Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells.
OBJECTIVE
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.
Bjarnadottir
M
M
Department of Clinical Chemistry, University of Lund, University Hospital, Sweden.
Wulff
B S
BS
Sameni
M
M
Sloane
B F
BF
Keppler
D
D
Grubb
A
A
Abrahamson
M
M
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D018929:Cell Culture Techniques; D016657:Cerebral Amyloid Angiopathy / Q000235:genetics / Q000378:metabolism*; D055316:Cystatin C; D015891:Cystatins / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D014162:Transfection
C525030:CST3 protein, human; C525031:Cst3 protein, mouse; D055316:Cystatin C; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors