THE OF BIO~ICAI. Vol. 268, No. 31, Issue of November 5, pp ... · THE JOURNAL OF BIO~ICAI. CHEMISTRY Vol. 268, No. 31, Issue of November 5, pp. 23148-23156, 1993 Printed in U.S.A

THE JOURNAL OF B I O ~ I C A I . CHEMISTRY Vol. 268, No. 31, Issue of November 5, pp. 23148-23156, 1993 Printed in U.S.A.

Overexpression of Fetal Human Pigment in Escherichia coli A FUNCTIONALLY ACTIVE NEUROTROPHIC FACTOR*

Epithelium-derived Factor

(Received for publication, May 21, 1993, and in revised form, July 9, 1993)

S. Patricia Becerralg, Ira Palmern, Amalendra Kumarll, Fintan SteeleS, Joseph Shiloach**, Vicente NotarioSS, and Gerald J. ChaderS From the Gaboratory of Retinal Cell and Molecular Biology, National Eye Institute, the IProtein Expression Laboratory, Office of Intramural Research, Office of the Director, and the **Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, the IlCenter for Molecular Sciences, University of Texas Medical Branch, Galveston, %as 77555, and the Swepartment of Radiation Medicine, Georgetown University Medical Center, Washington, D. C. 20007

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein present in low amounts in conditioned medium of cultured fetal human retinal pigment epithelial cells. Recently, the PEDF cDNA has been cloned from a fetal human cDNA library, and its derived amino acid sequence identified it as a member of the serine protease inhibitor (serpin) supergene family (Steele, E R., Chader, G. J., Johnson, L. V., and Tombran-Tink, J. (1993) Proc. Natl. Acad Sci. U. S. A. 90, 1526-1530). We have prepared recombinant expression constructs from the fetal human PEDF cDNA and obtained milligram amounts of biologically active PEDF from Escherichia coli. The full-length open reading frame (Met1-Pro418) and a truncated form (Aspu- Pro418) were used in our constructs. Induction from a vector containing the truncated PEDF version, named pEV-BH, produced a protein (BH) of expected size (M, 42,800) associated with inclusion bodies, which contained 2540% of expressed protein. After solubilization, BH was highly purified by gel filtration and cation exchange chromatography. The NH,-terminal sequence of the purified protein matched that of the pEV-BH construct. We have conducted neurite outgrowth assays in a human retinoblastoma Y-79 cell culture system. Recombinant PEDF (BH) demonstrated neurotrophic activity, as reported for the native PEDE Thus, unfolded and refolded in vitro BH retained a potent biological activity. In parallel experiments, protease inhibition assays were performed. Recombinant PEDF did not have an effect on trypsin, chymotrypsin, elastase, cathepsin G, endoproteinase Lys-C, endoproteinase Glu-C, or subtilisin activity, suggesting that inhibition of known serine proteases is not the biochemical pathway for the PEDF neurotrophic activity.

Pigment epithelium-derived factor (PEDF)’ is an extracellu- lar neurotrophic agent, first identified as a secreted product in

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 To whom correspondence should be addressed: Bldg. 6, Rm. 308, National Eye Institute, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-6514 Fax: 301-402-0750.

The abbreviations used are: PEDF, pigment epithelium-derived factor; rPEDF, recombinant pigment epithelium-derived factor; RPE, retinal pigment epithelium; ORF, open reading frame; Sn, soluble fraction;

from BamHI-Hind111 cDNA fragment construct; dNTp, deoxynucleotide pp, particulate fraction; BH, M, 42,820 PEDF polypeptide expressed

triphosphate; NLT, single letter code for amino acid sequence aspara- gine-leucine-threonine; LB medium, Luna-Bertani medium; pNA, pni-

conditioned medium from cultured fetal human retinal pigment epithelial (RPE) cells (Tombran-”ink and Johnson, 1989; lbmbran-”ink et al., 1991). Addition of nanogram amounts of purified human PEDF protein to cultures of human retinoblastoma cells induces a neuronal phenotype. Morphologically, the cells are induced to extend long dendrite-like structures. Neu- ron-specific enolase and the 200-kDa neurofilament protein increase with PEDF treatment. The importance of this protein lies on the hypothesis that PEDF could function in early devel- opment, signaling for retinal cell commitment and/or differentiation.

Recently, the PEDF cDNA has been cloned from a human fetal eye library, and the coding sequence identified PEDF as a member of the serine protease inhibitor (serpin) supergene family (Steele et al., 1993). The coding capacity for the PEDF open reading frame (ORF) is 418 amino acids, corresponding to a protein of 46,326 daltons and a reported apparent M , of about 50,000 on denaturing gels. From the derived amino acid sequence of PEDF, the following structural characteristics were reported: a homology to the prototype of the serpins, human al-antitrypsin; a sequence coding for a 17-amino acid signal peptide at the amino terminus, and a consensus sequence for N-glycosylation (NLT) a t position 285-287. Little is known about the biochemical activity and the structural features of PEDF necessary for its neurotrophic activity. Studies on other neurotrophic factors indicate distinct modes of action. For ex- ample, nerve growth factor (NGF) promotes neurite outgrowth on PC12 cells via a mechanism of signal transduction (Chao, 1990). NGF is produced in large quantities in the mouse sub- maxillary gland (Cohen, 1960) as a 7 S complex composed of three subunits a, 0, and y (Bradshaw, 1978; Greene and Shooter, 1980). Both the a- and y-chains are members of the kallikrein family of serine proteases; only the y subunit has proteolytic activity and the 0 subunit alone is responsible for its biological activity. Expression of cloned NGF in Escherichia coli cultures produced low levels of NGF protein (0.1-1% of total bacterial protein) exhibiting only low biological activity (Hu and Neet, 1988). In contrast, glia-derived nexin (GDN) secreted by rat glioma cells promotes neurite outgrowth in mouse neu- roblastoma cells (Monard et al., 1973) and behaves as a potent serine protease inhibitor (Guenther et al., 1985). The glia-de-

troaniline; Suc-AAF’F-pNA, succinyl-alanine-alanine-proline-phenyl- alanine-p-nitroaniline; NGF, nerve growth factor; GDN, glia-derived nexin; Dm, dithiothreitol; MEM, Eagle’s minimal essential medium with Earle’s salts; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylam- moniol-1-propanesulfonate.

23148

Recombinant Human PEDF 23149

rived protein inactivates thrombin and forms SDS-resistant complexes with urokinase, plasminogen activator, thrombin, or trypsin. Primary structures of coding sequences derived from cDNA clones of GDN revealed that GDN is a member of the serpin family (Gloor et al., 1986 and Sommer et al., 1987). The production of recombinant bacteria-derived GDN has not been reported yet. Studies on GDN so far suggest that the protease/ protease-inhibitor balance can modulate the rate of neurite outgrowth. However, inhibition of proteolytic activity, although necessary for neurite outgrowth, is not sufficient by itself to explain the mode of action of GDN (Monard, 1990).

Given both the low abundance of PEDF in conditioned medium of cultured fetal human W E cells and the rare availability of its source, we have used molecular biology and genetic engineering technologies to produce PEDF for further charac- terization. In the present work, we describe the overexpression, purification, and activity of human recombinant PEDF. We show that a bacterially produced truncated form of PEDF (amino acid residues exhibits neurotrophic activity; however, it behaves as a substrate rather than an inhibitor of previously described serine protease activities.

MATERIALS AND METHODS Buffers, Media, and Enzymes-Buffer TEN contained 10 ~ l l ~ Tris-C1,

pH 7.5,1 mM EDTA, and 100 mM NaC1. Buffer TED: 10 mM Tris-C1, pH 7.5, 1 mM EDTA, and 1 mM Dm. Buffer A. 50 mM sodium phosphate, 1 mM DTT, 20% glycerol, 1 mM EDTA, 1 pg/ml pepstatin, 1 mM benzamidine. Buffer B: 50 mM Tris-C1, pH 8, 1 mM DTT, 2 m EDTA. Buffer C: 4 M urea, 50 m sodium phosphate, pH 6.5,l mM benzamidine, 1 pg/ml pepstatin, 4 mM EDTA. Buffer D: 4 M urea, 50 mM sodium phosphate, pH 6.5. Buffer E: TEN buffer containing 1% SDS and 1 mM DTT. RPMI 1640 (without L-glutamine) was purchased from Mediatech. MEM, L-glutamine, sodium pyruvate, nonessential amino acids, and HEPES were from Life Technologies, Inc. Fetal bovine serum was purchased from Paragon Biotech, Inc. ITS mix: 5 pg/ml insulin, 5 pg/ml transferrin, 5 ndml selenium, was from Collaborative Research. Elastase and cathepsin G were from Calbiochem, other proteases were from Boehringer Mannheim.

Plasmids and Cells-Plasmids pRC23, pEV-vrfl, and pEV-vrf2 (Crow1 et al., 1985) containing the bacteriophage ApL promoter were used to construct expression vectors. The PEDF cDNA containing plasmid, 7FS17, is described in Steele et al. (1993). Human Y-79 retinoblastoma cells (HTB18) were purchased from American Type Culture Col- lection, Rockville, MD.

Construction of Plasmids for Expression of PEDF-Preparation of DNA fragments, ligation reactions, and bacterial transformations were performed as described before (Becerra et al., 1991). Plasmid rFS17 was digested with SfuNI and HindIII producing a DNA fragment (N107-1503) containing the PEDF ORF and 10 extra nucleotides at its 5' end. This fragment was subcloned into the EcoRI and HindIII sites of pRC23, to produce transformants containing plasmid pRC-SH. The SfaNI site and the EcoRI site from pRC23 were filled in with dNTPs and Klenow fragment for a blunt-end ligation at this junction. Similarly a SfaNI, blunt-endlHindII1 DNA fragment was subcloned into the EcoRI, blunt-endlHindII1 sites of pEV-vrfl to produce plasmid pEV-SH. A BamHI, blunt-endlHzndII1 fragment (N245-1503) was subcloned into the EcoRI, blunt-endlHindII1 sites of pEV-vrf2 to produce pEV-BH. Construct pRC-SH encompasses the entire PEDF ORF with its naturally occurring amino terminus ( M e t l - P r ~ ~ ~ ~ ) . Constructs pEV-SH and pEV-BH have fusion sequences at their amino end added during plasmid construction (see Fig. L4).

Expression of PEDF Peptides in E. coli-Expression of PEDF proteins was performed as described before (Becera et al., 1993) with the following modifications. Bacterial cells containing PEDF expression vectors were grown in LB medium containing 50 pg/ml ampicillin at 32 "C to early logarithmic phase such that OD,,, nm = 0.2. Temperature of the culture was rapidly raised to 42 "C, and the bacteria proceeded to grow at 42 "C for 3 h. Aliquots were taken for absorbance readings at 600 nm. Upon induction, E. coli-containing PEDF expression vectors grew and divided at a slower rate than bacterial cultures containing the parental plasmid; an indication of foreign gene expression in bacteria. This negative effect on bacterial growth was more prominent in cultures with constructs having the initiation codon closer to the Shine-Dalgarno (ribosomal binding site) sequence, pEV-SH and pEV-BH, than with

pRC-SH. For protein radiolabeling, ~-[~~Slmethionine (1040 Ci/mmol, Amersham Corp.) was added at 150 pCi/ml of culture at the moment of induction. Cells were harvested by centrifugation and washed with TEN. For large-scale production, bacterial cells containing PEDF expression vectors were grown in a %liter fermentor. Cultures were grown at 32 "C to an ODsoo nm = -20, the temperature of the culture was shifted to 42 "C, and incubation proceeded at this temperature for an additional 3 h (Bustin et el., 1991 and Hsiao et al., 1992). The cultures were chilled to 10 "C and the bacteria collected with a continuous-flow centrifuge. The yield was about 2540 g of wet E. colilliter of culture.

Subcellular fractionation of bacteria containing PEDF expression vectors was as follows. Cells induced for expression were collected by centrifugation and washed in TEN buffer. A total of 0.15 g of E. coli cells was resuspended in 5 ml of RPMI 1640 media and was probe-sonicated with five bursts, 15 s each at 50 watts (sonicator model W-225, Ultra- sonics, Inc.) with the tube immersed in an ice water bath. Soluble fraction, Sn, and particulate fraction, pp. (inclusion bodies) were separated by centrifugation. Fraction pp was resuspended in 2 ml of phosphate-buffered-saline (PBS). In order to solubilize the inclusion bodies, fraction pp was resuspended in different concentrations of urea in PBS or of SDS in TED buffer. Samples were incubated at 25 "C for 10 min. After centrifugation the soluble and insoluble materials were separated and analyzed by SDS-PAGE. The yield of BH was about 1.3 mg of proteidg of wet E. coli cells.

