Characterization of UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase V from a human hepatoma cell line Hep3B

UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltr ansferase V (GlcNAcT-V) has been purified from cell extracts of the human h epatoma cell line, Hep3B, with 8.7% recovery. The purified enzymes had mole cular masses of about 67 and 65 kDa on denaturated and natural conditions, respectively. The values of pI was 5.9. The GlcNAcT-V, when resolved by SDS -PAGE, was positive for Schiff staining, suggesting that the enzyme is glyc oprotein. When GlcN,GlcN-biant-PA and UDP-GlcNAc were used as substrates, t he enzyme displayed a temperature optimum of around 50 degrees C and optimu m an pH of 6.5, The enzyme was stable in response to incubation from pH 4.5 to pH 10.5 at 4 degrees C for 24 h, The presence of UDP-GlcNAc and GlcN,Gl cN-bi-PA protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was stimulated by Mn2+ ion; however, it was inhibited by Fe3+ The enzyme activity was inh ibited by another series of NDP-sugars including ADP-, CDP-, GDP-, and TDP- GlcNAc. Studies on the activity of the enzyme toward a variety of pyridylam inated sugars showed that the enzyme is most active toward biantennary (Glc N,GlcN-bi-PA) sugars. The enzymes had apparent K-m values of 1.28 and 5.8 m M for GlcN,GlcN-bi-PA and UDP-GlcNAc, respectively. In order to isolate the GlcNAcT-V gene, PCR primers of GNN-1 and GNN-8 were designed and the ampli fied PCR product carrying the gene was cloned and sequenced. Nucleotide seq uence analysis showed a 2220-bp open reading frame encoding a 740-amino-aci d protein. This was almost same as the previously reported human sequences, except for some sequence differences in three amino acids. The three amino acid changes were as follows: V-375 --> L, T-555 --> R, and (592)A --> G. These studies represent the detailed characterization of a purified GlcNAcT -V from human hepatoma cell Hep3B, (C) 1999 Academic Press.