Lysosomal Proteome and Secretome Analysis Identifies Missorted Enzymes and Their Nondegraded Substrates in Mucolipidosis III Mouse Cells

Abstract

Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (αβγ). Upon proteolytic cleavage by site-1 protease, the α/β-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the expression in mouse tissues, primary cultured cells, and in reporter mice , and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/β-precursor was not affected by deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in lysosomes. Incubation of fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.

The lysosomal storage disorders mucolipidosis type II, type III alpha/beta, and type III gamma: Update onGNPTABandGNPTGmutations

“…In the Golgi apparatus the α/β‐precursor is proteolytically cleaved into enzymatically active α‐ and β‐subunits by site‐1 protease (S1P; Figure a; Marschner, Kollmann, Schweizer, Braulke, & Pohl, ; Velho et al, ). The soluble γ‐subunit directly binds to the α‐subunit and enhances the GlcNAc‐1‐phosphotransferase activity for M6P modification of specific lysosomal enzymes (De Pace et al, ; Di Lorenzo et al, ; Velho, De Pace, Tidow, Braulke, & Pohl, ).…”

“…In addition, GNPTAB missense mutations in the γ‐subunit‐binding domain decrease the GlcNAc‐1‐phosphotransferase activity (Figure b), presumably by impairing the interaction between the α‐ and γ‐subunits and consequently the subunit assembly of the complex (De Pace et al, ; Velho, De Pace, et al, ). Likewise, loss‐of‐function mutations in GNPTG cause a significant reduction in enzymatic activity (De Pace et al, ; Di Lorenzo et al, ). Importantly, GNPTAB mutations in exons encoding binding domains for lysosomal enzymes, that is Notch repeat‐like and DMAP, and in those coding for the γ‐subunit‐binding domain were predominantly found in patients with MLIII alpha/beta, most likely because of residual GlcNAc‐1‐phosphotransferase activity that prevented a fast progressive and severe outcome of the disease (Figure a).…”

Combined in vitro and in silico analyses of missense mutations in GNPTAB provide new insights into the molecular bases of mucolipidosis II and III alpha/beta

“…In the Golgi apparatus, the α/β‐precursor is proteolytically cleaved between Lys 928 and Asp 929 by site‐1 protease (S1P) into the enzymatically active α‐ and β‐subunits (Figure a; Marschner, Kollmann, Schweizer, Braulke, & Pohl, ). We and others have previously shown that binding of the γ‐subunit to the α‐subunit enhances the GlcNAc‐1‐phosphotransferase activity for M6P modification of specific lysosomal enzymes (De Pace et al, ; Di Lorenzo et al, ; Qian et al, ).…”

Toward Engineering the Mannose 6-Phosphate Elaboration Pathway in Plants for Enzyme Replacement Therapy of Lysosomal Storage Disorders

“…It contains a M6P receptor homology (MRH) domain, a protein domain whose function is to bind high-mannose-type N-glycans [21]. The γ subunit enhances the PT catalysis to a subset of the lysosomal hydrolases [12,22].The UCE is a type I membrane-spanning glycoprotein of the trans-Golgi network (TGN); comprised of 515 amino acids, it contains a 25-amino acid signal peptide, a 24-amino acid propeptide, a luminal domain, a single transmembrane region, and a cytoplasmic tail [23][24][25]. Although it resides primarily in the TGN, it cycles between this compartment and the plasma membrane [26].…”

“…It contains a M6P receptor homology (MRH) domain, a protein domain whose function is to bind high-mannose-type N-glycans [21]. The γ subunit enhances the PT catalysis to a subset of the lysosomal hydrolases [12,22].…”