Journal qf Neurochemistry Raven Press, Ltd., New York 0 1991 International Society for Neurochemistry

Molecular Cloning and Expression of Biologically Active Human Glia Maturation Factor+ Ruth Kaplan, *Asgar Zaheer, Michael Jaye, and *Ramon Lim Rh6ne-Poulenc Rorer Central Research, King of Prussia, Pennsylvania; and *Department of Neurology (Division of Neurochemistry and Neurobiology), University of Iowa College of Medicine and Veterans Afairs Medical Center, Iowa City, Iowa, U.S.A.

Abstract: Glia maturation factor-& a protein found in the brains of all vertebrates thus far examined, appears to play a role in the differentiation, maintenance, and regeneration of the nervous system. Using oligonucleotide probes based on the sequences of three tryptic peptides derived from bovine glia maturation factor-p, we screened a human brainstem cDNA library in hgtl 1. A 0.7-kb clone was isolated, sequenced in its entirety, and found to encode a polypeptide of 142 amino acids which contained regions identical to the

three bovine peptides. This polypeptide, human recombinant glia maturation factor-& has been expressed in Escherichia coli and found to possess structural characteristics and biological activity indistinguishable from those of the native bomatvine protein. Key Words: Cloning-Expression-Glia uration factor-& Kaplan R. et al. Molecular cloning and expression of biologically active human glia maturation factor-& . I Neurochem. . 57, 483-490 (1991).

In 1972, Lim et al. reported an activity in bovine brain extracts that promoted the phenotypic expression of embryonic brain cells in culture. Although the proteinaceous nature of the active factor, designated glia maturation factor (GMF), was recognized early (Lim et al., 1972, 1973; Lim and Mitsunobu, 1974), complete characterization was not possible at that time due to lack of a homogeneous preparation. Recently, glia maturation factor$ (GMF-P), a 17-kDa single-chain polypeptide with an isoelectric point of 4.9, was purified from GMF, a less pure preparation (Lim et al., 1987a,b, 1988) and partially sequenced (Lim et al., 1989). GMF-/3promotes the differentiation of normal neurons and glial cells, and inhibits the proliferation of their derived tumor cells in vitro (Lim et al., 1989). The protein is endogenous to astrocytes (Lim et al., 1987a) and Schwann cells (Lim et al., 1988), and its presence in the latter is negatively regulated by axonal contact (Bosch et al., 1989). The application of GMF/3 to the injured brain promotes the appearance of large

neurons in the cerebral cortex (Lim and Huang, 1989), implying a role in neural regeneration. Knowledge of the amino acid sequences of three tryptic peptides derived from bovine GMF-0 permitted us to synthesize oligonucleotide probes for use in screening a human brainstem cDNA library. In this article we report the isolation of a cDNA clone for human GMF-/3, its nucleotide and deduced amino acid sequence, and expression of the biologically active recombinant protein in Escherichia coli. Our results indicate that the natural bovine protein (Lim et al., 1990~)and the recombinant human GMF-0 have identical physical and biological properties.

EXPERIMENTAL PROCEDURES Isolation of GMF-/3 cDNA clones Three oligonucleotides, T18, T24, and T28, were designed based on the known amino acid sequence of bovine brain GMF-@(Lim et al., 1989) and human codon usage (Lathe, 1985):

Abbreviations used: aFGF, acidic fibroblast growth factor: bFGF, basic fibroblast growth factor; GMF, glia maturation factor; GMF6, glia maturation factor-0; rGMF-0, recombinant glia maturation factor-& IPTG, isopropyl-0-D-thiogalactopyranoside;ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.

Received October 2, 1990; revised manuscript received January 9, 1991; accepted January 9, 1991. Address correspondence and reprint requests to Dr. M. Jaye at Rh6ne-Poulenc Rorer Central Research, 680 Allendale Road, King of Prussia, PA 19406, U.S.A. The nucleotide sequence reported in this paper has been submitted to GenBank with accession number M31742.

