Role of glycosylation in the expression of human procathepsin D

S. C. Fortenberry, J. S. Schorey, John Chirgwin

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Human procathepsin D carries two N-linked glycosylation sites at asparagine residues 70 and 199, widely separated on the surface of the folded protein. We created monoglycosylated procathepsin D molecules by site-directed mutagenesis in vitro of the individual glycosylation sites. With only two exceptions, all 12 mutants of this type were expressed efficiently in mammalian cells. The expressed proteins were stable, targeted to the lysosome, and partially secreted into the medium. When both glycosylation sites were eliminated, however, the expressed proteins (9 different mutants) were stable but most were not secreted and targeted poorly to the lysosome. Mammalian fibroblasts appear to sort nascent procathepsin D efficiently only if it is N-glycosylated. Procathepsin D monoglycosylated at N70 is readily distinguished from the endogenous protein in transfected human cells and thus provides an excellent substrate for studying lysosomal targeting in an homologous system.

Original languageEnglish (US)
Pages (from-to)2001-2006
Number of pages6
JournalJournal of Cell Science
Volume108
Issue number5
StatePublished - 1995
Externally publishedYes

Fingerprint

Glycosylation
Lysosomes
Proteins
Asparagine
Site-Directed Mutagenesis
Membrane Proteins
Fibroblasts
procathepsin D

Keywords

  • Lysosomal targeting
  • Procathepsin D
  • Protein glycosylation

ASJC Scopus subject areas

  • Cell Biology

Cite this

Role of glycosylation in the expression of human procathepsin D. / Fortenberry, S. C.; Schorey, J. S.; Chirgwin, John.

In: Journal of Cell Science, Vol. 108, No. 5, 1995, p. 2001-2006.

Research output: Contribution to journalArticle

Fortenberry, SC, Schorey, JS & Chirgwin, J 1995, 'Role of glycosylation in the expression of human procathepsin D', Journal of Cell Science, vol. 108, no. 5, pp. 2001-2006.
Fortenberry, S. C. ; Schorey, J. S. ; Chirgwin, John. / Role of glycosylation in the expression of human procathepsin D. In: Journal of Cell Science. 1995 ; Vol. 108, No. 5. pp. 2001-2006.
@article{34855b498f1e4c20b0309e251214a256,
title = "Role of glycosylation in the expression of human procathepsin D",
abstract = "Human procathepsin D carries two N-linked glycosylation sites at asparagine residues 70 and 199, widely separated on the surface of the folded protein. We created monoglycosylated procathepsin D molecules by site-directed mutagenesis in vitro of the individual glycosylation sites. With only two exceptions, all 12 mutants of this type were expressed efficiently in mammalian cells. The expressed proteins were stable, targeted to the lysosome, and partially secreted into the medium. When both glycosylation sites were eliminated, however, the expressed proteins (9 different mutants) were stable but most were not secreted and targeted poorly to the lysosome. Mammalian fibroblasts appear to sort nascent procathepsin D efficiently only if it is N-glycosylated. Procathepsin D monoglycosylated at N70 is readily distinguished from the endogenous protein in transfected human cells and thus provides an excellent substrate for studying lysosomal targeting in an homologous system.",
keywords = "Lysosomal targeting, Procathepsin D, Protein glycosylation",
author = "Fortenberry, {S. C.} and Schorey, {J. S.} and John Chirgwin",
year = "1995",
language = "English (US)",
volume = "108",
pages = "2001--2006",
journal = "Journal of Cell Science",
issn = "0021-9533",
publisher = "Company of Biologists Ltd",
number = "5",

}

TY - JOUR

T1 - Role of glycosylation in the expression of human procathepsin D

AU - Fortenberry, S. C.

AU - Schorey, J. S.

AU - Chirgwin, John

PY - 1995

Y1 - 1995

N2 - Human procathepsin D carries two N-linked glycosylation sites at asparagine residues 70 and 199, widely separated on the surface of the folded protein. We created monoglycosylated procathepsin D molecules by site-directed mutagenesis in vitro of the individual glycosylation sites. With only two exceptions, all 12 mutants of this type were expressed efficiently in mammalian cells. The expressed proteins were stable, targeted to the lysosome, and partially secreted into the medium. When both glycosylation sites were eliminated, however, the expressed proteins (9 different mutants) were stable but most were not secreted and targeted poorly to the lysosome. Mammalian fibroblasts appear to sort nascent procathepsin D efficiently only if it is N-glycosylated. Procathepsin D monoglycosylated at N70 is readily distinguished from the endogenous protein in transfected human cells and thus provides an excellent substrate for studying lysosomal targeting in an homologous system.

AB - Human procathepsin D carries two N-linked glycosylation sites at asparagine residues 70 and 199, widely separated on the surface of the folded protein. We created monoglycosylated procathepsin D molecules by site-directed mutagenesis in vitro of the individual glycosylation sites. With only two exceptions, all 12 mutants of this type were expressed efficiently in mammalian cells. The expressed proteins were stable, targeted to the lysosome, and partially secreted into the medium. When both glycosylation sites were eliminated, however, the expressed proteins (9 different mutants) were stable but most were not secreted and targeted poorly to the lysosome. Mammalian fibroblasts appear to sort nascent procathepsin D efficiently only if it is N-glycosylated. Procathepsin D monoglycosylated at N70 is readily distinguished from the endogenous protein in transfected human cells and thus provides an excellent substrate for studying lysosomal targeting in an homologous system.

KW - Lysosomal targeting

KW - Procathepsin D

KW - Protein glycosylation

UR - http://www.scopus.com/inward/record.url?scp=0029070829&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029070829&partnerID=8YFLogxK

M3 - Article

C2 - 7657720

AN - SCOPUS:0029070829

VL - 108

SP - 2001

EP - 2006

JO - Journal of Cell Science

JF - Journal of Cell Science

SN - 0021-9533

IS - 5

ER -