ADONIS 00 I928059100060C

Immunology 1991 72 350-354

Isolation and partial characterization of the structures of fibroblast activating factor-related proteins from U937 cells J. DEMETER, D. MEDZIHRADSZKY,* H. KHA, E. J. GOETZL & C. W. TURCK The Howard Hughes Medical Institute and Department of Medicine, University of California, Medical Center, San Francisco, California, U.S.A.

Acceptedfor publication 31 October 1990 SUMMARY Human cultured monocyte-like tumour cells of the U937 histiocyte derived line were stimulated with phorbol myristate acetate, and generated and released an 18,000 MW polypeptide fibroblastactivating factor (FAF). Based on recognition by an antiserum to a synthetic peptide representing the 17 amino-terminal amino acids of FAF, two proteins of 32,000 and 35,000 MW were identified in extracts of U937 cells. Purification of the intracellular FAF-related proteins to homogeneity allowed the generation and amino acid sequencing of nine tryptic fragments of 4-11 amino acids. Neither of the intracellular FAF-related proteins exhibited the fibroblast proliferation-stimulating activity of FAF, suggesting that they are biosynthetic precursors analogous to the inactive propeptides of interleukin- I beta and tumour necrosis factor-alpha.

INTRODUCTION Macrophages have central functions in wound-healing processes, which include phagocytic removal of components of damaged tissues and the generation and release of a wide variety of structurally distinct polypeptide growth factors for fibroblasts.',2 When stimulated with various agents in vitro, monocytes and macrophages produce and release platelet-derived growth factor (PDGF), interleukin-1 (IL-I), fibroblast growth factor (FGF), transforming growth factor (TGF)-alpha, TGFbeta, tumour necrosis factor (TNF)-alpha and fibroblastactivating factor (FAF).3-8 The stimulation of human cultured U937 monocyte-like cells with phorbol myristate acetate (PMA) in cell culture results in the appearance of intracellular FAF and the secretion of one predominant FAF mediator.89 The purification and partial determination of the amino acid sequence of the 18,000 MW secreted FAF made possible the generation of a polyclonal antiserum directed to a synthetic peptide, comprising the amino* Present Address: Dept. of Protein Chemistry, G. Richter Ltd, Budapest, Hungary. Abbreviations: EDTA, ethylenediamine tetraacetate; FAF, fibroblast-activating factor; FCS, foetal calf serum; FGF, fibroblast growth factor, HEPES, N-hydroxyethylpiperazine-N'-2-ethane sulphonic acid; HPLC, high-performance liquid chromatography, IgG, immunoglobulin G; IL-1, interleukin-1; MW, molecular weight; PBS, phosphatebuffered saline; PDGF, platelet-derived growth factor; PMA, phorbol myristate acetate; PMSF, phenylmethylsulphonyl fluoride; SDSPAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; Tfa, trifluoroacetic acid; TGF, transforming growth factor; TNF, tumour necrosis factor.

terminus of secreted FAF.9"'0 The macrophage-derived polypeptide growth factors stimulate human dermal fibroblast proliferation, synthetic functions, such as production of PGE2, proteoglycans, and collagen, and induction of collagen matrix with different relative potencies in a profile characteristic of each factor. 10 The present report describes the identification and purification of two intracellular FAF-immunorelated polypeptides, with apparent molecular weights (MW) of 32,000 and 35,000, from U937 cells stimulated with PMA. These factors appear to represent inactive cellular polypeptides which contain the amino-terminal sequence of secreted FAF. MATERIALS AND METHODS Protein concentration measurements were done with the Biorad Protein Assay (Biorad Laboratories Inc., Richmond, CA). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 12-5% gels under reducing conditions, according to Laemmli." The proteins were identified using a silver-staining technique, described by Morissey.'2 For protein preparation purposes the gels were polymerized 1 day in advance, and the cathodic buffer was supplemented with 0-1 mm sodium mercapto-acetate (Aldrich, Milwaukee, WI).'3 Phast-gels containing 10-15% gradient SDS-PAGE were run according to the manufacturer's recommendations (Pharmacia LKB Biotechnology Inc., Piscataway, NJ). Generation and purification of the intracellular forms of FAF U937 cells (Tissue Culture Facility, UCSF) were cultured in roller bottles in RPMI-1640 medium containing 10% (v: v) heatinactivated foetal calf serum (FCS), 100 jig/ml of streptomycin, 100 U/ml of penicillin G and 20 mm HEPES (pH 7 4), as

Correspondence: Dr C.Turck, The Howard Hughes Medical Institute and Dept. of Medicine, University of California, Medical Center,

U-426 San Francisco, CA 94143-0724, U.S.A.

