characterization of the biosynthesis, processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis iiic特征的生物合成、加工、动能的酶的作用机制缺乏黏多糖病iiic.pdfVIP

Characterization of the Biosynthesis, Processing and Kinetic Mechanism of Action of the Enzyme Deficient in Mucopolysaccharidosis IIIC 1 1,2 1 1 1,2 Xiaolian Fan , Ilona Tkachyova , Ankit Sinha , Brigitte Rigat , Don Mahuran * 1 Genetics and Genome Biology Program, The Hospital For Sick Children, Toronto, Canada, 2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Abstract Heparin acetyl-CoA:alpha-glucosaminide N-acetyltransferase (N-acetyltransferase, EC 8) is an integral lysosomal membrane protein containing 11 transmembrane domains, encoded by the HGSNAT gene. Deficiencies of N-acetyl- transferase lead to mucopolysaccharidosis IIIC. We demonstrate that contrary to a previous report, the N-acetyltransferase signal peptide is co-translationally cleaved and that this event is required for its intracellular transport to the lysosome. While we confirm that the N-acetyltransferase precursor polypeptide is processed in the lysosome into a small amino- terminal alpha- and a larger ß- chain, we further characterize this event by identifying the mature amino-terminus of each chain. We also demonstrate this processing step(s) is not, as previously reported, needed to produce a functional transferase, i.e., the precursor is active. We next optimize the biochemical assay procedure so that it remains linear as N- acetyltransferase is purified or protein-extracts containing N-acetyltransferase are diluted, by the inclusion of negatively charged lipids. We then use this assay to demonstrate that the purified single N-acetyltransferase protein is both nece