Synthesis and Assembly of a Functionally Active Recombinant Platelet ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1988 by The American Society for Biochemistryand Molecular Biology, Inc.

Vol. 263, No. 31, Issue of November 5, pp. 16202-16208,1988 Printed in U.S.A.

Synthesis and Assembly ofa Functionally Active Recombinant Platelet-derived Growth FactorAB Heterodimer” (Received for publication, January 21, 1988)

Arne OstmanS, Leslie Rallp, Annet HammacherS, Mary Ann Wormstead§, Doris CoitQ, Pablo Valenzuelat, Christer Betsholtzll, Bengt Westermarkll, and Carl-Henrik HeldinS From the $Ludwig Institutefor Cancer Research, Box 595, Biomedical Center, S-751 23 Uppsala, Sweden, the $Chiron Corporation, Emeryuille, California 94608, and the YDepartment of Pathology, University Hospital,S-75185 Uppsala, Sweden

A Chinese hamster ovary cell line that stably expresses transfected human platelet-derived growth factor (PDGF) A and B chain precursors was established. All three dimeric combinations of PDGFchains were produced by thiscell line; their biosynthesis, assembly, and processing were followed by pulse-chase analyses. PDGF-AA, PDGF-AB, and PDGF-BB were processed to M, values of about 30,000 and were accumulated in these forms in the medium. In addition, PDGF-BB was further processed to a 24-kDa component, which remained cell-associated. The major secreted component was PDGF-AB, which was purified and shown to have structural and functional characteristics indistinguishable from PDGF-AB purified from human platelets.

been produced in mammalian cells (15)* and in yeast ( 16).3 The fact that the two PDGF chains can be combined into threestructurallyand functionally different dimers raises questions related to the assembly and processing of PDGF chains in cells expressing both chaintypes. In order to address such questions, a CHO cell line that stably expresses transfected PDGF A and B chain precursors was established. This cell line secretes PDGF-AB into the medium; the factor was purified and shown to have structural andfunctional characteristics indistinguishable from PDGF-AB purified from human platelets. The cell line also produced PDGF-AA and PDGF-BB but at lower yields. EXPERIMENTAL PROCEDURES4 RESULTS

Platelet-derived growth factor (PDGF)’ is a major mitogen present in serum and stimulates the proliferation of connective tissue-derived cells in vitro (1, 2). PDGF is composed of two similar disulfide-bonded polypeptide chains, denoted A and B (3), and all three possible dimeric forms of PDGF chains have been identified. PDGF-AA has been purified from the conditioned media of several human tumor cell lines (46).The transforming product of simian sarcoma virus is structurally similar to PDGF-BB (7-9), as is PDGF purified from porcine platelets (10). Finally, the majority of PDGF purified from human platelets hasrecently been identified as a heterodimer, PDGF-AB (11).There is evidence that the various dimeric forms of PDGF have different functional activities. Thus, PDGF-AA has lower mitogenic activity, no chemotactic activity, and lower ability to cause actin rearrangement and membrane ruffling, as compared to PDGFAB (12). The functional differences maybe mediated by interaction with different receptors (12). The availability of cDNA clones for the A chain (13) and the B chain (14) has made it possible to produce recombinant PDGF; functionally active PDGF-AA and PDGF-BB have

Establishment of a CHOCell Line That Stably Expresses Transfected PDGF A and B Chains-The strategy for construction of a plasmid encoding the precursors of both PDGF A andBchains is shown in Fig. 1. cDNAs for the two precursors were linked in the same vector to assure the same gene dosage. This plasmid was transfected into CHO cells using the calcium phosphateprecipitation method. After clonal selection, a cell line, 106:3, was obtained that secreted PDGF receptor competing activity under serum-free conditions at a rate of 700 ng of PDGF-AB equivalents/24 h/106 cells (Fig. 2). In order to characterize the PDGF produced by cell line 106:3, immunoprecipitations were performed with rabbit antisera that recognize the native PDGF dimers (24), and either one of the two reduced chains (11).Cells were labeled for 6 h with [35S]cysteine;the medium and the cell lysate were then subjected to immunoprecipitations and analysis by SDS-gel electrophoresis and autoradiography. In the cell lysate, the PDGF antiserum recognized components of 33, 30, and 24 kDa; after reduction several components of the sizes 23, 17, 16, 14, and 12 kDa were seen (Fig. 3). Analysis by chain-specific antisera revealed that the species of23, 17, and 14 kDa corresponded to the A chain, whereas the B chain appeared as16 and 12 kDa. The components secreted into the medium differed some-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviationsused are: PDGF, platelet-derived growth factor; CHO, Chinese hamster ovary;dhfr-, dihydrofolate reductase negative; FCS, fetal calf serum; IMAC, immobilized metal ion affinity chromatography; PBS, phosphate-buffered saline; PTH, phenylthiohydantoin; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography.

