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 (α₂β₂γ₂). 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 Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, 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 Gnptg^ko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/β-precursor was not affected by Gnptg 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 Gnptg^ko 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 Gnptg^ko lysosomes. Incubation of Gnptg^ko 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.

Keywords: Affinity proteomics; Animal models; Enzyme modification; Gnptg-deficient mouse; Post-translational modifications; SILAC; arylsulfatase B; lysosomal missorting; lysosomal proteome; mannose 6-phospho-secretome.

Fig. 1.

In vivo expression of GlcNAc-1-phosphotransferase subunits. A, Gnptab and Gnptg mRNA expression in different tissues of 12 weeks old wild-type mice. The transcript level of Gnptg in bone marrow was assigned as 1 (mean ± S.D., n = 4). B, Gnptab and Gnptg mRNA expression in primary cultured cells of wild-type mice. The transcript level of Gnptab and Gnptg in chondrocytes was assigned as 1 (mean ± S.D., n = 3). C, Schematic presentation of murine Gnptg gene and Gnptg^lacZ reporter allele. The IRES lacZ cassette with a neomycin (neo) resistance gene in the intron 3 of Gnptg is flanked by FRT (blue) and loxP (green) sites. Primers for genotyping of mice are indicated with arrowheads. D, Representative X-gal staining of P0 wild-type and Gnptg^lacZ mice. Scale bar: 1 mm. E, Representative X-gal staining of different (a) brain regions, (b) olfactory epithelium, (c) maxilla and (d) mandible, (e) trigeminal ganglion, (f) first cervical vertebra (C1) and ganglia, (g) cervical vertebra and ganglia of P3 Gnptg^lacZ mice. Scale bar left panel: 1 mm, magnified right panel b-g: 200 μm.

Fig. 2.

Missorting of lysosomal enzymes in Gnptg^ko mice. A, Schematic presentation of Gnptg^ko strategy. Crossing of Gnptg^lacZ mice with Flp and Cre recombinase expressing mice leads to deletion of Gnptg exons 4–11 in Gnptg^ko mice using FRT and loxP sites, respectively (see also Fig. 1C). B, Representative Western blotting of Concanavalin A-Sepharose-enriched proteins from serum of 12 weeks old wild-type (wt), Gnptg^ko and Gnptab^ki mice using anti-γ-subunit antibodies. Transferrin (Tf) was used as loading control. C, Relative enzyme activities of β-hexosaminidase A/B (Hexa/b), β-galactosidase (Glb1), α-mannosidase (Man2b1), α-fucosidase (Fuca1) and arylsulfatase B (Arsb) in the serum of 12 weeks old wt (set as 1.0) and Gnptg^ko mice (mean ± S.D., ***p ≤ 0.001, n = 3). D, Relative mRNA expression levels of Gnptg in wild-type (wt, 100%) and Gnptg^ko MEF (mean ± S.D., ***p ≤ 0.001, n = 5). E, Representative γ-subunit Western blotting of cell extracts from wt and Gnptg^ko MEF. Gapdh was used as loading control. F, Relative enzyme activities of arylsulfatase B (Arsb), β-hexosaminidases A/B (Hexa/b), β-galactosidase (Glb1), α-mannosidase (Man2b1), α-fucosidase (Fuca1) and N-acetylgalactosamine-6-sulfatase (Galns) in extracts of wt (100%) and Gnptg^ko MEF (mean ± S.D., **p ≤ 0.01, ***p ≤ 0.001, n = 5). G, Relative GlcNAc-1-phosphotransferase activity in extracts of wt (100%) and Gnptg^ko MEF (mean ± S.D., **p ≤ 0.01, n = 3). H, M6P Western blotting of cell extracts and lysosome-enriched fractions (LF) from wt and Gnptg^ko MEF. Cellular Gapdh was used as loading control.

Fig. 3.

Proteolytic cleavage of the α/β-subunit precursor is independent of γ-subunits. A, Relative Gnptab mRNA expression in wild-type (wt, 100%) and Gnptg^ko MEF (mean ± S.D., n = 3). B, Representative Western blotting of cell extracts from wt and Gnptg^ko MEF transduced with lentiviral particles containing human α/β-subunit precursor-myc (α/β-myc) cDNA. Gapdh was used as loading control. C, Immunofluorescence microscopy of wt and Gnptg^ko cells transfected with human α/β-subunit precursor cDNA. Cells were stained with antibodies against the α-subunit (α/β, green), the marker proteins for the cis-Golgi apparatus (GM130, magenta) and the ER (PDI, magenta). Nuclei were visualized by DAPI (blue). In merged images, yellow indicates colocalization. Scale bars: 6 μm.

Fig. 4.

