Stable expression of protective protein cathepsin A-green fluorescent protein fusion genes in a fibroblastic cell line from a galactosialidosis patient - Model system for revealing the intracellular transport of normal and mutated lysosomal enzymes

Fibroblastic cell lines derived from a galactosialidosis patient, stably ex pressing the chimaeric green fluorescent protein variant (EGFP) gene fused to the wild-type and mutant human lysosomal protective protein/cathepsin A (PPCA) cDNA, were first established as a model system for revealing the sor ting and processing of lysosomal enzymes and for investigating the molecula r bases of their deficiencies. In the cell line expressing the wild-type PP CA-EGFP chimaera gene (EGFP-PPwild), an 81 kDa form (27 kDa EGFP fused to t he C-terminus of the 54 kDa PPCA precursor) was produced, then processed in to the mature 32/20 kDa two-chain form free of the EGFP domain. The intrace llular cathepsin A, alpha-N-acetylneuraminidase and beta-galactosidase acti vities, which are deficient in the parent fibroblastic cells, could also be significantly restored in the cells. In contrast with the uniform and stro ng fluorescence throughout the cytoplasm and nucleus in the mock-cell line expressing only EGFP cDNA, weak reticular and punctate fluorescence was dis tributed throughout the EGFP-PPwild cell line. Bafilomycin A(1), a potent i nhibitor of vacuolar ATPase and intracellular acidification, induced the di stribution of Golgi-like perinuclear fluorescence throughout the living and fixed cells, in which only the 81 kDa product was detected. After removal of the agent, time-dependent transport of the chimaeric protein from the Go lgi apparatus to the prelysosomal structure in living cells was monitored w ith a confocal laser scanning microscope system. Leupeptin caused the distr ibution of lysosome-like granular fluorescence throughout the cytoplasm in the fixed cells, although it was hardly observed in living cells. The latte r agent also dose dependently induced an increase in the intracellular amou nt of the 81 kDa product containing the EGFP domain and inhibited the resto ration of cathepsin A activity in the EGFP-PPwild cells after the removal o f bafilomycin A(1). In parallel, both the mature two-chain form and PPCA fu nction disappeared. These results suggested that the chimaera gene product was transported to acidic compartments (endosomes/lysosomes), where proteol ytic processing of the PPCA precursor/zymogen, quenching of the fluorescenc e, and random degradation of the EGFP portion occurred. A cell line stably expressing a chimaeric gene with a mutant PPCA cDNA containing an A(1184) - -> G (Y395C) mutation, commonly detected in Japanese severe early-infantile type of galactosialidosis patients, showed an endoplasmic reticulum (ER)-l ike reticular fluorescence pattern. The PPCA-immuno-reactive gene product w as hardly detected in this cell line. The mutant chimaeric product was sugg ested to be degraded rapidly in the ER before transport to post-ER compartm ents. A cell line expressing the chimaeric gene with a T-746 --> A (Y249N) PPCA mutation exhibited both ER-like reticular and granular fluorescence on the reticular structure that was stronger than that in the EGFP-PPwild cel ls. Some of them contained large fluorescent inclusion-body-like structures . The ineffectiveness of transport inhibitors in the distribution changes i n the two mutant chimaeric proteins suggested that they were not delivered to acidic compartments. Therefore this expression system can possibly be ap plied to the direct analysis of the sorting defects of mutant gene products in living cells and will be useful for the molecular investigation of lyso somal diseases, including galactosialidosis.