‘A. Ostman, L. Rall, A. Hammacher, M. A. Wormstead, D. Coit, P. Valenzuela, C. Betsholtz, B. Westermark, and C.-H. Heldin, unpublished data. A. Ostman, G. Backstrom, N. Fong, C. Betsholtz, C. Wernstedt, U. Hellman, B. Westermark, P. Valenzuela, and C.-H. Heldin, submitted for publication. “Experimental Procedures” are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

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what from those in thecell lysate. The PDGF immune serum precipitated components appearing as a broad zone in SDSgel electrophoresis in the M , range 35,000-28,000; after reduction, components of 23, 17, 16, 15, and 14 kDawere seen. Analysis by chain-specific antisera revealed that theA chain in the medium appeared to have the same sizes as in the lysate, but the relative amount of the 14-kDa species was considerably lower (Fig. 3). Less quantity of the B chain was found in themedium; it appeared as species of 16 and15 kDa, but the 12 kDa that constituted a major B chain component inthelysate was not seen. In conclusion, cell line 106:3 produces both PDGF A and B chains which are assembled into dimers,processed to the mature products, and secreted. The biosynthesis and processing of PDGF-like factors produced by cell line 106:3 was further examined by pulse-chase analysis. Cells received a 15-min pulse of [36S]cysteine;after various time periods, a lysate from the cells and the corresponding medium were subjected t o immunoprecipitation by PDGF antiserum, followed by analysis by SDS-gel electrophoresis in the absence or presence of reducing agents (Fig.

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14FIG. 3. Immunoprecipitations of cell lysate and conditioned medium of cell line 106:3 after metabolic labeling. 1063 cells were given a 6-h pulse of ["S]cysteine; thereafter thecell lysate (lanes 1-6) and the conditioned medium (lanes 7-12) weresubjected to immunoprecipitations by PDGFantiserum (lanes 1, 2, 7, and 8), PDGF A-peptide serum (lanes 3 and 9),PDGF A-peptide serum blocked by 90 nM of the corresponding peptide (lanes 4 and IO), PDGF B-peptide serum(lanes5 and IZ), and PDGF B-peptide serum blocked by 90 nM of the corresponding peptide(lanes6 and 12). SDSFIG.4. Pulse-chase analysis of the biosynthesis of PDGFgel electrophoresis was performed in the absence (lanes 1 and 7) or like factors in cell line 106:3. 106:3 cells were given a 15-min presence (lanes 2-6, 8-12) of reducing agents. The gel was then pulse of ["Slcysteine and subsequently chasedfor the indicated time subjected to fluorography. periods in amedium containing an excess of unlabeledcysteine. Immunoprecipitations were performed with a PDGF antiserum on 4). The component of highest M , seen in the lysate had an cell lysate (lanes 1-4, 7-10) and conditioned medium (lanes 5, 6, 11, unreduced size of 56 kDa; the amount of this component, as and 12). Samples were analyzed by SDS-gel electrophoresis in the well as othersof 52 and 42 kDa, decreased already after a 15- absence (lanes 1-6) or presence (lanes 7-12) of reducing agent, folmin chase period and appeared as components of lower mol- lowed by fluorography.

