localization of residues that confer antibody binding specificity

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 15, Issue of May 25, pp. 6511-8518, 1990 Printed in U.S. A.

Localization of Residues That Confer Antibody Binding Specificity Using Human Chorionic Gonadotropin/Luteinizing Hormone @ Subunit Chimeras and Mutants*

(Received for publication, December 28, 1989)

William R. Moyle$t, Martin M. MatzuknII , Robert K. Campbell& Ermes Cogliani$, Diana M. Dean-EmigS, Alex Krichevsky **, Richard W. Barnett#$ and Irving Boimell From the SDepartment of Obstetrics/Gynecology, Robert Wood Johnson (Rutgers) Medical School, Piscataway, New Jersey 08654, the llDepartment of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, the **Department of Medicine, Columbia University School of Medicine, New York, New York 10032, and $&Ilk& Biopharmaceuticals Inc., Mississauga, Ontario L4V lP1, Canada

The glycoprotein hormones are a family of conserved heterodimeric proteins which share a common (Y subunit but differ in their hormone-specific /3 subunits. We used chimeras of human chorionic gonadotropin (hCG) and luteinizing hormone (hLH) j3 subunits to identify residues which enable monoclonal antibodies (mAb) to distinguish the two hormones. The LH&CG/3 chimeras appeared to fold similar to hCGB, since they combined with hCGa and, depending on their sequences, were recognized by hCG-selective mAbs. Amino acid residues Arg’-Arg”, Gly4’-AlaS1, and Glnse-Leue2 form a major epitope region and appear to be adjacent to each other on the surface of hCGj3. Gly4’-Alas1 and Glnss- Leus2 are recognized by dimer-specific mAbs while Arg’-Arg” is recognized by mAbs which have highest affinity for the free B subunit. These observations suggest that the conformation of this region of the ,8 subunit changes when the (Y and /3 subunits combine. Res- idues which are C-teriminal of Asp112 form a second epitope domain. mAbs to the third domain distinguish hCGB and hLm by the presence of Asn77 in hCG@ and can be detected after hCG binds to receptors. These findings were used to develop a model of hCGj3 which predicts the locations of these residues and their positions relative to the cr subunit and receptor interfaces.

Human chorionic gonadotropin (hCG)’ and LH are glycoprotein hormone heterodimers which bind to LH receptors on gonadal tissues, activate adenyl cyclase, and promote ste- roidogenesis (1). The cy subunits of both hormones have an identical amino acid sequence whereas the 0 subunits are unique and confer the specific endocrine and biochemical characteristics of each hormone (2). Little is known about the tertiary structures of these subunits or how the fl subunit determines biological specificity. To devise a model of the /I

* This work was supported in part by National Institute of Health Grants HD14907, HD23398, and HD15454 and by a grant (to I. B.) from the Monsanto Co. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: RWJ-Medical School, Piscataway, NJ 08854.

II A participant in the Medical Scientist Training Prosram SUP- poked-by the-National Institute of General Medical-Sciences Grant GM-07200.

1 The abbreviations used are: hCG, human chorionic gonadotropin; hLH, human luteinizing hormone.

subunit, we are using an immunological approach that iden- tifies amino acid determinants which enable monoclonal antibodies to distinguish the p subunits of different glycoprotein hormones. By combining these data with maps which illustrate the relative positions of monoclonal antibody binding sites and receptors on hCGP (3-g), we should identify specific residues and/or regions of the protein which may be involved in subunit and receptor interactions.

The most common strategy for identifying hCG residues involved in antibody or receptor binding sites has been to compare the abilities of hCG, naturally occurring hCG analogs (i.e. LH from several species), or synthetic peptides derived from hCG to inhibit binding of radiolabeled hCG to antibodies (10-13) or receptors (14, 15). The success of this approach depends on the types of analogs which can be obtained and their ability to compete with hCG for binding. Unfortunately, many natural analogs differ from hCG@ throughout the region of the molecule (i.e. residues l-114) which appear to be involved in the binding sites of the LH receptor and most antibodies. Thus, comparisons of the abilities of the antibodies to bind LH from different species have provided relatively little information on the residues involved in the antibody binding sites. In addition, the antibodies in our panel are conformation-dependent and have low affinities for peptides derived from either native or reduced and carboxymethylated hCG. While the low affinity indicates that the antibodies probably recognize two or more noncontiguous sequences (16), it has prevented us from identifying the antibody binding sites.

To circumvent these problems, we prepared analogs of hCG/3 using gene transfer techniques (17-24) and compared their abilities to bind to hCG-selective monoclonal antibodies. Since hLHp and hCGp are identical to 97 of their 114 N- terminal residues, including the positions of all 12 cysteines (2), we anticipated that CG/3-LHfl chimeras prepared by sub- stituting homologous portions of hLHP for hCG@ and vice uersa would fold similar to hCGP and hLH@. Depending on the sequence, a given chimera should bind some but not all antibodies which recognize hCG@-specific conformational epitopes. We observed that CG@-LHP chimeras could be classed as either “hCG-” or “hLH-like” by their ability to bind hCG- specific monoclonal antibodies. Based on the primary sequence of the chimeras and their binding specificity, we identified residues of hCG/3 which are likely to comprise the antibody binding sites. These observations together with our epitope map enabled us to devise a model of the /3 subunit. The model is consistent with the presumed disulfide bond

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8512 Antibody Binding Sites on hCG

assignments (25) and with regions of the molecule which have been postulated to interact with LH receptors.