483

R. KAPLAN ET A L .

484

T18: 5'-AACACAGAGGACCTGACAGAGGAGTGGCT- 3' N T E D L T E E W L

T24 5'-GATGAGGAGCTGGAGGGCATCTCCCCTGATGAGCTG~GGATGAGCTGCCTGAG-3' D E E L E G I S P D E L K D E L P E T28: 5'-CCTGTGGGCTGCAAGCCTGAGCAGCAGATGATGTATGCTGG-3'

P

V

G

C

K

P

E

Q

Oligonucleotides were synthesized on an automatic synthesizer (Applied Biosystems Model 380A) using either methoxy or P-cyanoethyl phosphoramidites (House et al., 1987). Approximately 1 X lo6 plaques from a human brainstem cDNA library in Xgtll (ATCC #37432) were screened on nitrocellulose filters using oligonucleotide T28 as a probe; 100 pmol of T28 were kinased using [32P]ATPand T4 polynucleotide kinase. Duplicate filters were prepared essentially as described by Maniatis et al. ( I 982), baked at 80°C for 2 h, and hybridized overnight at 40°C in a solution of 5X SSPE (SSPE is 10 mM NaH2P04,pH 7.4, 0.18 M NaCl, and 1 M E D T A ) containing 0.25% nonfat dry milk, 0.1% sodium dodecyl sulfate (SDS), and 1 X lo6 cpm/ml of 32P-labeled T28. The filters were washed twice for 20-30 min each in 2X SSPE, 0.2% SDS at 45"C, and then processed for autoradiography. The 25 positive clones identified in this first round were replated at low density and rescreened with all three oligonucleotides (T18, T24, and T28). Five clones, which hybridized strongly with oligonucleotides T24 and T28, were purified by repetitious screening with oligonucleotide T28. The cDNA inserts of these clones were subcloned separately into the EcoRI site of M I3mp 18, and the ends sequenced by the chain termination method (Sanger et al., 1977) according to the directions accompanying the Sequenase Kit (United States Biochemical Corp., Cleveland, OH, U.S.A.). Sequence analysis indicated that one of these clones (8-3) contained nucleotide sequence that could be translated into known bovine GMF-0 peptide sequence. This clone was sequenced in its entirety using a series of oligonucleotide primers.

Northern blot analysis Samples containing 10 p g of total RNA were analyzed on a 1% agarose gel containing 2.2 M formaldehyde prepared essentially as described by Lehrach et al. (1977). The gels were run overnight at 15 mA in 1X MOPS buffer (20 mM morpholinepropanesulfonic acid, pH 7.0, 5 mM sodium acetate, and 1 m M EDTA). The RNA was transferred to nitrocellulose as described by Thomas (1980). Hybridization was carried out using a GMF-P cDNA probe prepared by random priming (Feinberg and Vogelstein, 1983). Prehybridization, hybridization, and washes were carried out as described by Soprano et al. (1985). The blots were then air dried and processed for autoradiography.

Production of recombinant GMF-fl To produce recombinant GMF-@in the T7 expression system (Rosenberg et al., 1987), a 5' NdeI site and a 3' BarnHI site were required to make the insert compatible with the PET-3b translation vector. (The plasmid PET-3b and the E. coli strain BL2 l(DE3)pLysS were provided by Dr. F. William Studier, Brookhaven National Laboratory.) Two oligonucleotides were designed, based on nucleotide sequence of clone 8-3. The 5' oligonucleotide: J. Neuroclicpm., Vol. 57, N o 2, 1991

Q

M

M

Y

A

G

5'-GCCGGAAGGCATATGAGTGAGTCTTTG-3'

* *

(nucleotides 28 through 54 of clone 8-3) contained two changes (*) relative to the GMF-(I nucleotide sequence that created the underlined NdeI site at the translation initiation ATG. The 3' oligonucleotide: 5'-TTATGTCTGGATCCAGTATGGTCA-3'

