Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands.
The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1-4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono-fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.
Britten
C J
CJ
Glycobiology Research Unit, GlaxoWellcome Research and Development Ltd., Medicines Research Centre, Stevenage, Herts, SG1 2NY, UK.
van den Eijnden
D H
DH
McDowell
W
W
Kelly
V A
VA
Witham
S J
SJ
Edbrooke
M R
MR
Bird
M I
MI
de Vries
T
T
Smithers
N
N
D000818:Animals; D002240:Carbohydrate Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007313:Insecta; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology* / Q000276:immunology*; D008024:Ligands; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019087:Selectins / Q000378:metabolism*; D000080506:Sialyl Lewis X Antigen; D013379:Substrate Specificity
D008024:Ligands; D009844:Oligosaccharides; D011994:Recombinant Proteins; D019087:Selectins; D000080506:Sialyl Lewis X Antigen; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase