title: Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.
abstract: Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.
authors.affiliation: Department of Medicinal Biotechnology, Institute for Medicinal Resources, Graduate School of Pharmaceutical Sciences, The University of Tokushima, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Itakura
authors.forename: Aya
authors.initials: A
authors.lastname: Kuroki
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Ishibashi
authors.forename: Daisuke
authors.initials: D
authors.lastname: Tsuji
authors.forename: Eri
authors.initials: E
authors.lastname: Kawashita
authors.forename: Yukari
authors.initials: Y
authors.lastname: Higashine
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sakuraba
authors.forename: Shoji
authors.initials: S
authors.lastname: Yamanaka
authors.forename: Kohji
authors.initials: K
authors.lastname: Itoh
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D005678:G(M2) Ganglioside / Q000378:metabolism*; D005822:Genetic Vectors; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D006801:Humans; D006868:Hydrolysis; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D012497:Sandhoff Disease / Q000201:enzymology / Q000378:metabolism*; D014162:Transfection; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D005678:G(M2) Ganglioside; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases