Increased body temperature accelerates aggregation of the Leu-68-->Gln mutant cystatin C, the amyloid-forming protein in hereditary cystatin C amyloid angiopathy.
Hereditary cystatin C amyloid angiopathy is a dominantly inherited disorder, characterized by dementia, paralysis, and death from cerebral hemorrhage in early adult life. A variant of the cysteine proteinase inhibitor, cystatin C, is deposited as amyloid in the tissues of the patients and their spinal-fluid level of cystatin C is abnormally low. The disease-associated Leu-68-->Gln mutant (L68Q) cystatin C has been produced in an Escherichia coli expression system and isolated by use of denaturing buffers, immunosorption, and gel filtration. Parallel physicochemical and functional investigations of L68Q-cystatin C and wild-type cystatin C revealed that both proteins effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation, 0.4 and 0.5 nM, respectively) but differ considerably in their tendency to dimerize and form aggregates. While wild-type cystatin C is monomeric and functionally active even after prolonged storage at elevated temperatures, L68Q-cystatin C starts to dimerize and lose biological activity immediately after it is transferred to a nondenaturing buffer. The dimerization of L68Q-cystatin C is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate. The aggregation at physiological concentrations is likewise increased at 40 compared to 37 degrees C, by approximately 60%. These properties of L68Q-cystatin C have bearing upon our understanding of the pathophysiological process of hereditary cystatin C amyloid angiopathy. They might also be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates.
Abrahamson
M
M
Department of Clinical Chemistry, University of Lund, University Hospital, Sweden.
Grubb
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D001831:Body Temperature; D016657:Cerebral Amyloid Angiopathy / Q000235:genetics* / Q000503:physiopathology; D015891:Cystatins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism
D015891:Cystatins; C085910:L68Q cystatin C; D011994:Recombinant Proteins