Characterization of the O-glycosylation sites in the chorionic gonadotropin beta subunit in vivo using site-directed mutagenesis and gene transfer.
Human chorionic gonadotropin (CG) is a member of a family of glycoprotein hormones which are heterodimers containing two nonidentical subunits: a common alpha and a hormone-specific beta subunit. One of the distinguishing features of the CGbeta subunit is the presence of four serine acceptors clustered within the last 25 amino 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 alpha or follitropin beta 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 vivo model for studying O-linked glycosylation in domains containing multiple serine recognition sites. A CGbeta 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 amino 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 amino 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 CGbeta sequence.
Sugahara
T
T
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Pixley
M R
MR
Fares
F
F
Boime
I
I
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006063:Chorionic Gonadotropin / Q000096:biosynthesis*; D006224:Cricetinae; D018014:Gene Transfer Techniques; D006023:Glycoproteins / Q000096:biosynthesis*; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship
D006063:Chorionic Gonadotropin; D006023:Glycoproteins; D011994:Recombinant Proteins; D012694:Serine