Molecular and Cellular Endocrinology, 8.5 (1992) 41-52 0 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/92/$05.00

41

MOLCEL 02740

Identification of assembled epitopes on the a-subunit of human follicle stimulating hormone

Russell S. Weiner a,b and James A. Dias a,b,c * Department of Biochemistry and Molecular Biology, Albany Medical College, Albany, NY 12208, USA, ’ Wadsworth Center

for Laboratories and Research, New York State Department of Health, Albany, NY 12201-0509, USA, and ’ Department of Biomedical Sciences, School of Public Health, University at Albany, State Unioersity of New York, Albany, NY 12201, USA

(Received 1 October 1991; accepted 2 January 1992)

Key rrzords: Follitropin; Alpha subunit; Epitope; Peptide; (Human)

Summary

In order to characterize further the antigenic structure of human follitropin (hFSH), BALB/c mice were immunized with hFSH and anti-hFSH monoclonal antibodies (mAbs) were generated. The hFSH subunit specificity of the mAbs was assessed by a solid-phase enzyme-linked immunosorbent assay (ELISA) and a solution-phase radioimmunoassay (RIA), each using hFSH, hFSHa, and hFSHP. Five mAbs bound hFSH and hFSHa in the ELISA and the RIA. In addition, some mAbs recognized hFSH/3, albeit to a much lower degree, as demonstrated by displacement of [‘251]hFSH binding to the mAbs by hFSHP, in the solution-phase RIAs. Next, synthetic peptides corresponding to the hFSH a-subunit sequence were used to identify sequences specific to the epitopes of each of the five mAbs. Using this epitope mapping strategy, two assembled epitopes were identified. mAbs 3A and 4B distinguish one discontinuous epitope comprised minimally of sequences a-16-21 and (u-66-92, whereas mAbs 5F and 2E distinguish a second discontinuous epitope comprised minimally of sequences (y-40-50 and a-66-72.

Introduction

Normal human fertility requires the coordi- nated interaction between gonadotropins. These include human follicle stimulating hormone (hFSH) and human luteinizing hormone (hLH) secreted from the anterior pituitary. hFSH in

Correspondence to: James A. Dias, Ph.D., Wadsworth Center for Laboratories and Research, New York State De-

partment of Health, Empire State Plaza, P.O. Box 509, Al-

bany, NY 12201-0509, USA. Tel. (518) 474-7592.

Supported by Grants HD 18407 and GM 4344301, NSF Grant DIR 891457 and 5K04-HD 00682 and a gift of human

pituitaries from NHPP.

particular acts on granulosa cells and is required for the stimulation of preovulatory ovarian estro- gen production and the initiation of follicle matu- ration in the female. In the male, hFSH acts on the Sertoli cells of the testis and is required for normal spermatogenesis (Labrie, 1990).

These two gonadotropins and human thyroid stimulating hormone (hTSH), also secreted from the anterior pituitary as well as human chorionic gonadotropin (hCG) secreted from the syncy- tiotrophoblastic cells of the placenta, constitute the family of structurally related glycoprotein hormones. The members of this family share a common o-subunit which, in non-covalent associ- ation with a unique hormone-specific P-subunit,

42

constitutes the physiologicahy active heterodimer (Pierce and Parsons, 1981: Baenziger and Green, 1988).

To date, there are no available crystallographic data on any of the glycoprotein hormones even though there have been reports of the crystalliza- tion of bovine TSHcu (McPherson et al., 1986), bovine LHa (McPherson et al., 19861, deglycosy- lated hCG (Harris et al, 1989; Lustbader et al., 19891, and desialated hCG (Lustbader et al., 1989). Identification of important structural fea- tures has thus relied on a variety of biochemical (Pierce and Parsons, 1981) and immunological investigations (Dias, 1992).

In this regard, antibodies have for many years been used to probe the surface topology of the glycoprotein hormones. The results of these stud- ies added to our knowledge of the spatial ar- rangement of epitopes but did not identify spe- cific amino acid residues involved in each epitope (Hojo and Ryan, 1985; Schwarz et al., 1986; Alonso-Whipple et al., 1988; Berger et al., 1988; Charlesworth et al., 1991a). In addition, immuno- chemical studies have been reported, the results of which impIicated the involvement of the Ly-sub- unit in receptor binding (Moyle et al., 1982; Dighe and Moudgal, 1983; Milius et al., 1983).

We have reported previously on seven mono- specific anti-hFSHa peptide antisera produced by immunizing rabbits with hFSHcu peptides cou- pled to hemocyanin (Weiner et al., 1990). These antisera were used to identify which sequences of hFSHa were on the surface of the hFSH het- erodimer and which sequences were involved in subunit contact. In addition, we have described studies in which synthetic ty- and p-subunit pep- tides were used to inhibit subunit recombination in an attempt to identify the subunit contact sites of hLHa! (Krystek et al., 1991) and hFSHP (Vakharia et al., 19911, respectively.

