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Title: Conversion of human choriogonadotropin into a follitropin by protein engineering

Abstract

Human reproduction is dependent upon the action of follicle-stimulating hormone (hFSH), luteinizing hormone (hLH), and chorionic gonadotropin (hCG). While the {alpha} subunits of these heterodimeric proteins can be interchanged without effect on receptor-binding specificity, their {beta} subunits differ and direct hormone binding to either LH/CG or FSH receptors. Previous studies employing chemical modifications of the hormones, monoclonal antibodies, or synthetic peptides have implicated hCG {beta}-subunit residues between Cys-38 and Cys-57 and corresponding regions of hLH{beta} and hFSH{beta} in receptor recognition and activation. Since the {beta} subunits of hCG or hLH and hFSH exhibit very little sequence similarity in this region, the authors postulated that these residues might contribute to hormone specificity. To test this hypothesis the authors constructed chimeric hCG/hFSH {beta} subunits, coexpressed them with the human {alpha} subunit, and examined their ability to interact with LH and FSH receptors and hormone-specific monoclonal antibodies. Surprisingly, substitution of hFSH{beta} residues 33-52 for hCG{beta} residues 39-58 had no effect on receptor binding or stimulation. However, substitution of hFSH{beta} residues 88-108 in place of the carboxyl terminus of hCG{beta} (residues 94-145) resulted in a hormone analog identical to hFSH in its ability to bind and stimulate FSH receptors. The altered binding specificity displayedmore » by this analog is not attributable solely to the replacement of hCG{beta} residues 108-145 or substitution of residues in the determinant loop located between hCD{beta} residues 93 and 100.« less

