Kinetic analysis of a Golgi UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-alpha-glucosaminyltransferase from Dictyostelium.
Mucin-type O-glycosylation in Dictyostelium is initiated in the Golgi by a UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-alpha-glucosaminyltransferase (Dd-pp alphaGlcNAcT2) whose sequence is distantly related to the sequences of animal polypeptide-Thr/Ser N-acetyl-alpha-galactosaminyltransferases, such as murine Mm-pp alphaGalNAcT1. To evaluate the significance of this similarity, highly purified Dd-pp alphaGlcNAcT2 was assayed using synthetic peptides derived from known substrates. Dd-pp alphaGlcNAcT2 strongly prefers UDP-GlcNAc 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 alphaGalNAcT1. Two selective competitive inhibitors of Mm-pp alphaGalNAcT1, retrieved from a screen of neutral-charge uridine derivatives, also inhibit Dd-pp alphaGlcNAcT1 competitively with only slightly less efficacy. Inhibition is specific for Dd-pp alphaGlcNAcT2 relative to two other Dictyostelium retaining glycosyltransferases. These data support a phylogenetic model in which the alphaGlcNAcT function in unicellular eukaryotes converted to an alphaGalNAcT function in the metazoan ortholog while conserving a similar reaction mechanism and active site architecture.
Ercan
Altan
A
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 937, Oklahoma City, OK 73104, USA.
West
Christopher M
CM
D000818:Animals; D004023:Dictyostelium / Q000201:enzymology*; D006056:Golgi Apparatus / Q000201:enzymology; D007700:Kinetics; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D013379:Substrate Specificity
D017350:N-Acetylgalactosaminyltransferases