To investigate how hFSH interacts with its receptor we have employed immunochemical techniques. Using polyclonal antisera to hFSHa and hFSHP we have demonstrated that both subunits have epitopes that are masked following receptor binding (Dias et al., 1984). We recently described two studies in which we determined the epitope specificity of anti-hFSH mAbs that were potent inhibitors of receptor binding in an at-

tempt to identify hFSH receptor binding se- quences. The studies concluded that residues within sequence ~~61-92 (Weiner et al, 1991) and residues within sequence /333-85 (Vakharia et al., 19901 were potentially involved in receptor binding. We hypothesized that monoclonal anti- bodies against hFSH would recognize discontinu- ous sequences in the context of an assembled epitope and through epitope mapping, neighbor- ing sequences could be identified.

In the present investigation, we describe re- sults that extend our previous epitope mapping reports to include the identification of two assem- bled epitopes comprised of a-subunit sequences. This was accomplished by epitope mapping a panel of five anti-hFSH mAbs which bind to hFSHa using synthetic cr-peptides in a solid- phase enzyme-linked immunosorbent assay (ELISAX

Materials and methods

Hormones Human pituitaries and hFSH preparation

10065 (80 IU/mg) were from the National Hor- mone and Pituitary Distribution Program (Bal- timore, MD, USA). Otherwise, hFSH, hFSHa, and hFSH@ were purified from frozen human pituitaries as previously described (Roes and Gemzell, 19641, omitting zone eiectrophoresis and finishing with immunoaffinity purification. Po- tency was verified as described previously (Weiner et al., 19901. We determined that hFSHP was not contaminated with hFSHa by ELISA, Western blot analysis and radioimmunoassay (RIA) (data not shown). In the ELISA, there was no de- tectable binding to our preparation of hFSHP by our alpha specific monoclonal antibodies CmAbs), alpha specific anti-hFSHcY-peptide antibodies, or by NIH anti-hFSHa: (batch 11 antisera. Using Western blot analysis, we eIectrophoresed 1 pg of hFSHcu and 10 fig of hFSH)3 in a 12% poly- acrylamide gel. The proteins in the gel were transferred to nitrocellulose, blocked with 1% gelatin, and incubated with rabbit anti-hFSH- all-27 antipeptide antisera (Weiner et al., 1990). To detect the binding of the antipeptide antisera we then incubated with [“‘IlProtein-G. Follow- ing autoradiography, the 1 pg of hFSHa had an

intense band; however, no band was visible for the 10 pg of hFSH/3. Therefore, if contamination did exist it would have to be less than 1%. In the RIA, using hFSHj3 AFP-4911B as a reference preparation which has a reported hFSHa con- tamination of 3.9% and NIH anti-hFSHa! (batch 1) antisera, a 0.2% hFSHa contamination of our hFSH@ preparation was calculated. Additionally, using NIH anti-hFSH@ (batch 4) and hFSH@ AFP-4911B as a reference preparation, a 0.8% hFSH@ ‘contamination’ of our hFSHcu prepara- tion was detected which we believe, in light of the other data, to be a result of the properties of the antisera.

hFSHcu and hFSH/3 peptide synthesis Table 1 illustrates the seven overlapping pep-

tides derived from the primary structure of the a-subunit of hFSH. The peptides were synthe- sized and purified as previously described (Weiner et al., 1991). The composition and purity of each peptide was confirmed by amino acid analysis using a Beckman System Gold amino acid ana- lyzer (Palo Alto, CA, USA, model 126AAA) em- ploying protocols supplied by Beckman.

Production of mAbs mAbs were prepared as previously described

(Weiner et al., 1991). The procedure used for fusion of myeloma and spleen cells was that of Claflin and Williams (1978). The myeloma cell line was re-cloned from P3X63-Ag8.653 and the spleen cells were from female BALB/c mice. The immunogen for the fusion which yielded 4B, 3A, and 5F (10.4B6, 10.3A6 and 10.5Fl.B4) and for the fusion which gave rise to 2E and 1E

43

(22.2E9.3 and 22.1E3.GlO) was intact hFSH (10065; 80 IU/mg).

Purification of mA bs mAbs were first purified from ascitic fluid by

precipitation with saturated ammonium sulfate to 50% saturation. The precipitate was recovered by centrifugation at 10,000 X g for 30 min and ex- tensively dialyzed against phosphate-buffered saline (PBS). The dialyzed mAbs were further purified by affinity chromatography on Protein-A Sepharose. Lastly, the mAbs were dialyzed against 0.01 M ammonium bicarbonate, pH 8.3, and lyophilized. The elution of mAb from the affinity column was monitored and protein was deter- mined by Bradford assay (Bradford, 1976) using a commercially available kit (Bio-Rad, Richmond, CA, USA; No. 500-00065).

Preparation and validation of monoclonal Fab fragments

The Fab fragments were prepared using a commercially available kit (Pierce, Rockford, IL, USA, No. 44885). First, 5 mg of lyophilized Pro- tein-A purified mAb were resuspended in 1 ml digestion buffer (19.8 mM cysteine-HCI in phos- phate buffer, pH 7.0). Next, 0.25 ml of immobi- lized papain were equilibrated with digestion buffer and added to the mAb solution. The diges- tion proceeded for 16 h at 37°C with vigorous shaking. The Fab fragments were then purified from the Fc portion of the mAb and undigested mAb by Protein-A affinity chromatography.

To confirm that the purified Fab preparations were not contaminated with intact mAb, dot blot assays were performed. Briefly, intact mAb or

TABLE 1

AMINO ACID SEQUENCE OF SYNTHETIC hFSHa! PEPTIDES

Peptide Sequence

l-15 A-P-D-V-Q-D-C-P-E-C-T-L-Q-E-D

11-27 T-L-Q-E-D-P-F-F-S-Q-P-G-A-P-I-L-Q

22-39 G-A-P-I-L-Q-C-M-G-C-C-F-S-R-A-Y-P-T

33-58 F-S-R-A-Y-P-T-P-L-R-S-K-K-T-N-L-V-Q-K-N-V-T-S-E~S-T

51-65 K-N-V-7-E-S-T-C-C-V-A-K-S-Y

61-78 V-A-K-S-Y-N-R-V-T-V-N-G-G-F-K-Y-E-N

73-92 G-F-K-V-E-N-H-T-A-C-H-C-S-T-C-Y-Y-H-K-S

44

Fab fragments were adsorbed onto nitrocellulose using a vacuum dot blot apparatus. Next, the blot was blocked with TBS (10 mM Tris, 150 mM NaCl, pH 8.0) containing 1% gelatin for 1 h at room temperature CRT). The blot was then washed (TBS containing 0.05% Tween 20) and incubated with [ 1251]Protein-G (250,000 cpm/ml) for 1 h at RT. Lastly, the blot was washed, air dried, and subject to autoradiography.

Reduction and alkylation of hFSHa (hFSHar/“) First, 50 pg hFSHa was resuspended in cys-

teine alkylation buffer (6 M guanidine HCl, 0.25 M Tris base, 1 mM EDTA, pH 8.2). Next, 2- mercaptoethanol was added to a final concentra- tion of 25 mM followed by iodoacetamide to a final concentration of 50 mM as previously de- scribed (Weiner et al., 1990).

Iodination of hormones Iodination of hFSH was performed as previ-

ously described (Dias et al., 1984; Weiner et al., 1991) with modifications. To 25 pug of hFSH in 25 ~1 0.01 M sodium phosphate, pH 7.0, were added 25 ~1 0.5 M sodium phosphate pH 7.0, 10 ~1 lactoperoxidase (50 unit/ml in 0.01 M sodium phosphate, pH 7.0) and 2 mCi of carrier-free Na1251 (New England Nuclear, Boston, MA, USA; No. NEZ033H). Next, 5 pl of H,O, (1: 30,000) was sequentially added at times 0, 2, 4, 6, 8, and 10 min. Twelve minutes following the first addi- tion the reaction was terminated by the addition of 100 ~1 of transfer solution (16% sucrose, 1% KI, and 0.01% bromphenol blue). Next, [‘251]hFSH was separated from unincorporated 12’1 on a Sephadex G50 column developed in PBS, while collecting 1 ml fractions into tubes containing 0.5 ml of PBS made 1% with bovine serum albumin (BSA). hFSHcu (5 pg) was iodin- ated using the same procedure with the exception that 1 mCi carrier-free Na1251 was used. The resulting specific activities were as follows: hFSH 70 pCi/pg, hFSHcu 95 pCi/pg.

ELISA for determining the hFSH subunit speci- ficity of mAbs

Briefly, hFSH, hFSHa, hFSH& and hFSHa’/” (60 pmol/ml) were diluted in 0.05 M Tris buffer, pH 9.5 and 100 PI/well were adsorbed onto a

96-well flat-bottomed Immulon I microtiter plate. Following a minimum 16 h incubation at 4°C the plate was emptied and the remaining solid phase was blocked by 200 pi/well of 1.0% BSA (im- munoglobulin G (IgG)-free) in the same Tris buffer for 2 h at RT. The wells were then washed with a PBS wash (PBS containing 0.01% NaN, and 0.025% Tween 20) and 100 pi/well of Pro- tein-A pure mAb diluted in ELISA binding buffer (PBS containing 0.25% IgG-free BSA, 0.01% NaN,, and 1 mM EDTA) were added. Following a 2 h incubation at RT, the wells were washed and bound mAb was detected by incubating with 100 ~1 of goat anti-mouse immunoglobulin-al- kaline phosphatase conjugate (Tago, Burlingame, CA, USA; No. 6543), diluted to 1: 1000 in ELISA binding buffer for 2 h at RT. The wells were washed, 100 PI/well of substrate (Bio-Rad, Rich- mond, CA, USA; No. 172-1063; p-nitrophenyl- phosphate in diethanolamine buffer) were added and absorbance (410 nm> was determined using a Dynatech microplate reader following develop- ment of substrate for 30 min to 1 h.

