Enzymatic transfer of a preassembled trisaccharide antigen to cell surfaces using a fucosyltransferase.
The Lewis alpha (1-->3/4)-fucosyltransferase (Le-FucT) is known to fucosylate both Type I (beta Gal(1-->3) beta GlcNAc) and Type II (beta Gal(1-->4) beta GlcNAc) sequences even when these are sialylated at OH-3 or fucosylated at OH-2 of the terminal Gal residues. These acceptor sequences are ubiquitous on mammalian cell-surface glycoproteins and glycolipids. The Le-FucT enzyme is therefore a potential candidate as a universal reagent for the modification of cell surfaces. We have found that a readily accessible, partially purified Le-FucT from human milk, which normally uses GDP-fucose (a 6-deoxy sugar) as the donor for the transfer of a single fucose residue, will also transfer a fucose residue substituted on C-6 by a very large sterically demanding structure, in this instance, a synthetic blood group antigen. As a demonstration of the ability of the Le-FucT to modify glycoconjugates in a mild and specific manner, we chemically synthesized the complex sugar-nucleotide alpha Gal(1-->3) [alpha Fuc(1-->2)]-beta Gal-O-(CH2)8COHN(6)-beta-L-fucose-GDP (13) which is a GDP-fucose analog where the human blood group B trisaccharide antigen is covalently linked to C-6 of fucose through an amino group. It is shown that, in enzyme-linked immunosorbent assays, the Le-FucT uses both immobilized beta Gal(1-->3) beta GlcNAc-bovine serum albumin conjugates and fetuin as acceptor substrates and renders them blood group B-active as detected by a monoclonal anti-B blood-grouping antibody. The fucose residue to which the B-trisaccharide is linked therefore becomes covalently attached to the acceptor oligosaccharide chains of those glycoproteins. Incubation of type "O" erythrocytes with the Le-FucT and complex donor 13 results in the covalent transfer of alpha Gal(1-->3) [alpha Fuc(1-->2)] beta Gal-O-(CH2)8COHN(6)-beta-L-Fuc to cell-surface acceptors since the cells become phenotypically "B" and are agglutinated by the same antibody. It is proposed that the Le-FucT represents a powerful new tool with the ability to label animal cell surfaces with preassembled oligosaccharide and possibly also other complex recognition markers.
Srivastava
G
G
Department of Chemistry, University of Alberta, Edmonton, Canada.
Kaur
K J
KJ
Hindsgaul
O
O
Palcic
M M
MM
D000017:ABO Blood-Group System / Q000737:chemistry*; D000954:Antigens, Surface / Q000737:chemistry*; D002240:Carbohydrate Sequence; D005647:Fucosyltransferases / Q000378:metabolism*; D006001:Glycoconjugates / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000276:immunology / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000017:ABO Blood-Group System; D000954:Antigens, Surface; D006001:Glycoconjugates; D009844:Oligosaccharides; D005647:Fucosyltransferases