Occurrence of Fragmentation of Free and Combined Forms of the β-Subunit of Human Chorionic Gonadotropin*

Abstract

In an attempt to further study various fragments of free and combined forms of hCG beta present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCG beta-(111-116) portion (monoclonal antibody FB12) antiserum to the hCG beta(8-16) portion or antiserum which was specific for fragments ending at residue 47. Results observed in a crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCG beta nicked subunit (beta NS) [hCG beta-(48-145)], three other fragments of mol wt 18,000 (F1), 16,500 (F2), and 12,000 (F3) were detectable after cleavage of disulfide bonds. Both the immunoreactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCG beta-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the beta NS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free beta-subunit contained beta NS. Furthermore, free hCG beta present in those urine samples appeared to be extensively, if not totally, nicked. Results observed in urine were confirmed using separation of hCG from its beta-subunit by a two-step chromatography procedure, identification of hCG and hCG beta immunoreactive peaks by specific monoclonal immunoradiometric assay, and analysis of resulting preparations by one-dimensional electrophoresis under reducing conditions, followed by Western immunoblotting with FB12. This latter protocol was also used to investigate the presence of beta NS in sera of four patients with choriocarcinoma tumors. In those sera, hCG appeared to be nicked. This study demonstrates that the beta-subunit of hCG is modified by multiple fragmentations.

Endocrinology: Clinical significance of invisible or partially visible luteinizing hormone

“…The authors concluded that 'invisible LH' was due to the existence of a fragmented form of LH where at least part of the 0 subunit was lost (Pettersson et al, 1992). Since similar observations were made with HCG leading to the discovery of nicked /3 HCG (Puisieux et al, 1990;Bischof et al, 1994), we postulate that the fragmented forms of LH could be nicked /3 LH.…”

Specific Measurement of O-Linked Core 2 Sugar-Containing Isoforms of Hyperglycosylated Human Chorionic Gonadotropin by Antibody B152

“…About a decade ago, we collaborated with the laboratory of Dr. Laurence Cole in an attempt to produce antibodies to 'nicked hCG' (hCGn) which is an isoform of hCG commonly observed in the urine and sometimes in the blood of patients with hCG-secreting tumors and other disorders [25][26][27][28][29][30] . hCGn is a proteolytically damaged form of hCG which has peptide bond cleavages in its ␤ -subunit between residues 43 and 49 [23,25,27,[29][30][31] .…”

“…hCGn is a proteolytically damaged form of hCG which has peptide bond cleavages in its ␤ -subunit between residues 43 and 49 [23,25,27,[29][30][31] . Figure 1 shows linear drawings of hCG ␤ which is the immunological focus of this review.…”

hCGbeta core fragment is a metabolite of hCG: evidence from infusion of recombinant hCG

“…Our observation of the appearance of hCG cf in the urine of four female subjects, after receiving a dose of r-hCG, supports the view that it is derived from the metabolic processing of hCG. Previous studies of pregnant subjects have shown that hCG cf concentrations are 2-10 times that of the hCG dimer, on a molar basis, in pregnancy urine , Blithe et al 1988, Krichevsky et al 1988, Wehmann et al 1989, 1990, de Medeiros et al 1992b. However, in this study, the hCG cf measured was only 12·2% of the amount of hCG excreted in urine, suggesting that, in pregnancy, there may be more than one pathway responsible for the production of hCG cf.…”

“…In addition to a wide range of heterogeneous forms of intact hCG, the urine of pregnant females contains free and free subunits, nicked hCG (missing linkages in the 41-54 region) (Puisieux et al 1990 and C-terminal forms, i.e. fragments of the chain (Amr et al 1983) and hCG cf (Kato & Braunstein 1988.…”