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

Timothy P. Skelton, Lora V. Hooper, Vandana Srivastava, Ole Hindsgaul, Jacques U. Baenziger

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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.

Original languageEnglish (US)
Pages (from-to)17142-17150
Number of pages9
JournalJournal of Biological Chemistry
Issue number26
StatePublished - Nov 7 1991


ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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