Characterization of the Biosynthesis, Processing and Kinetic Mechanism of Action of the Enzyme Deficient in Mucopolysaccharidosis IIIC

Heparin acetyl-CoA:alpha-glucosaminide N-acetyltransferase (N-acetyltransferase, EC 2.3.1.78) is an integral lysosomal membrane protein containing 11 transmembrane domains, encoded by the HGSNAT gene. Deficiencies of N-acetyltransferase 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 necessary and sufficient to express transferase activity, and that N-acetyltransferase functions as a monomer. Finally, the kinetic mechanism of action of purified N-acetyltransferase was evaluated and found to be a random sequential mechanism involving the formation of a ternary complex with its two substrates; i.e., N-acetyltransferase does not operate through a ping-pong mechanism as previously reported. We confirm this conclusion by demonstrating experimentally that no acetylated enzyme intermediate is formed during the reaction

Developing therapeutic approaches for metachromatic leukodystrophy

Shilpa A Patil,1 Gustavo HB Maegawa1,2 1McKusick-Nathans Institute of Genetic Medicine, 2Department of Pediatrics, The Johns Hopkins School of Medicine, Baltimore, MD, USA Abstract: Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal disorder caused by the deficiency of arylsulfatase A (ASA), resulting in impaired degradation of sulfatide, an essential sphingolipid of myelin. The clinical manifestations of MLD are characterized by progressive demyelination and subsequent neurological symptoms resulting in severe debilitation. The availability of therapeutic options for treating MLD is limited but expanding with a number of early stage clinical trials already in progress. In the development of therapeutic approaches for MLD, scientists have been facing a number of challenges including blood–brain barrier (BBB) penetration, safety issues concerning therapies targeting the central nervous system, uncertainty regarding the ideal timing for intervention in the disease course, and the lack of more in-depth understanding of the molecular pathogenesis of MLD. Here, we discuss the current status of the different approaches to developing therapies for MLD. Hematopoietic stem cell transplantation has been used to treat MLD patients, utilizing both umbilical cord blood and bone marrow sources. Intrathecal enzyme replacement therapy and gene therapies, administered locally into the brain or by generating genetically modified hematopoietic stem cells, are emerging as novel strategies. In pre-clinical studies, different cell delivery systems including microencapsulated cells or selectively neural cells have shown encouraging results. Small molecules that are more likely to cross the BBB can be used as enzyme enhancers of diverse ASA mutants, either as pharmacological chaperones, or proteostasis regulators. Specific small molecules may also be used to reduce the biosynthesis of sulfatides, or target different affected downstream pathways secondary to the primary ASA deficiency. Given the progressive neurodegenerative aspects of MLD, also seen in other lysosomal diseases, current and future therapeutic strategies will be complementary, whether used in combination or separately at specific stages of the disease course, to produce better outcomes for patients afflicted with this devastating inherited disorder. Keywords: metachromatic leukodystrophy, arylsulfatase A, enzyme replacement therapy, gene therapy, enzyme enhancement therapy, small molecule

A Large Intragenic Deletion in the ACADM Gene Can Cause MCAD Deficiency but is not Detected on Routine Sequencing

We report of a family who has three members affected by medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, one of whom sadly died in the neonatal period prior to diagnosis. Routine sequencing, available on a service basis in the UK, identified only a heterozygous mutation in ACADM gene (c.985A>G, p.Lys329Glu) in this family. Linkage analysis suggested a possible intragenic deletion which was confirmed by the use of array-based comparative genomic hybridization (aCGH). This second mutation was a large intragenic deletion encompassing at least exons 1–6 of the ACADM gene. Now that this deletion has been identified, several family members have come forward for carrier testing which was not possible previously. Larger deletions (20bp or more) have only previously been reported twice, but these may be a more frequent cause of MCAD deficiency than hitherto believed, due to fact that these are not anticipated and, therefore, the routine diagnostic techniques used will not identify them. This finding represents a useful learning point in the management of families with MCAD deficiency, and highlights that we should be routinely looking for larger deletions, when only one of the mutations can be identified on standard sequencing

Animal Model Contributions to Congenital Metabolic Disease

Genetic model systems allow researchers to probe and decipher aspects of human disease, and animal models of disease are frequently specifically engineered and have been identified serendipitously as well. Animal models are useful for probing the etiology and pathophysiology of disease and are critical for effective discovery and development of novel therapeutics for rare diseases. Here we review the impact of animal model organism research in three examples of congenital metabolic disorders to highlight distinct advantages of model system research. First, we discuss phenylketonuria research where a wide variety of research fields and models came together to make impressive progress and where a nearly ideal mouse model has been central to therapeutic advancements. Second, we review advancements in Lesch-Nyhan Syndrome research to illustrate the role of models that do not perfectly recapitulate human disease as well as the need for multiple models of the same disease to fully investigate human disease aspects. Finally, we highlight research on the GM2 gangliosidoses Tay-Sachs and Sandhoff Disease to illustrate the important role of both engineered traditional laboratory animal models and serendipitously identified atypical models in congenital metabolic disorder research. We close with perspectives for the future for animal model research in congenital metabolic disorders