The carboxy-terminal region of the beta-subunits of luteinizing hormone and chorionic gonadotropin differentially influence secretion and assembly of the heterodimers.

Muyan, Mesut; Furuhashi, Madoka; T, Sugahara; Boime, Irving

doi:10.1210/mend.10.12.8961276

Cited by 15 publications

(21 citation statements)

References 1 publication

(3 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The hydrophilic CTP of hCG contains determinants that favor the secretion of recombinant hLH and hFSH when fused to the C-terminus of their b-subunits (Fares et al 1992, Muyan et al 1996. We also showed that deletion of the CTP downstream of residue 111 in the dimeric eLH/CG (aebe111LH/CG) induces a 50% decrease in the secretion of the truncated hormone compared with the full-length aebeLH/CG (Galet et al 2000) as detected by ELISA.…”

Section: Introductionmentioning

confidence: 59%

The β104–109 sequence is essential for the secretion of correctly folded single-chain βα horse LH/CG and for its FSH activity

Galet¹,

Guillou²,

Foulon-Gauze³

et al. 2009

Journal of Endocrinology

View full text Add to dashboard Cite

The dual LH and FSH activity of the equine LH (eLH)/equine chorionic gonadotropin (eCG) in heterologous species makes eLH/CG a good model to study structure/ function relationships of gonadotropins. In order to bypass the problem of intracellular association of the heterodimer, a recombinant single-chain baeLH/CG was used to identify sequences in the b-subunit involved in the secretion and activities of the hormone. The C-terminal region of the b-subunit was progressively truncated. All resulting truncated single-chains were secreted in the media as detected by an anti-bpeptide antibody in reducing conditions. However, using a conformation sensitive ELISA we show that the truncated single-chains were differently recognized: deletion of the last 40 amino acids of the b-subunit (b109aeLH/CG) resulted in a 90% decrease in the recognized correctly folded hormone compared with the full-length baeLH/CG single-chain and no properly folded hormone was detected in the secretion medium when the last 46 amino acids of the b-subunit were deleted (b103aeLH/CG). We thus focused on the six amino acids sequence 104-109, which belongs to the seat-belt region. Mutation of the 104-109 sequence in alanines in the full-length baeLH/CG (b104-109Alaa) led to a 50% decrease in the production of properly folded hormone in COS-7 as well as in aT3 pituitary cells. Moreover, the FSH activity of this mutant was decreased by 70% whereas its LH activity remained intact. These data lead us to conclude that the 104-109 region of the b eLH/CG subunit is essential for the secretion of a fully folded baeLH/CG and for its FSH activity but not for its LH activity.

show abstract

Section: Introductionmentioning

confidence: 59%

The β104–109 sequence is essential for the secretion of correctly folded single-chain βα horse LH/CG and for its FSH activity

Galet¹,

Guillou²,

Foulon-Gauze³

et al. 2009

Journal of Endocrinology

View full text Add to dashboard Cite

show abstract

“…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). Although the hydrophobic carboxyl-terminal end of the hLH␤ subunit is involved in the intracellular retention of the protein, the hydrophilic CTP stretch of hCG␤ contains determinants that are favorable for secretion and, when fused to the ␤-subunits of human LH and human follitropin, enhance the release from transfected cells (14,16,17). In vivo, the storage and secretion profiles of the hormones differ; hLH is stored in secretagogue-sensitive granules, and there are data showing it is secreted from the basolateral surface of the pituitary gonadotropes (18 -20).…”

