Intracellular accumulation of the amyloidogenic L68Q variant of cystatin C in NIH/3T3 cells
(1998) In Molecular Pathology 51(6). p.317-326- Abstract
- AIM: To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Expression vectors for wild-type and L68Q cystatin C were constructed and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker... (More)
- AIM: To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Expression vectors for wild-type and L68Q cystatin C were constructed and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the endoplasmic reticulum, and evaluated by confocal microscopy. RESULTS: Concentrations of human cystatin C secreted from transfected NIH/3T3 cells were similar to those secreted from human cells in culture. In general, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wild-type human cystatin C. Both immunofluorescence cytochemistry and western blotting experiments showed an increased accumulation of cystatin C in cells expressing the gene encoding L68Q cystatin C compared with cells expressing the gene for the wild-type protein. The intracellularly accumulating L68Q cystatin C was insoluble and located mainly in the endoplasmic reticulum. CONCLUSIONS: The cellular transport of human cystatin C is impeded by the pathogenic amino acid substitution Leu68-- >Gln. The resulting intracellular accumulation and increased localised concentration of L68Q cystatin C might be an important event in the molecular pathophysiology of amyloid formation and brain haemorrhage in patients with HCCAA. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1113910
- author
- Bjarnadottir, M ; Wulff, B ; Keppler, D ; Moin, K ; Sloane, B ; Grubb, A and Abrahamson, Magnus LU
- organization
- publishing date
- 1998
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Molecular Pathology
- volume
- 51
- issue
- 6
- pages
- 317 - 326
- publisher
- BMJ Publishing Group
- external identifiers
-
- scopus:0032423707
- ISSN
- 1366-8714
- language
- English
- LU publication?
- yes
- id
- 176e3a5d-c9d8-4de8-b65c-d5cc2dd07f49 (old id 1113910)
- alternative location
- http://mp.bmj.com/cgi/reprint/51/6/317
- date added to LUP
- 2016-04-01 16:01:49
- date last changed
- 2022-01-28 08:49:45
@article{176e3a5d-c9d8-4de8-b65c-d5cc2dd07f49,
abstract = {{AIM: To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Expression vectors for wild-type and L68Q cystatin C were constructed and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the endoplasmic reticulum, and evaluated by confocal microscopy. RESULTS: Concentrations of human cystatin C secreted from transfected NIH/3T3 cells were similar to those secreted from human cells in culture. In general, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wild-type human cystatin C. Both immunofluorescence cytochemistry and western blotting experiments showed an increased accumulation of cystatin C in cells expressing the gene encoding L68Q cystatin C compared with cells expressing the gene for the wild-type protein. The intracellularly accumulating L68Q cystatin C was insoluble and located mainly in the endoplasmic reticulum. CONCLUSIONS: The cellular transport of human cystatin C is impeded by the pathogenic amino acid substitution Leu68-- >Gln. The resulting intracellular accumulation and increased localised concentration of L68Q cystatin C might be an important event in the molecular pathophysiology of amyloid formation and brain haemorrhage in patients with HCCAA.}},
author = {{Bjarnadottir, M and Wulff, B and Keppler, D and Moin, K and Sloane, B and Grubb, A and Abrahamson, Magnus}},
issn = {{1366-8714}},
language = {{eng}},
number = {{6}},
pages = {{317--326}},
publisher = {{BMJ Publishing Group}},
series = {{Molecular Pathology}},
title = {{Intracellular accumulation of the amyloidogenic L68Q variant of cystatin C in NIH/3T3 cells}},
url = {{http://mp.bmj.com/cgi/reprint/51/6/317}},
volume = {{51}},
year = {{1998}},
}