Occurrence of fragmentation of free and combined forms of the beta-subunit of human chorionic gonadotropin.
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.
Unité d'Immunochimie, Institut Gustave-Roussy, Villejuif, France.
A
A
Puisieux
D
D
Bellet
F
F
Troalen
A
A
Razafindratsita
C
C
Lhomme
C
C
Bohuon
J M
JM
Bidart
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002822:Choriocarcinoma / Q000652:urine; D006063:Chorionic Gonadotropin / Q000097:blood / Q000652:urine*; D018997:Chorionic Gonadotropin, beta Subunit, Human; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000097:blood / Q000652:urine*; D011247:Pregnancy; D014594:Uterine Neoplasms / Q000652:urine
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D010446:Peptide Fragments; D008623:Mercaptoethanol