title: Introduction of an N-glycan sequon into HEXA enhances human beta-hexosaminidase cellular uptake in a model of Sandhoff disease.
abstract: Human lysosomal beta-hexosaminidase A is a heterodimer composed of alpha- and beta-subunits encoded by HEXA and HEXB, respectively. We genetically introduced an additional N-glycosylation sequon into HEXA, which caused amino acid substitutions (S51 to N and A53 to T) at homologous positions to N84 and T86 in the beta-subunit. The mutant HexA (NgHexA) obtained from a Chinese hamster ovary (CHO) cell line co-expressing the mutated HEXA and wild-type HEXB complementary DNAs was demonstrated to contain an additional mannose-6-phosphate (M6P)-type-N-glycan. NgHexA was more efficiently taken up than the wild-type HexA and delivered to lysosomes, where it degraded accumulated substrates including GM2 ganglioside (GM2) when administered to cultured fibroblasts derived from a Sandhoff disease (SD) patient. On intracerebroventricular (i.c.v.) administration of NgHexA to SD model mice, NgHexA more efficiently restored the HexA activity and reduced the GM2 and GA2 (asialoGM2) accumulated in neural cells of the brain parenchyma than the wild-type HexA. These findings indicate that i.c.v. administration of the modified human HexA with an additional M6P-type N-glycan is applicable for enzyme replacement therapy (ERT) involving an M6P-receptor as a molecular target for HexA deficiencies including Tay-Sachs disease and SD.
authors.affiliation: Department of Medicinal Biotechnology, Institute for Medicinal Research, Graduate School of Pharmaceutical Sciences, The University of Tokushima, Tokushima 770-8505, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Matsuoka
authors.forename: Daisuke
authors.initials: D
authors.lastname: Tsuji
authors.forename: Sei-Ichi
authors.initials: S
authors.lastname: Aikawa
authors.forename: Fumiko
authors.initials: F
authors.lastname: Matsuzawa
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sakuraba
authors.forename: Kohji
authors.initials: K
authors.lastname: Itoh
mesh_terms: D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D006224:Cricetinae; D003412:Cricetulus; D005678:G(M2) Ganglioside / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D012497:Sandhoff Disease / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D054820:beta-Hexosaminidase alpha Chain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D054821:beta-Hexosaminidase beta Chain / Q000235:genetics / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use*
chemical_list: D011134:Polysaccharides; D005678:G(M2) Ganglioside; C518253:HEXA protein, human; C513386:HEXB protein, human; D054820:beta-Hexosaminidase alpha Chain; D054821:beta-Hexosaminidase beta Chain; D001619:beta-N-Acetylhexosaminidases