Protein Purification-A total of 1 g of E. coli cells containing pEV-BH, induced in a fermentor, was resuspended in 50 ml of 20 rn Tris-C1, pH 7.5,20% sucrose, 1 mM EDTA. After 10 min of incubation on ice, the cells were sedimented by centrifugation at 4000 x g and resuspended in 50 ml of ice-cold water for 10 min. Lysed outer cell walls were separated from spheroplasts by centrifugation at 8000 x g. The pelleted spheroplasts were resuspended in 10 ml of buffer P (PBS with 5 mM EDTA, 1 pg/ml pepstatin, and 20 pdml aprotinin). The suspension was probe- sonicated to lyse the cell membranes with three bursts, 30-s pulse, 30-s pause in an ice water bath. RNase TI (1300 units, Life Technologies, Inc.) and DNase I (500 pg, Life Technologies, Inc.) were added to the sonicated cell suspension and incubated at room temperature for 10 min. This suspension was diluted by the addition of 40 ml of washing buffer P and the crude inclusion bodies were sedimented by centrifugation at 13,000 x g for 30 min. The particulate material (inclusion bodies) was resuspended in 40 ml of PBS containing 25% sucrose, 5 mM EDTA, and 1% Triton X-100. incubated on ice for 10 min, and centrifuged at 25,000 x g for 10 min. This washing step was repeated three times. Finally, the inclusion bodies were resuspended in 10 ml of denaturation buffer (50 mM Tris-C1, pH 8.0, 5 M guanidine HCl, and 5 mM EDTA). The suspension was probe sonicated briefly for 5 s in an ice water bath. The resulting suspension (Pool I) was incubated on ice for an additional hour. After centrifugation at 12,000 x g for 30 min, the supernatant was added to 100 ml of renaturation buffer (50 nm Tris-C1, pH 8, 20% glycerol, 1 m DTT, 1 pg/ml pepstatin, and 20 pg/ml aprotinin) and stirred gently at 4 "C overnight to renature the protein. The soluble (IIa) and insoluble (IIb) fractions were separated by centrifugation at 13,500 x g for 30 min. Fraction IIa was concentrated to l ml using a Centricon 30 microconcentrator (Amicon Co.) and dialyzed against Buffer A at 4 "C for 3 h. The dialyzed extract was centrifuged at 14,000 rpm in an Eppendorf centrifuge (model 54150 for 10 min. The supernatant fraction was layered on an S-Sepharose fast flow (Phar- macia LKB Biotechnology Inc.) column (1-ml bed volume) equilibrated with buffer A. The column was washed with 2 column volumes of buffer A. Finally, BH protein was eluted with a step gradient of 50, 100, 150, 150, 200, 300, 400, 500, and 1000 mM NaCl in buffer A (one column volume per fraction). Fractions of 1 ml were collected by gravity flow. Only fraction 300 appeared to contain rPEDF and was stored at -20 "C (IIIa).

The particulate material of IIb contained 90% of the total recombinant PEDF protein in fraction I. It was dissolved in 10 ml of 6 M guanidine HCl in buffer B. This solution was centrifuged at 10,000 x g for 5 min. The supernatant was layered onto two Superose 12 (Phar- macia) columns attached in tandem (2.6 x 95 cm each) equilibrated with buffer B containing 4 M guanidine HC1. Flow rate was 3 mumin. Frac- tions containing BH were pooled (Pool IIIb) and dialyzed against buffer C in a recirculating tangential flow ultrafiltration system using an Amicon Sly3 spiral cartridge. The dialyzed fraction was passed through a 0.22-pm filter (MILLER-GV, Millipore Corp.). The filtered solution was layered on a Mono S (Pharmacia) column (1 x 10 cm) equilibrated with buffer C. The column was washed with buffer C, and elution of BH was accomplished with a gradient of 0-500 mM NaCl in buffer C at 0.5 ml/min. Fractions were of 2 ml. The peak fractions of BH were pooled (IVb) and stored at -20 "C. Recovery of rPEDF per g of

23150 Recombinant Human PEDF packed cells was 50 pg in pool IIIa and 0.5 mg in pool IVb. For a larger preparation, rPEDF was purified from 10 g of wet E. coli cells and by the same procedure.

Neurite Outgrowth Analyses-Human Y-79 retinoblastoma stock cells were grown in suspension in MEM supplemented with 15% fetal bovine serum and antibiotics (100 unitdml penicillin and 100 pg/ml streptomycin) at 37 "C in a humidified incubator under 5% COz. Cells were propagated for two passages after receipt and then frozen in the same MEM medium containing 10% dimethyl sulfoxide. Separate aliquots of cells were then used for each differentiation experiment. M e r thawing, cells were kept in suspension culture without further passag- ing in serum-containing MEM until the appropriate number of cells was available. Cells were collected by centrifugation, washed twice, and resuspended in PBS and counted. For treatment with recombinant PEDF, 2.5 x IO5 cells were seeded into each well in six-we11 plates (Nunc, Inc.) with 2 ml of serum-free medium consisting of MEM supplemented with 1 mM sodium pyruvate, 10 mM HEPES, 1 x nonessential amino acids, 1 mM L-glutamine, and 0.1% ITS mix and antibiotics as above. Approximately 12-16 h later, BH (at 50 ng of BH proteidml of media) and control samples were added to the medium. Additions were as follows: 1) fraction IVb in buffer E (BHsDs); 2) fraction IVb in buffer D (BHurea); 3) fraction IIIa; 4) inclusion bodies from E. coli [pEV-BH] in buffer E; 5) Buffer E; 6) buffer D; 7) fraction 400, a fraction collateral to IIIa free of rPEDF; 8) buffer A, final purification buffer for IIIa and 400; 9) inclusion bodies from E. coli [pEV-vrf21; and 10) no addition. The cultures were incubated and kept undisturbed for 7 days. Cells under these conditions remained in suspension.

On the 8th day after treatment, cells were transferred to six-well plates precoated with poly-D-lysine (Collaborative Research); once the cells attached to the substrate (about 6-8 h), the old medium was replaced with 2 ml of fresh serum-free medium. The cultures were maintained under these conditions for up to 11 days. Using an Olympus CK2 phase-contrast microscope, post-attachment cultures were examined daily for morphological differentiation and quantification of neurite outgrowth.

Enzyme and Inhibitor Assays-Protease activity was against /3-PO- lymerase as substrate. Control proteolysis was performed as reported by Kumar et al. (1990) (P-polymerase was kindly provided by Dr. S. Wilson). Briefly, proteolytic cleavage was carried out in a 15-pl reaction at 25 "C by incubating the substrate (200 pg/ml) with trypsin (0.3 pg/ ml), chymotrypsin (2.7 pg/mI), endoproteinase Lys-C (0.7 pdrnl), endoproteinase Glu-C (2.7 pg/ml), subtilisin (0.3 pg/ml), elastase (6.6 pg/ml), and cathepsin G (6.6 pg/ml). After 60 min, hydrolysis was terminated by addition of sample buffer, and products were analyzed by SDS, 15% PAGE. For inhibitor assays, samples containing increasing amounts of rPEDF were preincubated with a fixed quantity of protease at 25 "C for 5 min; substrate was then added and incubation proceeded a t 25 "C for 1 h. Heparin was added at 6.6 unitdml.