**

(inverse complement of nucleotides 508 through 53 1 of clone 8-3) contained two changes (*) that gave rise to the underlined BurnHI site. These two oligonucleotides were then used as primers in a polymerase chain reaction (Saiki et al., 1985). The reaction mix contained 4 ng of template (Xgt 1 1 recombinant GMF-/3 clone 8-3), 50-100 pmol of each oligonucleotide primer, 50 mM KCl, 1.5 mM MgC12, 0.0 1% gelatin, 2 mM each of dATP, dGTP, dCTP, and dTTP, 10 m M TrisHCl, pH 8.3, and 5 units of Taq DNA polymerase (Perkin Elmer Cetus, Nonvalk, CT, U.S.A.) in a final volume of 0.10 ml. Amplification was achieved by performing 30 cycles, each including denaturation at 94°C for 1.5 min followed by 2 min of annealing at 55°C and 5-min extensions at 72°C in an automated Perkiri Elmer Cetus DNA Thermal Cycler. The polymerase chain reaction product was ligated between the NdeI and BarnHI sites of PET-3b and the resultant plasmid was used to tranisform E. coli strain TG1 (Amersham). Plasmid DNA was prepared using an alkaline lysis miniprep procedure (Maniatis et al., 1982) and restricted to identify recombinant plasmids. One microliter of recombinant plasmid DNA was then used to transform E. coli strain BL2 I (DE3)pLysS (Moffatt and Studier, 1987; Rosenberg et al., 1987). Cultures of BL2 l(DE3)pLysS containing either the recombinant plasmid or the PET-3b vector alone were grown overnight at 37°C in Luria broth containing 100 pg/ml of ampicillin and 25 pg/ml of chloramphenicol. These cultures were then used to inoculate Luria broth containing both antibiotics. The resultant starting cuItures were grown at 37°C until A550reached 0.3-0.5, at which time isopropyl-/3-D-thiogalactopyranoside (IF'TG) was added to a concentration of 1.5 mM. The induced cells, as well as uninduced controls, were grown for an additional 3 h at 37"C, harvested by centrifugation, and stored frozen.

Isolation and purification of recombinant GMF-fl The frozen bacterial pellets were resuspended in 25 mM Tris-HC1, pH 8.0, 50 mM glucose, 10 mM EDTA and sonicated for 20 s on ice. Following centrifugation at 3,500 rpm for 20 min, the supernatant fraction was applied to a BioGel P30 fine column (2.6 X 100 cm) and eluted in 0.02 M TrisHCI, pH 7.4, and 0.15 M NaCl. The fractions were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970) and by imnnunoblottingwith monoclonal antibody G2-09 raised against bovine GMF-8 (Lim et al., 1985). Fractions containing essentially pure GMF-P were pooled. The

485

CLONING OF GLIA MATURATION FACTOR-0 yield was approximately 6 mg of GMF-P per I00 ml of bacterial culture. Further analysis of the recombinant protein was camed out using reverse-phase HPLC, performed on a pBondapak C18 column (3.9 mm X 30 cm, Waters). The solvents used were 0.1% trifluoroacetic acid (solvent A) and 100%acetonitrile containing 0. I % trifluoroacetic acid (solvent B). Recombinant GMF-/3 was injected under initial column conditions of 0% solvent B at a flow rate of 1.5 ml/min. After 2 min, a linear gradient of 0-80% solvent B in 40 min was established. Detection was made spectrophotometrically at 210 and 280 nm with a full scale absorbance of 0.3. The first run resulted in one major and two to three minor peaks (results not shown). The major protein peak, which reacted strongly with the monoclonal antibody G2-09, was collected and rechromatographed under identical conditions. The single protein peak obtained was used in the present studies. Activation of GMF-P before cell testing. Natural GMF-P purified from bovine brains was activated by gradual heating of the sample to 80°C followed by gradual cooling to 20°C, at a rate of 2°C per minute, as described previously (Lim et al., 1989). Purified recombinant GMF-/3 (rGMF-P) was treated by a procedure of reduction-oxidation (Morehead et al., 1984; Tsuji et al., 1987) as follows: rGMF-(3, at 0.5 mg/ ml in 0.1 M sodium phosphate buffer, pH 7.4, was mixed with an equal volume of 6 Mguanidine-HCI, pH 8.0, and subsequently with 10 mM reduced and 1 mM oxidized glutathione, both freshly weighed. After incubation at room temperature for at least 6 h, the mixture was diluted with 0.1 M sodium phosphate buffer, pH 7.4, to a rGMF-/3 concentration of 0.1 mg/ml and dialyzed overnight at 4°C in the same buffer. Peptide mapping. Trypsin digestion of extensively reduced and alkylated (Kasper, 1975) GMF-/3 was camed out at 37°C