Determination of mAb isotype and affinity The isotypes of the mAbs were determined

using a modification of the above ELISA proce- dure. hFSH was used as the capture antigen and goat anti-mouse isotype specific antibodies were used to identify isotype (Fisher Biotech, Orange- burg, NY, USA; No. OB1290-78). The affinity of each mAb was determined using competitive binding RIAs followed by data analysis using the LIGAND (Munson and Rodbard, 1980) program as previously described (Weiner et al., 19911.

Analysis of mAb hFSH subunit specificity using a competitive binding RIA

Briefly, 100 ~1 of Protein-A purified mAb was incubated for a minimum of 16 h at 4°C with 100 ~1 [‘251]hFSH or [‘251]hFSHa (50,000 cpm) and 100 ~1 of increasing concentrations of competing hormone or subunit (hFSH, hFSHa, hFSH@) to generate the displacement curves. Next, 200 ~1 bovine gamma globulin (5 mg/ml), and 500 ~1 ice-cold PBS were added. Lastly, 1 ml 25% polyethylene glycol (PEG) (w/v) was added, the tubes were vortexed and centrifuged at 2500 X g for 45 min to separate mAb bound from free

45

[ ‘251]hormone or [ ‘251]subunit. A concentration of mAb that resulted in 30% maximal binding of the radiolabeled hormone in the absence of unla- beled hormone or subunits was used.

~pitope happing of anti-hFSH m4bs using syn- thetic hFSHa peptides

Briefly, synthetic hFSHar peptides (2 nmol/ml) were diluted in coating buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, 3 mM sodium azide, pH 9.6) and 100 $/well were adsorbed onto a 96-well flat-bottomed Immulon I microtiter plate. Following a minimum 16 h incu- bation at 4°C the wells were washed (1.5 mM potassium phosphate, 20 mM sodium phosphate, 2.7 mM potassium chloride, 0.14 M sodium chlo- ride, pH 7.4, containing 0.05% Tween 20 (v/v) and the remaining solid phase was blocked by 200 PI/well of 5% Carnation non-fat dry milk (w/v) in the same coating buffer for 1 h at RT. The wells were washed and 100 ~l/well of mAb (10 fig/ml), or 100 ~l/well of mAb (10 pg,/ml) preincubated with hFSH (10 kg/ml), diluted in ELISA binding buffer, were added. Following a 16 h incubation at 4°C the wells were washed and bound mAb was detected as previously described.

FSH radioligand receptor assay (RRA) The receptor assay was performed as previ-

ously described (Weiner et aI., 1991). Briefly, 100

~1 [ ‘*‘I]hFSH (100,000 cpm; 30-35% bindable in the presence of excess receptor) was preincu- bated with increments (0.1-10 pg/lOO ,uI> of each Protein-A purified mAb or Fab fragment for 16 h at 4°C. Next, 200 ~1 of a 1: 10 dilution (w/v) of 30,000 x g bovine testes membranes was added, followed by a 16 h incubation at RT with shaking. Each tube contained 2.0 mg of bovine gamma globulin to obviate the possibility of non-specific association of mAbs with the membranes. Non- specific binding (3469 cpm) was determined in the presence of 2 pg of pure hFSH and total binding was determined in the absence of mAb (24,519 cpm), The assay was terminated by the addition of 1 ml of ice-cold 0.05 M Tris buffer, pH 7.5, followed by centrifugation at 2500 X g for 1 h to separate membrane-bound from free [‘251]hFSH.

The mAbs ( f hFSH) were incubated with each peptide in at least two experiments. Each reac- tion was in triplicate. The absorbance from the interaction of mAb and peptide (minus hFSH) was compared with the absorbance resulting from the interaction of the mAb (preincubated with hFSH) and peptide using the Student’s t-test (Glantz, 1981). Significance was obtained at P < 0.001.

1.0 c-\ m hFSH [7 hFSHa

48 5F 2E 1E

Monoclonot Antibody

Fig. 1. mAb specificity as determined by ELBA. Illustrated is a representative ELISA in which equimolar amounts (6 pmol) of hFSH, hFSHcu, hFSH@ were adsorbed onto an lmmulon I microtiter plate. Each Protein-A purified mAb was incubated at a final concentration of 10 pg/ml as described in Materials and methods. Non-specific binding values were determined by substituting mAb 4E for anti-hFSH mAbs. The values are expressed as the mean i SE following the subtraction of non-specific binding values.