Cathepsin G and chymotrypsin activities were assayed against Suc- AAPF-pNA as before (Barrett, 1981). Reactions were conducted a t 25 "C in 45 mM Tris-C1, 135 mM NaCI, 1.8 mM EDTA, pH 7.5, 10% dimethyl sulfoxide, and 1 pg/ml cathepsin G or 0.5 pg/ml chymotrypsin. Sub- strate concentration was 1.5 mM (eal5 = 14,000, DelMar et al. (1979)). The increase in absorbance ofp-nitroaniline (pNA) produced was monitored on a Beckman DU-30 spectrophotometer at 410 nm. The amount of pNA liberated was determined from its e410 of 8800. The rates of hydrolysis of substrate were found to be linear, at about 2-4 PM p N N min. For inhibition assays, the enzyme was preincubated with BH,,, at 25 "C before the addition of substrate (10 pl), and the concentration of enzyme and inhibitor given is that in the final reaction mixture. The preincubation mixtures (90 pl) were in 150 mM NaC1, 50 mM Tris-C1, 2 mM EDTA, pH 7.5, and contained 1 pl of enzyme and 10 pl of rPEDF solution with the appropriate amount of protein.

Assays for protease-inhibitor complex formation were performed essentially as described before (Travis et al., 1978). Protease (40 pg/ml) and rPEDF (70 pg/ml) or a-antitrypsin (80 pg/ml) were mixed in phosphate-buffered saline, pH 7.4, containing 1.3 M urea and incubated for 10 min a t 37 "C. PAGE-sample buffer minus 6-mercaptoethanol was added to the mixture and heated in a boiling water bath for 5 min. Samples were analyzed by SDS, 12% PAGE.

Other Methods-Protein concentration was determined by the method of Bradford (1976) using the Bio-Rad Protein Assay Kit (Bio- Rad). SDS-PAGE of proteins was performed according to Laemmli (1970). Protein markers were from Bio-Rad. Nucleotide sequence deter- mination was performed by Lofstrand Labs Ltd. (Gaithersburg, MD). NHz-terminal sequencing of protein sample bound to polyvinylidene difluoride membrane and purified protein in solution was conducted

using an Applied Biosystems instrument. Agar diffusion plates for protease activity assays were from Bio-Rad.

RESULTS

Expression of Fetal Human PEDF in E. coli-Construction of expression vectors was facilitated by the availability of a plasmid containing the full-length cDNA for fetal human PEDF (Steele et al., 1993). The PEDF coding sequence was placed under the control of the ApL promoter in three constructs. The full-length ORF (amino acids 1-418) was used in pRC-SH and pEV-SH and a truncated version (amino acids 44-418) in pEV- BH. A leader sequence provides an ATG initiator for the PEDF coding sequences in pEV-SH and pEV-BH which is located only eight nucleotides downstream from the Shine-Dalgarno sequence (see Fig. U). To monitor the emergence of newly syn- thesized proteins in bacteria, induced proteins were labeled with 135Slmethionine and analyzed by PAGE followed by fluorography (Fig. 1B). Bacterial extracts containing plasmids with full-length coding sequence did not produce a candidate polypeptide for PEDF ( M , -46,000). However, a M , 42,800 polypeptide (BH) was detected with the truncated version. The size of BH matches the expected coding capacity of the PEDF ORF placed in pEV-BH (379 amino acids). Rabbit reticulocyte ly- sates were programmed with a messenger RNA derived from the ~rFS17 cDNA plasmid and full-length PEDF mRNA trans- lated a protein of M, -46,000 (data not shown). Thus, proteolytic degradation of full-length PEDF by bacterial proteases is suggested.

Subcellular fractionation of bacteria containing BH revealed that the BH protein was associated with inclusion bodies, the particulate material obtained after sonication of bacterial cells (Fig. 2 A ) . BH constituted more than 25% of the protein in the particulate fraction, whereas most of the bacterial proteins par- titioned into the soluble extract. Solubilization of recombinant PEDF proteins was achieved with 4 M urea, as shown in Fig. 2 B . Polypeptides of M , 28,000 and M , 15,000 in size were also solubilized, but not major host proteins. BH was insoluble in a variety of neutral detergents, including Triton X-100, Nonidet P-40, octyl-P-D-thioglucopyranoside and CHAPS. However, SDS at concentrations as low as 0.1% was able to solubilize BH (Fig. 2C). The NH2-terminal sequence of BH was found to match that of the derived sequence from the pEV-BH ORF, i.e. MNRIDPFFKVPVNKLAAAVS. BH and the M, 28,000 and M, 15,000 proteins share an identical NH,-terminal sequence. Al- together, this indicates that BH corresponds to recombinant PEDF with an NH2-terminal truncation of 43 amino acids and a small leader sequence of 4 amino acids and that the M, 28,000 and M , 15,000 polypeptides correspond to COOH-terminal truncations of BH.

Purification of Recombinant PEDF-Our purification proto- col for the recombinant PEDF consists essentially of extracting BH from inclusion bodies. The recovery scheme involves isolation of inclusion bodies, solubilization, refolding, and purification (Fig. 3). After cell lysis, differential centrifugation sepa- rates the inclusion bodies from cellular debris. Denaturation with 5 M guanidine HCl and slow renaturation by 10-fold dilution of the guanidine HCI followed this extraction. Only 10% of the BH protein present in the inclusion bodies was renatured (fraction IIa), and fraction IIb contained BH that resisted renaturation. Fractions IIa and IIb were used to further purify BH by gel filtration and cation exchange chromatography. BH protein eluted from the S-Sepharose column at different salt concentrations in the presence or absence of urea. Based on Coomassie Blue staining of the gel, BH accounts for 25% of the total protein in IIIa. The other major component in IIIa was


A B SIaN t IN1011

Hrnd Il l IN15921 1 2 3 4 5 6 7 8 9

cDNA Mr x m Hsnd Ill

- S.D. b.e.