for 20 h at an enzyme/protein ratio of 1:lOO (wt/wt). The product was analyzed on a C 18 column as described above. Electrophoresis and electroblotting. SDS-PAGE was carried out under reducing conditions on 15% resolving gels according to the method of Laemmli (1970).Proteins were visualized by silver staining (Menil et al., 1981). The mobility of the protein samples was compared with that of known proteins: ovalbumin (44 kDa), chymotrypsinogen (25.7 kDa), 0-lactoglobulin ( 1 8.4 kDa), lysozyme (14.3 kDa), and bovine trypsin inhibitor (6.2 kDa). For transfer of proteins onto nitrocellulose membranes, electroblotting was performed at a constant voltage of 100 V for 1 h. Immunostaining of the blots with affinity-purified monoclonal antibody G2-09 (against bovine GMF-0) was camed out as described previously (Lim et a]., 1985).

RESULTS Knowledge of the amino acid sequences of three tryptic peptides of bovine GMF-P permitted us to synthesize oligonucleotide probes which were used to screen a human brainstem cDNA library in hgtl 1 (Young and Davis, 1983a,b). We identified and isolated one clone that hybridized to two of the oligonucleotide probes. Portions of the amino acid sequence deduced from the 0.7-kb cDNA insert of this clone were found to be identical to the three sequenced peptides of bovine GMF-P. Assuming initiation of translation at the methionine encoded by nucleotides 40-42 (Fig. I), the sequence predicts a polypeptide of 142 amino acids with a molecular mass of 16,693 Da, which does not

AAT TCG GGG GGC GAC AGG CCG CTG ACG GCC GGA AGG AM 10 20 MET S e r G l u S e r L e u V a l V a l C y s A s p V a l A l a G l u A s p L e u V a l G l u L y s L e u A r g L y s ATG AGT GAG TCT TTG GTT GTT TGT GAT GTT GCC GAA GAT TTA GTG GAA AAG CTG AGA AAG 30 40 P h e A r g P h e A r g Lys G l u T h r A s n A s n A l a A l a I l e I l e MET Lys I l e A s p L y s A s p L y s TTT CGT TTT CGC AAA GAA ACG M C M C GCT GCT ATT ATA ATG M G ATT GAC M G GAT AM

50 A r g L e u V a l V a l L e u A s p G l u G l u L e u G l u G l y I l e Ser P r o CGC CTG GTG GTA CTG GAT GAG GAG CTT GAG GGC ATT TCA CCA T24 70 L e u P r o G l u A r g Gln P r o A r g P h e I l e V a l T p r S e r T y r Lys CTA CCT G M CGA C M CCT CGC TTC ATT GTG TAT AGT TAT M A

60 Asp Glu Leu Lys Asp Glu GAT G M CTT AAA GAT G M

39

99

159

219

80

T y r Gln His A s p A s p G l y TAT C M CAT GAT GAT GGA

90 Arg Val S e r Tyr P r o Leu Cys Phe Ile Phe S e r S e r Pro Val Gly Cys AGA GTT TCA TAT CCT CTG TGC TTT ATT TTC TCC AGT CCT GTT GGA TGT T28 110 G l n MET KET T y r A l a G l y Ser L y s A s n Lys L e u V a l G l n T h r A l a G l u CAG ATG ATG TAT GCT GGA AGT M G M T M G CTA GTC CAG ACA GCT G M

279

100 Lys Pro Glu Gln AAG CCT G M CAA

339

120 Leu Thr Lys Val CTA ACC M G GTA

399

130 P h e G l u I l e AKg A s n T h r G l u A s p L e u T h r G l u G l u T r p L e u A r g G l u i p s L e u G l y TTT GAA ATA AGA M T ACC G M GAC CTA ACT G M G M TGG TTA CGT GAG AM CTT GGA T18 P h e His TTT CAC T M TGT G M CTT CTG TGT TTC TAA AGT ATT TAT GTA TTA ACC TGA CCA TAC

140 Phe TTT

459

TGG

519

M T CAG ACA T M ATA CTT ATT TAT GCC T M AAA TGC ACT GTT ACT TAC ACT TTG TTT CCT

579

GCA GTA M G AM M T TCT TCA TTT GTG CAA M T TTG M C A M GAG G M ATC ATC TTC ATA

639

GTA ATG AAA CTT TGT AM GTG TTT CCT TAT ATT GGT M T TGT TAG GTG GAC TAC TTT TCC

699

sequence and predicted amino acid sequence of human GMF-6. The cDNA clone 8-3 and its nucleotide sequence were obtained as described in Experimental Procedures. Th e amino acid sequence is numbered from t h e presumed initiator ATG. Both strands were sequenced in their entirety. The underlined regions indicate the poFIG. 1. N u c l e o t i d e

s i t i o n s of oligonucleotidesT 1 8 , T24, and T28 used in screening for t h e human cDNA clone.

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FIG. 2. Northern blot analysis. Total RNA from four cell lines, A204, NT2Dl , U563, and A1 72, was fractionated on a formaldehyde gel as described in Experimental Procedures. After transfer to nitrocellulose, samples were hybridized to 3'P-labeled full length GMFp cDNA, washed, and processed for autoradiography.

possess any potential N-linked glycosylation sites. Although we cannot rule out the possibility that initiation of translation occurs upstream of the proposed initiation methionine, we note that an A is present three bases upstream of the proposed start codon, thus satisfying the most stringent requirement for efficient initiation of protein synthesis in eukaryotes (Kozak, 1986). Northern blot analysis (Fig. 2) of total RNA from four human cell lines, A204 (rhabdomyosarcoma), NT2D 1 (teratocarcinoma),and two gliomas, U563 and A 172, revealed the presence of a single 3.7-kb transcript in three of the four samples. Thus it is clear that the 0.7-kb clone that we have isolated represents only a portion of the RNA transcript and is laclung approximately 3 kb. The absence of a poly(A) tail at the 3' end of the 0.7-kb cDNA clone indicates that at least a portion of the uncloned cDNA resides 3' to the sequence shown in Fig. 1. The results shown in Fig. 2 also demonstrate that transcription of the GMF-@gene is neither restricted to nor a marker of neural cells, as its RNA is detected in a rhabdomyosarcoma and in only one of two gliomas tested. Reprobing of the blot with an actin cDNA probe indicated that all of the RNA samples were intact, and thus the absence of GMF-/3in the U563 glioma cells could not be attributed to RNA degradation. The amount of GMF-/3 RNA relative to that of actin appeared to be approximately equal in all three positive cell lines. Two lines of evidence indicated that the 142-residue polypeptide predicted from the cDNA sequence corresponded to mature GMF-P. First, the predicted size of the recombinant protein, 16,693 Da, was similar to the size of bovine GMF-/3(17 kDa) estimated by SDSPAGE (Lim et al., 1989). Second, the amino acid compositions of the deduced 142-residue human protein and bovine GMF-P are nearly identical (Table 1). Thus, the cDNA sequence encoding the 142-residue polyJ. Nrurochem.. Vol. 57. No. 2, I991

peptide presumed to be mature GMF-p was cloned downstream of the inducible T7 promoter in PET-3b. Induction of the transformed E. coli cultures with IPTG resulted in the aplpearance of a major 17-kDa protein, which was purified by gel exclusion chromatography followed by reverse-phase HPLC. The purity of the isolated rGMF-P was evident in both the HPLC ]profile and the SDS-PAGE results, where a single peak and a single band, respectively, were observed (Fig. 3A and B). The protein comigrated with bovine GMF-P on SDS-PAGE and both proteins immunoblotted positively with the anti-GMF-p antibody G2-09 (Fig. 3C). Enzyme-linked immunosorbent assay analysis revealed superimposable immunoactivity curves (Fig. 4). 'Trypticdigestion resulted in identical peptide maps on HPLC (Fig. 5). The similarity in amino acid composition between recombinant and natural GMF-P is depicted in Table 1, where both are comparable to that predicted from the nucleotide sequence. To verify further the recombinant protein as the expression product of the cDNA, the terminal amino acid sequences of rGMF-P were determined. Gas-phase microsequencing with Edman degradation revealed an N-terminal segment of Ser-Glu-Ser-LeuVal-Val-Cys-Asp-Val-Ala, correspondingto amino acid residues 2 through. 11 in the deduced sequence. With carboxypeptidase A,a C-terminal sequence of Gly-PhePhe-His-OH was found, corresponding to residues 139 through 142, as predicted.

TABLE 1. Amino acid composition of GMF-p Residue per molecule Amino acid Asx Glx Ser G~Y His '4%

Thr Ala

Pro TYr Val Ile Leu Phe LYS CYs Met Trp Total no. Calculated MW

IZecombinant 14 22 8 5 2 9 6 5 6 5 10 6 14 8 13 3' 3' Id 140 16,443

Natural' 14 24 8 5 2 8 6

6 6 5 10 7 12 8 12

3' 3' Id

140 16,375

Deducedb 14 22 8 5 2 9 5 5 6 5 11 7

14 8 13 3 3 1 141 16,554

Data from previous publication (Lim et al., 1989). First amino acid residue (methionine) omitted for uniformity of comparison. Determined after per formic acid oxidation. Data from sequence analysis.

48 7

CLONING OF GLIA MATURATION FACTOR-@

FIG. 3. Purity and molecular mass analysis of recombinant [email protected]: Elution profileof pure recombinant GMF-P from reverse-phase C18 HPLC column; absorbance, 280 nm. B: SDS-PAGE pattern

of recombinant and natural GMF-P on a 15% gel (silver stain). Left to right: molecular weight standards; recombinant GMF-P (protein peak from “A”), 300 ng; GMF-P from bovine brain, 300 ng. C: lmmunostainingof proteins transferred from “ 6 ’ onto nitrocellulose membrane with monoclonal antibody G2-09.

The biological activity of recombinant human GMF-

p was compared with that of the bovine protein. Prior to testing, the recombinant protein was incubated with a mixture of oxidized and reduced glutathione. The biological activity of the recombinant GMF-p was absolutely dependent on this reduction/oxidation reaction with the glutathione. This is presumably due to inappropriate linkage among the three cysteine residues in the untreated polypeptide. Following the redox reaction, the biological activities of recombinant human GMF-p and natural bovine GMF-fl were compared. Figure 6 shows essentially identical dose-response curves for the inhibition of growth of N18 neuroblastoma and C6 glioma cells by the two proteins. The inhibition of cell number was accompanied by an altered cell morphology in the GMF-p-treated cells (Fig. 7) with obvious neurite outgrowth in the treated N18 cells (Fig. 7B and C). The results indicate that the biological activity of the 141-residue recombinant GMFp is indistinguishable from that of purified bovine GMF-B. 1.2

, , , , ,,,,

, , , , , ,,,,

, , , , , ,,,,

FIG. 5. Reverse-phase HPLC of tryptic peptides generated from recombinant and natural GMF-P. Top profile, rGMF-P; bottom, GMF-0. Absorbance, 210 nm.

