Expression and Processing of the Activin-A/Erythroid Differentiation Factor Precursor: A Member of the Transforming Growth Factor-/? Superfamily

Danny Huylebroeck, Kristien Van Nimmen, Abdul Waheed, Kurt von Figura, Anne Marmenout, Lucie Fransen, Peter De Waele, Jean-Marie Jaspar, Paul Franchimont, Henk Stunnenberg, and Hugo Van Heuverswijn Innogenetics S.A. Industriepark Zwijnaarde 7 (D.H., K.V.N., A.M., L.F., P.D.W., H.V.H.) B-9710 Ghent, Belgium European Molecular Biology Laboratory (D.H., H.S.) D-6900 Heidelberg, West Germany Biochemie II Georg-August Universitat (A.W., K.v.F.) D-3400 Gottingen, West Germany Laboratoire de Radioimmunologie B23 Institut de Pathologie Universite de Liege (J.-M.J., P.F.) B-4020 Liege, Belgium

The biosynthesis and intracellular processing of the polypeptide precursor of the #A-chain of the fertility hormone inhibin were assessed by infecting a wide spectrum of cell types with a recombinant vaccinia virus. Most cell lines, including follicular granulosa cells, secrete both prohormone and mature hormone as homodimers (activin) composed of disulfidelinked subunits of 54 kDa (proactivin-A) and 14 kDa (activin-A), respectively, and a small amount of prohormone-mature hormone heterodimers. Mature activin is secreted from mouse pituitary cells (AtT-20), while pig kidney cells [PK(15)j secrete mostly proactivin. More prohormone is secreted in the presence of NH4CI, suggesting that prohormone processing is facilitated by low pH. Proactivin-A is not a ligand for the mannose-6-phosphate/insulin growth factor-ll receptor. The recombinant activin stimulates FSH release from pituitary cells and differentiates erythroleukemia cell lines in vitro. (Molecular Endocrinology 4: 1153-1165, 1990)

dotropic hormones from the anterior pituitary (for review, see Ref. 1). Inhibin is a glycoprotein secreted by the granulosa cells of the follicle and inhibits the secretion of FSH by the anterior pituitary. In the male, inhibin is secreted by Sertoli cells within the testis. However, structurally related proteins have been purified from follicular fluid that preferentially inhibit (inhibins) or stimulate (activins) FSH secretion. Different forms of inhibin with mol wt ranging from 32-120 kDa have been isolated (1, 2). The smallest biologically active form of inhibin is a disulfide-linked dimer composed of an 18kDa a-chain which is glycosylated and a 14-kDa /?chain. Two /3-chains are known (/3A and /3B), which in combination with the a-chain yield inhibin-A and -B. Interestingly enough, activins are disulfide-linked homodimers of the jS-chains. The j8Aj8A 25-kDa dimer is also known as FSH-releasing protein (FRP) (3) or activin-A. The second form of activin is represented by the heterodimer /3A/?B (4). A third form of biologically active activin composed of /3B/3B (activin-B) possibly exists as well (5). Complex mechanisms for controlling FSH secretion are likely to operate at the level of expression, chain assembly, and/or processing of the inhibin and activin subunits because of their opposing biological activity.

INTRODUCTION Steroid hormones as well as proteins such as inhibins, activins, and follistatins regulate the release of gona-

Several groups have isolated cDNA clones encoding inhibin subunits (6-9) (Marmenout, A., L. Fransen, R. Ruysschaert, R. Wulgaert, H. Blockx, M.-T. Hazee-

0888-8809/90/1153-1165$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society

1153

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Vol 4 No. 8

MOL ENDO-1990 1154

Hagelstein, J.-M. Jaspar, P. Franchimont, and H. Van Heuverswijn, unpublished results). The /3-subunit precursor shows structural and sequence homology with the transforming growth factor-/? (TGF/3) family. All TGF/3 peptides (Refs. 10 and 11 and references therein), Mullerian inhibiting substance (12), glioblastoma-derived T-cell suppressor factor (13), cartilageinducing factor-A and -B (14, 15), mesoderm-inducing signal Vgl from Xenopus (16), Vg~\ -related protein (Vgr1) (17), bone morphogenetic protein-2 and -3 (18), and a gene product of the decapentaplegic complex from Drosophila (19) have been reported to have related, or some even identical, amino acid sequences. The homologies are most pronounced in the C-terminal part of the precursor polypeptide representing the mature form of these growth factors. The physiological implications of these findings have yet to be explained. A common receptor for TGF/3, activin, and inhibin has been identified in the cell line GH3 (20), but activin also binds to a specific receptor on granulosa cells (21) and to specific receptors on erythroleukemia and embryonic carcinoma cells (22). Activin-A is identical to erythroid differentiation factor (EDF) (23), modulates the proliferation of multipotential and erythroid hematopoietic progenitor cells (24), and is likely to be involved in the control of oxytocin secretion from neurosecretory neurons (25). This suggests that activin is a multifunctional protein and that many of its activities remain to be elucidated. The finding that /3A and /3B mRNAs are also detected in extragonadal tissues (26) supports this hypothesis. Since it is not known whether differential biological activities can be attributed to the precursor and mature forms of activin, it, therefore, seemed necessary to characterize the processing of proactivin in different cell types. Here we present data on the biosynthesis and posttranslational processing of the polypeptide precursor of bovine activin-A. Activin expressed by recombinant vaccinia virus was tested for its ability to stimulate FSH release and differentiate erythroid cells. In addition, we have investigated whether the prohormone is a ligand for the 215kDa mannose-6-phosphate/insulin growth factor-ll (M6P/IGF-II) receptor, as has been reported for TGF/^ (27).

