Determination of GDP-Fuc:Gal beta 1-4GlcNAc-R (Fuc to GlcNAc) alpha 1,3 fucosyltransferase activity by a solid-phase method.
We report the development of a solid-phase assay for the activity of the enzyme GDPFuc:Gal beta 1-4GlcNAc-R (Fuc to GlcNAc) alpha 1,3 fucosyltransferase (alpha 1,3FT). This enzyme generates the blood group antigen Lewis x (Lex)Gal beta 1-4(Fuc alpha 1-3)GlcNAc-R from the acceptor Gal beta 1-4GlcNAc-R. In our method, the tetrasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (lacto-N-neotetraose, LNnT) from human milk was chemically conjugated to bovine serum albumin (BSA) to generate LNnT-BSA. As a source of alpha 1,3FT to develop the assay, we used extracts of COS7 cells created to stably express the human FucTIII and FucTIV genes, both of which have alpha 1,3FT activity. LNnT-BSA was immobilized in microtiter wells and incubated with GDPFuc and cell extracts. The Lex antigen generated by alpha 1,3FT was detected with a monoclonal IgM antibody (anti-CD15). Binding of this IgM-type antibody to product was detected by one of two methods. Method 1 was based on the binding of alkaline phosphatase-conjugated goat anti-mouse IgM. Method 2 was based on the binding of a streptavidin conjugate of the recombinant bioluminescent protein aequorin to biotinylated goat anti-mouse IgM. The alpha 1,3FT assay was linear with respect to time (0-3 h), extract added (0-40 micrograms), and was dependent on GDPFuc (20 microM optimal) and LNnT-BSA. Both methods 1 and 2 allowed measurement of alpha 1,3FT in extracts of the human cell line HL-60.(ABSTRACT TRUNCATED AT 250 WORDS)
Yan
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Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Smith
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DF
Cummings
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RD
D002478:Cells, Cultured; D005647:Fucosyltransferases / Q000378:metabolism*; D006801:Humans
D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase