ABSTRACT
GM2 gangliosidoses, including Tay-Sachs and Sandhoff diseases, are neurodegenerative lysosomal storage diseases that are caused by deficiency of ß-hexosaminidase A, which comprises an αß heterodimer. There are no effective treatments for these diseases; however, various strategies aimed at restoring ß-hexosaminidase A have been explored. Here, we produced a modified human hexosaminidase subunit ß (HexB), which we have termed mod2B, composed of homodimeric ß subunits that contain amino acid sequences from the α subunit that confer GM2 ganglioside-degrading activity and protease resistance. We also developed fluorescent probes that allow visualization of endocytosis of mod2B via mannose 6-phosphate receptors and delivery of mod2B to lysosomes in GM2 gangliosidosis models. In addition, we applied imaging mass spectrometry to monitor efficacy of this approach in Sandhoff disease model mice. Following i.c.v. administration, mod2B was widely distributed and reduced accumulation of GM2, asialo-GM2, and bis(monoacylglycero)phosphate in brain regions including the hypothalamus, hippocampus, and cerebellum. Moreover, mod2B administration markedly improved motor dysfunction and a prolonged lifespan in Sandhoff disease mice. Together, the results of our study indicate that mod2B has potential for intracerebrospinal fluid enzyme replacement therapy and should be further explored as a gene therapy for GM2 gangliosidoses.
Subject(s)
Amino Acid Substitution , Brain/metabolism , Gangliosidoses, GM2/drug therapy , beta-Hexosaminidase beta Chain , Animals , Brain/diagnostic imaging , Disease Models, Animal , Gangliosidoses, GM2/diagnostic imaging , Gangliosidoses, GM2/metabolism , Humans , Mice , Mice, Knockout , Mutation, Missense , Peptide Hydrolases/chemistry , Proteolysis , beta-Hexosaminidase beta Chain/genetics , beta-Hexosaminidase beta Chain/pharmacologyABSTRACT
GM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA, HEXB and GM2A genes, which encode the human lysosomal ß-hexosaminidase (Hex) α- and ß-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A from the conditioned medium, which was taken up by fibroblasts derived from a patient with GM2A deficiency, and had the therapeutic effects of reducing the GM2 accumulated in fibroblasts when added to the culture medium. We also demonstrated for the first time that recombinant GM2A could enhance the replacement effect of human modified HexB (modB) with GM2-degrading activity, which is composed of homodimeric altered ß-subunits containing a partial amino acid sequence of the α-subunit, including the GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA (αß heterodimer) deficiency, caused by HEXA mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB ß-subunit to that in the native HexA α-subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses.
