Sulfotransferase and glycosyltransferase analyses using a 96-well filtration plate.
Previously, we identified and characterized a sulfotransferase responsible for the 4-O-sulfation of Asn-linked oligosaccharides terminating with the sequence GalNAc beta 1,4GlcNAc beta 1,2Man alpha (GGnM). Here we present a rapid and sensitive method to assay for this sulfotransferase which is significantly more convenient than existing procedures. The acceptor substrate in this assay is human transferrin (Tfn) which has been enzymatically modified such that its N-linked oligosaccharides bear the terminal sequence GGnM. GGnM-Tfn is biotinylated, and a fluid-phase transferase reaction is done which utilizes [35S]3'-phosphoadenosine 5'-phosphosulfate ([35S]PAPS) as the sulfate donor. The reaction product [35S]SGGnM-Tfn-biotin is separated from unreacted [35S]PAPS and radiolabeled endogenous acceptors by capture onto avidin immobilized onto a PVDF-based 96-well filtration plate. Sulfate incorporation in this assay is linear with respect both to the donor and to the acceptor substrates and is also dependent upon enzyme input and time. With this assay, we can detect as little as 0.1 pmol of sulfate transfer. In addition, by using asialo-Tfn-biotin as an acceptor and [3H]CMP-sialic acid as a donor substrate, we demonstrate that this assay can be modified for use with the alpha2,6-sialyltransferase.
Hooper
L V
LV
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Baenziger
J U
JU
D002240:Carbohydrate Sequence; D005069:Evaluation Studies as Topic; D016695:Glycosyltransferases / Q000032:analysis*; D006801:Humans; D008722:Methods; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000032:analysis*; D014168:Transferrin / Q000031:analogs & derivatives / Q000737:chemistry
D009844:Oligosaccharides; D014168:Transferrin; D016695:Glycosyltransferases; C498300:CHST9 protein, human; C092527:N-acetylgalactosamine-4-sulfotransferase; D015238:Sulfotransferases