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Cystatin C functions in vitro and in vivo. Studies on target enzyme inhibition by cystatin C variants and cystatin C deficient mice.

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Author

  • Katarina Håkansson

Summary, in English

Cystatin C variants with Arg8, Leu9 and/or Val10 replaced by Gly residues were produced by site-directed mutagenesis and E. coli expression. Their interactions with the target proteases, cathepsin B, L, H and S, were studied in vitro in order to provide basic structure-function information of use in the design of specific inhibitors for therapeutic use. Val10 was shown to be the most contributing residue in the N-terminal region in binding of all enzymes. By replacing Leu9 with a Gly residue a more specific inhibitor was obtained. Substitution of Gly for Trp106 in the second hairpin loop of cystatin C demonstrated a general importance of this region in binding to all enzymes. Studies on mouse and rat cystatin C were performed in order to clarify the normal physiological role of cystatin C in vivo. Mouse and rat cystatin C were produced, isolated and characterised. Quantitation of cystatin C demonstrated high overall similarities in distribution patterns in human, mouse and rat tissues and the inhibitory properties of the species variants were also alike. Rat and mouse cystatin C were observed to have slightly higher affinity for cathepsin B than human cystatin C, which could be explained by amino acid substitutions in the N-terminal segment and the first hairpin loop of the enzyme-interacting inhibitor surface. Mouse and rat were concluded to serve as relevant experimental animals for studies of cystatin C. Cystatin C deficient mice were generated. Injected melanoma cells were demonstrated to form fewer metastatic lung colonies in such mice than in wildtype mice. To allow an investigation of the mechanism leading to amyloid fibril formation in patients suffering from hereditary cystatin C amyloid agiopathy (HCCAA), vectors for homologous recombination were constructed to introduce the human wildtype and L68Q-cystatin C genes in the mouse genome. These constructs were functional in embryonic stem cells and should, therefore, be useful for the generation of gene targeted mice strains to accomplish an animal model for detailed studies of HCCAA.

Publishing year

1998

Language

English

Document type

Dissertation

Publisher

Department of Clinical Chemistry, Lund University

Topic

  • Medicinal Chemistry
  • Pharmacology and Toxicology

Keywords

  • amyloidosis
  • E.coli expression
  • Cysteine proteases
  • protease inhibitors
  • Clinical chemistry
  • Klinisk kemi

Status

Published

Supervisor

  • [unknown] [unknown]

ISBN/ISSN/Other

  • ISRN: LUMEDW/MECL-1014-SE

Defence date

12 May 1998

Defence time

10:15

Defence place

Segerfalkssalen at Neurocenter

Opponent

  • Birgitta Tomkinson (Doc)