A novel fluorescent assay for T-synthase activity.
Loss of T-synthase (uridine diphosphate galactose:N-acetylgalactosaminyl-α1-Ser/Thr β3galactosyltransferase), a key enzyme required for the formation of mucin-type core 1 O-glycans, is observed in several human diseases, including cancer, Tn syndrome and IgA nephropathy, but current methods to assay the enzyme use radioactive substrates and complicated isolation of the product. Here we report the development of a novel fluorescent assay to measure its activity in a variety of tumor cell lines. Deficiencies in T-synthase activity correlate with mutations in the gene encoding the molecular chaperone Cosmc that is required for folding the T-synthase. This new high-throughput assay allows for facile screening of tumor specimens and other biological material for T-synthase activity and could be used diagnostically.
Ju
Tongzhong
T
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA. tju@emory.edu
Xia
Baoyun
B
Aryal
Rajindra P
RP
Wang
Wenyi
W
Wang
Yingchun
Y
Ding
Xiaokun
X
Mi
Rongjuan
R
He
Miao
M
Cummings
Richard D
RD
D002138:Calibration; D045744:Cell Line, Tumor; D057075:Enzyme Assays / Q000379:methods*; D005700:Galactosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D018832:Molecular Chaperones / Q000235:genetics; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014468:Umbelliferones / Q000737:chemistry
C505369:C1GALT1C1 protein, human; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D014468:Umbelliferones; C505293:C1GALT1 protein, human; D005700:Galactosyltransferases; D006026:Glycoside Hydrolases