Cell-surface expression of a membrane-anchored form of the human chorionic gonadotropin alpha subunit.
We carried out experiments designed to generate a novel cell-surface protein from a small glycosylated secretory protein. DNA encoding the entire precursor of human chorionic gonadotropin (hCG, alpha subunit) was fused precisely to DNA encoding the transmembrane and cytoplasmic domains of the vesicular stomatitis virus glycoprotein. When expressed in animal cells this DNA encoded the 92-amino acid hCG-alpha subunit anchored in cellular membranes by an extension composed of the 49 carboxyl-terminal amino acids of vesicular stomatitis virus glycoprotein. This hybrid protein was transported efficiently to the plasma membrane of animal cells. The two asparagine-linked glycans on the anchored form of hCG-alpha were large and heterogeneous when compared to those on the secretory form. Experiments employing in vitro mutagenesis and the glycosylation inhibitor tunicamycin established that the presence of at least one of the two asparagine-linked glycans was required for expression of the anchored molecule on the cell surface. However, as reported previously, secretion of hCG-alpha occurred in the absence of glycosylation. Also, mutations eliminating the second glycosylation site (at amino acid 78) in both the anchored or secreted forms apparently led to partial denaturation or a conformational change interfering with transport of the protein.
D001693:Biological Transport, Active; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D015292:Glycoprotein Hormones, alpha Subunit; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008566:Membranes; D009844:Oligosaccharides / Q000494:pharmacology; D010908:Pituitary Hormones, Anterior / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D014415:Tunicamycin / Q000494:pharmacology; D014721:Vesicular stomatitis Indiana virus; D014760:Viral Fusion Proteins / Q000378:metabolism
D015292:Glycoprotein Hormones, alpha Subunit; D009844:Oligosaccharides; D010908:Pituitary Hormones, Anterior; D014760:Viral Fusion Proteins; D014415:Tunicamycin; D004247:DNA; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase