Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angio pathy, leading to stroke. Wild-type and variant cystatin C are cysteine pro teinase inhibitors which form concentration dependent inactive dimers; howe ver, variant cystatin C dimerizes at lower concentrations and has an increa sed susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cys tatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions i n which the wild-type protein forms amorphous aggregates. We also show by c ircular dichroism, steady-state fluorescence and Fourier-transformed infrar ed spectroscopy that the amino acid substitution modifies cystatin C struct ure by destabilizing a-helical structures and exposing the tryptophan resid ue to a more polar environment, yielding a more unfolded molecule. These sp ectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural diff erences between variant and wild-type cystatin C account for the susceptibi lity of the variant protein to unfolding, proteolysis and fibrillogenesis.