Studies on the relevance of the glycan at Asn-52 of the α-subunit of human chorionic gonadotropin in the αβ dimer

Abstract

Glycosylation of Asn-52 of the alpha-subunit (alphaAsn-52) is required for bioactivity of the alphabeta-dimeric human chorionic gonadotropin (hCG), although at a molecular level the effect of the glycan at alphaAsn-52 is not yet understood. To study the role of this glycan for heterodimer stability, the beta-subunit was recombined in solution with either the alpha-subunit or the alpha-subunit enzymically deglycosylated at alphaAsn-52. Enzymic deglycosylation avoids modification of the glycans at alphaAsn-78 and disturbing the protein folding. The efficiency of recombination after 16 h is 80%, independent of whether alphaAsn-52 is glycosylated or not. The dissociation constant of the hCG complex, with or without the glycan at alphaAsn-52, is less than 1 x 10(-5) s(-1), indicating that the glycan at alphaAsn-52 does not contribute significantly to the stability of the dimer. CD and NMR spectra indicate a local conformational difference between both alphabeta-dimeric hCG variants, most probably involving amino acids of the hCG beta-subunit close to the glycan at alphaAsn-52. These data explain the native-like receptor-binding abilities of hCG lacking the glycan at alphaAsn-52. It is proposed that for bioactivity the glycan at alphaAsn-52 is necessary for inducing and stabilizing a conformational change in hCG upon binding to the receptor, resulting in activation of the signal-transduction pathway.

Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris

“…The present study on non-O-glycosylated phCG also shows that the O-glycosylation of β-hCG seems to have little influence on receptor binding and signal transduction, in accordance with earlier studies on urinary hCG [56]. In several studies, the importance of the N-glycosylation at Asn52 of urinary α-hCG for steroidogenic activity has been shown [56,57], and it has been postulated that the bulky and extended glycan at this site could have a function in inducing and stabilizing a conformational change in hCG upon binding to the receptor [58]. It is suggested that the high-mannose-type N-glycans on αAsn52 in phCG accomplish the same function.…”

Consequences of Single-Chain Translation on the Structures of Two Chorionic Gonadotropin Yoked Analogs in α-β and β-α Configurations

“…5), indicating differences in their solution conformations. Heterodimeric hCG exhibits two prominent (negative) CD bands at 207 nm and 196 nm, in agreement with published spectra (29,30). The CD spectra for YhCG1 and YhCG3 also exhibit a similar negative band at 207 nm, but there are significant differences between the two fusion proteins, and each differs from that of hCG.…”

Single chain human chorionic gonadotropin, hCGαβ: Effects of mutations in the α subunit on structure and bioactivity

“…Hence, it appears that the carbohydrates are involved in subunit interaction and in maintaining a structure optimal for signal transduction. An alternate hypothesis suggested that the carbohydrates are required to stabilize a conformational change occurring as a consequence of receptor binding essential for signal transduction [30]. Our data indicate that the carbohydrates in the heterodimer may indeed be acting to stabilize the conformation of the heterodimer, be it a post-receptor binding acquired conformation or a conformation acquired due to subunit interaction.…”