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• W2804718596 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 Gnptgko 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 Gnptgko 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 Gnptgko lysosomes. Incubation of Gnptgko 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. 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 Gnptgko 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 Gnptgko 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 Gnptgko lysosomes. Incubation of Gnptgko 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. Lysosomes are acidic organelles of eukaryotic cells degrading extracellular and intracellular macromolecules as well as damaged organelles by the sequential activities of more than 70 different soluble lysosomal enzymes such as glycosidases, proteases, lipases, phosphatases, sulfatases, nucleases, and accessory proteins. For the directed delivery to lysosomes, newly synthesized lysosomal enzymes are equipped with mannose 6-phosphate (M6P) 1The abbreviations used are:M6Pmannose 6-phosphateCS/DSchondroitin sulfate/dermatan sulfateDTTdithiothreitolERendoplasmic reticulumGAGglycosaminoglycansGlcNAcN-acetylglucosamineGnptgγ-subunit of GlcNAc-1-phosphotransferaseMEFmouse embryonic fibroblastsMLmucolipidosisPDIprotein disulfide isomerasePSMpeptide-spectrum matchPNSpostnuclear supernatantS1Psite-1 proteaseSILACStable isotope labeling by amino acids in cell cultureTGNtrans-Golgi network. 1The abbreviations used are:M6Pmannose 6-phosphateCS/DSchondroitin sulfate/dermatan sulfateDTTdithiothreitolERendoplasmic reticulumGAGglycosaminoglycansGlcNAcN-acetylglucosamineGnptgγ-subunit of GlcNAc-1-phosphotransferaseMEFmouse embryonic fibroblastsMLmucolipidosisPDIprotein disulfide isomerasePSMpeptide-spectrum matchPNSpostnuclear supernatantS1Psite-1 proteaseSILACStable isotope labeling by amino acids in cell cultureTGNtrans-Golgi network. residues, which are generated by two enzymes. First, the cis-Golgi-resident GlcNAc-1-phosphotransferase catalyzes the transfer of GlcNAc-1-phosphate from UDP-GlcNAc to selected C6 hydroxyl groups of high-mannose type N-glycans on lysosomal enzymes. Second, the masking GlcNAc is removed by an α-N-acetylglucosaminidase, also called uncovering enzyme, in the trans-Golgi network (TGN) exposing the M6P residues (1Pohl S. Marschner K. Storch S. Braulke T. Glycosylation- and phosphorylation-dependent intracellular transport of lysosomal hydrolases.Biol. Chem. 2009; 390: 521-527Crossref PubMed Scopus (31) Google Scholar, 2Reitman M.L. Kornfeld S. Lysosomal enzyme targeting. N-Acetylglucosaminylphosphotransferase selectively phosphorylates native lysosomal enzymes.J. Biol. Chem. 1981; 256: 11977-11980Abstract Full Text PDF PubMed Google Scholar). Subsequently, M6P-containing lysosomal enzymes bind to M6P receptors mediating their vesicular transport from the TGN, via the endosomal compartment to lysosomes (3Braulke T. Bonifacino J.S. Sorting of lysosomal proteins.Biochim. Biophys. Acta. 2009; 1793: 605-614Crossref PubMed Scopus (571) Google Scholar). mannose 6-phosphate chondroitin sulfate/dermatan sulfate dithiothreitol endoplasmic reticulum glycosaminoglycans N-acetylglucosamine γ-subunit of GlcNAc-1-phosphotransferase mouse embryonic fibroblasts mucolipidosis protein disulfide isomerase peptide-spectrum match postnuclear supernatant site-1 protease Stable isotope labeling by amino acids in cell culture trans-Golgi network. mannose 6-phosphate chondroitin sulfate/dermatan sulfate dithiothreitol endoplasmic reticulum glycosaminoglycans N-acetylglucosamine γ-subunit of GlcNAc-1-phosphotransferase mouse embryonic fibroblasts mucolipidosis protein disulfide isomerase peptide-spectrum match postnuclear supernatant site-1 protease