Mouse C127 cells transfected with fucosyltransferase fuc-TIII express masked Lewisx but not Lewisx antigen.
To study human alpha1,3/1,4fucosyltransferase (Fuc-TIII) as an alpha1,3 fucosyltransferase, we constructed two cell clones, C127-FT and C127-T-FT, by transfecting cDNA in parental (C127) or Polyoma T antigen expressing (C127-T) mouse cells, respectively. Both C127-FT and C127-T-FT clones express high levels of a fucosyltransferase activity kinetically similar to Fuc-TIII and an RNA that is amplified by a Fuc-TIII-specific oligonucleotide primer pair after reverse transcription. Clone C127-FT is Lewisxpositive, by flow cytometry, only after alpha-galactosidase or sialidase treatment, and releases [3H]Fuc N-glycans which efficiently bind to immobilized Griffonia simplicifolia I and Sambucus nigra lectins. Immunoblotting confirms that C127-FT glycoproteins acquire Lewisxreactivity only after specific deglycosylation, and shows that a small subset of Griffonia simplicifolia I isolectin B4reactive glycoproteins bears masked Lewisx, suggesting fine substrate recognition by Fuc-TIII. Moreover, transient transfection of H type alpha1, 2fucosyltransferase in clone C127-T-FT directs synthesis of Lewisyantigen, as detected by flow cytometry. Results indicate that Fuc-TIII expressed in C127 cells synthesizes masked Lewisxantigen while Lewisxantigen is not detectable.
Valli
M
M
Department of Biochemistry, University of Pavia, via Taramelli 3B, 27100 Pavia, Italy.
Bardoni
A
A
Trinchera
M
M
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002846:Chromatography, Affinity; D003208:Concanavalin A; D006224:Cricetinae; D005434:Flow Cytometry; D005643:Fucose / Q000032:analysis; D005647:Fucosyltransferases / Q000235:genetics*; D006801:Humans; D037102:Lectins; D016256:Lewis X Antigen / Q000032:analysis* / Q000378:metabolism; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology*; D051379:Mice; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D037102:Lectins; D016256:Lewis X Antigen; D009844:Oligosaccharides; D003208:Concanavalin A; D005643:Fucose; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase