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

Abstract

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...

Modulation of the steroidogenic related activity according to the design of single-chain bovine FSH analogs

“…The three analogs contained N-linked glycans, and the hCGb-CTP linker, but not the boCTP sequence, appear to be efficiently O-glycosylated. The lack of O-glycans in the boCTP is consistent with our previous observation when this domain was fused to the bovine LHb and human CGb subunits as well used a linker in SC bovine LH analog (Nakav et al, 2006(Nakav et al, , 2005. The O-glycosylation of the hCGb-CTP linker in FSHbCTPa further support the notion that the O-glycosylation entity of the CTP domain is intrinsic, and is maintained when fused to various proteins from different species (Fares et al, 1992;Furuhashi et al, 1995;Nakav et al, 2005).…”

“…Two chimeric FSHb subunits, that contained at the carboxy end the CTP domain of the hCGb subunit (28 residues) and a CTP-like sequence previously decoded from the bovine LHb gene (Nakav et al, 2005) (denoted as boCTP; 33 residues) were genetically engineered, and the carboxyl terminus of the b subunit variants was fused to the N-terminus of the a subunit to generate the three SC variants (Scheme 1); (a) FSHba the SC analog that encoded the wild type subunit domains linked directly in tandem (206 residues), (b) FSHbboCTPa which included the boCTP sequence as a spacer between the tethered FSHb and a domains (this analog encods 234 residues). (c) For the construction of the FSHbCTPa analog, the CTP of the human CGb subunit was introduced between the two subunits domains (encoding 239 residues).…”

“…Expression of the SC variants was detected by Western blot and immunoprecipitation of metabolically labeled cells. To examine secretion, cells were grown in 12 or 6 well dishes, washed twice with PBS then continuously labeled for 8 or 16 h in cysteine and methionine-free medium containing Pro-mix (25 lCi/ml), as described (Muyan et al, 1994;Nakav et al, 2005). Briefly, media and cell lysate samples were collected, pre-cleared with normal rabbit serum (NRS) and immunoprecipitated overnight with anti o-FSH or NRS (control for non-specific precipitation).…”

“…Briefly, media and cell lysate samples were collected, pre-cleared with normal rabbit serum (NRS) and immunoprecipitated overnight with anti o-FSH or NRS (control for non-specific precipitation). The precipitates were resolved on 12.5% SDS-PAGE under reduced conditions, gels were soaked for 10 min in 1 M sodium salicylate, dried and autoradiographed, as described (Muyan et al, 1994;Nakav et al, 2005). The recovery of the secreted protein to the culture media was estimated as the fraction of the total synthesis (intracellular + secreted).…”

“…For this purpose, we designed SC FSH analogs, in the absence or presence of a linker sequence to space the tethered subunit domains, and generated analogs with differences in the backbone and glycosylation of the linker (Scheme 1). As linkers we used the carboxy terminal peptide of the human CGb subunit (denoted as CTP; (1)) and the cryptic CTP-like sequence we previously decoded from the bovine LHb DNA (denoted as boCTP; (Nakav et al, 2006(Nakav et al, , 2005. The ''linkerless'' NH 2 -FSHb-a-COOH (FSHba), and the spaced FSHbboCTPa (both had N-linked glycans) and FSHbCTPa (carrying N-and O-glycans) analogs were expressed in CHO cells.…”