EcoR I SIsN I b e rORF-

1. PRC-SH LTAAGGAGG AGGCCCCAGG ATG c M'

"200.0

-97.4 -8.0

-46.0 c

-30.0

- €COR I BamH I b e D r S.D. ,- ORF c

3. PEV-BH ~ T A 4 G G A G G A A T T A A l A T G A A l AGA A T 1 GAT CC M N R I 0" P'\

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FIG. 1. Expression vectors for PEDF. A, structure of the PEDF expression vectors. The cDNA for PEDF is illustrated at the top of the figure. Restriction enzyme sites used for the construction of expression vectors are given and their nucleotide numbers are in parentheses. The relative position of the PEDF open reading frame (ORF) is depicted as an open bar (N117-1373). Construction of pRC-SH, pEV-SH, and pEV-BH placed the PEDF coding sequences under the control of the ApL promoter. The structures and DNA sequences of the PEDF expression vectors at the 5' end of their coding sequences are given at the bottom. PL indicates the relative position for the ApL. S.D. corresponds to Shine-Dalgarno sequence or the ribosomal binding site. Nucleotide recognition sites for restriction enzymes are indicated under a line; b.e. indicates blunt ends. DNA sequence around this junction was confirmed and is given. Coding amino acids are indicated using the single letter code. Numbers over them correspond to the PEDF amino acid positions. B, protein synthesis de novo in E. coli containing PEDF bacterial expression vectors. Bacterial cells bearing expressions vectors were grown and induced for expression in the presence of L-['"Slmethionine as described under "Materials and Methods." 3"SS-labeled peptides from total bacterial extracts were analyzed by SDS, 12% PAGE followed by fluorography. Photograph of an autoradiogram is shown. Induction time for lunes 1-4 was 10 min and for lanes 6-9 was 30 min. Expression vectors were as follows: lune 1, pEV-vrfl; lune 2, pRC-SH; lune 3, pEV-SH; lune 4, pEV-BH; lune 6 , pEV-vrfl; lane 7, pRC-SH; lune 8, pEV-SH; lune 9, pEV-BH. Lune 5 contained rainbow 14C-methvlated Drotein markers: mvosin, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor. The arrow indicates t<e "S"l'abe1ed BH position:

A

1 2 3 4 5

B C Sn PP Sn PP

'0.5 1 1.5 2 3 4"0.51 1.5 2 3 4 ' ' 0 0.1 1 2 vi-7

PPb- ,

BSA-

OA -

CA-

c

*

TI -

c

*

c

*

FIG. 2. Subcellular fractionation of induced E. coli containing PEDF expression vectors A. Soluble ( S n ) and insoluble ( p p ) bacterial extracts obtained after sonication and centrifugation of induced cells were analyzed by SDS, 12% PAGE. Coomassie Blue-stained gel showing analysis of expressed proteins produced in E. coli [pEV-SHI and E. coli IpEV-BH]. An equal volume of each fraction, Sn and pp, was applied to the gel. Lanes are as follows: lune 1, protein size markers: PPb, rabbit muscle phosphorylase b (97,000); BSA, bovine serum albumin (66,200); OA, hen egg white albumin (45,000); bovine carbonic anhydrase (31,000); TI, soybean trypsin inhibitor (21,500); lune 2, fraction Sn from E. coli [pEV-SHI; lane 3, fraction Sn from E. coli [pEV-BH]; lane 4, fraction pp from E. coli [pEV-SH]; and lune 5 , fraction pp from E. coli [pEV-BHI. B and C, the insoluble bacterial extract obtained after sonication of E. coli [pEV-BH] cells was subjected to increasing concentrations of solubilizing agents. After centrifugation, the Sn (soluble) and pp (insoluble) were separated and analyzed by SDS, 12% PAGE. Photographs of gels stained with Coomassie Brilliant Blue are shown. Numbers at the top of each lane correspond to the concentration of urea (molarity) (B) and SDS (percentage) (C) . The arrow indicates the BH position, and the asterisk indicates two bands a t M, -28,000 and M, -15,000 positions.

aprotinin (M, -7000) as confirmed by NHz-terminal sequence hand, punty of fraction IVb was estimated to be 98% and de- analysis. Attempts to further purify the fraction IIIa BH pro- termination of the NHz-terminal sequence of BH,,, in solution tein by gel filtration on Superose 12 and TSK were not success- confirmed that it contained our recombinant PEDF and that ful, due either to protein degradation or to failure to be released BH was the main component of this sample. Removal of the from the column. Therefore, protease inhibition assays could urea present in samples containing BH protein was performed not be performed conclusively with fraction IIIa. On the other by stepwise dialysis against buffer containing decreasing urea

23152 Recombinant Human PEDF

A Crude Insoluble Extract (inclusion bodies)

PEDF from E. coli cells. A, purification FIG. 3. Purification of recombinant

scheme of PEDF expressed in E. coli containing pEV-BH as described under "Ma- terials and Methods." B and C shows protein patterns in two purification steps. Fractions were analyzed by SDS, 12% PAGE and stained with Coomassie Bril- liant Blue. Photographs of stained gels are shown. The arrow indicates the position for BH. B, numbers on top of each lane correspond to NaCl concentration (mM) after step gradient elution from S- Sepharose column; El: is flow-through; starting material was IIa. C, numbers on top of each lane correspond to column fractions for S-Sepharose column and the starting material ( IIIb) . Fractions 31-38 were pooled (Nb).

I 1 Gu-HCI Denaturation

Gu-HCCSoluble Fraction (100%)

Renaturation by Dilution

Ila Soluble Fraction (10%) Insoluble Fraction (90%) Ilb

I S-Sepharose Column I Superose 12 column in Gu-HCI f

Ilia 300 mM NaCl Eluate

B I I I a

"

Mr x

4 -97.4 , -66.2

-45.0 c

concentrations. The minimum concentration at which all of the BH protein remained in solution was 4 M urea; at 2 M urea, about 50% was precipitated, and without urea, most of the BH became insoluble (data not shown). Protein in fraction lVb dissolved in SDS-containing buffer (BHsDs) and in 4 M urea buffer (BH,,,) was used for our studies.

Biological Assay-In order to determine functionality of the PEDF protein produced in E. coli, we tested our rPEDF for morphological differentiation of retinoblastoma Y-79 cells. Neu- rotrophic activity was assessed by phase contrast microscopy of a culture of Y-79 cells attached to plates coated with poly-D- lysine after treatment with 1) partially purified BH (i.e. extract of inclusion bodies, fraction IIIa) and 2) highly purified BH (i.e. BH,,,,, BHsDs) in suspension. Only Y-79 cell cultures exposed to recombinant PEDF showed any significant evidence of neuronal differentiation. As shown in Fig. 4, no evidence of differentiation was found in untreated Y-79 cell cultures or in cells treated with the same buffer used for BH, i.e. the cells remained mostly round and refractile with few if any processes. Also, no differentiation was observed in cultures treated with Fraction 400, a side fraction of IIIa not containing BH, or with extracts of inclusion bodies from E. coli without recombinant PEDF (data not shown). Cells treated previously in suspension culture with 50 ng of BWml of medium showed that between 50 and 65% of the cell aggregates had neurite-like extensions by 3

