Comparison of the substrate specificities and catalytic properties of the sister N-acetylglucosaminyltransferases, GnT-V and GnT-Vb (IX)

N-Acetylglucosaminlyltransferase-V (GnT-V) synthesizes GlcNAc beta 1,6Man branched N-glycans both in vitro and in vivo. A paralog, GnT-Vb (or GnT-IX), has also been shown to synthesize both GlcNAc beta 1,6Man branched N- and O-glycans. GnT-V is expressed in most human and rodent tissues while GnT-Vb expression is limited mainly to neural tissue and testes. It is of interest, therefore, to compare the catalytic properties and reaction kinetics of these sister enzymes. The results demonstrate that while GnT-V was fully active without exogenous cation and in the presence of EDTA, the activity of GnT-Vb was stimulated over 4-fold in the presence of 10 mM Mn++. The pH optimum for GnT-V was in the range of 6.5-7.0, while that of GnT-Vb was 8.0. common for glycosyltransferases active in brain. Both enzymes transferred GlcNAc beta 1,6 to the Man residue of the GlcNAc beta 1,2Man moiety of glycan substrates, and both enzymes acted effectively on a synthetic GlcNAc beta 1,2Man alpha 1,2Glc-O-octyl trisaccharide acceptor. Moreover, although both enzymes utilized an N-linked asialo-agalacto-biantennary glycan as an acceptor, GnT-Vb displayed an almost 2.5-fold higher apparent K-m value compared to GnT-V. Conversely, GnT-Vb very efficiently glycosylated a synthetic glycopeptide, Ac-H2N-Val-Glu-Pro-(GlcNAc beta 1,2-Man-O-)Thr-Ala-Val-CO-Ac, while GnT-V showed relatively poor activity toward this O-Man-linked glycopeptide acceptor, with a K-m value of 20-fold higher than that of GnT-Vb. When the N-linked asialo-agalacto-biantennary glycan acceptor was utilized with GnT-Vb, the expected triantennary beta 1,6-branched product was observed up to 8 h incubation. An additional product with two beta 1,6-linked GlcNAc resides, however, was observed after prolonged (> 8 h) incubation, consistent with an earlier report. This unusual tetraantennary product was observed with GnT-Vb only after substantial accumulation of the first triantennary product and not during the early stages of incubation.