Carbohydrate and peptide structure of the α- and β-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma
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Human chorionic gonadotropin (hCG), purified from the urine of 14 individuals with normal pregnancy, diabetic pregnancy, hydatidiform mole, or choriocarcinoma, plus two hCG standard preparations, was examined for concurrent peptide-sequence and asparagine (N)- and serine (O)-linked carbohydrate heterogeneity. Protein-sequence analysis was used to measure amino-terminal heterogeneity and the “nicking” of internal peptide bonds. The use of high-pH anion-exchange chromatography coupled with the increased sensitivity of pulsed amperometric detection (HPAE/PAD) revealed that distinct proportions of both hCG α- and β-subunits from normal and aberrant pregnancy are hyperglycosylated, and that it is the extent of the specific subunit hyperglycosylation that significantly increases in malignant disease.
Peptide-bond nicking was restricted to a single linkage (β47–48) in normal and diabetic pregnancy, but occurred at two sites in standard preparations, at three sites in hydatidiform mole, and at three sites in choriocarcinoma β-subunit. In the carbohydrate moiety, α-subunit from normal pregnancy hCG contained non-fucosylated, mono-and biantennary N-linked structures (49.3 and 36.7%, means); fucosylated biantennary and triantennary oligosaccharides were also identified (7.3 and 6.9%). In choriocarcinoma α-subunit, the level of fucosylated biantennary increased, offset by a parallel decrease in the predominant biantennary structure of normal pregnancy (P<0.0001). The β-subunit from normal pregnancy hCG contained fucosylated and nonfucosylated biantennary N-linked structures; however, mono- and triantennary oligosaccharides were also identified (4.6 and 13.7%). For O-linked glycans, in β-subunit from normal pregnancy, disaccharidecore structure predominated, whereas tetrasaccharide-core structure was also detected (15.6%). A trend was demonstrated in β-subunit: the proportions of the nonpredominating N- and O-linked oligosaccharides increased stepwise from normal pregnancy to hydatidi-form mole to choriocarcinoma. The increases were: for monoantennary oligosaccharide, 4.6 to 6.8 to 11.2%; for triantennary, 13.7 to 26.7 to 51.5% and, for O-linked tetrasaccharide-core structure, 15.6 to 23.0 to 74.8%. For hCG from individual diabetic pregnancy, the principal N-linked structure (34.7%) was consistent with a biantennary oligosaccharide previously reported only in carcinoma; and sialylation of both N- and O-linked antennae was significantly decreased compared to that of normal pregnancy.
Taken collectively, the distinctive patterns of subunit-specific, predominant oligosaccharides appear to reflect the steric effect of local protein structure during glycosylation processes. The evidence of alternative or “hyperbranched” glycoforms on both α- and β-subunits, seen at low levels in normal pregnancy and at increased or even predominant levels in malignant disease, suggests alternative substrate accessibility for Golgi processing enzymes, α1,6fucosyltransferase andN-acetylglucosaminyltransferase IV, in distinct proportions of subunit molecules.
Key WordsHCG α- and β-subunits aberrant pregnancy diabetic pregnancy carcinoma N-and O-linked glycans
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