Free α subunit of human chorionic gonadotrophin: molecular basis of immunologically and biologically active domains

1994 ◽  
Vol 140 (1) ◽  
pp. 145-154 ◽  
Author(s):  
S Dirnhofer ◽  
O Lechner ◽  
S Madersbacher ◽  
R Klieber ◽  
R de Leeuw ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Receptor Binding ◽  
Solid Phase ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Biologically Active ◽  
Molecular Organization ◽  
Antigenic Determinants ◽  
Α Subunit

Abstract Immunochemical studies were undertaken to identify surface-orientated epitopes of the free α subunit of human chorionic gonadotrophin (hCG-α) at the amino acid sequence level. We investigated the molecular organization of these epitopes, resolved the immunological topography in terms of spatial arrangement of antigenic domains and related structures to functions such as subunit association or receptor binding. Overlapping synthetic peptides covering the entire amino acid sequence of hCG-α, an enzymatically digested hCG-α subunit, and a reduced and alkylated hCG-α preparation were assayed in a solid-phase one-site enzyme-linked immunoassay, and in a solution-phase competitive radioimmunoassay (RIA). The antigenic topography was mapped by monoclonal antibodies (MCAs) in two-site binding assays (sandwich RIA). On the surface of hCG-α, seven different epitopes (α1–α7), arranged in four spatially distinct domains, could be distinguished: A, α1,2,4; B, α3,5; C, α6; D, α7. The peptides spanning hCG-α(13–18), hCG-α(17–22) and hCG-α(33–42) appeared to contribute to the formation of epitopes α2, α4 and α6 respectively. Since epitope α6 is present only on the free non-assembled subunit of different species, we concluded that the region hCG-α(33–42), which is evolutionarily highly conserved, represents a subunit assembly site. All but one epitope (α7) are destroyed by reducing and alkylating hCG-α. In contrast, chymotryptic digestion of hCG-α, leading to release of the heptapeptide hCG-α(41–47), did not affect epitope expression, indicating that this sequence is not involved in the formation of antigenic determinants. Addressing the biological properties of hCG-α epitopes by radioreceptor assay revealed that the three hCG-α peptides corresponding to epitopes α2, α4 and α6 did not displace radiolabelled hCG from its receptor, whereas any of the MCAs directed against determinants (α1–α5), shared by hCG and hCG-α, totally inhibited binding. Consistent with this, the antibodies neutralized the biological activity of hCG in terms of testosterone production in a mouse Leydig cell in vitro bioassay. We therefore concluded that hormone antibody-binding sites differ from those of hormone receptor binding, revealing no essential congruence of immunologically and biologically active domains. Journal of Endocrinology (1994) 140, 145–154

The molecular basis for epitopes on the free β-subunit of human chorionic gonadotrophin (hCG), its carboxyl-terminal peptide and the hCGβ-core fragment

1994 ◽  
Vol 141 (1) ◽  
pp. 153-162 ◽  
Author(s):  
S Dirnhofer ◽  
S Madersbacher ◽  
J-M Bidart ◽  
P B W Ten Kortenaar ◽  
G Spöttl ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Basis ◽  
Tertiary Structure ◽  
Solid Phase ◽  
Critical Role ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Antigenic Determinants ◽  
Β Subunit ◽  
Carboxyl Terminal

Abstract The molecular basis for antigenic determinants on the free β-subunit of human chorionic gonadotrophin (hCGβ), its carboxyl-terminal peptide (hCGβCTP) and the hCGβcore fragment (hCGβcf) was elucidated by means of monoclonal antibodies (MCAs). The objective of the present study was to resolve the antigenic topography of these three molecules in terms of epitope identification at different levels of structural organization as well as analysis of their spatial arrangement. An hCGβcf preparation, a synthetic peptide corresponding to the hCGβCTP (β109–145), overlapping synthetic peptides spanning the entire amino acid sequence of hCGβ, and a reduced and alkylated hCGβ preparation were assayed in a solid-phase one-site enzyme-linked immunoassay and in a solublephase direct-binding radioimmunoassay (RIA) or competitive RIA. The antigenic topography was mapped by incorporating the MCAs into two-site binding assays. On the surface of free hCGβ, nine different epitopes (β1–β9), arranged in three spatially distinct domains, could be distinguished. Epitopes β1–β7 were located in a single large domain on both hCGβ and the hCGβcf whereas hCGβCTP contained two topographically distant determinants, designated β8 and β9 respectively. All but the two epitopes located on hCGβCTP (β8 and β9) were destroyed by reducing and alkylating hCGβ, suggesting that most antigenic determinants are predominantly non-contiguous and require an intact tertiary structure whereas the molecular structure of hCGβCTP is linear. At a molecular level, amino acid residues spanning hCGβ 45–52, hCGβ 137–144 and hCGβ 113–116 contributed to the formation of epitopes β5, β8 and β9 respectively. We have also shown that the hCGβcf represents the immunodominant part of the free β-subunit of hCG, containing seven mainly conformationally determined epitopes, one of which has a share of the sequence β45–52. The hCGβCTP does not play a critical role in the immunologically important tertiary structure of hCGβ and was itself found to be a predominantly continuous sequence also within the native hormone, expressing two spatially distant antigenic determinants located within residues β113–116 and β137–144 respectively. Journal of Endocrinology (1994) 141, 153–162

Lysine residues 2 and 104 of the human chorionic gonadotrophin β subunit influence receptor binding

1993 ◽  
Vol 10 (3) ◽  
pp. 337-343 ◽  
Author(s):  
H Xia ◽  
J Huang ◽  
T-M Chen ◽  
D Puett
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Basic Amino Acid ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Amino Acid Residues ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Lysine Residues

ABSTRACT Human chorionic gonadotrophin (hCG), like other members of the glycoprotein hormone family, contains a common α subunit and a hormone-specific β subunit. The latter is a 145 amino acid residue polypeptide with six sites of glycosylation. Positions 2 and 104 are occupied by basic amino acid residues in the 12 known amino acid sequences of mammalian β subunits from CG and LH, a related gonadotrophin that acts through the same receptor. Lysine residues are found in both these positions in hCG-β. Using site-directed mutagenesis, each of these two lysines in hCG-β was replaced with glutamic acid. The mutant and wild-type cDNAs were subcloned into a eukaryotic expression vector, which was then transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for the bovine α subunit. Holoprotein formation occurred with each of the two heterologous gonadotrophin mutants, i.e. the bovine α subunit bound to hCG-β (Glu2) and to hCG-β (Glu104), as well as with the control, i.e. the bovine α subunit bound to the hCG-β wild-type subunit. In two in-vitro assays, one a competitive binding assay with 125I-labelled hCG as bound ligand and the other based on stimulation of progesterone production in a transformed murine Leydig cell line, MA-10, both the heterodimers containing a mutant β subunit exhibited bioactivity, but their potencies were lower than that of the bovine α subunit bound to the hCG-β wild-type subunit. These results suggest that the basic amino acid residues at positions 2 and 104 in hCG-β participate, either directly or indirectly, in receptor binding.

Monoclonal antibodies against the free subunits of human chorionic gonadotrophin

1990 ◽  
Vol 125 (2) ◽  
pp. 301-309 ◽  
Author(s):  
P. Berger ◽  
R. Klieber ◽  
W. Panmoung ◽  
S. Madersbacher ◽  
H. Wolf ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cross Reactivity ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Antigenic Determinants ◽  
Α Subunit ◽  
Α Chain ◽  
Fluid Levels ◽  
Α Subunits

ABSTRACT Discordant results on body fluid levels of human chorionic gonadotrophin (hCG) free α- and β-subunits under physiological and pathophysiological conditions, prompted us to raise a total of 260 monoclonal antibodies (MCA) against free hCG-α, free hCG-β, holo-hCG, human follicle-stimulating hormone and bovine luteinizing hormone; 153 MCA recognizing the human α-subunit and 28 reacting with hCG-β were extensively analysed for their intra- and interspecies cross-reactivity with homologous hormones, and for the compatibility of epitopes recognized by them. The immunological topography of free hCG-α and free hCG-β was resolved by these MCA, and epitope maps were designed. Six antigenic determinants on the free α-chain (α1–α6), clustered in three spatially distinct domains, and seven epitopes on the surface of free hCG-β (β1–β7), could be distinguished. Strikingly, three α-chain epitopes (α4, α5 and α6) were shared between various species, which is in contradiction to the concept of immunological species-specificity of α-subunits. Three determinants were found to be present only on the free subunits but not on holo-hCG (α6, β6 and β7), and only two determinants (β1 and β7) were hormone-specific for hCG. Based on this information, an immunoenzymometric assay for the free α-subunit of human glycoprotein hormones was established, with a sensitivity of 1·3 pg/well and a cross-reactivity with holo-hCG of less than 0·005% Thus this assay provides the basis for detecting free α-subunits in the presence of extremely high levels of holo-hormones, which may assist in elucidating the role of free α-subunits in man. Journal of Endocrinology (1990) 125, 301–309

scholarly journals The Amino Acid Sequence of the A Protein (α Subunit) of the Tryptophan Synthetase of Escherichia coli

1967 ◽  
Vol 242 (22) ◽  
pp. 5397-5412 ◽  
Author(s):  
John R. Guest ◽  
Bruce C. Carlton ◽  
Charles Yanofsky
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Α Subunit ◽  
Tryptophan Synthetase

scholarly journals The Amino Acid Sequence of the A Protein (α Subunit) of the Tryptophan Synthetase of Escherichia coli

1967 ◽  
Vol 242 (22) ◽  
pp. 5442-5446 ◽  
Author(s):  
John R. Guest ◽  
Gabriel R. Drapeau ◽  
Bruce C. Carlton ◽  
Charles Yanofsky
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Α Subunit ◽  
Tryptophan Synthetase