Stable isotope labeling by amino acids in cell culture trans-Golgi network. GlcNAc-1-phosphotransferase is a hexameric complex consisting of two membrane-bound α- and β-subunits and two soluble γ-subunits (α2β2γ2) (4Bao M. Booth J.L. Elmendorf B.J. Canfield W.M. Bovine UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase. I. Purification and subunit structure.J. Biol. Chem. 1996; 271: 31437-31445Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The human α- and β-subunits are encoded by a single gene, GNPTAB, and synthesized as a common type III precursor membrane protein (5Kudo M. Bao M. D'Souza A. Ying F. Pan H. Roe B.A. Canfield W.M. The alpha- and beta-subunits of the human UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase [corrected] are encoded by a single cDNA.J. Biol. Chem. 2005; 280: 36141-36149Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 6Tiede S. Storch S. Lübke T. Henrissat B. Bargal R. Raas-Rothschild A. Braulke T. Mucolipidosis II is caused by mutations in GNPTA encoding the alpha/beta GlcNAc-1-phosphotransferase.Nat. Med. 2005; 11: 1109-1112Crossref PubMed Scopus (167) Google Scholar), whereas the soluble γ-subunits are encoded by GNPTG (7Raas-Rothschild A. Cormier-Daire V. Bao M. Genin E. Salomon R. Brewer K. Zeigler M. Mandel H. Toth S. Roe B. Munnich A. Canfield W.M. Molecular basis of variant pseudo-hurler polydystrophy (mucolipidosis IIIC).J. Clin. Invest. 2000; 105: 673-681Crossref PubMed Scopus (144) Google Scholar). After assembly of the GlcNAc-1-phosphotransferase in the ER, the inactive enzyme complex is transported to the Golgi apparatus that requires a combinatorial cytoplasmic sorting motif of the α/β-subunit precursor proteins (8Encarnação M. Kollmann K. Trusch M. Braulke T. Pohl S. Post-translational modifications of the gamma-subunit affect intracellular trafficking and complex assembly of GlcNAc-1-phosphotransferase.J. Biol. Chem. 2011; 286: 5311-5318Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 9Franke M. Braulke T. Storch S. Transport of the GlcNAc-1-phosphotransferase alpha/beta-subunit precursor protein to the Golgi apparatus requires a combinatorial sorting motif.J. Biol. Chem. 2013; 288: 1238-1249Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). On arrival in the cis-Golgi apparatus, the α/β-subunit precursor is proteolytically cleaved by the site-1 protease into the individual α- and β-subunits, which is a prerequisite for catalytic GlcNAc-1-phosphotransferase activity (10Marschner K. Kollmann K. Schweizer M. Braulke T. Pohl S. A key enzyme in the biogenesis of lysosomes is a protease that regulates cholesterol metabolism.Science. 2011; 333: 87-90Crossref PubMed Scopus (106) Google Scholar). The mature α- and β-subunits exhibit catalytic activity and binding sites for lysosomal enzymes (11Qian Y. Lee I. Lee W. Qian M. Kudo M. Canfield W. Lobel P. Kornfeld S. Functions of the alpha, beta, and gamma subunits of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase.J. Biol. Chem. 2010; 285: 3360-3370Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), whereas the function of the γ-subunits is poorly defined. It has been suggested that the γ-subunits enhance the recognition and M6P formation of distinct lysosomal enzymes by the α- and β-subunit (11Qian Y. Lee I. Lee W. Qian M. Kudo M. Canfield W. Lobel P. Kornfeld S. Functions of the alpha, beta, and gamma subunits of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase.J. Biol. Chem. 2010; 285: 3360-3370Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 12Lee W.S. Payne B.J. Gelfman C.M. Vogel P. Kornfeld S. Murine UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase lacking the gamma-subunit retains substantial activity toward acid hydrolases.J. Biol. Chem. 2007; 282: 