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Published in final edited form as: Am J Med Genet C Semin Med Genet. 2020 Oct 27;184(4):885–895. doi: 10.1002/ajmg.c.31849

Human recombinant lysosomal β-Hexosaminidases produced in Pichia pastoris efficiently reduced lipid accumulation in Tay-Sachs fibroblasts

Angela J Espejo-Mojica 1,*, Alexander Rodríguez-López 1, Rong Li 2, Wei Zheng 2, Carlos J Alméciga-Díaz 1, Cindy Dulcey-Sepúlveda 3, Germán Combariza 3, Luis A Barrera 1
PMCID: PMC8045741  NIHMSID: NIHMS1685281  PMID: 33111489

Abstract

GM2 gangliosidosis, Tay-Sachs and Sandhoff diseases, are lysosomal storage disorders characterized by the lysosomal accumulation of GM2 gangliosides. This accumulation is due to deficiency in the activity of the β-hexosaminidases Hex-A or Hex-B, which are dimeric hydrolases formed by αβ or ββ subunits, respectively. These disorders show similar clinical manifestations that range from mild systemic symptoms to neurological damage and premature death. There is still no effective therapy for GM2 gangliosidosis, but some therapeutic alternatives, as enzyme replacement therapy, have being evaluated. Previously, we reported the production of active human recombinant β-hexosaminidases (rhHex-A and rhHex-B) in the methylotrophic yeast Pichia pastoris. In this study, we evaluated in vitro the cellular uptake, intracellular delivery to lysosome, and reduction of stored substrates. Both enzymes were taken-up via endocytic pathway mediated by mannose and mannose-6-phosphate receptors and delivered to lysosomes. Noteworthy, rhHex-A diminished the levels of stored lipids and lysosome mass in fibroblasts from Tay-Sachs patients. Overall, these results confirm the potential of P. pastoris as host to produce recombinant β-hexosaminidases intended to be used in the treatment of GM2 gangliosidosis.

Keywords: GM2 gangliosidosis, recombinant hexosaminidases, enzyme replacement therapy, Pichia pastoris

1. INTRODUCTION

Tay-Sachs (TSD, OMIM #272800) and Sandhoff (SD, OMIM #268800) diseases are two lysosomal storage disorders (LSDs) classified into the GM2 gangliosidosis group (Gravel et al., 2001; Leal et al., 2020a). TSD and SD are caused by mutations in HEXA or HEXB genes, respectively, which encoded for α or β subunits of the lysosomal β-hexosaminidases (Hex, EC 3.2.1.52) (Breiden & Sandhoff, 2019). Hexosaminidase A (Hex-A) and hexosaminidase B (Hex-B) are lysosomal enzymes formed as heterodimer (αβ subunits) or homodimer (ββ subunits), respectively. These enzymes are involved in the hydrolysis of N-acetylglucosamine or N-acetylgalactosamine residues of glycosphingolipids (Leal et al., 2020a). TSD patients show decreased Hex-A activity levels while Hex-B remains normal. On the other hand, SD patients have decreased activity levels of both β-hexosaminidases. Clinical manifestations in TSD and SD are indistinguishable and include apathy, hyperacusia, motor weakness, hypotonia, poor head control, decreasing attentiveness, macular cherry-red spot in the retina, seizures, and progressive mental deterioration followed by blindness, deafness, and spasticity. However, in SD the central nervous system (CNS) and visceral organs are affected, whereas in TSD only the CNS is impaired. Patients’ lifespan varies from 1 to 5 years in the severe infantile form, to late teens for patients with the juvenile form; while it is still unclear if there is a reduction in the lifespan for the late-onset patients with the disease (Cachon-Gonzalez et al., 2018; Gravel et al., 2001; Tropak, M. & Mahuran, 2010).

