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. / Graduate / 2020-06-21

Human α-N-Acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in tthe rare metabolic storage disorder known as mucopolysaccharidosis type IIIB (MPS IIIB; also Sanfilippo syndrome B). Absence of this enzyme results in cytotoxic accumulation of heparan sulphate in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is not currently effective to treat neurological symptoms due to the inability of exogenous Naglu to access the brain. This laboratory uses a Spodoptera frugiperda (Sf9) insect cell system to express Naglu fused to a synthetic protein transduction domain with the intent to facilitate delivery of Naglu across the blood-brain barrier. The project described herein may be broken down into three main sections. Firstly, the impact of two cryptic splice sites on Naglu expression levels was analyzed in both transiently expressing Sf9 cultures and stably selected cell lines. Secondly, the effectiveness of the native Naglu secretion-signaling peptide in the Sf9 system was examined. Finally, purification of a Naglu fusion protein from suspension culture medium was performed using hydrophobic interaction chromatographic techniques. The ultimate goal of this research is to develop an efficient system for economical, large-scale production of a human recombinant Naglu fusion protein that has the potential to be successfully used for enzyme replacement therapy to treat MPS IIIB. / Graduate