The scope of this proposal is to study the function of mammalian lysosomal neuraminidase (NEU1) in normal cell metabolism, and the consequences of its loss in human diseases. NEU1 belongs to the ubiquitous superfamily of sialidases. Mammalian neuraminidases include cytosolic, lysosomal, and plasma membrane isoforms, and clues about the physiologic roles of these hydrolases, in particular NEU1, have emerged only recently. NEU1 initiates the hydrolysis of sialo-glyconjugates by removing terminal sialic acid residues. The enzyme is unique among sialidases in that it must be associated with protective protein/cathepsin A (PPCA) for intracellular routing and lysosomal activation. Finally, NEU1 is linked to 2 neurodegenerative diseases of metabolism: Sialidosis is caused by structural lesions in NEU1, and galactosialidosis (GS), a combined deficiency of NEU1 and (3-galactosidase, is caused by the absence of PPCA. The proposed studies are based on 3 Specific Aims.
In Aim 1, we will investigate the structure- function relationship between NEU1 and PPCA. The 3D structure of the PPCA precursor will be used to target mutagenesis of potential contact sites between the 2 proteins and NEU1 mutations identified in patients with sialidosis. We will also use overlapping peptides that span the full-length PPCA and NEU1 to identify domains crucial for NEU1/PPCA interaction, intracellular transport, and activation. These biochemical studies will be coupled to determine the 3D structure of the Neu1/PPCA complex.
In Aim 2, we will compare the characteristics of Neu1~*~ mice and PPCA^~ mice to determine the molecular bases of sialidosis and GS and to identify yet unknown Neu1 functions in normal cell physiology.
In Aim 3, we will implement various enzyme replacement therapy approaches in both models to assess the correction of the systemic phenotypes in these diseases. We are in position to develop this line of investigation, because we have established appropriate genetic and biochemical systems for the proposed studies and can rely on the expertise of an outstanding structural biologist for the crystallography part of the project. Our overall goal is to gain a broader understanding of NEU1 function in normal physiology and in the pathophysiology of the neurodegenerative diseases sialidosis and GS. These diseases affect primarily infants and children. Findings from these proposed studies should increase our knowledge about NEU1 function and improve the design of future therapies for pediatric patients with these catastrophic diseases.
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