Efficient preparation of glycoprotein hormones lacking an alpha-subunit oligosaccharide.
The oligosaccharide on alpha-subunit loop 2 (alpha 2) is needed for full glycoprotein hormone efficacy. Efforts to prepare glycoprotein hormone antagonists usually involve removing the alpha 2 oligosaccharide and are hampered by its requirement for efficient heterodimer secretion from mammalian cells. Here we show that hormones lacking this oligosaccharide can be produced by treating them at low pH to dissociate the heterodimer and permitting the subunits to re-associate in the presence of peptide N-glycosidase F (PNGase F). Re-assembly of human choriogonadotropin, human follitropin, and bovine lutropin occurred rapidly and efficiently following removal of the alpha 2 oligosaccharide by PNGase F. Consequently, virtually all heterodimers formed in the presence of this enzyme lacked this oligosaccharide. These findings support the notion that heterodimer assembly in vitro occurs by a threading mechanism that is impeded by the presence of the alpha 2 oligosaccharide. This procedure should facilitate the study of glycoprotein hormone structure and function.
Xing
Yongna
Y
Department of OBGYN, Robert Wood Johnson Medical School, Rutgers University, 675 Hoes Lane, Piscataway, NJ 08854, USA.
Moyle
William R
WR
D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002241:Carbohydrates / Q000737:chemistry; D002417:Cattle; D006063:Chorionic Gonadotropin / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005640:Follicle Stimulating Hormone / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D007986:Luteinizing Hormone / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000737:chemistry*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D002241:Carbohydrates; D006063:Chorionic Gonadotropin; D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase