Sulfation of lutropin oligosaccharides with a cell-free system.

Abstract

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 Asn-linked sugars can provide information regarding tissue specificity of oligosaccharide maturation. To characterize this post-translational modification, a reconstituted cell-free sulfation system was developed. Sulfate is incorported 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 hCT (AshCG) is sulfated by membranes from placenta and pituitary, but not liver, while the 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. 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 pituitary cells is present on a heterogeneous population of mono- and disulfated oligosaccharides. The cell-free sulfation system accurately duplicates the in vivo process.