Abstract | AIM: 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.
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Authors | M Bjarnadottir, B S Wulff, M Sameni, B F Sloane, D Keppler, A Grubb, M Abrahamson |
Journal | Molecular pathology : MP
(Mol Pathol)
Vol. 51
Issue 6
Pg. 317-26
(Dec 1998)
ISSN: 1366-8714 [Print] England |
PMID | 10193512
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)
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Chemical References |
- CST3 protein, human
- Cst3 protein, mouse
- Cystatin C
- Cystatins
- Cysteine Proteinase Inhibitors
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Topics |
- Animals
- Biological Transport
- Blotting, Western
- Cell Culture Techniques
- Cerebral Amyloid Angiopathy
(genetics, metabolism)
- Cystatin C
- Cystatins
(genetics, metabolism)
- Cysteine Proteinase Inhibitors
(genetics, metabolism)
- Fluorescent Antibody Technique
- Humans
- Mice
- Mice, Inbred Strains
- Transfection
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