-31 .O

-21.5

v -43,000-Mr Fraction lIIb

1 S-Sepharose Column in 4M Urea

125 mM NaCl Eluate IVb

C IIIb . IVb nt202224262830'32343638'4042

M r x

c

-66.2

-45.0 c

-31.0

- 14.4 1-

days post-attachment on poly-D-lysine. These processes appeared as short projections from pear-shaped cells at the edge of the cell aggregates (Fig. 4, C and D). The number of differentiating aggregates, the number of differentiating cells per aggregate, and the length of the neurite-like processes increased with post-attachment time. By day 5 post-attachment, 7585% of the aggregates showed signs of differentiation with neurites extending from most of their peripheral cells. BH- treated cell cultures reached the maximal extent of morphological differentiation by day 7 post-attachment with 8598% of the cell aggregates showing abundant neurite-like extensions. At that point, two types of neuronal processes were observed: (i) single neurites, 2-3-fold longer than those observed on day 3, extending from peripheral cells of individual aggregates (Fig. 4E) and (ii) much longer and thinner processes forming a branching network between neighboring cell aggregates (Fig. 4F). Beyond 10 days post-attachment, there was a decrease in the proportion of the network connections and no further growth of the single neurites. Viability of the cell aggregates was not markedly affected by BH addition; it remained at about 75-80% in different experiments. I t is worth mentioning here that additions of BH at concentrations as low as 1 ng/ml promoted significant differentiation (4040% at 7 days post-attachment). Moreover, it has been independently demonstrated that our purified recombinant PEDF (BH,,,) efficiently


FIG. 4. Recombinant PEDF induces neurite outgrowth in Y-79 human retinoblastoma cells. Y-79 cells were treated with recombinant PEDF (BHur,.,, a t 50 nglml), as described under “Materials and Methods.” The differentiation state of the cultures was monitored a t different intervals after attachment. Untreated cells (A) and cells treated with PEDF solubilization buffer ( B ) showed a typical undiffer- entiated morphology. C and D show the neuronal differentiation char- acteristic of 3-day post-seeding cultures. E and F show the two morphological varieties (see the text) most common in cultures 7 days post- attachment. All photographs are shown at x 40 magnification.

induces neuronal differentiation of Y-79 cells maintained in long term cultures.2

Protease Assay-Given the fact that the PEDF cDNA sequence placed PEDF within the serpin supergene family, we tested our purified recombinant PEDF preparations for their effect on proteolysis. Protease inhibition was examined in solution during controlled proteolysis of a well-characterized polypeptide substrate, P-polymerase (Kumar et al., 1990). Con- trolled proteolysis was accomplished by adding limiting amounts of protease, producing peptide products of discrete sizes. Reactions in the presence of SDS demonstrated stimula- tory effects when compared with proteolysis reactions without SDS. We selected the BH,,, sample for proteolysis studies in the presence of urea at concentrations of 0.5 or 4 M urea. As shown in Fig. 5, the ratio of BH:trypsin was augmented from

G. Seigel and M. del Cerro, personal communication.

A B

1 2 3 4 5 6 Mr x

1 2 3 4 5 6 7 Mr x

- 97.4 - 66.2 - 45.0

- 31.0

- TOP -Top - 97.4 - 66.2

- 45.0

-31.0

-21.5

-21.5

- 14.4

- 14.4

- Bottom - Bottom

FIG. 5. Trypsin activity in the presence of recombinant PEDF. Panels show SDS-PAGE analysis illustrating the effect of increasing concentrations of rPEDF on trypsin activity against P-polymerase. Pro- teolytic cleavage was camed out a t 25 “C by incubating p-polymerase (200 pg/ml) with trypsin (0.3 pg/ml). At 60 min, hydrolysis was terminated by addition of sample buffer, and samples were analyzed by SDS,

gel with Coomassie Brilliant Blue. In A, reactions were performed in 0.5 15% PAGE. Protein hydrolysis products were visualized by staining the

M urea, and amounts of BH,,. added to a 15-pl reaction were as follows: lane 1,25 ng; lane 2,50 ng; lane 3, 250 ng; lane 4, 500 ng; lane 5 .0 ng; and lane 6, 0 ng and no addition of trypsin. In B, reactions were performed in 4 M urea, amounts of BH.,. added to a 15-pl reaction were as follows: lane 1, 25 ng; lane 2, 50 ng; lane 3, 100 ng; lane 4, 250 ng; lane 5, 500 ng; lane 6, 0 ng and no addition of trypsin; and lane 7, 0 ng.

5:l to 1OO:l (w/w) without affecting proteolysis. Tryptic degradation was inhibited by al-antitrypsin (16:l) under the same reaction conditions. Other serine proteases were selected based on their specificity for cleavage at residues within the reactive site loop. Similarly, activities of chymotrypsin, endoproteinase Lys-C, endoproteinase Glu-C, subtilisin, and elastase were not affected when the same BH preparation that showed neurotrophic activity was incorporated in the reaction mixtures (Fig. 6). Heparin did not potentiate BHs putative inhibition. In contrast, for cathepsin G activity, products and/or disappearance of substrate were detectable, albeit low.

To better evaluate the effect of rPEDF on cathepsin G, a spectrophotometric assay was performed using Suc-AAPF-pNA as substrate. BH and protease were preincubated before the addition of substrate. As shown in Fig. 7, residual cathepsin G activity decreased linearly with preincubation time in the presence of 5 pg/ml BH although the rate of hydrolysis was not linear with respect to the amount of BH in the reaction. The minimum rate was found at a BH:cathepsin G ratio of 2:l (w/w); it increased to about 100% with a ratio of 20:l. However, in reactions containing 4 M urea, BH did not affect cathepsin G activity. In comparison, residual chymotrypsin activity was vir- tually unaffected by BH, remaining 95% of that without BH (data not shown).

I t was of interest then to determine the interaction between cathepsin G and rPEDF. Inhibitory serpins and their target proteases form a complex resistant to SDS. As shown in Fig. 7C, BH and cathepsin G (2:1, w/w) did not form such a complex, but BH was sensitive to cathepsin G proteolytic degradation. Similarly and as expected from our gel assays with P-polymerase, BH did not form a complex with trypsin, chymotrypsin, or elastase (data not shown), but acted as a substrate rather than an inhibitor of these proteases.

The presence of a protease activity in conditioned medium of Y-79 cell cultures was investigated. Protein components in serum-free medium conditioned by Y-79 cultures were concen-


A B

1 2 3 4 5 6 7 8 9 1 2 3 4 5

+ Substrate + Substrate

A

Preincubation Time (min)

PEDF. Panels show SDS-PAGE analysis illustrating the effect of FIG. 6. Proteolytic activity in the presence of recombinant

rPEDF on different proteolytic activities against P-polymerase, as described under “Materials and Methods.” Reactions contained a final concentration of 0.5 M urea. Protein hydrolysis products were visualized by staining the gel with Coomassie Brilliant Blue. A, a total of 80 ng of rPEDF was added to a reaction. Addition to reactions were as follows: lane I , no protease; lane 2, chymotrypsin; lane 3 , chymotrypsin and BH,,,,; lane 4, subtilisin; lane 5, subtilisin and BH,,,.; lane 6, endoproteinase Glu-C; lane 7, endoproteinase Glu-C and BH,,.; lane 8, endoproteinase Lys-C; and lane 9, endoproteinase Lys-C and BH,.,,. B, a total of 250 ng of rPEDF was added to a reaction. Lune I , elastase; lane 2, elastase and rPEDF; lane 3, elastase and heparin (6.6 unitdml); lane 4, elastase rPEDF and heparin (6.6 units/ml); lane 5, no additions.

trated and fractionated by cation exchange chromatography. Aliquots of each fraction were used as protease-containing samples in controlled proteolysis with P-polymerase and in agar diffusion plates with bovine casein as substrate. None of the samples showed the presence of protease activity (data not shown).