0.4 rGMF-P

, , ,

I

-

0.a

d

la

Q

10’ ‘ 0

0 c la

0.4

200

100

I

Growth Factor (nglml)

In 0

n 4

FIG. 6. Comparison of biological activity of recombinant and natural 0 0.2

1

10

100

500

Antigen (ng/well) FIG. 4. Immunologic comparison of recombinantand natural GMF@ by enzyme-linked immunosorbent assay using monoclonal antibody (32-09. rGMF-@,0; GMF-@,0.

GMF-6. The test was conducted on N18 neuroblastomaand C6 glioma lines as described previously (Lim et al., 1989). except that the cells were seeded at 5 X lo4 cells per well. Each point is the mean of triplicate wells with variability within 5%. Both naturaland recombinant GMF-8 show half-maximal activity at about 20 ng/ml for the two cell lines tested. A: N18 cells; 6: C6 cells. rGMF-@,0; GMF-@,0 . Note the growth inhibitionon these tumor cells by both samples of GMF-p.

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FIG. 7 . Morphologic differentiation elicited by recombinant and natural GMF-(3. Cells were grown and tested as described in Fig. 6, using a growth factor concentration of 250 ng/ml. Photographswere taken at the time of harvest (60h after stimulation).A N18 control cells; 8: N18 cells with rGMF-0; C: N18 cells with GMF-(3; D. C6 control cells; E: C6 cells with rGMF-0; F: C6 cells with GMF-0. Note larger cell size and neurite outgrowth in B and C,and spindle-shaped morphology in E and F. Bar = 40 pm.

DISCUSSION The complexity of the nervous system necessitates an intricate series of cellular interactions during its development. Agents that mediate these interactions include cell adhesion molecules, which are responsible for cell-cell recognition, and neurotrophic factors, which include growth factors that control the survival, proliferation, and/or differentiation of neural cells. Since the discovery of nerve growth factor in the 1950s (Levi-Montalcini and Hamburger, 1953), several other regulatory molecules active on neural cells have been .I Neurothem.,Vol 57, No. 2, 1991

detected in the brain. One of these regulatory proteins, GMF-6, has been purified recently from bovine brain and partially sequenced (Lim et al., 1989). We have screened a human brainstem cDNA library using oligonucleotide probes based on known bovine GMF-P peptide sequences. We report here the isolation of a cDNA clone for human GMF-P, its nucleotide and deduced amino acid sequence, and the expression of biologically active recombinant GMF-6 in E. coli. The cDNA sequence of clone 8-3 (Fig. 1) contains a single open reading frame (ORF) from nucleotide 1

48 9

CLONING OF GLIA MATURATION FACTOR-fi to 465, followed by a stop codon and 234 nucleotides of 3’ untranslated sequence. A potential translation initiation codon is found at nucleotides 40-42, which is in a favorable context for initiation of protein synthesis by virtue of the A situated three bases upstream (Kozak, 1986). The nearly identical deduced amino acid compositions and estimated molecular weights of the polypeptide encoded by the ORF between nucleotides 40 and 465 and bovine GMF-P strongly support the notion that the mature form of GMF-P is encoded by this segment of the ORF. Because the ORF in clone 8-3 extends amino terminal of the proposed start codon, we cannot eliminate the possibility that GMF-p is cleaved from a larger precursor polypeptide. This is unlikely, however, as hydrolysis of the peptide bond between Met’ and Ser2 would be required, and there is no known endopeptidase possessing this cleavage specificity. Thus we believe that mature GMF-/3is generated directly from translation of the ORF between nucleotides 40 and 465. If this is indeed the case, GMF/3 would join acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF), which are also brain-derived neurotrophic factors that lack NH2terminal hydrophobic signal peptide sequences facilitating secretion (Abraham et al., 1986; Jaye et al., 1986). The proposed lack of a signal peptide for GMFp is supported by the absence of GMF-/3 immunoreactivity in the conditioned media of producing cells and in human body fluids (Lim et al., 1987a,b). However, this finding does not preclude its role as an intercellular message, but rather implies that an unusual mechanism may exist for its mode of action. As mentioned previously, aFGF and bFGF also lack signal peptides, and indeed are absent from conditioned media, but they are found in the extracellular matrix in association with heparan sulfate proteoglycans (Baird and Ling, 1987; Vlodavsky et al., 1987). Unlike fibroblast growth factors, GMF-P does not bind heparin and has not been found in the extracellular matrix. Instead, we have detected the presence of GMF-P on the surface of the cells that synthesize this protein (Lim et al., 1990b). This observation supports a previously proposed hypothesis (Lim, 1977, 1980) in which two pools of GMF were postulated, a large, nonfunctioning intracellular pool, and a smaller but functional cell surface pool that mediates short-range cellular interactions whenever the GMF-producing cells and their targets are in direct contact. Finally, there is also the possibility that GMF-/3 is released following cell damage (Lim et al., 1987a,b; Nieto-Sampedro et al., 1988), as has been suggested for fibroblast growth factors (Abraham et al., 1986; Jaye et al., 1986) and interleukin-1 (Auron et al., 1984). GMF-P shows no major sequence homology with any other known proteins, including the recently described neurotrophic factors ciliary neurotrophic factor (Lin et al., 1989; Stockli et al., 1989), brain-derived neurotrophic factor (Leibrock et al., 1989), and neurotrophin-3 (Maisonpierre et al., 1990). A literature