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Fibroblast activating factor of macrophages described elsewhere.8'9 Batches of 1-6 x 1010 U937 cells from culture suspension were washed twice and resuspended at 2 x 106 cells/ml in serum-free medium, PMA was added at a 100 ng/ml concentration, which induces secretion of the highest amount of fibroblast proliferation-stimulating activity into cell culture supernatants, and incubation continued for 24 hr at 370 in 5% C02: 95% air. The suspension then was centrifuged at 10,000 g for 15 min at 40, the pelleted U937 cells were resuspended in phosphate-buffered saline (PBS) with 1 mM EDTA and 1 mm PMSF buffer at 2 x 108 cells/ml, and disrupted by sonication (Branson Sonifer 250; VWR, Philadelphia, PA) at 150 W for 4 min at 4°. The resulting disruptate was centrifuged at 18,000 g for 30 min at 4° and the pellet washed with PBS and centrifuged again. The two supernatants were combined to produce the aqueous extract. The remaining cell debris was incubated for 30 min at 370 twice with PBS, containing 10 mM SDS. The two supernatants were combined and are referred to as detergent extract. The aqueous and detergent extracts were separately subjected to 90% saturated (NH4)2SO4 precipitation, overnight at 40, and the precipitates were collected by centrifugation at 30,000g for 15 min at 40, suspended in 200 ml of 20 mm Tris-HCl (pH 8 2), and dialysed (MW cut-off: 6000-8000) (Spectrapor; Spectrum Medical Industries Inc., Los Angeles, CA) against the same buffer for 2 days at 4°. Gel filtration chromatography was carried out on a column of 5 cm x 76 cm Sephacryl S-200 (Pharmacia LKB Biotechnology Inc.) in 20 mm Tris-HCl-100 mm NaCl (pH 8 2) at a flow rate of 1 ml/min. The effluent was collected in 10 ml fractions. The sample load was 40 ml of the aqueous extract. the immunoreactive fractions then were combined and directly applied to the anion-exchange column. Anion-exchange chromatography was carried out on a preparative Mono Q column (Pharmacia LKB Biotechnology Inc.) (10 x 1 cm) in 20 mm Tris-HCI (pH 8 2) as starting buffer and the bound proteins were eluted with a linear gradient of 20 mm Tris-HCl, 500 mm NaCl (pH 8-2) at a flow rate of 4 ml/ min over 90 min. The UV absorbance was detected at 280 nm and 4-ml fractions were collected. The immunoreactive fractions were combined and either were subjected to preparative SDS-PAGE or, after dialysis against the starting buffer (MW cut-off: 6000-8000), were loaded onto the cation-exchange column. Cation-exchange chromatography was performed on an Aquapore X03-224 column (222 x 4-6 mm, 7 ,gm, 300 A; Brownlee Labs, Rainin Instrument Co., Woburn, MA) in 20 mM sodium acetate (pH 5-0) with a gradient of 0-1 M NaCl over 90 min, at a flow rate of 1 ml/min. The eluate was collected in 1-ml fractions. The immunoreactive fractions were pooled and further purified by preparative SDS-PAGE. The pooled fractions were concentrated on Centricon-10 ultrafilters (Amicon, Danvers, MA), electrophoresed on 12 5% SDS-PAGE, and electroeluted according to a published protocol.'3 The only modification in the protocol was that the dialysis step during the electroelution was carried out for 12 h to avoid precipitation of protein (Model ECU-040; C.B.S. Scientific Co., Del Mar, CA). Tryptic digestion After electroelution, the protein samples were dried on a SpeedVac concentrator (Savant Instruments Inc., Farmingdale,

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NY) and precipitated with 90% acetone for 3 hr, at -20°. The precipitate was redissolved in 50 ,A of 0-4 M NH4HCO3 with 8 M urea, and the protein was reduced with 5 mm dithiothreitol, at 500 for 15 min and carboxymethylated with 10 mm iodoacetic acid at room temperature for 15 min. Trypsin (sequencing grade; Boehringer Mannheim Biochemicals, Indianapolis, IN) was added after dilution of the sample to 200 ,ul with water in an enzyme to substrate ratio of 1: 10. The digestion was carried out at 370 for 24 hr. After digestion the mixture was acidified with Tfa, and loaded onto a reversed-phase HPLC column (LC-304, 5 x 4-6 cm, 5 tim; Supelco Inc., Bellefonte, PA), equilibrated with 01 % Tfa (sequenal grade; Pierce Chemical, Rockford, IL) in water, at a flow rate of 1 ml/min. The bound peptides were eluted with a 90 min gradient of 01% Tfa, 90% acetonitrile. The eluted peptides were monitored at 215 nm in UV and with fluorescent detection using a bypass technique with fluorescamine derivatization. 14 One-millilitre fractions were collected and the fractions containing peptide were concentrated by SpeedVac to approximately 30 Ml and loaded in the protein sequencer.