SILAC-based quantitative lysosomal proteome analysis revealed missorting of specific lysosomal enzymes in Gnptg^ko MEF. A, Schematic presentation of lysosomal proteome analysis. MEF were grown in light (Gnptg^ko) and heavy-isotope (wild-type) labeled medium, incubated with magnetite particles followed by a 36 h chase to allow magnetic beads accumulation in lysosomes. Equal amounts of postnuclear supernatants (PNS) proteins from both cell lines were combined, followed by magnetic isolation of lysosomes and MS analysis. B, Comparative proteomic data of soluble lysosomal proteins are displayed as Log2 ko/wt (light/heavy) ratio (mean ± S.D., n = 3, p values are given in supplemental Table S2). Significantly reduced lysosomal enzymes in Gnptg^ko samples are indicated by closed circles. C, Low concentrations of soluble lysosomal proteins in Gnptg^ko lysosomal fractions involved in different degradation pathways: putative phospholipase B-like 2 (Pldb2), palmitoyl-protein thioesterase 2 (Ppt2), group XV phospholipase A2 (Pla2g15), galactocerebrosidase (Galc), acid ceramidase (Asah1), lysosomal acid lipase (Lipa), arylsulfatases A and B (Arsa, Arsb), sialate O-acetylesterase (Siae), β-mannosidase (Manba), aspartylglucosaminidase (Aga), β-hexosaminidase A and B (Hexa, Hexb), α-mannosidase (Man2b2), β-galactosidase (Glb1), di-N-acetylchitobiase (Ctbs), N-acetylglucosamine-6-sulfatase (Gns), sialidase 1 (Neu1), dipeptidyl-peptidase 7 (Dpp7), tripeptidyl peptidase 1 (Tpp1), prolylcarboxypeptidase (Prcp), cathepsins C, Z, S and A (Ctsc, Ctsz, Ctss, Ctsa), serine carboxypeptidase 1 (Scpep1), Creg1 protein, γ-glutamyl hydrolase (Ggh) and neuronal ceroid-lipofuscin protein 5 (Cln5). D, Relative mRNA expression levels of indicated genes encoding soluble lysosomal enzymes were determined in wt (1.0) and Gnptg^ko MEF (mean ± S.D., *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, n = 3).

Fig. 5.

Missorting and altered lysosomal processing of specific soluble lysosomal proteins in Gnptg^ko MEF. Representative western blots of whole cell extracts (100 μg protein), aliquots (20%) of conditioned, corresponding media, and lysosome-enriched fractions (LF, 75 μg protein) from wild-type (wt) and Gnptg^ko (ko) MEF using antibodies against various soluble lysosomal proteins as indicated. Endogenous α-tubulin in cell extracts was used as loading control. The positions of precursors (non-filled arrowheads), intermediates (black arrowheads), and mature (black arrows) forms of the lysosomal enzymes are indicated.

Fig. 6.

Selective M6P formation on lysosomal enzymes in Gnptg^ko MEF. A, Schematic presentation of M6P proteome analysis. MEF were grown in light (Gnptg^ko) and heavy-isotope (wild-type, wt) labeled medium containing 10 mm NH₄Cl. Equal amounts of concentrated media from both cell lines were combined, followed by M6P affinity chromatography, trypsin digestion of bound proteins and MS analysis. B, Comparative M6P proteome data of identified soluble lysosomal proteins are displayed as Log2 ko/wt (light/heavy) ratio (mean ± S.D., n = 3, p values are given in supplemental Table S5). Significantly reduced lysosomal enzymes in Gnptg^ko samples are indicated by filled circles.

Fig. 7.

Loss of ARSB causes accumulation of sulfated GAGs in Gnptg^ko MEF. A, Relative [³⁵SO₄] incorporation in wild-type (wt) and Gnptg^ko MEF after 24 h pulse and 24 h chase in the absence (−) or presence (+) of human recombinant ARSB measured in the total lysate (non-treated wt = 1; n = 2; wt values experiment 1: 112 cpm/μg, experiment 2: 679 cpm/μg). B, Quantification of purified ³⁵SO₄-labeled GAGs (non-treated wt = 1; *p ≤ 0.05, n = 2; wt values experiment 1: 203 cpm/μg, experiment 2: 478 cpm/μg). C, Quantification of purified ³⁵SO₄-labeled HS and CS/DS after digestion with chondroitinase or heparinase, respectively (non-treated wt = 1; *p ≤ 0.05, n = 2; wt values experiment 1: HS: 21 cpm/μg, CS/DS: 17 cpm/μg; experiment 2: HS: 50 cpm/μg; CS/DS: 66 cpm/μg). C, Relative enzyme activities of Arsb in wt and Gnptg^ko MEF in the absence (−) or presence (+) of ARSB (non-treated wt = 1; mean ± S.D., n = 2). D, Overview of CS/DS and HS degradation. The numbers in brackets indicate the respective lysosomal enzymes. 1: Arsb, 2: Galns, 3: Gds, 4: Gusb, 5: Ids, 6: Idua, 7: Gns, 8: Naglu, 9: Sgsh.