MATERIALS AND METHODS

Antibodies-The sources of the natural hormones and the monoclonal antibodies have been described previously (3, 7, 26, 27). Anti- bodies B210 and SCTP will be described in a separate report.’ Additional monoclonal antibodies to hCGp (Bill and B112) and hCGa (A113) were obtained from Drs. Glenn Armstrong and Robert Wolfer? (Hybritech Corp., San Diego, CA). Monoclonal antibody All0 to the a-subunit was obtained from Drs. Richard Krogsrud and S. Berube of BioMega Diagnostics, Montreal, Canada. The Iodo-Gen- based procedure used to radioiodinate these proteins has been described (28). Studies described here were repeated using several different preparations of labeled antibodies. Most radioiodinated antibodies had a specific activity of about 50 rCi/pg, regardless of the labeled preparation employed.

Chimeras and Mutants-The amino acid sequences of the constructs used are listed in Fig. 1. To construct many of the chimeras, we took advantage of common endonuclease restriction sites in the hLH@ and hCGp genes (22). LCD41 was constructed by transferring the BglII-Sac1 fragment of the hLH@ gene (the 5’ portion of gene containing the first two exons) into the same sites of the hCG@ gene to form a gene which encoded the first 41 amino acids of hLHP and the remaining 104 residues of hCG@ CL@41 was constructed by making the opposite transfer and encoded a protein containing 121 residues. CGfiAT and LH@AT were constructed by inserting a pre- mature termination codon at position 115. LCp87AT and CLfl87AT were constructed by exchanging the BglII-PuuII restriction fragments of CG/3AT and LHPAT. Thus, LCP87AT and CLp87AT contained 114 amino acids, including 87 which were derived from the N terminus of hLH@ or hCG& respectively. CLCP41-87AT was constructed by inserting the SacI/PuuII fragment from the LHP gene into its corresponding region of CGpAT. LHfl15* and LCp41* were made by changing Ile’” to Thr’” thereby producing the Asni3-X-Thr” glycosylation signal found in hCG& Analogs LHPBAT, LH@8/lOAT, LH/342AT, LHP47/51AT, LHP58AT, LHp77AT, and LHP82/83AT were generated by site-directed mutagenesis using the LHPAT gene as template and methods described previously (21,22). These genomic constructs were expressed in Chinese hamster ovary cells using the eukaryotic expression vector pMZ as described (19-21). The cells were cultured in a-minimal essential medium + 10% fetal calf serum medium (23). Further details on the construction and expression of these clones has been reported (22).

CLCp42-58, CLCp77-96, and CL@96 were constructed by mutagenesis of the hCG/3 cDNA (29) subcloned into the Hind111 site of pTZ19 (30). These chimeras were sequenced using dideoxy methods, and cloned into the XhoI-BamHI sites of pSVL (Pharmacia LKB Biotechnology Inc.). Plasmids containing the inserts were transfected into COS-7 cells using a calcium phosphate procedure (31) and the proteins were expressed transiently in serum-free medium. The remaining constructs were prepared by cassette mutagenesis of hCGp cDNA using naturally occurring restriction sites and expressed in COS-7 cells with the pSVL vector as described above. The oligosac- charides on these proteins were not characterized since we have found previously that the antibodies had nearly the same affinity for deglycosylated hCG as for the intact hormones (39). The mammalian expression systems produced between 0.05 and 0.5 rg of protein/ml medium. In some cases, we concentrated the proteins by ultrafiltra- tion using an Amicon Centriprep 10 prior to further analysis.

Western Blotting-Free subunits and dimers in the culture media were separated by electrophoresis on 12% polyacrylamide gels containing 1% sodium dodecyl sulfate. Mercaptoethanol was omitted from the sample buffer to prevent dissociation of the dimer forms (32). The proteins were electroeluted from the gel onto nitrocellulose sheets as described (18) and detected using radiolabeled antibodies. When hCG is adsorbed to solid phase matrices, binding sites for antibodies near the BlOl, B107, B109, Bill, and B204 epitopes are obscured (28). To detect nanogram quantities of chimeras using these antibodies in Western blots, we first coated nitrocellulose sheets with antibodies to alternate epitopes (i.e. B105) by soaking the sheets in a solution of antibody (20 pg/ml) for 1 h at 37 “C. Next, we blocked remaining nonspecific adsorption sites by incubating the coated nitrocellulose in skim milk for 1 h at room temperature. After the proteins were electroblotted onto the coated nitrocellulose, we soaked

’ A. Krichevsky, manuscript in preparation.


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Antibody Binding Sites on hCG 8513

them in skim milk a second time to further reduce nonspecific binding. Detection of the glycoprotein analogs was performed with radiolabeled BlOl, B107, B109, or B204. To ensure that similar amounts of analog were present in each lane, results of all sandwich Western blotting were compared with those obtained using labeled B105 to detect LH, CG, or chimeras that had been adsorbed directly to nitrocellulose. B105 was used because it binds tightly to an exposed common epitope on hLH, hCG, and all LH-CG chimeras, even after they have been adsorbed to a variety of surfaces including nitrocellulose (Ref. 30, cf “Results”). The radiolabel bound to the nitrocellulose was detected by autoradiography. When radiolabeled B108, Bill, or B112 were used, we coated the nitrocellulose with an (Y subunit antibody (i.e. All0 or A113) instead of B105. In some cases in which we employed radiolabeled Bill, we substituted B201 for B105.

Sandwich Immunoassays-Since sandwich assays utilize considerably less antibody than the Western blots, we could screen the analogs with several different antibody combinations as a further test of antibody specificity. In these assays one antibody was adsorbed onto the surface of a plastic 96-well microtiter plate and used to “capture” chimeras from the culture medium (3). After free hormone or analog was washed from the surface, we compared the abilities of radiolabeled “detection” antibodies to bind to CG, LH, or the chimeras which had been “captured” by the antibody adsorbed to the plastic surface. Most sandwich assays were performed in both config- urations (i.e. by interchanging the capture and detection antibodies) and indicated that our results were independent of possible changes in antibody selectivity created when the antibodies were adsorbed to the plastic.

RESULTS

Properties of the p Subunit Antibodies Used

The properties of the antibodies used in this study are listed in Table I, and their relative binding sites are illustrated schematically in Fig. 2. Most hCG dimer or hCGp specific antibodies bind to one of three epitope regions. Two of these appear to be near regions of the p subunit which interact with the LH receptor since they block binding of hCG to receptor and do not bind to hCG-receptor complexes. Antibodies to one of these compete with one another for binding to the free /3 subunit (i.e. BlOl, B201, and B204) or to the hormone dimer (i.e. BlOl, B107, and B109). Blll binds to a different epitope region on hCG and hCGP subunit. Like antibodies to the first

domain, Blll inhibits hCG binding to receptors and does not bind to hCG-receptor complexes and therefore, this epitope also appears to be near the receptor interface. Antibodies to the third major epitope (i.e. B105, B108, BllO, B112, and B206) bind to hCG at the same time as antibodies to the two other domains. These antibodies bind to hCG-receptor complexes, albeit with lower affinity than they bind to hCG (8). This suggests that either 1) the third domain is not sufficiently near the receptor interface to block hCG binding completely, 2) the antibodies alter the shape of the /? subunit, or 3) the rate of association is reduced due to the size and/or charge of the complex relative to hCG. Since the affinities of some other antibodies (i.e. BllO) which bind to this domain are increased when hCG is bound to receptors, this region of the subunit likely undergoes a conformation change during receptor binding (8).

Characteristics of the Chimeras

Because the amino acid sequence differences in the p subunits of hCG and hLH are clustered in four regions, chimeras were prepared to study each region. CL/341 and LCD41 were constructed to examine the amino acids and Asn-linked oli- gosaccharides in the N-terminal portion of the 6 subunit (i.e. residues 2, 8, 10, 13, and 15). Chimera CL@43-58 permitted analysis of residues 42, 47, 51, and 58. Chimera CLp77-96 enabled us to study the roles of residues 77, 82,83,89,91,92, and 96 while chimera CLp96 enabled us to analyze residues 96, 112, and 114-121. Chimeras CLp87AT, LC/387AT, and CLCP41-87AT were used to estimate the relative roles of residues 88-114, l-87, and 42-87, respectively. By combining data from these latter chimeras with those obtained using CLC/377-96, we were able to distinguish the influence of residues 77,82, and 83 from that of residues 89,91, and 92 on antibody binding.

Using sandwich assays employing (Y subunit capture antibodies (not shown) or Western blotting (Fig. 3), we distin- guished antibody binding to dimers and free p subunit. Cells transfected with vectors coding for both CGa and either CG/3, LHP, or LHP-CGP chimeras secreted (Y and p subunits and

TABLE I Characteristics of the (3 subunit antibodies

Affinities were determined using competitive displacement assays for antibodies in solution (37). Values were calculated using the method of Scatchard (38) and were rounded to one significant figure. Some of the values were taken from earlier publications (3, 4, 7, 8, 26). The specificities of the antibodies in solid phase assays differs somewhat from that expected from these values. For example, B108 has greater ability to detect hLH bound to A105 and B107 has greater ability to bind to hLH bound to B105 than would be anticipated from their abilities to bind LH in solution. “Low” refers to less than lo7 M-' and was not measurable under these conditions without using large amounts of hCG@ or hLH. In many cases Scatchard analysis was not performed due to lack of materials (i.e. hLH) and/or the inability to detect the binding of antibody to the hCGp fragment.

Antibody bCG dimer bLH dimer

Approximate affinity

hCGl3 hLHR hCGfl fraement

BlOl/B204 domain BlOl B107 B109 B204

B108 domain B108 B112

Blll domain Blll

Antibodies that bind hCG and hLH B105 B201 B206

7 x 10s 4 x 1O’O 5 x 1O'O 1 x lo8

1 x 1O’O 1 x 1O’O

1 x 10’”

1 x 10” 1 x lo* 4 x IO"

1 x 10’ Low Low Low

2 x 10s Not done

Not done

1 x 10” 2 x 10s 2 x 1O’O

7 x lo7 Low Low 2 x lo9

6 x 10’ 9 x lo8

6 x lo8

1 x 10” 5 x 1O’O 2 x 10"

Not done Low Low Low

2 x lo8 Not done

Not done

1 x 10” 2 x 1O’O 2 x IO"

Low Not done Low 5 x 1O'O

5 x 1O’O Not done

Not done

3 x 1O'O 5 x 1O'O 1 x 10”


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8514 Antibody Binding Sites on hCG

6101

8105

lzz!!

8204 I3108 8201 8112 q 206

P

SCTP Bill

Blll

FIG. 2. Topological map describing the relative binding sites of the j3 subunit antibodies used in these studies. Antibod- ies whose epitopes are depicted within the same circle compete with one another for binding to hCG. When the subunits combine, the affinity for some of the antibodies increases substantially (e.g. B107 and B109) whereas that for other antibodies decreases (e.g. B204). Antibodies to the BlOl domain do not bind to hCG at the same time as many antibodies to the u subunit. This map was devised using Sandwich assays and has been modified from maps reported previously (3, 4).

(YP dimers that were recognized by antibodies which bound to both hLH and hCG. This suggested that the /3 subunit chimeras folded like the native /I subunit. Western blot analysis also revealed antibody binding to higher and lower molecular weight material (see Fig. 3). The large molecular weight species, detected mainly with B105, may be aggregates while the faster migrating species may be related to the p subunit fragment (27) since they were detected only by those antibodies which have high affinity for the @ subunit fragment (i.e. B210, not shown).

Although we did not purify the chimeric proteins from the culture medium and were therefore unable to obtain a quan- titative estimate of their affinity for B105, it is clear that Bl05 bound all the analogs (Fig. 3). The amount of material detected by B105 differed considerably between samples when equal volumes of medium were applied to the electrophoresis gels. This was due to differences in the amounts and rates of secretion of the hormones from Chinese hamster ovary cells (20, 22). To minimize potential difficulties caused by differences in the concentrations of the proteins, we determined the amount which gave approximately the same immunoreac- tivity with the internal standard B105 and used this amount in the other Western blotting experiments (Figs. 4 and 5). Thus, the relative antibody binding to an analog can be determined by comparing the blots.

Residues Responsible for CG Selectivity to the BlOl Domain

BlOl Epitope-With the sandwich immunoblotting procedure, we found that BlOl bound to all chimeras which contained hCGp subunit residues Gly47 and Alas1 (Fig. 4 and Table II). For example, BlOl bound to constructs containing CGP, LCp41, CL@37AT, CLCb77-96, CL/396, and CGpAT better than to those containing LHP, CLp41, LCP87AT, CLCp43-58, CLDAT, or CLCP41-87AT. While BlOl bound to LCp41* (Fig. 4), it did not bind to the LH analog containing Thr instead of Ile15 (not shown) indicating that the glycosylation at Asn13 in hCGp does not influence BlOl binding. BlOl recognized an LH analog in which residues 47 and 51 were changed to those of hCG (i.e. LHP47/51AT) but failed to recognize CG in which residues 47 and 51 were changed to LH (i.e. CG/347/51). Thus Gly47 and Ala51 significantly influence the selectivity of BlOl for hCG.

B107 Epitope-B107 binds hCG much better than hCG@ and recognized only those chimeras which were combined with the (Y subunit (Fig. 4). Although B107 competes with BlOl for binding to hCG, it binds to different residues. Whereas BlOl bound CLP87AT with higher affinity than

hCG-

hCGP-

c

FIG. 3. Western blots of CG-LH p subunit chimeras using B105. Portions of tissue culture media were subjected to electrophoresis as described under “Materials and Methods.” After blotting, the analogs were detected using radiolabeled B105. Note that analogs containing CGB migrate slower than those having LHP termini or those designated “A?“’ due to the presence of either 24 or 31 additional C-terminal residues which contain four O-linked glycosylation sites. We chose these blots for illustration because they demonstrate the largest quantities of higher molecular weight forms we obsered. Most other blots had considerably less. We attribute these to aggregates; however, this has not been tested. In other studies we observed smaller molecular weight forms, particularly when we used radiolabeled B210. B210 has a very high affinity for the naturally occurring p subunit fragment whose structure is illustrated in Fig. l.* Although we have not analyzed this material in detail, it has the same electrophoretic mobility as the (3 subunit fragment isolated from urine. The mobilities of CG and LH dimers and free p subunits were similar to those of the urinary and pituitary standards with minor exceptions which can probably be attributed to glycosylation (18). We observed multiple bands, particularly for the free subunit forms. These forms were more pronounced when the N-linked glycosylation signal was present at Asni3-Thri5 suggesting that some forms may not be glycosylated at AsniS and/or that processing of the carbohydrates may be altered.