Currently, the treatment of GM2 gangliosidosis is mainly focused on palliative interventions, and in the delay of disease progression in the late-onset form. However, several therapy approaches for TSD or SD are under development including enzyme replacement therapy (ERT), bone marrow or neural progenitor cell transplantation, gene therapy, and substrate reduction therapy (Leal et al., 2020a; Solovyeva et al., 2018). Nowadays, ERT is available for eleven LSDs treatment including Gaucher, Fabry, Pompe, neuronal ceroid lipofuscinoses type II, α-mannosidosis, acid lipase deficiency, and mucopolysaccharidoses type I, II, IVA, VI, and VII (Leal et al., 2020b). These recombinant proteins have been mainly produced in mammalian cells (Garbade et al., 2020). However, new eukaryotic platforms, as yeasts, have been also explored as platforms for the production of recombinant lysosomal enzymes (Espejo-Mojica et al., 2015). Among the different yeasts used for the development of novel ERT; Pichia pastoris has shown a great potential due to its well know genetics and metabolism and the about 70 recombinant proteins approved or in process to being approved for several conditions [(Karbalaei et al., 2020) and www.pichia.com]. In this sense, we have worked for several years in the production and characterization of recombinant lysosomal enzymes by using the yeast P. pastoris (Echeverri et al., 2018; Espejo-Mojica et al., 2015; Puentes-Tellez et al., 2020), which include iduronate-2-sulfatase (IDS) (Córdoba-Ruiz et al., 2009; Landázuri et al., 2009; Pimentel et al., 2018), N-acetylgalactosamine-6-sulfate sulfatase (GALNS) (Rodriguez-Lopez et al., 2016; Rodriguez-Lopez et al., 2019) and β-hexosaminidases (Espejo-Mojica et al., 2016; Vu et al., 2018). Noteworthy, these enzymes have shown to be biologically active and have the ability to be internalized and delivered to lysosomes, as well as to reduce the accumulated substrates in different cellular models. In addition, a single intravenous infusion of recombinant GALNS into wild-type mice showed that this enzyme was delivered to several organs and the immunogenicity caused in mice was slightly lower than to those obtained in other studies with GALNS produced in Chinese hamster ovary CHO cells (Rodriguez-Lopez et al., 2019). Nevertheless, no ERT, based on a P. pastoris-produced enzyme, has been approved yet, which could be associated to the differences in the N-glycans structures between proteins produced in mammalian cells and yeasts, which could be solved through glycoengineering approaches (Laukens et al., 2015). In addition, we have also observed that some recombinant lysosomal enzymes, specially sulfatases, may have a significant lower activity compared with the enzyme produced in mammalian cells (Echeverri et al., 2018). Nevertheless, other enzymes have shown enzyme activities similar or higher than those observed for the enzymes produced in mammalian cells (Echeverri et al., 2018; Espejo-Mojica et al., 2015).

In the case of GM2 gangliosidoses ERT, some of the recombinant enzymes have been modified to improve the cellular uptake and lysosome delivery (Leal et al., 2020a). For instance, the use of a highly mannosylated recombinant Hex-A produced in the yeast Ogataea minuta, and treated with α-mannosidase to expose the mannose-6-phosphate (M6P) residues on the N-glycans, resulted in a GM2 storage reduction and an improvement in the motor function and lifespan in a SD mouse model (Akeboshi et al., 2007; Tsuji et al., 2011). Similarly, the use of a recombinant chimeric Hex-B produced in CHO cells allowed a reduction of GM2 ganglioside storage in SD patient cells, as well as in the parenchyma of SD mice intracerebroventricularly injected with the enzyme (Matsuoka et al., 2011). Current studies have combined the structural features of α-subunit active site and stable β-subunit interface into a single hybrid subunit (μ-subunit) forming a recombinant hybrid protein (Hex-M). This hybrid protein showed a high cellular uptake in TSD fibroblasts, which is important property for the development of an ERT. The therapeutic potential of this enzyme was showed through the reduction of brain ganglioside accumulation in TSD mouse models through administration of scAAV9 vectors containing HEXM gene (Tropak, M. B. et al., 2016). Recently, it was proposed the use of a molecular Trojan horse through the fusing of Hex-A to either IgG-heavy or light chains (HIRMAb), which showed similar activity to that obtained by non-fused Hex-A (Boado et al., 2019). Similarly, we reported the production and characterization of human recombinant Hex-A and Hex-B (rhHex-A and rhHex-B) in the yeast P. pastoris GS115 (Espejo-Mojica et al., 2016). These recombinant enzymes, without any modification on the N-glycans structure, showed between 25- and 50-fold higher enzyme activity than that observed in enzymes from normal human leukocytes, as well as a high stability at different acidic pHs and temperatures (Espejo-Mojica et al., 2016). Noteworthy, rhHex-A normalized the lipid storage in neural stem cells derived from induced pluripotent stem cells (iPSCs) from TSD patient fibroblasts (Vu et al., 2018). In this study, we continue the characterization of these enzymes showing that rhHex-A and rhHex-B are taken up through both mannose and M6P receptors and are delivered to lysosomes. rhHex-A demonstrated reduction of lysosomal mass and lipids storage in TSD patient fibroblasts.

2. EXPERIMENTAL SECTION

2.1. Editorial Policies and Ethical Considerations

This study was approved by the Research and Ethics Committee from the Faculty of Science at Pontificia Universidad Javeriana, Bogotá D.C., Colombia.

2.2. Recombinant β-hexosaminidases.

Recombinant β-hexosaminidases (rhHex-A and rhHex-B) were produced in Pichia pastoris GS115 as previously reported (Espejo-Mojica et al., 2016). Briefly, the cDNA of α and β subunits of human β-hexosaminidases were cloned into pPICK9K expression vector and transfected into the yeast P. pastoris GS115 (Invitrogen, Thermo Fisher Scientific, San Jose, CA, USA). Production of both recombinant enzymes was carried out at 1.65 L scale using a modified FM22 saline medium under previously described conditions (Espejo-Mojica et al., 2016; Rodriguez-Lopez et al., 2016; Stratton et al., 1998). rhHex-A and rhHex-B were obtained from culture medium due an α-factor secretion signal present in vector and were purified by ion exchange chromatography (Espejo-Mojica et al., 2016).

