Characterization of EBV‐transformed B‐cells Established from an Individual Homozygously Mutated (G329A) in the FUT7α1,3‐fucosyltransferase Gene

The α1,3‐fucosyltransferase VII (Fuc‐TVII) is involved in the biosynthesis of E‐ and P‐selectin ligands such as sialyl Lewis x (SLex) on human leukocytes. Recently, individuals were characterized carrying a missense mutation (G329A; Arg110‐Gln) in the FUT7 gene encoding this enzyme. The mutated FUT7 construct produced a Fuc‐TVII enzyme with impaired activity compared with the wildtype enzyme. Polymorphonuclear granulocytes from an individual carrying this mutation homozygously also showed a reduced expression of SLex. In the present study, we have established Epstein–Barr virus‐transformed B‐cell lines from this individual (SIGN) and from an individual not carrying the mutation (IWO). The cell lines were confirmed to be of B‐cell origin by flow cytometry analysis. IWO cells interacted with E‐selectin in an in vitro flow chamber analysis whereas SIGN cell did not. However, when SIGN cell was transiently transfected with wildtype FUT7 cDNA, interaction with E‐selectin could be restored. Cell surface expression of the SLex‐related epitopes recognized by antibodies CSLEX‐1, KM‐93 and HECA‐452 was elevated on IWO cells compared with that on SIGN cells, consistent with a role of these antigens in E‐selectin recognition. These cell lines will be useful in further characterization of E‐selectin ligands and encourage further studies on the consequences of the FUT7‐G329A mutation in vivo.

[1]  A. Lundblad,et al.  Polymorphonuclear Leukocytes from Individuals Carrying the G329A Mutation in the α1,3-Fucosyltransferase VII Gene (FUT7) Roll on E- and P-Selectins , 2002, The Journal of Immunology.

[2]  J. Fornek,et al.  IgG plasma cells display a unique spectrum of leukocyte adhesion and homing molecules. , 2002, Blood.

[3]  A. Lundblad,et al.  Identification of a Missense Mutation (G329A; Arg110→ Gln) in the Human FUT7 Gene* , 2001, The Journal of Biological Chemistry.

[4]  Kangsheng Wang,et al.  Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction. , 2000, Analytical biochemistry.

[5]  F. Wolber,et al.  Alpha(1,3)-fucosyltransferase VII-dependent synthesis of P- and E-selectin ligands on cultured T lymphoblasts. , 1998, Journal of immunology.

[6]  M. Nimtz,et al.  In vivo specificity of human alpha1,3/4-fucosyltransferases III-VII in the biosynthesis of LewisX and Sialyl LewisX motifs on complex-type N-glycans. Coexpression studies from bhk-21 cells together with human beta-trace protein. , 1998, The Journal of biological chemistry.

[7]  W. McDowell,et al.  Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands. , 1998, Glycobiology.

[8]  H. Maaheimo,et al.  Complementary Acceptor and Site Specificities of Fuc-TIV and Fuc-TVII Allow Effective Biosynthesis of Sialyl-TriLex and Related Polylactosamines Present on Glycoprotein Counterreceptors of Selectins* , 1998, The Journal of Biological Chemistry.

[9]  A. Wagers,et al.  Expression of leukocyte fucosyltransferases regulates binding to E-selectin: relationship to previously implicated carbohydrate epitopes. , 1997, Journal of immunology.

[10]  J. Lowe.  Selectin ligands, leukocyte trafficking, and fucosyltransferase genes. , 1997, Kidney international.

[11]  G S Kansas,et al.  P-selectin glycoprotein ligand-1 is essential for adhesion to P-selectin but not E-selectin in stably transfected hematopoietic cell lines. , 1997, Blood.

[12]  R. Kannagi,et al.  Suppression of Sialyl Lewis X Expression and E-selectin-mediated Cell Adhesion in Cultured Human Lymphoid Cells by Transfection of Antisense cDNA of an α1→3 Fucosyltransferase (Fuc-T VII)* , 1996, The Journal of Biological Chemistry.

[13]  G S Kansas,et al.  Selectins and their ligands: current concepts and controversies. , 1996, Blood.

[14]  A. Wagers,et al.  An important role for the alpha 1,3 fucosyltransferase, FucT-VII, in leukocyte adhesion to E-selectin. , 1996, Blood.

[15]  Petr Malý,et al.  The α(1,3)Fucosyltransferase Fuc-TVII Controls Leukocyte Trafficking through an Essential Role in L-, E-, and P-selectin Ligand Biosynthesis , 1996, Cell.

[16]  A. Chang,et al.  The fucosyltransferase FucT-VII regulates E-selectin ligand synthesis in human T cells , 1996, The Journal of cell biology.

[17]  Richard D. Cummings,et al.  Post-translational Modifications of Recombinant P-selectin Glycoprotein Ligand-1 Required for Binding to P- and E-selectin (*) , 1996, The Journal of Biological Chemistry.

[18]  K. Sasaki,et al.  Expression cloning of a novel alpha 1,3-fucosyltransferase that is involved in biosynthesis of the sialyl Lewis x carbohydrate determinants in leukocytes. , 1994, The Journal of biological chemistry.

[19]  T. Springer.  Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm , 1994, Cell.

[20]  S. Watson,et al.  The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide , 1992, The Journal of cell biology.

[21]  F. Gaeta,et al.  ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Lex. , 1990, Science.

[22]  B. Seed,et al.  Recognition by ELAM-1 of the sialyl-Lex determinant on myeloid and tumor cells. , 1990, Science.

[23]  G. Lenoir,et al.  Human blymphocytes immortalization by epstein-barr virus in the presence of cyclosporin a , 1986, In Vitro Cellular & Developmental Biology.