Development of a novel method for β-hexosaminidase A purification and investigation of the transport of an engineered enzyme across the blood-brain barrier
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Date
2021
Authors
Bouma, Kristen
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Abstract
Tay-Sachs disease (TSD) and Sandhoff disease (SD) are severe autosomal recessive genetic disorders caused by loss-of-function mutations in the HEXA and HEXB genes encoding the heterodimeric enzyme β-hexosaminidase A (HexA). Deficiencies in HexA lead to the pathological accumulation of GM2 ganglioside (GM2) in the central nervous system (CNS). An engineered enzyme called HexM was previously developed based on the key features of the α- and the β-subunits of HexA as a potential therapeutic for these genetic disorders. Currently all treatments for TSD and SD are palliative in nature as there are no cures available. One of the biggest challenges in developing a therapeutic is the limited permeability of the blood-brain barrier (BBB), which impedes the delivery of molecules, including HexM, to the CNS. To this end, HexM variants were generated by genetically fusing the enzyme to peptide tags derived from apolipoprotein B (3371-3409) and E (159-167)2, known to facilitate the transport of lysosomal proteins across the BBB. The resulting HexM-ApoB and HexM-ApoE proteins were purified from a human embryonic kidney 293T cell line deficient in HEXA and HEXB (HEK293T-ABKO) after transfection with overexpression cassettes. The fusion proteins were kinetically active and stable in several buffer conditions, however, did not cross an in vitro BBB model consisting of human brain endothelial cells. To further therapeutic research on TSD and SD, a novel method of recombinantly producing HexA was developed. The HEK293T-ABKO cells were transfected with plasmids encoding HEXA and HEXB with a C-terminal FLAG-tag and a His6 tag, respectively. The enzyme could be isolated in a two-step purification procedure and was found to be kinetically active. This method provides a convenient means of obtaining HexA, rather than the traditional process of isolating the enzyme from human placentas, and can further be developed to generate HexA mutants in sufficient quantities for structural studies.
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Keywords
Tay-Sachs disease, Sandhoff disease, HexM, blood-brain barrier