Localization of the heparin binding site of follistatin

Molecular and Cellular Endocrinology, 90 (1992) l-6 0 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/92/$05.00

MOLCEL 02866

Localization of the heparin binding site of follistatin Satoshi Inouye, Nicholas Ling and Shunichi Shimasaki Department of Molecular Endocrinology, The Whittier Institute for Diabetes and Endocrinology, La Jolla, CA 92037, USA

(Received 12 June 1992; accepted 8 August 19921

Key words: Recombinant

human follistatin; Activin-binding protein; Staphylococcus aureus V8 digestion

Summary To define the heparin-binding site of follistatin, the reduced and S-carboxymethylated recombinant human follistatin containing 288 amino acids was digested by Staphylococcus aureus V8. The digestid product was subjected to sulfate cellufine column chromatography and the adsorbed peptide fragments eluted with a stepwise gradient of sodium chloride. The recovered column fractions were further purified by reversed-phase high-performance liquid chromatography (HPLC) and the HPLC peaks subjected to amino-terminal sequence analysis. All of the sulfate cellufine-retarded peptide fragments gave the same N-terminal amino acid sequence, which started at residue-68 of human follistatin, suggested that those fragments starting from residue-68 contain the heparin binding site. The multiple fragments might represent the oxidized, non-glycosylated or glycosylated forms of follistatin(68-113) resulting from the V8 digestion. A synthetic peptide corresponding to the region having the amino acid sequence 72-86 of follistatin was able to bind both heparin and sulfate cellufine, as well as compete with recombinant follistatin for binding to heparin. These findings further define the location of the heparin and heparan sulfate-binding site of follistatin at the basic amino acid-rich region comprising the amino acid sequence Lys75-Lys-CLs-Arg-Met-Asn-Lys-Lys-Asn-Lys-Pro-Arg8h.

Introduction Follistatin (FS), a gonadal polypeptide, was discovered in 1987 by two independent research groups as an inhibitor of follicle-stimulating hormone (FSH) secretion from the pituitary (Robertson et al., 1987; Ueno et al., 1987). Later, FS was shown to be a binding protein for activin (Nakamura et al., 1990), which was originally isolated in 1986 from porcine ovarian follicular fluid as a stimulator of FSH secretion (Ling et al., 1986; Vale et al., 1986). Besides its FSH releasing activity, activin was later found to have a wide spectrum of actions, including promotion of erythroleukemia cell differentiation (Eta et al., 19871, modulation of gonadal androgen biosynthesis (Hsueh et al., 1987), attenuation of growth hormone secretion (Bilezikjian et al., 1990), induction of mesoderm formation (Smith et al., 19901, maintenance of nerve cell

Correspondence to: Shunichi Shimasaki, Ph.D., Department of Molecular Endocrinology, The Whittier Institute, 9894 Genesee Avenue, La Jolla, CA 92037, USA. Tel. (619) 450-1280; Fax (6191 535-9473.

survival (Schubert et al., 1990) and control of oxytocin secretion from neurosecretory neurons (Sawchenko et al., 1988). Because of its ability to bind activin, FS was able to neutralize activin’s FSH stimulatory activity on pituitary cells Kogawa et al., 1991), attenuate the activin-induced mesoderm formation in Xenopus embryos (Asashima et al., 1991) and inhibit the mitogenic activity of activin on osteoblastic cells (Hashimoto et al., 1992). Therefore, FS may function as a modulator of activin’s action in various tissues and organs by virtue of its binding to activin. FS has also been shown to bind another gonadal polypeptide, inhibin, which is also an inhibitor of FSH secretion (Shimonaka et al., 1991). Because inhibin and activin share an identical P-subunit, it has been postulated that FS binds to both activin and inhibin through the common p-subunit (Shimonaka et al., 19911, but the ability of FS to neutralize inhibin’s activity has not been demonstrated. Besides binding activin and inhibin, FS associates with heparin as was described in its original isolation, in which a heparin-Sepharose affinity column was utilized to selectively adsorb FS from porcine follicular fluid (Ueno et al., 1987). Nakamura and co-workers

also reported that FS associated with the heparin sulfate moieties of proteoglycans on granulosa cells (Nakamura et al., 1991). In order to locate the region of the FS molecule responsible for binding to heparin, we have performed Staphylococcus aureus V8 digestion of recombinant FS with 288 amino acids and isolated the fragment that associated with heparan sulfate. Amino acid sequence analysis of the fragments showed that they correspond to the theoretical FS(68-113) fragment derived from V8 digestion. A synthetic peptide [Cys-SCHy]FS(72-86)NH,, incorporating the basic amino acid-rich region of the FS(68-113) sequence, possessed the ability to bind heparin and to displace bound FS from a heparin column, indicating that the heparin-binding site of FS is located in this region. Materials

and methods

Materials The sequencing grade endoproteinase from Staphylococcus aureus V8 was purchased from BoehringerMannheim Biochemica (Indianapolis, IN, USA). AffiGel heparin gel was obtained from Bio-Rad (Richmond, CA, USA) and sulfate cellufine gel was from Amicon (Danvers, MA, USA). Dithiothreitol (DTT) was purchased from Calbiochem (La Jolla, CA, USA) and iodoacetamide and urea were from Sigma (St. Louis, MO, USA). Ninhydrin was obtained from Pierce (Rockford, IL, USA) and silica gel 60 thin-layer chromatography (TLC) plates without fluorescent indicator, 0.25 mm layer thickness, were from Merck (Thomas Scientific, Swedesboro, NJ, USA). Recombinant human FS-288 and -315 were produced by expression in CHO cells as described (Inouye et al., 1991). ;The affinity purified rhFSs were further fractionated by reversed-phase HPLC on a 1 X 25 cm, 5 pm particle size, C, column (Vydac, Hesperia, CA, USA) using a linear gradient of 18-27% acetonitrile in 0.1% trifluoroacetic acid in 90 min at a flow rate of 3 ml/min before employed for this study. Enzyme digestion of rhFS-288 and separation of the digest on a sulfate cellufine column Recombination hFS-288 (6 nmol) purified from HPLC was dried down in a Speed-Vat concentrator (Savant, Hicksville, NY, USA). The protein was reduced with DTT (1 pmol) in 50 ~1 deoxygenated denaturing buffer (0.1 M Tris-HCl, pH 8.0, 10 mM EDTA, 6 M guanidine-HCl) at 37°C for 1 h and then S-carboxymethylated with iodoacetamide (5 pmol) at 37°C for 1 h. The S-carboxymethylated FS was concentrated with reversed-phase HPLC using a 0.46 X 25 cm, 5 pm particle size, C, column (Vydac) in the 0.1% trifluoroacetic acid/acetonitrile solvent system and the recovered protein fraction was brought to dryness in a Speed-Vat concentrator. Staphylococcus aureus V8 di-