search, however, revealed that the amino acid triplet “LRE’ (positions 134 to 136) near the carboxy end of GMF-P may be of potential interest. LRE is a component of S-laminin (Hunter et al., 1989b), a basal lamina protein present in the neuromuscular synapse, cerebral cortical capillaries,and kidney glomeruli which promotes neurite outgrowth. The sequence LRE within S-laminin was found to be the adhesive site of S-laminin for motor neurons (Hunter et al., 1989~).Nevertheless, the LRE triplet is also present in over a thousand other proteins in which it serves no apparent function (Hunter et al., 1989~).Therefore, whether LRE is involved in the neuronal effect of GMF-0 is currently a matter of speculation. While this work was in progress, the complete amino acid sequence of bovine GMF-/3 was established (Lim et al., 1990a). The sequence of the bovine protein is identical to that of the human except that its amino terminus, which corresponds to the Ser at residue 2 of the human sequence, is NH2-terminally blocked by an acetyl group. This supports the assumption of the first methionine as the initiation site for translation of the human protein. Furthermore, the complete conservation of the sequences of the human and bovine proteins suggests a very strong evolutionary constraint on the structure of this protein. Today, GMF-@joins a small but rapidly growing group of polypeptides isolated from the brain whose members regulate functions of neural cells. The availability of cloned probes for GMF-P will permit analysis of the regulation of its gene, and provide a ready source of GMF-P for structure/function studies of both the protein and its receptor. Although many unanswered questions remain, the elucidation of the sequence and the cloning of the biologically active protein constitute a major advance in GMF research and provide an additional tool with which to unravel the complex mechanisms controlling neural development and regeneration. Acknowledgment: We are grateful to Dr. David Givol for his support, to M. Maureen Whitman for technical assistance, and to Rosalie Ratkiewicz for excellent secretarial service. We are also grateful to Dr. David Hunvitz and Dr. George Ricca for critical review of the manuscript. This work was supported in part by the following grants to R.L.: Department of Veterans Affirs Merit Review Award, grant BNS-89 17665 from the National Science Foundation, and grant DK-25295 from the Diabetes-Endocrinology Research Center.

REFERENCES Abraham J. A., Mergia A., Whang J. L., Tumulo A., Friedman J., Hjemld K. A., Gospodarowicz D., and Fiddes J. C. (1986) Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science 233, 545-548. Auron P. E., Webb A. C., Rosenwasser L. J., Mucci S. F., Rich A,. Wolff S. M., and Dinarello C. A. (1984) Nucleotide sequence of human monocyte interleukin 1 precursor cDNA. Proc. Nutl. Acad. Sci. USA 81,7901-19 1 I . Baird A. and Ling N. (1987) Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: implications for a role of heparinase-like enzymes in the neo-

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