Characterization of a sulfotransferase responsible for the 4-O-sulfation of terminal beta-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 SO4-4GalNAc 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.
Skelton
T P
TP
Department of Pathology, Washington University Medical School, St. Louis, Missouri 63110.
Hooper
L V
LV
Srivastava
V
V
Hindsgaul
O
O
Baenziger
J U
JU
D000116:Acetylgalactosamine / Q000378:metabolism*; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002412:Cations; D002417:Cattle; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D010902:Pituitary Gland / Q000201:enzymology*; D010907:Pituitary Hormones / Q000378:metabolism*; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000378:metabolism*
D002412:Cations; D009844:Oligosaccharides; D010907:Pituitary Hormones; D010724:Phosphoadenosine Phosphosulfate; D001216:Asparagine; C092527:N-acetylgalactosamine-4-sulfotransferase; D015238:Sulfotransferases; D000116:Acetylgalactosamine