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• W3046217984 abstract "Mutations in the galactosidase β 1 (GLB1) gene cause lysosomal β-galactosidase (β-Gal) deficiency and clinical onset of the neurodegenerative lysosomal storage disease, GM1 gangliosidosis. β-Gal and neuraminidase 1 (NEU1) form a multienzyme complex in lysosomes along with the molecular chaperone, protective protein cathepsin A (PPCA). NEU1 is deficient in the neurodegenerative lysosomal storage disease sialidosis, and its targeting to and stability in lysosomes strictly depend on PPCA. In contrast, β-Gal only partially depends on PPCA, prompting us to investigate the role that β-Gal plays in the multienzyme complex. Here, we demonstrate that β-Gal negatively regulates NEU1 levels in lysosomes by competitively displacing this labile sialidase from PPCA. Chronic cellular uptake of purified recombinant human β-Gal (rhβ-Gal) or chronic lentiviral-mediated GLB1 overexpression in GM1 gangliosidosis patient fibroblasts coincides with profound secondary NEU1 deficiency. A regimen of intermittent enzyme replacement therapy dosing with rhβ-Gal, followed by enzyme withdrawal, is sufficient to augment β-Gal activity levels in GM1 gangliosidosis patient fibroblasts without promoting NEU1 deficiency. In the absence of β-Gal, NEU1 levels are elevated in the GM1 gangliosidosis mouse brain, which are restored to normal levels following weekly intracerebroventricular dosing with rhβ-Gal. Collectively, our results highlight the need to carefully titrate the dose and dosing frequency of β-Gal augmentation therapy for GM1 gangliosidosis. They further suggest that intermittent intracerebroventricular enzyme replacement therapy dosing with rhβ-Gal is a tunable approach that can safely augment β-Gal levels while maintaining NEU1 at physiological levels in the GM1 gangliosidosis brain. Mutations in the galactosidase β 1 (GLB1) gene cause lysosomal β-galactosidase (β-Gal) deficiency and clinical onset of the neurodegenerative lysosomal storage disease, GM1 gangliosidosis. β-Gal and neuraminidase 1 (NEU1) form a multienzyme complex in lysosomes along with the molecular chaperone, protective protein cathepsin A (PPCA). NEU1 is deficient in the neurodegenerative lysosomal storage disease sialidosis, and its targeting to and stability in lysosomes strictly depend on PPCA. In contrast, β-Gal only partially depends on PPCA, prompting us to investigate the role that β-Gal plays in the multienzyme complex. Here, we demonstrate that β-Gal negatively regulates NEU1 levels in lysosomes by competitively displacing this labile sialidase from PPCA. Chronic cellular uptake of purified recombinant human β-Gal (rhβ-Gal) or chronic lentiviral-mediated GLB1 overexpression in GM1 gangliosidosis patient fibroblasts coincides with profound secondary NEU1 deficiency. A regimen of intermittent enzyme replacement therapy dosing with rhβ-Gal, followed by enzyme withdrawal, is sufficient to augment β-Gal activity levels in GM1 gangliosidosis patient fibroblasts without promoting NEU1 deficiency. In the absence of β-Gal, NEU1 levels are elevated in the GM1 gangliosidosis mouse brain, which are restored to normal levels following weekly intracerebroventricular dosing with rhβ-Gal. Collectively, our results highlight the need to carefully titrate the dose and dosing frequency of β-Gal augmentation therapy for GM1 gangliosidosis. They further suggest that intermittent intracerebroventricular enzyme replacement therapy dosing with rhβ-Gal is a tunable approach that can safely augment β-Gal levels while maintaining NEU1 at physiological levels in the GM1 gangliosidosis brain. Correction: Intermittent enzyme replacement therapy prevents Neu1 deficiencyJournal of Biological ChemistryVol. 