Enzymatic basis for the accumulation of glycolipids with X and dimeric X determinants in human lung cancer cells (NCI-H69).

Many human carcinomas accumulate a large quantity of glycolipids having X (Gal beta 1----4[Fuc alpha 1----3] GlcNAc) as well as di- or trimeric X determinant (Gal beta 1----4 [Fuc alpha 1----3] GlcNAc beta 1----3Gal beta 1----4 [Fuc alpha 1----3]GlcNAc beta 1----3Gal) (e.g. Hakomori, S., Nudelman, E., Levery, S. B., and Kannagi, R. (1984) J. Biol. Chem. 259, 4672-4680). The enzymatic basis of this phenomenon has been investigated with human small cell lung carcinoma NCI-H69 cells, in which a series of these structures has been found to accumulate. An alpha 1----3 fucosyltransferase solubilized from the membrane fraction with Triton X-100 catalyzed not only the transfer of a fucosyl residue from GDP-fucose to the penultimate GlcNAc residue of lactoneotetraosylceramide (nLc4) and lactonorhexaosylceramide (nLc6), but also to the internal GlcNAc residue (III-GlcNAc) of y2 glycolipid (V3FucnLc6) and that of sialosyl2----6lactonorhexaosylceramide (VI6NeuAcnLc6). No transfer of fucose to the internal GlcNAc (III-GlcNAc) of lactonorhexaosylceramide occurred, unless the above substitutions (V3Fuc or VI6NeuAc) were present. Fucosylation at V-GlcNAc and III-GlcNAc of nLc6 could be catalyzed by the same enzyme, based on the following observations: (i) fucosylation at both III- and V-GlcNAc was competitively inhibited by V3FucnLc6 and III3V3Fuc2nLc6; (ii) the same conditions (pH, bivalent cation, detergent) were optimal for fucosylation at both III- and V-GlcNAc; (iii) the Km values of the enzyme for nLc4, nLc6, and V3FucnLc6 were approximately the same; and (iv) the activity of the enzyme catalyzing fucosylation at both III- and V-GlcNAc was adsorbed on GDP-hexanolamine-Sepharose and was not inhibited by N-ethylmaleimide. The enzyme preferentially transferred fucose to the penultimate VGlcNAc, followed by transfer to the internal III-GlcNAc of nLc6. Thus, the pathway for synthesis of dimeric X proceeds as follows: nLc6----V3FucnLc6----III3V3Fuc2nLc6. No mechanism was found to operate for chain elongation of the X hapten structure through addition of GlcNAc residues to the terminal Gal of the X hapten.