Expression of lysosomal protective protein/cathepsin A in a stably transformed human neuroblastoma cell line during bi-directional differentiation into neuronal and Schwannian cells.

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.
AuthorsKohji Itoh, Yurie Satoh, Yoshito Kadota, Yukako Oheda, Jun Kuwahara, Michie Shimmoto, Hitoshi Sakuraba
JournalNeurochemistry international (Neurochem Int) Vol. 44 Issue 6 Pg. 447-57 (May 2004) ISSN: 0197-0186 [Print] England
PMID14687610 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Recombinant Proteins
  • Cathepsin A
  • Cathepsin A (metabolism)
  • Cell Differentiation
  • Cell Transformation, Neoplastic
  • Electrophoresis, Polyacrylamide Gel
  • Fluorescent Antibody Technique, Indirect
  • Humans
  • Lysosomes (enzymology)
  • Neuroblastoma (metabolism, pathology)
  • Neurons (cytology)
  • Recombinant Proteins (metabolism)
  • Schwann Cells (cytology)

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