Glycobiology Advance Access originally published online on December 15, 2005
Glycobiology 2006 16(4):271-280; doi:10.1093/glycob/cwj069
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Elimination of abnormal sialylglycoproteins in fibroblasts with sialidosis and galactosialidosis by normal gene transfer and enzyme replacement
2 Department of Medicinal Biotechnology, Graduate School of Pharmaceutical Sciences, Institute for Medicinal Resources, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505, Japan; 3 Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, 3-18-22 Honkomagome, Bunkyo, Tokyo 113-8613, Japan; and 4 CREST, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
1 To whom correspondence should be addressed; e-mail: kitoh{at}ph.tokushima-u.ac.jp
Received on April 27, 2005; revised on November 22, 2005; accepted on December 9, 2005
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 ß-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.
Key words: galactosialidosis / Maackia amurensis lectin / molecular therapy / sialidosis / sialylglycoprotein