27198-27203Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), whereas other studies failed to show direct interactions of the γ-subunits with lysosomal enzymes (6Tiede S. Storch S. Lübke T. Henrissat B. Bargal R. Raas-Rothschild A. Braulke T. Mucolipidosis II is caused by mutations in GNPTA encoding the alpha/beta GlcNAc-1-phosphotransferase.Nat. Med. 2005; 11: 1109-1112Crossref PubMed Scopus (167) Google Scholar, 13Pohl S. Tiede S. Castrichini M. Cantz M. Gieselmann V. Braulke T. Compensatory expression of human N-Acetylglucosaminyl-1-phosphotransferase subunits in mucolipidosis type III gamma.Biochim. Biophys. Acta. 2009; 1792: 221-225Crossref PubMed Scopus (23) Google Scholar). Recently, we identified the γ-subunit binding domain in a previously uncharacterized luminal region of the α-subunit required for maximum GlcNAc-1-phosphotransferase activity (14De Pace R. Velho R.V. Encarnacao M. Marschner K. Braulke T. Pohl S. Subunit interactions of the disease-related hexameric GlcNAc-1-phosphotransferase complex.Hum. Mol. Genet. 2015; 24: 6826-6835Crossref PubMed Scopus (18) Google Scholar, 15Velho R.V. De Pace R. Tidow H. Braulke T. Pohl S. Identification of the interaction domains between alpha- and gamma-subunits of GlcNAc-1-phosphotransferase.FEBS Lett. 2016; 590: 4287-4295Crossref PubMed Scopus (10) Google Scholar). The significance of these data is demonstrated by the existence of the autosomal recessive lysosomal storage disease, mucolipidosis type III gamma (MLIII), caused by mutations in the GNPTG gene. To date 32 different GNPTG mutations are known including 12 frameshift, 6 nonsense, 5 missense, 2 small deletion and 7 splicing mutations (7Raas-Rothschild A. Cormier-Daire V. Bao M. Genin E. Salomon R. Brewer K. Zeigler M. Mandel H. Toth S. Roe B. Munnich A. Canfield W.M. Molecular basis of variant pseudo-hurler polydystrophy (mucolipidosis IIIC).J. Clin. Invest. 2000; 105: 673-681Crossref PubMed Scopus (144) Google Scholar, 13Pohl S. Tiede S. Castrichini M. Cantz M. Gieselmann V. Braulke T. Compensatory expression of human N-Acetylglucosaminyl-1-phosphotransferase subunits in mucolipidosis type III gamma.Biochim. Biophys. Acta. 2009; 1792: 221-225Crossref PubMed Scopus (23) Google Scholar, 16Barea J.J. van Meel E. Kornfeld S. Bird L.M. Tuberous sclerosis, polycystic kidney disease and mucolipidosis III gamma caused by a microdeletion unmasking a recessive mutation.Am. J. Med. Genet. A. 2015; 167A: 2844-2846Crossref PubMed Scopus (4) Google Scholar, 17Encarnação M. Lacerda L. Prata R.C. M.J. Coutinho M.F. Ribeiro H. Lopes L. Pineda M. Ignatius J. Galvez H. Mustonen A. Lima M.R. Alves S. Molecular analysis of the GNPTAB and GNPTG genes in 13 patients with mucolipidosis type II or type III - identification of eight novel mutations.Clin. Genet. 2009; 76: 76-84Crossref PubMed Scopus (44) Google Scholar, 18Gao Y. Yang K. Xu S. Wang C. Liu J. Zhang Z. Yuan M. Luo X. Liu M. Wang Q.K. Liu J.Y. Identification of compound heterozygous mutations in GNPTG in three siblings of a Chinese family with mucolipidosis type III gamma.Mol. Genet. Metab. 2011; 102: 107-109Crossref PubMed Scopus (6) Google Scholar, 19Liu S. Zhang W. Shi H. Meng Y. Qiu Z. Three novel homozygous mutations in the GNPTG gene that cause mucolipidosis type III gamma.Gene. 2014; 535: 294-298Crossref PubMed Scopus (15) Google Scholar, 20Persichetti E. Chuzhanova N.A. Dardis A. Tappino B. Pohl S. Thomas N.S. Rosano C. Balducci C. Paciotti S. Dominissini S. Montalvo A.L. Sibilio M. Parini R. Rigoldi M. Di Rocco M. Parenti G. Orlacchio A. Bembi B. Cooper D.N. Filocamo M. Beccari T. Identification and molecular characterization of six novel mutations in the UDP-N-acetylglucosamine-1-phosphotransferase gamma subunit (GNPTG) gene in patients with mucolipidosis III gamma.Hum. Mutat. 