gestion of the carboxymethylated FS was performed as follows. The dried protein dissolved in 10 ~1 8 M ureas was added to 90 ~1 50 mM sodium phosphate buffer (PB>, pH 7.8, and incubated with 2 pg enzyme (enzyme : substrate ratio = 1 : 75, w/w> at 25°C for 16 h. The digest was then diluted with 20 times volume PB and applied onto a 0.7 x 2.6 cm sulfate cellufine column (Vbed = 1 ml). A stepwise gradient elution was carried out by increasing the concentration of NaCl from 0 to 0.15, 0.5 and 2 M in 3 ml PB per step, and finally with 2 M guanidine-HCl. A 20% volume from each eluate fraction was analyzed by reversed-phase HPLC on a 0.46 X 25 cm, 5 pm particle size, C4 column (Vydac) with a linear gradient of O-80% acetonitrile in 0.1% trifluoroacetic acid in 80 min at a flow rate of 0.5 ml/min. The effluent from the HPLC column was monitored by ultraviolet WV> absorbance at 210 nm. Peptide sequencing Amino acid sequence analysis of the enzyme digested fragments recovered from HPLC purification was carried out using an Applied Biosystems (Foster City, CA, USA) model 470A gas-phase protein sequenator connected to an on-line model 120A phenylthiohydantoin amino acid analyzer (Applied Biosystems) (Esch, 1984). Peptide synthesis The peptide fragment corresponding to the amino acid sequence 72-86 of human FS, [Cys-SCHi’lhFS(72-86)NH 2, was synthesized by solid-phase methodology and purified by gel filtration, ion-exchange and partition chromatography procedures as described (Ling et al., 1984). Affi-Gel heparin gel and sulfate cellufine affinity chroma tography A mixture composed of 100 pg each of HPLC purified rhFS-288 and -315 was dissolved in 50 ~1 0.05 N acetic acid, diluted with 600 ~1 50 mM phosphate buffer, pH 7.2, and applied to a 1.2 X 2.7 cm Affi-Gel heparin gel column (Abed = 3 ml>. Elution of the adsorbed FS proteins was performed by a linear gradient from 0 to 3 M NaCl in 50 mM phosphate buffer, pH 7.2 at a flow-rate of 0.3 ml/min. The eluate fractions were dialyzed against water, concentrated in a SpeedVat concentrator, and then analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDSPAGE). To determine the binding ability of the synthetic [Cys-SCH:‘]hFS(72-86)NH2 fragment to an Affi-Gel heparin gel column and a sulfate cellufine column, 10 pug of peptide dissolved in 100 /*l 50 mM phosphate buffer, pH 7.2, was applied to a 0.7 X 2.6 cm column (Shed = 1 ml) packed with either one of the two gels

and then eluted stepwise with 3 ml of phosphate buffer containing increasing concentrations of NaCl at 0, 0.15, 0.5, 1.0, 2.0 M and finally with 2 M guanidine-HCl. The eluted peptide fractions were detected by ninhydrin spray (1.5 g ninhydrin, 50 ml acetic acid, 950 ml lbutanol), after spotting 10 ~1 of the eluate fractions on a TLC plate and heating the ninhydrin sprayed plate at 80°C for 3 min. The minimal amount of peptide detectable by the ninhydrin spray is 10 ng. To determine whether the synthetic peptide could displace the adsorbed FS from a sulfate cellufine column, a mixture containing 100 pg each of rhFS-288 and -315 dissolved in 100 ~1 50 mM phosphate buffer, pH 7.2, was applied to a 0.7 X 2.6 cm Affi-Gel heparin gel column (l/bed = 1 ml). Elution of the adsorbed proteins was performed first with 3 ml phosphate buffer and then with 3 ml 0.1 M synthetic peptide in phosphate buffer. The eluate fractions were subjected to SDS-PAGE followed by Western blotting with a specific antibody against FS (Inouye et al., 1991). Sodium dodecylsulfate /polyacrylamide sis

gel electrophore-

SDS/PAGE was performed according to the method described by Laemmli (1970) under non-reducing conditions using a 0.1 x 6 x 8 cm 12% polyacrylamide fractionating gel (Novex, Encinitas, CA, USA) and the electrophoresis was carried out at 25 mA for 2 h. The gel was either stained with Coomassie Brilliant Blue R-250 (Serva, Westbury, NY, USA) or blotted onto a nitrocellulose paper for Western blotting. Western blotting

After SDS/PAGE, the proteins were electroblotted onto a nitrocellulose filter (Bio-Rad) with the Mini-cell apparatus (Novex) at 150 mA for 2 h (Towbin et al., 1979). The filter was incubated with TBST-casein buffer (50 mM Tris-HCI, pH 7.4, 150 mM NaCl, 0.05% Tween 20 (Bio-Rad), 1% casein (Sigma)) for 1 h at room temperature, followed by incubation with an antiserum against FS diluted 1: 500 in TBST-casein buffer at 4°C for 16 h. The filter was then washed with TBST and incubated for 2 h at room temperature in a 1: 5000 dilution of alkaline phosphatase conjugated goat antirabbit IgG (Calbiochem, San Diego, CA, USA). The specifically bound antibody was visualized by treatment with 0.03% nitroblue tetrazolium chloride (Sigma) and 0.015% 5-bromo4-chloro3-indolyl phosphate p-toluidine salt (Sigma) in 100 mM Tris-HCl, pH 9.5, containing 100 mM NaCl and 5 mM MgCl,. Results