ecule masses, i.e. 40, 37, 33, and 30 kDa. After 105 min of chase a band of 24 kDa was also visible. The components of 37,33, and 30 kDa were secreted and accumulated in the medium. Under reducing conditions the predominating species, both in the lysate and in the medium, had molecular masses of 23, 17, and 16 kDa. These data show that dimerization occurs rapidly after synthesis and precedes proteolytic processing. Lacking antibodies that specifically recognize the different dimers, it is currently not possible to identify the dimer structure of the intermediates in the processing. However, the complex pattern of bands seen under nonreducing conditions in combination with the simpler pattern seen after reduction suggests that the A and B chains were assembled into multiple dimeric forms (see further below). Purification of PDGF-like Factors from Medium Conditioned by Cell Line 106:3"In order to structurally and functionally characterize PDGF secreted by cell line 106:3,2.5 liters of conditioned medium were collected and used as starting material for purification. The protocol developed for the purification of the osteosarcoma-derived growth factor (PDGF-AA) (4) was used, including concentration on a negatively charged matrix (Sulphadex),two consecutive gel chromatographies, and a final HPLC reversed-phase chromatography (Fig. 5). The elution position of the PDGF receptor competing activity in the last stepcorresponded to thatof an internal standardof '2sI-labeledPDGF-AB from human platelets, but the peak was broader and covered the region where all three dimeric forms of PDGF would be expected to elute (Fig. 5C and Ref. 11). Various fractions of the active peak were analyzed by SDS-gel electrophoresis and silver staining

(Fig. 6). Fractions 33-34 (pool I), 35-36 (pool 11), and 37-38 (pool 111)(Fig. 5C) all contained major unreduced components of about 30 kDa; after reduction bands of 16 and 17 kDa appeared (Fig. 6). Pool I1of the HPLC chromatogram was further analyzed by NH2-terminal amino acid sequencing, a double sequence, containing the sequences of PDGF A and B chains at approximately equimolar amounts, was found (Table I). Thus, thepurification protocol yielded pure PDGF; its size and chain composition were compatible with a heterodimer structure and/ora mixtureof homodimers of similar size. Dimeric Configuration of PDGF Purified from Conditioned Medium of Cell Line 106:3"Pools I, 11, and I11 of the HPLC chromatogram were labeled with '*'Iby the Bolton and Hunter (25) method, which labels both A and B chains. The '2sI-labeled material was then immunoprecipitated with PDGF chain-specific antisera (Fig. 7). Pool 11, coeluting with the internal standardof '2'I-labeled PDGF-AB purified from human platelets, contained aboutequal amounts of a 17-kDa A chain and a double band of 16 kDa corresponding to theB chain (Fig. 7B). Pool I contained the A chain but almost no B chain (Fig. 7A), whereas pool I11 contained both A and B chains, but more of the B chain (Fig. 7C). These data are compatible with the presence of a mixture of all three possible PDGF dimers, partly resolved in the HPLC reversed-phase system; the order of elution, PDGFAA first, followed byPDGF-AB and PDGF-BB, is consistent with previous findings (11). The amountsof the different dimers in pools I, 11, and I11 were determined by an IMAC system (27) capable of separat-

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TABLE I NH2-terminal amino acid sequencingof recombinant PDGF-AB 1 pg of protein from pool I1 (Fig. 5C) wassubjected to NH2terminal sequencing. The NHZ-terminal sequences of the A and B chains of PDGF purifiedfrom humanplatelets (28)are shownfor comparison. NHz-terminalsequence of platelet Amino acid(s)detected PDGF-AB no.

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FIG. 5. Purification ofPDGF-likefactors frommedium conditioned by cell line 106:3.The conditioned medium was first subjected to chromatography on a Sulphadex gel. After elution with 1.5 M NaCI, 0.01 M phosphatebuffer,pH 7.4, thematerial was concentrated and subjected to gel chromatography on Sephacryl S200 eluted in1 M NaCI, 0.01M phosphate buffer, pH 7.4 ( A ) .Fractions containing PDGF receptor competing activity (indicated by a bracket) were pooled, dialyzed against 1 M acetic acid, lyophilized, dissolved in 1 M acetic acid, and applied toa Bio-Gel P-150 column eluted with 1 M acetic acid ( B ) .Indicated fractions were pooled, concentrated by lyophilization, and applied to an Vydac C4 reversed-phase HPLC column eluted with a gradient of propanol in 1 M acetic acid, 2 M guanidine HCI ( C ) .The arrow indicates the elution position of 9 PDGF-AB. Active fractions were pooled as indicated intopools I, 11, and 111. Fractions of the different chromatograms were assayed for PDGF receptor competing activity a t 1:250 dilutions ( A and B ) or 1:500 dilutions (C) as described (21).