RESULTS Characterization of Activin by in Vitro Translation Bovine preproactivin (including the signal sequence) is 425 amino acids long (Marmenout, A., L. Fransen, R. Ruysschaert, R. Wulgaert, H. Blockx, M.-T. HazeeHagelstein, J.-M. Jaspar, P. Franchimont, and H. Van Heuverswijn, unpublished results) (Fig. 1). We assume the signal peptide to be 28 amino acids long because its sequence is identical to that of the human /?A-chain (28). Mature activin-A is identical in all species studied so far and has been shown to constitute the carboxyterminal 116 amino acids of the prohormone (4, 6).

Consequently, the amino-terminal pro part of bovine proactivin is 281 residues long. It contains one potential A/-glycosylation site at residue 137. T7-directed and capped /3A-RNA was translated in vitro, and the translocation of the polypeptide was studied by the addition of dog pancreas microsomal membranes. Unglycosylated but translocated proactivin was obtained by adding a short peptide (A/-benzoylAsn-l_eu-Thr-A/-methylamide) which can enter the microsomes and serves as a competing substrate for Nlinked glycosylation. Preproactivin migrates as a 53kDa protein (Fig. 2, lane 9). Upon the addition of microsomes, two proteins of 54 and 51 kDa appear (lane 10). Both arise by the removal of the signal sequence, but the 54-kDa form is A/-glycosylated proactivin. The peptide indeed causes all of the translocated proactivin chains to shift to 51 kDa (lane 11). Identification of Activin Secreted from Cells Infected with Vaccinia Virus Late in infection, three extra polypeptides are secreted from HeLa cells infected with /37 virus compared to those infected with wild-type virus. Their mol wt [in reducing sodium dodecyl sulfate (SDS)-polyacrylamide gels] are 54 kDa (pro/3A), 43 kDa [most likely representing the N-terminal part (pro) of proactivin], and (a doublet of about) 14-15 kDa (0A; Fig. 3, lane 2). The 54-kDa protein can be detected in pulsed cells and comigrates with translocated glycosylated proactivin synthesized in the microsome system (data not shown). Under nonreducing conditions (Fig. 3, lane 4), the proactivin and mature activin homodimers migrate at 110 and 24 kDa, respectively. A significant amount of a 67-kDa protein, only visible on nonreducing gels (lane 4), is indicative of the secretion of a dimer composed of one proactivin and one mature activin monomer that are disulfide linked. The 43-kDa pro part of the activin-A precursor does not form a disulfide-linked dimer. A 43kDa protein from vaccinia virus (lane 3) comigrates with the activin pro part in nonreducing gels, but migrates differently in reducing gels (lane 1). The medium from /37 virus-infected HeLa and AtT-20 mouse pituitary cells collected after the overnight infection-stimulated FSH release (and not LH release unless excessively high amounts of activin are added) from pituitary cells in a dose-dependent fashion (Table 1), indicating that the expression of biologically active activin was obtained. Activin Maturation Is not Cell Specific Because of the broad host range of vaccinia virus it is possible to assess the maturation of activin in a variety of cell types. Radiolabeled intracellular and secretory proteins obtained from pulse-chase experiments in virus-infected cells were characterized by SDS-polyacrylamide gel. Figure 4, A and B, shows the results using CV-1 (African Green monkey kidney cells) and primary granulosa cells, respectively.

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Processing of Recombinant Activin-A

1155

CHO NH0

I

- COOH

I

pro

pre

Fig. 1. Structure of Bovine Preproactivin-A Only the monomer chain is shown. The numbers represent amino acid residues and indicate the length of the respective polypeptide domains. The arrows, respectively, show the presumed site of signal peptide cleavage and the known processing site for generating the mature hormone (/9A) from the precursor hormone (pro/3A). The single A/-glycosylation site (CHO) in the whole polypeptide is at residue 137 in the N-terminal part of proactivin (pro).

No UNA PTZ19H pTZ19Rf)12 prZ19Rpi3

M

Wt

Mt-MBHANLS |-|| + | HI+ll+lFll + l l+l f-Tl + l l+l -(PR0RA)2

ACC. PEPTIDE H T 1 H I - l l + l H R I + l l - l l - l l + l

-(PR0n A -(3 A )

-PROpA -PRO

-PRO

-(f3 A ) 2

—prefi-lactamasp —fi-ladamase

12.5RED.

M

1

2

3

4

5

6

7

8

9

10 11

Fig. 2. Translation and Translocation of Proactivin in Vitro Sense RNA was obtained in vitro using plasmid pTZ19R/313; as controls, plasmids pTZ19R (no insert) and pTZ19R/312 (insert in the antisense orientation with regard to the T7 promoter) were also transcribed. RNA was translated in vitro (rabbit reticulocyte lysate) in the presence (+) or absence (-) of membranes and/or acceptor (ACC.) peptide for A/-glycosylation. Samples were analyzed on a 10% polyacrylamide gel. Lane 1 shows the proteins synthesized in a control translation mix without plasmid-specific RNA. Lane 2 is identical to lane 1, but microsomal membranes were included. * , A/-Glycosylated proactivin

Beta 7 virus-infected CV-1 cells secrete proactivin (54 kDa) together with two polypeptides representing mature activin (14-14.5 kDa). In a nonreducing polyacrylamide gel, this doublet is absent, and a /3A-dimer is visible as a 24-kDa protein as well as the proactivin dimer in the 100-kDa area (lane 16). The 43-kDa pro part is also secreted. These results agree with the observations of HeLa cells. While results obtained in granulosa cells (Fig. 4B) are essentially similar regarding

NON-RED.

