Characterization of a sulfotransferase responsible for the 4-O- sulfation of terminal β-N-acetyl-D-galactosamine on asparagine- linked oligosaccharides of glycoprotein hormones

The Asn-linked oligosaccharides on the glycoprotein hormones lutropin (LH) and thyrotropin terminate with the sequence SO₄-4GalNAcβ1-4GlcNAcβ1-2 Manα-. Using a chemically synthesized trisaccharide GalNAcβ1-4GlcNAcβ1-2Manα1-O(CH₂) ₈COOCH₃ (GGnM-MCO), we have developed a sensitive assay for the sulfotransferase responsible for the 4-O-sulfation of the terminal β-D-GalNAc. GGnM-MCO is incubated with a bovine pituitary membrane extract and [³⁵S]3′- phosphoadenosine 5′-phosphosulfate ([³⁵S]PAPS). The sulfated product [³⁵S]SGGnM-MCO is separated from [³⁵S]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 [³⁵S]SGGnM-MCO 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_m, (Henri-Michaelis-Menten equilibrium constant) for PAPS of 4 μM and for GGnM-MCO of 9 μM. Incorporation of sulfate into the trisaccharide is stimulated 3- fold 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 GalNAc- GlcNAc-Man- may modulate GGnM-4-sulfotransferase.