ing PDGF-AA, -AB, and -BB (11).Pool I1 consisted almost entirely of PDGF-AB, whereas pool I was mainly a mixture of PDGF-AA (72%)and PDGF-AB(24%), and pool I11 mainly a mixture of PDGF-AB (81%)and PDGF-BB (18%) (Fig. 8). The quantificationis based onthe observation that theincorporation of '1 afterBolton-Hunter labeling of PDGF is approximately equal into the two chains (Fig. 7B)." Estimation of the total amounts of the various dimers in the entire activepeak from the HPLC reversed-phase chromatogram rwealed 19% PDGF-AA, 69% PDGF-AB, and 12% PDGFBB.

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Characterization of Recombinunt PDGF-AB-The purification procedure described above allowed the preparation of highly purified PDGF-AB from the conditioned medium of cell line 106:3 (pool I1 of Fig. 5C). The recombinant PDGF-AB (pool 11) was compared with PDGF-AB purified from human platelets in two functional assays. The two factors competed forbinding to human fibroblasts in an equimolar fashion (Fig. 9), and both stimulated ["]thymidine incorporation in human fibroblasts with approximately the same dose dependence (Fig. 10). DISCUSSION

In this communication we show that a cell line that stably expresses both the A and B chains of PDGF produces functional PDGF dimers, mainly PDGF-AB, but also PDGF-AA and PDGF-BB. The structural andfunctional characteristics of therecombinantPDGF-AB was found to be virtually indistiguishable from those of PDGF-AB purified from human platelets. 'A. Ostman, A. Hammacher, and C-H. Heldin, unpublished obser- The PDGF A and B chain cDNAs that were used in the vations. plasmid construction encode precursor molecules of 196 and

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major PDGF-like componentof cells transformed with simian sarcoma virus (29). Since approximately equal quantities of the two chains were synthesized (Fig. 3), a random assembly without preference for homologous or heterologous combinations would yield50% PDGF-AB, and 25% each of PDGF-AA and PDGF-BB. However, quantification of the various dimers purified from the conditioned medium of the 1063cell line revealed that 19% was PDGF-AA, 69% PDGF-AB, and 12% PDGF-BB. The low amount of PDGF-BB may be related to thefact that the major part of this component remained cell-associated (Fig. 3). The recovered amount of PDGF-AB is almost four times that of PDGF-AA. This may indicate apreference for assembly of PDGF heterodimers in cell line 106:3. Alternatively, it may reflect e.g. differences in recovery of the various dimers during purification. The A chainmRNA occurs in two forms due to differential

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241 amino acids, respectively (13,14). Amino acid sequencing of A and B chains of PDGF purified from human platelets revealed that their amino termini correspond to amino acids 87 and 82 of their respective precursor molecules. Sequencing of peptides further suggested that the B chain is proteolytically processed in the COOH terminus around aminoacid 190 (28). There is no indication of COOH-terminal processing of the A chain. The biosynthesis, assembly, and processing of PDGF dimers wasfollowed in the 1063 cell line using pulse-chase analysis. The various steps that could be dissected are schematically illustratedin Fig. 11. A major 23-kDa A chain component, which was processed fairly slowly,was found (Figs. 3 and 4). This probably represents the undegraded A chain precursor after removal of the signal sequence. The component is larger than expected from the amino acid sequence (19 kDa), but thismight be due to glycosylation or to an anomalous migration in SDS-gel electrophoresis of the highly cationic protein. The 23-kDa component was chased to a 17-kDa component, which is the size of the mature A chainin SDS-gel electrophoresis (28). The cleavage most likely occurred after amino acid 86, yielding the normally occurring mature A chain, since the NH2-terminalamino acid sequence of the purified product contained the sequence SerLeu-Glu- (Table I). The B chain precursor seems to be processed more rapidly since the largest component detected had a size of only 16 kDa. This component was accumulated in the conditioned medium and was found to have the same size and NH2terminal amino acid sequence as PDGFB chainpurified from humanplatelets(28)(TableI). The B chain was further chased to a 12-kDa component, which remained associated with the cell (Figs. 3 and 4). Furthermore, a 24-kDa dimer was observed in the cell lysate but not in the conditioned medium (Figs. 3 and 4). This probably represents a homodimer of the 12-kDa B chain. Interestingly, a B chainhomodimer of similar size, which also remains cell associated, is the

