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

• Published in: The Journal of biological chemistry 1985-06, Vol.260 (12), p.7619-7627
• Main Authors: Holmes, E H, Ostrander, G K, Hakomori, S
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Cell Membrane - enzymology; Fucosyltransferases - isolation & purification; Fucosyltransferases - metabolism; Glycolipids - biosynthesis; Glycolipids - genetics; Glycolipids - isolation & purification; Hexosyltransferases - metabolism; Humans; Kinetics; Lung Neoplasms - metabolism; Medical sciences; Pneumology; Tumors of the respiratory system and mediastinum
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)39654-0

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.