295Issue 46PreviewVOLUME 295 (2020) PAGES 13556–13569 Full-Text PDF Open Access In humans autosomal recessive mutations in the GLB1 gene give rise to a deficiency of lysosomal β-galactosidase (β-Gal), a glycosidase that participates in the degradation of multiple galactose-containing substrates (1Bonten E.J. Annunziata I. d'Azzo A. Lysosomal multienzyme complex: pros and cons of working together.Cell Mol. Life Sci. 2014; 71 (24337808): 2017-203210.1007/s00018-013-1538-3Google Scholar). β-Gal deficiency gives rise to the accumulation of three biochemically distinct classes of substrates in lysosomes: GM1 and GA1 gangliosides, the glycosaminoglycan, keratan sulfate, and oligosaccharides derived from glycoprotein metabolism (2Lawrence R. Van Vleet J. Mangini L. Harris A. Martin N. Clark W. Chandriani S. LeBowitz J.H. Giugliani R. d'Azzo A. Yogalingam G. Crawford B.E. Characterization of glycan substrates accumulating in GM1 gangliosidosis.Mol. Genet. Metab. Rep. 2019; 21 (31720227): 10052410.1016/j.ymgmr.2019.100524Google Scholar). Absent or very low levels of residual β-Gal activity result in the lysosomal accumulation of these galactose-containing substrates throughout the brain, giving rise to the progressive and fatal neurodegenerative lysosomal storage disease, GM1 gangliosidosis (3Jarnes Utz J.R. Kim S. King K. Ziegler R. Schema L. Redtree E.S. Whitley C.B. Infantile gangliosidoses: mapping a timeline of clinical changes.Mol. Genet. Metab. 2017; 121 (28476546): 170-17910.1016/j.ymgme.2017.04.011Google Scholar, 4Brunetti-Pierri N. Scaglia F. GM1 gangliosidosis: review of clinical, molecular, and therapeutic aspects.Mol. Genet. Metab. 2008; 94 (28476546): 391-39610.1016/j.ymgme.2017.04.011Google Scholar). Substrate accumulation in several systemic tissues gives rise to additional chronic, debilitating symptoms (5Kannebley J.S. Silveira-Moriyama L. Bastos L.O. Steiner C.E. Clinical findings and natural history in ten unrelated families with juvenile and adult GM1 gangliosidosis.JMID Rep. 2015; 24: 115-122Google Scholar). Currently, no safe and effective treatment has yet been developed for GM1 gangliosidosis. A challenge for developing an effective enzyme replacement therapy (ERT) or gene therapy approach for GM1 gangliosidosis is that β-Gal has been reported to exist as a multienzyme complex in lysosomes with the chaperone protein, protective protein cathepsin A (PPCA), and neuraminidase 1 (NEU1) (1Bonten E.J. Annunziata I. d'Azzo A. Lysosomal multienzyme complex: pros and cons of working together.Cell Mol. Life Sci. 2014; 71 (24337808): 2017-203210.1007/s00018-013-1538-3Google Scholar). Although NEU1 is strictly dependent upon PPCA for trafficking from the Golgi to late endosomes and for catalytic activation in lysosomes, β-Gal is only partially dependent on PPCA (1Bonten E.J. Annunziata I. d'Azzo A. Lysosomal multienzyme complex: pros and cons of working together.Cell Mol. Life Sci. 2014; 71 (24337808): 2017-203210.1007/s00018-013-1538-3Google Scholar, 6Bonten E.J. Campos Y. Zaitsev V. Nourse A. Waddell B. Lewis W. Taylor G. d'Azzo A. Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization.J. Biol. Chem. 2009; 284 (19666471): 28430-2844110.1074/jbc.M109.031419Google Scholar, 7van der Spoel A. Bonten E. d'Azzo A. Processing of lysosomal β-galactosidase: the C-terminal precursor fragment is an essential domain of the mature enzyme.J. Biol. Chem. 2000; 275 (10744681): 10035-1004010.1074/jbc.275.14.10035Google Scholar). Bonten et al. (6Bonten E.J. Campos Y. Zaitsev V. Nourse A. Waddell B. Lewis W. Taylor G. d'Azzo A. Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization.J. Biol. Chem. 2009; 284 (19666471): 28430-2844110.1074/jbc.M109.031419Google Scholar) suggest that recombinant human NEU1 produced in insect cells self-associates and is rapidly degraded, whereas association of NEU1 with PPCA results in the formation of stable heterodimers that preserve the sialidase activity of NEU1 (6Bonten E.J. Campos Y. Zaitsev V. Nourse A. Waddell B. Lewis W. Taylor G. d'Azzo A. Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization.J. Biol. Chem. 2009; 284 (19666471): 28430-2844110.1074/jbc.M109.031419Google Scholar). We have recently demonstrated that recombinant human β-Gal (rhβ-Gal) produced in Chinese hamster ovary (CHO) cells exhibits pH-dependent dynamic self-association, with the enzyme more likely being a homogenous dimer under acidic conditions, which helps to increase its thermostability (8Chen J.C. Luu A.R. Wise N. De Angelis R. Agrawal V. Mangini L. Vincelette J. Handyside B. Sterling H. Lo M.J. Wong H. Galicia N. Pacheco G. Van Vleet J. Giaramita A. et al.Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.J. Biol. Chem. 2020; 295 (31481471): 13532-1355510.1074/jbc.RA119.009811Google Scholar). Our study also suggests that prolonged lentiviral-mediated GLB1 overexpression leads to accumulation of β-Gal in GM1 gangliosidosis patient fibroblasts in a prelysosomal compartment, presumably at neutral pH, which coincides with activation of an unfolded protein response and endoplasmic reticulum (ER) stress (8Chen J.C. Luu A.R. Wise N. De Angelis R. Agrawal V. Mangini L. Vincelette J. Handyside B. Sterling H. Lo M.J. Wong H. Galicia N. Pacheco G. Van Vleet J. Giaramita A. et al.Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.J. Biol. Chem. 2020; 295 (31481471): 13532-1355510.1074/jbc.RA119.009811Google Scholar). This gene therapy approach to augment β-Gal levels in GM1 gangliosidosis patient fibroblasts also results in overnormalization of the mature form of the enzyme in lysosomes (8Chen J.C. Luu A.R. Wise N. De Angelis R. Agrawal V. Mangini L. Vincelette J. Handyside B. Sterling H. Lo M.J. Wong H. Galicia N. Pacheco G. Van Vleet J. Giaramita A. et al.Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.J. Biol. Chem. 2020; 295 (31481471): 13532-1355510.1074/jbc.RA119.009811Google Scholar). However, it remains unknown whether chronically overnormalizing β-Gal levels in GM1 gangliosidosis patient lysosomes impacts directly on the PPCA–NEU1–β-Gal multienzyme complex and the activities of each enzyme. Here, we hypothesized that overnormalizing β-Gal levels in GM1 gangliosidosis patient fibroblasts may have the potential to displace NEU1 from the PPCA-containing multienzyme complex and promote secondary NEU1 deficiency. First, we tested an ERT approach, in which we monitored cell surface receptor–mediated endocytosis of purified rhβ-Gal and its delivery to lysosomes in primary cultures of skin fibroblasts. We compared this ERT approach with a lentiviral-mediated gene therapy approach to constitutively overexpress human GLB1 cDNA, with transcription driven from the cytomegalovirus (CMV) promoter (LV–CMV–GLB1). We found that continuous ERT with rhβ-Gal or sustained GLB1 overexpression both have the potential to promote a sustained secondary NEU1 deficiency in GM1 gangliosidosis patient fibroblasts. Given that NEU1 deficiency is associated with the related degenerative lysosomal storage disease sialidosis, we