| Abstrakt: |
An analysis of α1→3fucosyltransferase expression and enzyme properties has been conducted in human lung carcinoma NCI‐H69 and PC9 cells. The results indicate that multiple forms of α1→3 fucosyltransferase are found in these cells. RT‐PCR experiments using total RNA from NCI‐H69 and PC9 cells amplified transcripts for three of these enzymes, FucT‐IV, ‐VI, and ‐VII. Fucose transfer into glycolipid acceptors mediated by truncated chimeric and full length recombinant FucT‐IV and ‐VI enzymes was examined. Both enzymes were found to be type 2 chain specific, but only FucT‐VI efficiently transferred fucose to both neutral and sialylated acceptors. A truncated recombinant form of FucT‐VI was capable of fucose transfer to the internal Glc residue of a variety of glycolipid acceptors. This property was not observed with the recombinant full length enzyme, suggesting the N‐terminal portion of the protein, composed of the intracellular domain, transmembrane domain, and a part of the stem region, is involved in interactions with glycolipid acceptors. Using taurodeoxycholate as the detergent, the distribution of initial fucose transfer into nLc6 catalyzed by recombinant full length enzyme indicated 34% of the mono‐fucosyl product was fucosylated at the III‐GlcNAc and 66% at the V‐GlcNAc for FucT‐IV, and almost all of the FucT‐VI mono‐fucosyl product was III‐GlcNAc fucosylated. Similar experiments with VI2NeuAcnLc6 as the acceptor resulted in predominantly III‐GlcNAc monofucosylation, although detectable V‐GlcNAc monofucosylation was obtained with FucT‐VI. When the cationic detergent G‐3634‐A was used, substantially greater initial transfer into the V‐GlcNAc of both neutral and sialylated acceptors with FucT‐VI was observed. Using nonsialylated acceptors, total α1→3 fucosyltransferase activity in NCI‐H69 cells was analyzed and found to be diminished 25–30% by exposure to 30 mM NEM, which can be attributed to FucT‐VI inactivation. The remaining 70–75% of NEM‐resistant activity is attributed to FucT‐IV, an NEM‐resistant enzyme form capable of fucosylating nonsialylated acceptors. These results suggest that multiple forms of α1→3fucosyltransferase are expressed in NCI‐H69 and PC9 cells, which may account for the observed properties of enzyme derived from these cell lines. [ABSTRACT FROM PUBLISHER] |
