Lysosome function is essential for the normal development and good health of humans and other higher organisms. Patients with deficiencies in individual or multiple lysosomal enzymes exhibit acute and chronic health problems including mental retardation, developmental deformities, cardiac impairment, and premature death. Most lysosomal enzymes are delivered to the lysosome by the mannose 6-phosphate dependent targeting system. In this pathway, lysosomal enzymes are phosphorylated on selected mannose residues by a lysosomal enzyme phosphotransferase. The phosphotransferase is defective in patients with mucolipidosis II and III, which results in improper targeting of lysosomal enzymes. In normal individuals, the phosphorylated lysosomal enzymes bind to small and large mannose 6-phosphate receptors which then deliver the enzymes to lysosomes. the objectives of this research project are to understand the mechanism of lysosomal enzyme targeting and the molecular basis for mucolipidosis II and III. there are three specific aims. 1) cDNAs encoding the lysosomal enzyme phosphotransferase will be isolated by expression cloning, sequenced to determine the phosphotransferase's primary structure, and used to identify the molecular lesion in patients with mucolipidosis II and III. 2) Mammalian cell lines lacking either or both types of mannose 6-phosphate receptors will be created using targeted gene disruption and transfection experiments. The cells will be analyzed to determine the relative functions of the small and large mannose 6-phosphate receptors in lysosomal enzyme targeting. 3) The molecular basis for large mannose 6-phosphate receptor function will be investigated by transfecting cultured cells with mutant receptors and analyzing their ability to target lysosomal enzymes. In addition, the unique reagents created in this research will facilitate development of targeted enzyme replacement therapies to treat patients with many different lysosomal storage disorders.
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