Human lung adenocarcinoma α1,3/4-L-fucosyltransferase displays two molecular forms, high substrate affinity for clustered sialyl LacNAc type 1 units as well as mucin core 2 sialyl LacNAc type 2 unit and novel α1,2-L-fucosylating activity

Human lung tumor α1,3/4-L-fucosyltransferase (FT) was purified (2000-fold, 29% recovery) from 290 g of tissue by including a chromatography step on Affinity Gel-GDP. Two molecular forms (FTA, larger size carrying 15% α1,4-FT activity; FTB, the major form with 85% activity) were separated by further fractionation on a Sephacryl S-100 HR column. A difference in the electrophoretic mobilities of these two activities was also found on native polyacrylamide gel electrophoresis (PAGE). Both forms were devoid of typical α1,2-fucosylating activity but were associated with the novel α1,2-fucosylating ability of converting the Lewis a determinant to Lewis b. Based on percentage activity toward 2-O-MeGalβ1,3GlcNAcβ-O-Bn, both forms exhibited the same extent of activity toward various acceptors, which included sulfated, sialylated, or methylated LacNAc type 1 or type 2 as well as mucin core 2 acceptors. However, FTA and FTB exhibited a difference in their ability to act on mucin core 2 3-sialyl LacNAc (activities 24.2% and 40.8%, respectively, as compared to 2-O-MeGalβ1,3GlcNAcβ-O-Bn). The unsubstituted LacNAc type 1 acceptors were 15-20 times as active as the corresponding LacNAc type 2 acceptors. The 3-O-substitution on the β1,4-linked Gal (methyl, sulfate, or sialyl) in mucin core 2 acceptors increased the efficiency of these acceptors five- to eightfold. The most efficient acceptor for FTA and FTB was 3-O-sulfoGalβ1,3GlcNAcβ-O-Al (K_m 100 and 47 μM, respectively). The K_m (mM) values for 2-O-methyl Galβ1,3GlcNAcβ-O-Bn and 3-O-sialyl Galβ1,3GlcNAcβ-O-Bn were 0.40 and 2.5 (FTA) and 0.16 and 0.67 (FTB), respectively. The 35-kDa glycoprotein ancrod (from Malayan pit viper venom) containing 36% complex N-glycans with the antennae NeuAcα2,3Galβ1,3GlcNAcβ- acted as the best macromolecular acceptor substrate (K_m:45 μM), as examined with FTB. On desialylation the acceptor efficiency dropped to 50% (K_m for asialo ancrod: 167 μM). Sialylglycoproteins, such as carcinoembryonic antigen, fetuin, and bovine α₁-acid glycoprotein, were better acceptors than asialo fetuin. On the contrary, fetuin triantennary glycopeptide containing predominantly NeuAcα2,3Galβ1,4GlcNAcβ- was only 55% active as compared to the asialo glycopeptide (K_m: 1.43 and 0.63 mM, respectively). Thus, the human lung tumor α1,3/4-L-FT has the potential to generate clustered sialyl Lewis a and Lewis b determinants in N-glycans and sialyl Lewis determinant in mucin core 2 structures.