2.3. Enzymatic activity assays

β-hexosaminidase activity was assayed by using 4-methylumbelliferyl-β-D-acetyl-glucosaminide (MUG, Sigma-Aldrich, St. Louis, MO) or 4-methylumbelliferyl-β-D-acetyl-glucosaminide sulfate (MUGS, Calbiochem, San Diego, CA) substrates. One unit (U) was defined as the amount of enzyme hydrolyzing 1 nmol of substrate per hour. Specific hexosaminidase activity was expressed as U mg−1 of total protein determined by BCA assay (Shapira et al., 1989).

2.4. Cellular uptake of purified recombinant hexosaminidases

Cellular uptake assay was carried out as previously described (Mosquera et al., 2012; Pimentel et al., 2018; Rodriguez-Lopez et al., 2016). Briefly, 24 h before the experiment, 1×105 cells/well of wild-type human skin fibroblasts or HEK293 (ATCC CRL1573) were cultured in 12-well plates with Dulbecco’s modified medium (DMEM, Gibco, Carlsbad, CA), supplemented with fetal bovine serum 10%, penicillin 100 U mL−1 and streptomycin 100 U mL−1, at 37°C in a CO2 incubator. Two hours before the experiment, culture medium was replaced, and rhHex-A and rhHex-B were added to a final concentration of 10 or 50 nM. After 6 hours of incubation, culture medium was removed and stored at −20 °C. Cells were washed with PBS 1x (composition per liter: 8 g NaCl, 0.2 g KCl, 1.44 g NaH2PO4, 0.24 g KH2PO4, pH 7.2) and were lysed by resuspension in sodium deoxycholate 1% (Sigma-Aldrich, St. Louis, MO) (Rodriguez-Lopez et al., 2016). To evaluate if the protein uptake was mediated by endocytosis, cellular uptake assay was carried out at 4 °C or 37 °C with each recombinant protein (Rodriguez-Lopez et al., 2016). Enzyme activity was assayed in the culture medium and cell lysates. All assays were done in triplicate.

2.5. Inhibition of mannose and mannose-6-phosphate receptors.

To evaluate if rhHex-A and rhHex-B endocytosis process was mediated by membrane receptors, cellular uptake assays were carried out using 50 nM of recombinant proteins in presence or absence of 5 mM of mannose or mannose-6-phosphate (M6P) (Sigma-Aldrich, St. Louis, MO) (Pimentel et al., 2018; Rodriguez-Lopez et al., 2019). After 6 hours of incubation, the culture medium and cell lysate were treated as was described above. All assays were done in triplicate.

2.6. Protein-Lysosome colocalization assay

To evaluate if the recombinant enzymes were targeted to lysosomes, rhHex-A and rhHex-B were labeled with AlexaFluor 568® following the manufacturer’s protocol (Molecular Probes, Thermo Fisher Scientific, Eugene, OR, USA). HEK293 cells were grown on coverslips treated with 0.01% (W/V) type II collagen (Sigma-Aldrich, St. Louis, MO) and placed onto 12-well plates. Cells were seeded at a density of 2×104 cells/well. Once the cells were confluent, the culture medium was exchanged for fresh medium 1 h before adding the fluorescent-labeled enzymes to a final concentration of 50 nM (Pimentel et al., 2018; Rodriguez-Lopez et al., 2019). After 6 h of incubation, the cells were stained with 375 nM LysoTracker® Green DND-26 following the manufacturer’s protocol (Molecular Probes, Thermo Fisher Scientific, Eugene, OR, USA). Cells were fixed using freshly prepared 4% paraformaldehyde in 1x PBS for 20 min at room temperature. Finally, the cellular nucleus was stained with 4’,6-Diamidino-2-phenylindole dichydrochloride (DAPI, Thermo Fisher Scientific, San Jose, CA, USA). Cells were imaged using an Olympus FV1000 confocal microscope equipped with 405, 473, and 559 nm laser lines using a 63×/1.49 NA oil objective. DAPI (excitation 382 – 393, emission 417 – 477 nm), FITC (excitation 460–500 nm, emission 510–560 nm), and TRITC HyQ filter sets (excitation 530–560 nm, emission 590–650 nm) were applied to collect DAPI, LysoTracker® Green, and Alexa 568 signals, respectively. Images were processed and analyzed by using R software (R Core Team, 2018).

