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

Aim-To study the cellular transport of L68Q cystatin C, the cystatin varian t causing amyloidosis and brain haemorrhage in patients suffering from here ditary cystatin C amyloid angiopathy (HCCAA). Methods-Expression vectors for wildtype and L68Q cystatin C were constructe d and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolat ed after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzym e linked immunosorbent assay (ELISA), and for intracellular cystatin C by w estern blotting and immunofluorescence cytochemistry. Colocalisation studie s in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the e ndoplasmic reticulum, and evaluated by confocal microscopy. Results-Concentrations of human cystatin C secreted from transfected NIH/3T 3 cells were similar to those secreted from human cells in culture. In gene ral, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wildtype 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 w ild-type protein. The intracellularly accumulating L68Q cystatin C was inso luble and located mainly in the endoplasmic reticulum. Conclusions-The cellular transport of human cystatin C is impeded by the pa thogenic amino acid substitution Leu68-->Gln. The resulting intracellular a ccumulation 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.