Fig. 3. Comparison of secretory proteins from HeLa cells infected with /37 virus (lanes 2 and 4) or wild-type virus (lanes 1 and 3) after pulse-chase radiolabeling during the late phase of the viral infection. Pro/3A, proactivin (residues 1-397); /3A, mature activin (residues 282-397); pro, N-terminal part of pro/3A (residues 1-281); (/8A)2, homodimeric forms; RED., reduced samples; NON-RED., nonreduced samples; M, mol wt marker proteins.

the size of the different forms, the pro part is of heterogeneous size [42-39 kDa and visible as multiple bands; also the case in BHK-21 (C13) Syrian hamster kidney cells, data not shown]. Table 2 summarizes the results for the processing of proactivin-A in different cell types. The efficiency of processing was quantitated on autoradiographs from culture medium samples that were separated in reducing protein gels. The fact that the mature hormone contains only four methionine residues compared with 10 in the prohormone was taken into account. AtT-20 cells were found to secrete virtually no prohormone, whereas pig kidney PK(15) cells secreted it almost exclusively. Other cell types, including granulosa cells (site of ovarian synthesis of inhibin a- and 0-chains), secreted prohormone and mature hormone. Thus, the

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Vol 4 No. 8

MOL ENDO-1990 1156

Table 1. Dose-Dependent FRP Activity of Activin Polypeptides Expressed by Vaccinia Virus 07 FSH (ng/ml) % LH (ng/ml) Cell Line PBS HeLa Mock wt 07

ft (1:2) 07(1:4) 07(1:8) PBS + 5 fi\ BFF PBS + 1CMBFF PBS + 20 MI BFF PBS AtT-20 07

07(1:2) 07(1:4) PBS + 5 n\ BFF PBS+ 10^1 BFF PBS + 20 MI BFF

1423 ±72

1818 ± 3 7 6

1388 ±35 NS 1542 ±41 8 3316 ±91 133 3330 ±154 134 2972 ± 95 109 66 2361 ± 61 1239±173 13" 37" 898 ± 71 829 ±187 42"

1991 2322 3420 3220 2648 1921 2354 1984 2331

1142 ±78

1036 ± 2 0 9

±74 ±133 ± 422 ±516 ± 583 ±171 ±116 ±411 ± 224

2993 ± 371 162 1344 ± 6 3 2419 ±75 112 1249 ± 1 7 3 1593 ±191 40 1031 ± 2 7 747 ± 70 35" 48" 591 ± 69 64" 845 ± 78 408 ± 34

%

NS NS 88 77 NS NS NS NS NS

30 NS NS ND ND NS

One hundred microliters of 1 ml medium from about 106 cells were used and diluted 10-fold in the assay (see Materials and Methods), wt, wt vaccinia virus-infected cells; 07, recombinant vaccinia virus expressing (pro)activin (moi in Hela cells, 10; moi in AtT-20 cells, 20); BFF, bovine follicular fluid preparation at 1 mg/ml containing predominantly inhibin activity; NS, nonsignificant change compared with PBS at the 0.05 probability level according to the Mann-Whitney U test (see Materials and Methods); ND, not determined; %, percent change compared with PBS. " Inhibin activity of BFF.

processing of bovine proactivin is not cell type specific; the required protease(s) is present in all cell types and is a component of the constitutive secretory pathway. The low level of processing in PK(15) cells probably reflects low amounts of protease. All culture media from f37 virus-infected cells stimulate FSH release from pituitary cells (Table 3). Substantial stimulation is observed with activin secreted by PK(15) cells, although only about 5% of the secreted hormone is in the mature form. As the total expression in PK(15) cells is not expected to be much higher than that in other j87 virus-infected cells, this suggests that the secreted prohormone may have FRP activity. In contrast to natural TGF/3 (29) and recombinant TGF/? produced in Chinese hamster ovary (CHO) cells (30, 31), which have been shown to exist as latent TGF/3, recombinant vaccinia virus-encoded activin does not need to be exposed to acid pH (pH 2-3) to render it detectably active in the bioassay. Cleavage of Proactivin Is Low pH Facilitated during Exocytosis Intravesicular acidification in the endocytic and exocytic pathways has been studied in detail (for review, see Ref. 32). Acidophilic agents can change the pattern of

secretion. To test whether proactivin travels through low pH intracellular compartments during exocytosis and whether this is a prerequisite for maturation of activin, we analyzed the effect of NH4CI on the ratio of secreted proactivin to mature activin. HeLa cells, normally secreting both the prohormone and the mature hormone (Fig. 5, lane 6, and Table 2), secrete markedly more prohormone (consequently less of the mature hormone and pro part) when treated with 10 mM NH4CI (lane 12). This supports our assumption that the 43kDa polypeptide in fact represents the pro part of the prohormone. The amount of proactivin relative to mature activin in the medium from infected HeLa cells changes from about 25% to 70% (Fig. 5) or even close to 100% (data not shown) upon the addition of 10 mM NH4CI; with AtT-20 cells about 50-70% of the secreted activin is in the prohormone form under these conditions (data not shown). Secretion of the 67-kDa pro/V^A heterodimer is also increased by the addition of NH4CI, and its amount is quite substantial in the case of AtT20 cells (data not shown). An identical effect of NH4CI as that on HeLa cells has been observed in granulosa cells (data not shown). These results indicate that cleavage of proactivin is facilitated by low pH during exocytosis. Kinetics of Secretion and Endoglycosidase-H (EndoH) Experiments Most activin polypeptides are apparently secreted within 4-8 h (Fig. 6A), with an average secretion time of 5 h, as deduced from multiple experiments. A small but significant fraction of proactivin remains intracellularly as a proactivin monomer which contains an Asnlinked oligosaccharide of the high mannose type because it is sensitive to EndoH (data not shown). This reflects its presence in a premedial Golgi compartment, most likely the rough endoplasmic reticulum. The secreted precursor and pro part are resistant to EndoH, indicating that oligosaccharide of the complex type has been acquired during passage through the Golgi apparatus (Fig. 6B). Analogously to other oligomeric proteins, dimerization of proactivin must occur before leaving the rough endoplasmic reticulum. The pro part of activin is likely to play an important role in this dimerization/secretion process, since a mutant activin (deleted pro part but still containing signal peptide) was no longer found to be secreted (Goergen, M., and P. De Waele, unpublished results). The addition of tunicamycin and inhibitors for carbohydrate processing (1-deoxymannojirimycin and swainsonine are inhibitors for Golgi a-mannosidase-l and -II, respectively) does not influence dimerization of proactivin-A and does not inhibit activin secretion (Fig. 6B). Unlike TGF/3i (33), activin is secreted from tunicamycintreated cells. The glycosylated activin polypeptides secreted in the presence of 1-deoxymannojirimycin or swainsonine are EndoH sensitive, 100% and nearly 100%, respectively. The latter experiments provided the necessary controls for the determination of the

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Processing of Recombinant Activin-A

1157

CV-1 CELLS

mock P

wt

MEDIA b—M

Ch P Ch P Ch

m

wt

P

Ch P Ch P

ock

PROn A -

1

2

3

U 5 6 7

8

9

mock wt n^

fl7

Ch Ch Ch

Ch

dfc

»

-PR0n A

^

^

-PRO

10 11

12 13

-(PROn A ) 2 -PRonA-nA -PRO

14

RED.

15

16

NON-RED.

RAT GRANULOSA

B

MEDIA

Ch

Ch mock wt

mock wt

(I7

-(PRon A ) 2 •PRonA-nA

-PR0f3 A 3 PRO

-(n,'A'2

-0/

6.52 RED.

4

5 6 NON-RED.

Fig. 4. Pulse-Chase Labeling of Mock-Infected (mock), Wild-Type, or fr Virus-Infected CV-1 Cells (A) and primary granulosa cells (B). The protein patterns of pulsed (P) and chased (Ch) cells are shown only for CV-1 cells. RED., Reduced samples; NON-RED., nonreduced samples; M, mol wt marker proteins (92, 69, 43, 30, 2 0 , 1 4 , 1 2 . 5 , and 6.5 kDa).

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Vol 4 No. 8

MOL ENDO-1990 1158

Table 2. Comparison of Efficiencies of Cleavage of Proactivin (pro/3A) to Mature Activin (/3A) in Different Cell Lines Infected with a Recombinant Virus Cell Line

Pro/?A

Table 3. Stimulation of FSH Release from Cultured Primary Rat Pituitary Cells by Recombinant Activin Cell Line

Assay 1 Y-1 PK(15) BHK HeLaD98/AH2 AtT-20 PBS + 5 Ml BFF

BHK

80 77"

20 23

CV-1

55 60"

45 40

PK(15)

96

4

PBS+ 1 0 M ' B F F PBS + 20 MI BFF

HeLa S3

40 32"

60 68

Assay 2 CV-1 Mv1-Lu PBS + 5 n\ BFF PBS + 10/xl BFF PBS + 20 MI BFF Assay 3 HeLa S3"

HeLa D98/AH2

27

73

29"

71

AtT-20

2-0

Hep3B

51

49

Primary Rat Granulosa

47 s

53"

Mv1-Lu

46 41"

54 59a

98-100

Efficiency given as a percentage of all activin polypeptides secreted from pulse-chased cells. Values are calculated from the ratio of precursor/mature hormone as determined by densitometric scanning of autoradiographs (see Materials and Methods) from gels, as given in Fig. 4. The results are the mean value obtained from at least three independent experiments. " Calculated from the ratio of precursor/mature hormone plus pro part.

EndoH resistance of secreted proactivin-A and its pro part. Recombinant (Pro)Activin Is not a Ligand for the M6P/IGF-II Receptor precursor has recently been shown to be a phosphoglycoprotein (34) and a ligand for the M6P/ IGF-II receptor (27). These observations prompted us to assay whether this holds true for (pro)activin-A. Efficient cleavage of phosphomannosylated precursor TGF/3 polypeptides may be dependent on transport to an organelle (with low pH) rich in M6P/IGF-II receptors. This late endosome (35) could be equivalent to the prelysosome described by Griffiths et al. for NRK cells (36) and postulated by these researchers as the organelle at which the pathway for endogenous, newly synthesized lysosomal hydrolases and the endocytic pathway meet. To test this, two different experiments were performed. Labeled proteins secreted from uninfected and virusinfected HeLa cells and synthesized in the presence or absence of NH4CI were loaded onto a column to which the M6P/IGF-II receptor was covalently coupled. Bound proteins were eluted with M6P and compared with the

Sample

Granulosa6

FSH (%)

67 66 66 68 87

14" 36" 48" 88 122 13° 40" 49" Mock

Mock wt 07

NS NS

69 60" NS 42

For details, see Table 1. 07, Recombinant vaccinia virus expressing (pro)activin (moi 5); /34, tk~ virus. All changes in LH values were nonsignificant. 8 Inhibition of FSH release illustrating inhibin activity of bovine follicular fluid (BFF) or medium from uninfected granulosa cells. "Biological activity determined with another preparation of pituitary cells.

unbound protein fraction (Fig. 7; no NH4CI). In mockinfected cells (lane 15), a minor fraction of polypeptides was recovered in the M6P eluate. These are likely to represent newly synthesized lysosomal proteins which for unknown reasons have escaped binding to intracellular M6P/IGF-II receptors (for review, see Ref. 37). These proteins were not labeled in virus-infected cells, and proactivin and the pro part of activin did not bind to the column (lanes 16-18). The results obtained with media from NH4CI-treated cells were completely analogous (data not shown). Control experiments with extracts of cells infected with a recombinant vaccinia virus expressing the human lysosomal enzyme arylsulfataseA showed that 5-7% of arylsulfatase-A binds to the column in a M6P-dependent manner (data not shown). A second approach involved the addition, before and during the pulse-chase procedure, of an antireceptor antibody to the medium of (pro)activin-synthesizing cells. This addition has been shown to increase the secretion of lysosomal hydrolases (32, 37). If the conversion of proactivin to mature hormone is dependent upon M6P/IGF-II receptor interaction to reach the organelle in which efficient cleavage occurs, then results similar to those obtained upon the addition of NH4CI, e.g. more precursor relative to mature hormone, should be found. Figure 8 shows that the ratio of proactivin to mature hormone was similar to that found when using

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1159

Processing of Recombinant Activin-A

+NH 4 C I

-NH4CI

wt

mock

P7

wt

mock

P7 *1

iiffl :

P Ch P Ch P Ch P Ch P Ch P Ch

1

2

a

m

3

4

5

6

7

8

4

I -PROpA 1 -PRO

9

10 11 12 13

He l a

RED

Fig. 5. Effect of NH4CI on Maturation of Activin in HeLa Cells Compare lanes 6-12. Secreted proteins from labeled (P, pulse; Ch, chase) mock-infected or virus-infected cells were separated under reducing (RED.) conditions.

nonimmune serum. Vaccinia (pro)activin-A is thus not a ligand for the M6P/IGF-II receptor. Recombinant Activin Has EDF Activity One protein with EDF activity has recently been identified as activin-A (23). The EDF titers of activin produced in 5 x 105-106 AtT-20 and HeLa cells amount to 2560 and 640-1280 K562 U/ml, respectively. Addition of diluted (1:2) EDF activity (activin) leads to positivity in about 40% (AtT-20) and 30-50% (HeLa) of the K562 target cells after staining with benzidine compared to 30% when using 100 HM hemin. The benzidine-positive staining is due to production of hemoglobin (Fig. 9A), and the effect of activin is dose dependent (Fig. 9B). Our recombinant activin thus differentiates erythroid cells.

DISCUSSION

The biosynthesis of proactivin-A and the generation of mature hormone from its precursor were investigated using a vaccinia virus system that expresses proactivin, thereby taking advantage of the wide host range of the virus and the efficient shut-off of cell-specific protein synthesis in infected cells. Vaccinia virus has proven its

value in the study of the maturation of proenkephalin (38), factor VIII (39), and nerve growth factor (40). Intracellular and secreted prohormone were identified by the following criteria. 1) The 54-kDa A/-glycosylated prohormone synthesized in a microsome-supplemented in vitro translation system comigrates on electrophoresis with an additional protein synthesized in or secreted from cells infected with /37 virus. 2) Proteins secreted by j87 virus-infected cells contain extra virus-specific polypeptides compared with those infected with wildtype virus. These are activin related because of their protein size, the immunoreactivity of the 24-kDa /3Adimer with an activin-specific antiserum (Dello, C , P. De Waele, and D. Huylebroeck, unpublished results), and the ability of the respective media to stimulate FSH release and differentiate erythroid cells. 3) The prohormone and the mature hormone form disulfide-linked homodimers. The observations that j8A mRNA is detectable in extragonadal tissues (26) and that EDF is identical to activin-A (23), in addition to its sequence homology with TGF/3, indicate the importance of extending the study of activin biosynthesis to cell types other than granulosa and Sertoli cells, originally considered to be the only sources of activin. Most cells (except for AtT-20 cells) secrete a 110-kDa disulfide-linked proactivin dimer, a 24-kDa disulfide-linked mature activin dimer, a 67-kDa disulfide-linked proactivin-activin heterodimer (54 kDa plus 14-kDa subunit, respectively) and, as a consequence of the cleavage of the prohormone, the remaining amino-terminal part of the prohormone (43 kDa). An alternative explanation for the 67-kDa form of the protein has, in the case of activin-B, been suggested to occur via noncovalent interaction of a pro part with the mature homodimer (5). We find this prospect unlikely for the 67-kDa form of activin-A since the polypeptide persists in nonreducing gels (after heating proteins in SDS). Furthermore, this 67-kDa form is more abundant than mature activin and the pro part during treatment of the cells with NH4CI. The efficiency of processing varies with the cell type. Subtle differences in the cleavage efficiency of TGF/3', compared to that of TGFfo have been documented in CHO and COS cells (30, 41), in which the recombinant TGF/32 precursor is cleaved more efficiently. The pro parts of activin are secreted in an equimolar fashion compared with the secretion of mature activin (this follows from the comparison of two different calculations of cleavage efficiencies; Table 2), do not accumulate intracellularly in a post-Golgi compartment, and are not substantially cleaved at other dibasic amino acid residues in this polypeptide (except perhaps for granulosa cells and BHK cells). In contrast to TGF/?! (42), the pro regions of proactivin-A are not disulfide interlinked. Under reducing conditions in SDS-polyacrylamide gel electrophoresis, mature activin monomer from most cell lines can be separated into two protein bands. In the case of natural TGF/3, this has frequently been explained by the presence of two forms (for example, TGFfr and -/32) in the same cell. However, the cell lines

