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

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 gangliosidoses, 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; Pichia pastoris; enzyme replacement therapy; recombinant hexosaminidases.

Figure 1.. Cellular uptake of recombinant enzymes.

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).

Figure 2.. Endocytic pathway evaluation – Temperature evaluation.

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).

Figure 3.. Endocytic pathway evaluation - Receptor inhibition.

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.

Figure 4.. Intracellular delivery of recombinant hexosaminidases to lysosome.

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

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

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.