Purification, characterization, gene cloning and preliminary X-ray data of the exo-inulinase from Aspergillus awamori

2002 ◽  
Vol 362 (1) ◽  
pp. 131-135 ◽  
Author(s):  
Michael ARAND ◽  
Alexander M. GOLUBEV ◽  
J. R. Brandao NETO ◽  
Igor POLIKARPOV ◽  
R. WATTIEZ ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Solid Phase ◽  
Aspergillus Awamori ◽  
Glycoside Hydrolase Family ◽  
Orthorhombic Space Group ◽  
Ph Optimum ◽  
X Ray ◽  
Cell Parameters ◽  
Hydrolysis Of

Extracellular exo-inulinase has been isolated from a solid-phase culture of the filamentous fungus Aspergillus awamori var. 2250. The apparent molecular mass of the monomer enzyme was 69±1kDa, with a pI of 4.4 and a pH optimum of 4.5. The enzyme hydrolysed the β-(2 → 1)-fructan (inulin) and β-(2 → 6)-fructan (levan) via exo-cleavage, releasing fructose. The values for the Michaelis constants Km and Vmax in the hydrolysis of inulin were 0.003±0.0001mM and 175±5μmol·min−1·mg−1. The same parameters in the hydrolysis of levan were 2.08±0.04mg/ml and 1.2±0.02μmol/min per mg, respectively. The gene and cDNA encoding the A. awamori exo-inulinase were cloned and sequenced. The amino acid sequence indicated that the protein belongs to glycoside hydrolase family 32. A surprisingly high similarity was found to fructosyltransferase from Aspergillus foetidus (90.7% on the level of the amino acid sequence), despite the fact that the latter enzyme is unable to hydrolyse inulin and levan. Crystals of the native exo-inulinase were obtained and found to belong to the orthorhombic space group P212121 with cell parameters a = 64.726 Å (1Å = 0.1 nm), b = 82.041 Å and c = 136.075 Å. Crystals diffracted beyond 1.54 Å, and useful X-ray data were collected to a resolution of 1.73 Å.

A COMPARISON BETWEEN CHORIONIC GONADOTROPHINS EXTRACTED FROM HUMAN, RHESUS MONKEY AND MARMOSET PLACENTAE

1972 ◽  
Vol 55 (2) ◽  
pp. 363-368 ◽  
Author(s):  
BRUCE HOBSON ◽  
LEIF WIDE
Keyword(s):  
Biological Activity ◽  
Rhesus Monkey ◽  
Human Placenta ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Antigenic Determinants

SUMMARY Evidence is provided to show that chorionic gonadotrophins extracted from the human, rhesus monkey and marmoset placentae have antigenic determinants in common. Similar slopes were obtained for these gonadotrophins in a radioimmunoassay for human chorionic gonadotrophin (HCG). The biological activity of the monkey gonadotrophins was neutralized by anti-HCG serum. When the gonadotrophic activity of the monkey placental extracts was assayed biologically and immunologically, using HCG as a standard, similar results were obtained. Higher values were obtained by the immunoassay than by the bioassay when extracts of human placenta were assayed using the same HCG standard.

Specific enzyme immunoassay for human chorionic gonadotrophin

1981 ◽  
Vol 97 (4) ◽  
pp. 562-568 ◽  
Author(s):  
Tatsuhiro Sekiya ◽  
Yoshihito Furuhashi ◽  
Setsuko Goto ◽  
Shigeaki Kaseki ◽  
Yutaka Tomoda ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Serum Samples ◽  
Coefficients Of Variation ◽  
Highly Sensitive ◽  
Assay Tube ◽  
Sandwich Type ◽  
Serial Samples

Abstract. A sandwich-type enzyme immunoassay system specific for human chorionic gonadotrophin (hCG) was prepared with the antibody Fab'-β-D-galactosidase complex and the antibody F(ab')2-immobilized silicone rubber solid phase by using a purified antibody to β subunit of hCG (hCGβ). The assay system cross-reacted less than 4% with human luteinizing hormone (hLH) and human follicular stimulating hormone (hFSH), and proved to be highly sensitive with hCG measurable at levels as low as 0.3 mIU per assay tube. Using 50 μl of serum sample, 6–600 mIU/ml of hCG in serum could be determined specifically with the same degree of precision as in radioimmunoassay but without sample interference with the assay. The coefficients of variation within-run and between-run were 8.6–8.9%, and 4.9– 10.7%, respectively. Values obtained with the enzyme immunoassay correlated well with those of radioimmunoassay ([unk] = 0.98, slope = 0.94, y-intercept = 10.2 mIU/ml for 75 serum samples). Results of the immunoassay of hCG levels in serial samples of serum from healthy women and patients with choriocarcinoma show that this method is useful in the clinical diagnosis of trophoblastic disease.

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