To ascertain the relative binding affinity of rhFS-288 and rhFS-315 to heparin, a mixture composed of an equal amount of the two recombinant protein was

0.03

t 0.02 $ 8 0.01

3

55. 23 12

0.000

10

50 20 30 40 Fraction (1.6 ml/tube)

so0

Fig. 1. Affi-Gel heparin gel chromatography of rhFS-288 and -315. A mixture composed of 100 pg each of the two recombinant FS was applied to the column and then eluted with a linear gradient from 0 to 3 M NaCl in 50 mM phosphate buffer, pH 7.2. The eluate fractions were analyzed by SDS/PAGE followed by Coomassie staining of the gel (photo insert). Recombinant hFS-315 showed two bands at 38 and 35 kDa (a), whereas rhFS-288 showed two bands at 35 and 31 kDa (b). Lane M corresponded to the migration positions of the molecular mass markers in increasing mass size: trypsin inhibitor (21.5 kDa), carbonic anhydrase (31.0 kDa), ovalbumin (45 kDa), albumin (66.2 kDa) and phosphorylase B (97.4 kDa).

loaded onto an Affi-Gel heparin gel column and the adsorbed FS eluted with a linear gradient of NaCl in phosphate buffer. As shown in Fig. 1, both proteins were co-eluted from the column at a NaCl concentration between 0.8 and 1.0 M. However, SDS/PAGE analysis of each eluate fraction, followed by Coomassie staining of the gel revealed that the major component of fractions 15-19 was rhFS-315 with two protein bands at 38 and 35 kDa (Fig. 1 insert a) and the major component of fractions 22-23 was rhFS-288 with two protein bands at 35 and 31 kDa (Fig. 1 insert b), while fractions 20-21 contained both proteins in about equal amounts with three bands at 38, 35 and 31 kDa. A similar elution profile was obtained from a sulfate cellufine column but a concentration of 1.2-1.4 M NaCl was required to displace the proteins from the column (data not shown). These results suggest that the relative affinity of rhFS-288 to heparin or sulfate cellufine is slightly higher than that of rhFS-315. For subsequent studies, sulfate cellufine rather than heparin gel was employed, because the binding of FS to the former is stronger than the latter. To determine whether the binding of FS to heparin was dependent on the three-dimensional conformation of the intact protein, rhFS-288 was reduced and Scarboxymethylated and then subjected to sulfate cellufine affinity chromatography under the same conditions as above. The elution profile obtained was similar to the results shown in Fig. 1, suggesting that the intact three-dimensional structure of FS is not required for binding to heparin.

TABLE

I(a)

AMINO ACID SEQUENCE ANALYSIS OF REVERSED-PHASE RECOVERED FROM A SULFATE CELLUFINE COLUMN HPLC fraction

*

Amino

(A) 0.15 M NaCl 12 20 (B) 0.5 M NaCl 16 21 23 * Fraction

numbers

HPLC

V&DIGESTED

acid sequence

VQYQG

RCKKT

GNCWL

RQAKN

Sequence

CRD

location

FRAGMENTS

(FIG.

