Sulfation of lutropin oligosaccharides with a cell-free system.
Sulfate is covalently linked to the oligosaccharides on the alpha and beta subunits of bovine lutropin (luteinizing hormone; LH) but not to those on human chorionic gonadotropin (hCG). Since the amino acid sequences of the pituitary and placental alpha subunits are homologous, comparison of their asparagine-linked sugars can provide information regarding tissue specificity of oligosaccharide maturation. To characterize this post-translational modification, we have developed a reconstituted cell-free sulfation system. Sulfate is incorporated into exogenously added glycoproteins by sulfotransferases from Triton X-100-lysed Golgi membranes in the presence of 3'-phosphoadenosine 5'-phospho[35S]sulfate, which is generated from [35S]sulfate by a ribosome-free supernate from Krebs ascites tumor cells. LH is sulfated by pituitary and liver membranes but not by those from placenta. Desialylated hCG (AshCG) is sulfated by membranes from placenta and pituitary, but not liver, while hCG is not sulfated by any of these membranes. Endoglycosidase F releases all the incorporated sulfate from LH in the form of a heterogeneous mixture of mono- and disulfated oligosaccharides. In contrast, the sulfate added to AshCG is apparently attached to peptide rather than oligosaccharide. As found with the cell-free system, sulfate metabolically incorporated into LH by pituitary cells is present on a heterogeneous population of mono- and disulfated oligosaccharides. Thus the cell-free sulfation system accurately duplicates the in vivo process.
D000818:Animals; D002417:Cattle; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000235:genetics*; D009844:Oligosaccharides / Q000378:metabolism*; D010902:Pituitary Gland / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes
D009844:Oligosaccharides; D013431:Sulfates; D013462:Sulfur Radioisotopes; D007986:Luteinizing Hormone