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Purification and Characterization of Glia Maturation Factor F8: a Growth Regulator for Neurons and Glia RAMON LIM*, JOYCE F

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Purification and Characterization of Glia Maturation Factor F8: a Growth Regulator for Neurons and Glia RAMON LIM*, JOYCE F 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 L o Values are based on 1 kg of bovine brain. It is impractical to 20 40 60 estimate purification fold because of the presence of other growth factors in the cruder preparations that overlap the biological activ- FRACTION NUMBER (Sml) ities of GMF and because of a tendency for GMF-p to denature FIG. 1. Hydroxylapatite column chromatography. Only the pro- during the last two steps (see text). Protein was determined by the file ofthe phosphate gradient elution is shown. Fractions of5 ml were bicinchoninic acid method (11). collected. Protein concentration was measured at 280 nm. ELISA determination of GMF-/3 immunoreactivity with monoclonal anti- into four portions. A 10-ml aliquot was loaded on a Vydac C4 body G2-09 is expressed as absorbance at 415 nm. Vertical bars reverse-phase HPLC column (4.6 mm x 25 cm) (particle size, represent thymidine incorporation when tested on astrocytes. Dou- 5 ,um; pore size, 300 A) at a rate of 1.5 ml/min. Material from ble-headed arrow indicates fractions pooled. the charged HPLC column was eluted at the same speed taining 0.1% trifluoroacetic acid (F3CCOOH) over 45 min. using the following program: 0% acetonitrile for 4 min, 0-30% The amino acid sequence of the peptides were determined (vol/vol) acetonitrile gradient for 10 min, and finally 30-45% with an Applied Biosystems 477 gas-phase microsequencer (vol/vol) acetonitrile gradient for 15 min, all in the presence with on-line analysis of phenylthiohydantoin amino acid of0.1% TFA. The major peak that emerged at 40% (vol/vol) derivatives. acetonitrile and reacted positively with the monoclonal an- tibody G2-09 was designated GMF-p8 (Fig. 2A). The GMF-,B peaks from four HPLC runs were pooled and RESULTS purified once more through the same HPLC column. To do Final Purification of GMF-8. The partially purified GMF this the pool was diluted 1:1 with water containing TFA to sample (500 ml) from the Sephadex G-75 step was applied to achieve a final concentration of 20% (vol/vol) acetonitrile a hydroxylapatite (Bio-Gel HT) column having a gel volume and 0.1% TFA. The sample was loaded to the column and of 50 ml (5 cm diameter x 2.5 cm height). After washing the material was eluted as before. The GMF-/3 peak obtained at charged column with 1 column volume of0.15 M NaCl in 0.02 this time, representing the yield from one-third ofthe starting M Tris-HCI (pH 7.45), the bulk of the protein was eliminated material, was the final product (Fig.
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