2)

in FS

I-

NVDCG

PGKKC

RMNKK

NVDCG

PGKKC

RMNKK

NVDCG

PGKKC

RMNKK

6XNKPRC

VCAPD

CSN

6& 6%

were same as in Fig. 2A and 2B.

correspond to a peptide starting at the N-terminal (fraction 20) and another at residue-129 of FS (fraction 12). Since intact or linearized FS required a concentration of > 1 M NaCl to be eluted off the sulfate cellufine column, fragments that could be displaced from the column by I 0.15 M NaCl probably represent those that do not have a high affinity for sulfate cellufine. Elution by 0.5 M NaCl yielded three peptides, all of which have the same N-terminal starting at residue-68 of FS (fractions 16, 21, 23). Since no other peptides were recovered from the column at the higher concentration of NaCl or 2 M guanidine-HCl, the FS(68-113) fragment most likely comprises the region responsible for binding to heparin. Location of the heparin binding site in this region is buttressed by the stretch of basic amino acids presents in residues 75-86, because heparin and sulfate cellufine gels carry numerous negatively charged groups which would interact with the stretch of positively charged amino acids. To confirm that the basic amino acid-rich region is the heparin binding site, a synthetic peptide fragment

l(b)

THEORETICAL

rhFS-288

12%

Since the linearized FS could still bind to heparin, it was of interest to locate the region in the molecule responsible for this binding. To accomplish this purpose, the reduced and S-carboxylated rhFS-288 was digested with Staphylococcus aureus V8 and the digest applied to a sulfate cellufine column. After washing off the unbound fragments with phosphate buffer, the bound peptides were eluted with a stepwise gradient of 0.15, 0.5, 2.0 M NaCl and finally 2 M guanidine-HCl in phosphate buffer. The eluate fractions were analyzed by reversed-phase HPLC. As shown in Fig. 2, two peptide peaks were eluted off the column by 0.15 M NaCl (Fig. 2A), whereas three peptide peaks were recovered by elution with 0.5 M NaCl (Fig. 2B). No more peptide peaks were recovered from the sulfate cellufine column by the 2 M NaCl or 2 M guanidineHCl elution. Each of the recovered HPLC peaks was subjected to N-terminal amino acid sequence analysis and the results are presented in Table 1, together with the theoretical fragments derived from V8 digestion of rhFS-288. The FS fragments eluted by 0.15 M NaCl TABLE

PURIFIED

FRAGMENTS

DERIVED

FROM

V8 DIGESTION

Fragments

Amino

I- 20 21- 24 26- 38 40- 63 65- 67 68-l 13 114-126 127-128 129-169 170-175 176-207 208-217 218-243 244-252 253-265 266-270 271-280 281-288

GNCWLRQAKN

OF rhFS-288

acid sequence GRCQVLYKTE

LSKE CCSTGRLSTS

WTE

DVNDNTLFKW

MIFNGGAPNC

IPCKE

TCE NVDCGPKKC CALLKARCKE

RMNKKNKPRC

VCAPDCSNIT

WKGPVCGLDG

KTYRNE

QPE

LE VQYQGRCKKT

CRDVFCPGSS

TCVVDQTNNA

YCVTCNRICP

YSSACHLRKA

TCLLGRSIGL

AYE

LWDFKVGRGR

CSLCDE

PASSE QYLCGNDGVT GKCIKAKSCE DIQCTGGKKC LCPDSKSDE PVCASDNATY CAMKE ACSSGVLLE VKHSGSCN

ASE

E

5

A

B

-0.01 0'

I

‘\

O0 30 -firTIe(inin) Fig. 2. Reversed-phase HPLC analysis of the Staphylococcus aureus V8 digested rhFS-288 fragments recovered from a sulfate cellufine column. Panel A corresponds to the fraction recovered by 0.15 M NaCl elution of the column and panel B corresponds to the fractions recovered by 0.5 M NaCl elution of the column. 60

comprising residues 72-86 of FS was prepared with the free sulfhydryl group of the cysteine protected by a methyl group. As shown in Fig. 3, synthetic [CysSCHi7]FS(72-86)NH, binds to both sulfate cellufine column (row A> and Affi-Gel heparin gel column (row B) because the adsorbed peptide was eluted off the column by stepwise elution with an increasing concentration of NaCl at 1 M and 0.5 M, respectively, and detected with ninhydrin spray on a TLC plate. Moreover, the synthetic peptide was able to compete with rhFS-288 and -315 for binding to a sulfate cellufine column. As shown in Fig. 4, a mixture of rhFS-288 and -315 pre-adsorbed to a sulfate cellufine column was not washed off the column by phosphate buffer (lane A) but was eluted off the column by washing with a 0.1 M

Fig. 3. Ninhydrin spray detection on a TLC plate of the synthetic [Cys-SCHT]FS(72-86)NHz fragment eluted off a sulfate cellufine column (row A) and an Affi-Gel heparin gel column (row B) by a stepwise gradient of NaC1.