2009; 30: 978-984Crossref PubMed Scopus (26) Google Scholar, 21Pohl S. Encarnação M. Castrichini M. Müller-Loennies S. Muschol N. Braulke T. Loss of N-Acetylglucosamine-1-phosphotransferase gamma-subunit due to intronic mutation in GNPTG causes mucolipidosis type III gamma: Implications for molecular and cellular diagnostics.Am. J. Med. Genet. A. 2010; 152A: 124-132Crossref PubMed Scopus (23) Google Scholar, 22Raas-Rothschild A. Bargal R. Goldman O. Ben-Asher E. Groener J.E. Toutain A. Stemmer E. Ben-Neriah Z. Flusser H. Beemer F.A. Penttinen M. Olender T. Rein A.J. Bach G. Zeigler M. Genomic organisation of the UDP-N-acetylglucosamine-1-phosphotransferase gamma subunit (GNPTAG) and its mutations in mucolipidosis III.J. Med. Genet. 2004; 41: e52Crossref PubMed Scopus (50) Google Scholar, 23Schrader K.A. Heravi-Moussavi A. Waters P.J. Senz J. Whelan J. Ha G. Eydoux P. Nielsen T. Gallagher B. Oloumi A. Boyd N. Fernandez B.A. Young T.L. Jones S.J. Hirst M. Shah S.P. Marra M.A. Green J. Huntsman D.G. Using next-generation sequencing for the diagnosis of rare disorders: a family with retinitis pigmentosa and skeletal abnormalities.J. Pathol. 2011; 225: 12-18Crossref PubMed Scopus (22) Google Scholar, 24Tiede S. Cantz M. Raas-Rothschild A. Muschol N. Bürger F. Ullrich K. Braulke T. A novel mutation in UDP-N-acetylglucosamine-1-phosphotransferase gamma subunit (GNPTAG) in two siblings with mucolipidosis type III alters a used glycosylation site.Hum. Mutat. 2004; 24: 535Crossref PubMed Scopus (38) Google Scholar, 25Tüysüz B. Kasapcopur O. Alkaya D.U. Sahin S. Sozeri B. Yesil G. Mucolipidosis type III gamma: Three novel mutation and genotype-phenotype study in eleven patients.Gene. 2018; 642: 398-407Crossref PubMed Scopus (12) Google Scholar, 26Velho R.V. Alegra T. Sperb F. Ludwig N.F. Saraiva-Pereira M.L. Matte U. Schwartz I.V. A de novo or germline mutation in a family with Mucolipidosis III gamma: Implications for molecular diagnosis and genetic counseling.Mol. Genet. Metab. Rep. 2014; 1: 98-102Crossref PubMed Scopus (7) Google Scholar, 27Velho R.V. Ludwig N.F. Alegra T. Sperb-Ludwig F. Guarany N.R. Matte U. Schwartz I.V. Enigmatic in vivo GlcNAc-1-phosphotransferase (GNPTG) transcript correction to wild type in two mucolipidosis III gamma siblings homozygous for nonsense mutations.J. Hum. Genet. 2016; 61: 555-560Crossref PubMed Scopus (11) Google Scholar, 28Zrhidri A. Amasdl S. Lyahyai J. Elouardi H. Chkirate B. Raymond L. Egea G. Taoudi M. El Mouatassim S. Sefiani A. Next Generation Sequencing identifies mutations in GNPTG gene as a cause of familial form of scleroderma-like disease.Pediatr Rheumatol. Online J. 2017; 15: 72Crossref PubMed Scopus (4) Google Scholar). Biochemically, the cells from MLIII patients have reduced amounts of M6P residues on lysosomal enzymes leading to their missorting and hypersecretion into the extracellular compartment (29Kollmann K. Pohl S. Marschner K. Encarnação M. Sakwa I. Tiede S. Poorthuis B.J. Lübke T. Müller-Loennies S. Storch S. Braulke T. Mannose phosphorylation in health and disease. Eur. J.Cell. Biol. 2010; 89: 117-123Google Scholar, 30Raas-Rothschild A. Pohl S. Braulke T. Multiple enzyme deficiencies: Defects in transport: Mucolipidosis II alpha/beta; mucolipidosis III alpha/beta and mucolipidosis III gamma.in: Mehta A.B. Winchester B. Lysosomal storage diseases: A practical guide. WILEY-BLACKWELL, Oxford2012: 121-126Google Scholar). At present, however, it is unknown which lysosomal enzymes reach lysosomes and might be limiting for lysosomal function. The subsequent reduction of several lysosomal enzymes in lysosomes may result in the accumulation of non-degraded material, which impairs cellular homeostasis. The first clinical symptoms of MLIII patients are joint stiffness of fingers, hips and shoulders, and have been observed