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- Title
Lysosomal Cysteine Protease, Cathepsin B, Is Targeted to Lysosomes by the Mannose 6-Phosphate-Independent Pathway in Rat Hepatocytes: Site-Specific Phosphorylation in Oligosaccharides of the Proregion1.
- Authors
Tanaka, Yoshitaka; Tanaka, Rie; Kawabata, Takahiro; Noguchi, Youichiro; Himeno, Masaru
- Abstract
Cathepsin B, a lysosomal cysteine protease, is synthesized as a glycoprotein with two N-linked oligosaccharide chains, one of which is in the propeptide region while the other is in the mature region. When cultured rat hepatocytes were labeled with [32P] phosphate,32P -labeled cathepsin B was immunoprecipitated only in the proform from cell lysates and medium. Either Endo H or alkaline phosphatase treatment of 32P-labeled procathepsin B demonstrated the acquisition of a mannose 6-phosphate (Man 6-P) residue on high mannose type oligosaccharides. To identify the site of phosphorylation, immunoisolated 32S-or 32P-labeled procathepsin B was incubated with purified lysosomal cathepsin D, since cathepsin D cleaves 48 amino acid residues from the N-terminus of procathepsin B, in which one N-linked oligosaccharide chain was also included [Kawabata, T. et al (1993) J. Biochem 113, 389–394]. Treatment of intracellular 35S-label-ed procathepsin B with a molecular mass of 39-kDa with cathepsin D resulted in the production of the 31-kDa intermediate form, but the 32P-label incorporated into procathepsin B disappeared after treatment with cathepsin D. These results indicate that the phosphorylation of procathepsin B is restricted to an oligosaccharide chain present in the propeptide region. Interestingly, cathepsin B sorting to lysosomes was not inhibited by NH4C1 treatment and about 90%; of the intracellular procathepsin B initially phosphorylated was secreted into the medium without being dephosphorylated intrac-ellularly, and did not bind significantly to cation-independent-Man 6-P receptor, suggesting the failure of Man 6-P-dependent transport of procathepsin B to lysosomes. Additionally, about 50% of the newly synthesized 35S-labeled cathepsin B was retained in the cells in nature forms consisting of a 29-kDa single chain form and a 24-kDa two chain form, while part of the procathepsin B was associated with membranes in a Man 6-P-independent manner. Taken together, these results show that in rat hepatocytes, cathepsin B is targeted to lysosomes by an alternative mechanism(s) other than the Man 6-P-dependent pathway.
- Subjects
CATHEPSIN B; CYSTEINE proteinases; GLYCOPROTEINS; OLIGOSACCHARIDES; LYSOSOMES
- Publication
Journal of Biochemistry, 2000, Vol 128, Issue 1, p39
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/oxfordjournals.jbchem.a022728