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Glycobiology Advance Access published online on December 22, 2004
Glycobiology, doi:10.1093/glycob/cwi034
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© Oxford University Press 2004; all rights reserved.
Received August 29, 2004
Revised November 27, 2004
Accepted November 29, 2004

Article

Kinetic analysis of a Golgi UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl- alpha;-glucosaminyltransferase from Dictyostelium

Altan Ercan 1 and Christopher M. West 1*

1 Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 USA

* To whom correspondence should be addressed.
Christopher M. West, E-mail: Cwest2{at}ouhsc.edu


  Abstract

Mucin-type O-glycosylation in Dictyostelium is initiated in the Golgi by a UDP-GlcNAc:polypeptide- Thr/Ser N-acetyl-{alpha}-glucosaminyltransferase (Dd-pp {alpha}GlcNAcT2) whose sequence is distantly related to the sequences of animal polypeptide-Thr/Ser N-acetyl-{alpha}- galactosaminyltransferases such as murine Mm-pp {alpha}GalNAcT1. To evaluate the significance of this similarity, highly purified Dd-pp {alpha}GlcNAcT2 was assayed using synthetic peptides derived from known substrates. Dd-pp {alpha}GlcNAcT2 strongly prefers UDPGlcNAc over UDP-GalNAc, preferentially modifies the central region of the peptide, and modifies Ser- in addition to Thr-residues. Initial velocity measurements performed over a matrix of UDP-GlcNAc donor and peptide acceptor concentrations indicate that the substrates bind to the enzyme in ordered fashion before the chemical conversion. Substrate inhibition exerted by a second peptide, and the pattern of product inhibition exerted by UDP, suggest that UDP-GlcNAc binds first and the peptide binds second, consistent with data reported for Mm-pp {alpha}GalNAcT1. Two selective competitive inhibitors of Mm-pp {alpha}GalNAcT1, retrieved from a screen of neutral-charge uridine derivatives, also inhibit Dd-pp {alpha}GlcNAcT1 competitively with only slightly less efficacy. Inhibition is specific for Dd-pp {alpha}GlcNAcT2 relative to two other Dictyostelium retaining glycosyltransferases. These data support a phylogenetic model in which the {alpha}GlcNAcT function in unicellular eukaryotes converted to an {alpha}GalNAcT function in the metazoan ortholog while conserving a similar reaction mechanism and active site architecture.

Keywords: evolution/inhibitor/mucin type/O-glycosylation/reaction mechanism.
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[Abstract] [Full Text] [PDF]


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