Significance of the Glycan Moiety of the Rat Ovarian Luteinizing Hormone/Chorionic Gonadotropin (CG) Receptor and Human CG for Receptor-Hormone Interaction*

Abstract

The role of the glycan moiety of the rat ovarian LH/CG receptor and human CG (hCG) in high-affinity receptor-hormone interaction was investigated by cross-linking and quantitative binding experiments. hCG and its derivatives, desialylated hCG and deglycosylated hCG were labeled either to the alpha-subunit (125I) or the beta-subunit (3H). The ligands were attached to ovarian membrane particles, which were treated with neuraminidase or peptide-N-glycosidase F to remove terminal sialic acids or N-linked oligosaccharides of the receptor, respectively, and the complexes formed were solubilized, cross-linked with glutaraldehyde, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All of the ligands produced similar autoradiographic patterns with the native or glycosidase-treated receptor, and only the receptor-(alpha)hCG and receptor-(alpha, beta)hCG complexes were detected. Moreover, quantitative binding studies indicated that all of the hormone derivatives had similar affinities for the native or glycosidase-treated receptor. In addition, the orientation of the carbohydrate side chains on the receptor-hormone complex was studied by digesting the complex with the glycosidases. The molecular weight of the receptor, evidenced by ligand blotting, was reduced to the same extent, whether the membrane-bound free receptor or receptor-hormone complex was treated with the glycosidases, suggesting that the oligosaccharide side chains of the receptor are apart from the hormone binding region. As peptide-N-glycosidase F treatment reduced the size of the Mr 90,000 receptor first to about Mr 67,000 and finally to about Mr 62,000, there may possibly be 2 N-linked carbohydrate chains per receptor polypeptide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the glycosidase-treated receptor-[125I]hCG complex also revealed that neuraminidase was able to remove the sialic acids from both subunits of the receptor-bound hormone. In conclusion, the results suggest that hCG interacts with the polypeptide backbone of its ovarian receptor mainly through the peptide core of its alpha-subunit. Moreover, the carbohydrate side chains of both subunits of hCG are positioned on the outward face of the receptor-hormone complex.

Glycosylation of G-protein-coupled receptors for hormones central to normal reproductive functioning: its occurrence and role

“…The nature of the carbohydrates of the HCG/LHR has been elucidated using specific endo-, and exo-, glycosidases. It was demonstrated that the oligosaccharide chains were susceptible to cleavage by PNGaseF (as described above; Petäjä-Repo et al, 1991), and by neuraminidase (Rajaniemi et al, 1996). In contrast, EndoH, α-mannosidase and O-glycosidase (which cleave high mannose/hybrid N-linked sugars, mannose residues from high mannose oligosaccharides and O-linked oligosaccharides respectively) did not modify the HCG/LHR carbohydrate chains (Petäjä-Repo, 1994;Rajaniemi et al, 1996).…”

“…Exposing [ 125 I]-affinity-labelled, purified HCG/LHR to PNGaseF under limiting conditions generated a deglycosylated receptor of 62 kDa from the native 92 kDa receptor. As the fully deglycosylated receptor was produced via only 1-3 intermediates, it was concluded that not all six putative glycosylation sites were used (Petäjä-Repo et al, 1991;Petäjä-Repo, 1994). However, even if all six sites were utilized, it is unlikely that deglycosylation by PNGaseF and SDS-PAGE analysis would be sufficiently sensitive to reveal all the partially glycosylated intermediate states of the HCG/LHR.…”

Cloning and analysis of the cDNA for the common α-subunit of the donkey pituitary glycoprotein hormones

“…However, there are several lines of evidence to indicate that both subunits interact with target cell receptors. First, cross-linking experiments indicate that both subunits are in very close proximity to the receptor (Ji & Ji 1981) and that more of the a-subunit is actually involved in the receptor interaction than the ß-subunit (Kusuda & Dufau 1986, Petaja-Repo et al 1991. Secondly, both polyclonal and monoclonal antibodies against the a-subunit block binding of native human CG (hCG) to its receptor and fail to recognise native hCG once it has bound to its receptor; some antibodies against the ß-subunit have similar effects (Moyle et al 1982, Milius et al 1983).…”

The identification of secreted carbonic anhydrase VI as a constitutive glycoprotein of human and rat milk

“…Purified milk and salivary CA VI (1 g) were digested with PNGase F as described earlier (19), and the deglycosylated and nondeglycosylated proteins were subjected to SDS͞PAGE followed by Colloidal Coomassie staining (NOVEX, San Diego).…”