Fragmentation of the β-Subunit of Human Chorionic Gonadotropin Produced by Choriocarcinoma*

Nishimura, Ryuichiro; Ide, Kazuki; Utsunomiya, T; Kitajima, Takashi; Yuki, Yoshikazu; Mochizuki, Matsuto

doi:10.1210/endo-123-1-420

Cited by 31 publications

(12 citation statements)

References 24 publications

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“…Several groups, including ours, have reported peptide bond nicks in the 0-subunits of the pituitary glycoprotein hormones (1)(2)(3) and in hCG (4)(5)(6)(7)(8)(9). The reports of Puisieux et al (8) and Sakakibara et al (9) have found that hCG isolated from the urine of pregnant individuals has a peptide bond nick in the 0-subunit between residues 47-48, while our companion report (10) shows that some preparations of hCG are also nicked at 044-45 and 046-47.…”

mentioning

confidence: 71%

The Heterogeneity of Human Chorionic Gonadotropin (hCG). II. Characteristics and Origins of Nicks in hCG Reference Standards*

Birken

Gawinowicz²,

Kardana³

et al. 1991

Endocrinology

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hCG, the hormone produced by the trophoblast throughout pregnancy, has peptide bond cleavages, or nicks, in the beta-subunit. We sought to compare the nature of these nicks in standard reference preparations of hCG, to determine the enzymes that may be responsible for generating the peptide bond cleavages, and to devise means of separating nicked from intact hormone. The standard reference preparations of hCG, which are purified from a commercial product made from large pools of pregnancy urine, were found to have varying concentrations of nicked hormone. The preceding report showed that 11 of 13 hCG preparations isolated from individual pregnancy urine samples were nicked at the beta 47-48 bond, with 2 of 13 having a second nick at beta 44-45. As shown here, all of the hCG reference standards are nicked to similar extents at both the beta 47-48 bond and the beta 44-45 bond. The percentage of peptide bond nicking in the various hCG standard preparations ranged from 10-20% and appeared higher in the more recent preparations. We showed that human leukocyte elastase is capable of specifically cleaving the beta 44-45 bond, and in extended digests it can also cleave the beta 48-49 and beta 51-52 peptide bonds. Thus, human leukocyte elastase may be the origin of some of these cleavages in the individual samples and the reference standards. Furthermore, we report that a monoclonal antibody directed to hCG alpha-beta dimer binds preferentially to nonnicked hCG and much less to nicked hCG.

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mentioning

confidence: 71%

The Heterogeneity of Human Chorionic Gonadotropin (hCG). II. Characteristics and Origins of Nicks in hCG Reference Standards*

Birken

Gawinowicz²,

Kardana³

et al. 1991

Endocrinology

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“…Nishimura et al (4), Bidart and colleagues (5), Sakakibara et al (8), and Puisieux and colleagues (7) all used electrophoresis of reduced hCG and Western blotting with C-terminal peptide antisera to detect nicked molecules. Using this method we were able to demonstrate the broad occurrence of nicked molecules in pregnancy and trophoblast disease fluids, detecting nicks in 19 of 24 urine and 10 of 12 serum hCG samples.…”

Section: Discussionmentioning

confidence: 99%

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced hCG, followed by Western blotting and staining of the blot with antibodies to the 0-subunit Cterminal peptide region, permits visualization of nicked hCG (4)(5)(6)(7)(8). An example of this method is shown in Fig.…”

Section: Western Blots Of Nicked Hcgmentioning

confidence: 99%

“…Compari-1560 OCCURRENCE AND ACTIVITIES OF NICKED hCG Endo • 1991 Voll29«No3 sons of nicked and intact LH preparations have indicated that these nicks cause a 40% loss of biological activity (2). In 1988, Nishimura and colleagues (4) showed that nicks at undetermined sites were present on hCG from individuals with trophoblast disease. Since then, several groups, including ours, have confirmed this finding and located nicks at sites analogous to those found on LH (044-»45 and 047-48; see Fig.…”

mentioning

confidence: 99%

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The Heterogeneity of Human Chorionic Gonadotropin (hCG). III. The Occurrence and Biological and Immunological Activities of Nicked hCG*

