Immunodetection of alpha 1-3 fucosyltransferase (FucT-V).
The fucosyltransferases constitute a family of glycosyltransferases incorporating fucose residues into glycoprotein or glycolipid glycans. They afford one of the possible termination steps of glycoconjugate biosynthesis creating the sialyl Lewisx or sialyl Lewisa determinant, which play an important role in cell-cell interaction. While cDNA, chromosomal localization and kinetic properties of a number of fucosyltransferases are known, immunocytochemical localization and trafficking studies have been delayed because of the lack of specific antibodies due to the pronounced homology of alpha 1, 3 fucolsyltransferases III, V and VI. Here we report development and characterization of monospecific polyclonal antibodies to alpha 1-3 fucosyltransferase V (FucT-V) and their application for immunodetection in transfected cells. Antisera against FucT-V were raised in two different ways: first by producing a fusion protein beta-galactosidase-FucT-V in Escherichia coli, and by synthesizing a peptide stretch specific for FucT-V. Polyclonal antisera were raised against each of both antigens and characterized by enzyme-linked immunosorbent assay, neutralization of activity, immunoblotting, immunofluorescence and immunoprecipitation of metabolically labeled COS cells, transiently transfected with cDNA encoding FucT-V. Both antibodies recognized only FucT-V. No cross-reactivity to FucT-III or FucT-VI was observed. FucT-V was localized mainly to the Golgi apparatus by colocalization with beta 1, 4-galactosyltransferase, and to the cell surface of COS, CHO and HeLa cells. Expression of FucT-V in COS cells revealed three enzyme forms of 58, 53 and 50 kDa, respectively. These size differences arose by post-translational modifications, as shown by pulse-chase experiments. Our results indicate that alpha 1-3 fucosyltransferase is a Golgi-associated enzyme and suggest its possible occurrence on the cell surface.
Borsig
L
L
Institute of Physiology, University of Zürich, Switzerland.
Kleene
R
R
Dinter
A
A
Berger
E G
EG
D000818:Animals; D000918:Antibody Specificity; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D005647:Fucosyltransferases / Q000032:analysis* / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D011233:Precipitin Tests
D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase