Structural requirements of procathepsin D activation and maturation

G. R. Richo, Gregory E Conner

Research output: Contribution to journalArticle

76 Citations (Scopus)

Abstract

Cathepsin D biosynthesis involves several proteolytic events; however, the enzymology and sequence of these events are not known. Procathepsin D undergoes a pH-dependent, intramolecular proteolysis in vitro which removes 26 residues yielding an active form that is intermediate in size between procathepsin D and single-chain cathepsin D. This form, designated pseudocathepsin D, has not been shown to be an in vivo intermediate. The N- terminal sequence of the light chain of cathepsin D, isolated from human placenta, showed that 42 residues were removed as compared with 44 residues predicted by comparison with porcine cathepsin D. Site-directed mutations were generated at both processing sites within the propeptide of procathepsin D. Mutation at the autocatalytic site prevented in vitro autoactivation, but, after transfection of mouse Ltk- cells, the mutant procathepsin D was transported to the lysosome and processed normally to the mature enzyme despite its inability to autoactivate in vitro. Mutation at the mature N terminus of cathepsin D prevented in vivo formation of the single-chain form of the enzyme; however, the protein was still processed to the two-chain form of human cathepsin D. This change at the mature N terminus did not prevent in vitro autoactivation. Procathepsin D with mutations at both cleavage sites was processed to the two-chain form despite the inability to undergo removal of the propeptide. These results indicated that stepwise autoactivation and propeptide removal were not necessary for later processing or delivery of human cathepsin D to the lysosome. The results also suggested that pseudocathepsin D was not a normal intermediate of procathepsin D processing in vivo.

Original languageEnglish
Pages (from-to)14806-14812
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number20
StatePublished - Jan 1 1994

Fingerprint

Cathepsin D
Chemical activation
Mutation
Lysosomes
Processing
Proteolysis
Biosynthesis
Enzymes
procathepsin D
Placenta
Transfection
Swine
Light
In Vitro Techniques

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural requirements of procathepsin D activation and maturation. / Richo, G. R.; Conner, Gregory E.

In: Journal of Biological Chemistry, Vol. 269, No. 20, 01.01.1994, p. 14806-14812.

Research output: Contribution to journalArticle

@article{b6846228196942a3b1b8004816086cdb,
title = "Structural requirements of procathepsin D activation and maturation",
abstract = "Cathepsin D biosynthesis involves several proteolytic events; however, the enzymology and sequence of these events are not known. Procathepsin D undergoes a pH-dependent, intramolecular proteolysis in vitro which removes 26 residues yielding an active form that is intermediate in size between procathepsin D and single-chain cathepsin D. This form, designated pseudocathepsin D, has not been shown to be an in vivo intermediate. The N- terminal sequence of the light chain of cathepsin D, isolated from human placenta, showed that 42 residues were removed as compared with 44 residues predicted by comparison with porcine cathepsin D. Site-directed mutations were generated at both processing sites within the propeptide of procathepsin D. Mutation at the autocatalytic site prevented in vitro autoactivation, but, after transfection of mouse Ltk- cells, the mutant procathepsin D was transported to the lysosome and processed normally to the mature enzyme despite its inability to autoactivate in vitro. Mutation at the mature N terminus of cathepsin D prevented in vivo formation of the single-chain form of the enzyme; however, the protein was still processed to the two-chain form of human cathepsin D. This change at the mature N terminus did not prevent in vitro autoactivation. Procathepsin D with mutations at both cleavage sites was processed to the two-chain form despite the inability to undergo removal of the propeptide. These results indicated that stepwise autoactivation and propeptide removal were not necessary for later processing or delivery of human cathepsin D to the lysosome. The results also suggested that pseudocathepsin D was not a normal intermediate of procathepsin D processing in vivo.",
author = "Richo, {G. R.} and Conner, {Gregory E}",
year = "1994",
month = "1",
day = "1",
language = "English",
volume = "269",
pages = "14806--14812",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "20",

}

TY - JOUR

T1 - Structural requirements of procathepsin D activation and maturation

AU - Richo, G. R.

AU - Conner, Gregory E

PY - 1994/1/1

Y1 - 1994/1/1

N2 - Cathepsin D biosynthesis involves several proteolytic events; however, the enzymology and sequence of these events are not known. Procathepsin D undergoes a pH-dependent, intramolecular proteolysis in vitro which removes 26 residues yielding an active form that is intermediate in size between procathepsin D and single-chain cathepsin D. This form, designated pseudocathepsin D, has not been shown to be an in vivo intermediate. The N- terminal sequence of the light chain of cathepsin D, isolated from human placenta, showed that 42 residues were removed as compared with 44 residues predicted by comparison with porcine cathepsin D. Site-directed mutations were generated at both processing sites within the propeptide of procathepsin D. Mutation at the autocatalytic site prevented in vitro autoactivation, but, after transfection of mouse Ltk- cells, the mutant procathepsin D was transported to the lysosome and processed normally to the mature enzyme despite its inability to autoactivate in vitro. Mutation at the mature N terminus of cathepsin D prevented in vivo formation of the single-chain form of the enzyme; however, the protein was still processed to the two-chain form of human cathepsin D. This change at the mature N terminus did not prevent in vitro autoactivation. Procathepsin D with mutations at both cleavage sites was processed to the two-chain form despite the inability to undergo removal of the propeptide. These results indicated that stepwise autoactivation and propeptide removal were not necessary for later processing or delivery of human cathepsin D to the lysosome. The results also suggested that pseudocathepsin D was not a normal intermediate of procathepsin D processing in vivo.

AB - Cathepsin D biosynthesis involves several proteolytic events; however, the enzymology and sequence of these events are not known. Procathepsin D undergoes a pH-dependent, intramolecular proteolysis in vitro which removes 26 residues yielding an active form that is intermediate in size between procathepsin D and single-chain cathepsin D. This form, designated pseudocathepsin D, has not been shown to be an in vivo intermediate. The N- terminal sequence of the light chain of cathepsin D, isolated from human placenta, showed that 42 residues were removed as compared with 44 residues predicted by comparison with porcine cathepsin D. Site-directed mutations were generated at both processing sites within the propeptide of procathepsin D. Mutation at the autocatalytic site prevented in vitro autoactivation, but, after transfection of mouse Ltk- cells, the mutant procathepsin D was transported to the lysosome and processed normally to the mature enzyme despite its inability to autoactivate in vitro. Mutation at the mature N terminus of cathepsin D prevented in vivo formation of the single-chain form of the enzyme; however, the protein was still processed to the two-chain form of human cathepsin D. This change at the mature N terminus did not prevent in vitro autoactivation. Procathepsin D with mutations at both cleavage sites was processed to the two-chain form despite the inability to undergo removal of the propeptide. These results indicated that stepwise autoactivation and propeptide removal were not necessary for later processing or delivery of human cathepsin D to the lysosome. The results also suggested that pseudocathepsin D was not a normal intermediate of procathepsin D processing in vivo.

UR - http://www.scopus.com/inward/record.url?scp=0028238397&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028238397&partnerID=8YFLogxK

M3 - Article

VL - 269

SP - 14806

EP - 14812

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 20

ER -