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Glycobiology Advance Access published online on October 21, 2009
Glycobiology, doi:10.1093/glycob/cwp158
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© The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Gerardo Alvarez-Manilla, Karolyn Troupe, Maria Fleming, Erika Martinez-Uribe and Michael Pierce1

Complex Carbohydrate Research Center, University of Georgia Cancer Center, and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602

1 Corresponding author

Received on February 12, 2009; accepted on September 27, 2009

N-acetylglucosaminlyltransferase-V (GnT-V) synthesizes GlcNAc β1,6Man branched N-glycans both in vitro and in vivo. A paralog, GnT-Vb (or GnT-IX), has also been shown to synthesize both GlcNAcβ1,6Man 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 GlcNAcβ1,6 to the Man residue of the GlcNAcβ1,2Man moiety of glycan substrates, and both enzymes acted effectively on a synthetic GlcNAcβ1,2Man{alpha}1,2Glc-O-octyl trisaccharide acceptor. Moreover, although both enzymes utilized an N-linked asialo-agalacto-biantennary glycan as acceptor, GnT-Vb displayed an almost 2.5-fold higher apparent Km value compared to GnT-V. Conversely, GnT-Vb very efficiently glycosylated a synthetic glycopeptide, Ac-H2N-Val-Glu-Pro-(GlcNAcβ1,2-Man-O-)Thr-Ala-Val-CO-Ac, while GnT-V showed relatively poor activity toward this O-Man-linked glycopeptide acceptor, with a Km 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 β1,6 branched product was observed up to 8 hr. incubation. An additional product with two β1,6-linked GlcNAc resides, however, was observed after prolonged (>8hr) 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.


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