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Vol 4 No. 8

MOL ENDO1990 1160

n7vv P

0.5

1

1.5

2

22

4

chase(hours)

-(PRon A ) 2 "PROnA-nA -PRO

-in'A'2

B EndoH

wtVV - Tm Dmm - - -

-

+

n7vv Dmm sws +

+

Tm +

wtVV - Tm DmmSWS + + + +

M

Fig. 6. A, Accumulation of activin polypeptides secreted from vaccinia virus-infected HeLa cells (nonreducing SDS-polyacrylamide gel. B, analysis of susceptibility of activin to EndoH (•, proactivin; O, pro part; >, mature activin) secreted from /37 virus-infected HeLa cells (chase time, 4 h), including a comparison with activin polypeptides synthesized in the presence of 1 -deoxymannojirimycin (Dmm) and swainsonine (SWS). Tm, Tunicamycin.

used in this study do not secrete activin (except for granulosa cells; data not shown), and the vaccinia system shuts off the synthesis of host cell proteins. We do not know whether this doublet arises from differential posttranslational modification of the activin-A subunits. Putative cleavage of the mature activin at the lysine residues (position 102-103) near the C-terminus remains a possibility. Under nonreducing conditions, all mature activin is in the dimeric form, further suggesting that the one or both cysteine residues very close to the C-terminus (positions 113 and 115), if involved in dimerization of activin at all, would not be the only cysteine residues used for dimeric formation. Activin-A produced in all cell types, using identical experimental conditions, stimulates FSH release to comparable extents, and it should be noted that the

plateau values of stimulation are not reached in these assays. Consequently, this stimulation occurs at all degrees of maturation. Provided that the absolute amounts of activin polypeptides do not differ considerably, this would suggest that stimulation of FSH release, at least in vitro, can be attributed to both forms of the hormone. However, this does not exclude differential specific activities between these forms, as has been suggested for TGF/3 (31). Polypeptide hormones are synthesized through larger precursors that are cleaved on their way (via the Golgi complex) to the plasma membrane where the mature hormone is released through exocytosis. From the NH4CI experiments, it follows that a decrease in vesicular pH is a prerequisite for cleavage of proactivin, but not for its secretion. Although cleavage in trans-

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Processing of Recombinant Activin-A

1161

ELUTED

UNBOUND

M6P

G6P -

wt

M

-

wt

6

7

p7

(37

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-

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wt

92PRO[1A

69-

PRO - 4 3 -

30-

20-

12.53

4

5

9

10

11 12

13

14

15 16

17 18

RED. Fig. 7. Assay of Binding of Secretory Proteins from Pulse-Chased Uninfected (-) or Wild-Type Virus- or /37 Virus-Infected HeLa Cells to the M6P/IGF-II Receptor The pattern of the starting material is given in lanes 1 - 3 . The gel includes a sample of activin secreted in chase medium (see Materials and Methods) without BSA (lanes 4, 9, 14, and 18). The unbound and eluted (G6P and M6P) fractions from the column are separated on a reducing (RED.) SDS-polyacrylamide gel. M, Moi wt marker proteins.

Golgi was not tested, packaging of the prohormone in secretory vesicles and acidification of this latter compartment are critical steps in the proper maturation of activin. The finding that TGF/31 is a ligand for the 215-kDa M6P receptor (27) led us to test whether cleavage of proactivin molecules would be dependent on direct interaction with this receptor in order to reach a prelysosomal-like compartment where cleavage would then occur after ligand dissociation from the receptor. Our results indicate that proactivin is not a ligand for the M6P/IGF-II receptor since 1) proactivin (as well as mature activin) secreted from HeLa cells is not a specific ligand for the M6P/IGF-II receptor which is covalently bound to a column; 2) the addition of an antireceptor antibody to /37 virus-infected cells does not alter the ratio of precursor to mature hormone that is secreted; 3) the abundance of M6P/IGF-II receptors in PK(15) cells (data not shown) does not correlate with the low cleavage efficiency observed in these cells; and 4) proactivin could not be detectably pulse labeled with phosphate (data not shown). The vaccinia virus system has opened new perspectives for understanding the maturation and biological function(s) of activin, originally defined as a fertility hormone, but now also emerging as a growth/differen-

tiation factor. It is of interest to compare the actions of activin and TGF/3 in a number of biological systems. In addition, the experiments presented here can be carried out with the inhibin a-subunit and the methodology used to study the assembly of inhibin.

