|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1997 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1996 : ¥1,400,000 (Direct Cost : ¥1,400,000)
We examined galactosylceramidase (GALC) cDNA and gene in five Japanese patients with adult onset globoid cell 1eukodystrophy (Krabbe disease) (AO-GLD). We identified three missense mutations (I66M,G270D,L618S) and one exon 6 skipping (535-573de1). We constructed mutated GALC cDNAs and expressed them in COS-1 cells transiently and in CHO cell stably. In these experiences, it is shown that (1) AO-GLD mutations, including those found here, are located in the N (I66M,G270D,535-573de1) or C (L618S) terminus of the GALC enzyme. Whereas the reported mutations in the infantile form (IF-GLD) are in the central domain. This difference in mutation sites may affect the phenotype of the clinical features of GLD,(2) Although GALC (80kDa) is consistent with 50 and 30 kDa subunits, the former exits in extracellular, the latters in intrace11ular, both of which have enzymatic activity. However, mixture of these subunits after independent expression show no GALC activity, (3) According to the inhibition assay, GALC is considered to uptake into the lysosomal vesicle following with processing into two subunits in it and (4) In some mutation found in AO-GLD show inhibition of GALC processing. We also preparing to construct expression vectors for gene therapy using retroviral and adeno associated viral vectors.