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Recombinant PDGF-AB Heterodimer splicing (30), and theresulting products differ in their COOH termini. In the present study the shorter variant of the A chain, which seems to be more common in glioma cells (13) and endothelial cells (15,31), was expressed. It remains to be determined whetherexpression of the longer A chain variant, together with the B chain, would result in another pattern of dimers. Recent data indicate that PDGF-AA and PDGF-AB have different functional properties which are possibly mediated by different receptors (12). Thus, PDGF-AA has only a limited effect on cell growth and actin reorganization and no chemotactic activity. The effects of PDGF-BB are similar to that of PDGF-AB: but PDGF-BB is secreted less efficiently

'A. Ostman, K. Mellstrbm, and A. Siegbahn, unpublished data.

16207

than PDGF-AB (Fig. 3). Clearly, investigations of the pathophysiological function and mechanism of action of PDGF have to take into consideration that thedifferent dimers have different effects. The availability of recombinant PDGF-AB will be useful in these studies. The present communication describes a method to prepare pure and functionally active recombinant PDGF-AB. The size of the A and B chains of the heterodimer and the NH2terminal sequence of the mature products indicate that the recombinant PDGF-AB is very similar if not identical to PDGF-AB purified from human platelets. The procedure has two major advantages compared to the common method involving purification of PDGF-AB from human platelets (32); it does not involve the hazardous handling of human blood products, and the product is more homogenous since it is not exposed to the complex mixture of proteases in a platelet lysate. Acknowledgments-We thank Christer Wernstedt and Ulf Hellman for protein sequencing, Annika Hermansson, Marianne Kastemar, Diana Lee, and Ellen Freiberg for cell culturing and PDGF assays, Graeme Bell for his interest and comments on the inception of this work, and Linda Baltell for valuable help in the preparation of the manuscript.

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FIG. 9. Comparison of recombinant PDGF-AB and PDGFAB purified fromhuman platelets for binding human to fiFIG. 10. Comparison of recombinant PDGF-AB and PDGFbroblasts. Different concentrations of recombinant PDGF-AB AB purified from human platelets in a ['Hlthymidine incor(M and )PDGF-AB purified from human platelets (U) poration assay. Different concentrations of recombinant PDGFwere analyzed for their ability to compete with lmI-labeled platelet AB (W) and platelet PDGF-AB (U were ) tested for their PDGF-AB (A) or lZ6I-labeledrecombinant PDGF-AB ( B ) for binding ability to stimulate [3H]thymidine incorporation in foreskin fibroto fibroblasts. blasts.

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16208

Recombinant PDGF-AB Heterodimer REFERENCES

1. Heldin, C.-H., Wasteson, A,, and Westermark, B. (1985) Mol. Cell. Endocr. 39,169-187 2. Ross, R., Raines, E. W., and Bowen-Pope, D. F. (1986) Cell 4 6 , 155-169 3. Johnsson, A., Heldin, C.-H., Westermark, B., and Wasteson, A. (1982) Biochem. Biophys. Res. Commun. 104,66-74 4. Heldin, C.-H., Johnsson, A., Wennergren, S., Wernstedt, C., Betsholtz, C., and Westermark, B. (1986) Nature 319,511-514 5. Westermark, B., Johnsson, A., Paulsson, Y., Betsholtz, C., Heldin, C.-H., Herlyn, M., Rcdeck, U., and Koprowski, H. (1986) Proc. Natl. Acad. Sei. U.S. A. 83,7197-7200 6. Hammacher, A., Nistbr, M., Westermark, B., and Heldin, C. H. (1988) Eur. J. Biochem. 176,179-186 7. Waterfield, M D., Scrace, G. T., Whittle, N., Stroobant, P., Johnsson, A., Wasteson, A., Westermark, B., Heldin, C.-H., Huang, J. S., and Deuel, T.F. (1983) Nature 304,35-39 8. Doolittle, R. F., Hunkapiller, M. W., Hood, L. E., Devare, S. G., Robbins, K. C., Aaronson, S. A., and Antoniades, H. N. (1983) Science 221,275277 9. Robbins, K. C., Antoniades, H. N., Devare, S. G., Hunkapiller, M. W., and Aaronson, S. A. (1983) Nature 305,605-608 10. Stroobant, P., and Waterfield, M. D. (1984) EMBO J. 2,2963-2967 11. Hammacher, A., Hellman, U., Johnsson, A., Ostman, A., Gunnarsson, K., Westermark, B., Wasteson, A,, and Heldin, C.-H. (1988) J. Biol. Chem. 263, in press 12. Nistir, M., Hammacher, A., Mellstrom, K., Siegbahn, A,, Ronnstrand, L., Westermark, B., and Heldin C.-H. (1988) Cell 62,791-799 13. Betsholtz, C., Johnsson, A., Heldin, C.-H.,'Westermark, B., Lind, P., Urdea, M. S., Eddy, R., Shows, T. B., Philpott, K., Mellor, A. L., Knok, T. J., and Scott,J. (1986) Nature 320,695-699 14. Josephs, S. F., Gou, C., Ratner, L., and Wong-Staal, F. (1984) Science 2 2 3 , 487-490 15. Collins, T., Bonthron,D. T., and Orkin, S. H. (1987) Nature 328,621-624 16. Kelly, J. D., Raines, E. W., Ross, R., and Murray, M. J. (1985) EMBO J.

