0013-7227/91/1284-1797$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 4 Printed in U.S.A.
Determination of Subunit Contact-Associated Epitopes of the /?- Subunit of Human Follicle-Stimulating Hormone* DILIP D. VAKHARIA, JAMES A. DIAS, AND THOMAS T. ANDERSEN Wadsworth Center for Laboratories and Research, New York State Department of Health, and the School of Public Health, State University of New York (D.D. V., J.A.D.), Albany, New York 12201; and the Department of Biochemistry, Albany Medical College (T.T.A.), Albany, New York 12208
ABSTRACT. Three different experimental approaches were used to assess the regions on the /3-subunit of human FSH (hFSH/3) that may be altered or masked by its association with the a-subunit of hFSH (hFSHa) in the heterodimeric hFSH molecule. In a direct approach, we tested whether synthetic peptides corresponding to hFSH/3 sequences 1-20,16-36, 33-53, 49-67, 66-85, 81-100, and 98-111 inhibited association of hFSHa and hFSH/3 in an enzyme-linked immunosorbent assay. Synthetic peptides-(81-100), -(98-111), and -(66-85) caused greater than 50% inhibition of subunit association, whereas other peptides showed 26% or less inhibition. These data suggested that the C-terminal sequences of hFSH/3, particualrly 81100, are at a subunit interface with hFSHa in heterodimeric hFSH. In another approach we reasoned that antibodies with a higher affinity for free hFSH/3 than for heterodimeric hFSH bind to epitopes on hFSH/3 that are masked or altered by hFSHa subunit. To test this hypothesis, epitopes of hFSH/3 were mapped using synthetic peptides of hFSH/3 sequences, three monoclonal antibodies (3G3, 4D5, and 4G8), and a polyclonal antiserum (NIDDK anti-hFSHjS). Compared to 3G3 all the other antibodies
F
SH, LH, TSH, and CG are glycoproteins of identical a-subunits and hormone-specific /3-subunits. Determination of surfaces on the FSH/3 molecule that are accessible to antibody (epitopes) or to the FSH receptor (receptor-binding sites) or those that are altered or masked after association with a-subunit (subunit contact sites) is essential for elucidating the structure of FSH/3. Such information is necessary, but not sufficient for the development of either synthetic peptide-based contraceptive vaccines against FSH/3 or agonists and antagonists of the hormone. Rationale based on clinical usefulness notwithstanding, it is the task of modern biochemical and molecular endocrinology to correlate precise protein hormone structure with function. Until FSH is crystallized and x-ray crystallography Received September 27,1990. Address all correspondence and requests for reprints to: Dr. James Dias, Wadsworth Center for Laboratories and Research, P.O. Box 509, New York State Department of Health, Albany, New York 12201-0509. * This work was supported by US NIH Grants HD-18407, GM4344301, and NSF/DIR8914757 and Research Career Development Award HD-00682 (to J.A.D.).
exhibited minimal reactivity with hFSH, but bound strongly to hFSH/3. The epitope-mapping data with both 4D5 and NIDDK anti-hFSH/3 identified peptide 81-100, which was not recognized by 3G3. The epitope map with 4G8 identified the same three peptides as with 3G3. However, in the case of 4G8 its reactivity with peptide 33-53 was the least, whereas it was ranked first for 3G3. Since both 3G3 and 4G8 had an identical affinity for hFSH/3, it was hypothesized that sequences in peptide 33-53 may be altered or masked by hFSHa. To test this, we determined the specificity of anti-hFSH/3-(33-53) peptide antiserum for hFSH/3 and hFSH in an enzyme-linked immunosorbent assay. The antipeptide antiserum bound strongly to free hFSH/3 and weakly to hFSH, suggesting that part of the sequence in peptide(33-53) was masked or altered by association with hFSHa in heterodimeric hFSH. Taken together, the subunit association studies, the epitope-mapping data, and the specificity of antihFSH/3-(33-53) peptide antiserum have suggested that sequences in petpide-(81-100) and -(33-53) are masked or conformationally altered by hFSHa in heterodimeric hFSH. (Endocrinology 128: 1797-1804, 1991)
reveals its precise structure, the specific tertiary structural features of the individual subunits and heterodimeric FSH must be determined by other means. We have taken an epitope-mapping approach, using synthetic peptides corresponding to human FSH /3-subunit (hFSH/3) and monoclonal antibodies (mab) specific to hFSH/3. We have shown that an N-terminal region, specifically an assembled epitope comprising sequences contained in synthetic peptide-(33-53), -(49-67), and (66-85), is on the surface of the hFSH/3 molecule. This region also contains determinants for receptor binding (1, 2). In another approach, using antibodies to synthetic peptides corresponding to hFSH a-subunit (hFSHa), we have also shown that synthetic hFSHa peptide-(l-15), (11-27), and -(33-58) contain sequences that are near the a//3-subunit interface. Sequences in peptide-(73-92) and other distinct sequences in peptide-(11-27) and (33-58) are surface oriented (3). In the present study we have extended this work by using three different approaches to determine the subunit contact sites in hFSH/3. In the direct approach, we 1797
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hFSH/3 SUBUNIT CONTACT SITES
have tested the ability of synthetic peptides to prevent the association of a- and /3-subunits in a novel solid phase assay. We have also tested the ability of an antipeptide antibody generated against hFSH/3 peptide-(3353) to bind hFSH/3 and hFSHa-associated hFSH/3 and thereby determine whether the region 33-53 of hFSH/3 is at the subunit contact site. In another approach we have hypothesized that an antibody with a higher affinity for free hFSH/3 than for hFSHa-associated hFSH/3 would bind strongly, in addition to other epitopes on hFSH/3, to an epitope that is masked by hFSHa. Therefore, we mapped the epitopes recognized by such antibodies. From these data we have identified regions on hFSH/3 that are altered or masked by the association with hFSH«. Materials and Methods Hormone preparation Highly purified hFSH/3 (AFP-1194B), hFSH/3 (AFP-4911B), and hFSH (AFP-4822B; 3100 IU/mg) were obtained from the National Hormone and Pituitary Program (Baltimore, MD). The hFSHa used was immunoaffinity purified from human pituitaries (3). Mabs Details for the development of mab 3G3 have been reported previously (1). Mabs 41.4D5.4F6 (4D5) and 41.4G8.4Cl (4G8) were produced as follows. The procedure used for fusion of myeloma and spleen cells was that described by Claflin and Williams (4) with some modifications. The myeloma cell line was recloned from P3X63-Ag8.653. The spleen cells were from a female BALB/c mouse. The immunogen was human hFSH/3 (AFP-1194B). The animal was immunized ip with 25 Mg hFSH/3 emulsified in Freund's complete adjuvant. Four days before the fusion, the animal was boosted ip with 25 ng hFSH/3 in saline. A ratio of five splenocytes to one myeloma cell was used. Lysis of red cells with ammonium chloride was not performed. Spleen cells were isolated by mincing the spleen and expressing the cells through a stainless steel mesh screen using a 10-ml sterile syringe plunger. The screen was rinsed with Hanks' Buffered Salt Solution. The spleen cells were then triturated and passed through a sterile nylon sock. Then, 1 x 107 myeloma cells (cultured in log phase) and 2.07 X 108 spleen cells were combined in a round bottom 50-ml tube. The tube and contents were centrifuged for 5 min at 1200 rpm in a Beckman tabletop refrigerated centrifuge (Fullerton, CA). The supernatant was aspirated, and cells were resuspended by swirling in 0.2 ml 50% polyethylene glycol (American Type Culture Collection, Rockville, MD). The suspension was centrifuged at 700 rpm for 3 min at room temperature (RT). After a total time of exposure to polyethylene glycol of 5 min at 37 C, 5 ml Dulbecco's Modified Eagle's Medium were added without resuspending the cells. After 1-2 min the cell pellet was resuspended by gentle intermittent swirling for 3-4 min. Then, the suspension was centrifuged for 5 min at 1200 rpm. Next, 50 ml Dulbecco's Modified Eagle's Medium with 0.1 mM hypoxanthine and 16.0 mM thymidine (HT) were added, and the entire fusion was
Endo • 1991 Voll28«No4
plated in 5 96-well plates. After 24 h, 2 X HAT medium (HT medium containing 0.8 mM aminopterin) was added. A total of 156 wells contained colonies (32%) and were tested for antibody production using [125I]hFSH or [125I]hFSH/3. Two clones were identified that secreted antibodies specific for hFSH/?. These clones did not react appreciably with heterodimeric hFSH; they were subcloned to assure monoclonality, and the resultant subclones (41.4D5.4F6 and 41.4G8.4Cl) were used for ascites production. Immunoglobulins in the ascites fluid were further purifed by protein-A affinity chromatography according to manufacturer's instructions (Bio-Rad, Richmond, CA). Polyclonal rabbit NIDDK anti-hFSHfi antiserum NIDDK anti-hFSH/3-2 (AFP-2041889) antiserum was obtained from the National Hormone and Pituitary Program (Baltimore, MD). The antiserum was raised in a rabbit by immunization with immunoaffinity-purified hFSH/3. Polyclonal rabbit anti-hFSHP-(33-53) peptide antiserum Antiserum to hemocyanin-coupled peptide-(33-53) was developed in rabbits according to the method described previously (3). Synthetic peptide-(33-53) was coupled to hemocyanin (H1757, Sigma, St. Louis, MO) using the water-soluble heterobifunctional coupling reagent l-ethyl-3-(3-methylaminopropyl)carbodiimide HCl (Sigma, E 7750) according to the method previously described (3). Synthesis of hFSHfi peptides Solid phase synthesis of hFSH/3-peptides: iNSCELTNITIAIEKEECRFCzo, 16ECRFCLTINTTWCAGYCYTRD36, 33YTRDLVYKDPARPKIQKTCTF53, 49KTCTFKELVYETVRVPGCA67, 66CAHHADSLYTYPVATQCHCG85, sxQCHCGKCDSDSTDCTVRGLGxoo, and 98GLGPSYCSFGEMKQm, representing overlapping sequences of hFSH/3, was carried out. Details of the synthesis and purification of these peptides were given in detail previously (1). Peptide-(33-53), -(81-100), and -(98-111) were synthesized on an Applied Biosystems (Foster City, CA) 431A peptide synthesizer using Fmoc chemistry. Synthetic peptides were analyzed for their amino acid composition, and the mass of each peptide was derived from the calculations of the amino acid analysis (1). Radioligand assay Iodination of hFSH/3 was performed by the chloramine-T method. Briefly, 2 ng hFSH/3, 25 n\ phosphate buffer (0.5 M; pH 7.2). 0.25-0.5 mCi [125I]Na, and 10 Ml chloramine-T (1 mg/ ml; 0.05 M phosphate buffer, pH 7.2) were reacted together for 30 sec at RT. The reaction was stopped by the addition of 100 n\ sodium metabisulfite (2.5 mg/ml; 0.05 M phosphate buffer, pH 7.2). The separation of radiolabeled hFSH/3 from free iodine was effected by gel filtration, using a Sephadex G-50 column. A double antibody procedure was used to separate antibodybound hormone from free hormone. The assay procedure involved addition of 100 n\ diluted mab and [125I] hFSH/3 (50 /*!;
