waters Pico-Tag amino acid analyzer. wet weight)] were homogenized and centrifuged to obtain the Peptide Mapping and Sequence Determination. GMF-,8 was crude extract. The ammonium sulfate precipitate between extensively reduced and alkylated (10) and subsequently 45% and 70% saturation was dissolved in 100 ml ofwater and digested with trypsin using an enzyme/substrate ratio of dialyzed for two 6-hr periods against 10 liters of water. The 1:100 (wt/wt). The resulting tryptic peptides were separated sample was adjusted to contain 0.02 M Tris HCl (pH 7.45) and on a C8 reverse-phase HPLC column (2.1 mm x 10 cm, applied to a DEAE-Sephacel column (2.5 x 37 cm). After Applied Biosystems RP-300) at a flow rate of 200 ,u/min, eluting with 1.25 liters of a linear gradient of 0-0.3 M NaCl using a linear gradient of 2-80% (vol/vol) acetonitrile con- in the same buffer at 50 ml/hr, the fractions that showed mitogenic and morphologic activities on astrocytes were Abbreviations: DRG, dorsal root ganglion; FGF, fibroblast growth pooled (500 ml) and concentrated to 50 ml by Amicon PM10 factor; GMF, glia maturation factor; F3CCOOH, trifluoroacetic acid. filtration. The sample was applied to a Sephadex G-75 *To whom correspondence and reprint requests should be addressed at: Department of Neurology, University of Iowa College of Med- icine, Iowa City, IA 52242. The publication costs of this article were defrayed in part by page charge tLim, R., Li, W. K. P. & Mitsunobu, K. (1972) Abstracts of the payment. This article must therefore be hereby marked "advertisement" SecondAnnual Meeting ofthe Societyfor Neuroscience, Oct. 8-11, in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1972, Houston, TX, p. 181.

Downloaded by guest on September 25, 2021 3901 3902 Neurobiology: Lim et al. Proc. Natl. Acad. Sci. USA 86 (1989) Table 1. Purification steps and protein recovery

I_ Protein I Step recovered, mg E X o-0. Crude extract 13,900 c I 0 .E (NH4)2SO4 fraction 2,900 40 c c DEAE-Sephacel 627 0 VA Sephadex G-75 49 0.4 *0 -0 - .0 Hydroxylapatite 2.3 Ao0C Heparin-Sepharose 1.3 .0 HPLC 0.12 0 E

