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Physico-chemical properties of the N-terminally truncated L68Q cystatin C found in amyloid deposits of brain haemorrhage patients.

Abstract
Cystatin C, a major extracellular cysteine proteinase inhibitor, is deposited as amyloid in brain haemorrhage patients with hereditary cystatin C amyloid angiopathy (HCCAA). A disease-causing mutation on the genetic level results in the substitution Leu68-->Gln (L68Q) in cystatin C, which causes protein instability. Besides carrying the L68Q substitution, cystatin C in amyloid deposits isolated from patients is N-terminally truncated by 10 amino acids. To elucidate the role of the N-terminal truncation for protein stability and aggregation properties, (delta1-10,L68Q)-cystatin C was produced in an Escherichia coli expression system and characterised. Unlike wild-type cystatin C, this variant rapidly dimerised under physiological conditions. Two unfolding intermediates of (delta1-10,L68Q)-cystatin C were identified, under the same pH and ionic strength conditions as required to form intermediates of full-length L68Q cystatin C. No evidence was found that the N-terminal truncation per se alters protein stability and leads to higher forms of aggregation. Monomeric as well as dimeric L68Q cystatin C incubated with neutrophil elastase was truncated as in HCCAA patients' amyloid. A protein variant with a thrombin cleavage site placed in front of residue Gly11 in L68Q cystatin C was constructed and used to confirm that the N-terminal segment is similarly accessible to proteinases in the monomeric and dimeric states of L68Q cystatin C. Thus, the N-terminal segment of L68Q cystatin C is exposed to proteolytic attack and does not seem to be involved in intramolecular contacts leading to dimerisation or higher-order aggregation. We conclude that the N-terminal truncation likely is an event secondary to amyloid formation, and of no relevance for the development of HCCAA.
AuthorsBernd Gerhartz, Magnus Abrahamson
JournalBiological chemistry (Biol Chem) Vol. 383 Issue 2 Pg. 301-5 (Feb 2002) ISSN: 1431-6730 [Print] Germany
PMID11934268 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Cystatins
  • L68Q cystatin C
Topics
  • Cerebral Amyloid Angiopathy, Familial (genetics, metabolism)
  • Cerebral Hemorrhage (genetics, metabolism)
  • Cystatins (chemistry, genetics, metabolism)
  • Dimerization
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli (genetics)
  • Humans
  • Hydrogen-Ion Concentration
  • Protein Conformation

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