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hFSH/? SUBUNIT CONTACT SITES 50,000 cpm) to a sample in a reaction volume of 350 /xl. After 24-h incubation at 4 C, second antibody (rabbit antimouse immunoglobulin, 100 n\ of a 1:20 dilution) was added. After another 24-h incubation at 4 C, 2 ml RIA buffer (0.01 M phosphate buffer and 0.14 M sodium chloride, pH 7.4) were added, and tubes were centrifuged at 1,250 x g for 30-45 min. After aspirating the supernatant, the pellets were counted in a 7-counter. When using polyclonal antiserum NIDDK anti-hFSH/3, the antibody-bound hormone was separated from free hormone in the following manner. To the reaction volume (0.5 ml) containing sample, antibody, and [125I]hFSH/3, 100 n\ 1% (wt/vol) Staphylococcus aureus cells (Pansorbin, Calbiochem, La Jolla, CA) diluted in RIA buffer were added. After a 30-min incubation at RT, 2 ml RIA buffer were added, and tubes were centrifuged, aspirated, and counted in a 7-counter as described above. All reagents, with the exception of mabs, were diluted in RIA buffer containing 0.1% BSA. Mabs were diluted in 0.05 M EDTA-RIA buffer containing 1:200 normal mouse serum. Data from displacement curves were analyzed with the computer program NIHRIA to determine the ED50 value (5). Inhibition of hFSH-subunit association by hFSH/3 peptides Description of the method. Hormone subunits and peptides were diluted in bicarbonate buffer (15 mM sodium-carbonate, 35 mM sodium bicarbonate, and 3 mM sodium azide), pH 9.6. hFSHa (0.1 nmol) was incubated at RT with 1.0 nmol/100 /A of each of the synthetic peptides. After 24 h, 0.1 nmol/50 fi\ hFSH/3 was added to the reaction mixture. After another 24-h incubation at RT, 50 n\ of the reaction mixture were transferred per well in duplicate to Immulon I plates (Dynatech Laboratory, Chantilly, VA) previously coated with antibody to hFSHa (10.5F1) and incubated for another 24 h at RT. Immulon I plates were precoated with 1 ^tg/well protein-A-immunopurified mab 10.5F1 by overnight incubation at 4 C in 0.05 M Tris, pH 9.5. The characteristics of this mab were described previously (6). After the incubation period, the contents of the wells were discarded, and 2.5% nonfat milk solution (200 ^I/well; Carnation milk powder, Carnation Co., Los Angeles, CA) in bicarbonate buffer was added for 1 h at RT to block the free sites in the wells. hFSHa in the reaction mixture bound to mab 10.5F1 on the plate. The amount of hFSH/3 associated with hFSHa was determined using polyclonal NIDDK anti-hFSH/3 in the enzyme-linked immunosorbent assay (ELISA) test previously described in detail (1). In brief, after the incubation period, plates were washed three times, and NIDDK anti-hFSH/3 (50 /xl; 1:1500) was added to each well and incubated at RT for 2 h. After the incubation period, plates were washed three times, and then alkaline phosphatase-labeled goat antirabbit immunoglobulin, diluted 1:2000 in binding buffer, was added and incubated for 1 h at RT. The wells were washed again, and substrate (p-nitrophenylphosphate) was added. The yellow color that developed after the addition of substrate was quantitated after 1 h using a Dynatech plate reader, with a 410-nm filter setting. Wells containing only peptides (NSBpep) and only binding buffer (NSBbuff) served as negative controls (see below). The color quantitated in these wells was considered nonspecific. An estimate of the percent inhibition of association of hFSHa and hFSH/? subunits by each peptide was then obtained by the
1799
equation: 1 - [(absorbancy in wells containing a- + j8-subunits and peptide — NSBpep)/absorbancy in wells containing a- + /?subunits and no peptide - NSBbUff)] X 100. Proportion of a-/? association. The extent of a-/? association was determined by the optical density of wells containing a- and /?subunits and comparing it with the optical density of wells containing equimolar amounts of hFSH, similarly captured and treated thereafter. The proportion of a-/? association obtained was as high as 23% compared to that of equimolar heterodimeric hFSH. Although not used in this study, another mab, 10.3A6 (6), which binds poorly to free a-subunit in solution compared with binding of mab 10.5F1 was used to coat the plate then the proportion of a-/? association obtained was as high as 59% compared to that of equimolar heterodimeric hFSH. Incubation time and temperature. There was 30% less a-/? association if the coincubation of subunits was carried out at 37 C instead of RT. An incubation period of 24 h was used, since Reichert, Jr., et al. (7) had determined that an incubation time of 16 h or more was optimal for a-/? association. Further, more recent studies of Krystek, Jr., et al. (8), who determined the extent of subunit association by differential spectroscopy, reverse phase HPLC, and gel filtration chromatography, obtained optimal a-/? association when incubations were carried out at RT for 24 h. The efficiency of a-/? association differs, depending on the experimental approach used to determine the a-/? association. For Reichert, Jr., et al. (7) it was as high as 70%. For Krystek, Jr., et al. (8) it was 50-80%. Nonspecific binding controls. hFSHfi peptides added to the capture mab (10.5F1) that had been adsorbed to the plate (NSBpep): This control was included to ascertain whether mab nonspecifically bound any of the hFSH/? peptides. The optical density values obtained from these wells were subtracted from those from the corresponding wells containing subunits and the peptide. Only a-subunit or only fi-subunit added to the wells coated with the capture mab: The antiserum used to detect the presence of a-associated /3-subunit (NIDDK anti-hFSH/?) did not react with the free a-subunit bound to mab on the plate. Thus, the optical density measured in test wells was due to the interaction of antibody with a-associated /?-subunit. The optical density values in the wells containing only /?subunit suggested that adsorbed mab 10.5F1 did bind some of the free /?-subunit. However, this observation did not influence the results in test wells, as this was a constant variable in each of the different test wells and, therefore, was not subtracted from the test well values. Inhibitory effect of peptides on binding of a-/? heterodimer to mab adsorbed to the plate: To rule out the possibility that peptides interfere with the binding of associated a- and /?subunits to mab adsorbed to the plate, we conincubated peptides with 0.1 or 0.0125 nmol hFSH and then transferred the reaction mixture to plates preadsorbed with mab 10.5F1 or 10.3A6. The amount of peptide used, incubation time, temperature, and subsequent treatment of wells were identical to experiments described above where a- and /3-subunits were incubated together with peptides. A comparision of optical
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hFSH/3 SUBUNIT CONTACT SITES
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density values of wells containing both hFSH and peptides with wells containing only hFSH indicated that there was no appreciable (0-5%) inhibition of the binding of a-(3 heterodimer to mab adsorbed on the plate by different synthetic peptides. This observation assured us that peptides per se did not interfere with the binding of associated a-/?-subunits to mab on the plate.
Endo • 1991 Voll28«No4
100
Epitope mapping using synthetic peptides in an ELISA Mabs 3G3, 4D5, and 4G8 and polyclonal NIDDK antihFSHjS were tested for their binding to synthetic peptides, as previously described (1). Peptides were ranked for their potency to bind these antibodies by quantitating the product of the lowest amount of peptide and antibody required for a positive signal, as described previously (1). In brief, different concentrations (0.04-33 nmol) of each peptide were coated on the plate and tested against excess mabs or polyclonal antibody. The lowest concentration of each peptide that resulted in a positive signal (absorbance 0.1 or more, 1.5 h after adding the substrate) was determined. This concentration of each peptide was then used to titer out mabs (62.5-2000 ng) or polyclonal antibody (1:100 to 1:1500 dilution). The product of the lowest amount of peptide times the antibody titer was determined. The smallest value was ranked as first and signified the highest relative affinity of the peptide-antibody interaction. For every peptide, all the three mabs were tested in the same assay in duplicate. For NIDDK anti-hFSH/3, normal rabbit serum at dilutions corresponding to those of NIDDK antihFSH/3 was also used as a negative control. Binding of anti-hFSHP-(33-53) peptide antiserum to hFSH and hFSHfi The anti-hFSH/3-(33-53) peptide antiserum, the polyclonal anti-hFSH antiserum (used as positive control), and the corresponding preimmune sera (used as negative controls) were tested for their binding to 100 ng hFSH/Vwell and 200 ng hFSH/well coated on Immulon I plates in an ELISA according to the method described above for NIDDK anti-hFSH/3. Polyclonal anti-hFSH antiserum was raised in rabbits by immunization with hFSH (AFP-4822B). This antibody bound more strongly to hFSH and hFSHa than to hFSH/3.
Results Inhibition of hFSH-subunit association by hFSH(3 synthetic peptides As seen in Fig. 1. peptide-(66-85), -(81-100), and -(98111) inhibited by 52%, 72%, and 57% the association of a- and /3-subunits of hFSH. All other peptides showed 26% or less inhibition. To detect the association of hFSH/3 and hFSHa we used NIDDK anti-hFSH^ antiserum as the detection antibody. The epitope specificity of this antibody is described further below. Similar results were obtained if mab 10.3A6 was used instead of mab 10.5F1 to coat the plate. Antibody specificity Figure 2 illustrates the displacement curves of antibodies 3G3,4D5,4G8, and NIDDK anti-hFSH^, whereas
1 2 3 4 5 6 7 1-20 16-36 33-53 49-67 66-85 81-100 98-111 HFSH-BETA PEPTIDES
FIG. 1. Inhibition of hFSH subunit association by hFSH/3 synthetic peptides. As described in Materials and Methods, synthetic peptides were preincubated with hFSHa before the addition of hFSH/3. After further incubation, the reaction mixture was transferred to an Immulon I plate precoated with antibody specific to hFSHa. hFSHa in the reaction mixture bound to this antibody. Any hFSH/3 associated with hFSHa was detected in an ELISA test using NIDDK anti-hFSH/3 antiserum. The percent inhibition of the association of a-/3-subunits was determined by the equation described in Materials and Methods.
Table 1 summarizes their ED50 (hFSH) and ED50 (hFSH/3) values. These antibodies did not react with free hFSHa when tested by ELISA or RIA. Whereas mab 3G3 could bind [125I]hFSH, the other three antibodies showed no appreciable binding to [125I]hFSH (data not included) in solution phase assay. Therefore, specificity studies were performed using [125I] hFSH/3 as radioligand. Mab 3G3 required 7 times, whereas NIDDK anti-hFSH/3 and mabs (4D5 and 4G8) required 41 times and more than 90 times, respectively, the amount of hFSH than hFSH/3 to displace 50% of the antibody-bound [125I] hFSH/8. Notably, the latter three antibodies bound hFSH/3 with stronger affinity than 3G3. Therefore, the differences among these antibodies in their ability to bind heterodimeric hFSH compared with 3G3 were interpreted to be due to masking or a change in the conformation of epitopes on hFSH/3 by hFSHa. Epitope-mapping studies The results of the epitope mapping using mabs and synthetic peptides tested in an ELISA are illustrated in Fig. 3, which illustrates the values of the product of the lowest mass of peptide and antibody required in the ELISA to obtain a positive signal. Mab 4G8 bound primarily to peptide-(49-67) and less so to neighboring peptide-(33-53) and -(66-85). Only peptide-(66-85) was detected as a strong inhibitor of subunit association in the direct assay (Fig. 1). In contrast, 4D5 bound primarily to peptide-(33-53) and to a lesser extent to peptide-(81-100), suggesting that 4D5 binds to a discontinuous assembled epitope. As seen in Fig. 1, peptide-(81-100) was the strongest inhibitor of subunit association. Neither 4G8 nor 4D5 bound peptide-
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hFSH/3 SUBUNIT CONTACT SITES
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TABLE 1. hFSH and hFSH/3 binding characteristics of antibodies EDfio(M)
Antibody
hFSH
3G3 41.8 ± 3.7 X 4D5 >613 X 4G8 »613 X NIDDK anti-hFSHjS 0.41 ± 0.0024 X
hFSH/? 5.7 ± 0.40 x 10"' 10"' 6.8 ± 0.82 x 10"' 4.2 ± 0.31 x 10"' 0.010 ± 0.0016 x
10"' 10"' 10"' 10"'
The ED60 values are presented as molarity. The final assay volume was 0.5 ml. [126I]hFSH/3 was the radioligand. ED50 values were determined with the computer program NIHRIA (5).
strong inhibitor of subunit contact in the direct assay (Fig. 1). However, like 4D5, it bound intensly to 33-53, and like 4G8, it bound to 49-67, but these peptides were not identified as inhibitors of subunit contact. We also characterized a hFSH/3-specific polyclonal antiserum (NIDDK anti-hFSH/3) obtained from the National Pituitary Agency. This antiserum was reported to be /3 specific, and required 63 times the mass of heterodimeric hFSH compared with hFSH/3 to displace [125I] hFSH/3. This antiserum performed similarly in our hands (Fig. 2); therefore, it provided a potential tool to study the subunit contact sites of hFSH/3. As illustrated in Fig. 4, the solid phase synthetic ELISA demonstrated that NIDDK anti-hFSH/3 binds strongly to peptide-(33-53), -(66-85), and -(81-100). Peptide-(66-85) and -(81-100) were also capable of inhibiting the association of hFSHa and hFSH/3 (Fig. 1). Further, like all mabs, it bound to peptide-(33-53) strongly; however, this peptide did not inhibit subunit association. Topographic analysis of hFSH and hFSH(3 with antipeptide antisera
0.010
0.100
1.000
10.000
100.000 1000.000
NO HORMONE (LOO)
FlG. 2. Specificity of antibodies: inhibition of binding of [125I]hFSH/? to mabs 3G3, 4D5, and 4G8 and polyclonal antibody NIDDK antihFSH/3 by different concentrations of hFSH|8 (AFP-4911B) and hFSH (AFP-4822B) in a radioligand assay. The details of the radioligand assay are given in Materials and Methods. B/Bo, cpm boundeampie-NSP/ cpm boundMro-NSP.
(98-111), which also inhibited the association of hFSH subunits (Fig. 1). The peptides to which 4G8 and 4D5 did bind (49-67 and 33-53, respectively) with apparent greatest intensity were not identified as subunit contact sites by the results of the approach shown in Fig. 1. In the case of 3G3, this mab bound primarily to peptide-(33-53) and to a lesser extent to peptide-(49-67) and -(66-85) in descending order of potency. Like mab 4G8, 3G3 also identified epitope 66-85, which was a
It remained an enigma that all three mabs and the polyclonal antiserum bound to hFSH/3 peptide-(33-53), although this peptide was not identified as an inhibitor of subunit contact. To directly test the hypothesis that peptide hFSH/3-(33-53) includes a subunit contact site, we prepared an antipeptide antiserum against synthetic peptide-(33-53) and tested its ability to react with hFSH and hFSH/3 in the solid phase ELISA. As shown in Table 2, the antiserum reacted strongly with hFSH/3, but only weakly with heterodimeric hFSH. Thus, a significant portion of hFSH/3-(33-53) appears to be masked by the a-subunit. However, as observed from the results of the subunit association studies, this peptide sequence is not sufficient to prevent the association of a- and /3-subunits.
Discussion We have used three different approaches to identify subunit contact-associated epitopes in hFSH/3. In the direct approach we have ascertained the ability of syn-
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Endo«1991 Voll28«No4
0.300 40-
4GB
30-
0.20020-
10-
0.100-
0-
1
1 1-20
0.000
4D5
1
—H-
1—— —f-
1
—t—
2 3 4 5 6 7 16-36 33-53 49-67 66-85 81-100 98—111 HFSH-BETA SYNTHETIC PEPTIDES
FIG. 4. Epitope mapping of hFSH/3 using NIDDK anti-hFSH/3 antiserum in an ELISA. See Fig. 3 and Materials and Methods for the description of the ELISA. A value representing an inverse ratio of the highest serum dilution that gave a positive signal was used to determine the product.
8 § 0.050 a o
TABLE 2. Binding of antipeptide-(33-53) antibody to hFSH and hFSHjS
a: o.
Absorbance (410 nm) Time
Hormone
Antipeptide(33-53) antibody
Anti-hFSH antibody
lh
hFSH hFSH/3
0.00 0.43 ± 0.07
2.00 1.2 ± 0.05
4h
hFSH hFSH/3
0.18 ± 0.09 2.00
>2.00 >2.00
0.000 3G3 10.000 •
1.000 •
0.100 •
0.010
1 2 3 4 5 6 7 1-20 16-38 33-53 49-67 68-85 81-100 98-111 HFSH-BETA SYNTHETIC PEPTIDES
FIG. 3. Epitope mapping of hFSH/3 using mabs and synthetic peptides in a solid phase ELISA; the product of minimum amounts of peptide (nanomoles) and mabs (nanograms) required for positive signal in ELISA test is represented. Different concentrations of each peptide were coated on the plate and reacted with excess antibodies. The smallest concentration of each peptide that resulted in a positive signal was determined. This concentration of each peptide was then used to titer out each antibody in the ELISA. The product of these two values for each peptide was determined. The details of the experimental procedure were reported previously (1). Please note that for mab 3G3, the y-axis is a log scale.
thetic peptides of the hFSH/3 sequence to inhibit association of a- and /3-subunits of hFSH. The data have clearly shown that peptide-(81-100) exhibited the highest (72%) inhibition, whereas peptide-(66-85) and -(98111) exhibited more than 50% inhibition in the association of the two subunits. This has suggested to us that
Polyclonal antibodies to hFSH or synthetic hFSH/3-(33-53) peptide were tested for their binding to hFSH or hFSH/3 in an ELISA. Serum collected before the animals were immunized (preimmune) served as the negative control. The color developed in the wells containing preimmune serum was considered nonspecific, and these values were subtracted from the values obtained from the wells containing the corresponding antibody. The values represented in the table are corrected for the nonspecific color. hFSH/3 (100 ng) and hFSH (200 ng) were coated on the wells of Immunlon I plates. 0 Absorbance was determined 1 and 4 h after the addition of the substrate to the reaction mixture.
the C-terminal region of hFSH/3, specifically 81-100 and to a lesser extent 66-85 and 98-111, contains sequences involved in contact with the a-subunit in the heterodimeric hFSH molecule. In another approach we have hypothesized that an antibody with an higher affinity for free hFSH/3 than for hFSHa-associated hFSH/3 would bind strongly, in addition to other epitopes on hFSH/3, an epitope that is masked by hFSHa. To confirm this hypothesis we carried out epitope mapping of hFSH/3 using three monoclonal antibodies (3G3, 4D5, and 4G8) and one polyclonal antibody (NIDDK anti-hFSH|8) and identified peptides that bound the strongest to these antibodies. Compared with 3G3, all other antibodies required significantly higher amounts of hFSH than hFSH/3 to displace by 50% the antibody-bound [125I]hFSH/3. It was hypothesized
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hFSH/3 SUBUNIT CONTACT SITES that these antibodies would identify an epitope not recognized by 3G3 and that this epitope would be the one that is masked subsequent to the association with hFSHa in the heterodimeric hFSH. In the ELISA test, the epitope-mapping data for 3G3 described previously (1) and in the present study showed that 3G3 bound primarily to peptide-(33-53) and to a lesser extent to peptide-(49-67) and -(66-85). Compared with 3G3, 4D5 also bound strongly to peptide-(33-53), but was clearly distinguishable from 3G3 in that it did not bind peptide-(4967) and -(66-85) as 3G3 did. Instead, it bound to peptide(81-100). Mab 4G8 bound to the same peptides as 3G3. However, it too exhibited a unique pattern of binding compared with 3G3. It bound primarily to peptide-(4967), whereas 3G3 bound to peptide-(33-53). In spite of the fact that the affinity for peptide-(33-53) of 4G8 was lower than that of 3G3, both the mabs had near-identical affinity for hFSH/3. It is likely that masking of the region 33-53 or other determinants of the assembled epitope of hFSH/3 (comprising regions 33-53, 49-67, and 66-85) by hFSHa resulted in the inability of 4G8' and 4D5' to bind hFSH as strongly as 3G3. Thus, it is likely that after a-/? association, part of the surface-oriented epitopes (33-53, 49-67, and 66-85) are also masked, and therefore, these antibodies do not bind hFSHa-associated hFSH/3 as strongly as they do free hFSH/3. The anti-hFSH/3-(33-53) peptide antibody bound hFSH/3 more strongly than it did hFSH, thereby suggesting that part of the 33-53 region of hFSH/? is masked by hFSHa in a heterodimeric hFSH molecule. The epitope-mapping data of polyclonal NIDDK anti-hFSH/3 also suggest that regions 33-53, 66-85, and 81-100 are involved at the subunit contact site of hFSH/3. The results of subunit association studies, epitopemapping data, and hormone specificity of anti-hFSH/3(33-53) peptide antiserum have suggested that sequences of the surface-oriented assembled epitope comprising regions 33-53, 49-67, and 66-85 of hFSH/3 are partly masked or their conformation is altered subsequent to the association with hFSHa and that sequences in peptide-(81-100) and possibly sequences on either side of 81-100 of hFSH/? contained in peptide-(66-85) and -(98111) are situated at the subunit contact site. Earlier studies of Ward and Moore (9) postulated that the hCG/3-subunit loop region 93-100 may be the determinant loop of hormone specificity among the glycoprotein hormones. The region corresponding to this loop region in hFSH/3 is 87-94. Studies by Keutmann et al. (10) and Ryan et al. (11), using synthetic peptides, have suggested that the loop region 93-101 is a receptorbinding domain of hCG/3. More recent studies of Santa Coloma and Reichert, Jr. (12), indicate that hFSH/3 peptide-(81-95) binds to FSH receptor. Except for hFSH/3 peptide-(33-53), -(49-67), and -(66-85), we have
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not been able to inhibit binding of [125I]hFSH to receptor by peptide-d-20), -(16-36), -(81-100), or -(98-111) (1). The reason for this disparity is not clear. More recent studies by Bousfield and Ward (13) have indicated that the equivalent loop region in equine LH is involved in a-/3 association. They showed that enzyme (serine protease specific for Lys residues) nicked free /?subunit in less than an hour at positions Lys96-Thr97, whereas no significant nicking of ^-associated /3-subunit (intact eLH) occurred even after 6 h of incubation with the enzyme. Nicked /3-subunit did not reassociate with the free a-subunit. Studies of Ratanabanangkoon et al. (14), who employed a protein kinase to study the exposure of the 93-100 loop in hCG and hCG/3, also suggested that the 93-100 loop is shielded by the a-subunit in intact hCG. Earlier studies by Weare and Reichert, Jr. (15), have shown that Asp111 in hCG/3 cross-links with Lys49 of hCGa when treated with carbodiimide, thereby suggesting that regions on either side of Asp111 come in direct contact with regions around Lys49 of hCGa. In hFSH/3, this position is serine. It remains to be determined whether Asp88 or Asp93 in hFSH/3 could cross-link with Lys49 in the a-subunit or form a salt bridge in the native hormone. The balance of evidence in the literature suggests that the 93-100 determinant loop is at the a-/3subunit contact site in hCG/3; in the present studies the evidence suggests that the corresponding loop region in hFSH/3, contained within peptide 81-100, is also located at the subunit contact site. Selective proteolysis of ovine LH or its /?-subunit by Bousfield and Ward (16) using endoproteinase Arg-C, which preferentially nicks Lys residues adjacent to another basic residue, have shown that one of the peptide bonds on either side of Arg43 is nicked by Arg-C. Further, Arg-C does nick ovine (o) LH/3 when it is associated with oLHa:. This suggested that amino acid residues on either side of Arg43 are exposed to the surface. However, the rate of nicking of oLH/3 in the intact hormone was much slower than when the isolated subunit was nicked. They suggested that this could be due to either a change in the conformation of the 38-57 loop as a result of association with the a-subunit or partial shielding of this region by the a-subunit. From their observation that the nicked oLHjS-oLHa dimer is less stable in the presence of sodium dodecyl sulfate than either native oLH or recombined oLH subunits and that peptide bonds on either side of Arg43 are less susceptible to proteolysis when oLHjS is associated with oLHa, they have hypothesized that the hydrophobic region 42-49 of oLHjS would be in close proximity to the a-subunit and may be involved with a-/3-subunit association. The corresponding region in hFSH0 is 36-43. Our epitope-mapping study has shown that sequences within peptide-(33-53) are exposed on the surface of the molecule. As discussed previously,
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hFSH/3 SUBUNIT CONTACT SITES
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the epitope-mapping data of 4G8 and other antibodies have indirectly indicated that part of the assembled epitope, which comprises 33-53, 49-67, and 66-85, is masked by the a-subunit in the heterodimeric hFSH. Using anti-hFSH/3-(33-53) peptide antiserum, we have observed that this antibody binds more strongly to hFSH/? than to heterodimeric hFSH, thereby suggesting that a significant portion of hFSH/? is masked by hFSHa in the heterodimeric hFSH. Overall, the present study has indicated that surfaceoriented receptor-binding sequences within peptide-(3353) of hFSH/3 are partly masked or their conformation is altered by the a-subunit in heterodimeric hFSH. Further, the sequence 81-100 of the C-terminal region of hFSH/3 is situated at the subunit contact site. Thus, this study indicates that part of the receptor-binding site of hFSH/? is masked by the a-subunit in heterodimeric hFSH. It has been proposed by Bousfield and Ward (16), that receptor-binding and subunit association regions of both subunits are adjacent or overlapping. Further, it has been proposed that the /3-subunit acts as a template that reorders the structure of the a-subunit to put its receptor-binding components into the proper register with those contributed by the /3-subunit (9, 17). This possibility has to be considered.
3. 4. 5. 6. 7.
8. 9.
10.
11. 12. 13.
Acknowledgments The gifts of hormone, hormone subunits, and NIDDK antihFSH/3 antiserum from the National Pituitary Agency and the NIDDK (Baltimore, MD) are highly appreciated.
14. 15.
References 1. Vakharia DD, Dias JA, Thakur AN, Andersen TT, O'Shea A 1990 Mapping of an assembled epitope of human follicle stimulating hormone-j8 utilizing monoclonal antibodies, synthetic peptides, and hormone-receptor inhibition. Endocrinology 127:658-666 2. Santa Coloma TA, Dattatreyamurty B, Reichert Jr LE 1990 A synthetic peptide corresponding to human FSH beta subunit 33-
16. 17.
Endo*1991 Voll28«No4
53 binds to FSH receptor, stimulates basal estradiol biosynthesis and is a partial antagonist of FSH. Biochemistry 29:1194-1200 Weiner RS, Andersen TT, Dias JA 1990 Topographic analysis of the a-subunit of human follicle stimulating hormone using sitespecific antipeptide antisera. Endocrinology 127:573-579 Claflin L, Williams K 1978 Mouse myeloma-spleen cell hybrids: enhanced hybridization frequencies and rapid screening procedures. Curr Top Microbiol Immunol 81:107-109 Yanagishita M, Rodbard D 1978 Computer optimization of radioimmunoassays for hCG and estradiol: an experimental evolution. Anal Biochem 88:1-19 Weiner RS, Dias JA, Andersen TT 1991 Epitope mapping of human FSHa using monoclonal antibody 3A identifies a potential receptor binding sequence. Endocrinology 128:1485-1495 Reichert Jr LE, Lawson Jr GM, Leidenberger FL, Trowbridge CG 1973 Influence of a- and /3-subunits on the kinetics of formation and activity of native and hybrid molecules of LH human chorionic gonadotropin. Endocrinology 93:938-946 Krystek Jr SR, Dias JA, Andersen TT, Identification of subunit contact sites on the a-subunit of lutropin. Biochemistry, in press Ward DN, Moore Jr WT 1979 Comparative study of mammalian glycoprotein hormones. In: Alexander NJ (ed) Animal Models for Research on Contraception and Fertility. Harper Row, New York, pp 151-163 Keutmann HT, Charlesworth MC, Mason K, Ostrea T, Johnson L, Ryan RJ 1987 A receptor-binding region in human choriogonadotropin/lutropin beta unit. Proc Natl Acad Sci USA 84:20382042 Ryan RJ, Keutmann HT, Charlesworth MC, McCormick DJ, Milius RP, Calvo FO, Vutyavanich T 1987 Structure-function relationships of gonadotropins. Recent Prog Horm Res 43:383-429 Santa Coloma TA, Reichert Jr LE 1990 Identification of a follicle stimulating hormone receptor-binding region in hFSH/3-(81-95) using synthetic peptides. J Biol Chem 265:5037-5042 Bousfield GR, Ward DN 1989 Determinant loop involvement in a/3 association of equine LH. In: Serono Symposium: International Symposium on Glycoprotein Hormones. Serono Symposia, USA Press, pp 48 (Abstract) Ratanabanangkoon K, Keutmann HT, Kitzmann K, Ryan RJ 1983 Properties of the phosphorylated beta subunit of human choriogonadotropin. J Biol Chem 258:14527-14531 Weare JA, Reichert Jr LE 1979 Studies with carbodiimide crosslinked derivatives of bovine lutropin.II Location of the cross-link and implication for interaction with the receptors in testis. J Biol Chem 254:6969-6972 Bousfield GR, Ward DN 1988 Selective proteolysis of ovine lutropin or its /3-subunit by endoproteinase Arg-C. J Biol Chem 263:12602-12607 Gamier J 1978 Molecular aspects of the subunit assembly of glycoprotein hormones. In: McKerns LW (ed) Structure Function of the Gonadotropins. Plenum Press, New York, pp 381-414
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Vol. 128, No. 4 Printed in U.S.A.
Determination of Subunit Contact-Associated Epitopes of the /?- Subunit of Human Follicle-Stimulating Hormone* DILIP D. VAKHARIA, JAMES A. DIAS, AND THOMAS T. ANDERSEN Wadsworth Center for Laboratories and Research, New York State Department of Health, and the School of Public Health, State University of New York (D.D. V., J.A.D.), Albany, New York 12201; and the Department of Biochemistry, Albany Medical College (T.T.A.), Albany, New York 12208
ABSTRACT. Three different experimental approaches were used to assess the regions on the /3-subunit of human FSH (hFSH/3) that may be altered or masked by its association with the a-subunit of hFSH (hFSHa) in the heterodimeric hFSH molecule. In a direct approach, we tested whether synthetic peptides corresponding to hFSH/3 sequences 1-20,16-36, 33-53, 49-67, 66-85, 81-100, and 98-111 inhibited association of hFSHa and hFSH/3 in an enzyme-linked immunosorbent assay. Synthetic peptides-(81-100), -(98-111), and -(66-85) caused greater than 50% inhibition of subunit association, whereas other peptides showed 26% or less inhibition. These data suggested that the C-terminal sequences of hFSH/3, particualrly 81100, are at a subunit interface with hFSHa in heterodimeric hFSH. In another approach we reasoned that antibodies with a higher affinity for free hFSH/3 than for heterodimeric hFSH bind to epitopes on hFSH/3 that are masked or altered by hFSHa subunit. To test this hypothesis, epitopes of hFSH/3 were mapped using synthetic peptides of hFSH/3 sequences, three monoclonal antibodies (3G3, 4D5, and 4G8), and a polyclonal antiserum (NIDDK anti-hFSHjS). Compared to 3G3 all the other antibodies
F
SH, LH, TSH, and CG are glycoproteins of identical a-subunits and hormone-specific /3-subunits. Determination of surfaces on the FSH/3 molecule that are accessible to antibody (epitopes) or to the FSH receptor (receptor-binding sites) or those that are altered or masked after association with a-subunit (subunit contact sites) is essential for elucidating the structure of FSH/3. Such information is necessary, but not sufficient for the development of either synthetic peptide-based contraceptive vaccines against FSH/3 or agonists and antagonists of the hormone. Rationale based on clinical usefulness notwithstanding, it is the task of modern biochemical and molecular endocrinology to correlate precise protein hormone structure with function. Until FSH is crystallized and x-ray crystallography Received September 27,1990. Address all correspondence and requests for reprints to: Dr. James Dias, Wadsworth Center for Laboratories and Research, P.O. Box 509, New York State Department of Health, Albany, New York 12201-0509. * This work was supported by US NIH Grants HD-18407, GM4344301, and NSF/DIR8914757 and Research Career Development Award HD-00682 (to J.A.D.).
exhibited minimal reactivity with hFSH, but bound strongly to hFSH/3. The epitope-mapping data with both 4D5 and NIDDK anti-hFSH/3 identified peptide 81-100, which was not recognized by 3G3. The epitope map with 4G8 identified the same three peptides as with 3G3. However, in the case of 4G8 its reactivity with peptide 33-53 was the least, whereas it was ranked first for 3G3. Since both 3G3 and 4G8 had an identical affinity for hFSH/3, it was hypothesized that sequences in peptide 33-53 may be altered or masked by hFSHa. To test this, we determined the specificity of anti-hFSH/3-(33-53) peptide antiserum for hFSH/3 and hFSH in an enzyme-linked immunosorbent assay. The antipeptide antiserum bound strongly to free hFSH/3 and weakly to hFSH, suggesting that part of the sequence in peptide(33-53) was masked or altered by association with hFSHa in heterodimeric hFSH. Taken together, the subunit association studies, the epitope-mapping data, and the specificity of antihFSH/3-(33-53) peptide antiserum have suggested that sequences in petpide-(81-100) and -(33-53) are masked or conformationally altered by hFSHa in heterodimeric hFSH. (Endocrinology 128: 1797-1804, 1991)
reveals its precise structure, the specific tertiary structural features of the individual subunits and heterodimeric FSH must be determined by other means. We have taken an epitope-mapping approach, using synthetic peptides corresponding to human FSH /3-subunit (hFSH/3) and monoclonal antibodies (mab) specific to hFSH/3. We have shown that an N-terminal region, specifically an assembled epitope comprising sequences contained in synthetic peptide-(33-53), -(49-67), and (66-85), is on the surface of the hFSH/3 molecule. This region also contains determinants for receptor binding (1, 2). In another approach, using antibodies to synthetic peptides corresponding to hFSH a-subunit (hFSHa), we have also shown that synthetic hFSHa peptide-(l-15), (11-27), and -(33-58) contain sequences that are near the a//3-subunit interface. Sequences in peptide-(73-92) and other distinct sequences in peptide-(11-27) and (33-58) are surface oriented (3). In the present study we have extended this work by using three different approaches to determine the subunit contact sites in hFSH/3. In the direct approach, we 1797
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hFSH/3 SUBUNIT CONTACT SITES
have tested the ability of synthetic peptides to prevent the association of a- and /3-subunits in a novel solid phase assay. We have also tested the ability of an antipeptide antibody generated against hFSH/3 peptide-(3353) to bind hFSH/3 and hFSHa-associated hFSH/3 and thereby determine whether the region 33-53 of hFSH/3 is at the subunit contact site. In another approach we have hypothesized that an antibody with a higher affinity for free hFSH/3 than for hFSHa-associated hFSH/3 would bind strongly, in addition to other epitopes on hFSH/3, to an epitope that is masked by hFSHa. Therefore, we mapped the epitopes recognized by such antibodies. From these data we have identified regions on hFSH/3 that are altered or masked by the association with hFSH«. Materials and Methods Hormone preparation Highly purified hFSH/3 (AFP-1194B), hFSH/3 (AFP-4911B), and hFSH (AFP-4822B; 3100 IU/mg) were obtained from the National Hormone and Pituitary Program (Baltimore, MD). The hFSHa used was immunoaffinity purified from human pituitaries (3). Mabs Details for the development of mab 3G3 have been reported previously (1). Mabs 41.4D5.4F6 (4D5) and 41.4G8.4Cl (4G8) were produced as follows. The procedure used for fusion of myeloma and spleen cells was that described by Claflin and Williams (4) with some modifications. The myeloma cell line was recloned from P3X63-Ag8.653. The spleen cells were from a female BALB/c mouse. The immunogen was human hFSH/3 (AFP-1194B). The animal was immunized ip with 25 Mg hFSH/3 emulsified in Freund's complete adjuvant. Four days before the fusion, the animal was boosted ip with 25 ng hFSH/3 in saline. A ratio of five splenocytes to one myeloma cell was used. Lysis of red cells with ammonium chloride was not performed. Spleen cells were isolated by mincing the spleen and expressing the cells through a stainless steel mesh screen using a 10-ml sterile syringe plunger. The screen was rinsed with Hanks' Buffered Salt Solution. The spleen cells were then triturated and passed through a sterile nylon sock. Then, 1 x 107 myeloma cells (cultured in log phase) and 2.07 X 108 spleen cells were combined in a round bottom 50-ml tube. The tube and contents were centrifuged for 5 min at 1200 rpm in a Beckman tabletop refrigerated centrifuge (Fullerton, CA). The supernatant was aspirated, and cells were resuspended by swirling in 0.2 ml 50% polyethylene glycol (American Type Culture Collection, Rockville, MD). The suspension was centrifuged at 700 rpm for 3 min at room temperature (RT). After a total time of exposure to polyethylene glycol of 5 min at 37 C, 5 ml Dulbecco's Modified Eagle's Medium were added without resuspending the cells. After 1-2 min the cell pellet was resuspended by gentle intermittent swirling for 3-4 min. Then, the suspension was centrifuged for 5 min at 1200 rpm. Next, 50 ml Dulbecco's Modified Eagle's Medium with 0.1 mM hypoxanthine and 16.0 mM thymidine (HT) were added, and the entire fusion was
Endo • 1991 Voll28«No4
plated in 5 96-well plates. After 24 h, 2 X HAT medium (HT medium containing 0.8 mM aminopterin) was added. A total of 156 wells contained colonies (32%) and were tested for antibody production using [125I]hFSH or [125I]hFSH/3. Two clones were identified that secreted antibodies specific for hFSH/?. These clones did not react appreciably with heterodimeric hFSH; they were subcloned to assure monoclonality, and the resultant subclones (41.4D5.4F6 and 41.4G8.4Cl) were used for ascites production. Immunoglobulins in the ascites fluid were further purifed by protein-A affinity chromatography according to manufacturer's instructions (Bio-Rad, Richmond, CA). Polyclonal rabbit NIDDK anti-hFSHfi antiserum NIDDK anti-hFSH/3-2 (AFP-2041889) antiserum was obtained from the National Hormone and Pituitary Program (Baltimore, MD). The antiserum was raised in a rabbit by immunization with immunoaffinity-purified hFSH/3. Polyclonal rabbit anti-hFSHP-(33-53) peptide antiserum Antiserum to hemocyanin-coupled peptide-(33-53) was developed in rabbits according to the method described previously (3). Synthetic peptide-(33-53) was coupled to hemocyanin (H1757, Sigma, St. Louis, MO) using the water-soluble heterobifunctional coupling reagent l-ethyl-3-(3-methylaminopropyl)carbodiimide HCl (Sigma, E 7750) according to the method previously described (3). Synthesis of hFSHfi peptides Solid phase synthesis of hFSH/3-peptides: iNSCELTNITIAIEKEECRFCzo, 16ECRFCLTINTTWCAGYCYTRD36, 33YTRDLVYKDPARPKIQKTCTF53, 49KTCTFKELVYETVRVPGCA67, 66CAHHADSLYTYPVATQCHCG85, sxQCHCGKCDSDSTDCTVRGLGxoo, and 98GLGPSYCSFGEMKQm, representing overlapping sequences of hFSH/3, was carried out. Details of the synthesis and purification of these peptides were given in detail previously (1). Peptide-(33-53), -(81-100), and -(98-111) were synthesized on an Applied Biosystems (Foster City, CA) 431A peptide synthesizer using Fmoc chemistry. Synthetic peptides were analyzed for their amino acid composition, and the mass of each peptide was derived from the calculations of the amino acid analysis (1). Radioligand assay Iodination of hFSH/3 was performed by the chloramine-T method. Briefly, 2 ng hFSH/3, 25 n\ phosphate buffer (0.5 M; pH 7.2). 0.25-0.5 mCi [125I]Na, and 10 Ml chloramine-T (1 mg/ ml; 0.05 M phosphate buffer, pH 7.2) were reacted together for 30 sec at RT. The reaction was stopped by the addition of 100 n\ sodium metabisulfite (2.5 mg/ml; 0.05 M phosphate buffer, pH 7.2). The separation of radiolabeled hFSH/3 from free iodine was effected by gel filtration, using a Sephadex G-50 column. A double antibody procedure was used to separate antibodybound hormone from free hormone. The assay procedure involved addition of 100 n\ diluted mab and [125I] hFSH/3 (50 /*!;
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hFSH/? SUBUNIT CONTACT SITES 50,000 cpm) to a sample in a reaction volume of 350 /xl. After 24-h incubation at 4 C, second antibody (rabbit antimouse immunoglobulin, 100 n\ of a 1:20 dilution) was added. After another 24-h incubation at 4 C, 2 ml RIA buffer (0.01 M phosphate buffer and 0.14 M sodium chloride, pH 7.4) were added, and tubes were centrifuged at 1,250 x g for 30-45 min. After aspirating the supernatant, the pellets were counted in a 7-counter. When using polyclonal antiserum NIDDK anti-hFSH/3, the antibody-bound hormone was separated from free hormone in the following manner. To the reaction volume (0.5 ml) containing sample, antibody, and [125I]hFSH/3, 100 n\ 1% (wt/vol) Staphylococcus aureus cells (Pansorbin, Calbiochem, La Jolla, CA) diluted in RIA buffer were added. After a 30-min incubation at RT, 2 ml RIA buffer were added, and tubes were centrifuged, aspirated, and counted in a 7-counter as described above. All reagents, with the exception of mabs, were diluted in RIA buffer containing 0.1% BSA. Mabs were diluted in 0.05 M EDTA-RIA buffer containing 1:200 normal mouse serum. Data from displacement curves were analyzed with the computer program NIHRIA to determine the ED50 value (5). Inhibition of hFSH-subunit association by hFSH/3 peptides Description of the method. Hormone subunits and peptides were diluted in bicarbonate buffer (15 mM sodium-carbonate, 35 mM sodium bicarbonate, and 3 mM sodium azide), pH 9.6. hFSHa (0.1 nmol) was incubated at RT with 1.0 nmol/100 /A of each of the synthetic peptides. After 24 h, 0.1 nmol/50 fi\ hFSH/3 was added to the reaction mixture. After another 24-h incubation at RT, 50 n\ of the reaction mixture were transferred per well in duplicate to Immulon I plates (Dynatech Laboratory, Chantilly, VA) previously coated with antibody to hFSHa (10.5F1) and incubated for another 24 h at RT. Immulon I plates were precoated with 1 ^tg/well protein-A-immunopurified mab 10.5F1 by overnight incubation at 4 C in 0.05 M Tris, pH 9.5. The characteristics of this mab were described previously (6). After the incubation period, the contents of the wells were discarded, and 2.5% nonfat milk solution (200 ^I/well; Carnation milk powder, Carnation Co., Los Angeles, CA) in bicarbonate buffer was added for 1 h at RT to block the free sites in the wells. hFSHa in the reaction mixture bound to mab 10.5F1 on the plate. The amount of hFSH/3 associated with hFSHa was determined using polyclonal NIDDK anti-hFSH/3 in the enzyme-linked immunosorbent assay (ELISA) test previously described in detail (1). In brief, after the incubation period, plates were washed three times, and NIDDK anti-hFSH/3 (50 /xl; 1:1500) was added to each well and incubated at RT for 2 h. After the incubation period, plates were washed three times, and then alkaline phosphatase-labeled goat antirabbit immunoglobulin, diluted 1:2000 in binding buffer, was added and incubated for 1 h at RT. The wells were washed again, and substrate (p-nitrophenylphosphate) was added. The yellow color that developed after the addition of substrate was quantitated after 1 h using a Dynatech plate reader, with a 410-nm filter setting. Wells containing only peptides (NSBpep) and only binding buffer (NSBbuff) served as negative controls (see below). The color quantitated in these wells was considered nonspecific. An estimate of the percent inhibition of association of hFSHa and hFSH/? subunits by each peptide was then obtained by the
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equation: 1 - [(absorbancy in wells containing a- + j8-subunits and peptide — NSBpep)/absorbancy in wells containing a- + /?subunits and no peptide - NSBbUff)] X 100. Proportion of a-/? association. The extent of a-/? association was determined by the optical density of wells containing a- and /?subunits and comparing it with the optical density of wells containing equimolar amounts of hFSH, similarly captured and treated thereafter. The proportion of a-/? association obtained was as high as 23% compared to that of equimolar heterodimeric hFSH. Although not used in this study, another mab, 10.3A6 (6), which binds poorly to free a-subunit in solution compared with binding of mab 10.5F1 was used to coat the plate then the proportion of a-/? association obtained was as high as 59% compared to that of equimolar heterodimeric hFSH. Incubation time and temperature. There was 30% less a-/? association if the coincubation of subunits was carried out at 37 C instead of RT. An incubation period of 24 h was used, since Reichert, Jr., et al. (7) had determined that an incubation time of 16 h or more was optimal for a-/? association. Further, more recent studies of Krystek, Jr., et al. (8), who determined the extent of subunit association by differential spectroscopy, reverse phase HPLC, and gel filtration chromatography, obtained optimal a-/? association when incubations were carried out at RT for 24 h. The efficiency of a-/? association differs, depending on the experimental approach used to determine the a-/? association. For Reichert, Jr., et al. (7) it was as high as 70%. For Krystek, Jr., et al. (8) it was 50-80%. Nonspecific binding controls. hFSHfi peptides added to the capture mab (10.5F1) that had been adsorbed to the plate (NSBpep): This control was included to ascertain whether mab nonspecifically bound any of the hFSH/? peptides. The optical density values obtained from these wells were subtracted from those from the corresponding wells containing subunits and the peptide. Only a-subunit or only fi-subunit added to the wells coated with the capture mab: The antiserum used to detect the presence of a-associated /3-subunit (NIDDK anti-hFSH/?) did not react with the free a-subunit bound to mab on the plate. Thus, the optical density measured in test wells was due to the interaction of antibody with a-associated /?-subunit. The optical density values in the wells containing only /?subunit suggested that adsorbed mab 10.5F1 did bind some of the free /?-subunit. However, this observation did not influence the results in test wells, as this was a constant variable in each of the different test wells and, therefore, was not subtracted from the test well values. Inhibitory effect of peptides on binding of a-/? heterodimer to mab adsorbed to the plate: To rule out the possibility that peptides interfere with the binding of associated a- and /?subunits to mab adsorbed to the plate, we conincubated peptides with 0.1 or 0.0125 nmol hFSH and then transferred the reaction mixture to plates preadsorbed with mab 10.5F1 or 10.3A6. The amount of peptide used, incubation time, temperature, and subsequent treatment of wells were identical to experiments described above where a- and /3-subunits were incubated together with peptides. A comparision of optical
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hFSH/3 SUBUNIT CONTACT SITES
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density values of wells containing both hFSH and peptides with wells containing only hFSH indicated that there was no appreciable (0-5%) inhibition of the binding of a-(3 heterodimer to mab adsorbed on the plate by different synthetic peptides. This observation assured us that peptides per se did not interfere with the binding of associated a-/?-subunits to mab on the plate.
Endo • 1991 Voll28«No4
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Epitope mapping using synthetic peptides in an ELISA Mabs 3G3, 4D5, and 4G8 and polyclonal NIDDK antihFSHjS were tested for their binding to synthetic peptides, as previously described (1). Peptides were ranked for their potency to bind these antibodies by quantitating the product of the lowest amount of peptide and antibody required for a positive signal, as described previously (1). In brief, different concentrations (0.04-33 nmol) of each peptide were coated on the plate and tested against excess mabs or polyclonal antibody. The lowest concentration of each peptide that resulted in a positive signal (absorbance 0.1 or more, 1.5 h after adding the substrate) was determined. This concentration of each peptide was then used to titer out mabs (62.5-2000 ng) or polyclonal antibody (1:100 to 1:1500 dilution). The product of the lowest amount of peptide times the antibody titer was determined. The smallest value was ranked as first and signified the highest relative affinity of the peptide-antibody interaction. For every peptide, all the three mabs were tested in the same assay in duplicate. For NIDDK anti-hFSH/3, normal rabbit serum at dilutions corresponding to those of NIDDK antihFSH/3 was also used as a negative control. Binding of anti-hFSHP-(33-53) peptide antiserum to hFSH and hFSHfi The anti-hFSH/3-(33-53) peptide antiserum, the polyclonal anti-hFSH antiserum (used as positive control), and the corresponding preimmune sera (used as negative controls) were tested for their binding to 100 ng hFSH/Vwell and 200 ng hFSH/well coated on Immulon I plates in an ELISA according to the method described above for NIDDK anti-hFSH/3. Polyclonal anti-hFSH antiserum was raised in rabbits by immunization with hFSH (AFP-4822B). This antibody bound more strongly to hFSH and hFSHa than to hFSH/3.
Results Inhibition of hFSH-subunit association by hFSH(3 synthetic peptides As seen in Fig. 1. peptide-(66-85), -(81-100), and -(98111) inhibited by 52%, 72%, and 57% the association of a- and /3-subunits of hFSH. All other peptides showed 26% or less inhibition. To detect the association of hFSH/3 and hFSHa we used NIDDK anti-hFSH^ antiserum as the detection antibody. The epitope specificity of this antibody is described further below. Similar results were obtained if mab 10.3A6 was used instead of mab 10.5F1 to coat the plate. Antibody specificity Figure 2 illustrates the displacement curves of antibodies 3G3,4D5,4G8, and NIDDK anti-hFSH^, whereas
1 2 3 4 5 6 7 1-20 16-36 33-53 49-67 66-85 81-100 98-111 HFSH-BETA PEPTIDES
FIG. 1. Inhibition of hFSH subunit association by hFSH/3 synthetic peptides. As described in Materials and Methods, synthetic peptides were preincubated with hFSHa before the addition of hFSH/3. After further incubation, the reaction mixture was transferred to an Immulon I plate precoated with antibody specific to hFSHa. hFSHa in the reaction mixture bound to this antibody. Any hFSH/3 associated with hFSHa was detected in an ELISA test using NIDDK anti-hFSH/3 antiserum. The percent inhibition of the association of a-/3-subunits was determined by the equation described in Materials and Methods.
Table 1 summarizes their ED50 (hFSH) and ED50 (hFSH/3) values. These antibodies did not react with free hFSHa when tested by ELISA or RIA. Whereas mab 3G3 could bind [125I]hFSH, the other three antibodies showed no appreciable binding to [125I]hFSH (data not included) in solution phase assay. Therefore, specificity studies were performed using [125I] hFSH/3 as radioligand. Mab 3G3 required 7 times, whereas NIDDK anti-hFSH/3 and mabs (4D5 and 4G8) required 41 times and more than 90 times, respectively, the amount of hFSH than hFSH/3 to displace 50% of the antibody-bound [125I] hFSH/8. Notably, the latter three antibodies bound hFSH/3 with stronger affinity than 3G3. Therefore, the differences among these antibodies in their ability to bind heterodimeric hFSH compared with 3G3 were interpreted to be due to masking or a change in the conformation of epitopes on hFSH/3 by hFSHa. Epitope-mapping studies The results of the epitope mapping using mabs and synthetic peptides tested in an ELISA are illustrated in Fig. 3, which illustrates the values of the product of the lowest mass of peptide and antibody required in the ELISA to obtain a positive signal. Mab 4G8 bound primarily to peptide-(49-67) and less so to neighboring peptide-(33-53) and -(66-85). Only peptide-(66-85) was detected as a strong inhibitor of subunit association in the direct assay (Fig. 1). In contrast, 4D5 bound primarily to peptide-(33-53) and to a lesser extent to peptide-(81-100), suggesting that 4D5 binds to a discontinuous assembled epitope. As seen in Fig. 1, peptide-(81-100) was the strongest inhibitor of subunit association. Neither 4G8 nor 4D5 bound peptide-
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hFSH/3 SUBUNIT CONTACT SITES
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TABLE 1. hFSH and hFSH/3 binding characteristics of antibodies EDfio(M)
Antibody
hFSH
3G3 41.8 ± 3.7 X 4D5 >613 X 4G8 »613 X NIDDK anti-hFSHjS 0.41 ± 0.0024 X
hFSH/? 5.7 ± 0.40 x 10"' 10"' 6.8 ± 0.82 x 10"' 4.2 ± 0.31 x 10"' 0.010 ± 0.0016 x
10"' 10"' 10"' 10"'
The ED60 values are presented as molarity. The final assay volume was 0.5 ml. [126I]hFSH/3 was the radioligand. ED50 values were determined with the computer program NIHRIA (5).
strong inhibitor of subunit contact in the direct assay (Fig. 1). However, like 4D5, it bound intensly to 33-53, and like 4G8, it bound to 49-67, but these peptides were not identified as inhibitors of subunit contact. We also characterized a hFSH/3-specific polyclonal antiserum (NIDDK anti-hFSH/3) obtained from the National Pituitary Agency. This antiserum was reported to be /3 specific, and required 63 times the mass of heterodimeric hFSH compared with hFSH/3 to displace [125I] hFSH/3. This antiserum performed similarly in our hands (Fig. 2); therefore, it provided a potential tool to study the subunit contact sites of hFSH/3. As illustrated in Fig. 4, the solid phase synthetic ELISA demonstrated that NIDDK anti-hFSH/3 binds strongly to peptide-(33-53), -(66-85), and -(81-100). Peptide-(66-85) and -(81-100) were also capable of inhibiting the association of hFSHa and hFSH/3 (Fig. 1). Further, like all mabs, it bound to peptide-(33-53) strongly; however, this peptide did not inhibit subunit association. Topographic analysis of hFSH and hFSH(3 with antipeptide antisera
0.010
0.100
1.000
10.000
100.000 1000.000
NO HORMONE (LOO)
FlG. 2. Specificity of antibodies: inhibition of binding of [125I]hFSH/? to mabs 3G3, 4D5, and 4G8 and polyclonal antibody NIDDK antihFSH/3 by different concentrations of hFSH|8 (AFP-4911B) and hFSH (AFP-4822B) in a radioligand assay. The details of the radioligand assay are given in Materials and Methods. B/Bo, cpm boundeampie-NSP/ cpm boundMro-NSP.
(98-111), which also inhibited the association of hFSH subunits (Fig. 1). The peptides to which 4G8 and 4D5 did bind (49-67 and 33-53, respectively) with apparent greatest intensity were not identified as subunit contact sites by the results of the approach shown in Fig. 1. In the case of 3G3, this mab bound primarily to peptide-(33-53) and to a lesser extent to peptide-(49-67) and -(66-85) in descending order of potency. Like mab 4G8, 3G3 also identified epitope 66-85, which was a
It remained an enigma that all three mabs and the polyclonal antiserum bound to hFSH/3 peptide-(33-53), although this peptide was not identified as an inhibitor of subunit contact. To directly test the hypothesis that peptide hFSH/3-(33-53) includes a subunit contact site, we prepared an antipeptide antiserum against synthetic peptide-(33-53) and tested its ability to react with hFSH and hFSH/3 in the solid phase ELISA. As shown in Table 2, the antiserum reacted strongly with hFSH/3, but only weakly with heterodimeric hFSH. Thus, a significant portion of hFSH/3-(33-53) appears to be masked by the a-subunit. However, as observed from the results of the subunit association studies, this peptide sequence is not sufficient to prevent the association of a- and /3-subunits.
Discussion We have used three different approaches to identify subunit contact-associated epitopes in hFSH/3. In the direct approach we have ascertained the ability of syn-
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hFSH/3 SUBUNIT CONTACT SITES
Endo«1991 Voll28«No4
0.300 40-
4GB
30-
0.20020-
10-
0.100-
0-
1
1 1-20
0.000
4D5
1
—H-
1—— —f-
1
—t—
2 3 4 5 6 7 16-36 33-53 49-67 66-85 81-100 98—111 HFSH-BETA SYNTHETIC PEPTIDES
FIG. 4. Epitope mapping of hFSH/3 using NIDDK anti-hFSH/3 antiserum in an ELISA. See Fig. 3 and Materials and Methods for the description of the ELISA. A value representing an inverse ratio of the highest serum dilution that gave a positive signal was used to determine the product.
8 § 0.050 a o
TABLE 2. Binding of antipeptide-(33-53) antibody to hFSH and hFSHjS
a: o.
Absorbance (410 nm) Time
Hormone
Antipeptide(33-53) antibody
Anti-hFSH antibody
lh
hFSH hFSH/3
0.00 0.43 ± 0.07
2.00 1.2 ± 0.05
4h
hFSH hFSH/3
0.18 ± 0.09 2.00
>2.00 >2.00
0.000 3G3 10.000 •
1.000 •
0.100 •
0.010
1 2 3 4 5 6 7 1-20 16-38 33-53 49-67 68-85 81-100 98-111 HFSH-BETA SYNTHETIC PEPTIDES
FIG. 3. Epitope mapping of hFSH/3 using mabs and synthetic peptides in a solid phase ELISA; the product of minimum amounts of peptide (nanomoles) and mabs (nanograms) required for positive signal in ELISA test is represented. Different concentrations of each peptide were coated on the plate and reacted with excess antibodies. The smallest concentration of each peptide that resulted in a positive signal was determined. This concentration of each peptide was then used to titer out each antibody in the ELISA. The product of these two values for each peptide was determined. The details of the experimental procedure were reported previously (1). Please note that for mab 3G3, the y-axis is a log scale.
thetic peptides of the hFSH/3 sequence to inhibit association of a- and /3-subunits of hFSH. The data have clearly shown that peptide-(81-100) exhibited the highest (72%) inhibition, whereas peptide-(66-85) and -(98111) exhibited more than 50% inhibition in the association of the two subunits. This has suggested to us that
Polyclonal antibodies to hFSH or synthetic hFSH/3-(33-53) peptide were tested for their binding to hFSH or hFSH/3 in an ELISA. Serum collected before the animals were immunized (preimmune) served as the negative control. The color developed in the wells containing preimmune serum was considered nonspecific, and these values were subtracted from the values obtained from the wells containing the corresponding antibody. The values represented in the table are corrected for the nonspecific color. hFSH/3 (100 ng) and hFSH (200 ng) were coated on the wells of Immunlon I plates. 0 Absorbance was determined 1 and 4 h after the addition of the substrate to the reaction mixture.
