A novel bioluminescence-based solid-phase assay is described for the enzyme GDPFuc:Galβ1-3GlcNAc (Fuc to GlcNAc) α1,4-fucosyltransferase (α1,4FT), which generates the Lewis a blood group antigen (Lea) (Galβ1- 3[Fucα1-4]GlcNAc-R). Lacto-N-tetraose (LNT, Galβ1-3GlcNAcβ1-3Galβ1-4Glc) was chemically conjugated to bovine serum albumin (BSA) to generate the acceptor neoglycoprotein LNT-BSA. The Lea product of the reaction made in the presence of the donor GDPFuc is detected with a primary monoclonal IgG antibody to Lea and a secondary antibody coupled to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Recombinant human GDPFuc:Galβ1-3(4)GlcNAc (Fuc to GlcNAc) α1,4/α1,3- fucosyltransferase, which exhibits α1,4FT activity, was used to optimize the assay. With this assay α1,4FT activity is measurable in human serum, in human saliva, and in extracts of the human colon carcinoma cell line SW1116. Activity is absent, however, in extracts of human HL-60 and murine F9 cells, neither of which synthesize Lea antigen. Among 10 human donors tested, soluble α1,4FT activity, was measurable in serum and saliva of some, but not all donors. However, the presence of enzyme activity in sera does not correlate with Lewis blood group phenotype of erythrocytes. The saliva of one donor, which contained Lea antigens, exhibited no α1,4FT activity. That saliva was found to contain a heat-stable factor(s) capable of inhibiting the α1,4FT activity when mixed with donor saliva containing α1,4FT activity. This new assay should be useful in assessing the Lewis enzyme activity in body fluids and its relationship to the Lewis blood group status on cells and secreted glycoconjugates in normal and diseased states.
ASJC Scopus subject areas
- Molecular Biology