LCP87AT, B107 bound LCP87AT better than CLP87AT. B107 also recognized CLp43-58, CLp96, and CLP42-87AT better than CLp77-96. Thus, residues between l-87 and 96-121 do not appear to have a major role in the ability of B107 to distinguish hCG and hLH. Since B107 binds poorly to CLC/377-96, residues 89, 91, and 92 (i.e. the residues which differ between hLH/3 and hCGP in the region 88-95) are likely to be in the B107 binding site. Based on sandwich assays (Table II) residues near A$, and possibly Thr4’ also influence B107 binding. However, since the binding affinity of B107 to CLp41 and CLP87AT (lanes 5 and 7) was less than to LCp41 and LCP87AT (lanes 6 and 9) residues 8, 10, and 42 play a minor role in B107 binding compared to residues 89-92.

Bl09 Epitope-In contrast to BlOl and B107, B109 recognized both CLP87AT and LCP87AT suggesting that residues between l-87 and between 87-114 of the CG/3 subunit are responsible for the ability of this antibody to distinguish hCG from hLH. This is also supported by the observations that


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BlOl I

1 2 3 4 5 8 7 B 0 m 1112 13

hCG a

hCG8

- * ap *qp’tia

8107 I

1 2 3 4 5 8 7 8 9 10 11 12 13

BlOS

1 2 345 67 6 9 10 1, 12 13

hCG

hCW II) 0’

8204

FIG. 4. Western blots of CG-LH chimeras using antibodies to the BlOl/B204 domain. Portions of culture media were subjected to electrophoresis, electroblotted onto nitrocellulose which had been pre-coated with B105, and detected with radiolabeled BlOl (top row), B107 (second row), B109 (third row), or B204 (bottom row). Corresponding lanes in Figs. 3 and 4 received equal amounts of culture medium prior to electrophoresis. Note that B109 has much higher affinity for hCG than for hCG@ and recognized the free fl subunit in only about half of the blots. Nonetheless, the pattern of binding to the chimeras was consistent in all studies and the results illustrated here are typical of what we observed. Unlike the case in which B105 was used to detect hormones and analogs adsorbed to nitrocellulose in which we often observed higher molecular weight species, we rarely observed any such species in Western blots using other antibodies.

B109 bound to CL/342-58, CL@77-96, and CL@96 (Fig. 4), and that B109 recognized both LH/347/51AT and CGP47/51 (Table II). Thus, unlike BlOl and B107 where residues 47 and 51 and residues 89-92, respectively, are critical for binding, the presence of one of these regions seems to be sufficient for binding B109. B109 appears to bind better when both hCG/3 regions are present.

B204 Epitope-B204, an antibody prepared against the hCGP fragment (26), binds hCG/3 better than hCG (Table I). It retains this specificity in the chimeras and recognizes only the free P-subunit forms of CLp41, CLP87AT, CLCp43-58, CLCp77-96, CL@96, and CLC@41-87AT (Fig. 4). The binding of B204 to any analog having the first 15 amino acids of hCG/? subunit was comparable to that seen for hCGp. Thus, residues 2, 8, 10, and/or 15 play a role in B204 binding to LH. Substitution of Arg for hLH@ residue Trp’ enhanced binding to B204 (Table III) whereas the presence of oligosaccharide (at Asn’“) via a change of Thr’” to Ile’” had no effect (LC41*, lane 9). Since B204 binds to the hCGp fragment, which lacks the first five amino acids, with greater affinity than intact

TABLE II

Sandwich immunoassays to identify the BIOI, B107, and Bl09 binding specificities of LHp-CG/3 chimeras

Analogs were captured using B105 and detected with radiolabeled BlOl, B107, B109, or AllO. Values illustrated were corrected to exclude the influence of nonspecific binding (i.e. the amount of antibody which bound to wells that did not contain CG, LH, or chimeras) and are the means f S.E. of triplicates. Since All0 binds to CG, LH, and all the LH-CG chimeras, we assumed that the binding to All0 would indicate the amount of analog captured by B105. Therefore, we multiplied the net counts/min in each row by a factor which would normalize the binding of All0 to 4000 cpm. *, values illustrated for BlOl, B107, and B109 have been normalized in this fashion and should permit direct comparison of the relative abilities of these analogs to bind to the analogs shown. Values for All0 are the uncorrected net counts/min above the blank. Although not performed in this experiment, we have found that the ability of these antibodies to bind to (Y + LHBAT is similar to that of (Y + LH@.

Net antibody bound ArIalOgS

BlOl* B107* B109* All0

cpm cu+CGP 5906 + 65 1270 + 18 9370 k 125 3836 f 134 a+LHP 67 + 14 186 k 15 336 f 132 6914 k 112 01+ LHP-8AT 221+ 31 769 f 19 2320 + 43 4628 + 89 oi + LHP-42AT 4 f 11 616 f 27 1977 + 65 6148 f 206 (Y + LHP-47/ 5422 f 115 313 f 15 8090 + 34 4096 f 15

51AT (Y + CG/347/ 546 f 23 989 f 17 6960 + 292 6371 f 94

51AT (Y + LHP58A.T 301 f 11 543 5 16 1606 + 39 5392 + 57 (Y + LHP77AT 120 k 27 549 + 27 2188 + 39 4229 ? 42 a + LHP82/ 193 + 9 333 f 16 2509 + 51 5099 + 16

83A.T

TABLE III

Sandwich immunoassays to identify the B204 epitope binding specificity of CGfl-LHP chimeras

B204 was used as the capture antibody and radiolabeled B105 was used as a detection antibody. Values given are the means f S.E. of triplicates.

Net antibody Analog bound

B204

n+CGP n+LHP N + LHP8AT 01 + LH@8/lOAT (Y + LH/342AT LY + LHP47/51AT (Y + CG/J47/51AT (Y + LHP58AT (Y + LHP77AT 01+ LHfl82/83AT

wm 8,719 f 500

60 + 33 11,107 + 215 14,762 f 213

3,066 k 283 44 + 33

11,650 + 127 1,026 + 145

376 + 27 -6 + 48

hCGp (27), Lys* does not appear responsible for the ability of this antibody to discriminate hCG/3 from hLHP. Thus, Ar$ and possibly Arg” are important residues in the B204 binding sites. Since B204 competes with BlOl, residues 8-10 also appear to be near residues 47-51. Furthermore, since B204 binds to hCG@ much better than hCG and since B107 and B109 bind hCG much better than hCGj$ the data suggest that the relative positions of p subunit residues 8-10 (i.e. residues responsible for B204 selectivity), 47-51, and 89-92 (i.e. residues responsible for B107 and B109 selectivity) are changed when the subunits combine. This conformational change may be partly responsible for the enhanced ability of hCG to bind to receptors compared to hCG&


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Antibody Binding Sites on hCG

hCG-

hCGP- l

hCG-

FIG. 5. Western blots of CG-LH chimeras using Bill. Ra- diolabeled Blll was used to detect hormones and analogs that had been blotted onto nitrocellulose coated with B201. While Blll recognizes both dimer and monomer forms, due to the use of B201 as a capture antibody during electroblotting, the results seen here illustrate the binding of Blll to the p subunit.

TABLE IV

Sandwich immunoassays to identi/>l the RI08 and B112 epitope binding specificity for CG-LH b subunit chimeras

Net antibody bound Analog relative to B105 control

B108” B11P

n+CGP CY + LHn’ a + LHB81T o + LHB8/101T (Y + LH/X5*1T o + LHP42AT (Y + LH/347/5lAT cy + CGP47/51AT (Y + LH/358AT N + LHB771T

5,319 k 162 2,033 f 43 Not done Not done Not done 2,093 f 88 1,985 f 55 Not done’ 2,217 + 53 4,563 + 117

13,287 f 333 -178 f 61 -230 + 72 -119 f 75 -106 f 60

198 f 86 -133 f 66

18,773 t 473 -23 + 59

10,421 k 276 o( + LHP82/831T 1,520 + 66 70 + 72

’ Data for B108 were collected using A202 as the capture antibody and radiolabeled B108 and B105 as detection antibodies. Values illustrated represent the net counts/min B108 bound normalized to 10,000 cpm B105 bound.

*Data for Bl12 were obtained using B112 or BlO5 as capture antibodies and radiolabeled All0 as the detection antibody. Values illustrated represent the net counts/min All0 bound to B112 normalized to 10,000 cpm bound to B105.

‘In other studies we observed that B108 bound to this analog almost as well as it bound to u + CGB.

Identification of Residues Responsible for CG Selectivity of Bill

Bill did not bind to any derivatives lacking hCGP residues 115-145, nor did it bind to analogs such as CLp96 which contained the hLHp C terminus (Fig. 5). This suggests that Bill binds to residues between 112 and 145. Blll also binds

to hCG at the same time as SCTP, a monoclonal antibody directed against the hCG@ C terminus residues 115-145 (not shown). The higher affinity of Bill for hCG than for hCGP subunit (Table I) suggests that it recognizes residues closer to Asp”’ since this residue can be chemically cross-linked to the (Y subunit and thus this region would be influenced more by combination with the (Y subunit than the remainder of the C terminus.

Identification of Residues Which Are Exposed after CG Binds to Receptors

B108 and B112 compete with one another for binding to hCG and recognize all chimeras containing Asn” (not shown). Thus, B108 and B112 bound well to constructs containing CG& LCp41, CL/387AT, CLCp43-58, CLp96, and CGP?.T. They bound poorly to those containing LH& CLp41, LCP87AT, CLCp77-96, and CLCP42-87AT. B108 and B112 also bind LH in which AspT’ has been replaced by Asn, i.e. the corresponding residue found in CG (Table IV). These data suggest that this residue enables them to distinguish hCG from hLH and plays a large role in their binding sites.

DISCUSSION

The primary amino acid sequences of hCG, hLH, and other mammalian LH/3 subunits display considerable sequence di- versity (2). Since all /3 subunits combine with human or bovine (Y subunits, we assumed that p subunit chimeras were also likely to fold and combine with hCGol, particularly since the locations of the Cys residues would be conserved. The chimeras combined with hCGa and were recognized by monoclonal antibodies that have high affinity for both hLH and hCG. The chimeras also retained regions with significant properties of either hCG or hLH since they bind selectively to monoclonal antibodies which are specific for hCG. Thus once the /3 subunit has folded, the chimeras would maintain the characteristic domains of either hCG or hLH which would enable monoclonal antibody binding sites to be examined.

The chimeras enabled us to identify residues which are responsible for hCG antibody specificity. These residues are likely located in or near the antibody combining site. Based on the crystal structure of antibody-antigen complexes (16), the binding sites include several amino acid residues and occupy a surface of approximately 600-700 A’. Our data show that only one or two amino acid residues were sufficient to enable some antibodies to distinghish hLH from hCG. For example, an LHP construct with a change of Aspv7 to Asn, the corresponding residue in hCG/3, was recognized by the hCG-specific antibodies Bl08 and B112.

While we anticipate that residues which enable antibodies to distinguish hLH and hCG are part of the antibody binding site, it is possible that they may influence the binding through distant conformational changes. This may explain the differences in affinities of (Y subunit-directed antibodies for hCG, hLH, and human follicle-stimulating hormone (35). However, several observations suggest that residues which confer antibody binding specificity are likely to be near or within the antibody binding site. First, antibody binding sites in several different regions of the p subunit can be changed without influencing binding of antibodies to other domains. For example, we altered the binding of B108 and B112 without changing the binding to the BlOl or Blll domains. We were also able to influence binding of antibodies within the same epitope domain in a selective fashion. For example, we found that residues 47 and 51 had a major influence on the binding of BlOl and B109 but not on B107 or B204. Second, studies using synthetic peptides and the hCGp fragment, revealed


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hCG AN0 hLJl PESIDDES WJIICB DETERHINB ANIIBGDY BINDING SPECIE’ICITY

hCG hLB

hMi hLs

hCG hLB

hCG hI.a

hCG hLB

hCG hlJI

hCG hLB

hC!G hL.B

B204 20

S0rLy~GlrrPr0L~~rpPr0Arg~~~~Pr0Il~A~nAl~ThrL~~l~V~1GlrrLy~ -h,- - - - - Trp - Bi, - - - - Ilc - - - - -

40 GlnGlyCy~ProV~1CyrI1e~rV~lA~n~r~rIle~aAl~Gl~r~~Pro~r

-__-_- ----- - --------

BlOl/B107/B109 60 YetThrAtpV~1LeoOlnGlyV~lLerrProAlILonPrffilnV~1V~l~sA.nTyr~g

-Met- - _ -Ala- _ -pro- - - _ - -‘,,,r- -

BlOSlBll2 80 AapValArgPheGlnSerI1eAr6LerSroGlyCysProA~GlyV~lA~~roV~1V~l

_-_____-__-__- _ -Asp- _ -

B107/8109 100 SerTyrAl~V~1AlILenSerCyaGlnCysAllLenCyrArgArgSerThrThrAspCy.

- PhePro - - - - - Arg - GlyPro - - - - - Ser - -

Blll 120 GlyGlyProLyrAspBirProLanThrCyaA~pA~pProArgPheOlnAIpSerSerSer

- - - - - - - - - - - His - GlaLeuSerGlyLenLeuPhe

ProIleLeuProGln

FIG. 6. Sites that confer the abilities of hCG selective antibodies to distinguish KG from hLH. Based on the data obtained from a large number of studies similar to those illustrated in Tables II-IV and Figs. 4 and 5, we identified residues present in hCG which enable the molecule to be recognized by hCG-selective monoclonal antibodies. These residues are listed here and suggest that we have identified most regions of hCG which are responsible for antibody selectivity.

that BlOl binds with approximate dissociation constants of 10 and 100 PM to synthetic peptide loops containing hCG and hLH residues Cy~~s-Cys~~, respectively (13). Furthermore, based on the data presented here for the B204 binding site (i.e. residues near A$), a peptide was synthesized which was capable of binding to antibodies B204 and B210 with micro- molar aftinity.3 Third, based on the results obtained using chimeras, we identified how the antibodies would bind to the hCGB fragment. Thus, as predicted from the summary illustrated in Fig. 6, B108, B112, and B204 but not BlOl, B107, B109, or Blll bind to the hCG/3 fragment (cf primary sequence shown in Fig. 1).

Based on the data in Fig. 6 and our topological map of hCG (Fig. 2), we constructed a model of the structure of the hCGp subunit (Fig. 7). This model is useful for visualizing hCG/3 and its interactions with hCGa. Based on the importance of residues 8-10, 47-51, and 89-92 for binding of BlOl, B107, B109, and B204 to an overlapping region on hCG/3, we placed these residues adjacent to each other on the surface of the molecule. This region was located away from Asn77 and residues 112-121, the presumed binding sites comprising the B108/B112 and Blll epitopes. The model is consistent with the disulfide bond assignments reported by Bahl’s laboratory (25) although we cannot exclude a possible disulfide bond between Cysg and Cysss instead of between Cysg and Cysgo.

