The ELAM ligand fucosyltransferase, ELFT, directs E-selectin binding to a secreted scaffold protein: a method to produce and purify large quantities of specific carbohydrate structures.
Many biological processes depend on cell surface recognition of receptor-ligand pairs. Some receptors, such as the selectins, recognize specific carbohydrate structures as part of their ligands. The ability to synthesize such ligands for use in the study of cell adhesion mechanisms or as inhibitors of a variety of pathological conditions would be extremely useful. However, the chemical or enzymic in vitro synthesis of carbohydrate-based ligands has thus far been difficult and costly. We have used E-selectin and its carbohydrate ligand as a model system to test if it is possible to express specific carbohydrate structures on a secreted, glycosylated and easily purified scaffold protein and to use this newly modified protein as a functional adhesion molecule. We co-expressed a fucosyltransferase (ELFT) and a secreted immunoglobulin-LFA3 fusion protein (LFA31G) in the same cell to modify the carbohydrate structures on the secreted LFA3IG scaffold protein (we refer to this novel protein as X-LFA3IG). Using glycosidase digestion, lectin binding, carbohydrate composition analysis and antibody-binding assays, we show that approximately 50% of the potential N-linked carbohydrate sites on X-LFA3IG are, indeed, modified and that the modification is the addition of fucose. Furthermore, we show that X-LFA3IG contains epitopes recognized by anti-Slex antibodies, and, using an E-selectin-specific adhesion assay, we demonstrate that X-LFA3IG is a functional ligand for E-selectin. This in vivo approach for generating specific carbohydrate structures could be generalized to produce and purify large quantities of other biologically important carbohydrate structures.
Meier
W
W
Biogen Inc., Cambridge, MA 02142.
Leone
D R
DR
Miatkowski
K
K
Lobb
R
R
Goelz
S E
SE
D000906:Antibodies / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D018968:CD58 Antigens; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000302:isolation & purification*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D019040:E-Selectin; D005647:Fucosyltransferases / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011971:Receptors, Immunologic / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000906:Antibodies; D015703:Antigens, CD; D018968:CD58 Antigens; D002241:Carbohydrates; D015815:Cell Adhesion Molecules; D019040:E-Selectin; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D005647:Fucosyltransferases