Characterization of the O-glycosylation sites in the chorionic gonadotropin β subunit in vivo using site-directed mutagenesis and gene transfer

Tadashi Sugahara, Mary R. Pixley, Fuad Fares, Irving Boime

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Human chorionic gonadotropin (CG) is a member of a family of glycoprotein hormones which are heterodimers containing two nonidentical subunits: a common α and a hormone-specific β subunit. One of the distinguishing features of the CGβ subunit is the presence of four serine acceptors clustered within the last 25 amine acids. We previously demonstrated that this carboxyl-terminal region is important for maintaining its biologic half-life, and when the sequence was genetically fused to either the common α or follitropin β subunits, O-glycosylation was observed. Because this carboxyl-terminal sequence is located at the end of the subunit, we considered this region a convenient in vive model for studying O-linked glycosylation in domains containing multiple serine recognition sites. A CGβ gene was engineered in which the N-linked sites were inactivated to eliminate background from those carbohydrate groups. Using this construct, we made a series of truncation and amine acid substitutions of acceptor serines, and these mutants were transfected into Chinese hamster ovary cells. O- Glycosylation was determined by [3H]glucosamine incorporation and glycanase sensitivity of the products on SDS-polyacrylamide gels. We show that the O- linked sites comprise independent repetitive regions in which each acceptor serine has a recognition signal bounded by the next carboxy acceptor serine within four to five amine acids. It is also apparent that recognition of one site is not dependent on the glycosylation of another acceptor. Amino acid mutations in the acceptor regions demonstrated the importance of proline as a necessary feature for O-linked recognition in the CGβ sequence.

Original languageEnglish
Pages (from-to)20797-20804
Number of pages8
JournalJournal of Biological Chemistry
Issue number34
StatePublished - 1996
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry
  • Cell Biology


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