Kinetic properties and substrate specificities of two recombinant human N-acetylglucosaminyltransferase-IV isozymes |
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Authors: | Suguru Oguri Aruto Yoshida Mari T Minowa Makoto Takeuchi |
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Institution: | (1) Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido 099-2493, Japan;(2) Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawaku, Yokohama 236-0004, Japan;(3) Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawaku, Yokohama 236-0004, Japan;(4) Life Science Group, Hitachi, Ltd., 1-3-1 Minamidai, Kawagoe, Saitama 350-1165, Japan |
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Abstract: | N-acetylglucosaminyltransferase (GnT)-IV catalyzes the formation of the GlcNAcβ1-4 branch on the GlcNAcβ1-2Manα1-3 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 α1-6 arm. The Man α1-6 arm of the acceptors was not essential for their activities because a linear pentasaccharide
lacking this arm, GlcNAcβ1-2Manα1-3Manβ1-4GlcNAcβ1-4 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. |
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Keywords: | Human GnT-IVa Human GnT-IVb Kinetic properties Substrate specificities N-glycan biosynthesis |
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