Biosynthesis and processing of lysosomal cathepsin L in primary cultures of rat hepatocytes

Yukio Nishimura, Koji Furuno, Keitaro Kato

研究成果: ジャーナルへの寄稿記事

55 引用 (Scopus)

抄録

The biosynthesis and proteolytic processing of lysosomal cathepsin L was studied using in vitro translation system and in vivo pulse-chase analysis with [35S]methionine and [32P]phosphate in primary cultures of rat hepatocytes. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of a primary translation product of an immunoprecipitable 37.5-kDa cathepsin L in vitro. The 37.5-kDa form was converted to the 39-kDa form when translated in the presence of dog pancreas microsomes. During pulse-chase experiments with [35S]methionine in cultured rat hepatocytes, cathepsin L was first synthesized as a 39-kDa protein, presumably the proform, after a short time of labeling, and was subsequently processed into the mature forms of 30 and 25 kDa in the cell. On the other hand, considerable amounts of the proenzyme were found to be secreted into the culture medium without further proteolytic processing during the chase. The precursor and mature enzymes were N-glycosylated with high-mannose-type oligosaccharides, and the proenzyme molecule contained phosphorylated oligosaccharides. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, a 36-kDa unglycosylated polypeptide appeared in the cell and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results suggest that cathepsin L is initially synthesized on membrane-bound polysomes as a 37.5-kDa prepropeptide and that the cotranslational cleavage of the 1.5-kDa signal peptide and the core glycosylation convert the precursor to the 39-kDa proform, which is subsequently processed to the mature form during biosynthesis. Thus, the biosynthesis and secretion of lysosomal cathepsin L in rat hepatocytes seem to be analogous to those of the major excreted protein of transformed mouse fibroblasts [S. Gal, M. C. Willingham, and M. M. Gottesman (1985), J. Cell Biol., 100, 535-544] and the mouse cysteine proteinase of activated macrophages [D. A. Portnoy, A. H. Erickson, J. Kochan, J. V. Ravetch, and J. C. Unkeless (1986), J. Biol. Chem., 261, 14697-14703].

元の言語英語
ページ(範囲)107-116
ページ数10
ジャーナルArchives of Biochemistry and Biophysics
263
発行部数1
DOI
出版物ステータス出版済み - 5 15 1988

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Cathepsin L
Biosynthesis
Enzyme Precursors
Rats
Hepatocytes
Tunicamycin
Polyribosomes
Processing
Oligosaccharides
Methionine
Membranes
Cysteine Proteases
Chloroquine
Glycosylation
Mannose
Protein Sorting Signals
Microsomes
Proteins
Macrophages
Culture Media

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

これを引用

Biosynthesis and processing of lysosomal cathepsin L in primary cultures of rat hepatocytes. / Nishimura, Yukio; Furuno, Koji; Kato, Keitaro.

:: Archives of Biochemistry and Biophysics, 巻 263, 番号 1, 15.05.1988, p. 107-116.

研究成果: ジャーナルへの寄稿記事

Nishimura, Yukio ; Furuno, Koji ; Kato, Keitaro. / Biosynthesis and processing of lysosomal cathepsin L in primary cultures of rat hepatocytes. :: Archives of Biochemistry and Biophysics. 1988 ; 巻 263, 番号 1. pp. 107-116.
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abstract = "The biosynthesis and proteolytic processing of lysosomal cathepsin L was studied using in vitro translation system and in vivo pulse-chase analysis with [35S]methionine and [32P]phosphate in primary cultures of rat hepatocytes. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of a primary translation product of an immunoprecipitable 37.5-kDa cathepsin L in vitro. The 37.5-kDa form was converted to the 39-kDa form when translated in the presence of dog pancreas microsomes. During pulse-chase experiments with [35S]methionine in cultured rat hepatocytes, cathepsin L was first synthesized as a 39-kDa protein, presumably the proform, after a short time of labeling, and was subsequently processed into the mature forms of 30 and 25 kDa in the cell. On the other hand, considerable amounts of the proenzyme were found to be secreted into the culture medium without further proteolytic processing during the chase. The precursor and mature enzymes were N-glycosylated with high-mannose-type oligosaccharides, and the proenzyme molecule contained phosphorylated oligosaccharides. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, a 36-kDa unglycosylated polypeptide appeared in the cell and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results suggest that cathepsin L is initially synthesized on membrane-bound polysomes as a 37.5-kDa prepropeptide and that the cotranslational cleavage of the 1.5-kDa signal peptide and the core glycosylation convert the precursor to the 39-kDa proform, which is subsequently processed to the mature form during biosynthesis. Thus, the biosynthesis and secretion of lysosomal cathepsin L in rat hepatocytes seem to be analogous to those of the major excreted protein of transformed mouse fibroblasts [S. Gal, M. C. Willingham, and M. M. Gottesman (1985), J. Cell Biol., 100, 535-544] and the mouse cysteine proteinase of activated macrophages [D. A. Portnoy, A. H. Erickson, J. Kochan, J. V. Ravetch, and J. C. Unkeless (1986), J. Biol. Chem., 261, 14697-14703].",
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