46

Results

Determination of hFSH subunit specificity by a solid-phase ELISA

First, it was determined whether the mAbs could bind hFSH, hFSHa, hFSH/?, and hFSHa’/” adsorbed onto the solid-phase. As il- lustrated in Fig. 1, using equimolar concentra- tions of hormones, all five mAbs bound to hFSH and hFSHa. The binding to hFSH was approxi- mately 30-50% greater than to hFSHa. No bind- ing to hFSHP or hFSHa’/” (data not shown) was demonstrated for any of the five mAbs. The positive controls for hFSHp and hFSHa’/” were mAb 3G and anti-hFSHall-27 antipeptide anti- serum, respectively (Weiner et al., 1990). The positive controls performed as expected with 3G having a specific absorbance of 0.859 with hFSHP and anti-hFSHa!ll-27 having a specific ab- sorbance of 1.179 with hFSHa’/“, confirming that hFSHP and hFSHa’/” were adsorbed to the ELISA plate. Negative control mAb 4E (10.4E 4.5E38, anti-LHP) did not bind to any of the solid-phase hormones (data not shown).

Determination of hFSH subunit specificity by a solution-phase competitilre RIA

The solution-phase RIA was performed in or- der to take advantage of the higher sensitivity afforded by using a radioactive detection system. As illustrated in Fig. 2, hFSH displaces [‘251]hFSH from binding to all five mAbs. In contrast, hFSHa only displaced [ “‘I]hFSH from binding to mAbs 5E, 2E, and lE, at a higher dose than hFSH. Furthermore, hFSHP displaced [‘2”I]hFSH bind- ing to mAbs 3A, 4B, 5F, and lE, albeit at a much higher level than hFSH. This is in contrast to the results obtained with the ELISA; however, if the radioactive tracer is changed from [‘251]hFSH to [‘2”I]hFSHa then mAb 3A and 4B do recognize solution-phase hFSHa in agreement with the ELISA results, as illustrated in Fig. 3. There was no displacement of [‘251]hFSHa by hFSHP.

Analysis of mAb affinity and isotype The affinity constants of the mAbs were deter-

mined by analyzing binding isotherm data gener- ated by competitive binding RIAs with the pro- gram LIGAND (Munson and Rodbard, 1980).

100

60

60

40

20

0

100

60

60

40

20

0

0 100 0 - 60

X 60

0 40

20

0

100

00

60

40

20

0

0 hFSH V hFSHa

48 -

W--W--~----.

5F

“.f;yy%,,, 0.0001 0.001 0.01 0.1

nmol Hormone Added

Fig. 2. Competitive binding of [‘2s1]hFSH to Protein-A puri-

fied mAb. mAbs were coincubated with [“‘I]hFSH (50,000

cpm) and competing hormone (hFSH, hFSHcz, hFSH@) for

16 h at 4°C. Non-specific binding values were determined by

substituting mAb 4E for anti-hFSH mAbs; B, values were

determined in the absence of competing hormone or subunit.

The values are expressed as the meank:SE (non-specific

binding values subtracted).

The data were fit using both a one- and two-site model. As expected for a mAb, the data best fit the one-site model. K, values ranged from 0.10 to 1.70 X 10’ M-’ for heterodimer. The results of the data analysis and the isotype of the mAb are presented in Table 2.

47

t 0 hFSH V hFSHa I

60 -

0 40 -

0 - “-3A

X 0.’ b cllllll’

2o 40

0’ / .(.I,.,’ 0.001 0.01 0.1 1

nmol Hormone Added

Fig. 3. Competitive binding of [i2SI]hFSHa to Protein-A purified mAb 3A and 4B. mAbs were coincubated with [‘*“I]hFSHa (50,000 cpm) and competing hormone (hFSH, hFSHar, hFSHP) for 16 h at 4°C. Non-specific binding values were determined by substituting mAb 4E for anti-hFSH mAbs; B, values were determined in the absence of competing hormone or subunit. The values are expressed as the mean of-

SE (non-specific binding values subtracted).

Epiiope happing of mAbs using a ~nth~ti~ p~p~ide ELISA

Since all of the mAb recognize hFSHru, the peptide sequences involved in their epitopes were determined using synthetic hFSH a-subunit pep- tides in a peptide ELISA. The total binding of the mAbs was determined by incubating each mAb with each synthetic peptide. Non-specific binding was determined by preincubating each mAb with hFSH prior to the incubation with

TABLE 2

ISOTYPE AND AFFINITY CONSTANTS OF ANTI-hFSH MONOCLONAL ANTIBODIES

The affinities of the antibodies were determined by displacing [“‘I]hFSH from the mAbs with increasing levels of hFSH. The isotypes were determined by ELISA using isotype specific antibodies as described in Materials and methods.

