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

Abstract

Fibroblastic cell lines derived from a galactosialidosis patient, stably expressing 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 sorting and processing of lysosomal enzymes and for investigating the molecular bases of their deficiencies. In the cell line expressing the wild-type PPCA-EGFP chimaera gene (EGFP-PPwild), an 81 kDa form (27 kDa EGFP fused to the C-terminus of the 54 kDa PPCA precursor) was produced, then processed into the mature 32/20 kDa two-chain form free of the EGFP domain. The intracellular cathepsin A, α-N-acetylneuraminidase and β-galactosidase activities, which are deficient in the parent fibroblastic cells, could also be significantly restored in the cells. In contrast with the uniform and strong fluorescence throughout the cytoplasm and nucleus in the mock-cell line expressing only EGFP cDNA, weak reticular and punctate fluorescence was distributed throughout the EGFP-PPwild cell line. Bafilomycin A1, a potent inhibitor of vacuolar ATPase and intracellular acidification, induced the distribution 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 Golgi apparatus to the prelysosomal structure in living cells was monitored with a confocal laser scanning microscope system. Leupeptin caused the distribution of lysosome-like granular fluorescence throughout the cytoplasm in the fixed cells, although it was hardly observed in living cells. The latter agent also dose-dependently induced an increase in the intracellular amount of the 81 kDa product containing the EGFP domain and inhibited the restoration of cathepsin A activity in the EGFP-PPwild cells after the removal of bafilomycin A1. In parallel, both the mature two-chain form and PPCA function disappeared. These results suggested that the chimaera gene product was transported to acidic compartments (endosomes/lysosomes), where proteolytic processing of the PPCA precursor/zymogen, quenching of the fluorescence, and random degradation of the EGFP portion occurred. A cell line stably expressing a chimaeric gene with a mutant PPCA cDNA containing an A1184 → G (Y395C) mutation, commonly detected in Japanese severe early-infantile type of galactosialidosis patients, showed an endoplasmic reticulum (ER)-like reticular fluorescence pattern. The PPCA-immunoreactive gene product was hardly detected in this cell line. The mutant chimaeric product was suggested to be degraded rapidly in the ER before transport to post-ER compartments. A cell line expressing the chimaeric gene with a T746 → 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 cells. Some of them contained large fluorescent inclusion-body-like structures. The ineffectiveness of transport inhibitors in the distribution changes in the two mutant chimaeric proteins suggested that they were not delivered to acidic compartments. Therefore this expression system can possibly be applied to the direct analysis of the sorting defects of mutant gene products in living cells and will be useful for the molecular investigation of lysosomal diseases, including galactosialidosis.

A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C. elegans

“…However, none of the genes encoded the 91 amino acid pro-domain of CPY or the 2-kDa internal excision fragment of cathepsin A that are required for proper folding and activation, suggesting that the C. elegans proteins may be processed differently [17] . We cloned one of these wild-type genes (F13D12.6) and fused it to the N-terminus of YFP, as has been described for CPY-like or cathepsin A transgenes [18] , [19] . However, transgenic animals harboring the wild-type transgene, as well as those with a mutation corresponding to that in CPY* (G166R in F13D12.6), yielded a diffuse reticular pattern consistent with localization to the ER, but not the expected localization to lysosomal structures or dilated ER, respectively ( Figure S1B –I).…”

Peptides Presented by HLA-DR Molecules in Synovia of Patients with Rheumatoid Arthritis or Antibiotic-Refractory Lyme Arthritis

“…It appears that the endosomal cathepsins, which are important for antigen processing in APCs (44,45), may also be degraded and converted to antigens that are presented on the cell surface. Thus, the proteins that enter the HLA-DR pathway of APCs in inflamed synovia reflect the wide range of proteins found in the intracellular and extracellular milieu of the joint.…”

A Noninvasive Cell-Based Assay for Monitoring Proteolytic Activity within a Specific Subcellular Compartment

“…The relative instability of GFP at low pH suggests that it might be possible to develop a sensitive cell-based assay for a specific lysosomal protease, such as cathepsin L, by colocalizing GFP and cathepsin L within acidic lysosomes. GFP has been successfully expressed in lysosomes (15,16). In the present study, GFP has been delivered to lysosomes by fusing GFP to the C-terminus of preprocathepsin L. Overexpression of the cathepsin L-GFP fusion protein ensures colocalization of the enzyme and the substrate and can also minimize possible interference from other endogenous proteases present in the lysosome.…”