2.7. Nile Red and LysoTracker staining assays

Nile Red and LysoTracker assays were done as previously reported (Vu et al., 2018). Briefly, twenty-four hours before treatment, 4,000 cells/well of TSD fibroblasts (GM00221 and GM00515, Coriell Institute) were seeded in a 96-well black clear bottom plate (Greiner Bio-One, #655090, Monroe, NC, USA). On the day of the experiment, fibroblasts were treated with 50 and 100 nM rhHex-A and cultured on complete DMEM media. TSD and wild-type human skin fibroblasts treated with PBS 1x were used as controls. Twenty four, 48 and 72 h after treatment, the cells were treated with 50 nM LysoTracker Red DND-99 dye (Molecular Probes, Thermo Fisher Scientific, Eugene, OR, USA) in complete DMEM media at 37 °C for 1 h or 1 μM Nile Red in complete DMEM media at 37 °C for 10 min, followed by plate washing twice with DPBS. The plates were fixed and stained simultaneously in 3.2% paraformaldehyde solution with Hoechst dye at a 1:5,000 dilution for 30 min at room temperature. The plates were washed twice using DPBS and stored with 100 μl/well DPBS at 4 °C until imaging. The images (9 images/well) were acquired using the IN-Cell Analyzer 2200 imaging system, using a 20X objective lens (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The DAPI (excitation = 350 ± 50 nm, emission = 455 ± 50 nm), Texas Red (excitation = 545 ± 20 nm, emission = 593 ± 20 nm) and FITC (excitation = 490 ± 20 nm, emission = 525 ± 36 nm) filter sets were used to visualize Hoechst nuclear staining, LysoTracker Red DND-99, and Nile red staining, respectively. All assays were performed in triplicate.

2.8. Statistical analysis

The results are shown as mean ± standard deviation (± SD) and were analyzed by a one-way ANOVA, or by t test when appropriate. Differences between groups were considered significant when p < 0.05 on GraphPad PRISM 6.0.

3. RESULTS AND DISCUSSION

3.1. Cellular uptake

Conventional ERT for LSDs is based on the capacity of the recombinant lysosomal enzymes to be taken up and targeted to the lysosome, where they can catalyze the degradation of the stored substrates (Desnick & Schuchman, 2012). In this sense, we evaluated the ability of rhHex-A and rhHex-B to be internalized by HEK293 cells and human skin fibroblasts at 10 and 50 nM, as was previously reported for other recombinant enzymes produced in this host (Pimentel et al., 2018; Rodriguez-Lopez et al., 2016; Rodriguez-Lopez et al., 2019). Figure 1 shows that recombinant hexosaminidases were taken-up by HEK293 cells and fibroblasts, since the enzymatic activity in cell lysates was higher than that observed in non-treated cells. However, in HEK293 the highest increase in enzyme activity was observed with 10 nM, while a slight decrease on the enzyme activity was observed at 50 nM (Figure 1A and B). On the other hand, cellular uptake of the recombinant enzymes in fibroblasts was only observed with 50nM (Figure 1C and 1D), while at a lower concentration (10 nM) no differences were observed compared to the control. These results of cellular uptake are in agreement with reports about other recombinant lysosomal enzymes produced in P. pastoris without any modification in the N-glycans structure, such as α-glucosidase (Chen, Y. et al., 2000), acid lipase (phLA) (Du et al., 2005; Du et al., 2001), GALNS (Rodriguez-Lopez et al., 2016; Rodriguez-Lopez et al., 2019) and IDS (Pimentel et al., 2018). The N-glycosylation pattern of recombinant acid lipase and GALNS produced in the same strain P. pastoris used in this study, showed that both capped- and uncapped-phosphorylated mannoses were present in the recombinant enzymes, which could suggest that presence of those uncapped-phosphorylated mannoses could be the cause of the internalization of these enzymes into the cells (Du et al., 2005; Du et al., 2001; Rodriguez-Lopez et al., 2019). The differences in cellular uptake between both HEK293 cells and human skin fibroblasts could be related to variation in the expression of receptors. In this sense, the type and number of receptors present on HEK293 cell surface could be different than those found in fibroblasts (Chen, J. et al., 2017; Sly et al., 1978). Thus, at 50 nM receptors saturation may have occurred in HEK293 cells, causing the slight decreasing in protein internalization. It is expected that rhHex-A and rhHex-B may be also internalized by relevant cells for GM2 gangliosidosis, such as neurons, since the treatment with rhHex-A of neural stem cells derived from TSD iPSCs led to the normalization of lipid storage (Vu et al., 2018), suggesting that the enzyme was successfully internalized.

Figure 1. Cellular uptake of recombinant enzymes.

Figure 1.

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The cellular uptake was assayed in HEK293 cells (A, rhHex-A and B, rhHex-B) and normal human skin fibroblasts (C, rhHex-A and D, rhHex-B). The purified rhHex-A and rhHex-B were added to a final concentration of 10 and 50 nM. In all cases controls are cells without protein treatment. Results are expressed as mean ± SD Assays were done in triplicate (* p < 0.05, ** p < 0.01, *** p < 0.001).