-l-~ a..~~

_ IIC C' 4.2 L A . ° E _ 1.0 0.3 0.5 0.7 6 8 Ii0 i Relative Mobility Retention Time (min) _ c at FIG. 3. Determination ofmolecular mass. (A) SDS/PAGE under aC reducing conditions in 15% polyacrylamide gel (12). Bands: a, 0.5 ovalbumin (44 kDa); b, a-chymotrypsinogen (25.7 kDa); c, 68- . 0 lactoglobulin (18.4 kDa); d, lysozyme (14.3 kDa). Arrow pointing °0 ] - 42To upward indicates protein band of GMF-P (200 ng). Arrow pointing downward indicates position of GMF-f3 in the semilog plot. (B) 12 16 4 a Size-exclusion HPLC on a Bio-Sil TSK 125 column (Bio-Rad, 7.5 FRACTION NUMBER (1.5.1) mm x 30 cm), using 0.1 M Na2SO4/0.02 M NaH2PO4, pH 6.8, at 1 ml/min. Arrows: 1, thyroglobulin (670 kDa); 2, y-globulin (158 kDa); FIG. 2. HPLC fractionation of GMF-p by acetonitrile gradient 3, ovalbumin (44 kDa); 4, myoglobin (17 kDa). Elution profile shows elution. Symbols are as in Fig. 1. (A) Initial chromatography. (B) the position ofGMF-,B, which coincides with that ofmyoglobin. With Rechromatography of combined "a" peaks from four initial runs. both methods the molecular weight of GMF-.8 is estimated to be Fractions of 1.5 ml were collected. 17,250. Downloaded by guest on September 25, 2021 Neurobiology: Lim et A Proc. Natl. Acad. Sci. USA 86 (1989) 3903 Table 2. Amino acid composition of 10 GMF-P T12= ETNNAAIIMK T13= NKLVQTAELT K Amino Residue(s) per Amino Residue(s) per T14= VFEIR acid molecule acid molecule T15= FIVYSYK T17= LGFFH Asx 14 Tyr 5 T18= NTEDLTEEWL R 20 T24= LVVLDEELEG ISPDELKDEL PER Glx 24 Val 10 T28= VSYPLCFIFS SPVGCKPEQQ MMYAGSK Ser 8 Ile 7 Gly 5 Leu 12 FIG. 5. Amino acid sequence of eight of the tryptic peptides of His 2 Phe 8 GMF-3 (see Fig. 4). The single-letter code for the amino acids was Arg 8 Lys 12 used. Thr 6 Cys* 3 Ala 6 Met* 3 tested with monoclonal antibody G2-09, and has 2% of the Pro 6 Trpt 1 reactivity of GMF-,B, if tested with polyclonal antibody 88-02 (Fig. 7). The following growth factors, when tested with The total number of residues is 140 and the calculated molecular weight is 16,375. antiserum 88-02, all exhibited less than 1% the reactivity of *Determined after performic acid oxidation. GMF-P: basic FGF, interleukin 1, tumor necrosis factor, tData from sequence analysis. nerve growth factor, insulin, insulin-like growth factor II, epidermal growth factor, and S-100 protein. molecular weight of -16,000 and an isoelectric point of pH Biological Characterization. The biological effect ofGMF-,B 4.9. Further characterization has not been performed. was tested on normal and neoplastic cells derived from the Chemical Characterization of GMF-13. GMF-f3 behaves as nervous system. On normal astrocytes, GMF-,l promotes a single band on SDS/PAGE under reducing conditions, proliferation and morphological differentiation as described indicating a single polypeptide chain with an apparent mo- (8). In addition, GMF-f3 stimulates neuritic outgrowth from lecular weight of =17,000 (Fig. 3). The isoelectric point as intact DRGs in culture (Fig. 8). On dissociated DRGs where determined on an LKB Ampholine PAG plate is pH 4.9. The fibroblasts and glial cells have been eliminated by means of amino acid composition (Table 2) shows a predominance of cytosine arabinoside, GMF-,B promotes neuronal survival acidic amino acids and contains one tryptophan, three me- and stimulates the outgrowth ofneurites (Fig. 9). When tested thionines, and three cysteines (half cystines), with an esti- on the neuroblastoma line N-18 and the glioma line C6, mated length of 140 residues. The N terminus is blocked. GMF-p exhibits a strong antimitotic effect while enhancing Tryptic digestion releases 28 peptides (Fig. 4), 8 of which their phenotypic expression (Fig. 10). The N-18 cells respond have been sequenced (Fig. 5). The total number of known to GMF-,p by outgrowth of neuritic processes, whereas the residues adds up to more than two-thirds ofthe entire protein. C6 cells respond by transformation from a polygonal to a A homology search through the Protein Identification Re- spindle-shaped (fusiform) pattern, both consistent with mor- source database (November 1988; release no. 17) showed no phological maturation. Fig. 11 depicts the dose-response match with any ofthe known proteins, including other growth relationship of GMF-p with respect to the two tumor lines. factors isolated from the nervous system. The half-maximal dose is estimated to be =8 ng/ml for N-18 The secondary structure of GMF-,B was measured with cells and 40 ng/ml for C6 cells. far-ultraviolet circular dichroism (Fig. 6). The profile ob- tained at various temperatures indicates reversible thermal denaturation. The renatured GMF-f3, when obtained after DISCUSSION gradually heating to 80TC and cooling back to 20TC (both at a The current work is a continuation of an earlier effort in this rate of 20C/min), exhibits a higher helical content than the laboratory to isolate and identify GMF. Although we purified original protein. This process greatly increases the biological GMF to a single protein band on an SDS/polyacrylamide gel activity. For this reason the biological characterization of (8), subsequent attempts to sequence it proved unsuccessful, GMF-j3 in this report was conducted with the heat-treated, suggesting microheterogeneity. As an improvement to the renatured protein. earlier procedure, we now incorporated three additional Immunological Characterization. When GMF-,B was com- pared with acidic FGF for immunologic cross-reactivity using 0 ELISA, acidic FGF has 0.1% the reactivity of if GMF-0, V_ ~~~8000C

'? 4004 6000" L. 2i0

,[email protected]

0 .0 ,a ...... 220 nm 280 nm .0 -12000 - 0.4- 200 220 240 Nanometers

0.2 - FIG. 6. Circular dichroism spectra of GMF-,f in 50 mM sodium phosphate (pH 7.0) at various temperatures. At 20°C the native 0- protein shows major ellipticity bands around 210 nm and 220 nm, which represent a major contribution of the secondary structural Time (min) content. These bands disappear as the temperature is raised to 60°C and 80°C, consistent with protein denaturation. However, gradually FIG. 4. Tryptic peptide map of GMF-f3 determined on HPLC. heating and recooling the protein (80°C -. 20°C) results in renatur- Absorbance peaks are labeled from 1 to 28. ation, leading to a more active conformation than the original protein. Downloaded by guest on September 25, 2021 3904 Neurobiology: Lim et al. Proc. Natl. Acad. Sci. USA 86 (1989)

