title: Elimination of abnormal sialylglycoproteins in fibroblasts with sialidosis and galactosialidosis by normal gene transfer and enzyme replacement.
abstract: Sialidosis and galactosialidosis are lysosomal storage diseases caused by the genetic defects of lysosomal sialidase (neuraminidase-1; NEU1) and lysosomal protective protein/cathepsin A (PPCA), respectively, associated with a NEU1 deficiency, excessive accumulation of sialylglycoconjugates, and development of progressive neurosomatic manifestations; in addition, the latter disorder is accompanied by simultaneous deficiencies of beta-galactosidase and cathepsin A. We demonstrated that a few soluble N-glycosylated proteins carrying sialyloligosaccharides sensitive to glycopeptidase F (GPF) can be specifically detected in cultured fibroblasts from sialidosis and galactosialidosis cases by blotting with a Maackia amurensis (MAM) lectin. We also examined the therapeutic effects of normal gene transfer and enzyme replacement by evaluating the decreases in sialylglycoconjugates accumulated in fibroblasts with these NEU1 deficiencies. The specific N-glycosylated proteins detected on MAM lectin blotting as well as the granular lysosomal fluorescence due to an avidin-FITC/biotinylated MAM lectin conjugate in sialidosis and galactosialidosis fibroblasts disappeared in parallel with the restoration of the intracellular NEU1 activity after transfection of the recombinant NEU1 fused to HA tag sequence and the wild-type PPCA cDNA as well as administration of the recombinant PPCA precursor protein. The detection method for the abnormal sialylglycoproteins in cultured cells involving MAM lectin was demonstrated to be useful not only for biochemical and diagnostic analyses of NEU1 deficiencies but also for therapeutic evaluation of these conditions.
authors.affiliation: Department of Medicinal Biotechnology, Graduate School of Pharmaceutical Sciences, The University of Tokushima, Tokushima, Japan.
authors.forename: Yukako
authors.initials: Y
authors.lastname: Oheda
authors.forename: Masaharu
authors.initials: M
authors.lastname: Kotani
authors.forename: Mai
authors.initials: M
authors.lastname: Murata
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sakuraba
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Kadota
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Tatano
authors.forename: Jun
authors.initials: J
authors.lastname: Kuwahara
authors.forename: Kohji
authors.initials: K
authors.lastname: Itoh
mesh_terms: D043402:Cathepsin A / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D005069:Evaluation Studies as Topic; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D015316:Genetic Therapy; D006801:Humans; D009081:Mucolipidoses / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism; D009844:Oligosaccharides / Q000235:genetics / Q000378:metabolism; D046188:Protein Modification, Translational / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins; C016735:sialooligosaccharides; C486385:NEU1 protein, human; D009439:Neuraminidase; D043402:Cathepsin A