developed an intermittent ERT dosing regimen in GM1 gangliosidosis patient cells and in a mouse model of the disease to safely augment β-Gal levels without promoting secondary NEU1 deficiency. Collectively, our results suggest that β-Gal acts as negative regulator of NEU1 activity by displacing the labile sialidase from the PPCA chaperone in lysosomes. We have previously demonstrated that research-grade rhβ-Gal produced in CHO cells and purified in our laboratory exhibits CI-MPR–dependent cellular uptake in GM1 gangliosidosis patient fibroblasts (8Chen J.C. Luu A.R. Wise N. De Angelis R. Agrawal V. Mangini L. Vincelette J. Handyside B. Sterling H. Lo M.J. Wong H. Galicia N. Pacheco G. Van Vleet J. Giaramita A. et al.Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.J. Biol. Chem. 2020; 295 (31481471): 13532-1355510.1074/jbc.RA119.009811Google Scholar). In this study we also characterized the glycan profile of a commercial source of rhβ-Gal–His6 (R&D Systems) and compared this with our research-grade rhβ-Gal produced and purified in-house (8Chen J.C. Luu A.R. Wise N. De Angelis R. Agrawal V. Mangini L. Vincelette J. Handyside B. Sterling H. Lo M.J. Wong H. Galicia N. Pacheco G. Van Vleet J. Giaramita A. et al.Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.J. Biol. Chem. 2020; 295 (31481471): 13532-1355510.1074/jbc.RA119.009811Google Scholar). N-Linked oligosaccharide profiling of peptide N-glycosidase F–digested glycans by capillary zone electrophoresis demonstrate that commercial rhβ-Gal–His6 produced in CHO cells or rhβ-Gal produced and purified in our laboratory contains multiple glycan moieties (Fig. 1, A and B, black traces). Some of the glycans present on both enzymes can be digested to nonphosphorylated glycans after treatment with alkaline phosphatase (Fig. 1, A and B, blue traces). Both enzyme preparations contain similar amounts of bis-phosphorylated oligomannose (BPM7) and mono-phosphorylated oligomannose (Fig. 1, A and B). The overall amount of phosphorylated oligomannose present on commercial rhβ-Gal–His6 and in-house purified rhβ-Gal corresponds to 2.1 and 1.5 mol/mol of enzyme, respectively (Fig. 1C). These results suggest that both enzyme preparations contain similar amounts of phosphorylated oligomannose, the preferred glycan species for cation-independent mannose-6-phosphate receptor (CI-MPR)–dependent cellular uptake and lysosomal targeting. Newly synthesized β-Gal has been reported to undergo a C-terminal proteolytic maturation step in lysosomes, releasing a ∼20-kDa proteolytic fragment, a process that can be monitored by Western blotting and used as an indicator of enzyme delivery to lysosomes (8Chen J.C. Luu A.R. Wise N. De Angelis R. Agrawal V. Mangini L. Vincelette J. Handyside B. Sterling H. Lo M.J. Wong H. Galicia N. Pacheco G. Van Vleet J. Giaramita A. et al.Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.J. Biol. Chem. 2020; 295 (31481471): 13532-1355510.1074/jbc.RA119.009811Google Scholar). No mutant β-Gal protein can be detected in primary skin fibroblasts established from an infantile-onset GM1 gangliosidosis patient by Western blotting (GM05653; Fig. 1D). Following cellular uptake for 24 h in normal and GM1 gangliosidosis patient fibroblasts, the majority of internalized commercial rhβ-Gal–His6 is detected by Western blotting as a cleaved molecule of similar size to the endogenous mature lysosomal form of β-Gal detected in untreated normal fibroblasts (GM08399; Fig. 1D), with only a small amount of the precursor enzyme being detected (Fig. 1D). This would suggest that the majority of internalized