Fig. 4. Western blotting of the phosphate buffer wash (lane A) and 0.1 M synthetic peptide wash (lane B) of a rhFS-288 and -315 mixture pre-adsorbed to a sulfate cellufine column. The numbers on the left represent the migration positions of the prestained molecular mass markers (Bio-Rad): phosphorylase B (106 kDa), bovine serum albumin (80.0 kDa), ovalbumin (49.5 kDa), carbonic anhydrase (32.5 kDa), soybean trypsin inhibitor (27.5 kDa), and lysozyme (18.5 kDa).

solution of the synthetic peptide and detected by Western blotting with a FS specific antiserum (lane B). Discussion The present study demonstrated that the heparin binding domain of FS resides in the FS(68-113) fragment obtained by Staphylococcus aureus V8 digestion of the reduced and S-carboxymethylated rhFS-288. The heparin binding site in this fragment was further narrowed down to the stretch of basic amino acid-rich region of FS(72-861, because a synthetic replicate incorporating this sequence was able to bind heparin as well as to displace pre-adsorbed rhFS-288 and -315 from a sulfate cellufine column. That this basic amino acid-rich region of FS can bind heparin is in agreement with the heparin binding sites located in other proteins that associate with heparin or proteoglycans (Schwarzbauer et al., 1983). A helix wheel plot of the FS(72-86) fragment presented in Fig. 5 shows that the basic amino acid residues Lys7’, Arg78, Lys8*, and Arg86 can be sequestered on one side of the helix and close to each other to facilitate binding to the negatively charged sulfate groups on heparin. It is interesting to note that the binding of heparin to rhFS-288 is slightly stronger than that of rhFS-315, since it requires a higher concentration of NaCl to elute the former compound off the heparin column than the latter. The higher affinity of rhFS-288 in

Program Project Grant HD-09690 from the National Institutes of Health.

References

Fig. 5. A helix wheel plot of the FS(72-86) fragment showing the sequestration of basic residues at positions 75, 78, 82 and 86 clustered on one side of the helix.

comparison with rhFS-315 may be due to the difference in amino acid sequence between the two molecules, because rhFS-315 contains 27 additional amino acids at the C-terminal compared to rhFS-288. Within the 27 amino acid sequence, there is a stretch of acidic residues at positions 295-302. This stretch of acidic residues may fold back on the first 288 residues of FS and partially neutralizes the positive charges on the basic amino acid-rich regions of FS(72-86) and thus weakens its binding to heparin. The ability of FS to inhibit FSH secretion from the pituitary cells may be potentiated by the ability of FS to associate with heparin. Heparan sulfate proteoglycans are known to be present on the surface of a wide variety of cell types (Burgess and Maciag, 1989), including anterior pituitary cells. Since it has been demonstrated that the FS protein (Nakamura et al., 1990) as well as the mRNA of activin-B (Meunier et al., 1988) are present in pituitary, FS secreted from the pituitary cells could be trapped by the proteoglycan on the nearby cells or the extracellular matrix and lie in wait to capture the endogenously released activin to prevent its stimulation of FSH release. Thus FS and activin may act in an autocrine and/or paracrine mechanism to control the autonomous secretion of FSH from the pituitary. This hypothesis can be tested by preparing an FS mutant that does not bind heparin but still complexes with activin. Acknowledgements

We thank R. Schroeder, H.-P. Zhang and M. Regno-Lagman for their technical assistance and E. Exum for preparing the manuscript. This work was supported by a grant from the Andrew W. Mellon Foundation, NICHD contract NOl-HD-0-2902, and

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