title: Lysosomal neuraminidase. Catalytic activation in insect cells is controlled by the protective protein/cathepsin A.
abstract: Lysosomal N-Acetyl-alpha-neuraminidase is active in complex with the protective protein/cathepsin A (PPCA) and beta-galactosidase. The interaction with PPCA is essential for the correct intracellular routing and lysosomal localization of neuraminidase, but the mechanism of its catalytic activation is unclear. To investigate this process, we have used the baculovirus expression system to co-express neuraminidase and PPCA precursors in insect cells, which resulted in high enzymatic activity of neuraminidase. Both the 34- and 20-kDa PPCA subunits were required for the activation. We further demonstrated that when expressed alone, the neuraminidase precursor remained dimeric (114 kDa) and had low enzymatic activity, but when co-expressed with PPCA and beta-galactosidase, it multimerized in a complex of approximately 1350 kDa, together with the other two proteins. The fully active neuraminidase co-precipitated with full-length PPCA and beta-galactosidase precursors. However, when co-expressed with the individual PPCA subunits, neuraminidase co-precipitated only with the small 20-kDa polypeptide, which therefore must contain a neuraminidase-binding site. Our finding suggests a model of activation of neuraminidase dependent on its oligomerization at acidic pH that is mediated by interaction with PPCA.
authors.affiliation: St. Jude Children's Research Hospital, Department of Genetics, Memphis, Tennessee 38105, USA.
authors.forename: E J
authors.initials: EJ
authors.lastname: Bonten
authors.forename: A
authors.initials: A
authors.lastname: d'Azzo
mesh_terms: D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism*; D002384:Catalysis; D043402:Cathepsin A; D002460:Cell Line; D004789:Enzyme Activation; D008247:Lysosomes / Q000201:enzymology*; D009439:Neuraminidase / Q000378:metabolism*; D018411:Spodoptera / Q000201:enzymology
chemical_list: D009439:Neuraminidase; D002268:Carboxypeptidases; D043402:Cathepsin A