Murine aspartoacylase: cloning, expression and comparison with the human enzyme.
Canavan disease is caused by mutations in aspartoacylase, the enzyme that degrades N-acetylaspartate (NAA) into acetate and aspartate. Murine aspartoacylase (mASPA) was cloned using sequence information from mouse expressed sequence tags homologous to the human cDNA. The open reading frame was cloned into a thioredoxin fusion vector, overexpressed in bacteria, and the protein was purified using affinity chromatography to near homogeneity. Recombinant human ASPA (hASPA) was prepared by a similar method. Both recombinant enzymes were highly specific to NAA, with about 10% of the NAA activity toward N-acetylasparagine. More interestingly, the product of N-acetylasparagine was aspartate but not asparagine, indicating that ASPA catalyzed deacetylation as well as hydrolysis of the beta acid amide. Our success in preparing the recombinant ASPA in high purity should permit multiple lines of investigations to understand the pathogenic mechanisms of Canavan disease and the functional roles of NAA.
Namboodiri
M A
MA
Section on Neuroendocrinology, Laboratory of Developmental Neurobiology, NICHD, Bethesda, MD 20892, USA. anamboodiri@usuhs.mil
Corigliano-Murphy
A
A
Jiang
G
G
Rollag
M
M
Provencio
I
I
D000107:Acetylation; D000577:Amides / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000031:analogs & derivatives / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D017825:Canavan Disease / Q000201:enzymology / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D000577:Amides; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D001216:Asparagine; C000179:N-acetylaspartate; C026988:N-acetylasparagine; D000581:Amidohydrolases; C019751:aspartoacylase