Figure 4
From: Decreased levels of genuine large free hCG alpha in men presenting with abnormal semen analysis
hCGα purified from seminal plasma differs in glycosylation from both HEK293-derived large hCGα and hCGα dissociated from pregnancy-derived intact hCG. (A) Seminal plasma-derived hCGα (SMP) had an Mr,app of 24 kDa similar to the large free subunit purified from supernatant of HEK293 cells stably transfected with β2AR and stimulated with isoproterenol (HEK), whereas the pregnancy-derived dissociated subunit (Std; 1st IRP hCGα 99/720) had an apparent molecular mass (Mr,app) of 22 kDa. Digestion with PNGase F under non-reducing conditions (deglycosylation of Asn52) resulted in monoglycosyl hCGα subunits of identical size for HEK and Std (Mr,app = 18 kDa), while SMP migrated with an Mr,app of 19 - 20 kDa. (B) After digestion with PNGase F under denaturing conditions (deglycosylation of both N-glycosylation sites Asn52 and Asn78), hCGα from all three sources migrated identically (Mr,app = 15kDa), indicating identical amino acid backbones.