Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells
(1998) In Molecular Pathology 51. 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... (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)
- author
- Bjarnadottir, M LU ; Wulff, B S ; Sameni, M ; Sloane, B F ; Keppler, D ; Grubb, A LU and Abrahamson, M LU
- organization
- publishing date
- 1998
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Animals, Biological Transport, Blotting, Western, Cell Culture Techniques, Cerebral Amyloid Angiopathy/genetics, Cystatin C, Cystatins/genetics, Cysteine Proteinase Inhibitors/genetics, Fluorescent Antibody Technique, Humans, Mice, Mice, Inbred Strains, Transfection
- in
- Molecular Pathology
- volume
- 51
- pages
- 317 - 326
- publisher
- BMJ Publishing Group
- external identifiers
-
- pmid:10193512
- ISSN
- 1366-8714
- DOI
- 10.1136/mp.51.6.317
- language
- English
- LU publication?
- yes
- id
- ffef2ae8-ce4a-417e-a646-db67ea2f00d9
- date added to LUP
- 2021-10-29 12:23:06
- date last changed
- 2021-10-29 12:23:06
@article{ffef2ae8-ce4a-417e-a646-db67ea2f00d9, abstract = {{<p>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).</p><p>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.</p><p>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.</p><p>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.</p>}}, author = {{Bjarnadottir, M and Wulff, B S and Sameni, M and Sloane, B F and Keppler, D and Grubb, A and Abrahamson, M}}, issn = {{1366-8714}}, keywords = {{Animals; Biological Transport; Blotting, Western; Cell Culture Techniques; Cerebral Amyloid Angiopathy/genetics; Cystatin C; Cystatins/genetics; Cysteine Proteinase Inhibitors/genetics; Fluorescent Antibody Technique; Humans; Mice; Mice, Inbred Strains; Transfection}}, language = {{eng}}, pages = {{317--326}}, publisher = {{BMJ Publishing Group}}, series = {{Molecular Pathology}}, title = {{Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells}}, url = {{http://dx.doi.org/10.1136/mp.51.6.317}}, doi = {{10.1136/mp.51.6.317}}, volume = {{51}}, year = {{1998}}, }