mentioning

confidence: 99%

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

Nakav

Jablonka‐Shariff

Kaner

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

The expression of a previously untranslated carboxylterminal sequence is associated with the ancestral lutropin (LH) ␤ to the ␤-subunit gene evolution of choriogonadotropins (CG). The peptide extension (denoted as CTP) is rich in mucin-type O-glycans and confers new hormonal properties on CG relative to the LH. Although the LH␤ gene is conserved among mammals and only a few frameshift mutations account for the extension, it is merely seen in primates and equids. Bioinformatics identified a CTP-like sequence that is encrypted in the LH␤ gene of several mammalian species but not in birds, amphibians, or fish. We then examined whether or not decoding of the cryptic CTP in the bovine LH␤ gene (boCTP) would be sufficient to generate the LH␤ species of a ruminant with properties typical to the CG␤ subunit. The mutated bovine LH␤-boCTP subunit was expressed and N-glycosylated in transfected Chinese hamster ovary cells. However, unlike human (h) CG␤ CTP, the cryptic boCTP was devoid of mucin O-glycans. This deficiency was further confirmed when the boCTP domain was substituted for the natural CTP in the human CG␤ subunit. Moreover, when expressed in polarized Madin-Darby canine kidney cells, this hCG␤-boCTP chimera was secreted basolaterally rather than from the apical compartment, which is the route of the wild type hCG␤ subunit, a sorting function attributed to the Oglycans attached to the CTP. This result shows that the cryptic peptide does not orientate CG to the apical face of the placenta, to the maternal circulation as seen in primates. The absence of this function, which distinguishes CG from LH, provides an explanation as to why the LH␤ to CG␤ evolution did not occur in ruminants. We propose that in primates and equids, further natural mutations in the progenitor LH␤ gene resulted in the efficient O-glycosylation of the CTP, thus favoring the retention of an elongated reading frame.The family of glycoprotein hormones includes lutropin (LH), 1 follitropin, and thyrotropin, as expressed by the pituitary, and chorionic gonadotropin (CG), which is produced by the placenta of primates and equids (1). Each hormone is a noncovalent heterodimer composed of a common ␣-subunit and a unique ␤-subunit that confers receptor specificity. The ␤-subunits have substantial sequence similarities, including conserved location of cysteine residues that suggests an origin from a common ancestral gene (2). The human (h) CG␤ genes have evolved from an ancestral LH␤ gene by frameshift mutations that resulted in a readthrough into a previously untranslated region and the extension of the reading frame (3, 4) (Fig. 1A). As a result, a short carboxyl-terminal sequence of hLH␤ is replaced by a longer carboxyl-terminal peptide (CTP) in hCG␤ (3-6). In the equine (e), both the placental CG␤ and pituitary LH␤ subunits are products of the same gene (eLH/CG␤) and contain a CTP that is presumed to have evolved by a distinct mechanism (7) (for further explanations see "Results"). Except for primates and equids, no other identified animal species b...

show abstract

“…Moreover, the different ELISAs used an anti-mAb and this subunit was never mutated, so we could reasonably assume that its epitope is always intact. It is now well established that the CTP and the 26-110 disulphide bridge of the hCG subunit are involved in the intracellular trafficking and secretion of the hCG heterodimer (Suganuma et al 1989, Huth et al 1993, Muyan et al 1996. It seems to be the same for eLH/CG, particularly for the 26-110 disulphide bridge since its deletion induced a severe decrease in the heterodimer production but no decrease in the single chain production (Table 1).…”

Section: Discussionmentioning

confidence: 94%

“…The CTP of the hCG subunit has been shown to be involved in the half-life of the hormone (Matzuk et al 1990). It is not implicated in in vitro biological activity but is required for the correct intracellular folding of the dimer (Matzuk et al 1989, Muyan et al 1996, Muyan & Boime 1998.…”

Section: Introductionmentioning

confidence: 99%

Expression of an in vitro biologically active equine LH/CG without C-terminal peptide (CTP) and/or beta26-110 disulphide bridge

Galet¹,

Chopineau²,

Martinat³

et al. 2000

Journal of Endocrinology

View full text Add to dashboard Cite

The C-terminal region of the subunit of the human chorionic gonadotrophin (hCG) is implied in heterodimer stability ( 26-110 disulphide bridge), in vitro LH bioactivity (region 102-110) and in in vivo LH bioactivity ( CTP). Like the hCG , the equine eLH and eCG subunits, also possess a C-terminal extension (CTP). But, in contrast to hCG, eLH and eCG bind to both LH and FSH receptors in species other than the horse. This allows investigation of the roles of the subunit C-terminal region of a eLH/CG recombinant molecule on both LH and FSH activities. To do so, the CTP was deleted and/or the 26-110 disulphide bond was mutated and the resulting mutated subunits were transiently co-expressed with common subunit in COS7 cells. These regions were also deleted in a eLH/CG single chain also expressed in COS7 cells. The hormones produced were characterized by different ELISAs and in vitro LH and FSH bioassays. Mutation of the 26-110 disulphide bond and deletion of the CTP led to a decrease in eLH/CG heterodimer production. Double mutation promoted an additive effect on production of the heterodimer and of the corresponding tethered eLH/CG. The elimination of the 26-110 disulphide bond in the single chain had no effect on its production. However, neither the 26-110 disulphide bond nor the CTP mutations affected dimer stability and bioactivities of the secreted heterodimers and/or single chain molecules. Therefore, in contrast to hCG, the 26-110 S-S bond of the recombinant eLH/CG subunit does not seem to be essential for eLH/CG dimer stability upon secretion and expressing LH and FSH bioactivities.

show abstract

The carboxy-terminal region of the beta-subunits of luteinizing hormone and chorionic gonadotropin differentially influence secretion and assembly of the heterodimers.

Cited by 15 publications

References 1 publication

The β104–109 sequence is essential for the secretion of correctly folded single-chain βα horse LH/CG and for its FSH activity

The β104–109 sequence is essential for the secretion of correctly folded single-chain βα horse LH/CG and for its FSH activity

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

Expression of an in vitro biologically active equine LH/CG without C-terminal peptide (CTP) and/or beta26-110 disulphide bridge

Contact Info

Product

Resources

About