The Importance of O-Linked Glycans for Enhanced Circulatory Survival of the CGbeta Subunit: Further Insight on the Role of O-glycosylation in the LHbeta to CGbeta Evolution

Chorionic gonadotropins (CG) are distinguished from other gonadotropins because they have an O-glycosylated carboxyl terminal peptide (CTP) extension on their beta subunits that prolongs survival in the circulation to maintain early pregnancy. The CTP domain presumably evolved from a previously untranslated region of the ancestral luteinizing hormone (LH) β gene by a small set of mutations that shifted and elongated the reading frame. Although the LHβ to CGβ gene evolution occurred only in primates and equids, bioinformatics analysis previously identified an encrypted CTP-like sequence in the LHβ gene of several mammals. This raised the question why the LHβ to CGβ development is restricted among mammals, despite an apparent broader potential. To gain further insight on this issue, we continued in this report to study the structure and function of the CTP-like sequence that we have previously decoded from the '3 region of the bovine LHβ gene (denoted as boCTP). This cryptic sequence was fused to the human (h)CGβ subunit in place of the natural CTP, and the chimera (denoted as βboCTP), as well as the wild-type hCGβ subunit (βWT) and a truncated variant lacking the CTP domain (β117) were stably transfected into CHO cells. Carbohydrate analysis using lectin-array based technology suggested that N-glycosylation of the βboCTP chimera is similar to that of the βWT and β117 subunits. However, in contrast to the natural CTP, the cryptic boCTP appeared devoid of clustered O-glycans, as we have previously suggested. Pulse-chase experiments indicated that the secretion of the βboCTP variant was quantitative, but slightly slower than that of the βWT and β117 subunits. The clearance rate of β117 from the circulation of male rats increased compared to that of βWT (t1/2 of 18 vs. 47 min.), in accordance with the absence of the CTP. Of significance, the half-life of the chimeric subunit, lacking O-linked oligosaccharides in the CTP stretch was 25 min suggesting a longer circulatory survival than the truncated subunit, but still, much shorter (about 2 fold) than that of the WT β subunit. The βboCTP chimera combined with the common gonadotropin α subunit to form a heterodimer (CGboCTP), and the extent of the assembly was similar to that of the WT heterodimer. The CGboCTP heterodimer was active in a cell bioassay for LH/CG, and displayed a similar potency and efficacy to the WT heterodimer. No activity was detected in a FSH bioassay, suggesting that the cryptic CTP does not alter the specificity of the hormone. Thus, the boCTP stretch does not prevent the secretion and the assembly of the carboxyl extended β subunit with the α subunit to form a bioactive heterodimer. However, when translated, this cryptic domain lacks the hallmark function of prolonging the circulatory survival that is typical to the naturally expressed O-glycosylated CTP. These results support the hypothesis that the absence of new hormonal properties attributed to this post-translational modification provides an explanation as to why LH did not evolve into CG in ruminants, and possibly in additional species, that apply different strategies to delay luteolysis at the early stages of gestation. (poster)