Purification of human recombinant Naglu from Sf9 cells and uptake studies with MPS IIIB fibroblasts




Ashmead, Rhea

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Mucopolysaccharidosis IIIB (MPS IIIB) is a rare, metabolic disorder that results from a deficiency in the lysosomal hydrolase, α-N-acetylglucosaminidase (Naglu). Naglu is a housekeeping enzyme involved in the degradation pathway of heparan sulfate. A deficiency in active Naglu leads to an accumulation of heparan sulfate within the lysosome, initiating a pathological cascade within the cell. Patients with MPS IIIB experience progressive central nervous system degeneration and die within the first few decades of life. Presently, enzyme replacement therapy, which is a standard of care for other lysosomal storage disorders, is an ineffective treatment for MPS IIIB. This is due to impermeability of the blood-brain barrier (BBB) to exogenous recombinant enzymes. A promising approach to this therapeutic obstacle is protein transduction domains. Protein transduction domains have been shown to facilitate the delivery of active enzyme across the BBB in mice. Previously, our laboratory used Spodoptera frugiperda (Sf9) insect cell system to express human recombinant Naglu fused to a synthetic protein transduction domain (PTD4). The purpose was to use PTD4 to the facilitate the delivery of Naglu across biological membranes, including the blood-brain barrier. However, a missing stop codon following PTD4 limited its transducibility. The stop codon was re-introduced and the improved fusion enzyme, Naglu-PTD4X, was stably expressed in Sf9 cells. The overarching goal of this project is to create a large-scale production of human recombinant Naglu that has the potential to be used to treat the neuropathology of patients with MPS IIIB. This project used a three-step purification system to purify Naglu-PTD4X. Uptake of Naglu-PTD4X was assessed in MPS IIIB fibroblasts using a fluorogenic activity assay, immunoblotting, and immunocytochemistry. Our purification system was successful at purifying Naglu-PTD4X to homogeneity with a 26% yield and specific activity of 84,000 units/mg. An increase in Naglu activity was detected in MPS IIIB fibroblasts following incubation with Naglu-PTD4X. Future directions will focus on optimizing immunodetection and conducting BBB penetration studies in murine models.



Mucopolysaccharidosis, Mucopolysaccharidosis IIIB, MPS IIIB, α-N-acetylglucosaminidase, Spodoptera frugiperda, Naglu, blood-brain barrier, Protein transduction domains, enzyme replacement therapy, lysosomal disorders, lysosomal storage disorders, protein purification, cell-penetrating peptides