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Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells.

AbstractAIM:
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.
AuthorsM Bjarnadottir, B S Wulff, M Sameni, B F Sloane, D Keppler, A Grubb, M Abrahamson
JournalMolecular pathology : MP (Mol Pathol) Vol. 51 Issue 6 Pg. 317-26 (Dec 1998) ISSN: 1366-8714 [Print] England
PMID10193512 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)
Chemical References
  • CST3 protein, human
  • Cst3 protein, mouse
  • Cystatin C
  • Cystatins
  • Cysteine Proteinase Inhibitors
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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