FIG. 9. Effect of GMF-3 on neurons dissociated from DRGs. Ganglion cells were dissociated with trypsin and seeded at 6000 cells per well. The cells were grown in the presence of cytosine arabino- o '*- side (10 !LM) to eliminate nonneuronal cells. (A) Control without 0.2 1 10 100 600 GMF-/3. (B) GMF-13 at 50 ng/ml. Micrographs were taken after 48 hr Amount of Antigen (ng/well) of exposure to GMF-13. Note larger total cell number per field and larger percentage of neurite-bearing cells in the presence of GMF-.8. FIG. 7. Immunologic comparison by an ELISA between GMF-/3 (Phase-contrast. Bar = 40 (solid circles) and acidic FGF (open circles). (A) Mouse monoclonal jzm.) antibody G2-09 against GMF-P. (B) Rabbit polyclonal antiserum acidic and basic FGFs (13). The two proteins show 55% 88-02 against GMF-,8. homology and both are 30% homologous with interleukin 1. The FGFs exhibit a broad range of target-cell specificity, features in the scheme. (i) We enhanced protein resolution by including endothelial cells, fibroblasts, astrocytes, neurons, using a gradient elution for the hydroxylapatite column and and prostatic cells. In fact, their actions are so diverse that by increasing the length ofthe HPLC column from 5 cm to 25 many growth factor activities separately detected in brain cm. (ii) We included a heparin-Sepharose step to exclude the extracts turned out to be acidic FGF or a slight variant of it. FGFs (13) from the final product; these heparin-binding Therefore, the FGF family represents a large proportion of proteins were a source ofconfusion in the study ofGMF. (iii) growth-regulating activities in the brain. Since cruder prep- We utilized the monoclonal antibody G2-09 to monitor the arations of GMF showed functions overlapping those of final steps of purification. We have demonstrated (17) that FGF, a fact partly explainable in retrospect by cross- monoclonal antibody G2-09 recognizes a brain-specific pro- contamination, it became a pressing question as to whether tein localized in astrocytes and Schwann cells (14-16) and GMF was also another FGF in disguise. Obviously, a that the protein is expressed in Schwann cells only after nerve clearcut answer could only be obtained by comparing the injury, implying a role in axonal regeneration. The combina- tion of these improvements allowed us to isolate the brain protein GMF-3. A number of growth factors have been isolated from the nervous system, the most notable examples of which are the

FIG. 10. Effect of GMF-f3 on tumor cell lines. (A and A') N-18 neuroblastoma. (B and B') C6 glioma. (A and B) Controls without GMF-P. (A' and B') GMF-,3 added at 250 ng/ml. Cells were seeded FIG, 8. Effect of GMF-3 on an intact DRG. (A) GMF-f was at 2 x 105 cells per well and allowed to attach for 4 hr before testing. added at 50 ng/ml. (B) Control without GMF-P. Micrographs were Micrographs were taken after 60 hr of testing. (Phase-contrast. Bars taken after 48 hr of testing. (Phase-contrast montage. Bar = 1 mm.) = 40 jim.) Downloaded by guest on September 25, 2021 Neurobiology: Lim et al. Proc. Natl. Acad. Sci. USA 86 (1989) 3905 DRGs, where glial and fibroblast elements had been mostly AI 60 _ eliminated through the use of an antimitotic agent, GMF-P 40 2 enhanced neuronal survival and neurite outgrowth (Fig. 9). (n a) (ii) In the cloned neuroblastoma cell line N-18 where no glial 4040 $A cells existed, the neurite-promoting effect of GMF-13 still 0 20 0 showed up (Fig. 10). In any event, the direct and indirect I effects of GMF-,/ on neurons are not necessarily mutually v- 20 =a. I0 OC) exclusive. it In this paper, we also documented the antineoplastic effect x 00 of GMF-,/ on both neuronal and glial tumors. In both cases GMF-P arrests the proliferation and promotes the phenotypic a) L. expression ofthe cells-i.e., neurite outgrowth in neuroblas- 1 toma cells (Fig. 10A) and spindle-shaped morphology in en 7 glioma cells (Fig. lOB). It should be pointed out that the In antimitotic action of GMF-P on C6 cells is contrary to our 0 n earlier observation of a mitogenic effect ofcrude GMF on the same cell line (18). This discrepancy is probably due to contamination of the crude preparations by other growth