MATERIALS AND METHODS Plasmids A 1700-basepair long cDNA fragment containing the complete bovine/3A-inhibin coding sequence (Marmenout, A., L. Fransen, R. Ruysschaert, R. Wulgaert, H. Blockx, M.-T. Hazee-Hagelstein, J.-M. Jaspar, P. Franchimont, and H. Van Heuverswijn, unpublished results) was recloned into the EcoRI site of the plasmids pTZ19R (43) and pAta18 (44). Cell Lines and Cell Culture Hep3B and HeLa D98/AH2 cells were grown in Dulbecco's Modified Eagles's Medium (DMEM) containing 10% fetal bovine serum (FBS); granulosa cells in DMEM containing 20% FBS; AtT-20 in DMEM supplemented with 0.35% glucose and 10% horse serum (HS); CV-1 and 143 tk~ in Earle's Minimum Essential Medium (EMEM) containing 10% FBS; Mv1-Lu cells in DMEM supplemented with 10% FBS and 1 % nonessential amino acids (NEAA); HeLa S3 in EMEM containing 5% newborn calf serum (NBCS); PK(15) in EMEM supplemented with

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Vol 4 No. 8

MOL ENDO-1990 1162

CD 00

ro

o

-69

PROO A PRCT

-30

-20 -14

-12.5 1 Fig. 8. (Pro)Activin Is Not a Ligand for the M6P/IGF-II Receptor SDS-polyacrylamide gel electrophoresis of secreted proteins from /37 virus-infected HeLa cells radiolabeled in the presence of M6P/IGF-II receptor antiserum (lane 1) or nonimmune serum (lane 2).

1 % NEAA and 5% FBS; Y-1 in Ham's F-10 with 15% HS and 2.5% FBS; BHK-21 cells in Glasgow Minimum Essential Medium containing 10% tryptose phosphate broth, 8% NBCS, and 2% FBS; and K562 cells in HEPES-buffered RPMI-1640 medium supplemented with 10% FBS. In Vitro Transcription and Translation In vitro transcription and translation were carried out as described previously (45). Acceptor peptide for A/-glycosylation (A/-benzoyl-Asn-Leu-Thr-A/-methylamide; a gift from U. Tillman, University of Cologne, West Germany) was added at 100 ^M. Preparation of Recombinant Viruses Recombinant viruses were prepared essentially as previously described (44). Viruses were selected and plaque-purified on tk" cells in the presence of 100 ng 5-bromo-2'-deoxyuridine (BUdR)/ml. Virus /37 was selected for further expression studies. Analysis of Labeled Polypeptides Expressed by Recombinant Vaccinia Virus Subconfluent cells (10-cm2 dishes) were washed with PBS and infected with virus (moi 5-10) for 1 h at 24 C. The inoculum was removed, and the cells were incubated for 15 h at 37 C in 1 ml DMEM containing 20 ng BSA/ml. The medium was removed, filtered through a low protein-absorbing 0.22-/xm filter, and stored frozen until assay for biological activity. Cells were washed with methionine-free EMEM containing 20 ITIM

HEPES before starvation in this medium for 1 h at 37 C. The cells were pulse labeled for 30 min by adding 1 ml of this medium containing 50 nC\ [35S]methionine. In all experiments involving NH4CI, the medium had a minimal pH of 7.4. For the M6P/IGF-II receptor interaction studies, activin was labeled with [35S]methionine and [35S]cysteine in methionine- and cysteine-free medium prepared from a MEM Select-Amine kit (Gibco Europe, Ghent, Belgium) and chased for 4 h (or for 8 h after an infection of 12 h; the results of the column binding assay are identical; data not shown). The cells were chased in 1 ml DMEM supplemented with 20 ^g BSA/ml and containing a 10-fold molar excess of cold methionine (and cysteine) normally present in this medium. After 4 h at 37 C, the medium was collected and centrifuged, and the supernatant was frozen. The dishes with the cells were cooled on ice, washed with ice-cold PBS, and frozen. Samples were prepared for electrophoresis as follows. BSA (50 Mg/ml) was added to the medium, and the proteins were precipitated with 10% trichloroacetic acid (TCA). The protein pellet was neutralized and solubilized in 0.5 M Trizma (Sigma, St. Louis, MO) containing 6.25% SDS, diluted 4.5-fold with water, and adjusted to SDS-polyacrylamide gel electrophoretic conditions (46). Cells were lysed at 0 C with 1 % Nonidet P40, 50 ITIM Tris-HCI (pH 8.0), and 20 ng phenylmethylsulfonylfluoride/ml for 20 min; the nuclei were removed by centrifugation; and the cleared lysate was prepared for SDS-polyacrylamide gel electrophoresis (46). After electrophoresis, the 15% gels were fixed in 10% TCA, 30% methanol, and 10% acetic acid for 30 min and then prepared for autoradiography with 80% En3Hance (DuPont de Nemours, NEN, Dreieich, West Germany) and 20% methanol for 30 min, followed by 20% methanol-3% glycerol (20 min) and drying under vacuum at 55 C. 14C-Labeled proteins used as mol wt markers were obtained from NEN. Gels were scanned using an Ultrascan XL Enhanced Laser Densitometer (Pharmacia LKB, Uppsala, Sweden), and scanning data were processed with a Gelscan XL program (Pharmacia LKB). For digestions with EndoH (Genzyme, Boston, MA), cell extracts were diluted to contain 0.2% Nonidet P-40 and, after the addition of 0.5% SDS, heated for 5 min at 95 C. Digestions with EndoH (50 mlU/ml) were then performed at 37 C in 0.07% SDS, 0.03% Nonidet P-40,10 ITIM sodium phosphate (pH 6.0), and 100 mM NaCI for at least 17 h. Medium samples were precipitated with TCA, and the pellets were washed with cold acetone and solubilized in a minimal volume of 0.5% SDS-10 mM Tris-HCI, pH 7.4. After heating (5 min; 95 C), the samples were diluted 7.5-fold for digestion with EndoH (see above). Tunicamycin, swainsonine, and 1-deoxymannojirimycin were used according to the supplier's recommendation (Genzyme). M6P/IGF-II Receptor Binding Assays Medium samples were adjusted to 0.1 trypsin inhibitor units (TIU) aprotinin/ml and 25 M9/ml each of leupeptin, pepstatin, and chymostatin (all from Sigma). The samples were applied to a 0.5-ml column of M6P/IGF-II receptor-Affigel 10 (0.2 mg protein/ml gel) (Waheed, A., unpublished results) in 50 mM imidazole, pH 7.0, containing 150 mM NaCI, 5 mM Na-0glycerophosphate, 2 mM EDTA, 0.025% NaN3, and 0.05% Triton X-100 (buffer A). The samples were recirculated twice over the column. The columns were then washed five times with 0.5 ml buffer A, three times with 0.5 ml buffer A containing 5 mM glucose-6-phosphate (G6P), and three times with 0.5 ml buffer A containing 5 mM M6P. Fractions 1-3 of the wash, fractions 1 - 3 of the G6P, and fractions 1 - 3 of the M6P eluate were pooled, and the proteins were precipitated with TCA after the addition of 50 ^g BSA/ml. Electrophoresis was carried out as described above. Antibody Experiment HeLa cells infected with /37 virus were starved for 30 min in methionine-free medium; this medium was then replaced with

