Intermittent enzyme replacement therapy with recombinant human β-galactosidase prevents neuraminidase 1 deficiency

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.

Keywords: GM1 gangliosidosis; PPCA; beta-galactosidase; complex; enzyme replacement therapy; gene therapy; genetic disease; neuraminidase; sialidase.

Figure 1.

Cellular uptake of purified rhβ-Gal–His₆ and rhβ-Gal cannot be overnormalized in GM1 gangliosidosis patient cells. A–C, oligosaccharide analysis of rhβ-Gal–His₆ (A) or rhβ-Gal (B) treated without (black trace) or with (blue trace) alkaline phosphatase by capillary zone electrophoresis. The indicated tentative peak IDs assigned from co-migration with a known reference lysosomal enzyme standard were included in the run. BPM6 or BPM7, bisphosphorylated oligomannose 6 or 7. C, calculated molar ratio of total phosphorylated glycan to protein. D, representative Western blotting of primary skin fibroblasts from a normal individual (WT) or an infantile-onset GM1 gangliosiodosis patient. Fibroblasts were incubated with growth medium (control) or 50 nm enzyme for 24 h in the absence in the presence of Man6P, as indicated. Precursor, nonlysosomal β-Gal, and mature lysosomal β-Gal bands are indicated. For comparison 4 ng of purified rhβ-Gal–His₆ was included on the gel as an indicator of precursor, nonlysosomal enzyme. Enolase was used as a loading control. E, cellular enzyme uptake capacity for rhβ-Gal–His₆ and rhβ-Gal in normal fibroblasts. Enzymes were incubated at a dose of 50 nm for 24 h, at which time cells were assayed for β-Gal activity, with activity expressed as the fold increase above endogenous levels of β-Gal activity detected in untreated normal fibroblasts.

Figure 2.

rhβ-Gal–His₆ cellular uptake and stability in lysosomes is partially dependent upon PPCA. A, schematic representation of the lysosomal PPCA–NEU1–β-Gal multienzyme complex. Deficiency of each enzyme gives rise to a specific lysosomal storage disease (1). Deficiency of PPCA chaperone in galactosialidosis patients leads to secondary NEU1 deficiency, because of its strict dependence upon PPCA for stability and only partial β-Gal deficiency. B, rhβ-Gal cellular uptake (24 h) in GM1 gangliosidosis fibroblasts, normal fibroblasts, and galactosialidosis fibroblasts. In some instances GS cells were also preloaded with rhPPCA–His₆ prior to uptake with rhβ-Gal, as described under “Experimental procedures.” C, t_1/2 determination of rhβ-Gal–His₆ in galactosialidosis (GS) patient fibroblasts (closed circles) or GS fibroblasts preloaded for 16 h with 20 nm rhPPCA–His₆ (open circles), as described under “Experimental procedures.”

Figure 3.

NEU1 activation in lysosomes is strictly dependent upon PPCA and negatively regulated by β-Gal. β-Gal activity (A) and NEU1 activity (B) in PPCA-deficient galactosialidosis patient fibroblasts following cellular uptake (24 h) with rhβ-Gal–His₆ only (white bars). In some instances, the cells were preloaded with 1.56 nm rhPPCA–His₆ for 16 h prior to being incubated with rhβ-Gal (black bars). The dashed lines represent the level of β-Gal activity (A) or NEU1 activity (B) detected in fibroblasts from a normal individual cultured for the same period of time.

Figure 4.

Chronic cellular uptake of rhβ-Gal–His₆ in GM1 gangliosidosis patient fibroblasts coincides with secondary NEU1 deficiency, which cannot be rescued with exogenous rhPPCA. β-Gal activity (A) and NEU1 activity (B) in GM1 gangliosidosis patient fibroblasts following continuous (chronic) cellular uptake of rhβ-Gal–His₆ for 1 week (filled circles). Alternatively, GM1 gangliosidosis patient fibroblasts were preloaded with 20 nm rhPPCA–His₆ for 16 h prior to chronic rhβ-Gal–His uptake for 1 week (squares). In a third group, GM1 gangliosidosis patient fibroblasts were incubated for 24 h with rhβ-Gal–His₆ and then chased in growth medium without enzyme for a further 1 week (open circles). The dashed lines represent the level of β-Gal activity (A) or NEU1 activity (B) detected in fibroblasts from a normal individual cultured for the same period of time.

Figure 5.

Constitutive lentiviral-mediated GLB1 overexpression negatively regulates PPCA and NEU1 protein levels in a dose-dependent manner, whereas intermittent ERT with rhβ-Gal does not. A, Western blots of cell lysates prepared from three infantile-onset GM1 gangliosidosis patient fibroblast lines 8 days after being transduced with LV–CMV–GLB1 (24 h transduction followed by 8-day chase; multiplicity of infection (MOI) = 1.25, 2.5, of 5) or 8 days after being incubated with purified rhβ-Gal (24 h of enzyme uptake followed by 8 day chase; 6.25, 12.5, or 25 nm). Note that the β-Gal panel and enolase panel shown here were also included in an earlier companion article (7), and have now been integrated with the new PPCA and Neu1 immunoblot panels. B–E, quantification of precursor β-Gal (B), mature β-Gal (C), PPCA (D), and NEU1 (E) protein levels detected in the three patient lines, standardized to the enolase loading control and expressed as the means ± S.D.

Figure 6.

