Glycobiology Advance Access originally published online on July 31, 2006
Glycobiology 2006 16(12):1207-1218; doi:10.1093/glycob/cwl034
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Site-specific glycan analysis of human chorionic gonadotropin ß-subunit from malignancies and pregnancy by liquid chromatography—electrospray mass spectrometry
2 Department of Clinical Medicine, Division of Clinical Chemistry, Biomedicum, University of Helsinki, PO Box 63 (Haartmaninkatu 8), Helsinki FIN-00014, Finland; and
3 Department of Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, New York, NY 10032
1 To whom correspondence should be addressed; e-mail: leena.valmu{at}helsinki.fi
Received on May 12, 2006; revised on July 7, 2006; accepted on July 28, 2006
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 ß-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. hCGß 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 hCGß. 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 hCGß 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.
Key words: antibody B152 / cancer / hCG / hyperglycosylated hCG / mass spectrometry / N-glycosylation / O-glycosylation