N-acetylglucosaminyltransferase (
GnT)-IV catalyzes the formation of the GlcNAcbeta1-4 branch on the GlcNAcbeta1-2Manalpha1-3 arm of the core structure of N-
glycans. Two human
GnT-IV isozymes (GnT-IVa and GnT-IVb) had been identified, which exhibit different expression profiles among human tissues and
cancer cell lines. To clarify the enzymatic properties of the respective
enzymes, their kinetic parameters were determined using recombinant full-length
enzymes expressed in COS7 cells. The K (m) of human GnT-IVb for
UDP-GlcNAc was estimated to be 0.24 mM, which is 2-fold higher than that of human GnT-IVa. The K (m) values of GnT-IVb for pyridylaminated (PA) acceptor
sugar chains with different branch numbers were 3- to 6-fold higher than those of GnT-IVa. To compare substrate specificities more precisely, we generated recombinant soluble
enzymes of human GnT-IVa and GnT-IVb with N-terminal flag tags. Both
enzymes showed similar substrate specificities as determined using fourteen PA-
sugar chains. They preferred complex-type N-
glycans over hybrid-types. Among the complex-type N-
glycans tested, the relative activities of both
enzymes were increased in proportion to the number of GlcNAc branches on the Man alpha1-6 arm. The Man alpha1-6 arm of the acceptors was not essential for their activities because a linear pentasaccharide lacking this arm, GlcNAcbeta1-2Manalpha1-3Manbeta1-4GlcNAcbeta1-4 GlcNAc-PA, was a substrate for both
enzymes. These results indicate that human GnT-IVb exhibits the same acceptor substrate specificities as human GnT-IVa, although GnT-IVb has lower affinities for donors or acceptors than GnT-IVa. This suggests that GnT-IVa is more active than GnT-IVb under physiological conditions and that it primarily contributes to the biosynthesis of N-
glycans.