The Biological Role of the Carboxyl-Terminal Extension of Human Chorionic Gonadotroin β-Subunit

Abstract

hCG is a member of a family of glycoprotein hormones which share a common alpha-subunit, but differ in their hormone-specific beta-subunits. The CG beta-subunit is unique in that it contains a hydrophilic carboxyl-terminal extension with four serine O-linked oligosaccharides. To examine the role of the O-linked oligosaccharides and the carboxyl-terminal extension of hCG beta on receptor binding, steroidogenesis in vitro, and ovulation induction in vivo, site-directed mutagenesis and gene transfer methods were used. Wild-type hCG alpha and hCG beta expression vectors were transfected into an O-glycosylation mutant Chinese hamster ovary cell line to produce intact dimer hCG lacking the beta-subunit O-linked oligosaccharide units. In addition, a mutant hCG beta gene (CG beta delta T) was generated which contained a premature termination signal at codon 115. This gene was cotransfected with the hCG alpha gene into Chinese hamster ovary cells to produce hCG dimer which lacked the carboxyl-terminal amino acids 115-145 of hCG beta (truncated hCG). The O-linked oligosaccharide deficient or truncated hCG derivatives were examined for their ability to bind to the mouse LH/hCG receptor and stimulate cAMP and steroidogenesis in vitro. These studies show that the O-linked oligosaccharides and carboxyl-terminal extension play a minor role in receptor binding and signal transduction. In contrast, comparison of the stimulatory effects of truncated and wild-type hCG in a rat ovulation assay in vivo via either intrabursal or iv injection revealed that the truncated derivative was approximately 3-fold less active than wild-type hCG. These findings indicate that the carboxyl-terminal extension of hCG beta and associated O-linked oligosaccharides are not important for receptor binding or in vitro signal transduction, but are critical for in vivo biological responses.

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

“…Both glycohormones show the same receptorbinding potential, and stimulate testosterone production by mouse Leydig cells and progesterone production by MA-10 cells. 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].…”

The LHβ Gene of Several Mammals Embeds a Carboxyl-terminal Peptide-like Sequence Revealing a Critical Role for Mucin Oligosaccharides in the Evolution of Lutropin to Chorionic Gonadotropin in the Animal Phyla

“…1B). The attachment of multiple O-glycans is typical of the CTP domains of CG derived from humans and horses.This carboxyl-terminal extension prolongs the circulatory survival relative to LH (1,(12)(13)(14). Fusing the hCG␤-CTP to the bovine LH␤ subunit resulted in a hormone with a longer halflife and various abnormalities related to the LH excess when this LH␤-CTP chimera was expressed in a transgenic mouse model (15).…”

Expression of a bioactive, single‐chain choriogonadotropin in Dictyostelium discoideum

“…A unique feature of the hCG β-subunit is the carboxy-terminal peptide (CTP) which bears four serine-linked oligosaccharides. The major role of the glycosylated CTP seems to be the prolongation of the circulatory half-life of hCG [1].The biosynthesis of the glycoprotein hormones is a highly complex process. In the last decade, it has become clear that folding, assembly and secretion of gonadotropins is assisted by a large set of chaperones and folding enzymes, residing in the endoplasmic reticulum and the Golgi apparatus [2].…”

“…A unique feature of the hCG β-subunit is the carboxy-terminal peptide (CTP) which bears four serine-linked oligosaccharides. The major role of the glycosylated CTP seems to be the prolongation of the circulatory half-life of hCG [1].…”