Supplemental Material to "Synthesis and assembly of a functionally active recombinant PDGF-AB heterodlmer" by bstman. A,. R a l l . L.. Hammacher. A,. wormstead, M.A.. Coit, D., Valenzueld. P.. Betsholtz, C., Westermark. B. and C.-H. Heldin EXPERIMENTAL PROCEDURES Plasmid mnstructlons Methods were essentially as described 1" f l T , . Briefly. the EcoRI fragments encoding the A c h a m 1 1 3 ) and B chair (netsholtz et a l . , unpublished data) precursors of PDGF were cloned L:.-u the mammalian cell expression vector pSv7d (18). The two resulting plasmids, pSV7d-PDGF-Al02 and pSV7d-PDGF-81. were linearized with PvuI, pSV7d-PDGF-81 was treated wlth alkaline phosphatase and the fragments were ligated. E. coli s t r a m HBlOl was transformed with the ligation reaction and colonlesresisfsnt to ampicllin were screened with nicktranslated fragments encodmg the A and B chains of PDGF. The resultant chimeric plasmid. pSV7d-PDGF-A102-B1, 15 descrlbed in Figure 1A. Cell culture and trdnsfectlon Cell culture. transfection and selection procedures have been descrlbed in detall (19. 20). Brlefly, CHO, dhfr- cells (DxB11) were grown in Ham's F1Z supplemented with 10% FCS, 200 ilg/ml proline and antlbiotlcs. The cells were transfected using the calcium-phosphate procedure followed by a glycerol shock. 5 ug of pSV7d-PDGF-Al02-BI and 5 pg Of a plasmld (pAd) contalnlng mouse reductase dhfr =DNA and the adeno m a j o r late promoter were used per tha 10 cells. Cells were selected f o r functional dhfc v s m g DUlbeCCO's modlfled Eagle's medlum containing 10% dialyzed YCS. Colonies were Isolated, expanded and PDGF productmn was determined by [ Hlthymidine Incorporation into human foreskin fibroblasts cells. From one line (11102-81-106) a high producer. A102-B1-106-3. was isolated in 0.05 pM methotrexate. The secreted level of PDGF represented a 10-fold increase over the parental l m e . Pur~ficdtionof recombinant PDGF dmers. 2.5 llters of serum-free conditioned medium were pooled. cleared t r m cell debrls by centrifugation and Sublected PDGF-like activitv in the to the puriflcatlon Protocol descrlbed ln 141. . . various fractions vas-determined by a receptor carnpetlng assay (21).'Briefly, the conditioned medium was first applled to chromatography on a Sulfadex column. PDGF-like factors Yere eluted with 1.5 M NaCl, 0.01 M phosphate buffer. p~ 7 . 4 , concentrated by ammnnlum sulphate precipitatlon, and applied to a Sephacryl s-200 column. eluted with 1 M NaCl. 0.01 phosphate buffer. p~ dlalyred against 1 M acetic acid. 7.4. ACtlYe fractions were pooled, lyaphllined and applied to a Blocel P-I50 column. eluted wlth 1 M acetic acid. The material I" the active fractions was finally applied to an RPI HPLC reversed-phase Column equilibrated In 1 M acetic acid, 2 M guanidine-HCI and eluted with a gradlent of 2-propanol. The purlfled rnaterlal was desalted on a narrow bore HPLC column. eluted in 0.1% trifluaracetic acid with a gradient of acetonltrile. samples were then evaporated in a Speed vac Concentrator and dissolved I" 1 M acetic acld; aliquats (0.5 ug) were analyzed under reducing or "on-reduclng condltlons by SDS-gel electrophoresis using gradlent gels of 13.18% polyacrylamide. and silver Staining (23). i:ikin concentratron of pool 1 1 was determined by amino acid analysis. The over-all yleld of PDGF receptor competmg activlty was 4 % .