Monoclonal antibody

Isotype K, (M--l x 109)

3A IgGl, K 0.37 kO.06 4B IgGl, K 0.33 f 0.03 SF IgGl, ic 1.7OkO.28 2E IgGl, K 0.10+0.01 1E IgGl, K 0.22 i 0.04

solid-phase peptides. hFSH binds to the mAb and in so doing should block the mAb from binding to the peptides. If the interaction be- tween mAb and peptide is specific, a decreased binding shoufd be observed with the hFSH prein- cubated mAb. Although no method of determin- ing non-specific binding is perfect, we chose to use the hFSH preincubation as the control for non-specific binding for several reasons. First, using wells without peptide only determines non- specific binding which would result between the surface of the ELISA plate and the mAb, which if the ELISA plate is blocked properly is negligi- ble. This type of non-specific binding control ignores non-specific binding which could result between peptide and mAb. When one considers this a potential source of non-specific binding, the next level of control includes the use of a non-specific mAb for determining non-specific binding. However, this can bias the non-specific binding results toward different peptides since non-specific binding between a single peptide and a panel of mAb may often be different. There- fore, one could potentially obtain different epi- tope maps depending on which non-specific mAb was utilized. Therefore, the use of preincubation with hFSH appears to be a rigorous method for accurate determination of non-specific binding. This method takes into account contributions from non-specific mAb/ELISA plate and mAb/peptide interactions.

Fig. 4 illustrates the results of epitope map- ping the mAbs with hFSHa synthetic peptides. mAb ZE is not shown since there was no de- tectable binding of the mAb to any cr-peptides. The four remaining mAbs all recognized se- quence a61-78 (P < 0.001). In addition, the mAbs recognized the following sequences: 3A and 4B, ~~11-27, ~~73-92; 5F and 2E, (~33-58 (P< 0.001). A summary of the synthetic (Y- peptides and the minimal epitopes recognized by the mAbs is given in Table 3.

Two additional controls were incorporated. To insure that the peptides remain on the ELISA plate throughout the course of the assay, rabbit anti-peptide antisera to each of the seven pep- tides were included. All antipeptide antisera bound to their respective solid-phase peptide and had a resulting absorbance of greater than 2.0

48

(data not included). This also demonstrates that all seven synthetic peptides could be recognized by an antibody molecule. To insure that the preincubation with hFSH could block the mAb from further binding, hFSH was adsorbed onto

o.6 3A t * * 0.4 * 0.2

0.0 l.JJ-JJ 0.6 48 t *

0.4 L--J * * 0.2

0.0

o.6 - 5F

o.6 - 2E T

* 0.4 -

0.2 -

0.0 1-15 11-2, 22-39 a,-JI 51-65 61-78 73-9

hFSH Alpha Peptides

Fig. 4. Epitope mapping of hFSHa using a synthetic peptide

ELISA. Seven synthetic hFSHa peptides were adsorbed onto

Immulon I microtiter plates at a concentration of 2 nmol/ml.

Total binding of mAbs to the adsorbed synthetic hFSHa

peptides was determined by incubating with mAb at a concen- tration of 10 pg/ml. Non-specific binding of mAbs was deter-

mined by preincubating mAbs with hFSH prior to incubating

with the adsorbed synthetic hFSHa peptides as described in

Materials and methods. The graphs represent specific binding

to the synthetic hFSHa peptides following subtraction of

non-specific binding values. The values are expressed as the

mean f SE of triplicate determinations. mAb-peptide interac-

tions marked with an asterisk are considered positive (P <

0.001). mAb 1E is not shown since there was no detectable

binding of the mAb to any a-peptides.

TABLE 3

SYNTHETIC PEPTIDES AND MINIMAL hFSHa EPI-

TOPES RECOGNIZED BY ANTI-hFSH MONOCLONAL

ANTIBODIES

Monoclonal Synthetic

antibody a-peptides

3A ll-27,61-78,73-92

4B ll-27,61-78,73-92

5F 33-58,61-78

2E 33-58,61-78

1E NI a

a None identified.

Minimal

epitope

16-21,66-92

16-21,66-92

40-50,66-72

40-50, 66-72

NI a

the ELISA plate. Following the preincubation with hFSH, none of the mAbs bound to hFSH adsorbed to the ELISA plate.

Inhibition of [‘251]hFSH binding to FSH receptor in bovine testis membranes by Fab fragments

The Fab fragments of the mAbs were tested to determine whether they could immunoneutralize [lz51]hFSH (inhibit binding to receptor). Im- munoneutralization was previously demonstrated when the Protein-A purified mAbs 3A, 4B, 5F, 2E, and 1E were preincubated with [r2’I]hFSH prior to the addition of receptor. We now show that the much smaller (approximately 30 kDa as compared to 150 kDa) Fab fragments of these same mAbs can also inhibit [12’I]hFSH from binding to receptor, as illustrated in Fig. 5. In addition, the immunoneutralizing results ob- tained with the Fab fragments were similar to those obtained when using intact mAbs.

Discussion

This current investigation focused on the iden- tification of hFSH assembled epitopes which are comprised of a-subunit sequences. Two different assays were performed to determine accurately the subunit specificity of five anti-hFSH mAbs. When screened by solid-phase or solution-phase hormone format, all five mAbs bound to hFSH and hFSH&. Therefore, on the basis of these results, it would appear that all five mAbs recog- nize a-subunit sequences.

