INSTABILITY OF THE AMYLOIDOGENIC CYSTATIN C VARIANT OF HEREDITARY CEREBRAL-HEMORRHAGE WITH AMYLOIDOSIS, ICELANDIC TYPE

A cystatin C variant with L68Q substitution and a truncation of 10 NH2 -terminal residues is the major constituent of the amyloid deposited i n the cerebral vasculature of patients with the Icelandic form of here ditary cerebral hemorrhage with amyloidosis (HCHWA-I). Variant and wil d type cystatin C production, processing, secretion, and clearance wer e studied in human cell lines stably overexpressing the cystatin C gen es. Immunoblot and mass spectrometry analyses demonstrated monomeric c ystatin C in cell homogenates and culture media. While cystatin C form ed concentration-dependent dimers, the HCHWA-I variant dimerized at lo wer concentrations than the wild type protein. Amino-terminal sequence analysis revealed that the variant and normal proteins produced and s ecreted are the full-length cystatin C, Pulse-chase experiments demons trated similar levels of normal and variant cystatin C production and secretion. However, the secreted variant cystatin C exhibited an incre ased susceptibility to a serine protease in conditioned media and in h uman cerebrospinal fluid, explaining its depletion from the cerebrospi nal fluid of HCHWA-I patients. Thus, the amino acid substitution may i nduce unstable cystatin C with intact inhibitory activity and predispo sition to self-aggregation and amyloid fibril formation.