DISCUSSION

We have shown here that recombinant PEDF can be produced in bacteria from an expression vector containing a cDNA of human fetal origin. Both sequence and size of the bacterial product are in agreement with the BH polypeptide consisting of amino acid residues 44418 of the human PEDF protein preceded by an NH2-terminal leader of residues MNRI-. The apparent molecular mass of BH, as determined by its mobility in polyacrylamide gel electrophoresis, is 42,800 Da and matches the size for the protein deduced from the ORF present in expression construct pEV-BH (42,360 daltons). Most importantly, the bacterial recombinant PEDF is functionally active, being able to induce differentiation of Y-79 human retinoblastoma cells toward a neuronal phenotype. Native human fetal PEDF sources are scarce. Thus, for the first time a bacterial expression vector, pEV-BH, becomes an excellent source for the over- production of biologically active PEDF neurotrophic factor, with yields in the order of 1.3 mg/g of wet cells.

Intracellular expression of cloned eukaryotic genes in E. coli is a means of obtaining large amounts of biologically important proteins. However, in several cases, the overexpressed proteins accumulate in the cytoplasm in the form of inclusion bodies that account for a major fraction of the total expressed protein (Kane and Hartley, 1988). Despite their insolubility, inclusion bodies can be used as a source of proteins for further purification (Marston, 1986). Most of the bacterially-overexpressed BH protein is associated with inclusion bodies. Their isolation provides a convenient purification step because they contain highly concentrated expressed protein (2540% of the total protein in the cell lysis pellet). Reagents which solubilize BH par- tition the major components commonly associated with inclusion bodies into the insoluble fraction; this favors purification

C

1 2 3 4 5 6 7 8 91011

a

4 “

PEDF. A, cathepsin G residual activity versus preincubation time. BH FIG. 7. Cathepsin G activity in the presence of recombinant

(5 pg/ml) and cathepsin G (1 pg/ml) were preincubated a t 25 “C for a period of time before the addition of suc-AAF’F-pNA. Residual activity was determined as described under “Materials and Methods.” Percent- age of residual activity is referred to that without BH. B , cathepsin G activity versus BH concentration. Cathepsin G (1 pg/ml) was preincubated with increasing concentrations of BH a t 25 “C for 10 min before the addition of substrate. Hydrolysis was followed for 5 min. In the absence of BH, the residual rate of hydrolysis was 1.4 PM pNAlmin (10070). C , SDS-polyacrylamide gel electrophoresis of rPEDF/protease reaction mixtures. Enzymes and rPEDF or al-antitrypsin were incubated for 10 min a t 37 “C. The mixtures were then analyzed by SDS- PAGE under nonreducing conditions. Lune 1, rPEDF (2 pg); lane 2, cathepsin G (1 pg); lane 3 , cathepsin G (1 pg) and rPEDF (2 pg); lane 4, el-antitrypsin (2 pg); lane 5, chymotrypsin (1.5 pg); lane 6, trypsin (1.5 pg); lane 7, chymotrypsin (1.5 pg) and al-antitrypsin (2 pg); lane 8, trypsin (1.5 pg) and al-antitrypsin (2 pg); lane 9, chymotrypsin (1.5 pg) and rPEDF (2 pg); lane IO, trypsin (1.5 pg) and rPEDF (2 pg); and lane 11, protein size markers as in Fig 2.

and leaves the protein between 30 and 70% pure. Slow removal of guanidine HCl by dilution and dialysis allows refolding of the protein and favors formation of the correct intramolecular as- sociations. Chromatographic purification yielded a sample of soluble BH able to promote neurite outgrowth of Y-79 retinoblastoma cells. This indicates that individual BH molecules could be unfolded and refolded in vitro and yet retain biological activity.

Since most of BH was in the insoluble form, this fraction was used to purify large amounts of BH protein. Our final preparation of BH in 4 M urea yielded 5 mg of biologically active proteid10 g of packed cells. Importantly, when BH,,, is added to the Y-79 cell culture medium, the dilution factor of 1-1000 is equivalent to the effect of removing urea from the BH sample. Most of the BH protein precipitates when urea is removed from this preparation, and we estimate that about 10% of the protein would renature when first added to the media. Considering that only 1/10 of the protein added to the Y-79 cultures was responsible for the detected neurotrophic activity, the concentration of recombinant PEDF which promoted neurite outgrowth of more than 60% of the cells 3 days post-attachment would be estimated a t 5 ng/ml. These values are 10-fold less than the concentration of native PEDF previously used to elicit a neurotrophic effect (Tombran-Tink et al., 1991). Insolubility is usually due to improper folding or aggregation. Thus, some proper folding of BH must take place in the medium to allow for the expression of its differentiation effector potential. We have observed that 10-30% of the total radiolabeled BH,,, parti- tioned into the soluble fraction upon addition to serum-free medium conditioned for 5 days by Y-79 retinoblastoma cells (data not shown). At this point, we cannot rule out the action of


P12 Region P1/ P1’ P11’ Region

_ ” E X G Z Y A X 2 X Z ---(X),-----(X),.- - X N X P F L F X \ R E - - -

I I P12 P10 Pa P4 I P L I I I P15 I I P l 8 PEDF - - - E D G A G T T P S P G L Q P A H L - - - - T F P L D Y H L N Q P F I F V L R D - - -

P12 Anll Haminon A382T P12 Clinh Ma A434E P10 ATIII Charleville W P P10 Clinh b A 4 3 3 P10 ATlll Cambridge W P P10 Clinh Mo AUGT P11’ alATHeerlen p 3 6 9 L P11’ Anll Ufah P407L P12-P10 aZAP Enschede insenion of an additional

alanine between A354 and A357

P4 a1ATChristChurCh E363K P15’ Clinh M45W P l 8 alATM3E376D

Nmhhibimysapinr

P0 TBG lprolinel P12 AGTU (proline)

