Characterization of a Sulfotransferase Responsible for the 4-0-

The Asn-linked oligosaccharides on the glycoprotein hormones lutropin (LH) and thyrotropin terminate with the sequence SO4-4GalNAcpl-4GlcNAc@1-2 Mana-. Using a chemically synthesized trisaccharide GalNAc@1-4GlcNAc~l-2Manal-O(CH2)8COOCH3 (GGnM-MCO), we have developed a sensitive assay for the sulfotransferase responsible for the 4-0-sulfation of the terminal 6-D-GalNAc. GGnM-MCO is incubated with a bovine pituitary membrane extract and [35S]3’phosphoadenosine 5’-phosphosulfate ([35S]PAPS). The sulfated product [““SISGGnM-MCO is separated from [“%]PAPS, PAPS degradation products, and endogenous sulfated products by a two-step procedure utilizing an Ecteola cellulose column and a Sep-Pak (C18) cartridge. Characterization of the [“5S]SGGnM-MC0 produced in the assay indicates that sulfate is incorporated exclusively on the 4-position of GalNAc. Linear incorporation of sulfate into GGnM-MCO can be maintained for greater than 10 h. GGnM-4-sulfotransferase has a pH optimum of 7.2, requires the presence of a reducing agent, and is stimulated by, but does not require, divalent cations. Initial velocity studies indicate an apparent K , (Henri-Michaelis-Menten equilibrium constant) for PAPS of 4 I.LM and for GGnM-MCO of 9 PM. Incorporation of sulfate into the trisaccharide is stimulated 3fold by the presence of basic proteins including deglycosylated LH. The stimulation by deglycosylated LH suggests that the protein component of glycoproteins that bear oligosaccharides terminating with GalNAcGlcNAc-Manmay modulate GGnM-4-sulfotransferase .