Cellular Uptake of Cystatin C. Subcellular localisation and intracellular effects of a secreted cysteine protease inhibitor
Author
Summary, in English
Cystatin C is a cysteine protease inhibitor, aimed for secretion, as it is produced with a signal peptide. Its target enzymes are thought to be the lysosomal cysteine cathepsins and legumain. Cystatin C has been considered to excert its enzyme inhibiting functions extracellularly, as a defense against enzymes from leaking lysosomes or invading pathogens.
It was demonstrated by various techniques, including flow cytometry, confocal microscopy, ELISA and Western blotting, that cystatin C was internalised in cells of different cell lines after incubation with a physiological concentration of cystatin C. The internalised cystatin C was found in acidic endolysosomal vesicles and co-located with some potential target enzymes, in contrast to the endogenously produced inhibitor, which was mainly found in the endoplasmic reticulum. Cystatin C was non-degraded and still functional as an inhibitor of cysteine cathepsins after uptake, as the total enzyme inhibiting capacity of the cell lysates was increased, suggesting that intracellular cysteine protease activity can be regulated by the uptake. Invasion and migration of MCF-7 breast cancer cells were inhibited when cells were incubated in medium containing cystatin C.
To pin-point the structural requirements for cellular uptake, twelve variants of cystatin C, including wild-type, were produced by site-directed mutagenesis and cleaving of the N-terminal. Positively charged amino acid residues on the surface of the molecule, and the amino acid at position 106 were shown to be important for internalisation. In most cases the uptake was decreased after molecular engineering, but for the variant W106F-cystatin C it was increased. The substitution of W106 affects the cathepsin-inhibiting properties of cystatin C, but it is still an efficient inhibitor of legumain. The increased uptake of this variant also induced an increased inhibition of legumain in lysates of cells after uptake.
It was demonstrated by various techniques, including flow cytometry, confocal microscopy, ELISA and Western blotting, that cystatin C was internalised in cells of different cell lines after incubation with a physiological concentration of cystatin C. The internalised cystatin C was found in acidic endolysosomal vesicles and co-located with some potential target enzymes, in contrast to the endogenously produced inhibitor, which was mainly found in the endoplasmic reticulum. Cystatin C was non-degraded and still functional as an inhibitor of cysteine cathepsins after uptake, as the total enzyme inhibiting capacity of the cell lysates was increased, suggesting that intracellular cysteine protease activity can be regulated by the uptake. Invasion and migration of MCF-7 breast cancer cells were inhibited when cells were incubated in medium containing cystatin C.
To pin-point the structural requirements for cellular uptake, twelve variants of cystatin C, including wild-type, were produced by site-directed mutagenesis and cleaving of the N-terminal. Positively charged amino acid residues on the surface of the molecule, and the amino acid at position 106 were shown to be important for internalisation. In most cases the uptake was decreased after molecular engineering, but for the variant W106F-cystatin C it was increased. The substitution of W106 affects the cathepsin-inhibiting properties of cystatin C, but it is still an efficient inhibitor of legumain. The increased uptake of this variant also induced an increased inhibition of legumain in lysates of cells after uptake.
Department/s
- Division of Clinical Chemistry and Pharmacology
- BioCARE: Biomarkers in Cancer Medicine improving Health Care, Education and Innovation
Publishing year
2013
Language
English
Publication/Series
Lund University Faculty of Medicine Doctoral Dissertation Series
Volume
2013:85
Full text
Document type
Dissertation
Publisher
Division of Clinical Chemistry and Pharmacology, Faculty of Medicine, Lund University
Topic
- Pharmacology and Toxicology
- Medicinal Chemistry
Keywords
- cathepsin
- cell line
- co-localisation
- cystatin C
- internalisation
- legumain
Status
Published
Supervisor
ISBN/ISSN/Other
- ISSN: 1652-8220
- ISBN: 978-91-87449-57-4
Defence date
13 September 2013
Defence time
13:15
Defence place
Segerfalksalen, BMC, Sölvegatan 17, Lund
Opponent
- Elvar Theodorsson