between 5 and 10 years of life developing to moderate dysostosis multiplex with vertebral scoliosis (30Raas-Rothschild A. Pohl S. Braulke T. Multiple enzyme deficiencies: Defects in transport: Mucolipidosis II alpha/beta; mucolipidosis III alpha/beta and mucolipidosis III gamma.in: Mehta A.B. Winchester B. Lysosomal storage diseases: A practical guide. WILEY-BLACKWELL, Oxford2012: 121-126Google Scholar, 31Kelly T.E. Thomas G.H. Taylor Jr, H.A. McKusick V.A. Sly W.S. Glaser J.H. Robinow M. Luzzatti L. Espiritu C. Feingold M. Bull M.J. Ashenhurst E.M. Ives E.J. Mucolipidosis III (pseudo-Hurler polydystrophy): Clinical and laboratory studies in a series of 12 patients.Johns Hopkins Med. J. 1975; 137: 156-175PubMed Google Scholar, 32Smuts I. Potgieter D. van der Westhuizen F.H. Combined tarsal and carpal tunnel syndrome in mucolipidosis type III. A case study and review.Ann. N.Y. Acad. Sci. 2009; 1151: 77-84Crossref PubMed Scopus (19) Google Scholar). The skin may become thickened with time. Recently scleroderma-like symptoms were described in MLIII patients (28Zrhidri A. Amasdl S. Lyahyai J. Elouardi H. Chkirate B. Raymond L. Egea G. Taoudi M. El Mouatassim S. Sefiani A. Next Generation Sequencing identifies mutations in GNPTG gene as a cause of familial form of scleroderma-like disease.Pediatr Rheumatol. Online J. 2017; 15: 72Crossref PubMed Scopus (4) Google Scholar). Because skeletal dysplasia is the most prominent clinical complication in the MLIII disease, patients can survive into adulthood (33Umehara F. Matsumoto W. Kuriyama M. Sukegawa K. Gasa S. Osame M. Mucolipidosis III (pseudo-Hurler polydystrophy); clinical studies in aged patients in one family.J. Neurol. Sci. 1997; 146: 167-172Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In contrast, the total loss of GlcNAc-1-phosphotransferase activity caused by mutations in GNPTAB leads to complete failure to generate M6P residues on lysosomal enzymes and a fatal lysosomal disease, mucolipidosis II (MLII). The patients show progressive and severe dysostosis multiplex and cranofacial abnormalities, gingival hyperplasia, mental retardation, hepato- and cardiomegaly, immune defects and death in the first decade of life (30Raas-Rothschild A. Pohl S. Braulke T. Multiple enzyme deficiencies: Defects in transport: Mucolipidosis II alpha/beta; mucolipidosis III alpha/beta and mucolipidosis III gamma.in: Mehta A.B. Winchester B. Lysosomal storage diseases: A practical guide. WILEY-BLACKWELL, Oxford2012: 121-126Google Scholar, 34Köhne T. Markmann S. Schweizer M. Muschol N. Friedrich R.E. Hagel C. Glatzel M. Kahl-Nieke B. Amling M. Schinke T. Braulke T. Mannose 6-phosphate-dependent targeting of lysosomal enzymes is required for normal craniofacial and dental development.Biochim. Biophys. Acta. 2016; 1862: 1570-1580Crossref PubMed Scopus (12) Google Scholar, 35Otomo T. Schweizer M. Kollmann K. Schumacher V. Muschol N. Tolosa E. Mittrucker H.W. Braulke T. Mannose 6 phosphorylation of lysosomal enzymes controls B cell functions.J. Cell Biol. 2015; 208: 171-180Crossref PubMed Scopus (19) Google Scholar). However, in certain cell types (such as hepatocytes and leukocytes) and organs (liver, kidney and brain) in MLII patients and mice nearly normal level of selected lysosomal enzymes were observed, suggesting the existence of alternate M6P-independent targeting pathways (35Otomo T. Schweizer M. Kollmann K. Schumacher V. Muschol N. Tolosa E. Mittrucker H.W. Braulke T. Mannose 6 phosphorylation of lysosomal enzymes controls B cell functions.J. Cell Biol. 2015; 208: 171-180Crossref PubMed Scopus (19) Google Scholar, 36Owada M. Neufeld E.F. Is there a mechanism for introducing acid hydrolases into liver lysosomes that is independent of mannose 6-phosphate recognition? Evidence from I-cell disease.Biochem. Biophys. Res. Commun. 