Biosynthesis of Lewis fucolipid antigens in human colorectal carcinoma cells. Partial characterization of LcOse4Cer and H-1 fucolipid fucosyl transferase acceptor activities

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Abstract
Purified glycolipids were tested for their ability to serve as acceptors of [14C]fucose from GDP‐[14C]fucose as catalyzed by cell‐free extracts and purified membrane fractions of human colorectal carcinoma cells, SW1116, cultured in serum‐free medium. Purified lactotetraosyl ceramide (Galβ1→3GlcNAcβ1→3Galβ1→4Glc‐Cer or LcOse4Cer) and H‐1 glycolipid (Fucα1→2Galβ1→3GlcNAcβ1→3Galβ1→4Glc‐Cer or IV2 FucαLcOse4Cer) stimulated incorporation of radioactivity into lipid‐soluble glycolipid at a rate greater than ten times that of Lea glycolipid [Galβ1→3(Fucα1→4)GlcNAcβ1→3Galβ1→4Glc‐Cer or III4 FucαLcOse4Cer]. The enzymatic activities in crude and purified membrane fractions were optimized for substrate concentrations (glycolipid and GDP‐fucose), detergent requirement (taurocholate), pH, time and protein. The radioactive product of H‐1 fucosylation migrated as discrete and distinct bands on high‐performance thin‐layer chromatograms (HPTLC). Evidence for their identity with Leb fucolipid described previously [Fucα1→2Galβ1→3(Fucα1→4)GlcNAcβ1→3Galβ1→4Glc‐Cer or III4IV2 (Fucα) LcOse4Cer] is presented. The radioactive product of LcOse4Cer fucosylation was mainly Lea fucolipid as determined by co‐migration with authentic Lea fucolipid in three HPTLC systems as native and acetylated derivatives. Our results also indicated a low level of H‐1 and Leb glycolipid synthesis from LcOse4Cer. On the basis of the optima, linearity for time, and enzyme‐limiting conditions, we obtained a 12–19‐fold purification of the LcOse4Cer and H‐1 fucosyl transferase acceptor activities in three peaks of a sucrose gradient. The peak with the highest specific activity (peak 3) was highest in density and in Na+, K+, ATPase specific activity, although NADH–cytochrome‐c reductase and UDP‐GalNac transferase were also present in peak 3. The apparent Km values of LcOse4Cer acceptor activity and H‐1 acceptor activity in peak 3 were significantly different (p < 0.01) by statistical tests, 2.4 μM and 0.5 μM, respectively. These apparent Km values were much lower (103×) and the pH optima were lower (4.8–5.3), than the corresponding properties reported for the α1→3/α1→4 fucosyl transferase purified from human milk. Our results suggest a role for the non‐glycosidic moieties of the acceptors and/or the tissue‐specific or primitive expression of these fucosyl transferase activities.