Authors:
; ;  [1]
  1. (Univ. of Medicine and Dentistry of New Jersey, Piscataway (United States))
Publication Date:
OSTI Identifier:
5043488
Resource Type:
Journal Article
Resource Relation:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (United States); Journal Volume: 88:3
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; GONADOTROPINS; GENETIC ENGINEERING; PEPTIDE HORMONES; AMINO ACID SEQUENCE; DOSE-RESPONSE RELATIONSHIPS; FSH; IMMUNOLOGY; IODINE 125; LH; MONOCLONAL ANTIBODIES; PLASMIDS; REPRODUCTION; TESTOSTERONE; ANDROGENS; ANDROSTANES; ANTIBODIES; BETA DECAY RADIOISOTOPES; BIOTECHNOLOGY; CELL CONSTITUENTS; DAYS LIVING RADIOISOTOPES; ELECTRON CAPTURE RADIOISOTOPES; HORMONES; HYDROXY COMPOUNDS; INTERMEDIATE MASS NUCLEI; INTERNAL CONVERSION RADIOISOTOPES; IODINE ISOTOPES; ISOTOPES; KETONES; MOLECULAR STRUCTURE; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; PITUITARY HORMONES; PROTEINS; RADIOISOTOPES; STEROID HORMONES; STEROIDS; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Campbell, R.K., Dean-Emig, D.M., and Moyle, W.R.. Conversion of human choriogonadotropin into a follitropin by protein engineering. United States: N. p., 1991. Web. doi:10.1073/pnas.88.3.760.
Campbell, R.K., Dean-Emig, D.M., & Moyle, W.R.. Conversion of human choriogonadotropin into a follitropin by protein engineering. United States. doi:10.1073/pnas.88.3.760.
Campbell, R.K., Dean-Emig, D.M., and Moyle, W.R.. 1991. "Conversion of human choriogonadotropin into a follitropin by protein engineering". United States. doi:10.1073/pnas.88.3.760.
@article{osti_5043488,
title = {Conversion of human choriogonadotropin into a follitropin by protein engineering},
author = {Campbell, R.K. and Dean-Emig, D.M. and Moyle, W.R.},
abstractNote = {Human reproduction is dependent upon the action of follicle-stimulating hormone (hFSH), luteinizing hormone (hLH), and chorionic gonadotropin (hCG). While the {alpha} subunits of these heterodimeric proteins can be interchanged without effect on receptor-binding specificity, their {beta} subunits differ and direct hormone binding to either LH/CG or FSH receptors. Previous studies employing chemical modifications of the hormones, monoclonal antibodies, or synthetic peptides have implicated hCG {beta}-subunit residues between Cys-38 and Cys-57 and corresponding regions of hLH{beta} and hFSH{beta} in receptor recognition and activation. Since the {beta} subunits of hCG or hLH and hFSH exhibit very little sequence similarity in this region, the authors postulated that these residues might contribute to hormone specificity. To test this hypothesis the authors constructed chimeric hCG/hFSH {beta} subunits, coexpressed them with the human {alpha} subunit, and examined their ability to interact with LH and FSH receptors and hormone-specific monoclonal antibodies. Surprisingly, substitution of hFSH{beta} residues 33-52 for hCG{beta} residues 39-58 had no effect on receptor binding or stimulation. However, substitution of hFSH{beta} residues 88-108 in place of the carboxyl terminus of hCG{beta} (residues 94-145) resulted in a hormone analog identical to hFSH in its ability to bind and stimulate FSH receptors. The altered binding specificity displayed by this analog is not attributable solely to the replacement of hCG{beta} residues 108-145 or substitution of residues in the determinant loop located between hCD{beta} residues 93 and 100.},
doi = {10.1073/pnas.88.3.760},
journal = {Proceedings of the National Academy of Sciences of the United States of America; (United States)},
number = ,
volume = 88:3,
place = {United States},
year = 1991,
month = 2
}
  • We have devised a radioimmunoassay (RIA) for human choriogonadotropin (hCG) in first morning-voided urine specimens. Concanavalin A, a lectin, is used to extract and concentrate the hCG from urine. A high-affinity antiserum is used, directed to the hCG..beta.. carboxy-terminal peptide, a unique immunological determinant not shared by the beta subunit of human lutropin. This ensures that urinary human lutropin-related molecules, which interfere with RIAs involving antisera to the intact hCG..beta.. subunit, will not cross react in this assay. A concentration of hCG as low as 0.4 ..mu..g/L can be detected in the first morning-voided urine. The effective sensitivity of thismore » assay for the unequivocal detection of hCG production is somewhat better than that achieved with the serum hCG RIA involving antisera to the hCG..beta.. subunit. The improved specificity and sensitivity of this assay, and the greater convenience of collecting samples of urine rather than blood, are clinically useful advantages of this approach to assessing hCG production in humans.« less
  • We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptidesmore » spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.« less
  • Three monoclonal antibodies were raised against the free alpha subunit of choriogonadotropin (hCG); each recognized a different antigenic site on the molecule. One (antibody 42) preferentially bound to the alpha subunit when it was coupled to the beta subunit as dimeric choriogonadotropin (hCG), thyrotropin (TSH), lutropin (LH), or follitropin (FSH). Antibody 71 showed some cross-reaction with intact FSH; antibody 75 was more specific for the alpha subunit. All were of low affinity (10(-7) to 10(-8) mol/L), but when combined in immunoradiometric assays (IRMAS) they proved to be as sensitive as current radioimmunoassays involving polyclonal antibodies. Advantages of the combination ofmore » antibody 75 bound to the solid phase and antibody 71 as the radiolabeled antibody were: detection limit of at least 0.1 micrograms/L; linear dilution of serum and urine; insignificant cross-reaction with intact hCG, allowing direct assay in pregnancy fluids; and a coefficient of variation less than 3% over the reference interval for nonpregnant women. There was 4% cross-reaction with intact FSH, suggesting that the epitopes recognized by nos. 71 and 75 are more exposed in FSH and that perhaps there is less folding in this molecule than in intact hCG.« less
  • A comparison was made of 10 commercially available radioimmunoassay kits (American Diagnostics, Becton Dickinson, BioGenex, Clinical Assays, Hybritech, Leeco, Mallinckrodt, Microanalytic, Nuclear Medical Systems, and Radioassay Systems) for determination of human choriogonadotropin (hCG), using serum pools, hCG CR119 (NIH), 2nd I.S. (who), and 1st I.R.P. (who). Criteria were ease of performance, total assay time, sensitivity, potency, and parallelism as compared with reference standards and results for 15 serum pools. The Mallinckrodt kit exhibited the best overall performance, with good low-concentration sensitivity, parallelism with two of the three reference preparations, and good clinical correlation as compared with the reference kit frommore » NIH. Because the antibodies used in the kits are occasionally changed by the manufacturers, these results are necessarily valid only for kits that include reagents identical to those in the kits that we tested.« less
  • Luteal and interstitial/thecal elements of the ovary failed to show preferential accumulation of the label originally associated with the beta-subunit. Measurement of both radioactivities in crude subfractions of the ovarian tissues revealed that granulosa cells retain the excess of beta-subunit label in a plasma membrane/vesicular component. No such preferential retention of label was seen in any of the subfractions obtained from luteal or interstitial/thecal tissues. The radiolabeled components associated with the granulosa cells were shown to be mainly macromolecular by their precipitability with 13% trichloroacetic acid. Luteal tissue degraded the components associated with each label more rapidly than granulosa cells.more » In contrast, interstitial/thecal tissue degraded very little of the bound labeled components. The differential processing of individual hCG subunits by granulosa cells was shown not to result from different kinetics of binding of serum-borne hormone by two methods. Thus, changes over time in the ability of circulating hormone to bind to LH receptor in vitro were shown not to be a function of the hCG subunit having the label. Moreover, blockade of further radiolabel uptake by injection of a large excess of unlabeled hCG 30 min after radiolabel administration did not alter the rise in the ratio of beta-subunit label to alpha-subunit label normally observed in granulosa cells. The ability of kidney tissue to accumulate and metabolize hCG also varied with the physiological state. These studies demonstrate that differences exist in the metabolism of hCG by the various target cells of the ovary and that changes in processing occur during luteinization.« less