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

Bjarnadottir, M LU ; Wulff, B S ; Sameni, M ; Sloane, B F ; Keppler, D ; Grubb, A LU orcid and Abrahamson, M LU (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.

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author
; ; ; ; ; and
organization
publishing date
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--&gt;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}},
}