A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group alpha(1,3/1,4)fucosyltransferase.

Abstract

The stage-specific embryonic antigen SSEA-I is a cell-surface oligosaccharide molecule expressed with temporal precision during the murine preimplantation period and implicated in adhesive events involving the process of compaction. We used a mammalian transient expression system to isolate a cloned human cDNA that determines expression of the SSEA-I molecule. The cDNA sequence predicts a type II transmembrane protein with a domain structure similar to mammalian glycosyltransferases, but without primary sequence similarity to these enzymes. The carboxy-terminal domain of this protein was shown to be catalytically active as a fucosyltransferase when expressed in COS-I cells as a portion of a secreted protein A fusion peptide. The enzyme is an exceptional glycosyltransferase in that it can use both type I and type II oligosaccharides as acceptor substrates to generate subterminal Fucc~(l,4)-and Fuc~(l,3)-linkages, respectively, in a manner analogous to the human Lewis blood group fucosyltransferase. Southern blot analysis shows that the cDNA corresponds to sequences syntenic to the Lewis locus on chromosome 19. These results indicate that this cDNA is the product of the human Lewis blood group locus, provide genetic confirmation of the hypothesis that this enzyme can catalyze two distinct transglycosylation reactions, and outline an approach to the isolation of other sequences that determine expression of developmentally regulated oligosaccharide antigens.

Transfection of the c‐erbB2/neugene upregulates the expression of sialyl Lewis X, α1,3‐fucosyltransferase VII, and metastatic potential in a human hepatocarcinoma cell line

“…8B, they did not abolish the adhesion of 7721 cells to fibronectin, but significantly and slightly, respectively, inhibited cell adhesion to P-selectin. These phenomena were not only found in mock-transfected cells with 10 mg´mL 21 Ig, but also in c-erbB2/neu-transfected cells with 20 mg´mL 21 Ig.…”

“…Cells were homogenized in 0.1 mol´L 21 Mes buffer (pH 6.5)/150 mmol´L 21 NaCl/2% Triton X-100/25% glycerol/1 mg´100 mL leupeptin and pepstatin, and then centrifuged at 1000 g at 4 8C for 15 min. Protein concentration was determined by the Lowry method [33].…”

“…Western blotting was performed by a modified method of Marone et al [34]. Aliquots of 50 mg of protein samples were separated by SDS/PAGE (10% gel) and electroblotted on to a poly(vinylidene difluoride) membrane, which was then blocked with 5% fat-free dried milk in 0.1 mol´L 21 Tris buffer (pH 7.4)/0.15 mol´L 21 NaCl/0.05% Tween-20. The membrane was treated with a 1 : 200 dilution of rabbit anti-(human c-erbB2/neu) polyclonal Ig in 5% fat-free dried milk in Tris/NaCl/P i , followed by incubation with 1 : 200 diluted horseradish peroxidase-labeled goat anti-(rabbit IgG) Ig, and stained with enhanced chemiluminescence reagent.…”

“…Control, Cells not treated with any antibodies; CD15, cells treated with mAb to Le x ; KM93, cells treated with mAb to SLe x ; FH6, cells treated with mAb to SDLe x ; CA19-9, cells treated with mAb to SLe a . The final concentrations of antibodies were 10 ng´mL 21 and 20 ng´mL 21 for experiments with mock-transfected cells and c-erbB2/neu-transfected cells, respectively. *P , 0.01 compared with the control (n 3).…”

Significance of Individual Point Mutations, T202C and C314T, in the Human Lewis (FUT3) Gene for Expression of Lewis Antigens by the Human α(1,3/1,4)-Fucosyltransferase, Fuc-TIII

“…FUT3-7 encode for ␣(1,3)-Fuc-Ts. Two of these human enzymes, Fuc-TIII and Fuc-TV encoded by the FUT3 and FUT5 genes, respectively (10,14), also express ␣(1,4)-fucosyltransferase activities (15,19,20).…”