TY - JOUR
T1 - Characterization of a sulfotransferase responsible for the 4-O- sulfation of terminal β-N-acetyl-D-galactosamine on asparagine- linked oligosaccharides of glycoprotein hormones
AU - Skelton, Timothy P.
AU - Hooper, Lora V.
AU - Srivastava, Vandana
AU - Hindsgaul, Ole
AU - Baenziger, Jacques U.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1991/9/15
Y1 - 1991/9/15
N2 - 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 Km, (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.
AB - 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 Km, (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.
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M3 - Article
C2 - 1894609
AN - SCOPUS:0026044058
SN - 0021-9258
VL - 266
SP - 17142
EP - 17150
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 26
ER -