Murine aspartoacylase: cloning, expression and comparison with the human enzyme

Canavan disease is caused by mutations in aspartoacylase, the enzyme that d egrades N-acetylaspartate (NAA) into acetate and aspartate, Murine aspartoa cylase (mASPA) was cloned using sequence information from mouse expressed s equence tags homologous to the human cDNA. The open reading frame was clone d into a thioredoxin fusion vector, overexpressed in bacteria, and the prot ein was purified using affinity chromatography to near homogeneity. Recombi nant human ASPA (hASPA) was prepared by a similar method. Both recombinant enzymes were highly specific to NAA, with about 10% of the NAA activity tow ard N-acetylasparagine. More interestingly, the product of N-acetylasparagi ne was aspartate but not asparagine, indicating that ASPA catalyzed deacety lation as well as hydrolysis of the beta acid amide. Our success in prepari ng the recombinant ASPA in high purity should permit multiple lines of inve stigations to understand the pathogenic mechanisms of Canavan disease and t he functional roles of NAA. (C) 2000 Elsevier Science B.V. All rights reser ved.