On the other hand, these results contrast with those of rhHex-A produced in the yeast O. minuta, which required the treatment with α-mannosidase to expose the M6P residues and allows the cellular uptake by GM2 gangliosidosis patient-derived fibroblasts (Tsuji et al., 2011). A similar approach was necessary for human α-glucosidase produced in Y. lipolytica (Tiels et al., 2012) and α-galactosidase A produced in Saccharomyces cerevisiae (Chiba et al., 2002) to allow the cellular uptake of the recombinant protein both in in-vitro and in-vivo assays.

3.2. Endocytic pathway evaluation

It is well known that cellular internalization processes occurs at physiological temperatures, and low temperatures can affect or even inhibit the endocytic pathway (Jiao et al., 2009). In order to confirm that cellular uptake of rhHex-A and rhHex-B was mediated by an endocytic pathway; we evaluated the cellular uptake of these proteins at 4 °C and 37 °C. As is shown in Figure 2, the ratio of β-hexosaminidase activity between the intracellular (lysate) and extracellular (culture medium) fractions were higher at 37 °C than at 4 °C, suggesting that the cellular capture of these enzymes is mediated by an endocytic pathway (Jiao et al., 2009; Rodriguez-Lopez et al., 2016).

Figure 2. Endocytic pathway evaluation – Temperature evaluation.

Figure 2.

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Cellular uptake evaluation was carried out at 4 °C or 37 °C in HEK293 cells (A, rhHex-A and B, rhHex-B) and normal human skin fibroblasts (C, rhHex-A and D, rhHex-B). Results are reported as the relation of intracellular vs. extracellular activity. Assays were done in triplicate (* p < 0.05, ** p < 0.01, *** p < 0.001).

Lysosomal enzymes undergo several post-translational modifications during their trafficking from endoplasmic reticulum to the lysosome or extracellular compartments (Kornfeld, 1987). One of the main modifications is the addition of M6P-ended N-glycosylations in Golgi apparatus, which allows the delivery of the enzyme to the endosome/lysosome system through the interaction with M6P-receptors (M6PR) in lysosome or cell membrane (Braulke & Bonifacino, 2009). It is well known that the percentage of oligosaccharides added to lysosomal enzymes depends on the source and subcellular location of the enzyme (von Figura & Hasilik, 1986). In this sense, some oligomannose residues remain uncovered and exposed to be recognized by other receptors like mannose receptors (MR), which would favor the internalization of lysosomal enzymes (Martinez-Pomares, 2012). To evaluate the receptors used by rhHex-A and rhHex-B during internalization, we added the recombinant enzymes to cells in the presence or absence of mannose and M6P. Results showed that the cellular uptake of both rhHex-A and rhHex-B was inhibited in the presence of both mannose and M6P, suggesting that MR and M6PR mediate the uptake of these enzymes in wild-type fibroblasts (Figure 3). These results agreed with those reported for rhHex-A and rhHex-B produced in CHO cells, as well as for other recombinant lysosomal proteins produced in P. pastoris, such as IDS and GALNS (Pimentel et al., 2018; Rodriguez-Lopez et al., 2016; Rodriguez-Lopez et al., 2019), and even hexosaminidase produced in other yeasts, which showed an internalization via cation M6P independent manner (Akeboshi et al., 2007; Matsuoka et al., 2011; Ohsawa et al., 2005; Tsuji et al., 2011). The cellular uptake of rhHex-A and rhHex-B through both MR and M6PR could be associated to the presence of N-glycans with high-mannose chains and mannosyl phosphorylated derivatives thereof, as was described for the recombinant GALNS produced in P. pastoris under the same conditions used in the present study (Rodriguez-Lopez et al., 2019). Although the cellular uptake was demonstrated in HEK293 cells, the internalization pathway could not be demonstrated through the inhibition of MR and M6PR receptors, since the results showed no significant differences between treatments and controls (data not shown).

Figure 3. Endocytic pathway evaluation - Receptor inhibition.

Figure 3.

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Cellular uptake assayed in normal human skin fibroblasts after treatment with 50 nM rhHex-A (A) or 50 nM rhHex-B (B), with and without addition of mannose or mannose-6-phosphate (M6P). Results are expressed as mean ± SD.

These results may be related to recent findings about the identification of other receptors, as clathrin or caveolae, involved in the internalization of α-galactosidase in HEK293, HUVEC, primary fibroblasts, and UKEC cells (Ivanova et al., 2020), and the fact that apparently M6PR gene is low-expressed in HEK293 cell line. In this sense, other receptors or mechanisms could be involved in the cellular uptake of rhHex. However, these mechanisms have not been well described and remain poorly understood.