P1Z CBG (threonine) P1Z AGTH Iprolinel

FIG. 8. Amino acid sequence comparison around reactive sites for serine protease inhibitors (serpin) and PEDF. Conserved amino acid sequences flanking serpin reactive site loops are shown at the top. Sequences in the reactive site are represented at the center portion of the diagram (under P1 / P I ‘ ) ; they vary greatly in sequence and length. P1 designates the amino acid forming the new COOH terminus after proteolytic

highly conserved regions P12 and P11‘ are given to the left and right of Pl/Pl‘-region, respectively. Amino acid residues in the Pl2-region are cleavage and is preceded by P2, P3, etc. PI’ denotes the NH,-terminal amino acid following the cleavage site (al-antitrypsin numbering). The

proposed to play a critical role in determining inhibitodsubstrate status for serpins. Residue Z and Y correspond to amino acid residues with small side chains, e.g. threonine, serine, valine, and large charged residues, e.g. glutamic acid, glutamine, respectively. PEDF sequences are aligned at the bottom. A single-letter code is given for amino acid residues. Positions of PEDF amino acid residues shared with natural variants and noninhibitory serpins are indicated above the PEDF sequence. Natural serpin variants and noninhibitory serpins are listed at the bottom of the figure. Serpin abbreviations: ATIII, antithrombin 111; Clinh, C1 inhibitor; alAT, al-antitrypsin; and a2AP, a2-antiplasmin; TBG, thyroxine- binding globulin; AGTR, rat angiotensinogen; AGTH, human angiotensinogen; CBG, human corticosteroid-binding globulin. Sources of amino acid sequences are in Huber and Carrell (1989) and Bock (1991).

chaperon proteins nor the possibility that proper folding is not indispensable for activity.

From this study, several facts can be deduced about the structural features of PEDF required for its biological activity. First, absence of 43 amino acid residues derived from the NH2 terminus of the human PEDF ORF and presence of an NH2- terminal MNRI have no effect on biological activity. Determi- nation of amino acid sequences from the NH2 terminus of the native protein has not been reported yet. However, the sequence of the first 17 amino acids has similarity to a consensus signal peptide sequence and is expected to be cleaved for protein maturation and secretion (Steele et al., 1993). Thus, BH has a truncation of 27 amino acids from the supposedly mature protein which are not involved in the biochemical event that triggers differentiation. Second, N-glycosylation of human PEDF is not essential for neurotrophic activity. Since E. coli does not perform a number of eukaryotic post-translational modifications, recombinant PEDF is not acetylated, amidated, or glycosylated, although this does not preclude the possibility that the native protein may be naturally modified post-trans- lation. Third, the deduced amino acid sequence of PEDF re- veals cysteine residues at positions 9, 19, and 261. Our truncated rPEDF, BH protein, contains only the CysZ6l, suggesting that interactions based on disulfide bonds between domains containing these residues in the native protein are not essential for neurotrophic activity. However, these interactions might play a role in solubility. Fourth, the biochemical pathway for neurotrophic activity of PEDF cannot be explained by the inhibition of trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase Glu-C, subtilisin, elastase, and cathepsin G activities. The PEDF sequence exhibits 27% identity with a1- antitrypsin, the model of inhibitory serpins (Steele et al., 1993). This homology increases to 92% considering only the 51 conserved residues known to be necessary for structural integrity of serpins (Huber and Carrell, 1989).3 In spite of this similarity, the same BH preparation that demonstrated a potent neurotrophic activity did not behave as an antitrypsin or as an inhibitor of the other serine proteases but rather as a substrate of these.

Sequence comparison between PEDF and the known serpins

S. P. Becerra, unpublished observation,

at their reactive regions may explain PEDF’s inhibitor/ substrate status (see Fig. 8). The structural elements in the reactive region of a serpin can be divided into three functional regions, the Pl/€’l’-, P12- and P11”regions (Bock, 1991). The amino acid at the P1 position has an important role in dictating the specificity of inhibition (Patson et al., 1990; Bock, 1991). PEDF and the inhibitory serpins, human al-anti-chymotrypsin (Morii and Travis, 1983) and heparin cofactor I1 (Blinder et al., 19881, have a leucine at P1. Thus, PEDF has a reactive site specific for a leucyl-enteropeptidase such as the chymotrypsin- like proteases cathepsin G, chymase, and chymotrypsin itself. Although homology is increased at the flanking regions, the P11’- and P12-regions, the PEDF sequence is less homologous to serpin sequences at the Pl2-region than to sequences at the P11”region. Two variants with substitution mutations in the P11‘-region have amino acid residues as in PEDF and produce functional protein but at low circulating levels. However, the P12-region appears to have the structural determinant which enables a serpin to be an inhibitor rather than a substrate of its target protease (Bock, 1990). It has been suggested that residues in this region (the A4 strand) need to incorporate into the center of the Asheet of inhibitory serpins and that the presence of bulky or hydrophobic residues that affect this conformation are associated with the lack of protease inhibitor function in noninhibitor members (Stein et al., 1989) and in dysfunctional serpins (Bock, 1990). PEDF shares residues at P12 and P8 with the noninhibitory serpins corticosteroid-binding globulin and thyroxine-binding globulin, respectively, and furthermore, at P12 and P10 with variants producing dysfunctional inhibitors that behave as substrates rather than inhibitors of their target proteases. Thus, the failure of PEDF to behave as an inhibitor may be explained by the presence of unfavorable residues in its P12-region. Although unlikely, we cannot rule out the possibility that PEDF may be an inhibitor of a hitherto unidentified target serine protease in the developing retina.

In summary, recombinant PEDF isolated from bacterial inclusion bodies is a functional protein that exhibits a potent neurotrophic activity but does not behave as a classical anti- protease. Serine proteases have not yet been characterized in the fetal RPE-retinal interphotoreceptor space. Our finding that PEDF functions as a substrate rather than an inhibitor of the chymotrypsin-like proteases tested along with the failure to


detect serine proteases in media conditioned by retinoblastoma cells suggests that inhibition by PEDF may not be required for neurotrophic differentiation of retinal cells. A ligand binding function associated with some noninhibitory serpins may be in effect. The availability of large amounts of PEDF will facilitate detection of its target and enhance our understanding of structure-function relationships of serpins, as well as other possible biochemical events responsible for differentiation in which they are involved.

Acknowledgments-We thank Dr. Samuel Wilson for kindly provid- ing @-polymerase protein, Patricia Spinella for amino acid sequencing analysis, Dr. Steve Gaudet for TSK column chromatography, Dr. Wil- liam Beard for helpful comments, and Noreen Beavers for assistance in preparation of this manuscript.

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THE OF BIO~ICAI. Vol. 268, No. 31, Issue of November 5, pp ... · THE JOURNAL OF BIO~ICAI. CHEMISTRY Vol. 268, No. 31, Issue of November 5, pp. 23148-23156, 1993 Printed in U.S.A