Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells

G S Merz, Eirikur Benedikz, V Schwenk, T E Johansen, L K Vogel, J I Rushbrook, H M Wisniewski

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.
OriginalsprogEngelsk
TidsskriftJournal of Cellular Physiology
Vol/bind173
Udgave nummer3
Sider (fra-til)423-32
Antal sider10
ISSN0021-9541
DOI
StatusUdgivet - 1997

Fingeraftryk

Cystatin C
Cricetulus
Ovary
Brefeldin A
Dimerization
Cysteine Proteinase Inhibitors
Gel Chromatography
Half-Life

Citer dette

Merz, G S ; Benedikz, Eirikur ; Schwenk, V ; Johansen, T E ; Vogel, L K ; Rushbrook, J I ; Wisniewski, H M. / Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells. I: Journal of Cellular Physiology. 1997 ; Bind 173, Nr. 3. s. 423-32.
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abstract = "To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.",
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author = "Merz, {G S} and Eirikur Benedikz and V Schwenk and Johansen, {T E} and Vogel, {L K} and Rushbrook, {J I} and Wisniewski, {H M}",
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Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells. / Merz, G S; Benedikz, Eirikur; Schwenk, V; Johansen, T E; Vogel, L K; Rushbrook, J I; Wisniewski, H M.

I: Journal of Cellular Physiology, Bind 173, Nr. 3, 1997, s. 423-32.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells

AU - Merz, G S

AU - Benedikz, Eirikur

AU - Schwenk, V

AU - Johansen, T E

AU - Vogel, L K

AU - Rushbrook, J I

AU - Wisniewski, H M

PY - 1997

Y1 - 1997

N2 - To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.

AB - To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.

KW - Animals

KW - Brefeldin A

KW - CHO Cells

KW - Cells, Cultured

KW - Clone Cells

KW - Cricetinae

KW - Cycloheximide

KW - Cyclopentanes

KW - Cystatin C

KW - Cystatins

KW - Cysteine Proteinase Inhibitors

KW - Dimerization

KW - Endoplasmic Reticulum

KW - Enzyme-Linked Immunosorbent Assay

KW - Fluorescent Antibody Technique

KW - Humans

KW - Lysosomes

KW - Organelles

KW - Papain

KW - Recombinant Proteins

KW - Skin

KW - Transfection

U2 - 10.1002/(SICI)1097-4652(199712)173:3<423::AID-JCP15>3.0.CO;2-C

DO - 10.1002/(SICI)1097-4652(199712)173:3<423::AID-JCP15>3.0.CO;2-C

M3 - Journal article

VL - 173

SP - 423

EP - 432

JO - Journal of Cellular Physiology

JF - Journal of Cellular Physiology

SN - 0021-9541

IS - 3

ER -