3.3. Intracellular delivery of recombinant enzymes to lysosome

The intracellular location of rhHex-A and rhHex-B was carried out by treating the cells with fluorescent labeled recombinant enzymes. As is showed in Figure 4 the labeled proteins were observed inside the cell and co-localized with the LysoTracker staining, a phenotypic assay that detects acidic organelles (Lloyd-Evans et al., 2008; Xu et al., 2014). In this sense, these results confirm the internalization of the recombinant enzymes and their targeting to lysosomes (Figure 4A). Analyzed images showed that approximately 62.3% and 64.58% of rhHexA and rhHexB, respectively, overlapped with LysoTracker staining (acidic compartments / lysosomes) (Figure 4B). In addition, it was observed that a fraction of the internalized enzymes appears to be localized in the cytoplasm or possibly in earlier endosomal compartments. Similar results were reported for the recombinant GALNS (Rodriguez-Lopez et al., 2019) and IDS (Pimentel et al., 2018) produced in P. pastoris, as well as α-glucosidase produced in Y. lypolitica (Tiels et al., 2012), which support the use of P. pastoris in the development of an ERT for GM2 gangliosidosis. These results also agree with those of rhHex-A-treated neural stem cells (Vu et al., 2018), since the normalization of the stored substrates (GM2 gangliosides) in those cells suggest that the enzyme was efficiently delivered to the lysosomes.

Figure 4. Intracellular delivery of recombinant hexosaminidases to lysosome.

Figure 4.

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A. Fluorescent labeled-enzymes were added to HEK293 cells and colocalization with fluorescent labeled-target organelles were visualized by confocal microscopy. Upper panel: rhHex-A. Down-panel: rhHex-B. Scale bar 50μm. DAPI (nuclei); AlexaFluor 568 (recombinant enzymes); Lysotracker (acidic organelles/lysosomes); Overlapping (red/green signal overlapping). B. Images were analyzed by R software. Each image was normalized adjusting the threshold at 15% for DAPI and 5% for both AlexaFluor 568 and Lysotracker. Percentage of overlapping was stablished through the pixels counting by one filter over other using the R software. Nu: nuclei, Prot: protein, Lyso: lysosome

3.4. Ganglioside and lysosomal mass reduction

Once confirmed the internalization and cellular location of recombinant enzymes, we evaluated the effect of rhHex-A on the phenotype of TSD human skin-fibroblasts. First, the effect of rhHex-A on the lysosomal mass was evaluated by using the LysoTracker Red staining. This fluorescent dye accumulates in acidic organelles and fluoresces at low pH environment. Since an increase in LysoTracker staining, compared to normal cells, has been observed in LSDs patients cells accumulating different substrates (e.g. GAGs, lipids, or oligosaccharides), (Lloyd-Evans et al., 2008; Xu et al., 2014); it has been proposed its use in drug screening for LSDs (Xu et al., 2014). As observed in Figure 5A, both GM00221 and GM00515 TSD fibroblasts showed a 1.5-fold increase in the LysoTracker fluorescent intensity compared with the wild-type fibroblasts, consistent with previous findings in TSD fibroblasts (Xu et al., 2014) and neural stem cells (Vu et al., 2018). After 72 h of rhHex-A treatment, a reduction in the fluorescent intensity was observed for both TSD fibroblasts and leading to normalization of the intensity in GM00515 fibroblasts. We did not observe significant difference between 50 and 100 nM rhHex-A. In this sense, these results show that the TSD fibroblasts have an increased lysosomal mass that can be reduced, and even normalized, after treatment with rhHex-A.

Figure 5. Effect of rhHex-A on reduction of lysosomal mass and lipid accumulation in TSD fibroblasts.

Figure 5.

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TSD fibroblasts (GM00221 and GM00515) were treated with rhHex-A (50 and 100nM). Cells were either stained with LysoTracker Red DND-99 dye (A) or Nile Red (B). The images (9 images/well) were acquired using the IN-Cell Analyzer 2200 imaging system (left). Results, on right figure, are presented as cell intensity normalized to WT fluorescence level (dotted lines). All assays were performed in triplicate (ANOVA Sidak t test * p < 0.05, ** p < 0.01, **** p < 0.0001). Scale bar: 100 μm.

Since GM2 ganglioside buildup promotes the accumulation of others lipids such as phospholipids, cerebrosides, sphingomyelin, and cholesterol (Leal et al., 2020a), the staining of these lipids could be used as an assay for drug development. In this sense, it was reported that Nile Red staining offers a valid method for evaluating drug efficacy and compound screening in TSD patient neural stem cells (Vu et al., 2018). As observed in Figure 5B, the Nile Red staining intensity increased 1.7- and 2.1-fold in TSD fibroblasts compared to wild-type fibroblasts, consistent with previous findings in TSD neural stem cells (Vu et al., 2018). After treatment with rhHex-A a reduction in the fluorescent intensity was observed for both TSD fibroblasts, even during the first 24 h post-treatment. Noteworthy, wild-type levels of fluorescent intensity were observed for both TSD fibroblasts after 72 h treatment with 100 nM rhHex-A. Overall, these results show that rhHex-A allows the normalization of lipids storage in TSD fibroblasts with a subsequent reduction in the lysosomal mass. These results agree with those obtained after the treatment of TSD neural stem cells with the same rhHex-A, produced in P. pastoris, using neural stem cells derived from TSD iPSC (Vu et al., 2018). Nevertheless, normalization of lipids storage in the neural stem cells was observed after only 4 h treatment with 100 nM rhHex-A. In this sense, these results confirm the fact that there are differences in recombinant enzyme uptake depending on the cellular model, and suggest that neurons may be easy to treat with rhHexA. All together, these results show that rhHex-A produced in P. pastoris may catalyze the reduction accumulated GM2 ganglioside in different cellular models, which confirm the potential of this enzyme towards the development of an ERT for TSD.

Gangliosides reduction in patient fibroblasts has been also observed by other recombinant hexosaminidases, although using different assays. For instance, Akeboshi et al 2007, demonstrated that recombinant M6P-HexA produced in O. minuta diminished GM2 gangliosides in TSD fibroblasts using up to 1,800 nmol/h/well of recombinant M6P-HexA (Akeboshi et al., 2007). This enzyme was then improved by increasing level of a high M6P-type-N-glycan content (Om4HexA), producing and improvement in some motor functions and an increase in the survival rate in a SD mouse model after intracerebroventricularly administration (Tsuji et al., 2011). Similarly, Matsuoka et al 2011, used a chimeric Hex-B to treat TSD patients fibroblasts, showing a reduction of GM2 gangliosides (Matsuoka et al., 2011). A recent report using this chimeric Hex-B showed that stored GM2 was only significant reduced in TSD fibroblasts when the protein was co-administered with a recombinant GM2 ganglioside activator (GM2A) (Kitakaze et al., 2016).

4. CONCLUSIONS

We have extended the characterization of rhHex-A and rhHex-B produced in P. pastoris, showing that uncapping of the N-glycosylations ends was not necessary to facilitate the cellular uptake, and that these enzymes are highly active without the need of GM2A co-administration, as reported for other recombinant β-hexosamindases. Thus, rhHex-A and rhHex-B may have an advantage over other recombinant β-hexosamindases produced in different host, such as the yeast O. minuta, for which further modification on N-glycans are necessary. The results showed that the cellular uptake of rhHex-A and rhHex-B is mediated by MR and M6PR, which allows the delivery of those enzymes to the lysosome and the reduction of the stored lipids and lysosomal mass. Overall, these results continue offering valuable information towards the importance of P. pastoris as host to produce recombinant β-hexosaminidases for the design of an ERT for GM2 gangliosidoses. Further studies should be focused on the in-vivo evaluation of these recombinant enzymes on GM2 gangliosides levels, as well as the effect on the disease phenotype and the delivery of the enzyme to the CNS.

5. ACKNOWLEDGMENTS

AEM and ARL received a doctoral scholarship from the Ministry of Science, Technology and Innovation and the Pontificia Universidad Javeriana, respectively. CJAD received a scholarship from Fulbright Colombia. AEM and LAB were supported by Pontificia Universidad Javeriana (Grant ID 3637) and the Ministry of Science, Technology and Innovation (Grant contract No. 120352128575). AEM was supported by Pontificia Universidad Javeriana (Grant ID 8275). CJAD was supported by Pontificia Universidad Javeriana (Grant ID 7204 and 7520) and the Ministry of Science, Technology and Innovation [Grant ID 5174 - contract No. 120356933205, and Grant ID 5170 - contract No. 120356933427]. RL and WZ were supported by the Intramural Research Program of the Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.

Authoŕs biographies

Angela J. Espejo-Mojica: Bachelor of Industrial Microbiology and Doctor in Biological Sciences from Pontificia Universidad Javeriana in Bogotá Colombia. She is Assistant Professor of the Institute for the Study of Inborn Errors of Metabolism (IEIM) at Pontificia Universidad Javeriana. She has worked in the production and purification of IgG and IgY antibodies against lysosomal enzymes and house dust mite allergens, in rabbits and hens respectively. She used those antibodies in the development of proper ELISA tests for identification of those proteins. Additionally, she has worked in production and purification of different recombinant human lysosomal enzymes in microorganisms (bacteria and yeast), with promising results in the development of enzyme replacement therapy for inborn errors of metabolism. Her efforts have focused in evaluation of human recombinant lysosomal β-hexosaminidases, which were produced, purified and biochemically characterized and tested in vitro in different cell lines. Currently, she is working in the study of new strategies (nanoparticles) to delivery recombinant lysosomal enzymes through blood brain barrier in inborn errors of metabolism with central nervous system involvement.