the C-terminal region of hFSH/3, specifically 81-100 and to a lesser extent 66-85 and 98-111, contains sequences involved in contact with the a-subunit in the heterodimeric hFSH molecule. In another approach we have hypothesized that an antibody with an higher affinity for free hFSH/3 than for hFSHa-associated hFSH/3 would bind strongly, in addition to other epitopes on hFSH/3, an epitope that is masked by hFSHa. To confirm this hypothesis we carried out epitope mapping of hFSH/3 using three monoclonal antibodies (3G3, 4D5, and 4G8) and one polyclonal antibody (NIDDK anti-hFSH|8) and identified peptides that bound the strongest to these antibodies. Compared with 3G3, all other antibodies required significantly higher amounts of hFSH than hFSH/3 to displace by 50% the antibody-bound [125I]hFSH/3. It was hypothesized
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hFSH/3 SUBUNIT CONTACT SITES that these antibodies would identify an epitope not recognized by 3G3 and that this epitope would be the one that is masked subsequent to the association with hFSHa in the heterodimeric hFSH. In the ELISA test, the epitope-mapping data for 3G3 described previously (1) and in the present study showed that 3G3 bound primarily to peptide-(33-53) and to a lesser extent to peptide-(49-67) and -(66-85). Compared with 3G3, 4D5 also bound strongly to peptide-(33-53), but was clearly distinguishable from 3G3 in that it did not bind peptide-(4967) and -(66-85) as 3G3 did. Instead, it bound to peptide(81-100). Mab 4G8 bound to the same peptides as 3G3. However, it too exhibited a unique pattern of binding compared with 3G3. It bound primarily to peptide-(4967), whereas 3G3 bound to peptide-(33-53). In spite of the fact that the affinity for peptide-(33-53) of 4G8 was lower than that of 3G3, both the mabs had near-identical affinity for hFSH/3. It is likely that masking of the region 33-53 or other determinants of the assembled epitope of hFSH/3 (comprising regions 33-53, 49-67, and 66-85) by hFSHa resulted in the inability of 4G8' and 4D5' to bind hFSH as strongly as 3G3. Thus, it is likely that after a-/? association, part of the surface-oriented epitopes (33-53, 49-67, and 66-85) are also masked, and therefore, these antibodies do not bind hFSHa-associated hFSH/3 as strongly as they do free hFSH/3. The anti-hFSH/3-(33-53) peptide antibody bound hFSH/3 more strongly than it did hFSH, thereby suggesting that part of the 33-53 region of hFSH/? is masked by hFSHa in a heterodimeric hFSH molecule. The epitope-mapping data of polyclonal NIDDK anti-hFSH/3 also suggest that regions 33-53, 66-85, and 81-100 are involved at the subunit contact site of hFSH/3. The results of subunit association studies, epitopemapping data, and hormone specificity of anti-hFSH/3(33-53) peptide antiserum have suggested that sequences of the surface-oriented assembled epitope comprising regions 33-53, 49-67, and 66-85 of hFSH/3 are partly masked or their conformation is altered subsequent to the association with hFSHa and that sequences in peptide-(81-100) and possibly sequences on either side of 81-100 of hFSH/? contained in peptide-(66-85) and -(98111) are situated at the subunit contact site. Earlier studies of Ward and Moore (9) postulated that the hCG/3-subunit loop region 93-100 may be the determinant loop of hormone specificity among the glycoprotein hormones. The region corresponding to this loop region in hFSH/3 is 87-94. Studies by Keutmann et al. (10) and Ryan et al. (11), using synthetic peptides, have suggested that the loop region 93-101 is a receptorbinding domain of hCG/3. More recent studies of Santa Coloma and Reichert, Jr. (12), indicate that hFSH/3 peptide-(81-95) binds to FSH receptor. Except for hFSH/3 peptide-(33-53), -(49-67), and -(66-85), we have
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not been able to inhibit binding of [125I]hFSH to receptor by peptide-d-20), -(16-36), -(81-100), or -(98-111) (1). The reason for this disparity is not clear. More recent studies by Bousfield and Ward (13) have indicated that the equivalent loop region in equine LH is involved in a-/3 association. They showed that enzyme (serine protease specific for Lys residues) nicked free /?subunit in less than an hour at positions Lys96-Thr97, whereas no significant nicking of ^-associated /3-subunit (intact eLH) occurred even after 6 h of incubation with the enzyme. Nicked /3-subunit did not reassociate with the free a-subunit. Studies of Ratanabanangkoon et al. (14), who employed a protein kinase to study the exposure of the 93-100 loop in hCG and hCG/3, also suggested that the 93-100 loop is shielded by the a-subunit in intact hCG. Earlier studies by Weare and Reichert, Jr. (15), have shown that Asp111 in hCG/3 cross-links with Lys49 of hCGa when treated with carbodiimide, thereby suggesting that regions on either side of Asp111 come in direct contact with regions around Lys49 of hCGa. In hFSH/3, this position is serine. It remains to be determined whether Asp88 or Asp93 in hFSH/3 could cross-link with Lys49 in the a-subunit or form a salt bridge in the native hormone. The balance of evidence in the literature suggests that the 93-100 determinant loop is at the a-/3subunit contact site in hCG/3; in the present studies the evidence suggests that the corresponding loop region in hFSH/3, contained within peptide 81-100, is also located at the subunit contact site. Selective proteolysis of ovine LH or its /?-subunit by Bousfield and Ward (16) using endoproteinase Arg-C, which preferentially nicks Lys residues adjacent to another basic residue, have shown that one of the peptide bonds on either side of Arg43 is nicked by Arg-C. Further, Arg-C does nick ovine (o) LH/3 when it is associated with oLHa:. This suggested that amino acid residues on either side of Arg43 are exposed to the surface. However, the rate of nicking of oLH/3 in the intact hormone was much slower than when the isolated subunit was nicked. They suggested that this could be due to either a change in the conformation of the 38-57 loop as a result of association with the a-subunit or partial shielding of this region by the a-subunit. From their observation that the nicked oLHjS-oLHa dimer is less stable in the presence of sodium dodecyl sulfate than either native oLH or recombined oLH subunits and that peptide bonds on either side of Arg43 are less susceptible to proteolysis when oLHjS is associated with oLHa, they have hypothesized that the hydrophobic region 42-49 of oLHjS would be in close proximity to the a-subunit and may be involved with a-/3-subunit association. The corresponding region in hFSH0 is 36-43. Our epitope-mapping study has shown that sequences within peptide-(33-53) are exposed on the surface of the molecule. As discussed previously,
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the epitope-mapping data of 4G8 and other antibodies have indirectly indicated that part of the assembled epitope, which comprises 33-53, 49-67, and 66-85, is masked by the a-subunit in the heterodimeric hFSH. Using anti-hFSH/3-(33-53) peptide antiserum, we have observed that this antibody binds more strongly to hFSH/? than to heterodimeric hFSH, thereby suggesting that a significant portion of hFSH/? is masked by hFSHa in the heterodimeric hFSH. Overall, the present study has indicated that surfaceoriented receptor-binding sequences within peptide-(3353) of hFSH/3 are partly masked or their conformation is altered by the a-subunit in heterodimeric hFSH. Further, the sequence 81-100 of the C-terminal region of hFSH/3 is situated at the subunit contact site. Thus, this study indicates that part of the receptor-binding site of hFSH/? is masked by the a-subunit in heterodimeric hFSH. It has been proposed by Bousfield and Ward (16), that receptor-binding and subunit association regions of both subunits are adjacent or overlapping. Further, it has been proposed that the /3-subunit acts as a template that reorders the structure of the a-subunit to put its receptor-binding components into the proper register with those contributed by the /3-subunit (9, 17). This possibility has to be considered.
3. 4. 5. 6. 7.
8. 9.
10.
11. 12. 13.
Acknowledgments The gifts of hormone, hormone subunits, and NIDDK antihFSH/3 antiserum from the National Pituitary Agency and the NIDDK (Baltimore, MD) are highly appreciated.
14. 15.
References 1. Vakharia DD, Dias JA, Thakur AN, Andersen TT, O'Shea A 1990 Mapping of an assembled epitope of human follicle stimulating hormone-j8 utilizing monoclonal antibodies, synthetic peptides, and hormone-receptor inhibition. Endocrinology 127:658-666 2. Santa Coloma TA, Dattatreyamurty B, Reichert Jr LE 1990 A synthetic peptide corresponding to human FSH beta subunit 33-
16. 17.
Endo*1991 Voll28«No4
53 binds to FSH receptor, stimulates basal estradiol biosynthesis and is a partial antagonist of FSH. Biochemistry 29:1194-1200 Weiner RS, Andersen TT, Dias JA 1990 Topographic analysis of the a-subunit of human follicle stimulating hormone using sitespecific antipeptide antisera. Endocrinology 127:573-579 Claflin L, Williams K 1978 Mouse myeloma-spleen cell hybrids: enhanced hybridization frequencies and rapid screening procedures. Curr Top Microbiol Immunol 81:107-109 Yanagishita M, Rodbard D 1978 Computer optimization of radioimmunoassays for hCG and estradiol: an experimental evolution. Anal Biochem 88:1-19 Weiner RS, Dias JA, Andersen TT 1991 Epitope mapping of human FSHa using monoclonal antibody 3A identifies a potential receptor binding sequence. Endocrinology 128:1485-1495 Reichert Jr LE, Lawson Jr GM, Leidenberger FL, Trowbridge CG 1973 Influence of a- and /3-subunits on the kinetics of formation and activity of native and hybrid molecules of LH human chorionic gonadotropin. Endocrinology 93:938-946 Krystek Jr SR, Dias JA, Andersen TT, Identification of subunit contact sites on the a-subunit of lutropin. Biochemistry, in press Ward DN, Moore Jr WT 1979 Comparative study of mammalian glycoprotein hormones. In: Alexander NJ (ed) Animal Models for Research on Contraception and Fertility. Harper Row, New York, pp 151-163 Keutmann HT, Charlesworth MC, Mason K, Ostrea T, Johnson L, Ryan RJ 1987 A receptor-binding region in human choriogonadotropin/lutropin beta unit. Proc Natl Acad Sci USA 84:20382042 Ryan RJ, Keutmann HT, Charlesworth MC, McCormick DJ, Milius RP, Calvo FO, Vutyavanich T 1987 Structure-function relationships of gonadotropins. Recent Prog Horm Res 43:383-429 Santa Coloma TA, Reichert Jr LE 1990 Identification of a follicle stimulating hormone receptor-binding region in hFSH/3-(81-95) using synthetic peptides. J Biol Chem 265:5037-5042 Bousfield GR, Ward DN 1989 Determinant loop involvement in a/3 association of equine LH. In: Serono Symposium: International Symposium on Glycoprotein Hormones. Serono Symposia, USA Press, pp 48 (Abstract) Ratanabanangkoon K, Keutmann HT, Kitzmann K, Ryan RJ 1983 Properties of the phosphorylated beta subunit of human choriogonadotropin. J Biol Chem 258:14527-14531 Weare JA, Reichert Jr LE 1979 Studies with carbodiimide crosslinked derivatives of bovine lutropin.II Location of the cross-link and implication for interaction with the receptors in testis. J Biol Chem 254:6969-6972 Bousfield GR, Ward DN 1988 Selective proteolysis of ovine lutropin or its /3-subunit by endoproteinase Arg-C. J Biol Chem 263:12602-12607 Gamier J 1978 Molecular aspects of the subunit assembly of glycoprotein hormones. In: McKerns LW (ed) Structure Function of the Gonadotropins. Plenum Press, New York, pp 381-414
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