The α1,3-fucosyltransferase VII (Fuc-TVII) is involved in the biosynthesis of E- and P-selectin ligands such as sialyl Lewis x (SLe^x) 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 SLe^x. 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 SLe^x-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.

We recently identified several individuals carrying a missense mutation (G329A; Arg¹¹⁰-Gln) in the FUT7 gene encoding fucosyltransferase VII. This enzyme is involved in the biosynthesis of the sialyl Lewis x (Le^x) epitope on human leukocytes, which has been identified as an important component of leukocyte ligands for E- and P-selectin. No enzyme activity was measurable in expression studies in COS-7 cells using the mutated FUT7 construct. One of the identified individuals carried this mutation homozygously. Flow cytometry analysis of polymorphonuclear leukocytes (PMN) from this individual showed a nearly complete absence of staining with mAbs directed against sialyl Le^x and a diminished staining with an E-selectin IgG chimera. However, staining with P-selectin IgG chimera and Abs directed against P-selectin glycoprotein ligand-1 was not affected by the mutation. PMN from the homozygously mutated individual was further analyzed in an in vitro flow chamber assay. The number of rolling PMN and the rolling velocities on both E- and P-selectin were in the range of PMN from nonmutated individuals. FUT4 and FUT7 mRNA was quantified in PMN isolated from individuals carrying the FUT7 mutation. It was found that PMN from both FUT7 homozygously and heterozygously mutated individuals exhibited an elevated expression of FUT4 mRNA compared with PMN from FUT7 nonmutated individuals. The elevated expression of fucosyltransferase IV was reflected as an increased expression of the Le^x and CD65s Ags on PMN from these individuals. The significance of the mutation was supported by transfection of BJAB cells.

The human FUT7 gene codes for the α1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLe^x) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLe^x and SLe^x-related epitopes. One individual showed lower levels of SLe^x expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg¹¹⁰ → Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. TheFUT7 Arg¹¹⁰ is conserved in all previously cloned vertebrate α1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of ≤1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLe^x and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sjögren's syndrome but had no history of recurrent bacterial infections or leukocytosis.

The α1,3 fucosyltransferase VII (Fuc-TVII) is involved in the biosynthesis of E- and P-selectin ligands such as sialyl Lewis x 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 was not able to produce an active Fuc-TVII enzyme in transfection studies and polymorphonuclear granulocytes from an individual carrying the mutation homozygously showed severely reduced expression of sialyl Lewis x. In the present study we have established Epstein-Barr virus transformed B-celllines 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 were transfected with wild type FUT7 cDNA interaction with E-selectin could be restored. Cell surface expression of the sLe^x related epitopes recognized by antibodies CSLEX-1, KM-93 and HECA-452 was elevated on IWO cells compared to SIGN cells, consistent with a role of these antigens in E-selectin recognition. Despite high expression of PSGL-1 neither of the cell lines interacted with P-selectin under flow. These cell lines may be useful in the further characterization ofEselectin ligands and the obtained results encourage further studies on the consequences of the FUT7-G329A mutation in vivo.

According to the conventional wisdom leukocyte recruitment to an inflammatory site is initiated by selectin mediated capture and rolling along the activated endothelium. The galectins are members of another family oflectins with affinity for ß-galactosides. Expressed on endothelial cells, galectin-1 and galectin-3 have been proposed to mediate cell-cell interactions during inflammation and tumor cell metastasis, and hence act as an alternative to selectins/integrins under certain circumstances. To begin testing this hypothesis, we examined the interaction of neutrophils -with a galectin-1 or galectin-3 coated surface under shear flow conditions. Both galectins were found to trigger neutrophil arrest in a dose and carbohydrate dependent manner at coating concentrations of ≥ 200 nM, and 1 dynes/cm² wall shear stress. While, galectin-3 mediated neutrophil arrest was immediate, galectin-1 triggered a brief period of tethering before arrest. Rapidly following arrest neutrophils spread onto the galectin-coated surface. Cell spreading was accompanied by a redistribution of actin filaments, from an initial even staining with FITC-phalloidin to a more peripheral distribution in spread cells. These data suggest that galectin-1 and galectin-3 may act as adhesion molecules capturing and arresting neutrophils at sites knovvn to be less dependent on selectins and ß₂integrins. They behave in part like selectins in capturing the neutrophils, but also like the integrins in triggering firm adhesion and cell spreading.