Dicty News Electronic Edition Volume 24, number 1 January 14, 2005 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= A DICTYOSTELIUM MUTANT WITH REDUCED LYSOZYME LEVELS COMPENSATES BY INCREASED PHAGOCYTIC ACTIVITY Iris Mueller#, Ninon Subert~, Heike Otto#, Rosa Herbst¤, Harald Ruehling#, Markus Maniak# @, and Matthias Leippe& The Journal of Biological Chemistry, in press Lysozymes are bacteria-degrading enzymes and play a major role in the immune defense of animals. In free-living protozoa, lysozyme-like proteins are involved in the digestion of phagocytosed bacteria. Here, we purified a protein with lysozyme activity from Dictyostelium amoebae, which constitutes the founding member a novel class of lysozymes. By tagging the protein with GFP1 or the myc epitope, a new type of lysozyme containing vesicle was identified that was devoid of other known lysosomal enzymes. The most highly expressed isoform, encoded by the alyA gene, was knocked out by homologous recombination. The mutant cells had greatly reduced enzymatic activity and grew inefficiently when bacteria were the sole food source. Over time, the mutant gained the ability to internalize bacteria more efficiently, so that the defect in digestion was compensated by increased uptake of food particles. Submitted by: Markus Maniak [maniak@uni-kassel.de] ----------------------------------------------------------------------------- Kinetic analysis of a Golgi UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-alpha-glucosaminyltransferase from Dictyostelium Altan Ercan and Christopher M. West Department of Biochemistry and Molecular Biology, 940 Stanton L. Young Blvd., BMSB 937, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 USA; telephone: 405-271-2227 x1247; Fax 405-271-3139; email: Cwest2@ouhsc.edu Glycobiology, in press 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. Submitted by: Chris West [Cwest2@ouhsc.edu] ============================================================================== [End Dicty News, volume 24, number 1]