Kardana²,

et al. 1991

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Nicks, or missing peptide linkages, have been found in hCG beta-subunit between residues 44 and 45 and between residues 47 and 48. We examined the occurrence and biological and immunological activities of nicked hCG. As shown by sequence analysis, CR127 standard hCG is approximately 20% nicked, half at beta 44-45 and half at beta 47-48. Treatment with human leukocyte elastase increased the extent of nicking of CR127 standard hCG. The longer the incubation of CR127 standard with human leukocyte elastase (0, 2, and 21 h), the greater the extent of nicked hCG (20%, 46%, and 89%). As the extent of nicking increased, the receptor-binding ability diminished, as did the ability to stimulate progesterone production by rat corpus luteal cells in vitro (0.9, 0.74, and 0.29 microgram/microgram hCG, respectively). In a regression analysis, a linear relationship was indicated between the extent of nicking and receptor binding values (97% correlation) and between the extent of nicking and steroidogenic activity in vitro (99% correlation). From the intercepts of the regression lines, it was estimated that nicks reduced receptor binding by 11-fold and reduced the steroidogenic activity of hCG by 5-fold. We examined eight individual hCG preparations, three purified from pregnancy urine, three from urine from patients with hydatidiform mole, and two from urine from women with choriocarcinoma. In descending order, the eight individual hCG preparations were 100%, 100%, 85%, 76%, 42%, 41%, 0%, and 0% intact. Although no correlation was observed between the percent intact and the ability of the eight individual samples to displace 50% [125I]hCG in binding CG/LH receptor (r less than 0.5), a close correlation was noted between the percent intact and the steroidogenic activity in vitro (98% correlation). This separated the effects of nicking on receptor binding and steroidogenic activities and indicated that while multiple factors influence receptor binding, only nicking suppresses the steroidogenic activity of bound hCG. We examined the recognition of nicked hCG molecules by different hCG immunoassays. The Hybritech Tandem assay measured total hCG and did not distinguish nicked and intact hCG molecules (in a regression analysis, immunoactivity vs. percent intact hCG, r less than 0.5). In contrast, the immunometric assay using B109 hCG dimer-specific monoclonal antibody and anti-beta-peroxidase only detected the intact component of hCG (in a regression analysis, immunoreactivity vs. percent intact hCG, 98% correlation). We used these assays together to estimate the percentage of intact hCG and to deduce the extent of nicking.(ABSTRACT TRUNCATED AT 400 WORDS)

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“…Based on physico-chemical analyses of molar or tumor extracts, it was then shown that the thyroidal stimulator most probably was hCG (114 -116). It was also suggested that the thyroid-stimulating effects found in these pathological circumstances could be due not only to the extremely high circulating hCG levels, but perhaps also to the presence of molecular variants of hCG with particularly potent thyrotropic activity (117)(118)(119).…”

Section: Regulation Of Serum Tshmentioning

confidence: 99%

The Regulation of Thyroid Function in Pregnancy: Pathways of Endocrine Adaptation from Physiology to Pathology

1997

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Fragmentation of the β-Subunit of Human Chorionic Gonadotropin Produced by Choriocarcinoma*

Cited by 31 publications

References 24 publications

The Heterogeneity of Human Chorionic Gonadotropin (hCG). II. Characteristics and Origins of Nicks in hCG Reference Standards*

The Heterogeneity of Human Chorionic Gonadotropin (hCG). II. Characteristics and Origins of Nicks in hCG Reference Standards*

The Heterogeneity of Human Chorionic Gonadotropin (hCG). III. The Occurrence and Biological and Immunological Activities of Nicked hCG*

The Regulation of Thyroid Function in Pregnancy: Pathways of Endocrine Adaptation from Physiology to Pathology

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