U. factors, such as FGF. It is possible that some other biological differences between GMF-,B and crude GMF may exist, but this is beyond the scope of the present paper and will be the GMF-P (ng/ml) subject of future investigation. Inasmuch as pure GMF-P is now available, a thorough reevaluation of GMF function is in FIG. 11. Dose-response curves of GMF-,3. (A) N-18 neuroblas- order, and it is only after this that the complete range ofGMF toma line. (B) C6 glioma line. Cells were seeded at 2 x 105 cells per activity, in comparison with other growth factors, will be well and allowed to attach for 4 hr. The cells were then exposed to unfolded. GMF-f3 for 60 hr and scored. For N-18, cells with one or more neurites longer than one cell diameter were scored positive. For C6, cells with spindle shape were scored positive. Four hundred cells We thank the following for technical assistance: B. A. Baggens- from representative areas ofeach well were scored and the results are toss, M. A. Midthun, B. Fink, W. Zhong, and Y. Liu. Amino acid expressed as percent total population. Cell number per well was composition was carried out by A. Bergold at the University of Iowa determined with a Coulter counter after trypsin treatment. All values Protein Structure Facility. Protein sequencing was performed by W. are averages of at least four wells with a range of 5% or less. Lane at the Harvard Microchemistry Facility. This work was sup- ported by the following grants to R.L.: Veteran's Administration amino acid sequence of the two proteins. In the current work Merit Review Award, Grant BNS-8607283 from the National Science this has been accomplished. A search for homology using the Foundation, and Grant DK-25295 from the Diabetes-Endocrinology known sequence from GMF-f3 tryptic peptides demonstrated Research Center. that at least more than two-thirds of the amino acid sequence of GMF-,B is distinct from that of the FGFs. Furthermore, 1. Todd, J. T. (1823) J. Sci. Lit. Arts 16, 84-96. based on information from partial sequence, we have syn- 2. Trowell, 0. A. & Willmer, E. N. (1939) J. Exp. Biol. 16,60-70. thesized oligonucleotide probes and have screened and 3. Hoffman, R. S., Tenenbaum, E. & Doljanski, L. (1940) Growth cloned the cDNA for GMF-j3. The deduced complete se- 4, 207-221. quence of GMF-,6 again shows no homology with the FGFs 4. Hoffman, R. S. (1940) Growth 4, 361-376. and in fact with known 5. Lim, R., Mitsunobu, K. & Li, W. K. P. (1973) Exp. Cell Res. any protein entered in the Protein 79, 243-246. Identification Resource data base (Kaplan, R., Jaye, M. & 6. Lim, R. & Mitsunobu, K. (1974) Science 185, 63-66. R.L., unpublished results). 7. Lim, R. & Miller, J. F. (1984) J. Cell. Physiol. 119, 255-259. The lack of homology is corroborated by the absence of 8. Lim, R., Miller, J. F., Hicklin, D. J. & Andresen, A. A. (1985) immunologic cross-reactivity between GMF-,3 and the other Biochemistry 24, 8070-8074. proteins tested, using the polyclonal antibody developed 9. Groome, N. P. (1980) J. Neurochem. 35, 1409-1417. against GMF-,8. Polyclonal antibodies are a better measure of 10. Kasper, C. B. (1975) in Molecular Biology, Biochemistry and cross-reactivity than monoclonal antibodies because they are Biophysics, ed. Needleman, S. B. (Springer, New York), Vol. directed toward multiple epitopes distributed across the 8, pp. 114-161. entire length of the protein antigen. 11. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., One Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, important finding in the current work is that GMF-/3 N. M., Olson, B. J. & Klenk, D. C. (1985) Anal. Biochem. 150, undergoes reversible alterations in conformation during the 76-85. purification procedure; full biological activity can best be 12. Laemmli, U. K. (1970) Nature (London) 227, 680-685. demonstrated only after heat-treated renaturation. If this 13. Thomas, K. A. (1987) FASEB J. 1, 434-440. point were overlooked, one could have missed the biological 14. Lim, R., Hicklin, D. J., Ryken, T. C. & Miller, J. F. (1987) activity entirely and thereby failed to recognize the purified Dev. Brain Res. 33, 49-57. protein as a growth factor. Although GMF was originally 15. Lim, R., Hicklin, D. J., Miller, J. F., Williams, T. H. & detected as a growth factor for glial cells, we have now Crabtree, J. B. (1987) Dev. Brain Res. 33, 93-100. documented that the purified GMF-43 is a growth regulator for 16. Lim, R., Hicklin, D. J., Ryken, T. C., Miller, J. F. & Bosch, neurons as well. For a factor that E. P. (1988) Dev. Brain Res. 40, 277-284. growth has biological action 17. Lim, R., Zhong, W., Bosch, E. P. & Miller, J. F. (1988) Trans. on glial cells, it is always debatable as to whether the Am. Soc. Neurochem. 19, 83. observed neuronal effect is due to a direct action or an 18. Lim, R., Hicklin, D. J., Ryken, T. C., Han, X. M., Liu, K. N., indirect effect through glial cells. Two pieces of evidence Miller, J. F. & Baggenstoss, B. A. (1986) CancerRes. 46, 5241- favor a direct action. (i) In dissociated normal neurons from 5247. Downloaded by guest on September 25, 2021