Alexander Rodríguez-López: Bachelor of Chemistry from Universidad Distrital (Bogotá, D.C. - Colombia). Doctor in Biological Sciences from the Pontificia Universidad Javeriana (Bogotá D.C. - Colombia) and Doctor in Sciences, Biochemistry and Biotechnology from University of Ghent (Ghent, Belgium). Professor for more than 10 years in the Department of Chemistry and attached to the Institute for the Study in Inborn Errors of Metabolism, Faculty of Sciences at the Pontificia Universidad Javeriana. He has worked in the production, purification and characterization of recombinant enzymes obtained from microorganisms. He has worked in the glycoengineering of yeasts to allow the production of humanized recombinant enzymes. Research Interest in Applied Biotechnology to research in Biomedicine, cellular and molecular biology; biochemistry; genetic engineering; bioinformatics; bioprocesses and medicinal chemistry.

Rong Li: Bachelor in Pharmacy from West China University of Medical Science. Medical College, from Tsinghua University; Medicinal Chemisty and Doctor in Molecular Pharmacology from Peking Union Medical College, Tsinghua University. Research Scientist at National Center for Advancing Translational Sciences (NCATS). Research Fellow at National Institutes of Health. Postdoctoral Visiting Fellow at National Eye Institute (NEI). Project manager at Beijing Hua An Fu Medicine Research Center Co., Ltd. More than 15 years of basic and translational biomedical research experience. Broad interdisciplinary expertise in fields as cell and molecular biology, ophthalmology, stem cell, engineering antibody, pharmacology, biochemistry, microbiology, physiology and chemistry.

Wei Zheng: Bachelor in Pharmacy from the Zhejiang University College of Pharmacy in Hangzhou, China and Master in pharmacology from the same college. Doctor in pharmacology and toxicology from the University of Arizona in Tucson. Postdoctoral fellow in the State University of New York (SUNY) at Buffalo, where was trained in molecular biology and studied voltage-gated calcium channels. He obtained broad experience in early drug development including assay development, high throughput screening, and lead identification and optimization in the pharmaceutical industry (Berlex, Amgen and Merck). As a PI or co-Investigator on the NIH and foundation funded grants, he carried out probe identification and development by using a high throughput screening approach for a number of collaborative projects, and worked on a preclinical drug development program and drug repurposing screens for rare and neglected diseases in a team work and collaborative environment that led to advancement of over a dozen projects to clinical trials.

Carlos J. Alméciga-Díaz: Bachelor on Pharmacy from Universidad Nacional de Colombia, and Doctor of Biological Sciences from Pontificia Universidad Javeriana in Bogotá Colombia. Associate Professor and Director of the Institute for the Study of Inborn Errors of Metabolism at the same university. He works in three research lines 1) Cellular and molecular bases of inborn Errors of Metabolism, 2) Diagnosis of Inborn Errors of Metabolism, and 3) Biotechnological Tools in Inborn Errors of Metabolism. He carried out the first in vivo tests of gene therapy for Morquio A using adeno-associated viral vectors and lentiviral vectors, and currently is exploring the use of CRISPR/Cas9 as gene therapy tool for several inborn errors of metabolism. He has worked in the production and characterization of five recombinant lysosomal enzymes in microorganisms (bacteria and yeast), with great potential for the development of enzyme replacement therapies. He has worked in the glycoengineering of bacteria and yeasts to allow the production of enzymes with tailored N-glycosylations, and in the application of bioinformatics tools for the study of inborn errors of the metabolism, such as protein modeling, mutation effect, docking, and systems biology.

Cindy Dulcey-Sepúlveda: Bachelor in Mathematics from Pontificia Universidad Javeriana. She has worked on mathematics department on image processing with Python and R software using set theory methods to find overlapping in images under the supervision of Dr. German Combariza.

Germán Combariza: Bachelor in Mathematics and Master in Pure Mathematics from the Universidad de los Andes in Bogotá-Colombia. Doctor in Pure mathematics and postdoc in Computer Science from the University of Western Ontario, Canada. During his PhD he worked in Cohomology of Groups and currently works in Computer Algebra and Machine Learning. Research lines: Algebra and Computer Science. He has worked in Pontificia Universidad Javeriana as Assistant professor and Coordinator of Mathematics Program and now is Associate Professor and Coordinator of the new program Data Science, in the Faculty of Sciences.

Luis A. Barrera. Bachelor in Biology and Chemistry from Universidad Pedagógica y Tecnológica de Colombia, Tunja (Boyacá, Colombia). Master of Science in Clinical Chemistry from the State University of New York in Buffalo. Doctor in Biochemistry from University of Miami. Founder and ex-director of Center for Research in Biochemistry from Universidad de los Andes and Institute for the Study in Inborn Errors of Metabolism. Emeritus Professor from Pontificia Universidad Javeriana, Bogotá Colombia. He has had broad expertise and interest in Inborn Errors of Metabolism, Biochemistry, Clinic, Enzymology, Metabolism and Bioenergetics. He proposed the issuance of a law in favor of patients with Rare Diseases and their caregivers. The initiative was collected and promoted by some patient associations and was enacted as Law 1392 of 2010, most of which is still to be implemented.

Footnotes

7.

CONFLICTS OF INTERESTS

The authors declare that they have no competing interests.

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