The N-acetylglucosaminyl-1-phosphotransferase (termed phosphotransferase) catalyzes the initial step in the formation of mannose 6-phosphate (M6P) residues required for the efficient transport of soluble lysosomal enzymes. The phosphotransferase is a multisubunit enzyme composed of three subunits (alpha2beta2gamma2) that are products of two genes. The gene encoding the gamma-subunit (GNPTAG) appears to be defective in patients with mucolipidosis type III (ML III). We have analyzed the GNPTAG gene in two siblings with ML III showing elevated activities of several lysosomal enzymes in cultured fibroblasts serum and diminished activities in cultured fibroblasts. Immunoprecipitation of metabolically labeled cathepsin D (CtsD) from fibroblasts revealed that the sorting/transport of this lysosomal protease was affected. Addition of ammonium chloride inhibiting pH-dependent processes, such as the CtsD-M6P receptor interaction, indicated that 15 to 20% of the newly synthesized CtsD is transported in ML III fibroblasts in an M6P-dependent manner. By direct sequencing a novel homozygous mutation, c.347_349delACA (p.Asn116del), was identified affecting a potential N-linked glycosylation site. Western blot analysis of extracts from control fibroblasts detect a 97 kDa glycosylated dimer whereas ML III cells contain a GNPTAG dimer of reduced molecular mass. These data suggest that the loss of the used glycosylation site in the gamma subunit may affect the intracellular localization of GNPTAG and the overall efficiency of M6P formation.