1982; 105: 814-820Crossref PubMed Scopus (123) Google Scholar, 37Waheed A. Pohlmann R. Hasilik A. von Figura K. van Elsen A. Leroy J.G. Deficiency of UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase in organs of I-cell patients.Biochem. Biophys. Res. Commun. 1982; 105: 1052-1058Crossref PubMed Scopus (107) Google Scholar, 38Markmann S. Thelen M. Cornils K. Schweizer M. Brocke-Ahmadinejad N. Willnow T. Heeren J. Gieselmann V. Braulke T. Kollmann K. Lrp1/LDL receptor play critical roles in mannose 6-phosphate-independent lysosomal enzyme targeting.Traffic. 2015; 16: 743-759Crossref PubMed Scopus (39) Google Scholar). So far it is not clear whether tissue-specific expression of the γ-subunit or differences in the targeting efficiency of distinct lysosomal enzymes are responsible for the different clinical courses and features of MLII and MLIII patients. One study reports on similar GNPTG transcript level in human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas based on Northern blot analysis (7Raas-Rothschild A. Cormier-Daire V. Bao M. Genin E. Salomon R. Brewer K. Zeigler M. Mandel H. Toth S. Roe B. Munnich A. Canfield W.M. Molecular basis of variant pseudo-hurler polydystrophy (mucolipidosis IIIC).J. Clin. Invest. 2000; 105: 673-681Crossref PubMed Scopus (144) Google Scholar) but lacks however, information on the expression in specific cell types. In the present study, we determined the mRNA expression distribution of GlcNAc-1-phosphotransferase γ-subunits in a GnptglacZ reporter mouse at different ages in tissues that have previously not been reported to be affected in MLIII patients. In addition, the comparative lysosomal proteomes and M6P secretomes of fibroblasts from wild-type and Gnptgko mice led to the identification of a subset of lysosomal enzymes whose lysosomal targeting depend on the presence of γ-subunits and fail to use alternative M6P-independent pathways to lysosomes. The accumulation of chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycans (GAG) in Gnptgko fibroblasts correlated with low amounts of arylsulfatase B (Arsb), a key enzyme in the degradation of CS/DS. The CS/DS storage can be rescued for the most part by incubation of cells with recombinant arylsulfatase B that has been applied as enzyme replacement therapy for patients deficient for this enzyme (39Harmatz P. Shediac R. Mucopolysaccharidosis VI: pathophysiology, diagnosis and treatment.Front. Biosci. 2017; 22: 385-406Crossref PubMed Scopus (50) Google Scholar). The following antibodies were used: goat anti-Creg1, goat anti-Ctsc, goat anti-Ctsz, and goat anti-Ctsl from R&D (Minneapolis, MN); goat anti-Ctsb from Neuromics (Edina, MN); goat anti-Ctsd, mouse anti-Ctss, rabbit anti-Gapdh, rabbit anti-Npc2, and mouse anti-Ctsk from Santa Cruz Biotechnology (Dallas, TX); rabbit anti-Gba, rabbit anti-Pla2g15, goat anti-transferrin, and mouse anti-α-tubulin from Sigma-Aldrich (St. Louis, MO); mouse anti-GM130 from BD Bioscience (BD Biosciences, Franklin Lakes, NJ); mouse anti-myc and rabbit anti-PDI from Cell Signaling Technology (Cambridge, UK); rat anti-Lamp1 1D4B from the Hybridoma Bank, University of Iowa, USA. The polyclonal rabbit anti-γ-subunit and monoclonal rat anti-α-subunit of GlcNAc-1-phosphotransferase and myc-tagged single-chain M6P antibody fragment are described previously (8Encarnação M. Kollmann K. Trusch M. Braulke T. Pohl S. Post-translational modifications of the gamma-subunit affect intracellular trafficking and complex assembly of GlcNAc-1-phosphotransferase.J. Biol. Chem. 2011; 286: 5311-5318Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 40De Pace R. Coutinho M.F. Koch-Nolte F. Haag F. Prata M.J. Alves S. Braulke T. Pohl S. Mucolipidosis II-related mutations inhibit the exit from the endoplasmic reticulum and proteolytic cleavage of GlcNAc-1-phosphotransferase precursor protein GNPTAB.Hum. Mutat. 