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Processing of Recombinant Activin-A

1163

B

[N C0N1fENT

00414/ml

3 Growth medium + 100/uM hemin

0 3-

0 2-

O O o

0

1-

DILUTION OF TEST SAMPLE

Fig. 9. A, Photomicrograph of K562 Cells Incubated with Appropriately Diluted Media from Mock-Infected or /37 Virus-Infected HeLa Cells Cells were stained with benzidine and photographed under an invert microsocope. B, The dose-dependent EDF activity of two preparations of recombinant activin-A produced in /37 virus-infected AtT-20 cells, as measured by the hemoglobin content in K562 cell extracts, is shown together with the FRP activity (see Materials and Methods).

methionine-free medium containing rabbit anti-M6P/IGF-ll receptor antiserum or rabbit nonimmune serum. Both sera were used at a 10% final concentration throughout the experiment and were first dialyzed extensively against methionine-free medium. The cells were incubated for another 30 min, then pulse labeled for 30 min, followed by a 4-h chase in the presence of the respective antiserum. Samples were processed as described above.

bration. After a 3-day incubation, appropriately diluted culture media were assayed for FSH and LH by double antibody methods using NIAMDD rat pituitary gonadotropin reagents supplied by the National Pituitary Agency (NIH, Bethesda, MD). Data are presented as the mean ± SD and/or as the percent change in FSH and LH compared with PBS. Statistical analysis was performed using the Mann-Whitney U test. Results were considered significant when P < 0.05.

FRP Assay

EDF Assay

This assay is based on the activation of basal FSH secretion by anterior pituitary cells, as originally described for FSH inhibition by inhibin (47). Anterior pituitary cells from adult male Wistar rats were obtained by digestion with dispase. The cells (2.5 x 10s) were cultured at 37 C in 1 ml DMEM supplemented with 5% HS, 2.5% FBS, 1 % glutamine, and 1 % NEAA. After 2 days, the medium was replaced with 1 ml medium containing the diluted test material (1:10, so that 100 n\ medium were added) or a reference standard preparation of bovine follicular fluid. All samples were tested in triplicate with at least 10 PBS samples and five bovine follicular fluid samples used for cali-

Erythroid differentiation was tested using K562 cells, a human erythroleukemia cell line (ATCC CCL-243, Rockville, MD). Samples were subjected to 2-fold serial dilution in RPMI-1640 medium buffered with 25 ITIM HEPES and supplemented with 10% FBS. Dilutions were made in a 96-well plate, with final volumes of 100 fi\. Ten thousand K562 cells (in 100 n\) were then added to each well, and the cells were incubated for 3 days at 37 C before being stained with benzidine. Bovine hemin (type I, Sigma) was used as a positive control. The EDF titer (expressed as K562 units per ml) is defined as the reciprocal of the highest sample dilution in 1 ml at which

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MOL ENDO-1990 1164

differentiation of a significant percentage of the K562 cells is visible. Addition of 100 HM hemin differentiated 30% of the target cells, while spontaneous differentiation in complete RPMI-1640 medium accounted for 5%. Hemoglobin was quantitated by optical absorption at 414 nm in appropriately diluted cell lysates (48). For this purpose, the EDF assay was scaled up and performed with 8 x 105 K562 cells/test sample. Readings were taken at 403 and 425 nm to correct for nonspecific absorption due to light scattering.

Vol 4 No. 8

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Acknowledgments We thank Roos Ruysschaert for contributing to the cloning of activin cDNA; Andre Van De Voorde, Fons Bosman, Geert Maertens, Bernard Delaey, and Bettadapura Jayaram for reading the manuscript; Lucie De Moor for assistance during initial EDF assays; Luisa deMagistris for work with the vaccinia virus; Tony Briers for providing granulosa cells; Manco Zeynep and Andree Villers for performing the FSH and LH release assays; Dirk Pollet for help with computation of the gel scanning results; and Fred Shapiro for careful editing.

Received February 8,1990. Revision received May 4,1990. Accepted May 4,1990. Address requests for reprints to: Danny Huylebroeck, Innogenetics S.A., Industriepark Zwijnaarde 7, box 4, B-9710 Ghent, Belgium. This work was initiated while D.H. was holder of an EMBO Long-Term Fellowship at the EMBL.

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Processing of Recombinant Activin-A

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