Site-specific glycan analysis of human chorionic gonadotropin β-subunit from malignancies and pregnancy by liquid chromatography - Electrospray mass spectrometry

Abstract

Glycosylation is an important posttranslational modification in proteins, and aberrant glycosylation occurs in malignancies. Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced in high concentrations during pregnancy. It is also expressed as particular glycoforms by certain malignancies. These glycoforms, which are called "hyperglycosylated" hCG (hCGh), have been reported to contain more complex glycan moieties. We have analyzed tryptic glycopeptides of the β-subunit of hCG of various origins by liquid chromatography (LC) connected to an electrospray mass spectrometer. Site-specific glycan structures were visualized by the use of differential expression analysis software. hCGβ was purified from urine of two patients with testicular cancer, one with choriocarcinoma, one with an invasive mole, two pregnant women at early and late gestation, from a pharmaceutical preparation and culture medium of a choriocarcinoma cell line. N-glycans at Asn-13 and Asn-30 as well as O-glycans at Ser-121, Ser-127, Ser-132, and Ser-138 were characterized. In all samples, the major type of N-glycan was a biantennary complex-type structure, but triantennary structures linked to Asn-30 as well as fucosylation of the Asn-13-bound glycan are increased in cancer-derived hCGβ. There were significant site-specific differences in the O-glycans, with constant core-2 glycans at Ser-121, core-1 glycans at Ser-138, and putative sites unoccupied by any glycan. Core-2 glycans at either Ser-127 or Ser-132 were enriched in cancer. The glycans of free hCGβ were larger and had a higher fucose content of Asn-13-linked oligosaccharides than intact hCG. This may facilitate the detection of this malignancy-associated variant by a lectin assay. Analysis of hCGh affinity purified with antibody B152 confirmed that this antibody recognizes a core-2 glycan on Ser-132. © 2006 Oxford University Press.