3 S. Birken, R. K. Campbell, and W. R. Moyle, unpublished observations.

FIG. 7. Model of fl subunit. Based on the data illustrated in Figs. 2 and 6, we prepared a wire model of the hCG@ which is consistent with the locations of residues which strongly influence antibody binding. This view from the 01 subunit interface summarizes the data in a form which can be visualized in three dimensions and is useful for characterizing the approximate spatial positions of some @ subunit residues. It is also consistent with the disulfide bonds reported by Mise and Bahl(25). Disulfide bonds and residues which differ between hCG and hLH have been numbered.

The model is also useful for visualizing the relative positions of hCGa and hCG/3 and changes in hCG@ which might occur when the subunits combine. We have illustrated a view of hCGp which would be seen from the a subunit interface. Support for this location of the a subunit is based on the ability of hCG@ Asp”’ to be cross-linked to the a subunit of bovine LH (33), the low chemical reactivities of Tyr37, Tyr5’, and Tyr” in the dimer relative to those of free hCGB (2), and the inability of most a subunit antibodies to bind to hCG at the same time as BlOl, B107, and B109. This location is also consistent with the observation that As@ is important for subunit combination during synthesis (22). We postulate that the structure of hCG@ is changed when it combines with hCGcv so that residues near 47-51 and 89-92 can interact with B107 and B109. At the same time, this displaces the region of the molecule near Cysg and disrupts the B204 binding site. These hypotheses are consistent with the observation that B109 binds to two noncontiguous regions of hCG, namely one in the 38-57 loop and one near residues 89-92.

The model can also be used to visualize regions of the molecule which may be important for receptor interaction. Since B108 and B112 bind to hCG-receptor complexes, it seems likely that residues near Asn77 are not in the hormone- receptor interface. Antibodies to the BlOl and Blll domains do not bind to the hormone-receptor complex suggesting that these regions may be near the receptor interface or that they are altered when the hormone binds to receptors. Although the model does not show which specific residues interact with the receptors, the epitope map predicts that the BlOl/B204 and Blll domains are near the receptor interface. This result is in agreement with data from other laboratories. Keutmann et al. (15) found that oligopeptides Cy~~‘-Cys~~ and Cysg3- CydW inhibited binding of hCG to receptors as would be expected if these residues participated in receptor binding. Bidart et al. (12) suggested that the region of the molecule


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Antibody Binding Sites on hCG’

near Cys”’ is involved in receptor interaction consistent with our finding that Bill competes with receptor for binding hCG and fails to bind to hCG receptor complexes4

8. Moyle, W. R., Pressey, A., Dean-Emig, D., Anderson, D. M., Demeter, M., Lustbader. J.. and Ehrlich, P. (1987) J. Biol.

Many of the residues which differ between hCG and hLH are located adjacent to one another in space even though they are in different regions of the linear sequence. Indeed, half of the amino acid substitutions in the first 114 residues of hLH@ and hCG/3 are located in regions of the molecule recognized by antibodies which strongly inhibit binding of hCG to receptor complexes. This may account for the finding that antisera produced in men after hCG administration contain inhibitory antibodies which map to this region of the molecule (4). Since many of the differences between hCGP and hLH@ do not appear equally distributed on the surface of the molecule but occur in regions of the molecule which appear to be obscured when the hormones bind to receptors, they may have a phys- iological role.

In summary, LHP-CGP chimeras are a powerful tool for identifying residues which contribute to the binding sites of hLH- and hCG-specific monoclonal antibodies. We anticipate that chimeras derived from other members of this family will be necessary to identify the binding sites of antibodies which recognize hCG and hLH. Since epitope maps are available which illustrate the relative binding sites of the antibodies, these studies provide information needed for developing a model of the hormones and for identifying functional regions of hCG& Chemically deglycosylated hCG (36) and enzymati- tally desialylated hCG (40) have recently been crystallized and a high resolution structure is expected within the next few years. By combining the results of crystallography with those of mutagenesis, we should obtain an excellent model of the structure and function of these important hormones.

Acknowledgments-We would like to thank Dr. John Fiddes for the hCGa and fl subunit cDNA used in these studies. We thank Drs. Glenn Armstrong and Robert Wolfert of Hybritech Co. for some of the @ subunit antibodies and Drs. Richard Krogsrud and S. Berube of BioMega Co. for some of the 01 subunit antibodies used in these studies. We appreciate the expert help of Kathy Valentine and Carol Patterson in the preparation of this manuscript, Michael Bernard for suggestions of how Fig. 7 should be drawn, and Rong-Zeng Li for drawing the figure.

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R W Barnett and I BoimeW R Moyle, M M Matzuk, R K Campbell, E Cogliani, D M Dean-Emig, A Krichevsky,chorionic gonadotropin/luteinizing hormone beta subunit chimeras and mutants.

Localization of residues that confer antibody binding specificity using human

1990, 265:8511-8518.J. Biol. Chem.

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