Western blot assay Aliquots (1%) from all chromatography fractions were subjected to SDS-PAGE followed by electrotransfer to nitrocellulose membranes of 0-45 ,um pore size, (Schleicher & Schuell, Keene, NH) in a Biorad blotting system for 3 hr, 300 mA at 40. The transfer buffer contained 10 mm Tris-HCl, 77 mM glycine (pH 8 3), 20% methanol. After transfer, the filters were blocked by treatment with 5% non-fat dry milk in PBS (pH 7 0) for 2 hr at room temperature, and were incubated in the same buffer with a 1: 1000 dilution of a rabbit anti-FAF peptide antiserum'0 overnight. The filters were extensively washed four times with 0-5% Tween-20 (Biorad) in PBS for 120 min and incubated in 5% milk containing PBS with a 1:7500 dilution of alkaline phosphatasecoupled anti-rabbit IgG (Promega Corp., Madison, WI) for 2 hr, and washed four times in 0-5% Tween-20-PBS (4 x 30 min). The filters were developed with the alkaline phosphatase substrates nitroblue tetrazolium (NBT) and 5-bromo-4-chloro3-indolyl phosphate (BCIP) according to the manufacturer's recommendations (Promega, Madison, WI). Amino acid sequence analysis Amino-terminal sequence analysis was carried out on a model 470A sequencer equipped with a model 120A on-line PTHamino acid analyser and a model 900A data analysis station using Version 1.20 (Applied Biosystems Inc., Foster City, CA).

RESULTS Electrophoresis and Western blot analysis of the aqueous extracts of PMA-stimulated U937 cells, using an anti-peptide antiserum directed against the amino-terminal sequence of the 18,000 MW secreted FAF,9 revealed the presence of large quantities of two immunoreactive polypeptides (Fig. 1). The approximate molecular weights of the polypeptides were 32,000 and 35,000, respectively, independent of the PMA concentration that was used for stimulation. When the Western blot analysis was carried out using antiserum that had been preabsorbed with the FAF-derived peptide immobilized on Sepharose beads no immunoreactive polypeptides were detected (data not

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shown). The detergent extract contained no detectable immunoreactive polypeptides. The immunoreactive polypeptides were then purified from aqueous extracts of batches of 4 x 109 PMAstimulated U937 cells. Gel-filtration chromatography on Sephacryl S-200 separated the two polypeptides from each other in an anomalous order with the 32,000 MW polypeptide eluting earlier than the 35,000 MW polypeptide (Fig. 2). The effluents containing the 32,000 MW and 35,000 MW FAF-immunoreactive polypeptides then were pooled separately and loaded directly onto two different Mono Q ion-exchange columns. Unlike the 32,000 MW polypeptide the 35,000 MW polypeptide did not bind to the anion-exchange resin. The flow-through fraction, containing the FAF-immunoreactivity, was therefore dialysed against 20 mm sodium acetate (pH 5-0) and subjected to cationexchange chromatography. Both bound polypeptides were eluted with a linear NaCl gradient. The 35,000 MW polypeptide eluted as the last detectable peak of optical density from the cation-exchange column (Fig. 3a) and the 32,000 MW polypeptide eluted at 420 mm NaCl from the anion-exchange column (Fig. 3b). The chromatograms were highly reproducible in three

40 30 50Time (mun) Figure 3. Anion-exchange HPLC of the 32,000 MW (a) and cationexchange HPLC of the 35,000 MW (b) FAF-immunoreactive proteins after gel filtration of an aqueous extract. The shaded areas indicate the portion of the effluents containing FAF-immunoreactive polypeptides. 10

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separate experiments. Aliquots of the crude ammonium sulphate-precipitated material as well as effluent fractions after geland ion-exchange chromatographies, were assessed for fibroblast-stimulating activity using 1-2% of each respective fraction. In all cases no activity above the background level could be detected when using a dermal fibroblast proliferation assay9 (data not shown). After ion-exchange chromatography, the 32,000 MW and 35,000 MW polypeptides were purified to apparent homogeneity by SDS-PAGE. The ion-exchange eluates containing the 32,000 MW and 35,000 MW polypeptides were separately pooled, concentrated on Centricon-10 ultrafilters to 0-5 ml and then subjected to preparative SDS-PAGE. The appropriate bands were cut out from the Coomassie-stained gels, and were subjected to electroelution. Aliquots of the eluted samples were assessed for homogeneity and immunoreactivity using analytical SDS-PAGE and Western blot analysis (data not shown). Total material recovered after the electroelution step was approximately 150 pmol for each protein. Since neither polypeptide recovered from SDS-PAGE by electroelution or blotting on polyvinylidene difluoride membrane revealed any amino acid sequence from the aminoterminal end, it was necessary to cleave the proteins into fragments for structural studies. Isolated polypeptides were precipitated with acetone, cysteine residues were carboxymethylated, and the modified peptides subjected to tryptic digestion. The resulting mixtures of substituent peptides then were resolved on reversed-phase HPLC (Fig. 4) using a linear 0-90% acetonitrile gradient. The fluorescence of that portion of the eluate derivatized with fluorescamine was used to select peptides present in quantities sufficient for amino acid sequence analyses. The sequence of peptides in the indicated fractions are listed in Table 1. The internal sequences ofthese FAF-related peptides in the aqueous extracts of U937 cells bear no significant homology to each other, to known growth factors, or to proteins whose sequences currently are listed in the Dayhoff Protein Database. We therefore conclude that the peptides that were sequenced are derived from a portion of the FAF protein that is cleaved offand is not part of the secreted form of the protein which we had characterized earlier. We are currently in the process of utilizing

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