2014; 35: 368-376Crossref PubMed Scopus (18) Google Scholar, 41Müller-Loennies S. Galliciotti G. Kollmann K. Glatzel M. Braulke T. A novel single chain antibody fragment for detection of mannose 6-phosphate-containing proteins: Application in mucolipidosis type II patients and mice.Am. J. Pathol. 2010; 177: 240-247Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Polyclonal rabbit anti-Limp2, anti-Plbd2, anti-Clc7 and anti-Ppt1 antibodies were kindly provided by Dr. M. Schwake, University Bielefeld, Germany (42Zachos C. Blanz J. Saftig P. Schwake M. A critical histidine residue within LIMP-2 mediates pH sensitive binding to its ligand beta-glucocerebrosidase.Traffic. 2012; 13: 1113-1123Crossref PubMed Scopus (38) Google Scholar), Dr. T. Lübke, University Bielefeld, Germany (43Deuschl F. Kollmann K. von Figura K. Lubke T. Molecular characterization of the hypothetical 66.3-kDa protein in mouse: lysosomal targeting, glycosylation, processing and tissue distribution.FEBS Lett. 2006; 580: 5747-5752Crossref PubMed Scopus (23) Google Scholar), Dr. T. Jentsch, MDC, Berlin, Germany (44Kornak U. Kasper D. Bosl M.R. Kaiser E. Schweizer M. Schulz A. Friedrich W. Delling G. Jentsch T.J. Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man.Cell. 2001; 104: 205-215Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar) and Dr. S. Hofmann, University of Texas, South Western Medical Center, Dallas, TX, respectively. Horseradish peroxidase (HRP)-coupled secondary antibodies were from Dianova (Hamburg, Germany). Alexa Fluor® 546-coupled anti-mouse IgG and anti-rat IgG, Alexa Fluor® 488-coupled anti-rabbit IgG were from Thermo Fisher Scientific (Waltham, MA). For generation of GnptglacZ and Gnptgko mice, embryonic stem (ES) cells carrying the Gnptgtm1a(KOMP)Wtsi allele were obtained from the Knockout Mouse Program (KOMP, National Institute of Health, USA, #76092). The targeting vector used for electroporation of ES cells contained a floxed promotor-driven IRES lacZ neomycin cassette flanked by FRT sites, which was inserted into intron 3 of the murine Gnptg gene (Fig. 1A). The exons 4 to 11 are flanked by loxP sites. For generation of GnptglacZ mice, ES cells were injected into C57BL6/J blastocysts and subsequently implanted into the uterine horns of C57BL6/JxCBA foster mothers according to standard protocols. Resulting chimeric males from three clones were crossed with C57BL6/J mice to obtain heterozygous offspring, which were bred to generate homozygous GnptglacZ mice. For generation of Gnptgko mice, homozygous GnptglacZ mice were mated first with C57BL6/J Flp deleter mice (45Rodriguez C.I. Buchholz F. Galloway J. Sequerra R. Kasper J. Ayala R. Stewart A.F. Dymecki S.M. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP.Nat. Genet. 2000; 25: 139-140Crossref PubMed Scopus (894) Google Scholar) to excise the lacZ-neocassette (Fig. 2A) resulting in the tm1c allele. The offspring were mated with C57BL6/J Cre deleter mice (46Schwenk F. Baron U. Rajewsky K. A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells.Nucleic Acids Res. 1995; 23: 5080-5081Crossref PubMed Scopus (993) Google Scholar) to remove the floxed exons 4 to 11 through Cre-mediated recombination. Heterozygous Gnptgko mice were then mated to generate homozygous Gnptgko mice. For genotyping of mice, genomic DNA from tail biopsies were extracted using the KAPA Mouse Genotyping Hot Start Kit (" @default.
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• W2804718596 title "Lysosomal Proteome and Secretome Analysis Identifies Missorted Enzymes and Their Nondegraded Substrates in Mucolipidosis III Mouse Cells" @default.
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