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

Abstract

Purified glycolipids were tested for their ability to serve as acceptors of ['4C]fucose from GDP-['4C]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 (GalP1+3GlcNAcPl+ 3GalP1+4Glc-Cer or LcOse4Cer) and H-1 glycolipid (Fucal+2Gal~1+3GlcNAc~1+3Gal~1+4Glc-Cer or IV2 FucaLcOse4Cer) stimulated incorporation of radioactivity into lipid-soluble glycolipid at a rate greater than ten times that of Lea glycolipid [Gal~1+3(Fucal+4)GlcNAc~1+3Gal~1+4Glc-Cer or 1114 FucaLcOse4Cer].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 previouslyThe 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 K , values of LcOse4Cer acceptor activity and H-1 acceptor activity in peak 3 were significantly different 0) < 0.01) by statistical tests, 2.4 pM and 0.5 pM, respectively. These apparent K , values were much lower (lo3 x ) and the pH optima were lower (4.8 -5.3), than the corresponding properties reported for the a1 +3/ a1 4 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.

Purification and characterization of a CMP-sialic:LcOse4Cer sialyltransferase from human colorectal carcinoma cell membranes

“…Assay for Glycolipid-Dependent Sialyltransferase. Purified membrane fractions containing sialyltransferase activity were obtained from human colorectal carcinoma SW1116 cells by sucrose density centrifugation as previously described (Liepkans & Larson, 1987). Standard conditions for incubation were the following: 70 gg of taurocholate (sodium salt) dissolved in chloroform/methanol (2/1) was dried with 0.2-5 gg of LcOse4Cer or test glycolipid in a conical assay tube; 30-45 gL of (final concentrations) cacodylate (0.15 mM), sucrose (0.3 M), MgCl2 (1 mM), and CDP-choline or ATP (0.5 mM) were then added.…”

“…Two milliliters of affinity gel, CMP-agarose (Sigma) or LcOse4-fractogel (Bio-Carb, Lund, Sweden) were washed in CST buffer (0.025 M cacodylate, 0.050 M NaCl, 0.05% taurocholate) by suspension and centrifugation at lOOOg, 6 X 6 mL. Human colorectal carcinoma cells cultured in serum-free medium were obtained as described previously (Liepkans et al, 1985): the cells were homogenized and sonicated (15 s, 4 bursts at +4 °C) in CSM buffer (0.2 M cacodylate, 0.2 M sucrose, 1 mM MgCl2) and then centrifuged at lOOOg for 1 h. The pellet of nuclei unbroken cells and debris was discarded, and the supernatant was applied to a sucrose gradient (p = 1.10-1.18) in CSM buffer (Liepkans & Larson, 1987). After the sucrose gradient, the total membranes from this centrifugation (excluding the pellet) were recovered by dilution 2-fold with 0.15 M cacodylate, pH 6.2, and centrifugation at 150000g for 2 h. The resulting final microsomal pellet, which was devoid of mitochondria, was suspended in CST buffer by repeated gentle regurgitation with sterile Pasteur pipets.…”

“…In IV3NeuNAcnLcOse4Cer runs ahead of ganglioside GM, (II3NeuNAcGgOse4Cer), and GM, runs ahead of IV6NeuNAcnLcOse4Cer. Galactose oxidase treatment of LcOse4Cer was performed as previously described (Liepkans & Larson, 1987).…”

“…Our evidence indicates that the product is sialosyllactotetraosylceramide. This sialyltransferase may control the level of the sialylated Lewis3 because it synthesizes its direct precursor and appears to do so at a much lower rate of LcOse4Cer utilization than the fucosyltransferase activities for Lewis3 and Lewisb biosynthesis present in these cells (Liepkans & Larson, 1987). To our knowledge, this is the first characterization of this enzymatic activity in carcinoma cells from serum-free medium with a glycolipid acceptor of documented purity and structure (Karlsson & Larson, 1979).…”

Quantitative Analysis of Lea and Le^b Antigens in Human Saliva

“…Yazawa and Furukawa [28] showed no formation of Leb from Lea in the saliva from O secretor donors. It was also shown that the direct precursor of Leb fucolipid was H type 1 fucolipid and LeJ was not a good substrate for the synthesis of Leb fucolipid in extracts of purified membrane fractions of colorectal carcinoma cells, SW1116 [43]. On the other hand, it has been reported that extracts of microsomal fractions of human gastric mu cosa and gastric carcinoma cell line KATO III were active in forming Leb-glycolipid from Led-glycolipid [44,45], al though an activity to form Ley from Lex was not detected in KATO III cells [45], Recently, Yazawa et al [46] have also demonstrated the activity that catalyzed the forma tion of Leb from Lea and of Ley from Le' in a Triton X-100 extract of colon cancer tissue and suggested the presence of a new a-2-L-fucosyltransferase.…”