Role of the γ-subunit of GlcNAc-1-phosphotransferase in the pathogenesis of mucolipidosis type III

Abstract

Lysosomes contain about 70 soluble enzymes which have to be modified with mannose 6-phosphate (M6P) residues for efficient targeting to lysosomes. The key enzyme in the formation of M6P residues is the Golgi-resident hexameric GlcNAc-1-phosphotransferase complex (α2β2γ2). The α- and β-subunits exhibit catalytic activity, whereas the function of the -subunits encoded by the GNPTG gene is unknown. Mutations in the GNPTG gene cause the lysosomal storage disorder mucolipidosis type III (MLIII) that is mainly characterized by tissue-specific missorting of lysosomal enzymes and abnormalities of bone and cartilage. The experiments performed in this thesis revealed novel insights into the role of γ-subunits of the GlcNAc-1-phosphotransferase in the pathogenesis of the MLIII disease: 1. Analysis of GnptglacZ reporter mice demonstrated tissue and cell-specific expression, e.g. in bone and cartilage cells (osteoblasts, osteoclasts and chondrocytes), the major functionally impaired cell types in MLIII disease. 2. In fibroblasts of Gnptgko mice the loss of -subunits led to reduction of GlcNAc-1-phosphotransferase activity by 40%. SILAC-based M6P secretome and lysosomal proteome analyses in Gnptgko fibroblasts revealed that the M6P formation of 11 lysosomal enzymes is dependent on γ-subunits that impairs their intracellular targeting efficiency to lysosomes. Among these enzymes arylsulfatase B (Arsb), involved in the degradation of glycosaminoglycans (GAGs), was found to be missorted in primary cultured fibroblasts, osteoblasts and chondrocytes of Gnptgko mice accompanied by the accumulation of non-degraded GAGs in lysosomes. 3. The accumulation of storage material, a typical feature of MLIII patients, was surprisingly not associated with functional impairment of Gnptgko bone and cartilage cells. Therefore no skeletal abnormalities were detected in Gnptgko mice. 4. Incubation of chondroitin/dermatan sulfates-accumulating Gnptgko fibroblasts and chondrocytes with the human recombinant M6P-containing ARSB (Naglazyme®) partially rescued the lysosomal GAG storage, thereby identifying Arsb as a critical player in lysosome homeostasis in Gnptgko cells, and most likely in the MLIII disease. This result is a proof-of-principle that the approved Naglazyme® replacement therapy significantly reduces non-degraded GAGs in cultured Gnptgko cells. Further studies are needed to evaluate the efficiency of Naglazyme® in patients with MLIII.

Lysosomes contain about 70 soluble enzymes which have to be modified with mannose 6-phosphate (M6P) residues for efficient targeting to lysosomes. The key enzyme in the formation of M6P residues is the Golgi-resident hexameric GlcNAc-1-phosphotransferase complex (α2β2γ2). The α- and β-subunits exhibit catalytic activity, whereas the function of the -subunits encoded by the GNPTG gene is unknown. Mutations in the GNPTG gene cause the lysosomal storage disorder mucolipidosis type III (MLIII) that is mainly characterized by tissue-specific missorting of lysosomal enzymes and abnormalities of bone and cartilage. The experiments performed in this thesis revealed novel insights into the role of γ-subunits of the GlcNAc-1-phosphotransferase in the pathogenesis of the MLIII disease: 1. Analysis of GnptglacZ reporter mice demonstrated tissue and cell-specific expression, e.g. in bone and cartilage cells (osteoblasts, osteoclasts and chondrocytes), the major functionally impaired cell types in MLIII disease. 2. In fibroblasts of Gnptgko mice the loss of -subunits led to reduction of GlcNAc-1-phosphotransferase activity by 40%. SILAC-based M6P secretome and lysosomal proteome analyses in Gnptgko fibroblasts revealed that the M6P formation of 11 lysosomal enzymes is dependent on γ-subunits that impairs their intracellular targeting efficiency to lysosomes. Among these enzymes arylsulfatase B (Arsb), involved in the degradation of glycosaminoglycans (GAGs), was found to be missorted in primary cultured fibroblasts, osteoblasts and chondrocytes of Gnptgko mice accompanied by the accumulation of non-degraded GAGs in lysosomes. 3. The accumulation of storage material, a typical feature of MLIII patients, was surprisingly not associated with functional impairment of Gnptgko bone and cartilage cells. Therefore no skeletal abnormalities were detected in Gnptgko mice. 4. Incubation of chondroitin/dermatan sulfates-accumulating Gnptgko fibroblasts and chondrocytes with the human recombinant M6P-containing ARSB (Naglazyme®) partially rescued the lysosomal GAG storage, thereby identifying Arsb as a critical player in lysosome homeostasis in Gnptgko cells, and most likely in the MLIII disease. This result is a proof-of-principle that the approved Naglazyme® replacement therapy significantly reduces non-degraded GAGs in cultured Gnptgko cells. Further studies are needed to evaluate the efficiency of Naglazyme® in patients with MLIII.