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.