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

Abstract

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)

Molecular structures of glycoprotein hormones and functions of their carbohydrate components

“…Similar nicks have been reported in the corresponding regions of choriogonadotropin [128,129] and follitropin [91], and there is evidence that this peptide cleavage can result in decreased receptor binding activities of human lutropin [126,127] and choriogonadotropin [129].…”

Mitigation of septic shock in mice and rhesus monkeys by human chorionic gonadotrophin-related oligopeptides

“…'Missing' of loop 2 from the b-subunit (as in hCG b-core fragment) and 'nicking' of the b-subunit in the loop 2 region of the molecule, specifically between residues 44-49, can both reduce the biopotency of hCG. Cleavage of the peptide bonds in this area of the molecule also reduced biopotency and immunochemical recognition by monoclonal antibodies directed to the heterodimeric hormone [12][13][14]18,19]. Based on known preferential cleavage sites in loop 2 several oligopeptides were synthesized, including MTR, MTRV, LQG, LQGV, VLPALP and VLPALPQ.…”

“…1) of the b-subunit of hCG [12][13][14]17]. Oligopeptides selected for the in vivo studies reported in this publication were the most effective in pilot studies, i.e.…”

“…This selection was based on known nick sites in this loop [12][13][14]. The oligopeptides were first tested in a murine LPS infusion model for attenuation of septic shock.…”

How and Why Do Gestational Trophoblastic Neoplasms Overproduce Human Chorionic Gonadotropin?

“…The non trophoblast derived hCG is not glycosylated, which results in a rapid elimination from circulation [5]. Human placenta derived hCG exists in multiple forms due to differences in glycosylation [6]. These forms as well as free subunits, β-core fragment, etc.…”