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 approximate to 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.