Deficiency of Endothelin-Degrading Enzyme and Abnormality in Central Nervous System

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Protective protein/cathepsin A (PPCA) is a multifuctional glycoprotein that exerts the protective effects on lysososmal glycosidases and serine carboxypeptidase activity on a subset of neuropeptides. Galactosialidosis (GS) is a human PPCA deficiency with autosomal recessive genetic trait, accompanied by the simultaneous decrease of these enzyme activities and by the accumulation of their endogenous substrates. This disease shows very multiple clinial manifestations, including neurological abnormalities, such as cerebellar ataxia and myoclonus. From the research to elucidate the correlation between the catalytic functions of PPCA and the neurological abnormalities in the deficiency, new findings were obtained as follows ;1. Histochemical analysis of the autopsied neurvous tissues derived from three adult/juvenile type GS patients was performed with anti-human PPCA, anti-endothelin-1(ET-1), a putative endogenous substrate of the catalytic activtity of PPCA, and anti-big endothelin-1(bigE … More T-1), the metabolic precursor protein of ET-1, The immunoreactivity against PPCA was hardly detected in the GS tissues. However, neurons and glial cells in the central nervous system, including cerebellum, hippocampal formation, spinal cord, etc, were found to show the increase in immunoreactivity against both ET-1 and bigET-1. These cells were also observed to contain relative high amount of PPCA in the healthy controls.2. Antisense S-oligonucleotides, complementary to the 5' region of human and murine PPCA mRNA containing the translation initiation site, were selected to inhibit specifically the expression of PPCA functions in cultured cells by adding to the culture medium. The selected antisense S-oligonucleotides for murine PPCA gene was demonstrated to induce the increase of ET-1-like immunoreactivtiy in the type 1 astrocytes in cerebellar primary culture system.3. For the purpose of the visualization of PPCA gene expression in living cells, fibroblastic cell lines were established, expressing the chimeric green fluorescent protein (GFP) gene fused to the PPCA eDNA.Expression of the wild-type and mutant PPCA fusion genes showed the different intracellular sorting according to their kind of mutations.This model system was applicable to visualize the intracellulartransport of lysosomal enzymes and to investigate the molecular bases of their deficiencies. Less