Human lysosomal protective protein has cathepsin A-like activity distinct from its protective function

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• The protective protein is crucial for the stability and activity of lysosomal enzymes beta-galactosidase and neuraminidase.
• Deficiency of the protective protein causes the metabolic storage disorder galactosialidosis.
• Human and mouse protective proteins share homology with plant and yeast serine carboxypeptidases.

Purpose of the Study:

• To investigate the enzymatic activity of the protective protein.
• To determine if the protective protein possesses cathepsin A-like activity.
• To differentiate the catalytic function from the protective role of the protective protein.

Main Methods:

• Overexpression of human and mouse protective proteins in COS-1 cells.
• Assay of cathepsin A-like activity in patient fibroblasts and normal human fibroblasts.
• Site-directed mutagenesis of active site residues (serine, histidine) and Cys60.
• Intracellular routing, processing, and secretion analysis of wild-type and mutant proteins.
• Functional restoration assays in galactosialidosis fibroblasts.

Main Results:

• Overexpression led to a 3-4 fold increase in cathepsin A-like activity.
• Activity was reduced to approximately 1% in galactosialidosis patients.
• Antibodies against protective protein precipitated cathepsin A-like activity.
• Mutagenesis of active site residues abolished enzymatic activity but not protein processing or secretion.
• Mutant precursors lacking catalytic activity restored enzyme function in galactosialidosis fibroblasts.

Conclusions:

• The protective protein possesses distinct enzymatic activity similar to lysosomal cathepsin A.
• Catalytic activity is separable from the protein's essential protective function for lysosomal enzymes.
• Understanding this distinction is vital for galactosialidosis and related lysosomal storage disorders.