commercial rhβ-Gal–His6 is being rapidly delivered to lysosomes and converted to the mature lysosomal species following cellular uptake in both cell lines. Appearance of the mature lysosomal rhβ-Gal–His6 species can be completely abolished in the presence of mannose-6-phosphate (Man6P; Fig. 1D), a known inhibitor of CI-MPR–dependent cellular uptake (Fig. 1D). CI-MPR–mediated cellular uptake of commercial rhβ-Gal–His6 in GM1 gangliosidosis patient fibroblasts is sufficient to increase β-Gal protein levels to levels that are comparable with endogenous β-Gal protein levels present in control fibroblasts from a normal individual, as determined by Western blotting (Fig. 1D). Interestingly, cellular uptake of commercial rhβ-Gal–His6 or rhβ-Gal in normal fibroblasts results in β-Gal augmentation by only 1.56- and 2.67-fold above normal endogenous β-Gal activity levels, respectively (Fig. 1E). Collectively, these results suggest that our in-house purified rhβ-Gal (8Chen J.C. Luu A.R. Wise N. De Angelis R. Agrawal V. Mangini L. Vincelette J. Handyside B. Sterling H. Lo M.J. Wong H. Galicia N. Pacheco G. Van Vleet J. Giaramita A. et al.Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.J. Biol. Chem. 2020; 295 (31481471): 13532-1355510.1074/jbc.RA119.009811Google Scholar) and a commercial source of rhβ-Gal–His6 both exhibit CI-MPR–dependent cellular uptake in fibroblasts. Furthermore, the limited cellular uptake capacity for rhβ-Gal–His6 and rhβ-Gal in normal fibroblasts differs from other purified lysosomal enzymes utilizing the CI-MPR pathway, where overnormalization of lysosomal enzyme activity by severalfold above normal can be achieved (9Yogalingam G. Luu A.R. Prill H. Lo M.J. Yip B. Holtzinger J. Christianson T. Aoyagi-Scharber M. Lawrence R. Crawford B.E. LeBowitz J.H. BMN 250, a fusion of lysosomal α-N-acetylglucosaminidase with IGF2, exhibits different patterns of cellular uptake into critical cell types of Sanfilippo syndrome B disease pathogenesis.PLoS One. 2019; 14 (30657762): e020783610.1371/journal.pone.0207836Google Scholar). β-Gal has been proposed to associate with PPCA and NEU1 in lysosomes to form a multienzyme complex, with each enzyme deficiency being associated with a specific neurodegenerative lysosomal storage disease (summarized in Fig. 2A) (1Bonten E.J. Annunziata I. d'Azzo A. Lysosomal multienzyme complex: pros and cons of working together.Cell Mol. Life Sci. 2014; 71 (24337808): 2017-203210.1007/s00018-013-1538-3Google Scholar). In PPCA-deficient galactosialidosis patient cells, NEU1 is strictly dependent upon PPCA for delivery to lysosomes and activation in lysosomes, thus giving rise to secondary NEU1 deficiency in the absence of PPCA (Fig. 2A) (1Bonten E.J. Annunziata I. d'Azzo A. Lysosomal multienzyme complex: pros and cons of working together.Cell Mol. Life Sci. 2014; 71 (24337808): 2017-203210.1007/s00018-013-1538-3Google Scholar, 6Bonten E.J. Campos Y. Zaitsev V. Nourse A. Waddell B. Lewis W. Taylor G. d'Azzo A. Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization.J. Biol. Chem. 2009; 284 (19666471): 28430-2844110.1074/jbc.M109.031419Google Scholar, 7van der Spoel A. Bonten E. d'Azzo A. Processing of lysosomal β-galactosidase: the C-terminal precursor fragment is an essential domain of the mature enzyme.J. Biol. Chem. 2000; 275 (10744681): 10035-1004010.1074/jbc.275.14.10035Google Scholar). In contrast, β-Gal is partially dependent upon PPCA for stability in lysosomes, giving rise to only partial β-Gal deficiency in the absence of PPCA in galactosialidosis patient cells (Fig. 2A) (10Zhou X.Y. Morreau H. Rottier R. Davis D. Bonten E. Gillemans N. Wenger D. Grosveld F.G. Doherty P. Suzuki K. Grosveld G.C. d'Azzo A. Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells.Genes Dev. 1995; 9 (7590240): 2623-263410.1101/gad.9.21.2623Google Scholar). To determine whether the limited cellular uptake potential of rhβ-Gal–His6 in fibroblasts (Fig. 1E) is related to its dependence upon the multienzyme complex in lysosomes, we measured the cellular uptake and decay of lysosomal-delivered rhβ-Gal–His6 in GM1 gangliosidosis fibroblasts, normal fibroblasts, and PPCA-deficient galactosialidosis patient fibroblasts. The cellular uptake capacity of rhβ-Gal–His6 over 24 h is partially reduced in PPCA-deficient galactosialidosis fibroblasts (GM21262; Vmax = 2647 nmol/h/mg; Fig. 2B), when compared with GM1 gangliosidosis patient fibroblasts (GM05653; Vmax = 5484 nmol/h/mg; Fig. 2B) and WT fibroblasts (GM08399; Vmax = 4550 nmol/h/mg; Fig. 2B). The partial cellular uptake capacity of rhβ-Gal–His6 in galactosialidosis cells can be increased to normal levels, when galactosialidosis fibroblasts are preloaded with rhPPCA–His6 for 24 h prior to enzyme uptake (Vmax = 5265 nmol/h/mg; Fig. 2B). Following 24 h of cellular uptake, lysosome-delivered rhβ-Gal–His6 enzyme activity decays rapidly in PPCA-deficient galactosialidosis patient fibroblasts over a period of 40 days, with a t1/2 corresponding to 0.5 days (Fig. 2C). In contrast, rhβ-Gal–His6 stability is increased to 10 days in galactosialidosis patient fibroblasts preloaded with rhPPCA–His6 for 24 h, prior to performing the t1/2 experiment (Fig. 2C). Collectively, these experiments with PPCA-deficient galactosialidosis patient skin fibroblasts suggest that rhβ-Gal–His6 cellular uptake and stability in lysosomes is partially dependent upon PPCA and cannot be overnormalized. These results are in agreement with those of Zhou et al. (10Zhou X.Y. Morreau H. Rottier R. Davis D. Bonten E. Gillemans N. Wenger D. Grosveld F.G. Doherty P. Suzuki K. Grosveld G.C. d'Azzo A. Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells.Genes Dev. 1995; 9 (7590240): 2623-263410.1101/gad.9.21.2623Google Scholar), which indicate that β-Gal activity is only partially reduced in various tissues of PPCA KO mice. Previous studies have demonstrated that NEU1 is strictly dependent upon its association with chaperone PPCA protein for its trafficking to lysosomes and stability (1Bonten E.J. Annunziata I. d'Azzo A. Lysosomal multienzyme complex: pros and cons of working together.Cell Mol. Life Sci. 2014; 71 (24337808): 2017-203210.1007/s00018-013-1538-3Google Scholar, 6Bonten E.J. Campos Y. Zaitsev V. Nourse A. Waddell B. Lewis W. Taylor G. d'Azzo A. Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization.J. Biol. Chem. 2009; 284 (19666471): 28430-2844110.1074/jbc.M109.031419Google Scholar, 7van der Spoel A. Bonten E. d'Azzo A. Processing of lysosomal β-galactosidase: the C-terminal precursor fragment is an essential domain of the mature enzyme.J. Biol. Chem. 2000; 275 (10744681): 10035-1004010.1074/jbc.275.14.10035Google Scholar). In contrast, β-Gal, which also complexes with PPCA, is only partially dependent on PPCA (1Bonten E.J. Annunziata I. d'Azzo A. Lysosomal multienzyme complex: pros and cons of working together.Cell Mol. Life Sci. 2014; 71 (24337808): 2017-203210.1007/s00018-013-1538-3Google Scholar, 10Zhou X.Y. Morreau H. Rottier R. Davis D. Bonten E. Gillemans N. Wenger D. Grosveld F.G. Doherty P. Suzuki K. Grosveld G.C. d'Azzo A. Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells.Genes Dev. 1995; 9 (7590240): 2623-263410.1101/gad.9.21.2623Google Scholar). These observations led us to speculate that β-Gal mediates additional, novel roles in regulation of the multienzyme complex. A series of add-back experiments in PPCA-deficient galactosialidosis patient fibroblasts were designed to evaluate the role of β-Gal in the multienzyme complex. As expected, in the absence of PPCA, untreated galactosialidosis cells completely lack NEU1 activity (Fig. 3B). Cellular uptake of rhβ-Gal–His6 alone in galactosialidosis fibroblasts results in a dose-dependent increase in β-Gal activity, which is increased further in cells preloaded with rhPPCA–His6 (Fig. 3A). This result is not surprising in light of the partial dependence of β-Gal on PPCA for its stability in lysosomes (Fig. 2C) (1Bonten E.J. Annunziata I. d'Azzo A. Lysosomal multienzyme complex: pros and cons of working together.Cell Mol. Life Sci. 2014; 71 (24337808): 2017-203210.1007/s00018-013-1538-3Google Scholar, 10Zhou X.Y. Morreau H. Rottier R. Davis D. Bonten E. Gillemans N. Wenger D. Grosveld F.G. Doherty P. Suzuki K. Grosveld G.C. d'Azzo A. Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells.Genes Dev. 1995; 9 (7590240): 2623-263410.1101/gad.9.21.2623Google Scholar). Cellular uptake of rhβ-Gal–His6 in galactosialidosis patient fibroblasts does not augment NEU1 activity levels (Fig. 3B), suggesting that β-Gal does not positively regulate NEU1. In galactosialidosis patient fibroblasts preloaded with rhPPCA–His6, NEU1 activity levels are rescued close to NEU1 levels detected in fibroblasts from a normal individual (Fig. 3B). Interestingly, we show that this rescued NEU1 activity can be competitively reduced following endocytosis with increasing concentrations of rhβ-Gal–His6 (Fig. 3B). Given that NEU1 is strictly dependent upon its association with PPCA for its activation, one can presume that increasing concentrations of lysosome-delivered rhβ-Gal–His6 in galactosialidosis patient fibroblasts are directly or indirectly competing with and displacing the rescued NEU1 from PPCA. There are several lines of evidence to support a dynamic state, rather than a static state for the multienzyme complex. Bonten et al. (6Bonten E.J. Campos Y. Zaitsev V. Nourse A. Waddell B. Lewis W. Taylor G. d'Azzo A. Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization.J. Biol. Chem. 2009; 284 (19666471): 28430-2844110.1074/jbc.M109.031419Google Scholar) have reported that NEU1 exhibits weak self-association at acidic pH, which leads to its instability and rapid degradation, whereas association with PPCA leads to stable, long-lived PPCA-NEU1 heterodimers (6Bonten E.J. Campos Y. Zaitsev V. Nourse A. Waddell B. Lewis W. Taylor G. d'Azzo A. Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization.J. Biol. Chem. 2009; 284 (19666471): 28430-2844110.1074/jbc.M109.031419Google Scholar). In contrast to NEU1, our biophysical studies with rhβ-Gal suggest that β-Gal exhibits dynamic self-association and instability at neutral pH, with the enzyme being more stable as a dimer at acidic pH (8Chen J.C. Luu A.R. Wise N. De Angelis R. Agrawal V. Mangini L. Vincelette J. Handyside B. Sterling H. Lo M.J. Wong H. Galicia N. Pacheco G. Van Vleet J. Giaramita A. et al.Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.J. Biol. Chem. 2020; 295 (31481471): 13532-1355510.1074/jbc.RA119.009811Google Scholar). Collectively, these results suggest that β-Gal plays a previously undescribed role in the PPCA–NEU1–β-Gal multienzyme complex, where it competes with NEU1 in a concentration-dependent manner for association with PPCA and thereby has the potential to negatively regulate sialidase levels (Fig. 3, A and B; see also Fig. 8 for a summary). Our add-back studies in PPCA-deficient galactosialidosis fibroblasts (Fig. 3) suggest that β-Gal augmentation therapy has the potential to displace NEU1 from PPCA and promote secondary NEU1 deficiency. Because NEU1 deficiency is associated with the neurodegenerative lysosomal storage disease, sialidosis, we evaluated the potential for β-Gal augmentation therapy to promote secondary NEU1 deficiency in GM1 gangliosidosis patient fibroblasts. Chronic cellular uptake of rhβ-Gal–His6 in GM1 gangliosidosis patient fibroblasts over 1 week restores β-Gal activity (Fig. 4A), which coincides with a dose-dependent reduction in NEU1 activity levels (Fig. 4B). The highest concentration of rhβ-Gal–His6 cellular uptake tested (200 nm) coincides with almost complete loss of NEU1 activity (Fig. 4B), with the levels of NEU1 activity corresponding to 3% of normal NEU1 activity detected in fibroblasts from a normal individual (Fig. 4B). Interestingly, preloading GM1 gangliosidosis patient fibroblasts with rhPPCA–His6 does not further increase the uptake capacity of rhβ-Gal–His6 (Fig. 4A) or prevent secondary NEU1 reduction (Fig. 4B), suggesting that factors other than PPCA are required to augment β-Gal levels in GM1 gangliosidosis patient lysosomes (Fig. 4A) and prevent secondary NEU1 deficiency (Fig. 4B). Previous studies have suggested that the recycling time for the CI-MPR back to the cell surface for subsequent rounds of lysosomal enzyme targeting to be ∼15 min (11Cardone M. Porto C. Tarallo A. Vicinanza M. Rossi B. Polishchuk E. Donaudy F. Andria G. De Matteis M.A. Parenti G. Abnormal mannose-6-phosphate receptor trafficking impairs recombinant α-glucosidase uptake in Pompe disease fibroblasts.Pathogenetics. 2008; 1 (19046416): 610.1186/1755-8417-1-6Google Scholar), whereas we have previously shown that the rate of lysosomal-delivered β-Gal decay is much slower, with a t1/2 of ∼9 days (8Chen J.C. Luu A.R. Wise N. De Angelis R. Agrawal V. Mangini L. Vincelette J. Handyside B. Sterling H. Lo M.J. Wong H. Galicia N. Pacheco G. Van Vleet J. Giaramita A. et al.Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice.J. Biol. Chem. 2020; 295 (31481471): 13532-1355510.1074/jbc.RA119.009811Google Scholar). Therefore, with continuous exposure of GM1 gangliosidosis patient fibroblasts to rhβ-Gal over prolonged periods of time, the rate of β-Gal cellular uptake and delivery to lysosomes is likely to exceed the rate of β-Gal degradation, resulting in a net overall accumulation of β-Gal in lysosomes (Fig. 4A). To avoid inducing secondary NEU1 deficiency, we therefore developed an intermittent ERT dosing regimen to deliver a single pulsatile dose of rhβ-Gal–His6 to lysosomes of GM1 gangliosidosis patient fibroblasts, followed by a 1-week chase in the absence of enzyme. Cellular uptake with rhβ-Gal–His6 for 24 h, followed by enzyme withdrawal and a 1-week chase, is sufficient to augment β-Gal activity in GM1 gangliosidosis patient fibroblasts (Fig. 4A) without promoting secondary NEU1 deficiency (Fig. 4B). Collectively, our results suggest that chronic cellular uptake and delivery of β-Gal to lysosomes coincides with secondary NEU" @default.
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• W3046217984 date "2020-09-01" @default.
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• W3046217984 title "Intermittent enzyme replacement therapy with recombinant human β-galactosidase prevents neuraminidase 1 deficiency" @default.
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