With the exception of mAb 2E, the mAbs also appear to recognize, albeit to a much lower de-

L2 80

- 60 x

a 40

20

lntcct Antibody

o-

Fab Antibody

D tE

. 2E v 5F

0 3A l 48

i.. ..( /

0.1

Monocionoi .htibody ~~~,t”~~~

Fig. 5. Inhibition of [‘251]hFSH binding to calf testis mem- branes by Protein-A purified monoclonal anti-hFSH antibod- ies or their Fab fragments using an RRA. mAbs or Fab fragments were preincubated with [“*sI]hFSH prior to the addition of 30,000X g calf testis membranes as described in Materials and methods. Non-specific binding was determined in the presence of excess (2 pg) unlabeled hFSH and B, was determined in the absence of mAb. The values represent the mean f SE of triplicate determinations (non-specific binding

values have been subtracted).

gree, &subunit sequences. For example, the binding of [‘251]hFSH to mAbs 3A, 4B, 5F, and 1E was displaced by hFSHP. This recognition of hFSH@ sequences would explain why the ED,, for hFSH is less than that for hFSHau. Some of the differences in ED,, values could also be attributed to conformational changes in the tu- subunit following dissociation from the P-subunit. This may explain why mAb 2E also shows this difference in ED,, values between hFSH and hFSHu. Conformational differences between the heterodimer versus the isolated subunits have been suggested by others (Strickland and Puett, 1982; Hojo and Ryan, 1985; Dighe et al., 1990; Moyle et al., 1990).

The results obtained with the p-subunit could not be caused by a contamination of hFSHp with hFSHa. First, [iZI]hFSH is displaced from bind- ing to mAb 3A by hFSH@ but not hFSHa, there- fore, even if hFSHP were contaminated with hFSHa it would not change the result observed.

49

Secondly, using mAb 5F as an example, hFSH/3 would have to have a 15% contamination of hFSHa! to explain the results observed as deter- mined by comparing ED,, values for hFSHtu (100 ng) and hFSHP (687 ng). The hormone prepara- tions used in this study (with the exception of the immunogens) have all been immunopurified on subunit specific antibody affinity columns. Fur- thermore, the percent subunit cross-contamina- tion is less than 1% (see Materials and methods), far less than what would be required to explain the observed results.

The mAbs all recognize assembled epitopes on the cY-subunit as determined in the peptide ELISA. This finding is further supported by the inability of the mAbs to bind reduced and alkyl- ated hFSHcr. Two different assembled epitopes were identified. One was recognized by mAb 3A and 4B and the second was recognized by mAb 5F and 2E. It is generally believed that the epi- topes on protein antigens are mainly assembled in nature (Benjamin et al., 1984) which has been the case for the glycoprotein hormones (Bidart et al., 1987, 1989; Vakharia et al., 1990; Char- lesworth et al., 1991a). The a-subunit appears to contain an immunodominant region comprised of sequences between cr61 and ~78, as demon- strated by the specific binding of four mAbs to this region. This differs in the predicted immuno- dominant sequence within (~76-92 as determined by cross-species comparisons of the a-subunit (Bergert et al., 1991). It was not possible to map the epitope of mAb lE, which would suggest that its epitope is Confo~ationaIIy dependent, possi- bly owing to constraints enforced by disuifide bonds.

Recently, Charlesworth et al. (1991b) have re- ported on the epitope mapping of the human a-subunit. They found the epitopes to be assem- bIed as have we. They located an imnlunodomi- nant region between residues a70-87 which would correspond to our immunodominant re- gion between ~61-78. A significant difference between their approach and that presented here is their use of wetls without peptide as controls for non-specific binding. Using wells without pep- tide as controls assumes that the binding to the solid-phase peptides is always specific and that non-specific binding must be due to antibody-

plastic interactions. Under normal conditions, in our hands, if the solid phase is blocked properly, non-specific binding values obtained from incu- bating antibody in wells without peptide is virtu- ally zero. Competitive displacement controls, used in this study, appear to be the most accurate indicators of non-specific binding and therefore, we suggest that they be included when screening mAbs with synthetic peptides. Using the binding to wells without peptide, with scrambled peptide, or with other peptides included in the assay, may be misleading indicators of non-specific binding,

Previously, we reported that mAb 3A only recognized sequences ~~61-78 and ar73-92; how- ever, we now demonstrate specific binding to (~11-27. In the previous study our control for non-specific binding to synthetic peptides was Protein-A pure normal mouse Ig. This illustrates the importance of using displacement controls for determining non-specific interactions with syn- thetic peptides. This of course requires the avail- ability of large quantities of pure hormone.

These data are supported by our previous study using anti-hFSH~-peptide antisera in which we demonstrated that amino acid residues within all-27, a33-58 and all of ~~73-92 were on the surface of the hFSH heterodimer and thus acces- sible for antibody binding (Weiner et al., 1990). Our identification of sequence all-27 as part of an epitope corresponds to sequence al-35 which was identified by Birken et al. (1986) as contain- ing an epitope. With respect to subunit contact sites, none of the mAbs bound to sequence ~~1-15, a region which we and others have previously demonstrated to contain a potential subunit con- tact site in hFSH (Salesse et al., 1990; Weiner et al., 1990). Interestingly, none of our antibodies mapped to sequence (~51-65 which would be explained if this sequence were involved in sub- unit contact as has previously been demonstrated (Krystek et al., 1991) or possibly due to the carbo- hydrate at position 52 masking this region from antibody recognition.

