Characterization of bovine placental lactogen as a glycoprotein with N-linked and O-linked carbohydrate side chains.
In a previous report we showed that purified bovine placental lactogen (bPL) exists in two isoforms in the 31,000-33,000 Mr range, each with at least five isoelectric variants differing in approximately 2 orders of magnitude in isoelectric points (pI) 4-6. The multiple isoelectric variants are unique to the bovine hormone. In an effort to determine the nature of these variants endo- and exoglycohydrolase digestions were conducted to determine if this hormone was glycosylated. Analysis of peptide/N-glycosidase F and endoglycosidase F digests of radioiodinated bPL on one-dimensional gel electrophoresis showed a Mr decrease from 31,000 to 24,000 and 33,000 to 26,000 for the two isoforms. Digestion with a mixture of neuraminidase plus mixed exoglycosidases resulted in a Mr decrease of 4,000. Digestion with neuraminidase resulted in a Mr decrease of 2,000. Further analysis of peptide/N-glycosidase F- and neuraminidase-treated bPL by two-dimensional gel electrophoresis showed the isoelectric variants shifted from pI 4.4-6.3 to 4.9-8.0. The sialic acid residues on the N-linkage are responsible for the pronounced acidic character of bPL, but do not account for the residual charge heterogeneity as the different isoelectric variants persist after sialic acid removal. The apparent Mr of the protein after removal of N-linked carbohydrate residues is similar to that of PRL and GH. These enzymatic digestion results demonstrate the presence of N-linked complex oligosaccharide residues attached to the beta-amide group of an asparagine residue. Analyses of the sugar content of the molecule were consistent with the presence of one biantennary N-linked and two O-linked carbohydrate chains.
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002236:Carbohydrate Conformation; D002241:Carbohydrates / Q000032:analysis*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006026:Glycoside Hydrolases / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010928:Placental Lactogen / Q000032:analysis*
D002241:Carbohydrates; D010928:Placental Lactogen; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase