Insights into the hyperglycosylation of human chorionic gonadotropin revealed by glycomics analysis

Abstract

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that is essential for the maintenance of pregnancy. Glycosylation of hCG is known to be essential for its biological activity. "Hyperglycosylated" variants secreted during early pregnancy have been proposed to be involved in initial implantation of the embryo and as a potential diagnostic marker for gestational diseases. However, what constitutes "hyperglycosylation" is not yet fully understood. In this study, we perform comparative N-glycomic analysis of hCG expressed in the same individuals during early and late pregnancy to help provide new insights into hCG function, reveal new targets for diagnostics and clarify the identity of hyperglycosylated hCG. hCG was isolated in urine collected from women at 7 weeks and 20 weeks' gestation. hCG was also isolated in urine from women diagnosed with gestational trophoblastic disease (GTD). We used glycomics methodologies including matrix assisted laser desorption/ ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and MS/MS methods to characterise the N-glycans associated with hCG purified from the individual samples. The structures identified on the early pregnancy (EP-hCG) and late pregnancy (LP-hCG) samples corresponded to mono-, bi-, tri-, and tetra-antennary N-glycans. A novel finding was the presence of substantial amounts of bisected type N-glycans in pregnancy hCG samples, which were present at much lower levels in GTD samples. A second novel observation was the presence of abundant Lewis X antigens on the bisected N-glycans. GTD-hCG had fewer glycoforms which constituted a subset of those found in normal pregnancy. When compared to EP-hCG, GTD-hCG samples had decreased signals for tri-and tetra-antennary N-glycans. In terms of terminal epitopes, GTD-hCG had increased signals for sialylated structures, while Lewis X antigens were of very minor abundance. hCG carries the same Nglycans throughout pregnancy but in different proportions. The N-glycan repertoire is more PLOS ONE | https://doi.

Roles of N-linked glycosylation and glycan-binding proteins in placentation: trophoblast infiltration, immunomodulation, angiogenesis, and pathophysiology

“…At the same time, FcRn and FcγRIIIa expressed on the SCT cell surface collaborate to assist the trans-placental transport of IgGs modified with digalactosylated N-glycans across the villous wall to enter the fetal circulation. The depicted structures of N-glycans on hCG [ 10 ], PSG1 [ 95 ], and IgGs [ 13 ] correspond to findings from previous studies.…”

“…N-glycosylation is initiated in the endoplasmic reticulum (ER) with continued maturation in the Golgi apparatus, of which detailed pathways and kinetics have been illustrated elsewhere [ 8 ]. Several N-glycosylated proteins, such as integrins [ 9 ], human chorionic gonadotrophin (hCG) [ 10 ], human leukocyte antigens (HLAs) [ 11 ], pregnancy-specific glycoproteins (PSGs) [ 12 ], and immunoglobulin G (IgG) [ 13 ] are abundantly expressed at the maternal–fetal interface, and are involved in distinct trophoblastic activities. The role of N-glycan recognition for induction of tolerance during pregnancy has also been supported [ 14–16 ]; however, the depth of our understanding for how N-glycosylation is involved in human placental development requires further expansion.…”

Prevalence of high affinity naturally occurring IgG2 antibodies against human chorionic gonadotropin and its subunits in patients with ovarian cyst

“…In addition, the free form of the α subunit contains one O-glycosylation in position Thr-43 (Cole, 1987). The β subunit (hormone-specific) contains 145 amino acids and bears two N-glycosylation sites at Asn-13 and Asn-30 in its core region and in four sites of O-glycosylation at Ser 121, 127, 132 and 138 in its carboxy-terminal peptide (Ibeto et al, 2020;Kobata & Takeuchi, 1999). The mechanisms of the generation of these high avidity anti-carbohydrate antibodies in non-pregnant women need to be established.…”

The Immunology of Syncytialized Trophoblast

“…Alternatively, one or more of the glycoprotein hormones secreted by the trophoblast giant cells (e.g., human chorionic gonadotropin (hCG); human placental lactogen (hPL)) [ 33 ] and primitive STB (e.g., hCG) may alter local immune responses via glycan–ligand interactions. Consistent with this concept, it was recently shown that hCG isolated from the urine of pregnant women is decorated with bisected N-glycans, the majority of which are biantennary [ 34 ]. Human CG at levels well below those seen in the maternal circulation during early pregnancy have been shown to effectively inhibit the cytotoxic activities of peripheral NK cells against NK-sensitive K562 erythroleukemia cells in vitro [ 35 ].…”