| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
The specific accumulation of the hydrophobic protein, subunit c of ATP synthase in lysosomes from the cells with the late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by lysosomal proteolytic dysfunction. The defective gene in LINCL has recently been identified. The protein was thought to be endopeptidase. To elucidate the mechanism of lysosomal storage of subunit c, antibodies against the human CLN2 gene product (Cln2p) were prepared. Immunoblot analysis indicated that Cln2p is a 46kDa protein in normal control skin fibroblasts and carrier heterozygote cells, whereas it was absent in cells from patients with LINCL. RT-PCR analysis indicated the presence of mRNA for CLN2 in cells from the patients tested, suggesting a low efficiency of translation of mRNA or the production of the unstable translation products in these patient cells. Subcellular fractionation analysis and confocal immunomicroscopic analysis indicated that Cln2p is localized in the lysosomal fractio
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ns. The immunodepletion of CIn2p from normal fibroblasts extract caused a loss of degradative capacity of subunit c, but did not β subunit of ATP synthase, suggesting that the absence of CLN2p provokes a lysosomal accumulation of a hydrophobic protein, subunit c.
Recently, Vines and Warburton reported that tripeptidyl peptidase I (TPP-I) is identical to CIn2p, as evidenced by a comparison of TPP-I amino acid. LINCL fibroblasts are devoid of TPP-I activities. We investigated the relationship between endopeptidase activities and tripeptidyl peptidase activities of TPP-I. The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CHィイD22ィエD2Cl (specific inhibitor of TPP-I), but were not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin insensitive endopeptidase activity. Cln2p /TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity. Less
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