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

Open AccessPublished:September 15, 1991DOI:https://doi.org/10.1016/S0021-9258(19)47351-1
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      The Asn-linked oligosaccharides on the glycoprotein hormones lutropin (LH) and thyrotropin terminate with the sequence SO4-4GalNAc beta 1-4GlcNAc beta 1-2 Man alpha-. Using a chemically synthesized trisaccharide GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOCH3 (GGnM-MCO), we have developed a sensitive assay for the sulfotransferase responsible for the 4-O-sulfation of the terminal beta-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 Km (Henri-Michaelis-Menten equilibrium constant) for PAPS of 4 microM and for GGnM-MCO of 9 microM. 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.

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