4,3399-3405 17. Maniatis, T., Fritsch, E., and Sambrook, J. (1982) Mokculor Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N V*.

18. Truett, M.A., Blacher, R., Burke, R. L., Caput, D., Chu, C., Dina, D., Hartog, K., H., Kuo, C. Masiarz, R.,F. Merryweather, J. P., Najarian, R., Pachl, C., Potter, S. J., Puma, J., Quiroga, M., Rall, L. B., Randolph, A., Urdea, M. S., Valenzuela, P., Dahl, H. H., Favalaro, J., Hansen, J., Nordfang, O., and Ezban, M. (1985) DNA 4,333-349 19. Pachl, C., Burke, R. L., Stuve, L. L., Sanchez-Pescador, L., van Nest, G., Masiarz, F., and Dina, D. (1987) J. Virol. 6 1 , 315-325 20. Stuve, L.L., Brown-Shimer, C., Pachl, C., Najarian, R., Dina, D., and Burke, R. L. (1987) J. Virol. 61,326-335 21. Nistbr, M., Heldin, C.-H., Wasteson, A., and Westermark, B. (1984) Proc. NQtl.Acad. Sci. U.S. A. 81,926-930 22. Blobel, G., and Dobberstein, B. (1975) J. CeU Biol. 67,835-851 23. Morrisey, J. H. (1981) Anal. Biochem. 117,307-310 24. Heldin, C.-H., Westermark, B., and Wasteson, A. (1981) Ezp. Cell Res. 136,255-261 25. Bolton, A. E., and Hunter, W. M. (1973) Biochem. J. 133,529-539 26. Betsholtz, C., and Westermark, B. (1984) J. Cell. Physiol. 1 1 8 , 203-210 27. Belew,M.,Yip, T. T., Andersson, L., andEhnstrom, R. (1987) Anal Biochem. 164,457-465 28. Johnsson, A., Heldin, C.-H., Wasteson, A., Westermark, B., Deuel, T. F., Huang, J. S., Seeburg, P. H., Gray, A., Ullrich, A., Scrace, G.,Stroobant, P., and Waterfield, M. D. (1964) EMBO J. 3,921-928 29. Robbins, K. C., Leal, F., Pierce, J. H., and Aaronson, S. A. (1985) EMBO J. 4,1783-1792 30. Rorsman, F., Bywater, M., Knott, T. J., Scott, J., and Betsholtz, C. (1988) Mol. Cell. Biol. 8 , 571-577 31. Tong, B. D., Auer, D. E., Jaye, M., Kaplow, J. M., Ricca, G., McConathy, E., Drohan, W., and Deuel, T. F. (1987) Nature 328,619-621 32. Heldin, C.-H., Johnsson, A., Ek, B., Wennergren, S., Ronnstrand, L., Hammacher, A., Faulders, B., Wasteson, A., and Westermark, B. (1987) Methods Enzymol. 147,3-13

Immunoprecipltation was performed with a rabbit PDGF antiserum ( 2 4 ) . and With rabbit peptide antisera specifically recognlring PDGF A and B chains. respectively (11). Before precipitation with the latter antlsera cell culture lysafoltes and medium were treated with 10 mM dithiothreitol for 2 h at 37'C. lowed by 50 nu( lodoacetarnide for 0 . 5 h at neutral pH at room temperature. Lysates and cell culture media were first incubated with 100 "1 ofta 50% Slurry proteln A-Sepharose In PBS for I h at 4%. After the protein A-Sepharose had been collected by centrifugation. the cell lysates and conditioned medla recelved 50 "1 of normal rabbit Serum. After incubation at 4°C for 4 h, protem A-Sepharose was added and collected as above. Cell lysates and medium then received 50 p l of antlserum, and were incubated over-night at IOC before prateln A-Sepharose was added and collected as above. Imunoprecipitates were then washed 4 tlmes in 0.5 H NaC1, 0.01 M phosphate buffer. pH 7.4. 5 mg/ml BSA. 1% Triton. 0.1% SDS. and once i n 20 mH TriS-HC1, pH 7.4. In cases where PDGF antiserum had been used. immunocomplexes Yere extracted by adding 200 u1 of non-reducmg SDS-sample buffer and Incubating at 95°C far 3 min. Half of the extracted immunocomplexes where then reduced by a d d m g dithiothreitol to a final concentration of 10 mM and incubating at 95OC for 3 mi". Samples that had received A and B chain speclfic antisera were extracted by adding 100 u l All reduced of reducing SDS-Sample buffer and incubating at 95% far 3 mi". samples were alkylated by adding iodoacetamide to a final concentration of 50 m ~ . samples were analyzed by SDS-gel electrophoresis 1221 u s i n g gradient gels polyacrylamide, and fluorography. of 13.18% Pulse Chase experiments were performed using the Same labeling medium as described above, and cysteine-free F-lo medium supplemented with 150 pg/ml of cysteine, 10% dialyzed FCS, 200 p g l r n l Of proline and antibiotics as chase medlum. SamDles were ImmunoDreciDitated with PDGF antiserum and analvzed bu SDS-gel ele&rophoresis and fluorbgraphy. as described above.

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Immunoprecipitation of 125~-1abe1ed factors. 0.5-1 ug of the material from each of €he three pools from the final HPLC purification step were radiolabeled by the method of Bolton and Hunter (25); 5.000-13.000 cpm/ng of protein was incorporated. The iodinated factors were treated with 4 M quanldine-HCl 1 M TriS-HCI. pH 8 . 10 m M EDTA, 10 mM dithiothreitol, for 10 minutes at 95'C'after which iodoacetamide was added to a final concentration of 50 mM. The samples were then diluted 40 times in PBS with 3 mglml of BSA and 0.01% Triton x-loo, 50,000 cpm of iodinated factor in a volume Of 40 p l was incubated With 2 p 1 of antiserum overnight at 4%. samples were then incubated with 40 p l Of a 50% slurry protein A-Sepharose in PBS for 1 h at room temperature. The beads were washed and extracted as described above and samples were analyzed by SDS-gel electrophoresis on gradient gels (13-18% polyacrylamide) and autoradiography. Characterization of PDGF-llke dimers by IHIIC. Each of the three iodinated HPLC pools were precipitated by trichloroacetic acid: 150.000 cpm of each was dissolved in 1 mM imidazole, 1 H NaCI. 0.01 M DhosDhate buffer. DH 7 . 4 , and applied to an IMC column (1 ml: kind gift from Pharmacia, uppsala) equilibrated with the same buffer. The Column was eluted by a linear gradient of imidazole ( 1 mM/rnin) and an inverse gradient of NaCl ( l o mM/mln) at a flow rate of 0.512yl/min as described 1 1 1 ) . AS markers far PLGF-M, -AB. and -88 were used I-labeled glioma-derlved growth factor-I, PDGF purified from platelets and a recombinant B c h a m hornodmer purified from medium conditioned by a CHO cell line that expresses a transfected human PDGF B Chain. respectively. Assa of mitogenic activity. Mitogenic activity was determined by a [3H]thymiinneYincorporatian assay using human foreskin fibroblasts performed as described as 126). Trichloroacetic acid precipitable radioactivity was determined by liquid scintillation counting.

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phate buffer, pH 7:4, 0.5% -Tritan.'l% Trasyloi, 1 mN phenylmethylsulphony fluoride. The cell lysates W e r e centrifuged for 1 5 min at 1 0 . 0 0 0 x g and the Supernatant subjected to Immunoprecipitation.

Protein sequencing. Automated Edman degradation was performed using an Applied Biosystems Moael 4 7 0 A gas phase sequenator. The PTH amino acid residues were determined on-line by an Applied BiosyStemS 120 A PTH analyzer.