Sequences within these epitopes have been previously implicated in hLH/hCG receptor binding. Peptides a26-46 and &l-92 can inhibit hCG, hLH and bovine TSH from binding to receptor (Bellet et al., 1986; Cinarlesworth et al., 1987; Morris et al., 1988). In the latter case, there

are no reports of these peptides inhibiting the binding of human TSH to receptor. Recently, the ~~26-46 sequence has been narrowed down to a30-44 as determined by inhibition of hCG bind- ing to receptor by carboxyl- and amino-terminal truncated versions of ~~26-46 (Reed et al,, 1991). In addition, peptide 71-85 can stimulate testos- terone production in vitro (Erickson et al., 1990). Interestingly, our attempts to directly inhibit hFSH from binding to receptor by synthetic LY- peptides have been unsuccessful (data not shown) as have attempts by others (Ryan et al., 1988). This may be due to differences in the presenta- tion of hFSIIa (in the context of heterodimer) to the FSH receptor. Due to this inability to directly inhibit hFSH from binding to receptor by syn- thetic cu-peptides, we have no choice but to rely on other methods for the identification of recep- tor binding sequences on hFSHa. Although we fully expect the receptor binding sequences of hFSHa to be similar to those of the other three glycoprotein hormone ff-subunits, there is no rea- son for the receptor binding sequences to be identical. This is based on the studies in which hCG and bovine TSH were inhibited from bind- ing to receptor by synthetic a-peptides. The stud- ies demonstrated that the receptor binding re- gions of.hCG and bovine TSH are similar but not identical (Charlesworth et al., 1987; Morris et al., 1988).

Previously, we demonstrated that these five mAbs were potent inhibitors of receptor binding (Weiner et al., 1991). It would appear these anti- genie sites may overlap with receptor binding sequences. We of course recognize that due to the relatively large size of the mAb (1.50 kDa) with respect to hFSH (32 kDa), inhibition of receptor binding could simply be due to steric hindrance. Therefore, using inhibition of hor- mone binding to receptor does not provide definitive evidence that the epitope to which the mAb binds is comprised of peptide sequences important for receptor binding. To lessen the potential problem of steric inhibition of receptor binding we used the much smaller Fab fragments (approximately 30 kDa) to inhibit receptor bind- ing. The non-steric inhibition of receptor binding is further supported by Fab/antigen crystalliza- tion studies. For example, crystallization of Hy-

51

HEL-1OFab with lysozyme demonstrated that the Fab only interacts with 19 amino acids on lysozyme demonstrating that the Fab does not completely surround the antigen (Fischmann et al., 1991). In addition, three Fab fragments can attach simultaneously to lysozyme (Davies and Padlan, 1990). This demonstrates that following the binding of a single Fab, a significant number of amino acids remain free to interact with an- other Fab or in the case of hFSH, free to interact with receptor.

Without the advantage of direct a-peptide in- hibition of hFSH binding to receptor, the identifi- cation of assembled epitopes on heterodimeric hFSHa would allow the placement of specific sequences on the surface of the heterodimer. This method of analysis does not directly identify receptor binding sequences but instead provides crucial tertiary structural information by juxtapos- ing several sequences and thus narrowing down the potential receptor binding sites on hFSHcu. The ability of a mAb to bind to and immunoneu- tralize hormone is not mutually exclusive, even though all of the mAbs in this study could im- munoneutralize hFSH. For example, anti-TSH mAbs have been reported which do not inhibit hTSH from binding to FRTL-5 cells (Endo et al., 1990). If our Fab fragments had failed to inhibit receptor binding, the identification of their epi- tope specificity would have eliminated such se- quences as potential receptor binding sites.

Since these hormones share a common cu-sub- unit, elucidation of its tertiary structure should enhance our understanding of the mechanism of action of all four glycoprotein hormones. This will provide a rational basis for designing alterna- tive structures which could function as modula- tors (agonist or antagonists) of glycoprotein hor- mone activity, and thus, regulate normal repro- ductive functions. In addition, these results should assist in the evaluation of future crystal structures of hFSH.

In summary, we have identified assembled antigenic determinants associated with hFSHcu. On the basis of these data the two minimal as- sembled epitopes recognized by the mAbs in- clude a16-21/a66-92 and CY~O-50/a66-72. The sequences within the mapped epitopes have been separately implicated in receptor binding in pre-

vious studies by other groups. Whether these a-sequences (epitopes) are or are not involved in hFSH binding to receptor remains to be deter- mined by other experimental approaches. How- ever, since the glycoprotein hormones share a common a-subunit, it would then follow that the receptor binding sequences of the a-subunit would in turn be similar.

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