Constitutive lentiviral-mediated GLB1 overexpression promotes secondary NEU1 deficiency, whereas an intermittent ERT dosing regimen does not. A–D, GM1 gangliosidosis patient fibroblasts were transduced with LV–CMV–GLB1 for 24 h (A and C) or incubated with rhβ-Gal for 24 h (B and D). After 24 h, the cells were washed; chased for 1, 7, or 21 days; and then assayed for β-Gal activity (A and B) or NEU1 activity (C and D). A red dashed line on each graph represents the theoretical threshold, where the majority of lysosomal enzyme deficiencies are associated with onset of lysosomal storage disease. E, GM1 gangliosidosis patient fibroblasts (GM05653) were transduced for 24 h with LV–CMV–GFP or LV–CMV–GLB1 (multiplicity of infection (MOI) = 2.5) or incubated with rhβ-Gal (50 nm) for 24 h. The cells were then washed; chased in growth medium for 1, 7, or 21 days; and then analyzed by Western blotting for β-Gal and NEU1 protein levels, along with the GFP reporter and enolase loading control. rhβ-Gal (6.25 nm) was also included on the gel as an indicator of the precursor nonlysosomal form of β-Gal (arrow). The mature lysosomal β-Gal band is also indicated with an arrow. Note that the β-Gal, GFP, and enolase panels shown here were also included in an earlier companion manuscript (7) and have now been integrated with the new Neu1 immunoblot panel.

Figure 7.

NEU1 activity is up-regulated in GLB1 KO mice, which can be restored to normal levels with weekly ICV dosing with rhβ-Gal. A, summary of the 8-week short-term proof-of-concept (PoC) ICV-ERT studies evaluated in the GM1 gangliosidosis mouse model. The 8-week proof-of-concept study commenced at 12 weeks of age, with mice receiving weekly ICV dosing (100 μg/dose) with rhβ-Gal or vehicle for 8 weeks. The mice were taken down 24 h after the final ICV dose of enzyme. B, correlation between NEU1 activity (y axis) and β-Gal activity (x axis) in brain homogenates prepared from individual mice in the 8-week proof-of-concept study. WT vehicle, n = 4; KO vehicle, n = 5; KO-rh β-Gal, n = 9. C, Western blotting of β-Gal protein levels and NEU1 protein levels in pooled brain homogenates prepared from the left hemisphere of WT or GLB1 KO mice treated with vehicle or rhβ-Gal, as summarized for A. For comparison, 4 ng of purified rhβ-Gal l is included on the gel as an indicator of precursor, nonlysosomal enzyme. Also included is 5 μg of cell lysate prepared from WT human fibroblasts, as an indicator of mature β-Gal successfully delivered to lysosomes. Enolase was used as a loading control. Note that the β-Gal and enolase panels shown here was also included in an earlier companion article (7), which has now been integrated with the new Neu1 immunoblot panel. D, Neu1 activity expressed as fold above normal levels in the three test groups. E, quantification of Neu1 protein levels in brain homogenates from Western blots of individual samples, standardized to an enolase loading control. WT vehicle, n = 4; KO vehicle, n = 5; KO-rhβ-Gal, n = 9. ***, statistically significant with p values indicated. NS, not significant.

Figure 8.

Summary of the findings of this study. A, β-Gal is reported to exist in a multienzyme complex with PPCA and NEU1 in lysosomes, with NEU1 being strictly dependent upon PPCA chaperone for its stability and activity in lysosomes and β-Gal only being partially dependent upon PPCA (1). However, we have recently demonstrated that β-Gal is most likely to be a stable dimer in the acidic environment of the lysosome (7), suggesting that the multienzyme complex may in fact be more dynamic and heterogeneous than represented here. B–D, our cellular uptake results suggest that NEU1 and β-Gal compete with each other for association with PPCA, resulting in two pools of PPCA, either associated with β-Gal dimer or NEU1. B, our results suggest that in normal cells β-Gal dimer and NEU1 are expressed at levels where both enzymes can associate with PPCA, resulting in physiological levels of β-Gal and NEU1. C, in the absence of β-Gal in GM1 gangliosidosis patient cells, more PPCA can associate with NEU1, resulting in the potential for NEU1 to be overnormalized. In support of this we observed a significantly increased level of NEU1 activity (Fig. 7D) and NEU1 protein (Fig. 7E) in GLB1 KO mouse brain homogenates above normal WT controls. D, in contrast, in GM1 gangliosidosis patient fibroblasts continually overexpressing β-Gal from the CMV promoter (chronic GLB1 gene therapy; Fig. 6A, 6C) or in cells chronically endocytosing rhβ-Gal from the cell surface (chronic rhβ-Gal ERT; Fig. 4), lysosome-delivered β-Gal has the potential to displace NEU1 from PPCA chaperone and promote secondary NEU1 deficiency. Furthermore, in our gene therapy studies in patient cells, chronic overexpression of β-Gal coincides with a reduction in PPCA levels as well as NEU1 levels (Fig. 5A, 5D), suggesting that overexpressed β-Gal has the potential to also negatively regulate PPCA chaperone, in addition to NEU1. E, to avoid disrupting NEU1 from PPCA, we developed an intermittent ERT dosing strategy to augment β-Gal levels in GM1 gangliosidosis patient cells without promoting secondary NEU1 deficiency. We show in GM1 gangliosidosis patient cells that cellular uptake of rhβ-Gal over 24 h can normalize β-Gal activity levels (Fig. 6B), which coincides with a partial reduction in NEU1 activity (Fig. 6D). Following withdrawal of the rh β-Gal from the uptake medium and a 3-week chase, the lysosomal-targeted rhβ-Gal slowly decays in GM1 gangliosidosis patient skin fibroblasts (Fig. 6B), which coincides with restoration of NEU1 activity to normal levels (Fig. 6D). Weekly ICV-ERT dosing with rhβ-Gal is also sufficient to normalize β-Gal levels in brain tissue of a mouse model of GM1 gangliosidosis and helps to restore NEU1 activity to normal levels (Fig. 7).